U.S. patent application number 10/667200 was filed with the patent office on 2005-08-11 for secondary binding site of dipeptidyl peptidase iv (dpiv).
Invention is credited to Bar, Joachim, Brandt, Wolfgang, Demuth, Hans-Ulrich, Heiser, Ulrich, Hoffmann, Torsten, Kuhn-Wache, Kerstin.
Application Number | 20050176622 10/667200 |
Document ID | / |
Family ID | 32072828 |
Filed Date | 2005-08-11 |
United States Patent
Application |
20050176622 |
Kind Code |
A1 |
Kuhn-Wache, Kerstin ; et
al. |
August 11, 2005 |
Secondary binding site of dipeptidyl peptidase IV (DPIV)
Abstract
The present application relates to the secondary binding site of
dipeptidyl peptidase IV, its relationship amongst substrates and to
the modulation of substrate specificity of dipeptidyl peptidase IV
(DP IV, synonym: DPP IV, CD26, EC 3.4.14.5). The application
relates further to compounds that bind to the secondary binding
site of DP IV and their use to modulate the substrate specificity
of DP IV; methods of treatment of various DP IV mediated disorders;
and screening methods for the identification of secondary binding
sites on DP IV and DP IV-like enzymes.
Inventors: |
Kuhn-Wache, Kerstin;
(Halle/Saale, DE) ; Bar, Joachim; (Halle/Saale,
DE) ; Demuth, Hans-Ulrich; (Halle/Saale, DE) ;
Hoffmann, Torsten; (Halle/Saale, DE) ; Heiser,
Ulrich; (Halle/Saale, DE) ; Brandt, Wolfgang;
(Halle/Saale, DE) |
Correspondence
Address: |
BROWN, RUDNICK, BERLACK & ISRAELS, LLP.
BOX IP, 18TH FLOOR
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
32072828 |
Appl. No.: |
10/667200 |
Filed: |
September 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60443417 |
Jan 29, 2003 |
|
|
|
Current U.S.
Class: |
514/1.9 ;
514/11.7; 514/15.7; 514/18.2; 514/18.6; 514/25; 514/340; 514/355;
514/369; 514/4.8; 514/423; 514/592; 514/6.7; 514/6.9; 514/635;
514/7.4 |
Current CPC
Class: |
A61K 38/00 20130101;
C12Q 1/37 20130101; G01N 2500/04 20130101; A61K 31/401 20130101;
C07K 7/06 20130101; A61P 3/00 20180101 |
Class at
Publication: |
514/002 ;
514/025; 514/340; 514/003; 514/369; 514/592; 514/635; 514/017;
514/355; 514/423 |
International
Class: |
A61K 038/28; A61K
038/22; A61K 031/704; A61K 031/455; A61K 031/401; A61K 031/155;
A61K 031/175; A61K 038/08 |
Claims
1-25. (canceled)
26. A method for the treatment of metabolic diseases in a mammal
comprising co-administration to said mammal of (i) a compound
capable of binding to a secondary binding site of DPIV and DPIV
like enzymes and (ii) at least one anti-diabetic agent.
27. A method for the treatment of metabolic diseases in a mammal
comprising co-administration to said mammal of (i) a compound
capable of binding to a secondary binding site of DPIV and DPIV
like enzymes and (ii) at least one anti-diabetic agent selected
from the group consisting of: DP IV inhibitors; PPAR agonists;
biguanides, e.g. metformin, phenformin or buformin; protein tyrosin
phosphatase-1B (PTP-1B) inhibitors; insulin and insulin mimetics;
sulfonylureas and other insulin secretagogues; .alpha.-glucosidase
inhibitors or acarbose; glucagon receptor agonists; GLP-1, GLP-1
mimetics, and GLP-1 receptor agonists; GLP-2, GLP-2 mimetics, and
GLP-2 receptor agonists or teduglutide; exendin-4, exendin-4
mimetics, exenatide; GIP, GIP mimetics, and GIP receptor agonists;
PACAP, PACAP mimetics, and PACAP receptor 3 agonists; PYY, PYY
mimetics, PYY receptor agonists, and PYY receptor antagonists; one
or more cholesterol lowering agents selected from the group
consisting of: HMG-CoA reductase inhibitors, sequestrants,
nicotinyl alkohol, nicotinic acid and salts thereof, PPAR.alpha.
agonists, PPAR.gamma. agonists, PPAR.alpha./.gamma. dual agonists,
inhibitors of cholesterol absorption, acyl CoA:cholesterol
acyltransferase inhibitors, and antioxidants; PPAR.delta. agonists;
anti-obesity compounds; an ileal bile acid transporter inhibitor;
and anti-inflammatory agents.
28. The treatment method according to claim 27 wherein the compound
is selected from the group comprising: a consensus sequence of the
GRF-peptide family, TFTSDY (SEQ ID NO: 1), TFTDDY (SEQ ID NO:4),
H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH, and compounds of formulas
a) to d): 25
29. The treatment method according to claim 27 wherein the
anti-diabetic agent is selected from DPIV inhibitors, metformin,
exenatide, exendin-4, acarbose, insulin, and sulfonylureas.
30. The treatment method according to claim 27 wherein the
metabolic disease is selected from Syndrome X, impaired glucose
tolerance, glucosuria, lipid disorders, dyslipidemia,
hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL
levels, high LDL levels, metabolic acidosis, hyperglycemia,
diabetes mellitus, diabetic neuropathy and nephropathy and of
sequelae caused by diabetes mellitus in mammals, metabolism-related
hypertension and cardiovascular sequelae caused by hypertension in
mammals.
31. The treatment method according to claim 27 for the prophylaxis
and/or treatment of skin diseases, diseases of the mucosa,
autoimmune diseases, inflammatory conditions, psychosomatic,
neuropsychiatric and depressive illnesses, such as anxiety,
depression, sleep disorders, chronic fatigue, schizophrenia,
epilepsy, nutritional disorders, spasm and chronic pain,
atherosclerosis and its sequelae, vascular restenosis, irritable
bowel syndrome, inflammatory bowel disease, including Crohn's
disease and ulcerative colitis, other inflammatory conditions,
pancreatitis, abdominal obesity, neurodegenerative disease,
retinopathy, nephropathy, ovarian hyperandrogenism (polycystic
ovarian syndrome), growth hormone deficiency, neutropenia, tumor
metastasis, benign prostatic hypertrophy, gingivitis, osteoporosis,
and other conditions.
32. A pharmaceutical composition comprising a compound capable of
binding to a secondary binding site of DP IV and DP IV like
enzymes, at least one anti-diabetic agent and a pharmaceutically
acceptable carrier therefore.
33. The pharmaceutical composition of claim 32 wherein said at
least one anti-diabetic agent is selected from the group consisting
of: DP IV inhibitors; PPAR agonists; biguanides, e.g. metformin,
phenformin or buformin; protein tyrosin phosphatase-1B (PTP-1B)
inhibitors; insulin and insulin mimetics; sulfonylureas and other
insulin secretagogues; .alpha.-glucosidase inhibitors or acarbose;
glucagon receptor agonists; GLP-1, GLP-1 mimetics, and GLP-1
receptor agonists; GLP-2, GLP-2 mimetics, and GLP-2 receptor
agonists or teduglutide; exendin-4, exendin-4 mimetics, exenatide;
GIP, GIP mimetics, and GIP receptor agonists; PACAP, PACAP
mimetics, and PACAP receptor 3 agonists; PYY, PYY mimetics, PYY
receptor agonists, and PYY receptor antagonists; one or more
cholesterol lowering agents selected from the group consisting of:
HMG-CoA reductase inhibitors, sequestrants, nicotinyl alkohol,
nicotinic acid and salts thereof, PPAR.alpha. agonists, PPAR.gamma.
agonists, PPAR.alpha./.gamma. dual agonists, inhibitors of
cholesterol absorption, acyl CoA:cholesterol acyltransferase
inhibitors, and antioxidants; PPAR.delta. agonists; anti-obesity
compounds; an ileal bile acid transporter inhibitor; and
anti-inflammatory agents.
34. The pharmaceutical composition of claim 32 wherein the compound
is selected from the group comprising: a consensus sequence of the
GRF-peptide family, TFTSDY (SEQ ID NO: 1), TFTDDY (SEQ ID NO:4),
H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH, and compounds of formulas
a) to d): 26
35. The pharmaceutical composition of claim 32 wherein said
compound is TFTSDY (SEQ ID NO:)1 or TFTDDY (SEQ ID NO:4).
36. The pharmaceutical composition of claim 32 wherein said
compound is H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH.
37. The pharmaceutical composition of claim 32 wherein said
compound capable of binding to a secondary binding site of DP IV
and/or DP IV-like enzymes modulates the selectivity and/or activity
of DP IV or DP IV-like enzymes in a mammal.
38. The pharmaceutical composition of claim 32 wherein said
compound capable of binding to a secondary binding site of DP IV
and/or DP IV-like enzymes substantially prevents of the interaction
of DPIV or DPIV-like enzymes with their binding proteins in a
mammal.
39. The pharmaceutical composition of claim 32 wherein said
secondary binding site of DPIV and DPIV like enzymes comprises the
amino acid residues L90, E91, T152, W154, W157, R310, Y330, R318,
Y416, S460, K463, E464 and R560 of DP IV.
40. The pharmaceutical composition of claim 32 wherein said
secondary binding site of DPIV and DPIV like enzymes comprises the
amino acid residues Glu361 and Ile407 and N.epsilon.2 of His363 of
DP IV.
41. The treatment method according to claim 27 wherein the compound
blocks the product release site of DP IV and/or DP IV-like
enzymes.
42. The treatment method according to claim 27 wherein the compound
substantially prevents the tetramerization of DP IV and/or DP
IV-like enzymes.
43. The treatment method according to claim 27 wherein the compound
comprises 3 to 20 amino acid residues.
44. The treatment method according to claim 27 wherein the compound
comprises 5 to 12 amino acid residues.
45. The treatment method according to claim 27 wherein the compound
comprises 5 to 7 amino acid residues.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority of U.S. patent application
Ser. No. 10/246,817, filed Sep. 18, 2002 and also claims priority
of U.S. Provisional Patent Application Ser. No. 60/443,417 filed
Jan. 29, 2003 both of which are incorporated herein by reference in
their entirety.
FIELD OF THE APPLICATION
[0002] The present application relates to the secondary binding
site of dipeptidyl peptidase IV, its relationship with any type of
substrates and to the modulation of substrate specificity of
dipeptidyl peptidase IV (DP IV, synonym: DPP IV, CD26, EC
3.4.14.5).
[0003] The application relates further to compounds that bind to
the secondary binding site of DP IV and their use to modulate the
substrate specificity of DP IV.
[0004] Furthermore, the present invention provides a method for
treating DP IV mediated disorders, selected from but not restricted
to, impaired glucose tolerance, glucosuria, lipid disorders,
dyslipidemia, hyperlipidaemia, hypertriglyceridemia,
hypercholesterolemia, low HDL levels, high LDL levels, metabolic
acidosis, hyperglycemia, diabetes mellitus, diabetic neuropathy and
nephropathy and of sequelae caused by diabetes mellitus in mammals,
metabolism-related hypertension and cardiovascular sequelae caused
by hypertension in mammals, for the prophylaxis or treatment of
skin diseases and diseases of the mucosae, autoimmune diseases and
inflammatory conditions, and for the treatment of psychosomatic,
neuropsychiatric and depressive illnesses, such as anxiety,
depression, sleep disorders, chronic fatigue, schizophrenia,
epilepsy, nutritional disorders, spasm and chronic pain,
atherosclerosis and its sequelae, vascular restenosis, irritable
bowel syndrome, inflammatory bowel disease, including Crohn's
disease and ulcerative colitis, other inflammatory conditions,
pancreatitis, abdominal obesity, neurodegenerative disease,
retinopathy, nephropathy, Syndrome X, ovarian hyperandrogenism
(polycystic ovarian syndrome), growth hormone deficiency,
neutropenia, tumor metastasis, benign prostatic hypertrophy,
gingivitis, osteoporosis, and other conditions, in mammals.
[0005] The present application also provides a screening method for
the identification of agents, which bind to the secondary binding
site of dipeptidyl peptidase IV.
[0006] Further on, a screening method for the identification and
determination of one or more secondary binding sites of DP IV-like
enzymes is provided.
BACKGROUND OF THE INVENTION
[0007] The exopeptidase dipeptidyl peptidase IV (DP IV, CD26, EC
3.4.14.5) is involved in a number of physiological regulation
processes. On the one hand, DP IV is a peptidase which can change
the activity of a number of peptide hormones, neuropeptides and
chemokines in a very specific manner (Mentlein, Reg. Pep. 85, pp.
9-24 (1999) while on the other hand the DP IV protein molecule
exerts protein-protein interactions, so mediating the regulation of
intracellular signaling cascades. A growing number of peptide
substrates containing proline, alanine or serine in the penultimate
position are identified as substrates of DP IV in vitro and in
vivo. Bioactive peptides which are substrates for DP IV and members
of such regulation cascades are, among others, NPY, GIP, GLP-1,
glucagons, VIP and PACAP. Furthermore, many DP IV-inhibitors
belonging to different structural classes are known.
[0008] It is known that DP IV-Inhibitors may be useful for the
treatment of impaired glucose tolerance and diabetes mellitus
(International Patent Application, Publication Number WO 99/61431,
Pederson R A et al, Diabetes. 1998 Aug; 47(8):1253-8 and Pauly R P
et al, Metabolism 1999 Mar; 48(3):385-9). In particular WO 99/61431
discloses DP IV-Inhibitors comprising an amino acid residue and a
thiazolidine or pyrrolidine group, and salts thereof, especially
L-threo-isoleucyl thiazolidine, L-allo-isoleucyl thiazolidine,
L-threo-isoleucyl pyrrolidine, L-allo-isoleucyl thiazolidine,
L-allo-isoleucyl pyrrolidine, and salts thereof.
[0009] Further examples of low molecular weight dipeptidyl
peptidase IV inhibitors are agents such as
tetrahydroisoquinolin-3-carboxamide derivatives, N-substituted
2-cyanopyroles and pyrrolidines, N-(N'-substituted
glycyl)-2-cyanopyrrolidines, N-(substituted glycyl)-thiazolidines,
N-(substituted glycyl)-4-cyanothiazolidines,
amino-acyl-borono-prolyl-inhibitors, cyclopropyl-fused pyrrolidines
and heterocyclic compounds. Inhibitors of dipeptidyl peptidase IV
are described in U.S. Pat. No. 6,380,398, U.S. Pat. No. 6,011,155;
U.S. Pat. No. 6,107,317; U.S. Pat. No. 6,110,949; U.S. Pat. No.
6,124,305; U.S. Pat. No. 6,172,081; WO 95/15309, WO 99/61431, WO
99/67278, WO 99/67279, DE 198 34 591, WO 97/40832, DE 196 16 486 C
2, WO 98/19998, WO 00/07617, WO 99/38501, WO 99/46272, WO 99/38501,
WO 01/68603, WO 01/40180, WO 01/81337, WO 01/81304, WO 01/55105, WO
02/02560 and WO 02/14271, WO 02/04610, WO 02/051836, WO 02/068420,
WO 02/076450; WO 02/083128, WO 02/38541, WO 03/000180, WO
03/000181, WO 03/000250, WO 03/002530, WO 03/002531, WO
03/002553,WO 03/002593, WO 03/004496, WO 03/004498, WO 03/024965,
WO 03/024942, WO 03/035067, WO 03/037327, WO 03/035057, WO
03/045977, WO 03/055881, WO 03/68748, WO 03/68757, WO 03/057666, WO
03057144, WO 03/040174 and WO 03/033524, the teachings of which are
herein incorporated by reference in their entirety, especially
concerning these inhibitors, their definition, uses and their
production.
[0010] Definitions
[0011] The term "active site" as used in the claims and in the
description is generally known to a person skilled in the art and
means the catalytical site or region of DP IV and/or DP IV-like
enzymes, which is responsible for the cleavage or biodegredation of
the natural substrates of these enzymes.
[0012] The term "secondary binding site" as used in the claims and
in the description means a site or region of DP IV and/or DP V-like
enzymes, which is different from the active site, e.g. a) a
receptor site or b) a substrate recognition site or c) a regulatory
site or allosteric site. The secondary binding site can a) affect
the receptor function of DP IV and/or DP IV-like enzymes or b)
affect the catalytic activity of DP IV and/or DP IV-like enzymes,
especially the selectivity and/or specificity of these enzymes
toward their substrates. Some secondary binding sites are
complementary to the structure of the substrate of the enzymes,
co-enzymes, co-factors and other compounds, which are involved in
the activity and function of the enzyme. The enzymes may even have
one or more secondary binding sites.
[0013] The secondary binding site is an element of the enzyme
distinct from the catalytic site with a different form of
regulation than the competition between substrates and inhibitors
at the catalytic site (Darnell, J., Lodish, H. and Baltimore, D.
1990, Molecular Cell Biology 2.sup.nd Edition, Scientific American
Books, New York, page 63).
[0014] The term "DP IV and/or DP IV-like enzymes" means DP IV or DP
IV-like enzymes or both.
[0015] The term "activity modifying" as used in the claims and in
the description means both the modification of the enzymatic
activity as well as the modification of the selectivity or
specificity of DP IV and/or DP IV-like enzymes. Especially
preferred is the modification of the selectivity or specificity of
DP IV and/or DP IV-like enzymes toward their natural
substrates.
[0016] "Effectors", as that term is used herein, are defined as
molecules or ligands that interact with a secondary binding site of
DP IV and/or DP IV-like enzymes, thereby changing their catalytical
behaviour in vitro and/or in vivo. Effectors can increase or
decrease the catalytical activity of the enzymes. Examples of
effectors are activators or inhibitors. The effectors as used
herein do not act at the active sites of enzymes, but at at least
one secondary binding site, e.g. a regulatory site, or an
allosteric site. The term "effectors" is used herein synonymously
with "agent" or "compound".
[0017] The term "DP IV-inhibitor" is generally known to a person
skilled in the art and means enzyme inhibitors, which interact with
the active site or catalytical site of DP IV or DP IV-like enzymes
or DP IV and/or DP IV-like enzymes and inhibit the catalytical
activity of these enzymes.
[0018] The "use of effectors" encompasses one single effector or
two or more effectors together. Preferred is the use of two
effectors. Especially preferred is the use of one single
effector.
[0019] "Conditions associated with diabetes mellitus" itself
include hyperglycaemia, insulin resistance, including acquired
insulin resistance and obesity. Further conditions associated with
diabetes mellitus itself include hypertension and cardiovascular
disease, especially atherosclerosis and conditions associated with
insulin resistance. Conditions associated with insulin resistance
include polycystic ovarian syndrome and steroid induced insulin
resistance and gestational diabetes.
[0020] "Complications associated with diabetes mellitus" includes
renal disease, especially renal disease associated with Type 2
diabetes, neuropathy and retinopathy.
[0021] Renal diseases associated with Type 2 diabetes include
nephropathy, glomerulonephritis, glomerular sclerosis, nephrotic
syndrome, hypertensive nephrosclerosis and end stage renal
disease.
[0022] Diabetes mellitus is preferably Type 2 diabetes.
[0023] Classification of Diabetes
[0024] Clinical diabetes may be divided into four general
subclasses, including (1) type 1 (caused by beta cell destruction
and characterized by absolute insulin deficiency) (2) type 2
(characterized by insulin resistance and relative insulin
deficiency (3) other specific types of diabetes (associated with
various identifiable clinical conditions or syndromes) and (4)
gestational diabetes mellitus. In addition to these clinical
categories, two conditions--impaired glucose tolerance and impaired
fasting glucose--refer to a metabolic state intermediate between
normal glucose homeostasis and overt diabetes. These conditions
significantly increase the later risk of diabetes mellitus and may
in some instances be part of its natural history. It should be
noted that patients with any form of diabetes might require insulin
treatment at some point. For this reason the previously used terms
insulin-dependent diabetes (for type I diabetes mellitus) and
non-insulin-dependent diabetes (for type 2) have been
eliminated.
[0025] Diabetes is currently classified as follows:
[0026] Clinical Diabetes
[0027] 1. Type 1 diabetes, formerly called insulin-dependent
diabetes mellitus (IDDM) or "juvenile-onset diabetes"
[0028] 2. Type 2 diabetes, formerly called non-insulin-dependent
diabetes (NIDDM) or "adult-onset diabetes"
[0029] 3. Other specific types
[0030] a) Genetic defects of .beta.-cell function (e.g.,
maturity-onset diabetes of the young [MODY] types 1-3 and point
mutations in mitochondrial DNA)
[0031] b) Genetic defects in insulin action
[0032] c) Disease of the exocrine pancreas (e.g., pancreatitis,
trauma, pancreatectomy, neoplasia, cystic fibrosis,
hemochromatosis, fibrocalculous pancreatopathy).
[0033] d) Endocrinopathies (e.g. acromegaly, Cusing's syndrome,
hyperthyroidism, pheochromocytoma, glucagonoma, somatostinoma,
aldosteronoma)
[0034] e) Drug or chemical induced (e.g., glucocorticosteroids,
thiazides, diazoxide, pentamidine, vacor, thyroid hormone,
phenytoin [Dilantin], .beta.-agonists, oral contraceptives)
[0035] f) Infections (e.g., congenital rubella,
cytomegalovirus)
[0036] g) Uncommon forms of immune-mediated diabetes (e.g.,
"stiff-man", syndrome, anti-insulin receptor antibodies)
[0037] h) Other genetic syndromes (e.g., Down, Klinefelter's,
Turner's syndrome, Huntington's disease, myotonic dystrophy,
lipodystrophy, ataxia-telangiectasia)
[0038] 4. Gestational diabetes mellitus
[0039] Risk Categories
[0040] 1. Impaired fasting glucose
[0041] 2. Impaired glucose tolerance
[0042] Type 1 Diabetes Mellitus
[0043] Patients with this disorder have little or no insulin
secretory capacity and depend on exogenous insulin to prevent
metabolic decompensation (e.g., ketoacidosis) and death.
[0044] Commonly but not always, diabetes appears abrubtly (i.e.,
over days and weeks) in previously healthy non-obese children or
young adults; in older age groups it may have a more gradual onset.
At the time of initial evaluation the typical patient often appears
ill, has marked symptoms (e.g., polyuria, polydipsia, polyhagia,
and weight loss), and may demonstrate ketoacidosis. Type 1 diabetes
is believed to have a long a symptomatic p reclinical stage often
lasting years, during which pancreatic beta cells are gradually
destroyed by an autoimmune attack that is influenced by HLA and
other genetic factors, as well as the environment. Initially,
insulin therapy is essential to restore metabolism toward normal.
However, a so-called honeymoon period may follow and last weeks or
moths, during which time smaller doses of insulin are required
because of partial recovery of beta cell function and reversal of
insulin resistance caused by acute illness. Thereafter, insulin
secretory capacity is gradually lost (over several years). The
association of type 1 diabetes with specific immune response (HLA)
genes and the presence of antibodies to islet cells and their
constituents provides strong support for the theory that type 1
diabetes is an autoimmune disease. This syndrome accounts for lese
than 10% of diabetes in United States.
[0045] Type 2 Diabetes Mellitus
[0046] Type 2, by far the most common form of the disease, is found
in over 90% of the diabetic patient population. These patients
retain a significant level of endogenous insulin secretory
capacity. However, insulin levels are low relative to the magnitude
of insulin resistance and ambient glucose levels. Type 2 patients
are not dependent on insulin for immediate survival and ketosis
rarely develops, except under conditions of great physical stress.
Nevertheless, these patients may require insulin therapy to control
hyperlgycemia. Type 2 diabetes typically appears after the age of
40 years, has a high rate of genetic penetrance unrelated to HLA
genes, and is associated with obesity. The clinical features of
type 2 diabetes may be mild (fatigue, weakness, dizziness, blurred
vision, or other non-specific complaints may dominate the picture)
or may be tolerated for many years before the patient seeks medical
attention. Moreover, if the level of hyperglycemia is insufficient
to produce symptoms, the disease may become evident only after
complications develop.
[0047] Other Specific Types of Diabetes
[0048] This category encompasses a variety of diabetic syndromes
attributed to a specific disease, drug, or condition. Genetic
research has provided new insights into pathogenesis of MODY, which
was formerly included as a form of type 2 diabetes. MODY
encompasses several genetic defects of beta cell function, among
which mutations at several genetic loci on different chromosomes
have been identified. The most common forms--MODY type 3--is
associated with a mutation for a transcription factor encoded on
chromosome 12 named hepatocyte nuclear 1.alpha. (HNF 1, also known
as TCF1) and--MODY type 2 is associated with mutations of the
glucokinase gene (on chromosome 7) Mutations of the HNF-4.alpha.
gene (on chromosome 20) are responsible for type 1 of MODY. Each of
these conditions is inherited in an autosomal dominant pattern. Two
new rare forms of MODY are associated with mutations of the
HNF-1.beta. (on chromosome 17) and an insulin gene transcription
factor termed PDX-1 or 1DX-1 (on chromosome 13).
[0049] The distinction between the various subclasses of diabetes
mellitus is usually made on clinical grounds. However, a small
subgroup of patients are difficult to classify, that is, they
display features common to both type 1 and 2 diabetes. Such
patients are commonly non-obese and have reduced insulin secretory
capacity that is not sufficient to make them ketosis prone. Many
initially respond to oral agents but, with time , require insulin.
Some appear to have a slowly evolving form of type 1 diabetes,
whereas others defy easy categorization.
[0050] Gestational Diabetes
[0051] The term gestational diabetes describes women with impaired
glucose tolereance that appears or is first detected during
pregnancy. Gestational diabetes usually appears in the 2.sup.nd or
3.sup.rd trimester, a time when pregnancy-associated insulin
antagonistic hormones peak. After delivery, glucose tolerance
generally (but not always) reverts to normal.
[0052] Diagnosis
[0053] The diagnosis of diabetes is usually straightforward when
the classic symptoms of polyuria, polydipsia, and weight loss are
present. All that is required is a random plasma glucose
measurement from venous blood that is 200 mg/dL or greater. If
diabetes is suspected but not confirmed by a random glucose
determination, the screening test of choice is overnight fasting
plasma glucose level. The diagnosis is established if fasting is
equal to or greater than 126 mg/dL on at least two separate
occasions.
[0054] Related Conditions
[0055] Impaired Glucose Tolerance and Impaired Fasting Glucose
[0056] Impaired glucose tolerance (IGT) and impaired fasting
glucose (IFG) are terms applied to individuals who have glucose
levels that are higher than normal, (under fed or fasting
conditions, respectively) but lower than those accepted as
diagnostic for diabetes mellitus. Both conditions are associated
with an increased risk for cardiovascular disease, but do not
produce the classic symptoms or the microvascular and neuropathic
complications associated with diabetes mellitus. In a subgroup of
patients (about 25 to 30%), however, type 2 diabetes eventually
develops.
[0057] Impaired Glucose Metabolism
[0058] Impaired Glucose Metabolism (IGM) is defined by blood
glucose levels that are above the normal range but are high enough
to meet the diagnostic criteria for type 2 diabetes mellitus. The
incidence of IGM varies from country to country, but usually occurs
2-3 time more frequently than overt diabetes. Until recently,
individuals with IGM were felt to be pre-diabetics, but data from
several epidemiological studies argue that subjects with IGM are
heterogeneous with respect to their risk of diabetes and their risk
of cardiovascular morbidity and mortality. The data suggest that
subjects with IGM , in particular, those with impaired glucose
tolerance (IGT), do not always develop diabetes, but whether they
are diabetic or not, they are, nonetheless, at high risk for
cardiovascular morbidity and mortality. Among subjects with IGM,
about 58% have Impaired Glucose tolerance (IGT), another 29% have
impaired fasting glucose (IFG), and 13% have both abnormalities
(IFG/IGT). As discussed above, IGT is characterized by elevated
post-prandial (post-meal) hyperglycemia while IFG has been defined
by the ADA (American Diabetes Association) on the basis of fasting
glycemic values.
[0059] The categories of (a) normal glucose tolerance (NGT), (b)
impaired glucose metabolism (IGM) and (c) overt type 2 diabetes
mellitus are periodically revised and adopted by the Expert
Committee of the American Diabetes Association (ADA). The actual
values as defined in "Report of the Expert Committee on the
Diagnosis and Classification of Diabetes Mellitus. Diabetes Care
(26) 1, 2003, 5-20" and "The Diabetes Ready-Reference Guide for
Health Care Professionals, 2000, published by the American Diabetes
Association" are:
[0060] a) Normal Glucose Tolerance (NGT)=fasting glucose level
<6.1 mmol/L or less than 110 mg/dl and a 2 h post-prandial
glucose level of <7.8 mmol/L or <140 mg/dl.
[0061] b) Impaired Glucose Metabolism (IGM) is impaired fasting
glucose (IFG) defined as IFG=fasting glucose level of 6.1-7.0
mmol/L or 110-126 mg/dl and/or impaired glucose tolerance (IGT)=a 2
h post-prandial glucose level (75 g OGTT) of 7.8-11.1 mmol/L or
140-200 mg/dl).
[0062] c) Type 2 diabetes=fasting glucose of greater than 7 mmol/L
or 126 mg/dl or a 2 h post-prandial glucose level (75 g OGTT) of
greater than 11.1 mmol/L or 200 mg/dl.
[0063] These criteria were defined using the WHO recommended
conditions for administration of an oral glucose tolerance test (75
g 0 GTT) i. e., the oral administration of a glucose load
containing the equivalent of 75 g of anhydrous glucose dissolved in
water with a blood sample taken 2 hours later to analyze to
post-prandial glucose. Other OGTT test conditions have confirmed
the associated risks of the IGT and IFG categories including: 1)
using 50 g glucose instead of 75 g, 2) using a casual (non-fasting)
glucose sample as the analyte, and 3) analysing the post-prandial
glucose at 1 hour rather than 2 hours post-glucose load. Under all
of these conditions, the glycemic categories defined above have
been linked to the increased risks described below, but the
standardized OGTT is preferred in order to minimize variations in
test results.
[0064] Insulin resistance is not primarily due to a diminished
number of insulin receptors but to a post-insulin receptor binding
defect that is not yet understood. This resistance to insulin
responsiveness results in insufficient insulin activation of
glucose uptake, oxidation and storage in muscle and inadequate
insulin repression of lipolysis in adipose tissue and of glucose
production and secretion in the liver.
[0065] The term "subject" as used herein, refers to an animal,
preferably a mammal, most preferably a human, who has been the
object of treatment, observation or experiment.
[0066] The term "therapeutically effective amount" as used herein,
means that amount of active compound or pharmaceutical agent that
elicits the biological or medicinal response in a tissue system,
animal or human, being sought by a researcher, veterinarian,
medical doctor or other clinician, which includes alleviation of
the symptoms of the disease or disorder being treated.
[0067] As used herein, the term "pharmaceutically acceptable"
embraces both human and veterinary use: for example the term
"pharmaceutically acceptable" embraces a veterinarily acceptable
compound or a compound acceptable in human medicine a health
care.
[0068] Throughout the description and the claims the expression
"acyl" can denote a C1-20 acyl residue, preferably a C1-8 acyl
residue and especially preferred a C1-4 acyl residue; "cycloalkyl"
can denote a C3-12 cycloalkyl residue, preferably a C4, C5 or C6
cycloalkyl residue; and "carbocyclic" can denote a C3-12
carbocyclic residue, preferably a C4, C5 or C6 carbocyclic residue.
"Heteroaryl" is defined as an aryl residue, wherein 1 to 4, and
more preferably 1, 2 or 3 ring atoms are replaced by heteroatoms
like N, S or O. "Heterocyclic" is defined as a cycloalkyl residue,
wherein 1, 2 or 3 ring atoms are replaced by heteroatoms like N, S
or O. "Peptides" are selected from dipeptides to decapeptides,
preferred are dipeptides, tripeptides, tetrapeptides and
pentapeptides. The amino acids for the formation of the "peptides"
can be selected from those listed above.
[0069] Throughout the description and the claims the expression
"alkyl" can denote a C.sub.1-50 alkyl group, preferably a
C.sub.6-30 alkyl group, especially a C.sub.8-12 alkyl group; for
example, an alkyl group may be a methyl, ethyl, propyl, isopropyl
or butyl group. The expression "alk", for example in the expression
"alkoxy", and the expression "alkan", for example in the expression
"alkanoyl", are defined as for "alkyl"; aromatic compounds are
preferably substituted or optionally unsubstituted phenyl, benzyl,
naphthyl, biphenyl or anthracene groups, which preferably have at
least 8 C atoms; the expression "alkenyl" can denote a C.sub.2-10
alkenyl group, preferably a C.sub.2-6 alkenyl group, which has the
double bond(s) at any desired location and may be substituted or
unsubstituted; the expression "alkynyl" can denote a C.sub.2-10
alkynyl group, preferably a C.sub.2-6 alkynyl group, which has the
triple bond(s) at any desired location and may be substituted or
unsubstituted; the expression "substituted" or substituent can
denote any desired substitution by one or more, preferably one or
two, alkyl, alkenyl, alkynyl, mono- or multi-valent acyl, alkanoyl,
alkoxyalkanoyl or alkoxyalkyl groups; the afore-mentioned
substituents may in turn have one or more (but preferably zero)
alkyl, alkenyl, alkynyl, mono- or multi-valent acyl, alkanoyl,
alkoxyalkanoyl or alkoxyalkyl groups as side groups; organic
amines, amides, alcohols or acids, each having from 8 to 50 C
atoms, preferably from 10 to 20 C atoms, can have the formulae
(alkyl).sub.2N- or alkyl-NH-, --CO--N(alkyl).sub.2 or
--CO--NH(alkyl), -alkyl-OH or -alkyl-COOH.
SUMMARY OF THE INVENTION
[0070] The inventors of the present application unexpectedly show,
that the biodegradation of different substrates, which bind to the
same catalytic domain of DP IV and/or DP IV-like enzymes, can be
modulated in an unexpected very specific manner.
[0071] The invention provides a method to identify the site in the
DP IV protein or in DP IV-like enzymes or in both, DP IV and DP
IV-like enzymes which is responsible for the modulation of the
substrate specificity of DP IV and also provides new compounds,
which regulate the substrate specificity of DP IV and which are
useful for the treatment of, for example, impaired glucose
tolerance, glucosuria, lipid disorders, dyslipidemia,
hyperlipidaemia, hypertriglyceridemia, hypercholesterolemia, low
HDL levels, high LDL levels, metabolic acidosis, hyperglycemia,
diabetes mellitus, diabetic neuropathy and nephropathy and of
sequelae caused by diabetes mellitus in mammals, metabolism-related
hypertension and cardiovascular sequelae caused by hypertension in
mammals, for the prophylaxis or treatment of skin diseases and
diseases of the mucosae, autoimmune diseases and inflammatory
conditions, and for the treatment of psychosomatic,
neuropsychiatric and depressive illnesses, such as anxiety,
depression, sleep disorders, chronic fatigue, schizophrenia,
epilepsy, nutritional disorders, spasm and chronic pain,
atherosclerosis and its sequelae, vascular restenosis, irritable
bowel syndrome, inflammatory bowel disease, including Crohn's
disease and ulcerative colitis, other inflammatory conditions,
pancreatitis, abdominal obesity, neurodegenerative disease,
retinopathy, nephropathy, Syndrome X, ovarian hyperandrogenism
(polycystic ovarian syndrome), growth hormone deficiency,
neutropenia, tumor metastasis, benign prostatic hypertrophy,
gingivitis, osteoporosis, and other conditions, in mammals.
[0072] Other Potential target diseases and the actual stage of
research are summarized in table 1.
1TABLE 1 Target diseases for DP IV-inhibition Target disease
Development stage Comments AIDS cell culture mechanism not fully
understood Autoimmune cell culture and animal high doses necessary
diseases models Rheumatoid animal models Arthritis Multiple
sclerosis animal experiments Psoriasis cell culture and animal
experiments Graft rejection animal experiments Wound healing
Anxiety effective in animal models Diabetes type II Phase II
studies Cancer cell culture, animal models DP IV and FAP are
involved Obesity animal experiments NPY, GLP-1 and orexine
mediated
[0073] The problem of the invention is solved by using a prolyl
oligopeptidase (POP) based computer-generated model of DP IV and of
the crystal structure of DP IV for the identification of secondary
binding sites of DP IV and by providing specific compounds, which
bind to at least one secondary binding site and are able to modify
very differently and/or specifically the DP IV-catalyzed truncation
of substrates of DP IV and DP IV-like enzymes, e.g. bioactive
peptides. The overall result is a significant increase of substrate
dependent DP IV-selectivity by such compounds and thereby
minimization of side reactions with other substrates and as such of
potential side effects after complete inhibition of DP
IV-activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] Further understanding of these and other aspects of the
instant invention may be had by reference to the figures
wherein:
[0075] FIG. 1 shows a plot of the distribution of the backbone
dihedral angles of the complete model of human DP IV. There are no
residues in disallowed regions, but some residues are located in
only generously allowed areas. Most of them represent residues in
loops at the surface of the propeller domain.
[0076] The plot statistics is a measure for the plot quality. The
plot statistics for FIG. 1 is as follows:
2 Residues in most favoured regions 435 62.8% Residues in
additionally allowed regions 226 32.6% Residues in generously
allowed regions 32 4.6% Residues in disallowed regions 0 0.0%
Number of non-glycine and non-proline residues 693 100.0% Number of
end-residues (excl. Gly and Pro) 1 Number of glycine residues
(shown as triangles) 43 Number of proline residues 29 Total number
of residues 766
[0077] FIG. 2 shows the analysis of the quality of the model of
human DP IV with regard to some essential stereo-chemical
parameters. The plot statistics is as follows:
3 No. of Comparison band Values widths Stereochemical No. of
Parameter Typical Band from parameter data points value value width
mean a. %-tage residues in 693 63.9 83.8 10.0 -2.0 WORSE A, B, L b.
Omega angle 765 4.7 6.0 3.0 -0.4 Inside standard deviation c. Bad
contacts/100 19 2.5 4.2 10.0 -0.2 Inside residues d. Zeta angle
standard 723 3.5 3.1 1.6 0.3 Inside deviation e. H-bond energy 389
0.9 0.8 0.2 0.5 Inside standard deviation f. Overall G-factor 766
-0.6 -0.4 0.3 -0.5 Inside
[0078] FIG. 3 shows the computer-assisted structure model of DP IV
and the ADA-binding site (indicated by the arrows and amino acid
residue numbers);
[0079] FIG. 4 shows the active site of DP IV docked with the active
site DP IV-inhibitor isoleucyl pyrrolidine (Ile-Pyr) (dark
gray);
[0080] FIG. 5 shows the interaction of Lys-Z-nitro-pyrrolidine with
the active site of DP IV;
[0081] FIG. 6 shows the tetrahedral intermediate of Asp-Pro-pNA
bound to DP IV;
[0082] FIG. 7 shows the interaction of the HIV-tat(1-9) protein
with DP IV;
[0083] FIG. 8 shows the docking of the N-terminal nonapeptide of
the tromboxane receptor;
[0084] FIG. 9 shows the 3D-structure model of the interaction
between GIP (black thread) and human DP IV;
[0085] FIG. 10 shows the docking arrangement of GIP (black) to the
active site of DP IV;
[0086] FIG. 11 shows the molecular dynamic simulation based model
of the tertiary structure of GIP (middle part), bound to DP IV.
Important amino acid residues from the enzyme are shown in light
gray, those from GIP are shown in black, respectively;
[0087] FIG. 12 shows the docking of VIP (black) to the active site
of DP IV;
[0088] FIG. 13 shows the docking of the C-terminal part of VIP to
DP IV;
[0089] FIG. 14 shows the docking of glucagon (black) to the active
site of DP IV;
[0090] FIG. 15 shows the molecular dynamic simulation based model
of the hexapeptide TFTSDY, bound to the secondary binding site of
DP IV. Important amino acid residues from the enzyme are light
gray, those from the hexapeptide are marked in dark gray,
respectively;
[0091] FIG. 16 shows the prolongation of the half-lifes of GIP,
Glucagon, PACAP-27 and PACAP-38 by the hexapeptide TFTSDY in a DP
IV (porcine and recombinant human) catalyzed peptide truncation
test;
[0092] FIG. 17 shows the DP IV-catalyzed hydrolysis of RANTES1-15
with (black solid triangle or broken line) or without TFTSDY (black
solid square or straight line);
[0093] FIG. 18 shows the DP IV-catalyzed hydrolysis of GIP with
(black solid triangle) or without TFTSDY (black solid square);
[0094] FIG. 19 shows the DP IV-catalyzed hydrolysis of glucagon
with (black solid circle) or without TFTSDY (black solid
triangle);
[0095] FIG. 20 shows a plot of the distribution of the backbone
dihedral angles of the complete model of porcine DP IV. All
residues are in most favored and additional allowed regions. The
plot statistics is as follows:
4 Residues in most favoured regions (A, B, L) 457 66.0% Residues in
additional allowed regions (a, b, l, p) 235 34.0% Residues in
generously allowed regions (.about.a, .about.b, .about.l, .about.p)
0 0.0% Residues in disallowed regions 0 0.0% Number of non-glycine
and non-proline residues 692 100.0% Number of end-residues (excl.
Gly and Pro) 2 Number of glycine residues (shown as triangles) 44
Number of proline residues 31 Total number of residues 769
[0096] FIG. 21 shows the analysis of the quality of the model of
porcine DP IV with regard to some essential stereo-chemical
parameters of the main chain. The plot statistics is as
follows:
5 No. of Comparison band No. of Values widths data Parameter
Typical Band from Stereochemical parameter points value value width
mean a. %-tage residues in A, B, L 692 66.0 83.8 10.0 -1.8 WORSE b.
Omega angle standard 765 8.5 6.0 3.0 0.8 Inside deviation c. Bad
contacts/100 residues 2 0.3 4.2 10.0 -0.4 Inside d. Zeta angle
standard deviation 725 2.1 3.1 1.6 -0.6 Inside e. H-bond energy
standard 400 0.8 0.8 0.2 -0.1 Inside deviation f. Overall G-factor
769 -0.4 -0.4 0.3 -0.1 Inside
[0097] FIG. 22 shows the analysis of the quality of the model of
porcine DP IV with regard to some essential stereo-chemical
parameters of the side chains. The plot statistics is as
follows:
6 No. of band No. of Comparison Values widths data Parameter
Typical from Stereochemical parameter points value value Band width
mean a. Chi-1 gauche minus 156 16.2 18.1 6.5 -0.3 Inside standard
deviation b. Chi-1 trans standard 218 17.9 19.0 5.3 -0.2 Inside
deviation c. Chi-1 gauche plus standard 279 17.6 17.5 4.9 0.0
Inside deviation d. Chi-1 pooled standard 653 18.0 18.2 4.8 0.0
Inside deviation e. Chi-2 trans standard 144 16.3 20.4 5.0 -0.8
Inside deviation
[0098] FIG. 23 shows soluble DP IV from prorcine kidney, which
forms a 2-2-2 symmetric assembly as dimer of dimers. The view is
along one two-fold axis. Potential glycosylation sites are
indicated as grey spheres, black spheres are the sites modified in
the crystal structure. The transmembrane helices and their
orientation to the membrane were modeled to illustrate how
tetramerization of DP IV can mediate cell-cell contacts. The figure
was prepared by using the program MOLSCRIPT and RASTER3D.
[0099] FIG. 24 shows a topology diagram illustrating the domain
structure of porcine kidney DP IV. Blade IV of the propeller is
involved in both the dimer contact (IV A-IV B: L235-P255, together
with the highlighted C-terminal three secondary structure elements
F713-C762) and the tetramerization of DP IV (IV A-IV C and IV B-IV
C, not shown).
[0100] FIG. 25 shows oligomerization interfaces. (A) Detailed view
perpendicular to the dimer two-fold axis. The experimental electron
density after phase extension to 2.0 .ANG. resolution is
superimposed on key residues mediating the contact. (B) View along
the two-fold axis on the tetramerization interface. Blades IV of
each subunit align to form an eight-bladed antiparallel .beta.
sheet. The highlighted Leu294 and Val341 are involved in ADA
binding. The figure was prepared using BOBSCRIPT, MOLSCRIPT and
RASTER3D.
[0101] FIG. 26 shows substrate recognition by procine kidney DP IV.
(A) The peptidomimetic active-site inhibitor p-Iodo-Phe-Pyr-CN is
bound to active site. The accessible surface is indicated and
cut-open (dark gray area at the top of the figure) for better
visibility. (B) Schematic representation of the active site access
in tricom and DP IV. The figure was prepared by using the programs
MAIN , MOLSCRIPT, GRASP and RASTER3D.
[0102] FIG. 27 shows the DP IV-catalyzed hydrolysis of GIP.sub.1-42
with (black solid squares) or without the heptapeptide
H-Ser-D-Glu-Thr-Gly-D-V- al-D-Lys-D-Val-OH (black solid
triangles).
DETAILED DESCRIPTION OF THE INVENTION
[0103] The inventors of the present application unexpectedly show,
that the biodegradation of substrates, which bind to the same
catalytic domain of DP IV, can be modulated very specifically.
[0104] One aspect of the invention is to identify the site in the
DP IV protein, which is responsible for the modulation of the
substrate specificity and selectivity of DP IV and DP IV-like
enzymes and to provide new compounds, which regulate the substrate
selectivity and/or activity of DP IV and DP IV-like enzymes and
which are useful for the treatment of, for example, impaired
glucose tolerance, glucosuria, lipid disorders, dyslipidemia,
hyperlipidaemia, hypertriglyceridemia, hypercholesterolemia, low
HDL levels, high LDL levels, metabolic acidosis, hyperglycemia,
diabetes mellitus, diabetic neuropathy and nephropathy and of
sequelae caused by diabetes mellitus in mammals, metabolism-related
hypertension and cardiovascular sequelae caused by hypertension in
mammals, for the prophylaxis or treatment of skin diseases and
diseases of the mucosae, autoimmune diseases and inflammatory
conditions, and for the treatment of psychosomatic,
neuropsychiatric and depressive illnesses, such as anxiety,
depression, sleep disorders, chronic fatigue, schizophrenia,
epilepsy, nutritional disorders, spasm and chronic pain,
atherosclerosis and its sequelae, vascular restenosis, irritable
bowel syndrome, inflammatory bowel disease, including Crohn's
disease and ulcerative colitis, other inflammatory conditions,
pancreatitis, abdominal obesity, neurodegenerative disease,
retinopathy, nephropathy, Syndrome X, ovarian hyperandrogenism
(polycystic ovarian syndrome), growth hormone deficiency,
neutropenia, tumor metastasis, benign prostatic hypertrophy,
gingivitis, osteoporosis, and other conditions.
[0105] Usually, DP IV is inhibited by compounds mimicking the
N-terminal dipeptide part of a DP IV-substrate. This leads to
potent compounds which are inhibitors of DP IV and DP IV-like
enzymes and inhibit at sufficient concentrations (e.g. 5.times.
K.sub.i-dose) the DP IV-catalyzed hydrolysis of small chromogenic
or higher molecular weight peptide substrates. In the present
invention it is demonstrated that compounds interacting with DP
IV-binding sites far distant from the catalytic center are capable
to differentiate the degradation of different substrates, e.g.
peptide substrates, or even discriminate DP IV-catalyzed hydrolysis
completely.
[0106] The substrate properties of the peptides of the growth
hormone releasing factor (GRF) family against DP IV were
examined.
[0107] The GRF family consists of the following peptide
hormones:
[0108] Gastrin-releasing peptide (GRP)
[0109] Enterostatin
[0110] Peptide histidine methionine (PHM)
[0111] Cholecystokinin
[0112] Glucagon-like peptide-2 (GLP-2)
[0113] Glucose-dependent insulinotropic polypeptide (GIP)
[0114] Glucagon-like peptide-1 (GLP-1)
[0115] Growth-hormone releasing factor (GRF)
[0116] Pituitary-adenylate cyclase activating polypeptide (PACAP
(27 und 38))
[0117] Vasoactive intestinale peptide (VIP)
[0118] Exendin-1
[0119] Exendin-2
[0120] Exendin-3
[0121] Exendin-4
[0122] Secretin
[0123] Glucagon
[0124] In particular, the capability of purified DP IV from human,
from porcine kidney, of recombinant human DP IV and the DP IV
activity of the human serum to truncate the peptides of the GRF
family were analyzed. The half-life of the peptides were determined
using matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) whereas the kinetic constants
K.sub.m and k.sub.cat/K.sub.m were calculated using capillary zone
electrophoresis. All peptides were hydrolyzed by porcine DP IV,
recombinant human DP IV or DP IV activity of the human serum. The
resulting K.sub.m-values were independent from the amino acid in
the P.sub.1-position. That means that the binding of substrates to
DP IV is not mainly affected by the P.sub.1-residue rather than by
secondary interactions between substrate and DP IV protein.
[0125] The same surprising phenomenon of different substrate
properties was shown with GIP-fragments of different chain lengths.
V.sup.2GIP( 1-6) and G.sup.2GIP( 1-6) were not hydrolyzed by DP IV.
V.sup.2GIP(1-30) and G.sup.2GIP(1-30) were accepted as substrates
and both S.sup.2GIP(1-6) and S.sup.2GIP(1-30) were truncated by DP
IV (Table 2). These findings prove the existence of a secondary
binding site in the DP IV protein, which is responsible for
substrate recognition and which modulates the biodegradation of
substrates and, therefore forms the basis for the management of
substrate selectivity and specificity of DP IV and/or DP IV-like
enzymes.
7TABLE 2 Truncation half life of various bioactive peptides which
are substrates for DP IV substance half-life [min] GIP.sub.1-30
2.68 S.sup.2GIP.sub.1-30 137.14 V.sup.2GIP.sub.1-30 298.04
G.sup.2GIP.sub.1-30 150.02 GIP.sub.1-6 <7.5 S.sup.2GIP.sub.1-6
79.04 V.sup.2GIP.sub.1-6 no degradation G.sup.2GIP.sub.1-6 no
degradation
[0126] The amino acid sequences of natural GIP.sub.1-30 and
GIP.sub.1-6 are:
8 GIP.sub.1-30: YAEGTFISDYSIAMAKIHQQAFVNWLLAQK GIP.sub.1-6:
YAEGTF
[0127] To identify the secondary binding site, a hexapeptide
derived from a consensus sequence of the amino acid sequences of
GRF-family peptides was synthesized and its influence on the
substrate specificity of DP IV was measured. The selected consensus
sequence corresponds to glucagon.sub.5-10, comprising the amino
acid sequence TFTSDY. As expected this peptide had only weak
influence on the GP-4-Nitroanilide hydrolysis (K.sub.i=0.71
mM).
[0128] In support of the results achieved with the GRF family
peptides, the truncation half-lifes of GIP, GLP-1, NPY, glucagon or
PACAP by DP IV were also changed after preincubation with 160 .mu.M
TFTSDY (Table 3). No differences could be detected between
incubation of Rantes.sub.1-15 and DP IV with or without the
hexapeptide TFTSDY (Table 3). The latter finding shows that the
peptide Rantes.sub.1-15 is too short to reach the secondary binding
site and therefore TFTSDY has no effect on its hydrolysis rate. The
half-lives of GIP and glucagon in presence of DP IV were prolonged
by TFTSDY, the strongest influence had TFTSDY on the DP
IV-catalyzed truncation of glucagon.
[0129] Further, a modified variant of the hexapeptide TFTSDY,
TFTDDY was synthesized, studied for docking in the DP IV 3D
structural model and tested for its regulatory efficacy to modulate
substrate specificity of DP IV.
9TABLE 1 Inhibitory effect of TFTSDY on DP IV-catalyzed peptide
truncation expressed in K.sub.i-values K.sub.i [.mu.M] peptide rec.
human DP IV porcine DP IV PACAP-27 26.7 n.d. PACAP-38 2.8 n.d. GIP
14.0 65.9 glucagon 3.7 6.8 RANTES.sub.1-15 n.d. 12307.7 GLP-1 n.d.
13.7 NPY n.d. 17.2 n.d.--not determined
[0130] The hexapeptides TFTSDY and TFTDDY were found to be instable
in biological fluids, e.g. humen serum or human plasma and/or they
were rapidly degraded by proteolytic enzymes in the serum or
plasma. Therefore, and in another embodiment of the present
invention, a heptapeptide of the sequence
10 H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH
[0131] was synthesized. This heptapeptide is not enzymatically
degraded in human serum or plasma and is stable in these fluids. A
stabilization of the heptapeptide was especially achieved by the
incorporation of D-amino acids in the molecule. It was further
shown to be very effective in improving the substrate specificity
of DP IV compared to control experiments without the
heptapeptide.
[0132] The existence of a secondary binding site was proven, e.g.
using a set of dipeptide compounds, coupled to a chromogenic
(p-nitroaniline, pNA) or fluorogenic (aminomethylcoumarine, AMC)
group. The dipeptides His-Pro, His-Ala, His-Ser, His-Val, His-Gly,
and His-Thr represent the first two amino acids from the N-terminus
of the following peptide hormones: GIP, GLP-1, GLP-2, PACAP, VIP,
PHM and glucagon. The peptide hormones GIP, GLP-1, GLP-2, PACAP,
VIP, PHM and glucagon are substrates of DP IV. DP IV hydrolizes
these peptide hormones and the respective N-terminal dipeptides are
released. In contrast, the dipeptides are much slower released,
when they are coupled to the chromogenic (p-nitroaniline, pNA) or
fluorogenic (aminomethylcoumarine, AMC) group. Secondary
interactions of the peptide hormones GIP, GLP-1, GLP-2, PACAP, VIP,
PHM and glucagon far from the DP IV active site must exist as a
prerequisite for substrate recognition. Data supporting the
existence of a secondary binding site of DP IV are shown in table 4
below
11TABLE 4 DP IV-catalyzed hydrolysis of His-Ser-, His-Gly and
His-Val-dipeptides compared to the full-lenght substrates glucagon
and NPY. Compound K.sub.m [M] His-Ser-AMC 2.1 * 10.sup.-2 Glucagon
3.8 * 10.sup.-6 His.sup.1-Ser.sup.2-NPY 6.8 * 10.sup.-5 His-Gly-AMC
4.1 * 10.sup.-4 [Gly].sup.2-glucagon 2.2 * 10.sup.-5 His-Val-AMC
1.9 * 10.sup.-2
[0133] The Prolyl Oligopeptidase (POP) Based Computer-Generated
Models of Human DP IV
[0134] Prolyl oligopeptidase (POP) based computer-generated models
of human DP IV and porcine DP IV and the crystal structure of
porcine DP IV were used according to the present invention to
predict enzyme-substrate-interactions and to identify the
interaction site in the DP IV protein structure.
[0135] Since the sequence homology between DP IV and the template
POP is not very high, standard methods for homology modeling such
as the application of COMPOSER gave only very crude preliminary
models which needed a lot of manual modification and improvements.
These improvements were made by inspection of the conformation and
spatial position of each of the 766 amino acid residues with regard
to forming sheets or helices and favored intra-residual
interactions such as hydrogen bonds, salt bridges and hydrophobic
interactions as well. All modifications made were examined by using
PROCHECK, which allows the analysis of the stereochemical quality
of the model (dihedral angles in favored areas of a Ramachandran
Plot, see FIG. 1 for human DP IV and FIG. 20 for porcine DP IV),
bond angles and bond length, hydrogen bonds (see FIG. 2 for human
DP IV and FIGS. 21 and 22 for porcine DP IV), and by PROSA which
analyzes its energy in comparison to native folded proteins. All
these residues show that some residues are located in unfavorable
areas but all belong to loop regions of the propeller domain which
is not of essential importance for docking studies and predictions
of new ligands.
[0136] In summary of this part, the model of DP IV used herein is
in a state where the overall fold is correct and highly useful for
the explanation of experimental results and to allow predictions of
recommendations for positions of site directed mutagenesis,
development of ligands based on the identified second binding site
or selective ligands to bind at the closer active site.
[0137] In order to identify essential amino acids for the secondary
interaction independently from the active site, site-directed
mutageneses were performed using human DP IV cDNA. The mutation
sites were: W629A and R560A. The characterization of these mutants
showed that both mutations have no influence on the enzyme
catalyzed hydrolysis of GP-4-nitroanilide and the kinetic
parameters of short and/or low molecular weight inhibitors, which
are directed to the active site of DP IV (see table 5). Another
mutated enzyme variant, R310A, was expressed as inactive protein.
This mutation resulted in the appearance of three DP IV fragments.
Based on the computer generated model was shown that an
intramolecular salt bridge is formed between R310 and D332 and that
this intramolecular salt bridge is crucial for the formation and
stabilization of the DP V tertiary protein structure.
12TABLE 5 Kinetic characterization of DP IV-catalyzed substrate
hydrolysis by mutants of DP IV in the secondary binding site Test
K.sub.m K.sub.i kcat k.sub.cat/K.sub.m Mutation compound [M] [M]
[s.sup.-1] [M.sup.-1 * s.sup.-1] mu 15 DP IV Gly-Ser-AMC Not
hydrolyzed mu 15 DP IV Gly-Pro-AMC 4.66E-05 1.00E+06 2.15E+10 mu 15
DP IV V.sup.2GIP(1-4)* no inhibition mu 15 DP IV S.sup.2GIP(1-6)*
no inhibition mu 15 DP IV Glucagon (1-14)* no inhibition mu 15 DP
IV Leu-hia-Fum* 6.81E-08 mu 15 DP IV TFTSDY* no inhibition mu 15 DP
IV PACAP(1-38)* 3.67E-05 mu 15 DP IV Transp 01* 7.69E-08 mu 15 DP
IV YAESTF amide* 1.14E-06 mu 16 DP IV Gly-Ser-AMC Not hydrolyzed mu
16 DP IV Gly-Pro-AMC 5.02E-05 1.44E+06 2.86E+10 mu 16 DP IV
V.sup.2GIP(1-4)* no inhibition mu 16 DP IV S.sup.2GIP(1-6)* no
inhibition mu 16 DP IV Glucagon (1-14)* no inhibition mu 16 DP IV
PACAP(1-38)* 3.21E-05 mu 16 DP IV Transp 01* 8.55E-08 mu 16 DP IV
YAESTF amide* 1.06E-06 mu 16 DP IV TFTSDY* no inhibition mu 16 DP
IV Leu-Thia Fum* 6.57E-08 rh wt DP IV Gly-Ser-AMC 4.4E-04 rh wt DP
IV Gly-Pro-AMC 3.53E-05 1.66E+06 4.7E+10 rh wt DP IV
V.sup.2GIP(1-4)* no inhibition rh wt DP IV S.sup.2GIP(1-6)* no
inhibition rh wt DP IV Glucagon (1-14)* no inhibition rh wt DP IV
PACAP(1-27)* 2.28E-04 1.13E-04 rh wt DP IV PACAP(1-38)* 3.83E-05 rh
wt DP IV Transp 01* 5.08E-08 rh wt DP IV YAESTF amide* 3.51E-08 rh
wt DP IV TFTSDY* no inhibition rh wt DP IV Leu-Thia Fum* 4.26E-05
6.58E-08 p wt DP IV Leu-Thia-Fum* 5.98E-05 7.29E-08 p wt DP IV
PACAP(1-27)* 1.22E-04 5.43E-05 *The Ki-values were determined in
competition of the test compound to the standard substrate GP-4NA
(see examples). No inhibition means that the compound doesn't
influence the DP IV-catalyzed hydrolysis of the standard substrate
GP-4NA.
[0138] Definitions in table 5:
13 mu 15 recombinant human DP IV, mutation R560A mu 16 recombinant
human DP IV, mutation W629A rh wt recombinant human DP IV, wild
type p wt porcine kidney DP IV, wild type Transp 01
RRLSYSRRRF-E-Thia
[0139] In the present invention a region was identified in the DP
IV-protein, which is responsible for the interaction with a
hexapeptide, e.g. TFTSDY or TFTDDY, or more suitably, a degadation
resistent heptapeptide, e.g.
H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH. The most important amino
acids for the formation of the secondary binding site on DP IV for
the GRF family of peptide hormones were found to be but are not
restricted to L90, E 91, T 152, W154, W157, R310, Y330, R318, Y416,
S460, K463, E464 and R560.
[0140] The Cyrstal Structure of DP IV
[0141] In a further embodiment of the present invention, the
sequence and 1.8 .ANG. crystal structure of native DP IV prepared
from porcine kidney were determined. The crystal structure reveals
a 2-2-2 symmetric tetrameric assembly which depends on the natively
glycosylated .beta.-propeller blade IV. The crystal structure
indicates that tetramerization of DP IV is a key mechanism to
regulate its interaction with other components. Each subunit
comprises two structural domains, the N-terminal eight-bladed
.beta.-propeller with open Velcro topology and the C-terminal
.alpha./.beta.-hydrolase domain. Analogy with the structurally
related POP and tricom protease suggests that substrates access the
buried active site through the .beta.-propeller tunnel while
products leave the active site through a separate side exit. A
dipeptide mimicking inhibitor complexed to the active site
discloses key determinants for substrate recognition, including a
Glu-Glu motif which distinguishes DP IV as an aminopeptidase and an
oxyanion trap which binds and activates the P.sub.2-carbonyl oxygen
necessary for efficient post-proline cleavage.
[0142] Sequence of the Porcine DP IV
[0143] Sequence comparison of the porcine DP IV with the human and
other mammalian DP IV-sequences reveals a very high degree of
sequence conservation. In particular, there is not a single
sequence insertion or deletion between the porcine and the human
sequence. The overall sequence identity between these two species
is 88%. (and 92% within the catalytic domain). Noteworthy, Ser339
in the porcine sequence substitutes for Cys339 in the human
sequence, causing the absence of an extra disulfide bond
(Cys328-Cys339) in human DP IV. In comparison to the human sequence
the potential glycosylation site at Asn520 is lost because of an
Asn-His exchange. On the other hand there is an additional
potential glycosylation site at Asn179 which is not found in the
human sequence. Interestingly, the glycosylation site at Asn279 is
found at Asn281 in the human sequence.
[0144] Recently, the structure of human DP IV was published (PDB
entry code 1N1M) (Rasmussen H. B., Branner, S., Wiberg, F. C.,
Wagtmann, N. (2002) http://www.nature.com/naturestructuralbiology,
published online 16 Dec. 2002.) The model of porcine DP IV
according to the present invention was refined by using the program
CNS with current R-values of 21.7% (working set) and 24.9% (test
set) and deviations from ideality of 0.008 .ANG. (bond length) and
1.4 degree (angle deviation).
[0145] Overall Structure and Subunit Assembly
[0146] The monomer comprises an N-terminal .beta.-propeller domain
(Arg54-Asn497) followed by the catalytic domain Gln508-Pro766.
Notably, the crystal structure reveals a dimer of DP IV dimers in
the crystallographic unit cell obeying a 222 symmetry with all axes
intersecting, FIG. 23. The by far more extensive dimer contact is
predominantly mediated by residues of the catalytic domain with a
contact area of 2270 .ANG..sup.2 versus the dimer-to-tetramer
interface of 2.times.570 .ANG..sup.2=1140 .ANG..sup.2. The dimer
interface is dominated by hydrophobic interactions, with a central
and well shielded aromatic stacking involving Trp734 and Phe713 of
both monomers. The hydrophobic contacts are complemented by polar
interactions such as Asp729 with His754 and His757, FIG. 25A.
Notably, the Gln731(O.epsilon.1)-Gln731(N.epsilon.2) contact
resembles a subtle (atomic) break of the exact two-fold symmetry
within the dimer. The residues critical to the dimerization are
strictly conserved throughout all species.
[0147] The dimer-dimer interface has a more hydrophilic character.
In its center, the strands Asn279-Gln286 of each DP IV-dimer form
an antiparallel .beta.-sheet, thus extending propeller blade IV to
an eight-stranded antiparallel sheet, FIG. 25B. An additional
contribution to the tetrameric assembly in our crystals arises from
the outer strands of blade V. The residues contributing to the
dimer-dimer contact are similar over different species, albeit
especially in rodents not strictly identical. More importantly,
there are no insertions or deletions in the outer .beta. strands of
blade IV with a contact area of 570 .ANG..sup.2 per monomer.
Significantly, Asn279 is located at the tetramerization interface
and is glycosylated (FIGS. 23, 25B). Thus, glycosylation of Asn279
might provide one missing regulatory link which was proposed to
control the assembly of a 900 kDa oligomer (Lambeir, A. M.,
Pereira, J. F. D., Chacon, P., Vermeulen, G., Heremans, K.,
Devreese, B., VanBeeumen, J., Demeester, I. & Scharpe, S.
(1997) Biochim. Biophys. Acta 1340, 215-226.).
[0148] Subdomain Structure
[0149] The .beta.-propeller. The N-terminal .beta.-propeller domain
contains eight blades with four antiparallel strands each.
Typically for .beta. propeller structures, the first and the last
blade of a .beta.-propeller is clamped together either covalently
by disulfide bond formation (four-bladed .beta.-propellers) or by
strand exchange between the first and last blade (five to
eight-bladed propellers). So far there are three exceptions to this
closed propeller topology rule, namely the seven bladed
.beta.-propeller of POP (Fulop, V., Bocskei, Z. & Polgr, L.
(1998) Cell 94, 161-170.), the seven and the six bladed propellers
of the tricom protease (Brandstetter, H., Kim, J. -S., Groll, M.
& Huber, R. (2001) Nature 414, 466-469.), and the five bladed
propeller of .alpha.-L-arabinase 43A (Nurizzo, D., Turkenburg, J.
P., Charnock, S. J., Roberts, S. M., Dodson, E. J., McKie, V. A.,
Taylor, E. J., Gilbert, H. J. & Davies, G. J. (2002) Nature
Struct. Biol. 9, 665-668.). The .beta.8-propeller of DP IV can also
be classified as an open Velcro-type topology, because no segment
C-terminal to blade VIII interacts with the first propeller blade.
Interestingly, however, the interaction of the first strand within
blade I is limited to Thr59-Ile63 while its N-terminal extension
Phe53-Tyr58 tightens up the propeller structure by interacting with
the immediate C-terminal extension to the fourth strand of blade
VIII (Glu499-Met503). A similar, yet shorter, external clamp has
been described for the .delta.7-propeller of the tricorn protease
(Brandstetter, H., Kim, J. -S., Groll, M. & Huber, R. (2001)
Nature 414, 466-469.).
[0150] With the exception of Cys649-Cys762 all disulfide bonds are
located in the .beta. propeller domain where they form intra-blade
stabilizing crosslinks exclusively, FIG. 24. Cys339 in the human
sequence is replaced by Ser339 in the porcine sequence which causes
the absence of the disulfide bond connecting strand 3 and 4 of
blade V (Cys328-Cys339 in human DP IV), although the backbone
conformation of the strands would readily allow a disulfide bond to
be formed. Similarly, all glycosylation sites but Asn685 are
located on the .beta.-propeller. Five of in total 10 potential
glycosylation sites cluster at the top surface side oriented away
from the catalytic domain. Four are positioned on the loops
connecting strand 3 and 4 of blade I (Asn85), III (Asn179), IV
(Asn279) and VI (Asn393), one on the loop connecting strand 1 and 2
of blade IV (Asn219), FIGS. 23, 24. Intriguingly, out of these five
potential glycosylation sites only Asn279 is actually
posttranslationally modified which is involved in the
tetramerization of DP IV. Further glycosylation sites are located
on blade I (Asn92 at the end of strand 4), blade IV (Asn229 close
to the tetramerization motif), blade V (Asn321 on the loop
connecting strand 2 and 3), FIG. 23, 24. Asn150 on the exit strand
of blade II is not actually modified in the crystals. The shape of
the DP IV-.beta.8-propeller is asymmetric where blades VI, VII,
VIII, I, and blades II, III, IV, V form more compact subdomains,
respectively (FIGS. 23, 25B). The structural division of the
.beta.-propeller fits physico-chemical data which indicated a three
domain organization of DP IV (Lambeir, A. M., Pereira, J. F. D.,
Chacon, P., Vermeulen, G., Heremans, K., Devreese, B., VanBeeumen,
J., Demeester, I. & Scharpe, S. (1997) Biochim. Biophys. Acta
1340, 215-226.). The ellipsoidal tunnel through the propeller is
continuously open. At the solvent exposed opening, its diameter
measures 9 .ANG. and 15 .ANG. from blade IV to VIII and from blade
II to VI, respectively. The tunnel widens towards the catalytic
domain with opening diameters of 15 .ANG. and 25 .ANG. between the
same pairs of propeller blades. By its dimensions, the tunnel
allows for direct passage of an extended peptide, but not for a
folded .alpha.-helix.
[0151] Only few solvent molecules are visible in the tunnel, most
remarkable a sulfate is bound to the oxyanion pocket formed by the
amide nitrogens of Glu361 and Ile407 and N.epsilon.2 of His363.
[0152] The Catalytic domain. The catalytic domain is located at the
C-terminus of DP IV and spans residues Gln509 to Pro766. It adopts
a typical .alpha./.beta.-hydrolase fold with a central 8 stranded
.beta. sheet, where only the second strand Thr522-Pro531 deviates
from the otherwise parallel strand polarity, FIG. 23. The
.beta.-sheet exerts a significant twist of more than 90 degrees, in
line with observations on related .alpha./.beta.-hydrolases.
[0153] Within the catalytic domain a single disulfide bond
Cys649-Cys762 crosslinks the C-terminal helix Met746-Ser764 with
the sixth strand of the .beta.-sheet (Lys648-Ala654), thus
stabilizing its tertiary arrangement. Helix Met746-Ser764, together
with helix Gln714-Asp725 and strand Asp729-Thr736 from the
C-terminal region, constitutes the central dimerization motif which
is further stabilized through an interaction contributed by the
oligomerization blade IV of the .beta.-propeller.
[0154] The .beta.-sheet is sandwiched by several a-helices,
including helix Tyr631-Ala642 immediately succeeding the catalytic
Ser630. Ser630 is embedded in the surrounding secondary structure
framework where it participates both in the preceding strand 4
(Arg623-Trp629) as well as in the following helix Tyr631-Ala642.
This causes a strained backbone conformation of the active site
Ser630. The high energy conformation of Ser630 is reflected by its
dihedral angles (.PHI., .psi.)=(61.4,-115.7) and presumably
provides a reservoir needed for catalysis (Goettig, P., Groll, M.,
Kim, J. -S., Huber, R. & Brandstetter, H. (2002) EMBO J 21,
5343-5352.).
[0155] Active site and substrate recognition. The sequential and
three-dimensional arrangement of the catalytic residues Ser630,
His740, Asp708 corresponds to that of related
.alpha./.beta.-hydrolases. The oxyanion hole is formed by the amide
Tyr631 and the hydroxyl O.eta. of Tyr547 and serves to recognize
and activate the carbonyl oxygen of the P.sub.1-residue. It is
occupied by a water molecule in the uninhibited structure. To
detail the exact mechanism of substrate recognition, the structure
of a dipeptid mimetic, the iodinated Phe-cyanopyrrolidide inhibitor
in complex with DP IV was determined (FIG. 26A). The active site
nucleophile, the hydoxyl residue of Ser630, forms a covalent bond
with the scissile carbonyl carbon of the cyanopyrrolidine of the
inhibitor. The bending of the linkage (FIG. 26A) indicates the
formation of a stable carbaminic acid adduct. The pyrrolidine ring
is accommodated by a hydrophobic pocket formed by side chains of
Tyr666, Tyr662, Val711, Val656 and Trp659. While this environment
is almost perfectly suited for the imino acid proline as
P.sub.1-residue, the hydroxyl O.eta. of Tyr662 would be correctly
positioned to interact with the normal amide nitrogen of an amino
acid in P.sub.1. The inhibitor also unambiguously maps the
S.sub.2-site. The P.sub.2-carbonyl oxygen gets trapped in an
electrostatic sink formed by the side chains of Arg125 and Asn710.
Glu205 and Glu206, and to a lesser extent the carbonyl oxygen of
Glu205, interact with the free amino terminus of the
P.sub.2-residue, thus determining the dipeptidyl "amino"-peptidase
activity of the enzyme. It is, therefore, the .beta.-propeller
which provides essential determinants for P.sub.2-recognition,
namely Arg125, which is positioned on the hairpin loop between
strands 2 and 3 of blade II and Glu205-Glu206, positioned on a
short helical insertion within strand 1 of the .beta. propeller
blade IV. Ample space is available to accommodate voluminous side
chains such as Tyr or Trp in P.sub.2, FIG. 26A. In our inhibitor
the phenyl ring of the P.sub.2-residue is iodated rather than
hydroxylated. It forms an ionic interaction with Arg358, FIG.
26A.
[0156] Substrate access to and product egress from the active site.
The .beta.-propeller domain covers the active site and thereby
restricts the substrate access to it. There are two possible routes
to the active site, namely through the tunnel of the
.beta.-propeller and through a side opening. Similar as the
propeller tunnel, the shape of the side entrance is oval with
dimensions of 15 .ANG. and 22 .ANG.. The side opening to the active
site is generated by the kinked blade arrangement of blade I and
II, FIG. 23. In POP, blades I and II are arranged more regularly
and there is no side opening to the active site chamber. The
distance from the protein surface to the active site measures 20
.ANG. and 37 .ANG. through the side opening and the propeller
tunnel, respectively. From its dimensions, both routes give active
site access to unfolded peptidic substrates, but the side entrance
is significantly shorter and less winded. Once the substrate has
been cleaved, two products have to leave the active site chamber.
Clearly, the product exit route differs from the entrance to the
active site.
[0157] Based on the crystal structure model new functional
characteristics of DP IV were identified and are part of the
present invention. These new features of DP IV are:
[0158] Oligomerization of membrane-bound and soluble DP IV.
Tetramerization on the cell surface involves, for geometric
reasons, a membrane bound and a soluble DP IV dimer pair or dimers
located on the surface of two different cells, as illustrated on
FIG. 23. DP IV is known as a cell-cell communication molecule.
Thus, the way DP IV is involved in mediating such cell-cell
contacts may be by tetramerization of two homodimers present on the
surface of interacting cells. Alternatively, soluble dimers can
assemble to form a homotetramer, as observed in the crystal
structure described above. The tetramer assembles to enclose a
large cavity. Since tetramerization of DP IV depends on the
correctly glycosylated propeller blade IV, glycosylation could
function as a quality control unit.
[0159] Dimerization is mediated by the three C-terminal secondary
structure elements positioned on the catalytic domain, and a finger
like insertion motif within strand 2 and 3 of propeller blade IV.
Furthermore, DP IV is known to form heterodimers with fibroblast
activation protein .alpha. (FAP.alpha., seprase). Like DP IV
(FAP.beta.), FAP.alpha. lacks an N-terminal extension as found in
POP. Moreover, the essential elements of the DP IV-dimerization
motif are also present in FAP.alpha., including the extension of
strand 2 and 3 of propeller blade IV.
[0160] Functional role of oligomerization. The crystal structure
shows that dimerization is not required to complete the active site
architecture of DP IV, as for example in the case of tricorn
(Brandstetter, H., Kim, J. -S., Groll, M. & Huber, R. (2001)
Nature 414, 466-469.). Instead, dimerization and tetramerization
will affect interaction with other components, including
proteolytic substrates and ADA and mediate cell-cell contacts.
Moreover, dimerization of DP IV is likely to enhance the
receptor-ligand affinity by bivalent interaction. Finally, it is
likely that dimerization is critical for signal transduction into
the cell.
[0161] Substrate preference and catalytic mechanism. The
hydrophobic S.sub.1-pocket visualizes that proline is perfectly
suited as a P.sub.1-residue, although it will also fit other small
uncharged residues such as alanine or serine. Interestingly, the
S.sub.1-site implements a mechanism to adapt to both imino and
amino acids in P.sub.1-position. The hydroxyl O.theta. of Tyr662 is
able to form a hydrogen bond with the P.sub.1-amide nitrogen and
thus optimally presents the substrate for catalysis. By contrast,
the proper orientation of proline in P.sub.1-position is achieved
by its side chain interaction in addition to the binding to the
oxyanion pocket. In this situation, the hydroxyl of Tyr662 can
slightly reorient to form a hydrogen bond with the side chain
O.delta.1 of Asn710, FIG. 26A. The recognition of the
P.sub.2-residue is dictated by main chain interactions with two
prominent anchor sites, namely Glu205-Glu206 which form a
twin-single salt bridge with the free amino terminus of the
P.sub.2-residue; and Arg125 together with N.delta.2 of Asn710 which
stabilize and activate the P.sub.2-carbonyl oxygen. The
Glu205-Glu2O6 motif is highly conserved in the DP IV gene family
and it has been shown by site directed mutagenesis to be essential
for enzymatic activity. The role of the P.sub.1oxyanion hole in
activating the substrate's scissile bond is well established for
all proteases. In the case of DP IV as a post-proline processing
enzyme an additional requirement has to be met to achieve efficient
catalysis. Proline containing peptides can adopt in solution also
cis-peptide bond as well as trans-peptide bond conformation.
However, as highlighted by the inhibitor used in this invention,
only a peptide in trans-conformation is able to productively bind
to the active site.
[0162] .beta.-propeller architecture: The tunnel through the
eight-bladed .beta.-propeller widens from the surface towards the
active site of DP IV.
[0163] Substrate access to and product egress from the active site.
Two openings of similar diameter, but differing length, give access
to the active site. The situation in DP IV is most closely
resembled by the tricom protease where a seven-bladed and
six-bladed .beta.-propeller provide a separate entrance to and exit
from the active site, respectively. Tricom protease is a serine
protease with low but significant structural homology to the family
of .alpha./.beta.-hydrolase- s. This similarity suggests that the
.beta.8-propeller provides substrate access to and the side opening
product release from the DP IV active site. This tricom-derived
model is able to explain the high substrate selectivity critical
for DP IV-function to activate or inactivate regulatory peptides.
Passage through the .beta.-propeller tunnel requires the substrates
to unfold thereby providing their "finger print" to DP IV. Once the
amino terminus of the peptide approaches the active site, it is
still held in place by its C-terminus interacting with the
.beta.-propeller which then contributes to conformationally
activate the substrate for cleavage. After the nucleophilic attack
the acyl enzyme intermediate forms, while the primed product is
directly released through the side exit.
[0164] Interaction with other components. DP IV binds adenosine
deaminase (ADA) to the T-cell surface, thereby preventing the cell
from adenosine mediated inhibition of proliferation. DP IV-ADA
complex formation is presumably hydrophobically driven, as the
complex dissociates at very low ionic strength. By using
site-directed mutagenesis, Leu294 and Val341 were identified as two
ADA binding sites (Abbott, C. A., McCaughan, G. W., Levy, M. T.,
Church, W. B. & Gorrell, M. D. (1999) Eur. J Biochem. 226,
798-810.). Leu294 and Val341 are positioned at the outer strand of
the tetramerization blade IV and blade V, respectively. Therefore,
ADA-binding will interfere with tetramerization. Similarly, the
glycosylation of Asn279 (Asn281 in the human sequence) is likely to
influence ADA-binding. This teaches that tetramerization of DP IV
and proper glycosylation of Asn279 serve as major control mechanism
for ADA-binding
[0165] DP IV as a target for drug design. The inhibitor structure
used to establish the crystal structure of DP IV in the present
invention identified important recognition elements at DP IV's
active site and represents an excellent starting point for rational
design of active site directed inhibitors. However, DP IV's
involvement in a great variety of physiological processes poses a
high challenge to avoid unwanted side effects for any DP IV drug
development program. Ideally, it is possible now to target a
particular DP IV substrate rather than the complete DP IV activity.
Active site directed DP IV inhibitors, however, will interfere with
the complete DP IV proteolytic activity and might even interfere
with structurally related members of the .alpha.-.beta. hydrolase
family. Non-active site directed inhibition strategies depict a
solution to this problem. The sulfate bound to the oxyanion pocket
within the .beta. propeller tunnel formed by the amide nitrogens of
Glu361 and Ile407 and N.epsilon.2 of His363, as indicated in FIG.
26A, identified an excellent target point for the development of
inhibitors that block substrate passage through the .beta.
propeller tunnel.
[0166] In the peptides, proteins and mutants shown, each encoded
residue where appropriate is represented by a one-letter or a
three-letter designation, corresponding to the trivial name of the
amino acid, in accordance with usual practice. Examples of usual
definitions are given in the following conventional list:
14 Amino Acid One-Letter Symbol Three-Letter Symbol Alanine A Ala
Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys
Glutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H His
Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met
Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr
Tryptophan W Trp Tyrosine Y Tyr Valine V Val Selenocysteine Sec
[0167] In a preferred embodiment of the present invention, a
secondary binding site in the DP IV protein and/or DP IV-like
enzymes is identified. More preferred, the existence of this
secondary binding site can be used to influence the selectivity of
the DP IV-catalyzed biodegradation of DP IV-substrates, e.g.
alanine (GIP), proline (GRP) or serine (glucagon) substrates,
dependent on the amino acid residue in the PI position and
dependent on the tertiary structure of the DP IV-substrates.
Preferred DP IV-substrates, the biodegradation whereof shall be
regulated according to the invention with compounds, which bind to
the secondary binding site, are serine substrates.
[0168] The regulation of the biodegradation of DP IV-substrates due
to compounds, which bind to the secondary binding site, is further
dependent on the chain length of the substrates. Preferably, DP
V-substrates have a chain length of more than 5 amino acid
residues, more preferably more than 10 amino acid residues. Most
preferred are substrates with more than 15 amino acid residues up
to 70 amino acid residues.
[0169] Currently known substrates of DP IV are:
[0170] Xaa-Pro peptides
[0171] Tyr-melanostatin
[0172] Endomorphin-2
[0173] Enterostatin
[0174] .beta.-Casomorphin
[0175] Trypsinogen pro-peptide
[0176] Bradykinin
[0177] Substance P
[0178] Corticotropin-like intermediate lobe peptide
[0179] Gastrin-releasing peptide
[0180] Neuropeptide Y
[0181] Peptide YY
[0182] Aprotinin
[0183] RANTES
[0184] GCP-2
[0185] SDF-1.alpha.
[0186] SDF-1.beta.
[0187] MDC
[0188] MCP-1
[0189] MCP-2
[0190] MCP-3
[0191] Eotaxin
[0192] IP-10
[0193] Insulin-like growth factor-I
[0194] Pro-colipase
[0195] Interleukin-2
[0196] Interleukin-1.beta.
[0197] .alpha..sub.1-Microglobulin
[0198] Prolactin
[0199] Trypsinogen
[0200] Chorionic gonadotropin
[0201] Xaa-Ala peptides
[0202] PHM
[0203] GRH-(1-29)
[0204] GRH-(1-44)
[0205] GLP-1
[0206] GLP-2
[0207] Gastric inhibitory peptide
[0208] Orexin B
[0209] Xaa-Ser peptides
[0210] Orexin A
[0211] In the most preferred embodiment of the present invention,
compounds for the modulation of DP IV-catalyzed biodegradation of
DP IV-substrates are provided, which compounds bind to the
secondary binding site of DP IV or DP IV-like enzymes. Such
compounds are e.g. selected from the compounds of the formulas
a)-d): 1
[0212] Furthermore, the present invention provides agents, which
bind to both the active site and the secondary binding site of DP
IV and DP IV-like enzymes and thereby simultaneously modulate the
enzyme activity and substrate specificity of DP IV or DP V-like
enzymes.
[0213] DP IV is present in a wide variety of mammalian organs and
tissues e.g. the intestinal brush-border ( Gutschmidt S . et al.,
"In situ"--measurements of protein contents in the brush border
region along rat jejunal villi and their correlations with four
enzyme activities. Histochemistry 1981, 72 (3), 467-79), exocrine
epithelia, hepatocytes, renal tubuli, endothelia, myofibroblasts
(Feller A. C. et al., A monoclonal antibody detecting
dipeptidylpeptidase IV in human tissue. Virchows Arch. A. Pathol.
Anat. Histopathol. 1986; 409 (2):263-73), nerve cells, lateral
membranes of certain surface epithelia, e.g. Fallopian tube, uterus
and vesicular gland, in the luminal cytoplasm of e.g., vesicular
gland epithelium, and in mucous cells of Brunner's gland (Hartel S.
et al., Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of
immunohistochemistry and activity histochemistry. Histochemistry
1988; 89 (2): 151-61), reproductive organs, e.g. cauda epididymis
and ampulla, seminal vesicles and their secretions (Agrawal &
Vanha-Perttula, Dipeptidyl peptidases in bovine reproductive organs
and secretions. Int. J. Androl. 1986, 9 (6): 435-52). In human
serum, two molecular forms of dipeptidyl peptidase are present
(Krepela E. et al., Demonstration of two molecular forms of
dipeptidyl peptidase IV in normal human serum. Physiol. Bohemoslov.
1983, 32 (6): 486-96), the serum high molecular weight form of DP
IV is expressed on the surface of activated T cells (Duke-Cohan J.
S. et al., Serum high molecular weight dipeptidyl peptidase IV
(CD26) is similar to a novel antigen DPPT-L released from activated
T cells. J. Immunol. 1996, 156 (5): 1714-21). It is also a goal of
the present invention to minimize possible side effects of
currently available DP IV-inhibitors by the control and management
of the DP IV substrate specificity for the selective treatment of a
DP IV mediated disease.
[0214] In another preferred embodiment of the present invention,
all molecular forms, homologues and epitopes of proteins showing DP
IV or DP IV-like enzyme activity, from all mammalian tissues and
organs, also of those, which are undiscovered yet, are intended to
be embraced by the scope of this invention.
[0215] Among the rare group of proline-specific proteases, DP IV
was originally believed to be the only membrane-bound enzyme
specific for proline as the penultimate residue at the
amino-terminus of the polypeptide chain. However, other molecules,
even structurally non-homologous with the DP IV but bearing
corresponding enzyme activity, have been identified. DP IV-like
enzymes, which are identified so far, are e.g. fibroblast
activation protein .alpha., dipeptidyl peptidase IV .beta.,
dipeptidyl aminopeptidase-like protein, N-acetylated .alpha.-linked
acidic dipeptidase, quiescent cell proline dipeptidase, dipeptidyl
peptidase II, attractin and dipeptidyl peptidase IV related protein
(DPP 8), DPL1 (DPX, DP6), DPP 9 and DPL2 (DPP 10) are described in
the review articles by Sedo & Malik (Sedo & Malik,
Dipeptidyl peptidase IV-like molecules: homologous proteins or
homologous activities? Biochimica et Biophysica Acta 2001, 36506:
1-10) and Abbott & Gorrell (Abbott, C. A. & Gorrell, M. D.,
The family of CD26/DP IV and related ectopeptidases. In: Langner
& Ansorge (ed.), Ectopeptidases. Kluwer Academic/Plenum
Publishers, New York, 2002, pp. 171-195), and in Qi, S. Y., Cloning
and characterization of dipeptidyl peptidase 10, a new member of an
emerging subgroup of serine proteases.
[0216] Another preferred embodiment of the present invention
comprises screening methods for agents which bind to the secondary
binding site and/or modulate the selectivity and/or the activity of
DP IV and/or DP IV-like enzymes. An agent according to the
invention preferably binds to at least one secondary binding site
of the DPIV or DP IV-like enzyme proteins.
[0217] The screening method for agents of the secondary binding
site comprises the following steps:
[0218] a) Contacting at least one of that effectors with DP IV
and/or a DP IV-like enzyme, preferably under conditions which
permit binding there between;
[0219] b) Adding a substrate of DP IV and/or DP IV-like enzymes to
said DP IV and/or DP IV-like enzyme;
[0220] c) Monitoring the biodegradation of the substrate and/or
measuring the residual DP IV and/or DP IV-like enzyme activity;
[0221] d) Correlating changes in the biodegradation and/or enzyme
activity with the binding of said effectors to DP IV and/or DP
IV-like enzymes; and
[0222] e) Identification of selectivity and/or activity modifying
effectors.
[0223] The agents selected by the above described screening method
can work by regulating (increasing or decreasing) the
biodegradation of at least one substrate of DP IV or the DP IV-like
enzyme, preferably by the prolongation of the half-life of such
substrate, most preferably by the inhibition of the biodegradation
of such substrate.
[0224] Conditions, under which binding between compounds and DP IV
or DP IV-like enzymes are permitted, are described, e. g. in
example 2.
[0225] DP IV or DP IV-like enzymes as used in the screening method
described above mean purified DP IV or DP IV-like enzymes from
mammals, selected from but not restricted to human, monkey, mouse,
rat etc., or DPIV or DP IV-like enzyme containing cells and cell
lines from mammals, selected from but not restricted to human,
monkey, mouse, rat etc., or DP IV or DP IV-like enzyme containing
cell extracts or body liquids e.g. liver extracts, blood plasma
samples, blood serum samples, brain extracts etc., from such
mammals.
[0226] Preferably, an agent increases the selectivity and/or
activity of DP IV or DP IV-like enzymes towards substrates by at
least about 10, preferably about 50, more preferably about 75, 90
or 100% relative to the absence of the agent. More preferably, an
agent increases the selectivity and/or activity of DP IV or DP
IV-like enzymes towards specific substrates by at least about 10,
preferably about 50, more preferably about 75, 90 or 100% and
prolongs the half live of the substrates in the serum or in the
plasma of a mammal at least about 1 fold, preferably about 2fold,
more preferably about 3fold, 4fold or higher relative to the
absence of the agent. Most preferably, an agent increases the
selectivity and/or activity of DP IV or DP IV-like enzymes in such
a way that the half live of at least one substrate in the serum or
in the plasma of a mammal is increased at least about 1fold,
preferably about 2fold, more preferably about 3fold, 4fold or
higher, most preferably complete inhibition of the degradation of
such a substrate is achieved, relative to the absence of the
agent.
[0227] It is also preferred according to the invention that the
agents modulate the interaction between DP IV or DP IV-like enzymes
and binding proteins thereof. Binding proteins are proteins that
bind other proteins in a non-covalent manner and thereby modulate
their activity or serve as carriers of these proteins. Binding
proteins of DP IV (CD26) identified so far include adenosine
deaminase, two proteins of HIV, transactivator protein (tat) and
the gp120 envelope protein, CD45, a membrane located tyrosine
phosphatase, extracellular matrix proteins, such as collagen and
fibronectin, plasminogen and streptokinase, mannose
6-phosphat/insulin-like growth factor II receptor, the isoform NH3
of the Na.sup.+/H.sup.+ exchanger from renal microvilly membranes
and the thromboxane A2 receptor.
[0228] Especially preferred are compounds or agents that prevent
and/or inhibit the interaction between DP IV and/or DP IV-like
enzymes and binding proteins of these enzymes.
[0229] According to another embodiment of the present invention,
the selectivity and/or activity modifying effectors block the
product release site of DP IV and/or DP IV-like enzymes.
[0230] Further preferred are selectivity and/or activity modifying
effectors, which prevent the tetramerization of DP IV and/or DP
IV-like enzymes at the cell surface between a soluble DP IV dimer
pair or dimers located on the surface of two different cells in a
mammal.
[0231] Agents (also called compounds herein) can be pharmacological
agents already known in the art or can be compounds previously
unknown to have any pharmacological activity. The compounds can be
naturally occurring or designed in the laboratory. They can be
isolated from microorganisms, animals, or plants, and can be
produced recombinantly, or synthesized by chemical methods in the
art. If desired, agents can be obtained using any of the numerous
combinatorial library methods known in the art, including but not
limited to, biological libraries, spatially addressable parallel
solid phase or solution phase libraries, synthetic library methods
requiring deconvolution, the "one-bead-one-compound" library
method, and synthetic library methods using affinity chromatography
selection. The biological library approach is limited to
polypeptide libraries, while the other four approaches are
applicable to polypeptide, non-peptide oligomer, or small molecule
libraries of compounds. See Lam, Anticancer Drug Des., 12, 145,
1997.
[0232] Methods for the synthesis of molecular libraries are well
known in the art (see, for example, De Witt et al., Proc. Natl.
Acad. Sci. USA 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci.
USA 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678,
1994; Cho et al., Science 261, 1303, 1993 ; Carell et al., Angew.
Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem.
Int. ed. engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233,
1994). Libraries of compounds can be present in solution (see, e.g.
Houghten, Bio Techniques 13, 412421, 1992) or on beads (Lam, nature
354, 824, 1991) chips (Fodor, Nature 364, 555556, 1993) bacteria or
spores (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al.,
Proc. Natl. Acad. Sci. USA 89, 198651869, 1992), or phage (Scott
& Smith, Science 249, 386390, 1990; Devlin, Science 249,
404406, 1990); Cwirla et la., Proc. Natl. Acad. Sci. 97, 63786382,
1990; Felici, J. Mol. Biol. 222, 301310, 1991; and Ladner, U.S.
Pat. No. 5,223,409).
[0233] High Throughput Screening
[0234] Agents can be screened for the ability to bind to DP IV or
DP IV-like enzymes or to affect DP IV or DP IV-like enzyme activity
using high throughput screening. Using high throughput screening,
many discrete compounds can be tested in parallel so that large
numbers of agents can be quickly screened. The most widely
established techniques utilize 96-well microtiter plates. The well
of the microtiter plates typically require assay volumes that range
from 50 to 500 .mu.l. In addition to the plates, many instruments,
materials, pipettors, robotics, plate washers, and plate readers
are commercially available to fit the 96-well format.
[0235] Alternatively, "Free format assays", or assays that have no
physical barrier between samples, can be used. For example, an
assay using pigment cells (melanocytes) in a simple homogeneous
assay for combinatorial peptide libraries is described by
Jayawickreme et al., Proc. Natl. Acad. Sci. USA 19, 161418
(1994).
[0236] Another example of a free format assay is described by
Chelsky, "Strategies for Screening Combinatorial Libraries: Novel
and Traditional Approaches," reported at the First Annual
conference of The Society for Biomolecular Screening in
Philadelphia, Pa. (Nov. 710, 1995). Chelsky placed a simple
homogenous enzyme assay for carbonic anhydrase inside an agarose
gel such that the enzyme in the gel would cause a color change
throughout the gel. Thereafter, beads carrying combinatorial
compounds were partially released by UV LIGHT. Compounds that
inhibited the enzyme were observed as local zones of inhibition
having less color change.
[0237] Yet another example is described by Salomon et al.,
Molecular Diversity 2, 5763 (1996). In this example, combinatorial
libraries were screened for compounds that had cytotoxic effects on
cancer cells growing in agar.
[0238] Another high throughput screening method is described in
Beutel et al., U.S. Pat. No. 5,976,813. In this method, test
samples are placed in a porous matrix. One or more assay components
are then placed within, on top of, or at the bottom of a matrix
such as a gel, a plastic sheet, a filter, or other form of easily
manipulated solid support. When samples are introduced to the
porous matrix they diffuse sufficiently slowly, such that the
assays can be performed without the test samples running
together.
[0239] Binding Assays
[0240] For binding assays, the agent is preferably a small molecule
which binds to and occupies, the secondary binding site of DP IV or
DP IV-like enzymes, such that normal biological activity is changed
or prevented. Examples of such small molecules include, but are not
limited to, small peptides or peptide like molecules.
[0241] In binding assays, either the agent of DP IV or the DP
IV-like enzyme can comprise a detectable label, such as a
fluorescent, radioisotopic, chemiluminescent, or the enzyme is
labeled, such as horseradish peroxidase, alkaline phosphatase, or
luciferase. Detection of an agent, which is bound to DP IV or the
DP IV-like enzyme can then be accomplished, for example, by direct
counting of radioemmission, by scintillation counting, or by
determining conversion of an appropriate substrate to a detectable
product.
[0242] Alternatively, binding of an agent to DP IV or a DP IV-like
enzyme can be determined without labelling either of the
interactants. For example, a microphysiometer can be used to detect
binding of an agent with DP IV or a DP IV-like enzyme. A
microphysiometer (e.g., Cytosensor.TM.) is an analytical instrument
that measures the rate at which a cell acidifies its environment
using a light-addressable potentiometric sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the
interaction between an agent and DP IV or a DP IV-like enzyme
(McConnel et al., Science 257, 19061912, 1992).
[0243] Determining the ability of an agent to bind to DP IV or a DP
IV-like enzyme also can be accomplished using a technology such as
real-time Biomolecular Interaction Analysis (BIA) (Sjolander &
Urbaniczky, Anal. Chem. 63, 23382345, 1991, and Szabo et al., Curr.
Opin. Struct. Biol. 5, 699705, 1995) BIA is a technology for
studying biospecific interactions in real time, without labelling
any of the interactants (e.g. BIAcore.TM.). Changes in the optical
phenomenon surface plasmon resonance (SPR) can be used as an
indication of real-time reactions between biological molecules.
[0244] In yet another aspect of the invention, DP IV or a DP
IV-like enzyme can be used as a "bait protein" in a two hybrid
assay or three-hybrid assay (see, e.g. U.S. Pat. No. 5,283,317;
Zervos et al., Cell 72, 223232, 1993; Madura 920924, 193; Iwabuchi
et al., Oncogene 8, 16931696, 1993; and Brent WO94/10300), to
identify other proteins which bind to or interact with the DP IV or
the DP IV-like enzyme and modulate its activity.
[0245] The two hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. For example, in one construct, a polynucleotide
encoding DP IV or a DP IV-like enzyme can be fused to a
polynucleotide encoding the DNA binding domain of a known
transcription factor (e.g. GAL4). In the other construct a DNA
sequence that encodes an unidentified protein ("prey" or "sample")
can be fused to a polynucleotide that c odes for the activation
domain of the known transcription factor. If the "bait" and the
"prey" proteins are able to interact in vivo to form an protein
dependent complex, the DNA binding and activation domains of the
transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g. LacZ),
which is operably linked to a transcriptional regulatory site
responsive to the transcription factor. Expression of the reporter
gene can be detected, and cell colonies containing the functional
transcription factor can be isolated and used to obtain the DNA
sequence encoding the protein which interacts with the
dipeptidyl-peptidase IV-like enzyme polypeptide.
[0246] It may be desirable to immobilize either the DP IV or DP
IV-like enzyme or the agent to facilitate separation of bound from
unbound forms of one or both of the interactants, as-well-as to
accommodate automation of the assay. Thus, either DP IV or the DP
IV-like enzyme or the agent can be bound to a solid support.
Suitable solid supports include, but are not limited to, glass or
plastic slices, tissue culture plates, microtiter wells, tubes,
silicon chips, or particles such as beads (including, but not
limited to latex, polysterene, or glass beads). Any method known in
the art can be used to attach DP IV or the DP IV-like enzyme or
agent to a solid support, including use of covalent and
non-covalent linkages, passive absorption, or pairs of binding
moieties attached respectively to the polypeptide or agent and the
solid support. Agents are preferably bound to the solid support in
an array, so that the location of individual test compounds can be
tracked. Binding of a test compound to a DP IV or a DP IV-like
enzyme can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and microcentrifuge tubes.
[0247] In one embodiment, the DP IV or DP IV-like enzyme is a
fusion protein comprising a domain that allows the DP IV or DP
IV-like enzyme to be bound to a solid support. For example,
glutathione-S-transferase fusion proteins can be absorbed onto
glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microtiter plates, which are then combined
with the agent and the non-absorbed DP IV or DP IV-like enzyme; the
mixture is then incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads or microtiter plate wells are
washed to remove any unbound components. Binding of the
interactants can be determined either directly or indirectly, as
described above. Alternatively, the complexes can be dissociated
from the solid support before binding is determined.
[0248] Other techniques for immobilizing proteins on a solid
support also can be used in the screening assays of the invention.
For example, either DP IV or a DP IV-like enzyme or an agent can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated DP IV or DP IV-like enzymes or agents can be prepared
from biotin-NHS-(N-hydroxysuccinimide) using techniques well known
in the art (e.g. biotinylation kit, Pierce Chemicals, Rockford,
Ill.) and immobilized in the wells of streptavidin-coated 96 well
plates (Pierce chemical). Alternatively, antibodies which
specifically bind to DP IV, a DP IV-like enzyme or an agent, but
which do not interfere with a desired binding site, such as
secondary binding site or the active site of DP IV or the DP
IV-like enzyme, can be derivatized to the wells of the plate.
Unbound targets or proteins can be trapped in the wells by antibody
conjugation.
[0249] Examples for commercial available antibodies against DP IV
or CD26 are for instance:
15 Species Company Clone (antigen) Application Host Coulter Ta1
human IF, FACS Mouse Ba5 human FACS Biozol TA59 human ICH*
(Endogen) Pharmingen M-A216 human IF, FACS Mouse Biotrend 13.4 rat
ICH, IF Mouse M-T099 human ICH, IF Mouse 134-2C2 human IF, FACS
Mouse LT-27 human IF, FACS Mouse Biozol MRCOX-61 rat FC Mouse
Biozol 236.3 rat IF, IPrep, IHstaining Mouse Research 202.36 human
IF Mouse Diagnostics Research 134-2C2 human T-cell signaling, HIV
Mouse Diagnostics infection
[0250] Methods for detecting such complexes, in addition to those
described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies which specifically
bind to DP IV or DP IV-like enzymes or the agent, enzyme linked
assays which rely on detecting an activity of the DP IV or the DP
IV-like enzyme, and SDS gel electrophoresis under non-reducing
conditions.
[0251] Screening for agents which bind to DP IV or a DP IV-like
enzyme also can be carried out in an intact cell. Any cell which
comprises DP IV or a DP IV-like enzyme can be used in a cell-based
assay system. DP IV or a DP IV-like enzyme can be naturally
occurring in the cell or can be introduced using techniques such as
those described above. Binding of the agents to DP IV or a DP
IV-like enzyme is determined as described above.
[0252] Enzyme Assays
[0253] Agents can be tested for the ability to increase or decrease
the activity of a mammalian DP IV or DP IV-like enzyme. DP IV
activity can be measured, for example, as described in U.S. Pat.
No. 5,601,986 and, specific for the present invention, in examples
1 to 3.
[0254] Further on, a screening method for the identification and
determination of one or more secondary binding sites on DP IV
and/or DP IV-like enzymes is provided.
[0255] The screening method for secondary binding site(s) of DP IV
and/or DP IV-like enzymes comprises the following steps:
[0256] a) Providing two or more different substrates, each having
an amino acid sequence, which binds to DP IV and/or DP IV-like
enzymes and aligning the amino acid sequences of said
substrates;
[0257] b) Identifying at least one consensus sequence amongst said
substrate amino acid sequences;
[0258] c) Synthesizing a peptide having said consensus
sequence;
[0259] d) Contacting said synthesized peptide with DP IV and/or a
DP IV-like enzyme;
[0260] e) Adding a substrate of DP IV and/or a DP IV-like enzyme to
the DP IV and/or DP IV-like enzyme;
[0261] f) Monitoring the biodegradation of the substrate and/or
measuring the residual DP IV and/or DP IV-like enzyme activity;
and
[0262] g) Correlating changes in said biodegradation and/or enzyme
activity with the presence of a secondary binding site capable of
modulating the substrate specificity of DP IV and/or DP IV-like
enzymes.
[0263] Consensus sequences are highly conserved sequence segments.
Preferred according to the invention are consensus sequences with
the length of 3 to 20 amino acids, more preferred of 5 to 12 amino
acids, most preferred 5 to 7 amino acids.
[0264] In another illustrative embodiment of the present invention,
the agents, which bind to the secondary binding site, e.g. obtained
or selected by the screening method described herein, can be used
alone or in combination with DP IV-inhibitors for the treatment of
any type of DP IV mediated disorders, selected but not restricted
to, impaired glucose tolerance, glucosuria, lipid disorders,
dyslipidemia, hyperlipidaemia, hypertriglyceridemia,
hypercholesterolemia, low HDL levels, high LDL levels, metabolic
acidosis, hyperglycemia, diabetes mellitus, diabetic neuropathy and
nephropathy and of sequelae caused by diabetes mellitus in mammals,
metabolism-related hypertension and cardiovascular sequelae caused
by hypertension in mammals, for the prophylaxis or treatment of
skin diseases and diseases of the mucosae, autoimmune diseases and
inflammatory conditions, and for the treatment of psychosomatic,
neuropsychiatric and depressive illnesses, such as anxiety,
depression, sleep disorders, chronic fatigue, schizophrenia,
epilepsy, nutritional disorders, spasm and chronic pain,
atherosclerosis and its sequelae, vascular restenosis, irritable
bowel syndrome, inflammatory bowel disease, including Crohn's
disease and ulcerative colitis, other inflammatory conditions,
pancreatitis, abdominal obesity, neurodegenerative disease,
retinopathy, nephropathy, Syndrome X, ovarian hyperandrogenism
(polycystic ovarian syndrome), growth hormone deficiency,
neutropenia, tumor metastasis, benign prostatic hypertrophy,
gingivitis, osteoporosis, and other conditions.
[0265] Agents such as N-(N'-substituted
glycyl)-2-cyanopyrrolidines, L-threo-isoleucyl thiazolidine
(P32/98), L-allo-isoleucyl thiazolidine, L-threo-isoleucyl
pyrrolidine, and L-allo-isoleucyl pyrrolidine have been developed
which inhibit the enzymatic activity of DP IV and are described in
U.S. Pat. No. 6,001,155, WO 99/61431, WO 99/67278, WO 99/67279, DE
198 34 591, WO 97/40832, DE 196 16 486 C 2, WO 98/19998, WO
00/07617, WO 99/38501, and WO 99/46272. Further examples of low
molecular weight dipeptidyl peptidase IV inhibitors are agents such
as tetrahydroisoquinolin-3-carboxamide derivatives, N-substituted
2-cyanopyroles and--pyrrolidines, N-(N'-substituted
glycyl)-2-cyanopyrrolidines, N-(substituted glycyl)-thiazolidines,
N-(substituted glycyl)-4-cyanothiazolidines,
amino-acyl-borono-prolyl-inh- ibitors and cyclopropyl-fused
pyrrolidines. Inhibitors of dipeptidyl peptidase IV are described
in U.S. Pat. No. 6,380,398, U.S. Pat. No. 6,011,155; U.S. Pat. No.
6,107,317; U.S. Pat. No. 6,110,949; U.S. Pat. No. 6,124,305; U.S.
Pat. No. 6,172,081; WO 95/15309, WO 99/61431, WO 99/67278, WO
99/67279, DE 198 34 591, WO 97/40832, DE 196 16 486 C 2, WO
98/19998, WO 00/07617, WO 99/38501, WO 99/46272, WO 99/38501, WO
01/68603, WO 01/40180, WO 01/81337, WO 01/81304, WO 01/55105, WO
02/02560 and WO 02/14271, WO 02/04610, WO 02/051836, WO 02/068420,
WO 02/076450; WO 02/083128, WO 02/38541, WO 03/000180, WO03/000181,
WO 03/000250, WO 0 3/002530, WO 03/002531, WO 03/002553, WO
03/002593, WO 03/004496, WO 03/004498, WO 03/024965, WO 03/024942,
WO 03/035067, WO 03/037327, WO 03/035057, WO 03/045977, WO
03/055881, WO 03/68748, WO 03/68757, WO 03/057666, WO 03057144, WO
03/040174 and WO 03/033524, the teachings of which are herein
incorporated by reference in their entirety concerning these
inhibitors, their uses, definition and their production. The goal
of these agents is to inhibit DP IV, and by doing so, to relieve
effectively any type of DP IV-mediated disease. The inventors of
the present invention have surprisingly found that such agents can
be advantageously employed for an entirely different therapeutic
purpose, then previously known by those skilled in the art.
[0266] Preferred for the use in combination with agents binding to
the secondary binding site of DP IV or DP IV-like enzymes are DP
IV-inhibitors such as valine pyrrolidide (Novo Nordisk),
NVP-DPP728A
(1-[[[2-[{5-cyanopyridin-2-yl}amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrro-
lidine) (Novartis) as disclosed by Hughes et al., Biochemistry, 38
(36), 11597-11603, 1999, LAF-237
(1-[(3-hydroxy-adamant-1-ylamino)-acetyl]-pyrr-
olidine-2(S)-carbonitrile); disclosed by Hughes et al., Meeting of
the American Diabetes Association 2002, Abstract no. 272 or
(Novartis), TSL-225
(tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid),
disclosed by Yamada et. al., Bioorg. & Med. Chem. Lett. 8
(1998), 1537-1540, 2-cyanopyrrolidides and 4-cyanopyrrolidides as
disclosed by Asworth et al., Bioorg. & Med. Chem. Lett., 6, No.
22, pp 1163-1166 and 2745-2748 (1996), FE-999011, disclosed by
Sudre et al., Diabetes 51 (5), pp 1461-1469 (2002) (Ferring) and
the compounds disclosed in WO 01/34594 (Guilford), employing
dosages as set out in the above references.
[0267] In one especially illustrative embodiment, the present
invention relates to the use of agents, which bind to the secondary
binding site(s) of DP IV or DP IV-like enzymes in combination with
dipeptide-like compounds and compounds analogous to dipeptide
compounds that are formed from an amino acid and a thiazolidine or
pyrrolidine group, and salts thereof, referred to hereinafter as
dipeptide-like compounds. Preferably the amino acid and the
thiazolidine or pyrrolidine group are bonded with an amide
bond.
[0268] Especially suitable for that purpose according to the
invention are dipeptide-like compounds in which the amino acid is
preferably selected from a natural amino acid, such as, for
example, leucine, valine, glutamine, glutamic acid, proline,
isoleucine, asparagines and aspartic acid.
[0269] The dipeptide-like compounds used according to the invention
exhibit at a concentration (of dipeptide compounds) of 10 .mu.M, a
reduction in the activity of plasma dipeptidyl peptidase IV or DP
IV-analogous enzyme activities of at least 10%, especially of at
least 40%. Frequently a reduction in activity of at least 60% or at
least 70% is also required. Preferred agents may also exhibit a
reduction in activity of a maximum of 20% or 30%.
[0270] Preferred compounds are N-valyl prolyl, O-benzoyl
hydroxylamine, a lanyl pyrrolidine, isoleucyl thiazolidine like
L-allo-isoleucyl thiazolidine, L-threo-isoleucyl pyrrolidine and
salts thereof, especially the fumaric salts, and L-allo-isoleucyl
pyrrolidine and salts thereof. Especially preferred compounds are
glutaminyl pyrrolidine and glutaminyl thiazolidine of formulas 1
and 2: 2
[0271] Further preferred compounds are given in Table 6.
[0272] The salts of the dipeptide-like compounds can be present in
a molar ratio of dipeptide (-analogous) component to salt component
of 1:1 or 2:1. Such a salt is, for example, (Ile-Thia).sub.2
fumaric acid.
16TABLE 6 Structures of further preferred dipeptide compounds DP
IV-inhibitor H-Asn-pyrrolidine H-Asn-thiazolidine H-Asp-pyrrolidine
H-Asp-thiazolidine H-Asp(NHOH)-pyrrolidine H-Asp(NHOH)-thiazolidine
H-Glu-pyrrolidine H-Glu-thiazolidine H-Glu(NHOH)-pyrrolidine
H-Glu(NHOH)-thiazolidine H-His-pyrrolidine H-His-thiazolidine
H-Pro-pyrrolidine H-Pro-thiazolidine H-Ile-azididine
H-Ile-pyrrolidine H-L-allo-Ile-thiazolidine H-Val-pyrrolidine
H-Val-thiazolidine
[0273] In another preferred embodiment, the present invention
provides the use of agents binding to the secondary binding site(s)
of DP IV or DP IV-like enzymes in combination with substrate-like
peptide compounds of formula 3 useful for competitive modulation of
dipeptidyl peptidase IV catalysis: 3
[0274] wherein
[0275] A, B, C, D and E are independently any amino acid moieties
including proteinogenic amino acids, non-proteinogenic amino acids,
L-amino acids and D-amino acids and wherein E and/or D may be
absent.
[0276] Further conditions regarding formula (3):
[0277] A is an amino acid except a D-amino acid,
[0278] B is an amino acid selected from Pro, Ala, Ser, Gly, Hyp,
acetidine-(2)-carboxylic acid and pipecolic acid,
[0279] C is any amino acid except Pro, Hyp,
acetidine-(2)-carboxylic acid, pipecolic acid and except
N-alkylated amino acids, e.g. N-methyl valine and sarcosine,
[0280] D is any amino acid or missing, and
[0281] E is any amino acid or missing,
[0282] or:
[0283] C is any amino acid except Pro, Hyp,
acetidine-(2)-carboxylic acid, pipecolic acid, except N-alkylated
amino acids, e.g. N-methyl valine and sarcosine, and except a
D-amino-acid;
[0284] D is any amino acid selected from Pro, Ala, Ser, Gly, Hyp,
acetidine-(2)-carboxylic acid and pipecolic acid, and
[0285] E is any amino acid except Pro, Hyp,
acetidine-(2)-carboxylic acid, pipecolic acid and except
N-alkylated amino acids, e.g. N-methyl valine and sarcosine.
[0286] Examples of amino acids which can be used in the present
invention are: L and D-amino acids, N-methyl-amino-acids; allo- and
threo-forms of Ile and Thr, which can, e.g. be .alpha.-, .beta.- or
.OMEGA.-amino acids, whereof .alpha.-amino acids are preferred.
[0287] Examples of amino acids throughout the claims and the
description are: aspartic acid (Asp), glutamic acid (Glu), arginine
(Arg), lysine (Lys), histidine (His), glycine (Gly), serine (Ser)
and cysteine (Cys), threonine (Thr), asparagine (Asn), glutamine
(Gln), tyrosine (Tyr), alanine (Ala), proline (Pro), valine (Val),
isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine
(Phe), tryptophan (Trp), hydroxyproline (Hyp), beta-alanine
(beta-Ala), 2-amino octanoic acid (Aoa), azetidine-(2)-carboxylic
acid (Ace), pipecolic acid (Pip), 3-amino propionic, 4-amino
butyric and so forth, alpha-aminoisobutyric acid (Aib), sarcosine
(Sar), ornithine (Orn), citrulline (Cit), homoarginine (Har),
t-butylalanine (t-butyl-Ala), t-butylglycine (t-butyl-Gly),
N-methylisoleucine (N-MeIle), phenylglycine (Phg),
cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) and
methionine sulfoxide (MSO), Acetyl-Lys, modified amino acids such
as phosphoryl-serine (Ser(P)), benzyl-serine (Ser(Bzl)) and
phosphoryl-tyrosine (Tyr(P)), 2-aminobutyric acid (Abu),
aminoethylcysteine (AECys), carboxymethylcysteine (Cmc),
dehydroalanine (Dha), dehydroamino-2-butyric acid (Dhb),
carboxyglutaminic acid (Gla), homoserine (Hse), hydroxylysine
(Hyl), cis-hydroxyproline (cisHyp), trans-hydroxyproline
(transHyp), isovaline (Iva), pyroglutamic acid (Pyr), norvaline
(Nva), 2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid (3-Abz),
4-aminobenzoic acid (4-Abz), 4-(aminomethyl)benzoic acid (Amb),
4-(aminomethyl)cyclohexanecarboxylic acid (4-Amc), Penicillamine
(Pen), 2-Amino-4-cyanobutyric acid (Cba), cycloalkane-carboxylic
aicds.
[0288] Examples of .OMEGA.-amino acids are e.g.: 5-Ara
(aminoraleric acid), 6-Ahx (aminohexanoic acid), 8-Aoc
(aminooctanoic aicd), 9-Anc (aminovanoic aicd), 10-Adc
(aminodecanoic acid), 11-Aun (aminoundecanoic acid), 12-Ado
(aminododecanoic acid).
[0289] Further amino acids are: indanylglycine (Igl),
indoline-2-carboxylic acid (Idc), octahydroindole-2-carboxylic acid
(Oic), diaminopropionic acid (Dpr), diaminobutyric acid (Dbu),
naphtylalanine (1-Nal), (2-Nal), 4-aminophenylalanin
(Phe(4-NH.sub.2)), 4-benzoylphenylalanine (Bpa), diphenylalanine
(Dip), 4-bromophenylalanine (Phe(4-Br)), 2-chlorophenylalanine
(Phe(2-C1)), 3-chlorophenylalanine (Phe(3-C1)),
4-chlorophenylalanine (Phe(4-C1)), 3,4-chlorophenylalanine (Phe
(3,4-C1.sub.2)), 3- fluorophenylalanine (Phe(3-F)),
4-fluorophenylalanine (Phe(4-F)), 3,4- fluorophenylalanine
(Phe(3,4-F2)), pentafluorophenylalanine (Phe(F.sub.5)),
4-guanidinophenylalanine (Phe(4-guanidino)), homophenylalanine
(hPhe), 3-jodophenylalanine (Phe(3-J)), 4 jodophenylalanine
(Phe(4-J)), 4-methylphenylalanine (Phe(4-Me)), 4-nitrophenylalanine
(Phe-4-NO.sub.2)), biphenylalanine (Bip),
4-phosphonomehtylphenylalanine (Pmp), cyclohexyglycine (Ghg),
3-pyridinylalanine (3-Pal), 4-pyridinylalanine (4-Pal),
3,4-dehydroproline (A-Pro), 4-ketoproline (Pro(4-keto)),
thioproline (Thz), isonipecotic acid (Inp),
1,2,3,4,-tetrahydroisoquinolin-3-carboxyl- ic acid (Tic),
propargylglycine (Pra), 6-hydroxynorleucine (NU(6-OH)),
homotyrosine (hTyr), 3-jodotyrosine (Tyr(3-J)), 3,5-dijodotyrosine
(Tyr(3,5-J.sub.2)), d-methyl-tyrosine (Tyr(Me)),
3-NO.sub.2-tyrosine (Tyr(3-NO.sub.2)), phosphotyrosine
(Tyr(PO.sub.3H.sub.2)), alkylglycine, 1-aminoindane-1-carboxy acid,
2-aminoindane-2-carboxy acid (Aic),
4-amino-methylpyrrol-2-carboxylic acid (Py),
4-amino-pyrrolidine-2-carbox- ylic acid (Abpc),
2-aminotetraline-2-carboxylic acid (Atc), diaminoacetic acid
(Gly(NH.sub.2)), diaminobutyric acid (Dab),
1,3-dihydro-2H-isoinole-- carboxylic acid (Disc),
homocylcohexylalanin (hCha), homophenylalanin (hPhe oder Hof),
trans-3-phenyl-azetidine-2-carboxylic acid,
4-phenyl-pyrrolidine-2-carboxylic acid,
5-phenyl-pyrrolidine-2-carboxylic acid, 3-pyridylalanine (3-Pya),
4-pyridylalanine (4-Pya), styrylalanine,
tetrahydroisoquinoline-1-carboxylic acid (Tiq),
1,2,3,4-tetrahydronorharm- ane-3-carboxylic acid (Tpi),
.beta.-(2-thienryl)-alanine (Tha).
[0290] Other amino acid substitutions for those encoded in the
genetic code can also be included in peptide compounds within the
scope of the invention and can be classified within this general
scheme.
[0291] Proteinogenic amino acids are defined as natural
protein-derived .alpha.-amino acids. Non-proteinogenic amino acids
are defined as all other amino acids, which are not building blocks
of common natural proteins.
[0292] The resulting peptides may be synthesized as the free
C-terminal acid or as the C-terminal amide form. The free acid
peptides or the amides may be varied by side chain modifications.
Such side chain modifications include for instance, but are not
restricted to, homoserine formation, pyroglutamic acid formation,
disulphide bond formation, deamidation of asparagine or glutamine
residues, methylation, t-butylation, t-butyloxycarbonylation,
4-methylbenzylation, thioanysilation, thiocresylation,
benzyloxyrnethylation, 4-nitrophenylation, benzyloxycarbonylation,
2-nitrobencoylation, 2-nitrosulphenylation,
4-toluenesulphonylation, pentafluorophenylation,
diphenylmethylation, 2-chlorobenzyloxycarbonylation,
2,4,5-trichlorophenylation, 2-bromobenzyloxycarbonylation,
9-fluorenylmethyloxycarbonylation, triphenylmethylation,
2,2,5,7,8,-pentamethylchroman-6-sulphonylation, hydroxylation,
oxidation of methionine, formylation, acetylation, anisylation,
benzylation, bencoylation, trifluoroacetylation, carboxylation of
aspartic acid or glutamic acid, phosphorylation, sulphation,
cysteinylation, glycolysation with pentoses, deoxyhexoses,
hexosamines, hexoses or N-acetylhexosamines, famesylation,
myristolysation, biotinylation, palmitoylation, stearoylation,
geranylgeranylation, glutathionylation, 5'-adenosylation,
ADP-ribosylation, modification with N-glycolylneuraminic acid,
N-acetylneuraminic acid, pyridoxal phosphate, lipoic acid,
4'-phosphopantetheine, or N-hydroxysuccinimide.
[0293] In the compounds of formula (3), the amino acid moieties A,
B, C, D, and E are respectively attached to the adjacent moiety by
amide bonds in a usual manner according to standard nomenclature so
that the amino-terminus (N-terminus) of the amino acids (peptide)
is drawn on the left and the carboxyl-terminus of the amino acids
(peptide) is drawn on the right. (C-terminus).
[0294] Until the present invention by Applicants, known peptide
substrates of the proline-specific serine protease dipeptidyl
peptidase IV in vitro are the tripeptides Diprotin A (Ile-Pro-Ile),
Diprotin B (Val-Pro-Leu) and Diprotin C (Val-Pro-Ile). Applicants
have unexpectedly discovered that the compounds disclosed herein
above and below act as substrates of dipeptidyl peptidase IV in
vivo in a mammal and, in pharmacological doses, improve insulin
sensitivity and islet signaling and alleviate pathological
abnormalities of the metabolism of mammals such as glucosuria,
hyperlipidaemia, metabolic acidosis and diabetes mellitus by
competitive catalysis.
[0295] Preferred peptide compounds are listed in table 7.
17TABLE 7 Examples of peptide substrates Mass (exp.).sup.1 Peptide
Mass (calc.) [M + H.sup.+] 2-Amino octanoic acid-Pro-Ile 369.5
370.2 Abu-Pro-Ile 313.4 314.0 Aib-Pro-Ile 313.4 314.0 Aze-Pro-Ile
311.4 312.4 Cha-Pro-Ile 381.52 382.0 Ile-Hyp-Ile 356.45 358.2
Ile-Pro-allo-Ile 341.4 342.0 Ile-Pro-t-butyl-Gly 341.47 342.36
Ile-Pro-Val 327.43 328.5 Nle-Pro-Ile 341.45 342.2 Nva-Pro-Ile
327.43 328.2 Orn-Pro-Ile 342.42 343.1 Phe-Pro-Ile 375.47 376.2
Phg-Pro-Ile 361.44 362.2 Pip-Pro-Ile 338.56 340.0 Ser(Bzl)-Pro-Ile
405.49 406.0 Ser(P)-Pro-Ile 395.37 396.0 Ser-Pro-Ile 315.37 316.3
t-butyl-Gly-Pro-D-Val 327.4 328.6 t-butyl-Gly-Pro-Gly 285.4 286.3
t-butyl-Gly-Pro-Ile 341.47 342.1 t-butyl-Gly-Pro-Ile-amid- e 340.47
341.3 t-butyl-Gly-Pro-t-butyl-Gly 341.24 342.5 t-butyl-Gly-Pro-Val
327.4 328.4 Thr-Pro-Ile 329.4 330.0 Tic-Pro-Ile 387.46 388.0
Trp-Pro-Ile 414.51 415.2 Tyr(P)-Pro-Ile 471.47 472.3
Tyr-Pro-allo-Ile 391.5 392.0 Val-Pro-allo-Ile 327.4 328.5
Val-Pro-t-butyl-Gly 327.4 328.15 Val-Pro-Val 313.4 314.0 .sup.1[M +
H.sup.+] were determined by Electrospray mass spectrometry in
positive ionization mode.
[0296] t-butyl-Gly is defined as: 4
[0297] Ser(Bzl) and Ser(P) are defined as benzyl-serine and
phosphoryl-serine, respectively. Tyr(P) is defined as
phosphoryl-tyrosine.
[0298] Further preferred compounds, which can be used according to
the present invention in combination with agents binding to the
secondary binding site(s) of DP IV or DP IV-like enzymes, are
peptidylketones of formula 4: 5
[0299] and pharmaceutically acceptable salts thereof, wherein:
[0300] A is selected from the following structures: 6
[0301] wherein
[0302] X.sup.1 is H or an acyl or oxycarbonyl group including an
amino acid residue, N-protected amino acid residue, a peptide
residue or a N-protected peptide residue,
[0303] X.sup.2 is H, --(CH).sub.m--NH--C.sub.5H.sub.3N--Y with
m=2-4 or --C.sub.5H.sub.3N--Y (a divalent pyridyl residue) and Y is
selected from H, Br, Cl, I, NO.sub.2 or CN,
[0304] X.sup.3 is H or selected from an alkyl-, alkoxy-, halogen-,
nitro-, cyano- or carboxy-substituted phenyl or from an alkyl-,
alkoxy-, halogen-, nitro-, cyano- or carboxy-substituted pyridyl
residue,
[0305] X.sup.4 is H or selected from an alkyl-, alkoxy-, halogen-,
nitro-, cyano- or carboxy-substituted phenyl or from an alkyl-,
alkoxy-, halogen-, nitro-, cyano- or carboxy-substituted pyridyl
residue,
[0306] X.sup.5 is H or an alkyl, alkoxy or phenyl residue,
[0307] X.sup.6 is H or an alkyl residue,
[0308] for n=1
[0309] X is selected from: H, OR.sup.2, SR.sup.2, NR.sup.2R.sup.3,
N.sup.+R.sup.2R.sup.3R.sup.4, wherein:
[0310] R.sup.2 stands for acyl residues, which are optionally
substituted with alkyl, cycloalkyl, aryl or heteroaryl residues, or
for amino acid residues or peptidic residues, or alkyl residues,
which are optionally substituted with alkyl, cycloalkyl, aryl or
heteroaryl residues,
[0311] R.sup.3 stands for alkyl or acyl residues, wherein R.sup.2
and R.sup.3 may be part of a saturated or unsaturated carbocyclic
or heterocyclic ring,
[0312] R.sup.4 stands for alkyl residues, wherein R.sup.2 and
R.sup.4 or R.sup.3 and R.sup.4 may be part of a saturated or
unsaturated carbocyclic or heterocyclic ring,
[0313] for n=0
[0314] X is selected from: 7
[0315] wherein
[0316] B stands for: O, S or NR.sup.5, wherein R.sup.5 is H, alkyl
or acyl,
[0317] C, D, E, F, G, Y, K, L, M, Q, T, U, V and W are
independently selected from alkyl and substituted alkyl residues,
oxyalkyl, thioalkyl, aminoalkyl, carbonylalkyl, acyl, carbamoyl,
aryl and heteroaryl residues, and
[0318] Z is selected from H, or a branched or straight chain alkyl
residue from C.sub.1-C.sub.9, a branched or straight chain alkenyl
residue from C.sub.2-C.sub.9, a cycloalkyl residue from
C.sub.3-C.sub.8, a cycloalkenyl residue from C.sub.5-C.sub.7, an
aryl or heteroaryl residue, or a side chain selected from all side
chains of all natural amino acids or derivatives thereof.
[0319] In preferred compounds of formula 4, A is 8
[0320] wherein
[0321] X.sup.1 is H or an acyl or oxycarbonyl group including an
amino acid residue, N-acylated amino acid residue, a peptide
residue from di- to pentapeptides, preferably a dipeptide residue,
or a N-protected peptide residue from di- to pentapeptides,
preferably a N-protected dipeptide residue
[0322] X.sup.2 is H, --(CH).sub.m--NH--C.sub.5H.sub.3N--Y with
m=2-4 or --C.sub.5H.sub.3N--Y (a divalent pyridyl residue) and Y is
selected from H, Br, Cl, I, NO.sub.2 or CN,
[0323] for n=1
[0324] X is preferably selected from: H, OR.sup.2, SR.sup.2,
NR.sup.2R.sup.3, wherein:
[0325] R stands for acyl residues, which are optionally substituted
with alkyl, cycloalkyl, aryl or heteroaryl residues, or for amino
acid residues or peptidic residues, or alkyl residues, which are
optionally substituted with alkyl, cycloalkyl, aryl or heteroaryl
residues,
[0326] R.sup.3 stands for alkyl or acyl residues, wherein R.sup.2
and R.sup.3 may be part of a saturated or unsaturated carbocyclic
or heterocyclic ring,
[0327] for n=0
[0328] X is preferably selected from: 9
[0329] wherein
[0330] B stands for: O, S or NR , wherein R.sup.5 is H, alkyl or
acyl,
[0331] C, D, E, F, G, Y, K, L, M and Q are independently selected
from alkyl and substituted alkyl residues, oxyalkyl, thioalkyl,
aminoalkyl, carbonylalkyl, acyl, carbamoyl, aryl and heteroaryl
residues, and
[0332] Z is selected from H, or a branched or straight chain alkyl
residue from C.sub.1-C.sub.9, preferably C.sub.2-C.sub.6, a
branched or straight chain alkenyl residue from C.sub.2-C.sub.9, a
cycloalkyl residue from C.sub.3-C.sub.8, a cycloalkenyl residue
from C.sub.5-C.sub.7, an aryl or heteroaryl residue, or a side
chain selected from all side chains of all natural amino acids or
derivatives thereof
[0333] In more preferred compounds of formula 4, A is 10
[0334] wherein
[0335] X.sup.1 is H or an acyl or oxycarbonyl group including an
amino acid residue, N-acylated amino acid residue or a peptide
residue from di- to pentapeptides, preferably a dipeptide residue,
or a N-protected peptide residue from di- to pentapeptides,
preferably a N-protected dipeptide residue
[0336] for n=1,
[0337] X is preferably selected from: H, OR.sup.2, SR.sup.2,
wherein:
[0338] R.sup.2 stands for acyl residues, which are optionally
substituted with alkyl or aryl residues,
[0339] for n=0
[0340] X is preferably selected from: 11
[0341] wherein
[0342] B stands for: O, S or NR.sup.5, wherein R.sup.5 is H, alkyl
or acyl,
[0343] C, D, E, F, G, Y, K, L, M and Q are independently selected
from alkyl and substituted alkyl residues, oxyalkyl, thioalkyl,
aminoalkyl, carbonylalkyl, acyl, carbamoyl, aryl and heteroaryl
residues, and
[0344] Z is selected from H, or a branched or straight chain alkyl
residue from C.sub.1-C.sub.9, preferably C.sub.2-C.sub.6, a
branched or straight chain alkenyl residue from C.sub.2-C.sub.9, a
cycloalkyl residue from C.sub.3-C.sub.8, a cycloalkenyl residue
from C.sub.5-C.sub.7, an aryl or heteroaryl residue, or a side
chain selected from all side chains of all natural amino acids or
derivatives thereof.
[0345] In most preferred compounds of formula 4, A is 12
[0346] wherein
[0347] X.sup.1 is H or an acyl or oxycarbonyl group including an
amino acid residue, N-acylated amino acid residue or a dipeptide
residue, containing a Pro or Ala in the penultimate position, or a
N-protected dipeptide residue containing a Pro or Ala in the
penultimate position,
[0348] for n=1,
[0349] X is H,
[0350] for n=0
[0351] X is preferably selected from: 13
[0352] wherein
[0353] B stands for: O or S, most preferably for S
[0354] C, D, E, F, G, Y, K, L, M, Q, are H and
[0355] Z is selected from H, or a branched or straight chain alkyl
residue from C.sub.3-C.sub.5, a branched or straight chain alkenyl
residue from C.sub.2-C.sub.9, a cycloalkyl residue from
C.sub.5-C.sub.7, a cycloalkenyl residue from C.sub.5-C.sub.7, an
aryl or heteroaryl residue, or a side chain selected from all side
chains of all natural amino acids or derivatives thereof.
[0356] Most preferred for Z is H.
[0357] According to a preferred embodiment the acyl groups are
C.sub.1-C.sub.6-acyl groups.
[0358] According to a further preferred embodiment the alk(yl)
groups are C.sub.1-C.sub.6-alk(yl) groups, which may be branched or
unbranched.
[0359] According to a still further preferred embodiment the alkoxy
groups are C.sub.1-C.sub.6-alkoxy groups.
[0360] According to yet another preferred embodiment the aryl
residues are C.sub.5-C.sub.12 aryl residues that have optionally
fused rings.
[0361] According to a still further preferred embodiment the
cycloalkyl residues (carbocycles) are C.sub.3-C.sub.8-Cycloalkyl
residues.
[0362] According to another preferred embodiment the heteroaryl
residues are C.sub.4-C.sub.11 aryl residues that have optionally
fused rings and, in at least one ring, additionally from 1 to 4
preferably 1 or 2 hetero atoms, such as O, N and/or S.
[0363] According to a further preferred embodiment peptide residues
are corresponding residues containing from 2 to 50 amino acids.
[0364] According to another preferred embodiment the heterocyclic
residues are C.sub.2-C.sub.7-cycloalkyl radicals that additionally
have from 1 to 4, preferably 1 or 2 hetero atoms, such as O, N
and/or S.
[0365] According to astill further preferred embodiment the carboxy
groups are C.sub.1-C.sub.6 carboxy groups, which may be branched or
unbranched.
[0366] According to yet another preferred embodiment the
oxycarbonyl groups are groups of the formula
--O--(CH.sub.2).sub.1-6COOH.
[0367] The amino acids can be any natural or synthetic amino acid,
preferably natural alpha amino acids.
[0368] Preferred compounds of formula (4) are
2-Methylcarbonyl-1-N-[(L)-Al- anyl-(L)-Valinyl]-(2S)-pyrrolidine
hydrobromide; 2-Methyl)carbonyl-1-N-[(L-
)-Valinyl-(L)-Prolyl-(L)-Valinyl]-(2S)-pyrrolidine hydrobromide;
2-[(Acetyl-oxy-methyl)carbonyl]-1-N-[(L)-Alanyl-(L)-Valinyl]-(2S)-pyrroli-
dine hydrobromide;
2-[Benzoyl-oxy-methyl)carbonyl]-1-N-[{(L)-Alanyl}-(L)-V-
alinyl]-(2S)-pyrrolidine hydrobromide;
2-{[(2,6-Dichlorbenzyl)thiomethyl]c-
arbonyl}-1-N-[{(L)-Alanyl}-(L)-Valinyl]-(2S)-pyrrolidine;
2-[Benzoy-loxy-methyl)carbonyl]-1-N-[Glycyl-(L)-Valinyl]-(2S)-pyrrolidine
hydrobromide;
2-[([1,3]-thiazole-2-yl)carbonyl]-1-N-[{(L)-Alanyl}-(L)-Val-
inyl]-(2S)-pyrrolidine trifluoracetat;
2-[(benzothiazole-2-yl)carbonyl]-1--
N-[N-{(L)-Alanyl}-(L)-Valinyl]-(2S)-pyrrolidin trifluoracetat;
2-[(-benzothiazole-2-yl)carbonyl]-1-N-[{(L)-Alanyl}-Glycyl]-(2S)-pyrrolid-
ine trifluoracetat;
2-[(pyridin-2-yl)carbonyl]-1-N-[N-{(L)-Alanyl}-(L)-Val-
inyl]-(2S)-pyrrolidine trifluoracetat.
[0369] Further, according to the present invention compounds of
formula (5) including all stereoisomers and pharmaceutical
acceptable salts thereof can be used in combination with agents
binding to the secondary binding site(s) of DP IV or DP IV-like
enzymes:
B--(CH--R.sup.1).sub.n--C(.dbd.X.sup.2)--D (5)
[0370] wherein
[0371] n is 0 or 1,
[0372] R.sup.1 stands for H, C.sub.1-C.sub.9 branched or straight
chain alkyl, preferably H, n-butan-2-yl, n-prop-2-yl or isobutyl,
C.sub.2-C.sub.9 branched or straight chain alkenyl, C.sub.3-C.sub.9
cycloalkyl, preferably cyclohexyl, C.sub.5-C.sub.7 cycloalkenyl,
aryl, heteroaryl or a side chain of a natural amino acid or
mimetics thereof,
[0373] X.sup.2 stands for O, NR.sup.6, N.sup.+(R.sup.7).sub.2, or
S,
[0374] B is selected from the following groups: 14
[0375] where X.sup.5 is H or an acyl or oxycarbonyl group including
amino acids,
[0376] R.sup.5 is H, C.sub.1-C.sub.9 branched or straight chain
alkyl, preferably H, n-butan-2-yl, n-prop-2-yl or isobutyl,
C.sub.2-C.sub.9 branched or straight chain alkenyl, C.sub.3-C.sub.9
cycloalkyl, preferably cyclohexyl, 3-hydroxyadamant-d-yl,
C.sub.5-C.sub.7 cycloalkenyl, aryl, heteroaryl or a side chain of a
natural amino acid or derivatives thereof, or a group of the
formula --(CH).sub.m--NH--C.sub.5H- .sub.3N--Y where m is an
integer of 2-4, --C.sub.5H.sub.3N--Y is a divalent pyridyl moiety
and Y is a hydrogen atom, a halogen atom, a nitro group or a cyano
group,
[0377] R.sup.6, R.sup.7 R.sup.8 and R.sup.9 are independently
selected from H, optionally substituted C.sub.1-C.sub.9 branched or
straight chain alkyl, preferably an optionally substituted
C.sub.2-C.sub.5 branched or straight chain alkyl; or optionally
substituted C.sub.2-C.sub.9 branched or straight chain alkenyl,
preferably an C.sub.2-C.sub.5 branched or straight chain alkenyl;
or optionally substituted C.sub.3-C.sub.8 cycloalkyl, preferably an
optionally substituted C.sub.4-C.sub.7 cycloalkyl; or an optionally
substituted C.sub.5-C.sub.7 cycloalkenyl, or an optionally
substituted aryl residue,
[0378] Z is selected from H, pyridyl or optionally substituted
phenyl, optionally substituted alkyl groups, alkoxy groups,
halogens, nitro, cyano and carboxy groups,
[0379] W is selected from H, pyridyl or optionally substituted
phenyl, optionally substituted alkyl groups, alkoxy groups,
halogens, nitro, cyano and carboxy groups,
[0380] W.sup.1 is H or optionally substituted alkyl, alkoxy or
optionally substituted phenyl, and
[0381] Z.sup.1 is H, or optionally substituted alkyl,
[0382] R.sup.3 and R.sup.4 are independently H, hydroxy, alkyl,
alkoxy, aralkoxy, nitro, cyano or halogen,
[0383] D is an optionally substituted compound of the formula
15
[0384] which can be saturated, or can have one, two or three double
bonds,
[0385] wherein
[0386] X.sup.8 to X.sup.11 are independently CH, N,
N.sup.+(R.sup.7), or CR.sup.8, if unsaturated, or
[0387] X.sup.8 to X.sup.11 are independently CH.sub.2, NH,
NH.sup.+(R.sup.7), O, or S if saturated,
[0388] X.sup.12 is CHA, NA, CH.sub.2, NH, NH.sup.+(R.sup.7), or
CHR.sup.8, if saturated or
[0389] X.sup.12 is CA, NA.sup.+, CH, N, N.sup.+(R.sup.7), or
CR.sup.8, if unsaturated and
[0390] A is H or an isoster of a carboxylic acid such as CN,
SO.sub.3H, CONOH, PO.sub.3R.sup.5R.sup.6, a tetrazole, an amide, an
ester or an acid anhydride.
[0391] Throughout the application, D contains preferably at most
two, further preferred at most one hetero atom in the ring.
[0392] According to preferred embodiments of the present invention,
D stands for optionally substituted C.sub.4-C.sub.7 cycloalkyl,
preferably C.sub.4-C.sub.6 cycloalkyl, optionally substituted
C.sub.4-C.sub.7 cycloalkenyl, or optionally substituted
(hetero)cycloalkyl of the formulae 16
[0393] wherein the residues are as defined above,
[0394] or 17
[0395] that is, a five-membered ring containing one or two double
bonds in the ring,
[0396] wherein the residues are as defined above, 18
[0397] wherein the residues are as defined above, 19
[0398] wherein the residues are as defined above, 20
[0399] that is a six-membered ring containing one or two double
bonds in the ring,
[0400] wherein the residues are as defined above, 21
[0401] wherein the residues are as defined above.
[0402] According to a preferred embodiment, B has the following
formula: 22
[0403] wherein the residues are as defined above.
[0404] According to another preferred embodiment, B has the
following formula: 23
[0405] wherein the residues are as defined above.
[0406] Preferred compounds according to formula (5) are
[0407] 1-cyclopentyl-3-methyl-1-oxo-2-pentanaminium chloride,
[0408] 1-cyclopentyl-3-methyl-1-oxo-2-butanaminium chloride,
[0409] 1-cyclopentyl-3,3-dimethyl-1-oxo-2-butanaminium
chloride,
[0410] 1-cyclohexyl-3,3-dimethyl-1-oxo-2-butanaminium chloride,
[0411] 3-(cyclopentylcarbonyl)-1,2,3,4-tetrahydroisoquinolinium
chloride, and N-(2-cyclopentyl-2-oxoethyl)cyclohexanaminium
chloride.
[0412] Because of the wide distribution of the protein in the body
and the wide variety of mechanisms involving DP IV, DP IV-activity
and DP W-related proteins, systemic therapy (enteral or parenteral
administration) with DP IV-inhibitors can result in a series of
undesirable side-effects.
[0413] The problem to be solved was moreover, to provide compounds
that can be used, in combination with agents binding to the
secondary binding site(s) of DP IV or DP IV-like enzymes, for
targeted influencing of locally limited patho-physiological and
physiological processes. The problem of the invention especially
consists in obtaining locally limited and highly specific
inhibition of DP IV or DP IV-analogous activity for the purpose of
targeted intervention in the regulation of the activity of locally
active substrates.
[0414] This problem is solved according to the invention by the use
compounds of the general formula (6) in combination with agents
binding to the secondary binding site(s) of DP IV or DP IV-like
enzymes: 24
[0415] A is an amino acid having at least one functional group in
the side chain,
[0416] B is a chemical compound covalently bound to at least one
functional group of the side chain of A,
[0417] C is a thiazolidine, pyrrolidine, cyanopyrrolidine,
hydroxyproline, dehydroproline or piperidine group amide-bonded to
A.
[0418] In accordance with a preferred embodiment of the invention,
pharmaceutical compositions are used comprising at least one
compound of the general formula (6) and at least one customary
adjuvant appropriate for the site of action.
[0419] Preferably A is an .alpha.-amino acid, especially a natural
.alpha.-amino acid having one, two or more functional groups in the
side chain, preferably threonine, tyrosine, serine, arginine,
lysine, aspartic acid, glutamic acid or cysteine.
[0420] Preferably B is an oligopeptide having a chain length of up
to 20 amino acids, a polyethylene glycol having a molar mass of up
to 20 000 g/mol, an optionally substituted organic amine, amide,
alcohol, acid or aromatic compound having from 8 to 50 C atoms.
[0421] Despite an extended side chain function, the compounds of
formula (6) can still bind to the active centre of the enzyme
dipeptidyl peptidase IV and analogous enzymes but are no longer
actively transported by the peptide transporter PepT1. The
resulting reduced or greatly restricted transportability of the
compounds according to the invention leads to local or site
directed inhibition of DP IV and DP IV-like enzyme activity.
[0422] By extending/expanding the side chain modifications, for
example beyond a number of seven carbon atoms, it is accordingly
possible to obtain a dramatic reduction in transportability. With
increasing spatial size of the side chains, there is a reduction in
the transportability of the substances. By spatially and sterically
expanding the side chains, for example beyond the atom group size
of a monosubstituted phenyl radical, hydroxylamine radical or amino
acid residue, it is possible according to the invention to modify
or suppress the transportability of the target substances.
[0423] Preferred compounds of formula (6) are compounds, wherein
the oligopeptides have chain lengths of from 3 to 15, especially
from 4 to 10, amino acids, and/or the polyethylene glycols have
molar masses of at least 250 g/mol, preferably of at least 1500
g/mol and up to 15 000 g/mol, and/or the optionally substituted
organic amines, amides, alcohols, acids or aromatic compounds have
at least 12 C atoms and preferably up to 30 C atoms.
[0424] The compounds of the present invention can be converted into
and used as acid addition salts, especially pharmaceutically
acceptable acid addition salts. The pharmaceutically acceptable
salt generally takes a form in which an amino acids basic side
chain is protonated with an inorganic or organic acid.
Representative organic or inorganic acids include hydrochloric,
hydrobromic, perchloric, sulfuric, nitric, phosphoric, acetic,
propionic, glycolic, lactic, succinic, maleic, fumaric, malic,
tartaric, citric, benzoic, mandelic, methanesulfonic,
hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic,
2-naphthalenesulfonic, p-toulenesulfonic, cyclohexanesulfamic,
salicylic, saccharinic or trifluoroacetic acid. All
pharmaceutically acceptable acid addition salt forms of the
compounds of formulas (1) to (6) are intended to be embraced by the
scope of this invention.
[0425] In view of the close relationship between the free compounds
and the compounds in the form of their salts, whenever a compound
is referred to in this context, a corresponding salt is also
intended, provided such is possible or appropriate under the
circumstances.
[0426] The present invention further includes within its scope
prodrugs of the compounds of formulas (1) to (6). In general, such
prodrugs will be functional derivatives of the compounds which are
readily convertible in vivo into the desired therapeutically active
compound. Thus, in these cases, the present invention shall
encompass the treatment of the various disorders described with
prodrug versions of one or more of the claimed compounds, which
convert to the above specified compound in vivo after
administration to the subject. Conventional procedures for the
selection and preparation of suitable prodrug derivatives are
described, for example, in "Design of Prodrugs", ed. H. Bundgaard,
Elsevier, 1985 and the patent applications DE 198 28 113 and DE 198
28 114, which are fully incorporated herein by reference.
[0427] Where the compounds or prodrugs according to this invention
have at least one chiral center, they may accordingly exist as
enantiomers. Where the compounds or prodrugs possess two or more
chiral centers, they may additionally exist as diastereomers. It is
to be understood that all such isomers and mixtures thereof are
encompassed within the scope of the present invention. Furthermore,
some of the crystalline forms of the compounds or prodrugs may
exist as polymorphs and as such are intended to be included in the
present invention. In addition, some of the compounds may form
solvates with water (i.e. hydrates) or common organic solvents, and
such solvates are also intended to be encompassed within the scope
of this invention.
[0428] The compounds, including their salts, can also be obtained
in the form of their hydrates, or include other solvents used for
their crystallization.
[0429] A further preferred embodiment of the present invention
comprises compounds capable of binding to the secondary binding
site of DP IV or DP IV-like enzymes according to any one of the
embodiments of the present invention
[0430] in combination with acarbose, or
[0431] in combination with metformin; or
[0432] in combination with acarbose and metformin.
[0433] In a further preferred embodiment, the compounds capable of
binding to the secondary binding site of DP IV and/or DP IV-like
enzymes of the present invention, can be used in combination with
at least one antidiabetic agent selected from the group consisting
of:
[0434] insulin sensitizers selected from the group consisting
of
[0435] PPAR agonists,
[0436] biguanides, and
[0437] protein tyrosin phosphatase-1B (PTP-1B) inhibitors;
[0438] insulin and insulin mimetics;
[0439] sulfonylureas and other insulin secretagogues;
[0440] .alpha.-glucosidase inhibitors, e.g. acarbose;
[0441] glucagon receptor agonists;
[0442] GLP-1; GLP-1 mimetics, and GLP-1 receptor agonists;
[0443] GLP-2; GLP-2 mimetics, and GLP-2 receptor agonists, e.g.
ALX-600 (teduglutide from NPS Allelix Corp.);
[0444] exendin-4 and exendin-4 mimetics, e.g. exenatide (AC-2993,
synthetic exendin-4 from Amylin/Eli Lilly);
[0445] GIP, GIP mimetics, and GIP receptor agonists;
[0446] PACAP, PACAP mimetics, and PACAP receptor 3 agonists;
[0447] PYY, PYY mimetics, PYY receptor agonists, and PYY receptor
antagonists,
[0448] cholesterol lowering agents selected from the group
consisting of
[0449] HMG-CoA reductase inhibitors,
[0450] sequestrants,
[0451] nicotinyl alkohol, nicotinic acid and salts thereof,
[0452] PPAR.alpha. agonists,
[0453] PPAR.gamma. agonists,
[0454] PPAR.alpha./.gamma. dual agonists,
[0455] inhibitors of cholesterol absorption,
[0456] acyl CoA:cholesterol acyltransferase inhibitors, and
[0457] antioxidants;
[0458] PPAR.delta. agonists;
[0459] antiobesity compounds;
[0460] an ileal bile acid transporter inhibitor; and
[0461] anti-inflammatory agents.
[0462] A further preferred embodiment of the present invention
comprises compounds capable of binding to the secondary binding
site of DP IV or DP IV-like enzymes according to any one of the
embodiments of the present invention mentioned above
[0463] in combination with a gene therapeutic expression system for
GLP-1 comprising a viral vector comprising
[0464] (a) a polynucleotide sequence encoding GLP-1 (gluacogen like
peptide-1); and
[0465] (b) a polynucleotide sequence encoding a signal sequence
upstream of (a); and
[0466] (c) a polyadenylation signal downstream of (a); and
[0467] (d) a polynucleotide sequence encoding a proteolytic
cleavage site located between the polynucleotide sequence encoding
GLP-1 and the polynucleotide sequence encoding the signal sequence;
and
[0468] (e) wherein the expression of GLP-1 underlies a constitutive
promoter or is controlled by a regulatable promotor;
[0469] (f) wherein, optionally, the viral vector comprises a
polynucleotide sequence encoding GIP (glucose dependent
insulinotropic peptide);
[0470] (g) wherein, optionally, the viral vector is encompassed by
a mammalian cell. and /or
[0471] in combination with a gene therapeutic expression system for
GIP comprising a viral vector comprising
[0472] (a) a polynucleotide sequence encoding GIP (glucose
dependent insulinotropic peptide); and
[0473] (b) a polynucleotide sequence encoding a signal sequence
upstream of (a); and
[0474] (c) a polyadenylation signal downstream of (a); and
[0475] (d) a polynucleotide sequence encoding a proteolytic
cleavage site located between the polynucleotide sequence encoding
GIP and the polynucleotide sequence encoding the signal sequence;
and
[0476] (e) wherein the expression of GIP underlies a constitutive
promoter or is controlled by a regulatable promotor;
[0477] (f) wherein, optionally, the viral vector comprises a
polynucleotide sequence encoding GLP-1 (glucagon like peptide
1);
[0478] (g) wherein, optionally, the viral vector is encompassed by
a mammalian cell.
[0479] A further preferred embodiment of the present invention
comprises the compounds capable of binding to the secondary binding
site of DP IV or DP IV-like enzymes in combination with a gene
therapeutic expression system for GLP-1 and/or GIP according to any
one of the embodiments of the present invention mentioned above
wherein
[0480] the signal sequence upstream of the gene of interest (GLP-1;
GIP) is the murine immunoglobulin K signal sequence or the glia
monster exendin signal sequence; and/or
[0481] the polyadenylation signal downstream of the gene of
interest (GLP-1; GIP) is derived from simian viraus 40 (SV 40); and
/or
[0482] the proteolytic cleavage site is cleaved by furin preotease;
and/ or
[0483] the gene delivery vector for expression the gene of interest
is an adenoviral, retroviral, leniviral, adeno associated viral
vector; and /or
[0484] the constitutive promoter is a cytomegalovirus (CMV)
promotor, or a Rous sarcoma long-terminal repeat (LTR) sequence,
and the SV 40 early gene gene promoter; and the inducible promoter
is the Tet-On.TM./Tet-Off.TM. system available from Clontech; and
/or
[0485] the mammalian cell is a primate or rodent cell, preferably a
human cell, more preferably a human hepatocyte.
[0486] In a further illustrative embodiment, the present invention
provides formulations for agents binding to the secondary binding
site of DP IV or DP IV-like enzymes allone or in combination with
DP IV-inhibitors, e.g. the compounds of formulas (1) to (6), and
their corresponding pharmaceutically acceptable prodrugs and acid
addition salt forms, in pharmaceutical compositions. 7
[0487] To prepare the pharmaceutical compositions of this
invention, one or more compounds capable of binding to the
secondary binding site and/or DP IV-inhibitors or salts thereof of
the invention can be used as the active ingredient(s). The active
ingredient(s) is intimately admixed with a pharmaceutical carrier
according to conventional pharmaceutical compounding techniques,
which carrier may take a wide variety of forms depending of the
form of preparation desired for administration, e.g., oral or
parenteral such as intramuscular. In preparing the compositions in
oral dosage form, any of the usual pharmaceutical media may be
employed. Thus, for liquid oral preparations, such as for example,
suspensions, elixirs and solutions, suitable carriers and additives
include water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents and the like; for solid oral
preparations such as, for example, powders, capsules, gelcaps and
tablets, suitable carriers and additives include starches, sugars,
diluents, granulating agents, lubricants, binders, disintegrating
agents and the like. Because of their ease in administration,
tablets and capsules represent the most advantageous oral dosage
unit form, in which case solid pharmaceutical carriers are
obviously employed. If desired, tablets may be sugar coated or
enteric coated by standard techniques. For parenterals, the carrier
will usually comprise sterile water, through other ingredients, for
example, for purposes such as aiding solubility or for
preservation, may be included.
[0488] Injectable suspensions may also prepared, in which case
appropriate liquid carriers, suspending agents and the like may be
employed. The pharmaceutical compositions herein will contain, per
dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful
and the like, an amount of the active ingredient(s) necessary to
deliver an effective dose as described above. The pharmaceutical
compositions herein will contain, per dosage unit, e.g., tablet,
capsule, powder, injection, suppository, teaspoonful and the like,
from about 0.03 mg to 100 mg/kg (preferred 0.1-30 mg/kg) and may be
given at a dosage of from about 0.1-300 mg/kg per day (preferred
1-50 mg/kg per day) of each active ingredient or combination
thereof The dosages, however, may be varied depending upon the
requirement of the patients, the severity of the condition being
treated and the compound being employed. The use of either daily
administration or post-periodic dosing may be employed.
[0489] Preferably these compositions are in unit dosage forms from
such as tablets, pills, capsules, powders, granules, sterile
parenteral solutions or suspensions, metered aerosol or liquid
sprays, drops, ampoules, autoinjector devices or suppositories; for
oral parenteral, intranasal, sublingual or rectal administration,
or for administration by inhalation or insufflation. Alternatively,
the composition may be presented in a form suitable for once-weekly
or once-monthly administration; for example, an insoluble salt of
the active compound, such as the decanoate salt, may be adapted to
provide a depot preparation for intramuscular injection. For
preparing solid compositions such as tablets, the principal active
ingredient is mixed with a pharmaceutical carrier, e.g.
conventional tableting ingredients such as corn starch, lactose,
sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g. water, to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention, or a
pharmaceutically acceptable salt thereof. When referring to these
preformulation compositions as homogeneous, it is meant that the
active ingredient is dispersed evenly throughout the composition so
that the composition may be readily subdivided into equally
effective dosage forms such as tablets, pills and capsules. This
solid preformulation composition is then subdivided into unit
dosage forms of the type described above containing from 0.1 to
about 500 mg of each active ingredient or combinations thereof of
the present invention.
[0490] The tablets or pills of the compositions of the present
invention can be coated or otherwise compounded to provide a dosage
form affording the advantage of prolonged action. For example, the
tablet or pill can comprise an inner dosage and an outer dosage
component, the latter being in the form of an envelope over the
former. The two components can be separated by an enteric layer
which serves to resist disintegration in the stomach and permits
the inner component to pass intact into the duodenum or to be
delayed in release. A variety of material can be used for such
enteric layers or coatings, such materials including a number of
polymeric acids with such materials as shellac, cetyl alcohol and
cellulose acetate.
[0491] This liquid forms in which the compositions of the present
invention may be incorporated for administration orally or by
injection include, aqueous solutions, suitably flavoured syrups,
aqueous or oil suspensions, and flavoured emulsions with edible
oils such as cottonseed oil, sesame oil, coconut oil or peanut oil,
as well as elixirs and similar pharmaceutical vehicles. Suitable
dispersing or suspending agents for aqueous suspensions, include
synthetic and natural gums such as tragacanth, acacia, alginate,
dextran, sodium carboxymethylcellulose, methylcellulose,
polyvinylpyrrolidone or gelatin.
[0492] Where the processes for the preparation of the compounds
according to the invention give rise to mixture of stereoisomers,
these isomers may be separated by conventional techniques such as
preparative chromatography. The compounds may be prepared in
racemic form, or individual enantiomers may be prepared either by
enantiospecific synthesis or by resolution. The compounds may, for
example, be resolved into their components enantiomers by standard
techniques, such as the formation of diastereomeric pairs by salt
formation with an optically active acid, such as
(-)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-1-tartaric
acid followed by fractional crystallization and regeneration of the
free base. The compounds may also resolved by formation of
diastereomeric esters or amides, followed by chromatographic
separation and removal of the chiral auxiliary. Alternatively, the
compounds may be resolved using a chiral HPLC column.
[0493] During any of the processes for preparation of the compounds
of the present invention, it may be necessary and/or desirable to
protect sensitive or reactive groups on any of the molecules
concerned. This may be achieved by means of conventional protecting
groups, such as those described in Protective Groups in Organic
Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W.
Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis,
John Wiley & Sons, 1991. The protecting groups may be removed
at a convenient subsequent stage using conventional methods known
from the art.
[0494] The method of treating conditions modulated by the
dipeptidyl peptidase IV or dipeptidyl peptidase IV-like enzymes
described in the present invention may also be carried out using a
pharmaceutical composition comprising any compound or any
combination of the compounds as defined herein and a
pharmaceutically acceptable carrier. The pharmaceutical composition
may contain between about 0.01 mg and 100 mg, preferably about 5 to
50 mg, of each compound, and may be constituted into any form
suitable for the mode of administration selected. Carriers include
necessary and inert pharmaceutical excipients, including, but not
limited to, binders, suspending agents, lubricants, flavorants,
sweeteners, preservatives, dyes, and coatings. Compositions
suitable for oral administration include solid forms, such as
pills, tablets, caplets, capsules (each including immediate
release, timed release and sustained release formulations),
granules, and powders, and liquid forms, such as solutions, syrups,
elixirs, emulsions, and suspensions. Forms useful for parenteral
administration include sterile solutions, emulsions and
suspensions.
[0495] Advantageously, compounds of the present invention may be
administered in a single daily dose, or the total daily dosage may
be administered in divided doses of two, three or four times daily.
Furthermore, compounds for the present invention can be
administered in intranasal form via topical use of suitable
intranasal vehicles, or via transdermal skin patches well known to
those of ordinary skill in that art. To be administered in the form
of transdermal delivery system, the dosage administration will, of
course, be continuous rather than intermittent throughout the
dosage regimen.
[0496] For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic pharmaceutically acceptable inert carrier such
as ethanol, glycerol, water and the like. Moreover, when desired or
necessary, suitable binders; lubricants, disintegrating agents and
coloring agents can also be incorporated into the mixture. Suitable
binders include, without limitation, starch, gelatin, natural
sugars such as glucose or betalactose, corn sweeteners, natural and
synthetic gums such as acacia, tragacanth or sodium oleate, sodium
stearate, magnesium stearate, sodium benzoate, sodium acetate,
sodium chloride and the like. Disintegrators include, without
limitation, starch, methyl cellulose, agar, bentonite, xanthan gum
and the like.
[0497] The liquid forms in suitable flavored suspending or
dispersing agents such as the synthetic and natural gums, for
example, tragacanth, acacia, methyl-cellulose and the like. For
parenteral administration, sterile suspensions and solutions are
desired. Isotonic preparations which generally contain suitable
preservatives are employed when intravenous administration is
desired.
[0498] The compounds of the present invention can also be
administered in the form of liposome delivery systems, such as
small unilamellar vesicles, large unilamellar vesicles, and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, such as cholesterol, stearylamine or
phosphatidylcholines.
[0499] Compounds of the present invention may also be delivered by
the use of monoclonal antibodies as individual carriers to which
the compound molecules are coupled. The compounds of the present
invention may also be coupled with soluble polymers as targetable
drug carriers. Such polymers can include polyvinylpyrrolidone,
pyran copolymer, polyhydroxypropylmethacrylamidephenol,
polyhydroxyethylaspartamid-ephenol- , or polyethyl
eneoxidepolyllysine substituted with palmitoyl residue.
Furthermore, the compounds of the present invention may be coupled
to a class of biodegradable polymers useful in achieving controlled
release of a drug, for example, polyactic acid, polyepsilon
caprolactone, polyhydroxy butyeric acid, polyorthoesters,
polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked
or amphipathic block copolymers of hydrogels.
[0500] Compounds of this invention may be administered in any of
the foregoing compositions and according to dosage regimens
established in the art whenever treatment of the addressed
disorders is required.
[0501] The daily dosage of the products may be varied over a wide
range from 0.01 to 1.000 mg per mammal per day. For oral
administration, the compositions are preferably provided in the
form of tablets containing, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0,
10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 and 500 milligrams of
each active ingredient or combinations thereof for the symptomatic
adjustment of the dosage to the patient to be treated. An effective
amount of the drug is ordinarily supplied at a dosage level of from
about 0.1 mg/kg to about 300 mg/kg of body weight per day.
Preferably, the range is from about 1 to about 50 mg/kg of body
weight per day. The compounds may be administered on a regimen of 1
to 4 times per day.
[0502] Optimal dosages to be administered may be readily determined
by those skilled in the art, and will vary with the particular
compound used, the mode of administration, the strength of the
preparation, the mode of administration, and the advancement of
disease condition. In addition, factors associated with the
particular patient being treated, including patient age, weight,
diet and time of administration, will result in the need to adjust
dosages.
EXAMPLES
Example 1
[0503] Determination of the Half-Life (t.sub.1/2)
[0504] Matrix-assisted laser-desorption ionization time of flight
mass spectrometry (MALDI-TOF MS) experiments were carried out at
30.degree. C. at pH 7.6 in 0.1 M Tris/HCl (Sigma-Aldrich,
Deisenhofen, Germany) buffer with 25 .mu.M peptide solution. The
degradation fate of peptides was measured by monitoring the signal
intensity of the pseudomolecular ion peaks of parent peptides and
N-terminal shorted peptides versus time when incubated with 40 mU
procine DP IV, recombinant human DP IV or serum DP IV activity. The
enzyme was preincubated with hexapeptide TFTSDY or TFTDDY or the
heptapeptide H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH (15 min,
30.degree. C., 0,016M, 1:1 with DP IV, the concentration of the
hexapeptide or the heptapeptide in the reaction mixture was 160
.mu.M). As control served the preincubation of DP IV with 0.01M
Tris-buffer (Sigma-Aldrich, Deisenhofen, Germany). The mass
spectrometer employed was a Hewlett-Packard G2025 model with a
linear time of flight analyzer; samples (4 .mu.L) were mixed 1:1
v/v with matrix (44 mg diammonium-hydrogen-citrate and 30 mg
2',6'-dihydroxyacetophenone in 1 ml aqueous solution containing 50%
acetonitrile and 0.05% trifluoroacetic acid; Sigma-Aldrich),
transferred to a probe tip and immediately evaporated using the
Hewlett-Packard G2024A (Hewlett-Packard, Waldbronn, Germany) sample
preparation vacuum chamber. 250 single laser-shot spectra were
accumulated. This method of monitoring biodegradation has been
validated and allows the general comparison of half-degradation
times (t.sub.1/2) under various conditions.
[0505] The t.sub.1/2- calculation followed this procedure:
[0506] The height of the substrate peak was measured and set as
100% at time=0. During the reaction course the sum of substrate and
product peak height were set as 100% and the percentage of the
remaining substrate peak (also expressed as relative concentration)
was determined. Diagrammed relative substrate concentration versus
time t.sub.1/2 can be calculated based on first order exponential
decay reaction course. 1 A k l B v = - [ A ] [ t ] = k l * [ A ] -
A 0 A 1 [ A ] [ A ] = t 0 t k 1 t [ A ] = [ A ] 0 - k l t k l ln 2
t 1 / 2
[0507] Legend:
[0508] A substrate (bioactive peptide)
[0509] B product (N-terminal truncated bioactive peptide)
[0510] K.sub.i first order rate constant
[0511] K.sub.m Michaelis-Menten-constant
[0512] v.sub.i initial rate of the reaction
[0513] V.sub.max maximal rate of the reaction
[0514] [S] substrate concentration
Example 2
[0515] Determination of K.sub.i:
[0516] In order to measure the inhibition constant K.sub.i a
photometric assay was used The peptides were measured as
competitors of the standard substrate GP-4-Nitroanilide. Three
different substrate concentrations (0.4 mM to 0.05 mM) were
combined with 8 different competitor concentrations (0.5 mM to 2
.mu.M). The reaction was started by addition of 3.5 nM DP IV.
Experiments were carried out under standard conditions: 30.degree.
C. in pH 7.6 40 mM HEPES (Sigma-Aldrich) buffer. Nitroaniline
production was monitored using a HTS 7000+ microplate reader
(PerkinElmer, Uberlingen, Germany). The K.sub.i-values were
calculated via non-linear regression using the enzyme kinetic
program Grafit 4.016 (Erithacus Ltd, UK).
[0517] For a reversible competitive inhibition is to assumed: 2 v i
= V max * K m [ S ] + K m ( 1 + [ I ] K i )
[0518] Legend.
[0519] [I] inhibitor concentration
[0520] K.sub.i inhibition constant
Example 3
[0521] MALDI-TOF Approach
[0522] In order to investigate directly the influence of the test
compounds TFTSDY, TFTDDY and
H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH on the DP IV-catalyzed
peptide hydrolysis the MALDI-TOF assay was used.
[0523] As described before (determination of t.sub.1/2) DP IV and
the test compounds were preincubated and the reaction was started
by adding the enzyme/hexapeptide mixture to substrate/buffer mix.
The control reaction mixture consisted of buffer, enzyme and
substrate. From the curves of the first order exponential the
initial rate (v.sub.i) for the control and the reversible inhibited
reaction was calculated.
[0524] For the uninfluenced reaction the Michaelis-Menten-equation
was used.
[0525] V.sub.i was calculated from plotting the relative substrate
concentration versus time.
[0526] K.sub.m is given, also the substrate concentration. 3 v i =
V max * K m K m + [ S ]
[0527] For the reversible inhibited reaction the following reaction
was used to calculate K.sub.i: 4 v i = V max * K m [ S ] + K m ( 1
+ [ I ] K i )
Example 4
[0528] Determination of K.sub.m
[0529] Experiments were carried out with a capillary zone
electrophoresis apparatus (MDQ, Beckmann, Muinchen, Germany).
[0530] The reaction mixture contained 50 .mu.l Gly-Gly (100 mM as
standard), 50 .mu.M substrate solved in 0.01 M sodium phosphat
buffer (pH 7.6) and 10 .mu.l DP IV (40 mU/ml) stored at 30.degree.
C. Six substrate concentrations varying from 1 .mu.M to 60 .mu.M
were measured. As running buffer 0.1 M sodium phosphat buffer, pH
2.5 was used. A sample from the reaction mixture was injected with
0.5 psi over 5 s at predefined time points. Separation was carried
out in a capillary with 50 .mu.M inner diameter and 20 cm effective
length. The following separation parameters were used:
18 Separation voltage: 16 kV Separation time: 12 min Separation
temperature: 25.degree. C. Detection wave length: 200 nm
[0531] The maximal rate was calculated by plotting product
concentration versus time. The K.sub.m-value was calculated
transferrng the data in the Michaelis-Menten-equation (GraFit
4.0.16, Erithacus Ltd., UK).
Example 5
[0532] Expression, Fermentation and Purification of Human DP IV and
its Mutant Variants
[0533] Strains and Plasmid:
[0534] P. pastoris strain X-33 and the vector pPIC.alpha.C were
purchased from Invitrogen (USA). E. coli XL-10 cells were provided
from Stratagene (USA).
[0535] Plasmid Construction and DNA Sequencing
[0536] The DP IV encoding region (.DELTA.1-36) plus his.sub.6-tag
contained in a pcDNA-3.1 vector was amplified using primers DP
IV-21 (TCATCGATGCATCATCATCATCATCAT) and DP IV-22
(TAGGTACCGCTAAGGTAAAGAGAAAC) while implementing the restriction
sites for KpnI and BspD1. This fragment was digested with the
restriction enzymes KpnI and BspD1 as well as the vector
pPCR-ScriptCam (Stratagene, USA), afterwards vector and PCR product
were ligated and transformed into the E. coli-strain XL-10.
Insertion and orientation was confirmed applying restriction enzyme
analysis and partial sequencing. That was followed by excision of
the DP IV encoding region from the pPCR-ScriptCam vector with the
same restriction enzymes KpnI and BspD1 and its ligation into the
Pichia vector pPIC.alpha.C, which was also treated with the same
restriction enzymes before.
[0537] Site Directed Mutagenesis:
[0538] Single amino acid mutations were carried out with the Quick
Change Site-directed Mutagenesis Kit from Stratagene (USA).
Following primers were used to introduce the mutations:
19 R310A-DP IV: DP IV-84 GACATGGGCAACACAAGAAGCAATTTCTTTGCA- GTGGC
DP IV-85 GCCACTGCAAAGAAATTGCTTCTTGTGTTGCCCATGTC R560A-DP IV: DP
IV-73: GCAGACACTGTCTTCGCACTGAACTGGGCCAC- TTACC DP IV-74b:
GGTAAGTGGCCCAGTTCAGTGCGAAGACAGTGTCTGC W629A-DP IV: DP IV-75:
GCAATTTGGGGCTGGTCATAGCGAGGGTACG- TAACC DP IV 76:
GGTTACGTACCCTCGCTATGACCAGCCCCAAATTGC.
[0539] Transformation of P. pastoris X-33:
[0540] The vector pPIC.alpha.C containing the DP IV-variants was
linearized using the restriction enzyme Sac I. Transformation was
carried out with an electroporation system from BioRad (Germany)
according to the Invitrogen Pichia expression kit manual.
[0541] Media and Buffers:
[0542] YPD, BMMY, and BMGY for shake flask expression were prepared
as described in the Invitrogen Pichia expression kit manual using
reagents obtained from Difco. Media for fermentation were composed
as described in the Invitrogen Pichia fermentation process
guidelines using chemicals purchased from Sigma (Deisenhofen,
Germany).
[0543] Small-Scale Expression Studies:
[0544] Single colonies were grown in BMGY at 250 rpm, 28.degree. C.
overnight. Induction of gene expression was initiated after a media
exchange to BMMY. DP IV activity in the expression medium was
assayed after 48 hours. Clones displaying highest activity were
further monitored in a shaking flask culture (15 ml BMGY and 15 ml
BMMY respectively) regarding growth rate and expression rate.
[0545] Fermentation:
[0546] The clone displaying the highest DP IV activity was used to
inoculate 5 ml of BMGY. After 16-18 h of growth at 250 rpm and
28.degree. C. 1 ml of the culture was used to start a 200 ml BMGY
flask shake preculture. The cells were grown for 16-18 h at
28.degree. C. A 21 fermentation was started with the 200 ml
inoculum according to the Invitrogen Pichia fermentation process
guidelines.
[0547] Purification of DP IV
[0548] Expression medium was centrifuged at 40,000*g for 20 minutes
to pellet the yeast cells. The supernatant was filtered to remove
any residual solids using a 45 .mu.M cellulose acetate filter from
Satorius (Germany). Medium was adjusted to pH 7,6 while adding 300
mM NaCl and 50 mM sodium phosphate buffer.
[0549] Affinity chromatography was carried out at 4.degree. C. with
a Ni-NTA sepharose column (Qiagen, Germany). The column was
pre-equilibrated with 300 mM NaCl, 50 mM NaH.sub.2PO.sub.4-buffer
pH 7,6. The enzyme was eluted with 250 mM imidazole. DP IV assay
and SDS-PAGE monitored the purification process. The fractions with
the highest DP IV content was further concentrated by
ultra-filtration in an Amicon apparatus (cut off 10 kDa) to 0,5
ml.
[0550] Gel Filtration:
[0551] The 0.5 ml ultra-filtrate were applied to a Superdex 200
HiLoad 26/60 column (Pharmacia, Upsalla, Sweden) with a flow rate
at 0.25 ml /min using a 300 mM NaCl, 50 mM NaH.sub.2PO.sub.4-buffer
pH 7,6 at 4.degree. C. The purification process was monitored via
SDS-PAGE and activity assay.
[0552] DP IV Assay:
[0553] DP IV activity assays were performed spectrofluorimetrically
using H-Gly-Pro-AMC (Bachem, Heidelberg, Germany) as substrate and
a 0.1M HEPES buffer pH 7.6 plus 0.05 M NaCl (Sigma, Deisenhofen,
Germany) while monitoring the releasing of AMC by DP IV
(.lambda..sub.excitation=380 nm; .lambda..sub.emission=460 nm).
[0554] SDS-PAGE Analysis:
[0555] Proteins were analysed by SDS-PAGE using 12% separating gels
with 3% stacking gel. Gels were stained applying Coomassie
brilliant blue R-250.
[0556] Protein Determination:
[0557] Protein concentrations were determined using the BioRad
(Germany) Bradford assay kit according to the instructions of the
manufacturer.
[0558] Western Blot Analysis:
[0559] Analytical gel electrophoreses in SDS-polyacrylamid gels
were performed according to Laemmli [1] with seperation gels
containing 12% acrylamide. The seperated proteins were transferred
to a nitrocellulose membrane (Schleicher&Schuell, Germany)
following standard procedures. To detect his-tagged protein a
penta-his-tag-antibody and a secondary antibody provided from
Qiagen (Germany) (1:2000) was used. Chemo-luminescence was assayed
according to the manufacturers protocol (SuperSignal.TM. West Pico,
PIERCE).
[0560] Substrates:
[0561] All investigated bioactive peptides were obtained from
Bachem (Heidelberg, Germany), with exception of glucagon, GIP and
its analogs and fragments. These peptides were synthesized at
applicant's laboratories.
Example 6
[0562] Synthesis of DP IV-Substrates
[0563] Glucagon, GIP and the GIP analogs were synthesized with an
automated synthesizer SYMPHONY (RAININ) using a modified
Fmoc-protocol. Cycles were modified by using double couplings from
the 15.sup.th amino acid from the C-terminus of the peptide with
five-fold excess of Fmoc-amino acids and coupling reagent. The
peptide couplings were performed by TBTU/NMM-activation using a
0.23 mmol substituted NovaSyn TGR-resin or the corresponding
preloaded Wang-resin at 25 .mu.mol scale. The cleavage from the
resin was carried out by a cleavage-cocktail consisting of 94.5%
TFA, 2.5% water, 2.5% EDT and 1% TIS.
[0564] Analytical and preparative HPLC were performed by using
different gradients on the LiChrograph HPLC system of
Merck-Hitachi. The gradients were made up from two solvents: (A)
0.1% TFA in H.sub.2O and (B) 0.1% TFA in acetonitrile. Analytical
HPLC were performed under the following conditions: solvents were
run (1 ml/min) through a 125-4 Nucleosil RP18-column, over a
gradient from 5%-50% B over 15 min and then up to 95 % B until 20
min, with UV detection (.lambda.=220 nm). Purification of the
peptides was carried out by preparative HPLC on either a 250-20
Nucleosil 100 RP8-colunm or a 250-10 LiChrospher 300 RP18-column
(flow rate 6 ml/min, 220 nm) under various conditions depending on
peptide chain length. For the identification of the peptide
analogues, laser desorption mass spectrometry was employed using
the HP G2025 MALDI-TOF system of Hewlett-Packard.
Example 7
[0565] Computer-assisted Model for Specificity Examinations of
Proline-Specific Proteases
[0566] By means of homology modeling approaches a
tertiary-structure-model- s of human DP IV and porcine DP IV have
been developed.
[0567] The structure of prolyl oligopeptidase (Fulobp, V., et al.
(1998) Prolyl Oligopeptidase: An unusual .beta.-propeller domain
regulates proteolysis. Cell 94, 161-170) (Brookhaven Protein Data
Bank entry: 1 qfm) was used as a target to model the structure of
DP IV.
[0568] COMPOSER (Blundell, T. L.; Sibanda, B. L.; Sternberg, M. J.
E.; Thornton, J. M. Knowledge-based prediction of protein
structures and the design of novel molecules. Nature 1987, 326,
347-352; Blundell, T. L.; Carney, D.; Gardner, S.; Hayes, F.;
Howlin, B.; Hubbard, T.; Overington, J.; Singh, D. A.; Sibanda, B.
L.; Sutcliffe, M. Knowledge-based protein modelling and design.
Eur. J. Biochem. 1988, 172, 5 13-520) a program for homology
modeling which is included in the molecular graphics program
package SYBYL (TRIPOS Associates Inc., 1699 S. Hanley Road, Suite
303, St. Louis, Mo. 63144) (TRIPOS Associates Inc.) was used to
generate the model of DP IV. The amino acid sequences were aligned
using the BLOSUM30 matrix (Henikoff, S.; Henikoff, J. G. Amino acid
substitution matrices from protein blocks. Proc. Natl. Acad. Sci.
USA, (1992), 89, 10915-10919). Afterwards, the modeling procedure
consisted of the following steps: structurally conserved regions
(SCRs) were identified and a framework of conserved regions was
defined as mean positions of structurally equivalent
C.quadrature.-atoms. Structurally variable regions (SVRs, loops)
were selected from a program attached database of peptide fragments
in order to satisfy end-to-end distances of the SCRs already
positioned in the framework. Loops which could not be formed with
this procedure were added manually to complete the structure. The
conformations of these loops (mainly in the propeller domain) were
determined by simulated annealing techniques in heating the
temperature to 700 K and subsequently cooling to 100 K by fixing
the remaining part of the structure. This procedure was repeated 30
times. All resulting low temperature structures were minimized
using the Kollman all-atom force field (Weiner, S. J.; Kollman, P.
A.; Case, D. A.; Singh, U. C.; Ghi, C.; Alagona, G.; Profeta, S.;
Weiner, P. A new Force Field for molecular mechanical simulation of
nucleic acids and proteins, J. Am. Chem. Soc., 1984, 106,765-784).
Loop conformations with the lowest energy which fulfill all
criteria by analyzing the stereo-chemical quality of the protein
structure by means of PROCHECK (Laskowski, R. A. et al. (1993)
PROCHECK: a program to check the stereochemical quality of protein
structures, J. Appl. Cryst. 26, 283-289) were used.
[0569] Small molecule ligands such as substrates of the type
Xaa-Pro-p-Nitroanilide were docked with the "automatic" docking
program GOLD (C. Bissantz, G. Folkers, D. Rognan; J. Med. Chem. 43,
4759-4767, 2000) to the catalytically active site of DP IV to
inspect and analyze the principal correctness of the tertiary
structure. Ligands such as GIP or glucagon and longer peptides of
the GRF family were docked by application of molecular dynamics
simulations. These simulations were started to form a random
conformation of these compounds, manual positioned at the outer
side of the pore formed by the propeller domain. A low force
constant between the protonated N-terminus of the ligands and the
side chain of Glu668, which is proposed to be the responsible
residue for the recognition of the N-terminus of DP IV was added.
Molecular dynamics simulations at 300 K for 100 ps using the
Kollman all-atom force field were performed by fixing the backbone
atoms of DP IV. All these longer peptides reached the catalytically
active site (amino acid position S630), showing that ligands are
penetrating through the propeller domain to dock to the active
site. The resulting docking structures were optimized and
subsequently analyzed to define the so called second binding site
of DP IV-substrates.
Example 8
[0570] Validation of the Computer-Assisted Model of DP IV
[0571] Glycosylation Sites
[0572] The following residues are assumed to be glycosylated and
are therefore placed at the surface of the protein: Asn85, Asn92,
Asn150, Asn299, Asn229, Asn281, Asn321, Asn520 and Asn685, which
are displayed in FIG. 3. All these amino acid residues are
accessible except Asn150 and 321, which are slightly buried but may
become accessible by thermal moving of the loop region close to
this position.
[0573] ADA-Binding Site
[0574] Site directed mutagenesis studies proved that the residues
L294, V341 and R343 play an important role in ADA binding to DP IV.
Therefore, these residues have to be accessible too. These amino
acid residues are displayed in FIG. 3. All these residues are
situated at the surface of the protein and interact with ADA.
[0575] Binding of Small Inhibitors to the Active Site of DP IV
[0576] A number of Xaa-Pyrrolidine and Xaa-Proline dipeptides where
docked to DP IV and their preferred interaction with the active
site was examined (FIG. 4). One of the most important region is the
proline recognition site. In POP this site is formed by the two to
three amino acid residues. In analogy to POP the proline binding
pocked in DP IV is formed also by two aromatic side chains, the two
tyrosine residues Y670 and Y631 and by the hydrophobic residue
V711.
[0577] The S2-binding site in DP IV must be responsible for the
recognition of the protonated and positively charged N-terminus of
DP IV ligands and preferred interactions of hydrophobic residues
such as Val or Ile. The model shows that the side chain of Glu668
is able to form a salt bridge to the N-terminus of ligands. The
recognition of the side chains is realized by interactions with the
side chains of two other tyrosine residues (Y211 and Y330) and
explains the preferred hydrophobic P2-residues of inhibitors.
[0578] Another DP IV-inhibitor, Lys(Z-nitro)-Pyrrolidine, which
carries not a completely hydrophobic P2-side chain, was also docked
to DP IV. The result is represented in FIG. 5. In the most stable
docking arrangement a scorpion like conformation of the Lys-Z-nitro
group can be observed, which finally leads to the formation of a
strong hydrogen bond to R453. This additional interaction in
comparison to usual dipeptide related ligands explains the high
affinity and action of this compound.
[0579] Substrate Interactions and Aspects of the Catalytic
Mechanism
[0580] The mode of interaction of substrates to DP IV is shown in
FIG. 6. First, the substrates dock exactly in the following
conformation: A hydrogen bond is formed between the N-H group and
the carbonyl group (torsional angle .psi.2.about.80.degree.) of the
first amino acid residue (C7-conformation) and the N-terminal amino
group is turned out of a .psi.1 torsion of 180.degree. to about
120.degree.. The scissile bond or better plane of the peptide bond
to be cleaved is in a perpendicular orientation to the active
serine side chain (S630) and allows the reactive attack of the
serine to the peptide bond.
[0581] Of main importance is the side chain of Y547. The phenolic
hydroxyl group forms a hydrogen bond to the carbonyl group of the
scissile peptide bond. This interaction plays a very important role
in the stabilisation of the tetrahedral intermediate and therefore
in the catalytic mechanism in particular in the acylation step.
Another interesting finding by Heins et al. (heins et al., Biochim.
Biophys. Acta, 1988, 954(2),161-169) was the fact that in the case
of proline (in P1) substrates usually the deacylation is the rate
limiting step except, when in P2-position an Asp is introduced. A
possible docking arrangement of such a substrate is displayed in
FIG. 6. The aspartate side chain forms a hydrogen bond to the
phenolic OH-group of Y547. This strong interaction prevents the
cleaved dipeptide to move out of the binding site and thus shifts
the thermodynamic equilibrium and the activation barrier somewhat
to the tetrahedral intermediate site and consequently the acylation
rate is considerably reduced and becomes rate limiting.
[0582] Docking Behavior of Ligands with Biological Importance
[0583] It has been demonstrated that the N-terminal nonapeptide of
the HIV-tat protein shows inhibitory effects to DP IV. Docking
studies of this compound were done with the complete DP IV model as
described above. The resulting most stable binding arrangement is
shown in FIG. 7.
[0584] There are some important interactions. Similar to the
already discussed interaction of the substrate Asp-Pro-PNA D2 of
Tat forms a hydrogen bond with Y330 and furthermore as seen for
Lys-Z-nitro-Pyrrolidine, D5 forms a salt bridge with R453. Further
considerable hydrophobic interactions occur between I8 and Y330 and
another salt bridge is observed between the C-terminal E9 and R310
of DP IV.
[0585] Another similar peptide that was used for docking studies is
the N-terminal nonapeptide of the tromboxane receptor (FIG. 8).
Similar interactions as seen for HIV-tat were detected.
Additionally important is the hydrophobic interaction between W2
and I742.
Example 9
[0586] Docking of GIP; VIP and Glucagons to DP IV
[0587] Several oligopeptides such as GIP, VIP, glucagon and others
are hydrolysed by DP IV and therefore it is clear, that these
substrates are docking to DP IV and reaching the active site.
Extensive docking investigations by means of molecular dynamics
simulations were done using the old model. From these studies the
amino acid sequences of the hexapeptides, TFTSDY and TFTDDY and the
degradation stabilized heptapeptide
H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH were derived and its
ability to protect oligopeptide substrates from their interaction
with a secondary binding site.
[0588] Results
[0589] The binding and hydrolysis of small dipeptide substrates
were only slightly influenced when DP IV was preincubated with the
hexapeptides TFTSDY or TFTDDY or the degradation stabilized
heptapeptide H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH but the
affinity of larger oligopeptides such as GIP, VIP, glucagon and
others was considerably reduced. These experiments clearly prove
the existence of a secondary binding site.
[0590] How these rather long peptides reach the active site of DP
IV without essential steric hindrance was investigated. GIP was
placed at the top of the propeller domain with the N-terminus
pointing to the direction middle to DP IV. A small constraint
(additional force constant) was placed between the N-terminal
nitrogen atom of GIP and a carboxyl oxygen atom of E668. Then a
molecular dynamics simulation over 50.000 fs at 300 K was started
with fixed backbone atoms of DP IV in the gas phase. Surprisingly
it was shown that GIP moved in the pore rapidly without any
considerable steric hindrance and was indeed able to reach the
active site. Finally starting from the end structure of this
"constrained" dynamic model, dynamic simulations with GIP already
situated inside DP IV were repeated. The final optimized docking
arrangement is shown in FIGS. 9 to 12 and the most important
interactions are summarized in Tables 8 to 11.
20TABLE 8 Most important interactions of GIP with DP IV GIP DP IV
typ of interaction NT E668 salt bridge Y1--CO N710 H-bond S2--OH
Y631 H-bond S2--CO Y547 H-bond (Catalysis!) E3 R560 salt bridge I7
Y330 hydrophob D9 R310 salt bridge Y10 W154 hydrophob I12 W157
hydrophob D15 K463 (R318) salt bridge K16 E464 (E91) salt
bridge
[0591]
21TABLE 9 Most important interactions of VIP with DP IV VIP DP IV
typ of interaction NT E668 salt bridge H1--CO N710 H-bond H1-side
chain I742 hydrophob S2--OH Y631 H-bond S2--CO Y547 H-bond
(catalysis!) D3 R560 salt bridge D8 R310 salt bridge Y10 W154
hydrophob Y10--CO S460 H-bond K15 E464 salt bridge
[0592]
22TABLE 10 Most important interactions of Glucagon with DP IV
Glucagon DP IV typ of interaction NT E668 salt bridge H1--CO N710
H-bond H1-side chain I742 hydrophob A2-CO Y547 H-bond Q3 R560
H-bond T5 T152 H-bond T7 Y416 H-bond S8 Y330 H-bond D9-CO Y416
H-bond D9 R310 salt bridge Y10 W154 hydrophob Y13 L90 hydrophob D15
R318 salt bridge R17 E91 salt bridge
[0593]
23TABLE 11 Most important interactions of the hexapeptide
T(5)-F-T-S-D-Y with DP IV hexapeptide DP IV type of interaction T5
T152 H-bond T7 Q153(CO-backbone) H-bond S8 S552 H-bond D9 R310 salt
bridge Y10 W154 hydrophobic Y10(OH) T152(OH) H-bond Y10(OH)
T152(CO-backbone) H-bond Y10(CT) S460(OH) H-bond
[0594] As can be seen in FIGS. 9 and 11, GIP is able to reach the
active site of DP IV, but the C-terminal tail is still at the
surface of the propeller domain.
[0595] The scissile peptide bond after Ser2 is exactly in an
orientation required for optimal hydrolysis (FIG. 10). A number of
important interactions which explain the affinity of GIP to DP IV
were detected. These attractive interactions are summarized in
Table 6. Interestingly a number of interactions were observed,
which were already discussed for other ligands (see above).
[0596] Based on these results analogous docking studies were
performed with VIP, glucagon and the hexapeptide TFTSDY (FIGS. 12
to 15) The results are summarized by listing the most attractive
interactions in Tables 7 to 9.
[0597] These results prove that the oligopeptide ligands penetrate
through the propeller domain to dock to the active site.
Furthermore, some highly attractive interactions between the
oligopeptide ligands and DP IV were shown, which explain the
affinity of the calculated compounds and which were used to predict
the structure of non-peptidic ligands for the secondary binding
site of DP IV. Some preliminary structures of such non-peptidic
ligands are provided in the description above.
[0598] Moreover, the results of these studies confirm the proposed
docking of Lys-Z-nitro-Pyrrolidine, e.g. the interaction of the
nitro-group with AR560. Wher the oligopeptide ligands have an Asp
in third or fourth position in their amino acid sequence, a salt
bridge with R560 is formed. By docking arrangement of the
hexapetide TFTSDY (FIG. 15), it was proven that this hexapeptide
indeed prevents binding of oligopeptide ligands to the active
site.
Example 10
[0599] Preparation of Porcine DP IV
[0600] DP IV was purified from porcine kidney. Approximately 2 kg
cortex was removed from pig kidneys and cut in small pieces. This
tissue material was washed with 0.9% NaCl solution overnight at
4.degree. C. to remove remaining blood. The washed cortex was
homogenized using an ultraturrax. During homogenization an equal
amount of a 0.02 M sucrose solution containing 0.2% Triton X-100
was added. After homogenization the DP IV-protein was released from
the membrane by an 18 h autolysis step at 30.degree. C. Insoluble
particles were removed by centrifugation at 15900.times. g for 30
min. After a first precipitation and centrifugation step (60%
(NH.sub.4).sub.2SO.sub.4-saturation, 3 h, room temperature,
centrifugation: 39200.times. g, 30 min) DP IV-activity remains in
the supernatant. Second precipitation was conducted overnight at
4.degree. C. and 85% saturation. After centrifugation the DP
IV-containing pellet was dissolved in a minimal volume of a 25 mM
phosphate buffer, pH 6.8 and dialyzed against 3.times.21 of this
buffer over night. After additional centrifugation (30 min,
39200.times. g) the solution was concentrated to approximately 45
ml using an Amicon ultrafiltration cell (cut-off 100 kDa) and again
centrifuged at 44000.times. g. A preparative size exclusion
chromatography was used as second purification step. 15 ml of the
DP IV-containing solution were applied to a Sepharose 6B
(Pharmacia) column (100 cm.times.2.6 cm) and eluted with the
phosphate buffer, pH 6.8. The pooled fractions from 3 runs were
further purified by anion exchange chromatography on a
DEAE-Sephacel (Pharmacia) column (17 cm.times.2.5 cm). For binding
the 25 mM phosphate buffer pH 6.8 was used and DP IV was eluted
with a salt gradient from 39 mM to 150 mM NaCl in 5 column volumes.
The final separation step was a high resolution anion exchange
chromatography on a Uno Q column (6 ml, BioRad). The DP
IV-containing fraction was diluted with an equal volume of H.sub.2O
and applied to the column using a 25 mM Bis-Tris buffer pH 6.8.
Elution was performed with a NaCl gradient from 0 to 60 mM. Final
purification of the naturally glycosylated protein to homogeneity
was achieved by preparative isoelectric focusing using the Rotofor
system (BioRad). One run of the above procedure yields roughly 60
mg of total protein, purified by a factor of approximately 280 and
exhibiting a specific activity of always above 42 U/mg.
Example 11
[0601] Sequencing of Porcine DP IV cDNA
[0602] To obtain the cDNA sequence of porcine DP IV, total RNA was
extracted from porcine kidney and RT-PCR was performed as described
elsewhere. The sequence was submitted to GenBank (accession number:
AY198323).
Example 12
[0603] Synthesis of p-Iodo-Phe-Pyr-CN *TFA
[0604] Synthesis of p-Iodo-Phe-Pyr-CN *TFA, an slow-tight binding
inhibitor of DP IV was performed according to known chemical
protocols (Ashworth, D. M., Atrash, B., Baker, G. R., Baxter, A.
J., Jenkins, P. D., Jones, D. M. & Szelke, M. (1996) Bioorg.
Medicinal Chem. Letter 6, 1163-1166).
[0605] Boc-p-Iodo-Phe-Pro-NH.sub.2. Triethylamine (163.8 ml, 1.17
mmol) was added to a solution of H-ProNH.sub.2*HCl (118.5 mg, 0.782
mmol) in dry DMF (10 ml). Boc-p-Iodo-Phe-OSu (0.42 g, 0.86 mmol)
was added in one portion and the mixture stirred for 16 h under an
argon atmosphere. The solvent was evaporated and the residue
treated in a standard way, i.e. the residue was partitioned between
ethylacetate (60 ml) and 0.3N KHSO.sub.4 solution (10 ml). The
organic layer was further washed with saturated NaCHO.sub.3
solution (10 ml), water (10 ml)and brine (5 ml). The solution was
dried and evaporated at reduced pressure.
[0606] Boc-p-Iodo-Phe-Pyr-CN. Imidazole (38.96 mg, 0.572 mmol) was
added to a solution of Boc-p-Iodo-Phe-Pro-NH.sub.2 in dry pyridine
(5 ml) under an argon atmosphere. The solution was cooled to
-35.degree. C., before the dropwise addition of POCl.sub.3 (0.105
ml, 1.13 mmol). The reaction was stirred at -30.degree. C.--to
-20.degree. C. for 60 min. The solution was then evaporated and the
crude residue subjected to column chromatography (silica gel) to
yield 180 mg (94%) of
2-(S)-cyano-1-[tert-(butoxycarbonyl)(p-Iodo-phenylalanyl)-pyrrolidine
as a colourless oil.
[0607] p-Iodo-Phe-Pyr-CN *TFA. Deprotection was carried out by
stirring with trifluoro acetic acid for 60 min. Evaporation and
lyophilisation from water afforded 82.7 mg of
2-(S)cyano-1-(p-Iodo-phenylalanyl)pyrrolid- ine as a white
solid.
[0608] ESI-MS: calculated 369.0, found (M+H).sup.+=370.0
[0609] .sup.1H-NMR: (D.sub.2O), d (ppm): 1.55-1.61 (m, 1H),
1.7-1.82 (m, 1H), 1.91-2.19 (m, 2H), 2.49-2.62 (m, 1H), 2.89-3.09
(m, 1H), 3.19-3.21 (m, 1H), 3.21-3.34 (m, 1H), 4.31-4.39 (m, 1H),
4.61-4.69 (m, 4H), 6.91-7.00 (m, 2H), 7.60-7.71 (m, 2H)
.sup.13C-NMR: (D.sub.2O), d (ppm); 167.832, 131.656, 118.055,
93.173, 65.934, 52.250, 47.061, 46.428, 36.322, 29154, 24.063,
Example 13
[0610] Crystallization and Crystal Transformation
[0611] Triclinic crystals were obtained at room temperature within
several days by mixing equal volumes of protein at concentration of
20 mg/ml with the reservoir solution (20-22% PEG2K, 0.1 M ammonium
sulfate, and 0.1 M Tris/HCl pH 8.0) using the sitting drop vapor
diffusion method. The crystals were very sensitive towards manual
handling and oxygen. Opening of the crystallization vials led to
protein precipitation which was only partly reversible. These
problems were solved by piercing the cover tape of the
crystallization plates with a syringe and immediate covering of the
crystallization drop with perfluoropolyether (PFPE) oil. By
harvesting the crystals using a loop with humidity control most of
the surrounding mother liquor gets replaced with the PFPE oil.
Crystals were mounted on an in-house rotating anode. Crystals
initially diffracted very weakly, typically below 10 .ANG.. The
humidity was then ramped down from 96.5% to 86.5% using a gradient
of 0.5% (150 s).sup.-1 which induced a phase transition in the
crystalline lattice order reflected by a dramatically improved
diffraction pattern. At an optimal relative humidity crystals were
flash frozen in the cold nitrogen stream and transported to the
synchrotron for data collection. For ligand complex studies, DP
IV-crystals were soaked with the inhibitor prior to the crystal
transformation procedure. Data were processed and scaled using
DENZO and SCALEPACK (Otwinowski, Z. & Minor, W. (1997) in Meth.
Enzym., eds. Carter, C. W. J. & Sweet, R. M. (Academic Press,
Vol. 276, pp. 307-326.).
Example 14
[0612] Structure Determination
[0613] The structure was determined by multiple wavelength
anomalous dispersion (MAD) using a mercury derivative and
subsequent non-crystallographic symmetry (NCS) electron density
averaging. Briefly, local two-fold axes were determined by using
the program GLRF (Tong, L. & Rossmann, M. G. (1990) Acta Cryst.
A46, 783-792.). Next, a local Harker section perpendicular to the
molecular dimer axis was cut out of the three-dimensional anomalous
Patterson map using the program MAIN (Turk, D. (1992) in Chemistry
(Technische Universitat, Munchen.), averaged along the orthogonal
local two-fold axes, and subsequently input to RSPS (Knight, S. D.
(2000) Acta Cryst. D52, 42-47) for automatic local doublet sites
detection. We estimate this procedure to enhance the signal to
noise ratio for about 50-100 fold. The relative position of the two
symmetry-related Hg-doublets was determined by translational search
(Knight, S. D. (2000) Acta Cryst. D52, 42-47). By construction, the
resulting sites follow the local symmetry and determine the
translational NCS parameters. After heavy atom refinement and
phasing (program MLPHARE) and solvent flipping (SOLOMON)
(Collaborative Computational Project Number 4 (1994) Acta Cryst.
D50, 760-763; Abrahams, J. P. & Leslie, A. G. W. (1996) Acta
Cryst. D52, 30-42.), phases were extended to 2.0 .ANG. resolution
by NCS averaging using the program MAIN (Turk, D. (1992) in
Chemistry (Technische Universitat, Munchen.) which rendered the
electron density readily interpretable, FIG. 25.
[0614] Model Building and Refinement
[0615] Using the program MAIN, we placed the catalytic domain of
POP in the electron density which served as a jump start in model
building and sequence assignment of the DP IV-structure. The model
was refined by using the program CNS (Bruinger, A. T., Adams, P.
D., Clore, G. M., Delano, W. L., Gros, P., Grossekunstleve, R. W.,
Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J.,
Rice, L. M., Simonson, T. & Warren, G. L. (1998) Acta Cryst.
D54, 905-921.) with current R-values of 21.7% (working set) and
24.9% (test set) and deviations from ideality of 0.008 .ANG. (bond
length) and 1.4 degree (angle deviation).
Sequence CWU 1
1
14 1 6 PRT Artificial synthetic peptide 1 Thr Phe Thr Ser Asp Tyr 1
5 2 30 PRT Homo sapiens 2 Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr
Ser Ile Ala Met Ala Lys 1 5 10 15 Ile His Gln Gln Ala Phe Val Asn
Trp Leu Leu Ala Gln Lys 20 25 30 3 6 PRT Homo sapiens 3 Tyr Ala Glu
Gly Thr Phe 1 5 4 6 PRT Artificial synthetic peptide 4 Thr Phe Thr
Asp Asp Tyr 1 5 5 6 PRT Artificial synthetic peptide, with
C-terminal amide 5 Tyr Ala Glu Ser Thr Phe 1 5 6 11 PRT Artificial
synthetic peptide thiazolidine 6 Arg Arg Leu Ser Tyr Ser Arg Arg
Arg Phe Glu 1 5 10 7 27 DNA Artificial synthetic probe/primer 7
tcatcgatgc atcatcatca tcatcat 27 8 26 DNA Artificial synthetic
probe/primer 8 taggtaccgc taaggtaaag agaaac 26 9 38 DNA Artificial
synthetic probe/primer 9 gacatgggca acacaagaag caatttcttt gcagtggc
38 10 38 DNA Artificial synthetic probe/primer 10 gccactgcaa
agaaattgct tcttgtgttg cccatgtc 38 11 37 DNA Artificial synthetic
probe/primer 11 gcagacactg tcttcgcact gaactgggcc acttacc 37 12 37
DNA Artificial synthetic probe/primer 12 ggtaagtggc ccagttcagt
gcgaagacag tgtctgc 37 13 36 DNA Artificial synthetic probe/primer
13 gcaatttggg gctggtcata gcgagggtac gtaacc 36 14 36 DNA Artificial
synthetic probe/primer 14 ggttacgtac cctcgctatg accagcccca aattgc
36
* * * * *
References