U.S. patent application number 11/626140 was filed with the patent office on 2007-09-27 for method of identifying inhibitors of dhodh.
Invention is credited to Katharina Aulinger-Fuchs, Roland Baumgartner, Bernd Kramer, Johann Leban, Stefan Tasler.
Application Number | 20070224672 11/626140 |
Document ID | / |
Family ID | 37695206 |
Filed Date | 2007-09-27 |
United States Patent
Application |
20070224672 |
Kind Code |
A1 |
Leban; Johann ; et
al. |
September 27, 2007 |
Method of Identifying Inhibitors of DHODH
Abstract
The present invention provides a compound capable of binding to
the ubiquinone binding site of DHODH which contains a non-aromatic
ring system as a core structure, a group capable of interacting
with structural elements of subsite 2 or 3 of the ubiquinone
binding site of DHODH and a group capable of interacting
hydrophobically with structural elements of subsite 1 of the
ubiquinone binding site of DHODH. Furthermore, the present
invention provides a compound capable of binding to the ubiquinone
binding site of DHODH which contains an aromatic ring system as a
core structure, a group capable of interacting with residues His 56
and/or Tyr 356 of subsite 3 of the ubiquinone binding site of DHODH
and a group capable of interacting hydrophobically with structural
elements of subsite 1 of the ubiquinone binding site of DHODH.
Inventors: |
Leban; Johann; (Germering,
DE) ; Kramer; Bernd; (Aachen, DE) ;
Baumgartner; Roland; (Munchen, DE) ; Aulinger-Fuchs;
Katharina; (Neuried, DE) ; Tasler; Stefan;
(Gilching, DE) |
Correspondence
Address: |
Womble Carlyle Sandridge & Rice, PLLC;Attn: Patent Docketing 32nd Floor
P.O. Box 7037
Atlanta
GA
30357-0037
US
|
Family ID: |
37695206 |
Appl. No.: |
11/626140 |
Filed: |
January 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
10736739 |
Nov 10, 2004 |
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11626140 |
Jan 23, 2007 |
|
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|
60526992 |
Dec 5, 2003 |
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60435258 |
Dec 23, 2002 |
|
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|
60435285 |
Dec 23, 2002 |
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Current U.S.
Class: |
435/189 ;
702/19 |
Current CPC
Class: |
C07C 233/60 20130101;
Y02A 50/30 20180101; C07D 333/24 20130101; C07C 2601/10 20170501;
A61K 31/44 20130101; A61K 31/401 20130101; Y02A 50/411
20180101 |
Class at
Publication: |
435/189 ;
702/019 |
International
Class: |
C12N 9/02 20060101
C12N009/02; G06F 19/00 20060101 G06F019/00 |
Claims
1. A method for determining the three dimensional structure of a
crystalline polypeptide comprising an ubiquinone binding site of
DHODH complexed with at least one ligand, said method comprising:
obtaining at least one crystal of the polypeptide comprising the
ubiquinone binding site of DHODH complexed with a ligand; obtaining
x-ray diffraction data for said crystal; and solving the crystal
structure of said crystal using said x-ray diffraction data and
atomic coordinates for the DHODH complex with the ligand.
2. A method of identifying a compound which is capable of
inhibiting DHODH, said method comprising: obtaining at least one
crystal of a polypeptide comprising an ubiquinone binding site of
DHODH complexed with a ligand; obtaining the atomic coordinates of
the polypeptide in said crystal; using said atomic coordinates to
define the ubiquinone binding site of DHODH complexed with a
ligand; and identifying at least one compound which fits the
ubiquinone binding site.
3. A method of claim 2, further comprising obtaining or
synthesizing the compound to inhibit at least one biological
activity of DHODH.
4. A method of claim 3, wherein said activity is enzymatic
activity.
5. A method of identifying a compound that is capable of inhibiting
DHODH comprising: determining the ability of one or more functional
groups and/or moieties of the compound, when present in, or bound
to, an ubiquinone binding site of DHODH to interact with at least
one subsite of the ubiquinone binding site of DHODH, wherein the
ubiquinone binding site of DHODH is defined by atomic coordinates
of a polypeptide comprising the ubiquinone binding site of DHODH,
and assessing whether said compound is able to interact with said
at least one subsite, and/or has a calculated interaction energy
with a desired or preselected range to determine a potential
thereof for inhibiting DHODH.
6. A method of claim 2, wherein the atomic coordinates of the
polypeptide are used to generate a three-dimensional structure of
the ubiquinone binding site of DHODH, and said structure is used to
assess the ability of said compound to fit into the ubiquinone
binding site.
7. A method of claim 2, wherein the atomic coordinates of the
polypeptide and ligand are used to generate a three-dimensional
structure of the ligand in a binding conformation thereof, and
wherein said structure is used to assess the ability of said
compound to exhibit a similar spatial orientation, electrostatic
and/or van der Waals interaction as the ligand and to fit into the
binding site.
8. A method of claim 5, wherein said determining the ability to
interact, comprises determining whether said compound is of a size
and shape to physically reside in the ubiquinone binding site,
and/or whether said compound has a shape which is complementary to
the ubiquinone binding site and can reside in the ubiquinone
binding site without significant unfavorable sterical and/or van
der Waals interaction.
9. A method of claim 8, wherein said compound possesses an
energetically stabilizing interaction with at least one moiety
within the subsite.
10. A method of claim 9 wherein said compound possesses an
energetically stabilizing interaction with at least two
complementary moieties within the subsite and said moieties are
capable of participating in an attractive and/or stabilizing
interaction.
11. A method of claim 10, where in said interaction comprises an
electrostatic and/or a van der Waals interaction.
12. A method of claim 10, wherein said interaction is an ion-ion, a
salt bridge, ion-dipole, dipole-dipole, hydrogen bond, pi-pi
interaction, hydrophobic interaction, and/or a covalent bond.
13. A compound identified by the method of claim 1.
14. A compound identified by the method of claim 2.
15. A compound identified by the method of claim 5.
16. A method of claim 5, wherein said compound is capable of
interacting with at least two subsites.
17. A method of claim 5, wherein said compound is capable of
interacting with at least three subsites.
18. A method of identifying a compound that is capable of
inhibiting DHODH comprising: identifying at least one functional
group capable of interacting with at least one subsite of the DHODH
ubiquinone binding site; identifying a scaffold which presents the
functional group in a suitable orientation for interacting with
said at least one subsite of the DHODH ubiquinone binding site, and
attaching the identified functional group to the scaffold to
identify a compound, wherein said compound is capable of inhibiting
DHODH.
19. A method of claim 18, wherein said ubiquinone binding site is
defined by the atomic coordinates of a polypeptide comprising the
DHODH ubiquinone binding site.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/736,739 filed Nov. 10, 2004, which claims priority to U.S.
Provisional Application Nos. 60/526,992 filed Dec. 5, 2003,
60/435,258 filed Dec. 23, 2002 and 60/435,285 filed Dec. 23, 2002,
the contents of which are all incorporated herein by reference in
their intireties.
DESCRIPTION
[0002] The present invention relates to a polypeptide which
comprises the ligand binding domain of human dihydroorotate
dehydrogenase (DHODH), the crystalline forms of this polypeptide
complexed with new antiproliferative agents and the use of these
crystalline forms to determine the three dimensional structure of
the ubiquinone binding site of DHODH complexed with the ligands.
The invention also refers to the use of the three dimensional
structure of the ubiquinone binding site of DHODH in methods of
designing and/or identifying potential inhibitors of dihydroorotate
dehydrogenase (DHODH), for example, compounds which are inhibitors
of the ubiquinone binding site, for example, compounds which
inhibit the binding of a native substrate to the ubiquinone binding
site of DHODH.
[0003] Inhibitors of DHODH, an enzyme of the pyrimidine
biosynthesis, and pharmaceutical compositions containing them, are
useful, for example, for the treatment of rheumatoid arthritis
(RA). Its treatment with usual medications as for example
non-steroid anti-inflammatory agents is not satisfactory. In view
of the increasing ageing of the population, especially in the
developed Western countries or in Japan, the development of new
medications for the treatment of RA is urgently required.
[0004] The DHODH inhibiting leflunomide (ARAVA) [EP 780128, WO
97/34600] is the first medicament of this class of compounds
(leflunomides) for the treatment of RA. Leflunomide has
immunomodulatorial as well as anti-inflammatorial properties [EP
217206, DE 2524929].
[0005] In the body, DHODH catalyzes the synthesis of pyrimidines,
which are necessary for cell growth. An inhibition of DHODH
inhibits the growth of (pathologically) fast proliferating cells,
whereas cells which grow at normal speed may obtain their required
pyrimidine bases from the normal metabolic cycle. The most
important types of cells for the immune response, the lymphocytes,
use exclusively the synthesis of pyrimidines for their growth and
react particularly sensitively to DHODH inhibition. Substances that
inhibit the growth of lymphocytes are important medicaments for the
treatment of auto-immune diseases.
[0006] WO 99/45926 is a further reference that discloses compounds
which act as inhibitors of DHODH. A further object of the present
invention is to provide alternative effective agents which can be
used for the treatment of diseases which require the inhibition of
DHODH.
[0007] In Structure, 2000, Vol. 8, No. 1, pages 25-33, the
structure of human DHODH in complex with the antiproliferative
agents brequinar and leflunomide are described.
[0008] In Structure, 2000, Vol. 8, No. 1, pages 1227-1238, crystal
structures of DHODH B and its product complex are determined. In
Pharmaceutical Research, 1998, Vol. 15, No. 2, pages 286-295, and
in Biochemical Pharmacology, 1990, Vol. 40, No. 4, pages 709-714,
the structure-activity relationship of leflunomide and quinoline
carboxylic acid analogues is analyzed.
[0009] In the Journal of Medicinal Chemistry, 1999, Vol. 42, pages
3308-3314, virtual combinatorial syntheses and computational
screening of new potential anti-Herpes compounds are described. In
Table 3 on page 3313 experimental results regarding IC.sub.50 and
cytotoxicity are presented for
2-(2,3-difluorophenylcarbamoyl)-1-cyclopentene-1-carboxylic acid,
2-(2,6-difluorophenylcarbamoyl)-1-cyclopentene-1-carboxylic acid
and 2-(2,3,4-trifluorophenyl-carbamoyl)-1-cyclopentene-1-carboxylic
acid.
[0010] In one embodiment, the present invention relates to a
polypeptide comprising the ligand binding domain of human
dihydroorotate dehydrogenase (DHODH), crystalline forms of this
polypeptide complexed with a ligand, and the three dimensional
structure of the polypeptide, including the three dimensional
structure of the ubiquinone binding site of DHODH.
[0011] In another embodiment, the present invention provides a
method of determining the three dimensional structure of a
crystalline polypeptide comprising the ubiquinone binding site of
DHODH complexed with the ligands. The method comprises the steps of
(1) obtaining a crystal of the polypeptide comprising the
ubiquinone binding site of DHODH complexed with a ligand; (2)
obtaining x-ray diffraction data for said crystal; and (3) solving
the crystal structure of said crystal by using said x-ray
diffraction data and the atomic coordinates for the DHODH complex
with the ligand.
[0012] The invention further relates to a method of identifying a
compound which is a potential inhibitor of DHODH. The method
comprises the steps of (1) obtaining a crystal of the polypeptide
comprising the ubiquinone binding site of DHODH complexed with a
ligand; (2) obtaining the atomic coordinates of the polypeptide in
said crystal; (3) using said atomic coordinates to define the
ubiquinone binding site of DHODH complexed with a ligand; and (4)
identifying a compound which fits the ubiquinone binding site. The
method can further include the steps of obtaining or synthesizing
the compound to inhibit at least one biological activity of DHODH,
such as enzymatic activity.
[0013] In another embodiment, the method of identifying a potential
inhibitor of DHODH comprises the step of determining the ability of
one or more functional groups and/or moieties of the compound, when
present in, or bound to, the ubiquinone binding site of DHODH; to
interact with one or more subsites of the ubiquinone binding site
of DHODH. Generally, the ubiquinone binding site of DHODH is
defined by the atomic coordinates of a polypeptide comprising the
ubiquinone binding site of DHODH. If the compound is able to
interact with a preselected number or set of subsites, or has a
calculated interaction energy with a desired or preselected range,
the compound is identified as a potential inhibitor of DHODH.
[0014] The human DHODH enzyme is composed of two domains, namely a
large C-terminal domain (Met78 to C-terminus) and a small
N-terminal domain (Met30 to Leu68), connected by an extended loop.
The large C-terminal domain can be described best as an
.alpha./.beta.-barrel fold with a central barrel of eight parallel
.beta. strands surrounded by eight .alpha. helices. The redox site,
formed by the substrate binding site and the site of the cofactor
flavine mononucleotide (FMN), is located on this large C-terminal
domain. The small N-terminal domain, on the other hand, consists of
two .alpha. helices, .alpha.1 and .alpha.2, connected by a short
loop. This small N-terminal domain contains the binding site for
the cofactor ubiquinone. The helices .alpha.1 and .alpha.2 span a
slot of about 10.times.20 .ANG. in the so-called hydrophobic patch,
with the short .alpha.1-.alpha.2 loop at the narrow end of that
slot. The slot forms the entrance to a tunnel that ends at the FMN
cavity nearby the .alpha.1-.alpha.2 loop. This tunnel is narrowing
towards the proximal redox site and ends with several charged or
polar sidechains (Gln47, His56, Tyr356, Thr360 and Arg136). It is
evident that ubiquinone which can easily diffuse into the
mitochondrial inner membrane uses this tunnel to approach the FMN
cofactor for a redox reaction.
[0015] The structural knowledge mentioned above can be used to
design potential inhibitors of the human DHODH activity targeting
the tunnel mentioned above and competing with ubiquinone for the
ubiquinone binding site. Potential inhibitors were co-crystallized
with human DHODH (Met30 to Arg396) and the three dimensional
structures were solved by protein X-ray crystallography techniques,
ten of the solved structures being three dimensional structures of
human DHODH (Met30 to Arg396) in complex with compounds 1, 2, 3, 4,
5, 6, 7, 8, 9, and 10. These crystal structures were solved at
atomic resolution and the binding modes of the ten compounds were
analyzed. The structural formulars of the co-crystallized compounds
are given below. ##STR1## ##STR2##
[0016] Detailed analysis of the three dimensional structure of the
DHODH small N-terminal domain as well as the three dimensional
structure of DHODH in complex with certain inhibitors designed to
target the ubiquinone binding site revealed the presence of a
number of subsites. Each subsite includes molecular functional
groups or moieties capable of forming stabilizing interactions with
complementary functional groups or moieties of an inhibitor.
[0017] The found subsites are characterized below according to the
properties of functional groups or chemical moieties they are
complementary to, or they can interact with in a stabilizing way,
for example, groups or moieties capable of hydrogen bond formation
or groups or moieties with hydrophobic (=lipophilic) character. A
hydrogen bond is formed between a hydrogen atom covalently bond to
an electronegative element (proton donor or hydrogen bond donor)
and a lonely electron pair of a second electronegative atom (proton
acceptor or hydrogen bond acceptor). Hydrogen bonds typically occur
when the hydrogen bond donor and the hydrogen bond acceptor are
separated by about 2.5 .ANG. and 3.5 .ANG..
[0018] Stabilizing hydrophobic or lipophilic interactions occur if
two groups or moieties with hydrophobic/lipophilic character, for
example, aliphatic chains or aromatic systems, are separated by
distances close to their van der Waals radii.
[0019] The method of identifying a potential inhibitor of DHODH
comprises the step of determining the ability of one or more
functional groups and/or moieties of the compound, when present in
the ubiquinone binding site, to interact with one or more subsites
of the ubiquinone binding site. Preferably, the ubiquinone binding
site is defined by the atomic coordinates of a polypeptide
comprising the ubiquinone binding site of DHODH. If the compound is
able to interact with a preselected number or set of subsites, the
compound is identified as a potential inhibitor of DHODH.
[0020] A functional group or moiety of the compound is said to
"interact" with a subsite of the ubiquinone binding site if it
participates in an energetically favourable, or stabilizing,
interaction with one or more complementary moieties within the
subsite.
[0021] Two chemical moieties are "complementary" if they are
capable, when suitably positioned, of participating in an
attractive, or stabilizing, interaction, such as an electrostatic
or an van der Waals interaction. Typically, the attractive
interaction is an ion-ion, a salt bridge, ion-dipole,
dipole-dipole, hydrogen bond, pi-pi or hydrophobic interaction. An
extreme case of attractive interaction is the formation of a
covalent bond by a chemical reaction between the test compound and
the enzyme. For example, a negatively charged moiety and a
positively charged moiety are complementary because, if suitably
positioned, they can form a salt bridge. Likewise, a hydrogen bond
donor and a hydrogen bond acceptor are complementary if suitably
positioned.
[0022] Preferably, the groups capable of hydrogen bond formation
("HB") are selected from halogen, such as fluorine, chlorine,
bromine and iodine, NO.sub.2, haloalkyl, haloalkyloxy, CN,
hydroxyl, amino, hydroxylamine, hydroxamic acid, carbonyl, carbonic
acid, sulfonamide, amide, sulfone, sulfonic acid, alkylthio,
alkoxy, ester, hydroxyalkylamino group, and other groups including
a heteroatom having at least one lone pair of electrons, such as
groups containing trivalent phosphorous, di- and tetravalent
sulfur, oxygen and nitrogen atoms;
[0023] Preferably, hydrophobic groups ("H") are selected from
groups, such as linear, branched or cyclic alkyl groups; linear,
branched or cyclic alkenyl groups; linear, branched or cyclic
alkynyl groups; aryl groups, such as mono- and polycyclic aromatic
hydrocarbyl groups and mono- and polycyclic heteroaryl groups;
[0024] Preferably, negatively charged groups ("N") are selected
from groups, such as carboxylate, sulfonamide, sulfamate, boronate,
vanadate, sulfonate, sulfinate and phosphonate groups. A given
chemical moiety can contain one or more of these groups.
[0025] In the following a detailed description of identified
subsites is provided. Residue numbering and atom labeling is
identical to the numbering and labeling in FIGS. 2, 3 and 4.
[0026] Subsite 1: Hydrophobic pocket; interacting chemical
moieties: H;
[0027] Residues involved: Leu 42; Met 43; Leu 46; Ala 55; Ala 59;
Phe 98; Met 111; Leu 359; Pro 364;
[0028] Non-hydrogen atoms which interact with H: Leu 42 CB, CG,
CD1, CD2; Met 43 SD, CE; Leu 46 CB, CG, CD1, CD2; Ala 55 CB; Ala 59
CA, CB; Phe 98 CG, CD1, CD2, CE1, CE2; Met 111 SD, CE; Leu 359 CA,
CB, CG, CD1, CD2; Pro 364 CB, CD, CG;
[0029] Preferably for the hydrophobic interacting with subsite 1,
the group is selected from aryl groups, such as an aromatic group
having five to fifteen carbon atoms, which can optionally be
substituted by one or more substituents R'. More preferably the
aryl group is a phenyl group, such as --CH.sub.2Ph,
--C.sub.2H.sub.4Ph, --CH.dbd.CH-Ph, --C.ident.C-Ph,
-o-C.sub.6H.sub.4--R', -m-C.sub.6H.sub.4--R',
-p-C.sub.6H.sub.4--R', -o-CH.sub.2--C.sub.6H.sub.4--R',
-m-CH.sub.2--C.sub.6H.sub.4--R', -p-CH.sub.2--C.sub.6H.sub.4--R';
or a biphenyl group, in which the phenyl rings can optionally be
substituted by one or more substituents R', such biphenyl groups
are --C.sub.6H.sub.4--C.sub.6H.sub.5;
--C.sub.6H.sub.4--C.sub.6H.sub.4--R';
--C.sub.6H.sub.3--R'--C.sub.6H.sub.5;
--C.sub.6H.sub.3--R'--C.sub.6H.sub.4--R';
[0030] --C.sub.6H.sub.3--R'--C.sub.6H.sub.4--R';
--C.sub.6H.sub.4--O--C.sub.6H.sub.5;
--C.sub.6H.sub.3--R'--O--C.sub.6H.sub.4--R';
--C.sub.6H.sub.4--O--C.sub.6H.sub.4--R';
--C.sub.6H.sub.3--R'--O--C.sub.6H.sub.5;
--C.sub.6H.sub.4--O--CH.sub.2--C.sub.6H.sub.5;
--C.sub.6H.sub.3--R'--O--CH.sub.2--C.sub.6H.sub.4--R';
--C.sub.6H.sub.4--O--CH.sub.2--C.sub.6H.sub.4--R;
--C.sub.6H.sub.3--R'--O--CH.sub.2--C.sub.6H.sub.5;
[0031] R' is independently H, --CO.sub.2R'', --CONHR'', --CR''O,
--SO.sub.2NR'', --NR''--CO-haloalkyl, --NO.sub.2,
--NR''--SO.sub.2-haloalkyl, --NR''--SO.sub.2-alkyl,
--SO.sub.2-alkyl, --NR''--CO-alkyl, --CN, alkyl, cycloalkyl,
aminoalkyl, alkylamino, alkoxy, --OH, --SH, alkylthio,
hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkyloxy,
aryl, arylalkyl or heteroaryl;
[0032] R'' is independently hydrogen, haloalkyl, hydroxyalkyl,
alkyl, cycloalkyl, aryl, heteroaryl or aminoalkyl;
[0033] R' is preferably F, Cl, Br, I, CF.sub.3, OCF.sub.3, or
OCH.sub.3;
[0034] Subsite 2: First anion binding site; interacting with HB, N,
HB and N, HB and HB, or N and N;
[0035] Residues involved: Gln 47; Arg 136; one conserved water
molecule
[0036] Non-hydrogen atoms which interact with HB and N: Glu 47 OE1,
NE2; Arg 136 NE, NH1, NH2; conserved water molecule OH2.
[0037] preferably for one or two hydrogen bond formations with
subsite 2 the group is selected from halogen, such as fluorine,
chlorine, bromine and iodine, NO.sub.2, haloalkyl, haloalkyloxy,
CN, hydroxyl, amino, hydroxylamine, hydroxamic acids, carbonyl,
carbonic acid, sulfonamide, amide, sulfone, sulfonic acid,
alkylthio, alkoxy, such as methoxy, ester, hydroxyalkylamino,
carboxylate, tetrazole, sulfonamide, sulfamate, boronate, vanadate,
sulfonate, sulfinate and phosphonate group, more preferably from a
carboxylate, sulfonamide, sulfamate, sulfonate, carbonyl or
carbonic acid group.
[0038] Subsite 3: Second anion binding site; interacting with HB,
N, HB and N, HB and HB, or N and N;
[0039] Residues involved: His 56; Tyr 356; Tyr 147 (interacting via
a conserved water molecule);
[0040] Non-hydrogen atoms which interact with HB and N: His 56 N,
ND1; Tyr 356 OH; Tyr 147 OH (interacting via a conserved water
molecule);
[0041] preferably for one or two hydrogen bond formations with
subsite 2 the group is selected from halogen, such as fluorine,
chlorine, bromine and iodine, NO.sub.2, haloalkyl, haloalkyloxy,
CN, hydroxyl, amino, hydroxylamine, hydroxamic acids, carbonyl,
carbonic acid, sulfonamide, amide, sulfone, sulfonic acid,
alkylthio, alkoxy, such as methoxy, ester, hydroxyalkylamino,
carboxylate, tetrazole, sulfonamide, sulfamate, boronate, vanadate,
sulfonate, sulfinate and phosphonate group, more preferably from a
carboxylate, sulfonamide, sulfamate, sulfonate, carbonyl or
carbonic acid group.
[0042] Subsite 4: Remote hydrophobic pocket; interacting chemical
moieties: H;
[0043] Residues involved: Pro 52; Val 134; Arg 136; Val 143; Thr
360; FMN;
[0044] Non-hydrogen atoms which interact with H: Pro 52 CB, CG, CD;
Val 134 CB, CG1, CG2; Val 143 CB, CG1, CG2; Thr 360 CG2; FMN C7M,
C8M;
[0045] Preferably for the hydrophobic interacting with subsite 4,
the group is selected from such as linear, branched or cyclic
C.sub.1-C.sub.6-alkyl groups; such as methyl, ethyl, propyl, butyl,
tert. butyl, linear, branched or cyclic C.sub.1-C.sub.6-alkenyl
groups; linear, branched or cyclic C.sub.1-C.sub.6-alkynyl groups;
aryl groups, such as mono- and bi aromatic hydrocarbyl groups, such
as --CH.sub.2Ph, --C.sub.2H.sub.4Ph, --CH.dbd.CH-Ph,
--C.ident.C-Ph, -o-C.sub.6H.sub.4--R', -m-C.sub.6H.sub.4--R,
-p-C.sub.6H.sub.4--R, --O--CH.sub.2--C.sub.6H.sub.4--R,
-m-CH.sub.2--C.sub.6H.sub.4--R, -p-CH.sub.2--C.sub.6H.sub.4--R and
mono- and bicyclic heteroaryl groups, such as thiazol-2-yl,
thiazol-4-yl, thiazol-5-yl, isothiazol-3-yl, isothiazol-4-yl,
isothiazol-5-yl, 1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl,
1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl, 1,2,5-oxadiazol-3-yl,
1,2,5-oxadiazol-4-yl, 1,2,5-thiadiazol-3-yl, 1-imidazolyl,
2-imidazolyl, 1,2,5-thiadiazol-4-yl, 4-imidazolyl, 1-pyrrolyl,
2-pyrrolyl, 3-pyrrolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrazinyl,
1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, indolyl, indolinyl,
tetrazolyl, benzo-[b]-furanyl, benzo[b]thiophenyl, benzimidazolyl,
benzothiazolyl, quinazolinyl, quinoxazolinyl, or preferably
isoxazol-3-yl, isoxazol-4-yl, isoxazol-5-yl, quinolinyl,
tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl; all
this groups can optionally be substituted by one or more
substituents R, such as H, amino, alkoxy, OH, SH, alkylthio,
hydroxyalkyl, haloalkyl, haloalkyloxy hydroxyalkylamino, halogen; R
is preferably F, Cl, Br, I, CF.sub.3, OCF.sub.3, or OCH.sub.3;
[0046] Core: chemical moiety connecting the different moieties
interacting with Subsite 1, Subsite 2, Subsite 3, and Subsite
4;
[0047] Preferably, the core is selected from cyclic alkyl groups;
cyclic alkenyl groups; cyclic alkynyl groups; aryl groups, such as
mono- and polycyclic aromatic hydrocarbyl groups and mono- and
polycyclic heteroaryl groups; more preferably it is selected from
mono-, or bicyclic aromatic or non-aromatic ring systems, most
preferably from 5-membered mono-, or bicyclic aromatic or
non-aromatic ring systems, such as trans-cyclopentan-1,2-diyl,
trans-cyclohexan-1,2-diyl, cis-cyclopentan-1,2-diyl,
cis-cyclohexan-1,2-diyl, 1-cyclopenten-1,2-diyl,
2-cyclopenten-1,2-diyl, 3-cyclopenten-1,2-diyl,
4-cyclopenten-1,2-diyl, 5-cyclopenten-1,2-diyl,
1-cyclopenten-1,3-diyl, 1-cyclopenten-1,4-diyl,
1-cyclohexen-1,2-diyl, 1-cyclohepten-1,2-diyl or
1-cycloocten-1,2-diyl, 2,5-dihydrothiophene-3,4-diyl,
2,5-dihydro-furan-3,4-diyl, 2,5-dihydro-1H-pyrrole-3,4-diyl,
2,5-dihydro-1-methyl-pyrrole-3,4-diyl,
2,5-dihydro-1-ethyl-pyrrole-3,4-diyl,
2,5-dihydro-1-acetyl-pyrrole-3,4-diyl,
2,5-dihydro-1-methylsulfonyl-pyrrole-3,4-diyl, thiazol-2-yl,
thiazol-4-yl, thiazol-5-yl, isothiazol-3-yl, isothiazol-4-yl,
isothiazol-5-yl, 1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl,
1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl, 1,2,5-oxadiazol-3-yl,
1,2,5-oxadiazol-4-yl, 1,2,5-thiadiazol-3-yl, 1-imidazolyl,
2-imidazolyl, 1,2,5-thiadiazol-4-yl, 4-imidazolyl, 1-pyrrolyl,
2-pyrrolyl, 3-pyrrolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrazinyl,
1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, indolyl, indolinyl,
tetrazolyl, benzo-[b]-furanyl, benzo[b]thiophenyl, benzimidazolyl,
benzothiazolyl, quinazolinyl, quinoxazolinyl, or preferably
quinolinyl, tetrahydro-quinolinyl, isoquinolinyl,
tetrahydroisoquinolinyl or from a group comprising of: ##STR3##
[0048] Bridge: chemical moiety connecting the core with Subsite
1;
[0049] Preferably, the bridge is selected from --NH; --O; --CO--NH;
--NH--CO; --NH--CO--NH; alkyl;
[0050] --O--CH.sub.2; --CH.sub.2--O; --O--CH.sub.2--CH.sub.2;
--CH.sub.2--CH.sub.2--O; --NH--CH.sub.2; --CH.sub.2--NH;
--NH--CH.sub.2--CH.sub.2; --CH.sub.2--CH.sub.2--NH;
--CH.sub.2--CO--NH; --CH.sub.2--NH--CO;
[0051] Subsite 5: Solvent anchor; interacting chemical moieties:
HB
[0052] Residues involved: Met 30; Tyr 38; Leu 67;
[0053] Non-hydrogen atoms which interact with HB: Met 30 O, SD, CE;
Tyr 380H, CE2, CD2; Leu 67 O;
[0054] preferably for the hydrogen bond formation with subsite 5,
the group is selected from F, Cl, Br, I, CF.sub.3, OCF.sub.3, or
OCH.sub.3
[0055] Subsite 6: Solvent anchor; interacting chemical moieties:
H;
[0056] Residues involved: Leu 68;
[0057] Non-hydrogen atoms which interact with H: Leu 68 CB, CG,
CD1, CD2;
[0058] Preferably for the hydrophobic interacting with subsite 6,
the group is selected from such as linear, branched or cyclic
C.sub.1-C.sub.6-alkyl groups; such as methyl, ethyl, propyl, butyl,
tert. butyl, linear, branched or cyclic C.sub.1-C.sub.6-alkenyl
groups; linear, branched or cyclic C.sub.1-C.sub.6-alkynyl groups;
aryl groups, such as mono- and bi aromatic hydrocarbyl groups, such
as --CH.sub.2Ph, --C.sub.2H.sub.4Ph, --CH.dbd.CH-Ph,
--C.ident.C-Ph, -o-C.sub.6H.sub.4--R', -m-C.sub.6H.sub.4--R,
--P--C.sub.6H.sub.4--R, --O--CH.sub.2--C.sub.6H.sub.4--R,
-m-CH.sub.2--C.sub.6H.sub.4--R, -p-CH.sub.2--C.sub.6H.sub.4--R and
mono- and bicyclic heteroaryl groups.
[0059] An alkyl group, if not stated otherwise, denotes a linear or
branched C.sub.1-C.sub.6-alkyl, preferably a linear or branched
chain of one to five carbon atoms, a linear or branched
C.sub.1-C.sub.6-alkenyl or a linear or branched
C.sub.1-C.sub.6-alkinyl group, which can optionally be substituted
by one or more substituents R', preferably by halogen;
[0060] the C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-alkenyl and
C.sub.1-C.sub.6-alkinyl residue may be selected from the group
comprising --CH.sub.3, --C.sub.2H.sub.5, --CH.dbd.CH.sub.2,
--C.ident.CH, --C.sub.3H.sub.7, --CH(CH.sub.3).sub.2,
--CH.sub.2--CH.dbd.CH.sub.2, --C(CH.sub.3).dbd.CH.sub.2,
--CH.dbd.CH--CH.sub.3, --C.ident.C--CH.sub.3,
--CH.sub.2--C.ident.CH, --C.sub.4H.sub.9,
--CH.sub.2--CH(CH.sub.3).sub.2, --CH(CH.sub.3)--C.sub.2H.sub.5,
--C(CH.sub.3).sub.3, --C.sub.5H.sub.11, --C.sub.6H.sub.13,
--C(R').sub.3, --C.sub.2(R').sub.5, --CH.sub.2--C(R').sub.3,
--C.sub.3(R').sub.7,
[0061] --C.sub.2H.sub.4--C(R').sub.3,
--C.sub.2H.sub.4--CH.dbd.CH.sub.2, --CH.dbd.CH--C.sub.2H.sub.5,
--CH.dbd.C(CH.sub.3).sub.2, --CH.sub.2--CH.dbd.CH--CH.sub.3,
[0062] --CH.dbd.CH--CH.dbd.CH.sub.2, --C.sub.2H.sub.4--C.ident.CH,
--C.ident.C--C.sub.2H.sub.5, --CH.sub.2--C.ident.C--CH.sub.3,
--C.ident.C--CH.dbd.CH.sub.2,
[0063] --CH.dbd.CH--C.ident.CH, --C.ident.C--C.ident.CH,
--C.sub.2H.sub.4--CH(CH.sub.3).sub.2,
--CH(CH.sub.3)--C.sub.3H.sub.7,
--CH.sub.2--CH(CH.sub.3)--C.sub.2H.sub.5,
--CH(CH.sub.3)--CH(CH.sub.3).sub.2,
--C(CH.sub.3).sub.2--C.sub.2H.sub.5, --CH.sub.2--C(CH.sub.3).sub.3,
--C.sub.3H.sub.6--CH.dbd.CH.sub.2,
[0064] --CH.dbd.CH--C.sub.3H.sub.7,
--C.sub.2H.sub.4--CH.dbd.CH--CH.sub.3,
--CH.sub.2--CH.dbd.CH--C.sub.2H.sub.5,
--CH.sub.2--CH.dbd.CH--CH.dbd.CH.sub.2,
[0065] --CH.dbd.CH--CH.dbd.CH--CH.sub.3,
--CH.dbd.CH--CH.sub.2--CH.dbd.CH.sub.2,
--C(CH.sub.3).dbd.CH--CH.dbd.CH.sub.2,
[0066] --CH.dbd.C(CH.sub.3)--CH.dbd.CH.sub.2,
--CH.dbd.CH--C(CH.sub.3).dbd.CH.sub.2,
--CH.sub.2--CH.dbd.C(CH.sub.3).sub.2,
C(CH.sub.3).dbd.C(CH.sub.3).sub.2, --C.sub.3H.sub.6--C.ident.CH,
--C.ident.C--C.sub.3H.sub.7, --C.sub.2H.sub.4--C.ident.C--CH.sub.3,
--CH.sub.2--C.ident.C--C.sub.2H.sub.5,
--CH.sub.2--C.ident.C--CH.dbd.CH.sub.2,
--CH.sub.2--CH.dbd.CH--C.ident.CH,
--CH.sub.2--C.ident.C--C.ident.CH,
--C.ident.C--CH.dbd.CH--CH.sub.3,
--CH.dbd.CH--C.ident.C--CH.sub.3,
[0067] --C.ident.C--C.ident.C--CH.sub.3,
--C.ident.C--CH.sub.2--CH.dbd.CH.sub.2,
--CH.dbd.CH--CH.sub.2--C.ident.CH,
--C.ident.C--CH.sub.2--C.ident.CH,
[0068] --C(CH.sub.3).dbd.CH--CH.dbd.CH.sub.2,
--CH.dbd.C(CH.sub.3)--CH.dbd.CH.sub.2,
--CH.dbd.CH--C(CH.sub.3).dbd.CH.sub.2,
--C(CH.sub.3).dbd.CH--C.ident.CH, --CH.dbd.C(CH.sub.3)--C.dbd.CH,
--C.ident.C--C(CH.sub.3).dbd.CH.sub.2,
--C.sub.3H.sub.6--CH(CH.sub.3).sub.2,
--C.sub.2H.sub.4--CH(CH.sub.3)--C.sub.2H.sub.5,
--CH(CH.sub.3)--C.sub.4H.sub.9,
--CH.sub.2--CH(CH.sub.3)--C.sub.3H.sub.7,
--CH(CH.sub.3)--CH.sub.2--CH(CH.sub.3).sub.2,
--CH(CH.sub.3)--CH(CH.sub.3)--C.sub.2H.sub.5,
--CH.sub.2--CH(CH.sub.3)--CH(CH.sub.3).sub.2,
--CH.sub.2--C(CH.sub.3).sub.2--C.sub.2H.sub.5,
--C(CH.sub.3).sub.2--C.sub.3H.sub.7,
--C(CH.sub.3).sub.2--CH(CH.sub.3).sub.2,
--C.sub.2H.sub.4--C(CH.sub.3).sub.3,
--CH(CH.sub.3)--C(CH.sub.3).sub.3,
--C.sub.4H.sub.8--CH.dbd.CH.sub.2, --CH.dbd.CH--C.sub.4H.sub.9,
--C.sub.3H.sub.6--CH.dbd.CH--CH.sub.3,
--CH.sub.2--CH.dbd.CH--C.sub.3H.sub.7,
--C.sub.2H.sub.4--CH.dbd.CH--C.sub.2H.sub.5,
--CH.sub.2--C(CH.sub.3).dbd.C(CH.sub.3).sub.2,
--C.sub.2H.sub.4--CH.dbd.C(CH.sub.3).sub.2,
--C.sub.4H.sub.8--C.ident.CH, --C.ident.C--C.sub.4H.sub.9,
--C.sub.3H.sub.6--C.ident.C--CH.sub.3,
[0069] --CH.sub.2--C.ident.C--C.sub.3H.sub.7,
--C.sub.2H.sub.4--C.dbd.C--C.sub.2H.sub.5;
[0070] R' is independently H, --CO.sub.2R'', --CONHR'', --CR''O,
--SO.sub.2NR'', --NR''--CO-haloalkyl, --NO.sub.2,
--NR''--SO.sub.2-haloalkyl, --NR''--SO.sub.2-alkyl,
--SO.sub.2-alkyl, --NR''--CO-alkyl, --CN, alkyl, cycloalkyl,
aminoalkyl, alkylamino, alkoxy, --OH, --SH, alkylthio,
hydroxyalkyl, hydroxyalkylamino, halogen, haloalkyl, haloalkyloxy,
aryl, arylalkyl or heteroaryl;
[0071] R'' is independently hydrogen, haloalkyl, hydroxyalkyl,
alkyl, cycloalkyl, aryl, heteroaryl or aminoalkyl;
[0072] a cycloalkyl group denotes a non-aromatic ring system
containing four to eight carbon atoms, preferably four to eight
carbon atoms, wherein one or more of the carbon atoms in the ring
can be substituted by a group X, X being as defined above; the
C.sub.4-C.sub.8-cycloalkyl residue may be selected from the group
comprising -cyclo-C.sub.4H.sub.7, -cyclo-C.sub.5H.sub.9,
-cyclo-C.sub.6H.sub.11, -cyclo-C.sub.7H.sub.13,
-cyclo-C.sub.8H.sub.15;
[0073] an alkoxy group denotes an O-alkyl group, the alkyl group
being as defined above; the alkoxy group is preferably a methoxy,
ethoxy, isopropoxy, t-butoxy or pentoxy group;
[0074] an alkylthio group denotes an S-alkyl group, the alkyl group
being as defined above.
[0075] an haloalkyl group denotes an alkyl group which is
substituted by one to five halogen atoms, the alkyl group being as
defined above; the haloalkyl group is preferably a
--C(R.sup.10).sub.3, --CR.sup.10(R.sup.10').sub.2,
--CR.sup.10(R.sup.10')R.sup.10'', --C.sub.2(R.sup.10).sub.5,
--CH.sub.2--C(R.sup.10).sub.3,
--CH.sub.2--CR.sup.10(R.sup.10').sub.2,
--CH.sub.2--CR.sup.10(R.sup.10')R.sup.10'',
--C.sub.3(R.sup.10).sub.7 or --C.sub.2H.sub.4--C(R.sup.10).sub.3,
wherein R.sup.10, R.sup.10', R.sup.10'' represent F, Cl, Br or I,
preferably F;
[0076] a hydroxyalkyl group denotes an HO-alkyl group, the alkyl
group being as defined above;
[0077] an haloalkyloxy group denotes an alkoxy group which is
substituted by one to five halogen atoms, the alkyl group being as
defined above; the haloalkyloxy group is preferably a
--OC(R.sup.10).sub.3,
--OCR.sup.10(R.sup.10').sub.2--OCR.sup.10(R.sup.10')R.sup.10'',
--OC.sub.2(R.sup.10).sub.5, --OCH.sub.2--C(R.sup.10).sub.3,
--OCH.sub.2--CR.sup.10(R.sup.10').sub.2,
--OCH.sub.2--CR.sup.10(R.sup.10')R.sup.10'',
--OC.sub.3(R.sup.10).sub.7 or --OC.sub.2H.sub.4--C(R.sup.10).sub.3,
wherein R.sup.10, R.sup.10', R.sup.10'' represent F, Cl, Br or I,
preferably F;
[0078] a hydroxyalkylamino group denotes an (HO-alkyl).sub.2-N--
group or HO-alkyl-NH-- group, the alkyl group being as defined
above;
[0079] an alkylamino group denotes an HN-alkyl or N-dialkyl group,
the alkyl group being as defined above;
[0080] a halogen group is chlorine, bromine, fluorine or iodine,
fluorine being preferred;
[0081] an aryl group preferably denotes an aromatic group having
five to fifteen carbon atoms, which can optionally be substituted
by one or more substituents R', where R' is as defined above; the
aryl group is preferably a phenyl group, --CH.sub.2Ph,
--C.sub.2H.sub.4Ph, --CH.dbd.CH-Ph, --C.ident.C-Ph,
-o-C.sub.6H.sub.4--R', -m-C.sub.6H.sub.4--R',
-p-C.sub.6H.sub.4--R', -o-CH.sub.2--C.sub.6H.sub.4--R',
-m-CH.sub.2--C.sub.6H.sub.4--R',
-p-CH.sub.2--C.sub.6H.sub.4--R';
[0082] a heteroaryl group denotes a 5- or 6-membered heterocyclic
group which contains at least one heteroatom like O, N, S. This
heterocyclic group can be fused to another ring. For example, this
group can be selected from a thiazol-2-yl, thiazol-4-yl,
thiazol-5-yl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl,
1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl, 1,2,4-thiadiazol-3-yl,
1,2,4-thiadiazol-5-yl, 1,2,5-oxadiazol-3-yl, 1,2,5-oxadiazol-4-yl,
1,2,5-thiadiazol-3-yl, 1-imidazolyl, 2-imidazolyl,
1,2,5-thiadiazol-4-yl, 4-imidazolyl, 1-pyrrolyl, 2-pyrrolyl,
3-pyrrolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl,
3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl,
3-pyridazinyl, 4-pyridazinyl, 2-pyrazinyl, 1-pyrazolyl,
3-pyrazolyl, 4-pyrazolyl, 1H-tetrazol-2-yl, 1H-tetrazol-3-yl,
tetrazolyl, indolyl, indolinyl, benzo-[b]-furanyl,
benzo[b]thiophenyl, benzimidazolyl, benzothiazolyl, quinazolinyl,
quinoxazolinyl, or preferably quinolinyl, tetrahydroquinolinyl,
isoquinolinyl, tetrahydroisoquinolinyl group. This heterocyclic
group can optionally be substituted by one or more substituents R',
where R' is as defined above.
[0083] In another embodiment, the present invention provides DHODH
inhibitors, and methods of use thereof, which are capable of
binding to the ubiquinone binding site of DHODH, for example,
compounds which are identified as inhibitors of DHODH or which are
designed by the methods described above to inhibit DHODH. For
example, the invention includes compounds which interact with one
or more, preferably two or more, and more preferably, three or more
of DHODH subsites 1 to 6.
[0084] Preferably an inhibitor of DHODH should have a core-unit and
interact with subsite 1, 2, 3 and 5 or an inhibitor of DHODH should
have a core-unit and interact with subsite 1, 2 and 5, or an
inhibitor of DHODH should have a core-unit and interact with
subsite 1, 3 and 5.
[0085] More preferably an inhibitor of DHODH should have a
core-unit and interact with subsite 1, 2 and 3, or an inhibitor of
DHODH should have a core-unit and interact with subsite 1 and
3.
[0086] In FIG. 1, the spatial arrangement of the subsites is
depicted schematically.
[0087] The three dimensional structure published by Shenpig et al.
shows human DHODH(Met30-Arg396) in complex with brequinar and the
leflunomide metabolite A771726, respectively. The main interaction
in the binding of brequinar to DHODH is the formation of a salt
bridge between the carboxy group of brequinar and the sidechain of
Arg136. In particular, the salt bridge is formed between the
carboxylic group and the atoms NE, NH1 or NH2. More precisely, the
above mentioned subsite 2, the first anion binding site, is
addressed in this kind of interaction. In the following, this type
of interactioned will be termed "brequinar-like binding mode".
[0088] Analysis of the three dimensional structures of human DHODH
in complex with ligands presented here clearly shows a new binding
mode for inhibitors containing a carboxylic acid group. This
binding mode differs from the brequinar-like binding mode in
interacting not with subsite 2 but with subsite 3, termed the
second anion binding site. In particular this is true for inhibitor
compounds 1, 4, 5, 7 and 8 as can be seen from FIG. 2. This so far
unobserved binding mode will be termed "non-brequinar-like" binding
mode in the following.
[0089] The "non-brequinar-like" binding mode is characterized by a
number of hydrogen bonds formed between the ligand and protein
residues belonging to subsite 3. In particular this residues are
His 56, Tyr 356 and Tyr 147. Non-hydrogen atoms involved in the
formation of hydrogen bonds are N and ND1 of His 56, the oxygen of
the hydroxyl group of Tyr 356 and the oxygen of the hydroxyl group
of Tyr 147. The latter interaction involves a conserved water
molecule bridging the space between the carboxyl function of the
ligand molecule and the hydroxyl group of the tyrosine residue
147.
[0090] Similar findings can be seen in the three dimensional
structure of human DHODH in complex with the compounds 2, 6 and 10.
As can be seen clearly from the electron density map, the compounds
2, 6 and 10 are able to utilize both anion binding sites (subsite 2
and 3) by adopting two alternative conformations. Therefore, both a
brequinar-like and a non-brequinar-like binding mode can be
utilized. In the brequinar-like binding mode the carboxy group of
compounds 2, 6 and 10 forms hydrogen bonds to the sidechains of
residues Gln 47 and Arg 136. In the non-brequinar-like binding mode
the five membered ring of compounds 2, 6 and 10 containing the
carboxy group is rotated by almost 180 degrees and forms hydrogen
bonds to residues His 56 and Tyr 356. Non-hydrogen atoms involved
in the formation of hydrogen bonds are N and ND1 of His 56 and the
oxygen of the hydroxyl group of Tyr 356.
[0091] The compounds 2, 3 and 4 are particularly interesting for a
structure-activity-relationship (SAR) analysis. These molecules
differ only in the degree of ring substitution (see structures
above). Clearly, one can observe a correlation between the number
of fluorinated positions at the aromatic ring in the middle of the
molecules and the corresponding IC.sub.50 values. The higher the
number of ring substituents the lower the IC.sub.50. Interestingly
compound 2 and compound 3 display both the brequinar-like and
non-brequinar-like binding mode in the crystal structure (see table
27). It is quite reasonable to speculate whether the ring
substituents exhibit a steering effect on the five membered ring
and by such facilitate the formation of the more favourable
brequinar-like binding mode. Therefore, the presence of both
binding modes might explain the increased affinity of this
compounds. TABLE-US-00001 TABLE 27 Relation of inhibitor binding
mode and degree of ring substitutions. Structures of the compounds
are shown above. Compound Brequinar-like Non-Brequinar-like 3 X X 2
X X 4 X
[0092] A similar structure-activity-relationship can be deduced
from the crystal structures of human DHODH in complex with
compounds 9 and 10. These compounds carry a sulfur atom at an ortho
position with respect to the carboxylic group in the five membered
ring. Compound 10 is single substituted with fluorine at the biaryl
ring system, whereas compound 9 bears two substituents.
Interestingly, compound 9 exhibits a pure brequinar-like binding
mode whereas compound 10 shows both alternatives. Additionally, the
sulfur atom in the ortho position on the five membered ring can
favourably interact with the protein's subsite 4 (remote
hydrophobic pocket). The activity data correlate to a very high
degree with the presence of a particular binding mode (Table 28).
Obviously, not only the degree of ring substitution but also ring
planarity might contribute to the formation of a particular binding
mode. TABLE-US-00002 TABLE 28 Relation of inhibitor binding mode
and degree of ring substitutions. Structures of the compounds are
shown above. Compound Brequinar-like Non-Brequinar-like 9 X 10 X
X
[0093] From the discussion above several possibilities for further
synthesis of compounds emerge. First, one could try to stabilize
the Brequinar-like conformation by a more elaborate variation of
substitution patterns at the aromatic ring system. A second way to
improve on the affinity might comprise the addition of a second
functional group, which is able to form hydrogen bonds or salt
bridges to the five membered ring opposite to the position of the
carboxy group. Thus the molecule should be able to address both
anion subsites and utilize brequinar-like as well as
non-brequinar-like binding modes at the same time. This is highly
supported by the evidence of structural data. Mobility at the site
of Gln47 and Arg136 indicates that the protein should be able to
exhibit sufficient conformational flexibility to adopt ligand
molecules displaying more demanding sterical requirements.
[0094] Another interesting finding is that the DHODH binding pocket
is able to selectively discriminate between enantiomeres. Compounds
5 and 6 were synthesized as a racemic mixtures caused by the
presence of a stereo centre at the five membered ring (see above).
The racemic mixtures were used for crystallization experiments. In
both cases the refined structures unequivocally showed the
inhibitor bound in its R-form. It is not possible to fit the
S-enantiomer into the electron density.
[0095] The invention further provides a method of designing a
compound which is a potential inhibitor of DHODH. The method
includes the steps of (1) identifying one or more functional groups
capable of interacting with one or more subsites of the ubiquinone
binding site of DHODH; and (2) identifying a scaffold which
presents the functional group or functional groups identified in
step 1 in a suitable orientation for interacting with one or more
subsites of the ubiquinone binding site of DHODH. The compound
which results from attachment of the identified functional groups
or moieties to the identified scaffold is a potential inhibitor of
DHODH. The DHODH ubiquinone binding site is, generally, defined by
the atomic coordinates of a polypeptide comprising the DHODH
ubiquinone binding site.
[0096] The present invention also provides several advantages. For
example, the invention provides a new three dimensional structure
of a crystalline polypeptide comprising the ubiquinone binding site
of DHODH complexed with the ligands. This structure enables the
rational development of inhibitors of DHODH by permitting the
design and/or identification of molecular structures having
features which facilitate binding to the ubiquinone binding site of
DHODH. The methods of use of this structure disclosed herein, thus,
permit more rapid discovery of compounds which are potentially
useful for the treatment of conditions which are mediated, at least
in part, by DHODH activity.
[0097] The polypeptide preferably comprises the ubiquinone binding
site of a mammalian DHODH. More preferably the polypeptide
comprises the ubiquinone binding site of human DHODH. In a
preferred embodiment, the polypeptide is a polypeptide of the
present invention, as described above.
[0098] The polypeptide can be crystallized using methods known in
the art, such as the methods described in Structure, 2000, Vol. 8,
No. 1, pages 25-33, to afford polypeptide crystals which are
suitable for x-ray diffraction studies. A crystalline
polypeptide/ligand complex can be produced by co-crystallizing the
polypeptide with a solution including the ligand.
[0099] The atomic coordinates of the polypeptide and the ligand can
be determined, for example, by x-ray crystallography using methods
known in the art. The data obtained from the crystallography can be
used to generate atomic coordinates, for example, of the
polypeptide and ligand, if present. As is known in the art,
solution and refinement of the x-ray crystal structure can result
in the determination of coordinates for some or all of the
non-hydrogen atoms.
[0100] The atomic coordinates of the polypeptide can be used, as is
known in the art, to generate a three-dimensional structure of the
ubiquinone binding site of DHODH. This structure can then be used
to assess the ability of any given compound, preferably using
computer-based methods, to fit into the ubiquinone binding
site.
[0101] The atomic coordinates of the polypeptide/ligand complex can
be used, as is known in the art, to generate a three-dimensional
structure of the ligand in its binding conformation. This structure
can then be used to assess the ability of any given compound,
preferably using computer-based methods, to exhibit a similar
spatial orientation and electrostatic and/or van der Waals
interactions as the ligand and therefore, to fit into the addressed
binding site.
[0102] A compound fits into the ubiquinone binding site if it is of
suitable size and shape to physically reside in the ubiquinone
binding site, that is if it has a shape which is complementary to
the ubiquinone binding site and can reside in the ubiquinone
binding site without significant unfavorable sterical or van der
Waals interactions. Preferably, the compound includes one or more
functional groups and/or moieties which interact with one or more
subsites within the ubiquinone binding site. Computational methods
for evaluating the ability of a compound to fit into the ubiquinone
binding site, as defined by the atomic coordinates of the
polypeptide, are known in the art, and representative examples are
provided below.
[0103] In another embodiment, the method of identifying a potential
inhibitor of DHODH comprises the step of determining the ability of
one or more functional groups and/or moieties of the compound, when
present in the DHODH ubiquinone binding site, to interact with one
or more subsites of the DHODH ubiquinone binding site. Preferably,
the DHODH ubiquinone binding site is defined by the atomic
coordinates of a polypeptide comprising the DHODH ubiquinone
binding site. If the compound is able to interact with a
preselected number of subsites, the compound is identified as a
potential inhibitor of DHODH.
[0104] In yet another embodiment, the method of identifying a
potential inhibitor of DHODH comprises the steps of (1) identifying
the size and shape of the ligand co-crystallized in the
polypeptide/ligand complex and/or identifying functional groups or
moieties of the ligand which are capable to form stabilizing
interactions with the polypeptide, and (2) by comparison with
these, identifying one or more functional groups and/or moieties of
any given compound which have similar size and shape as the
cocrystallized ligand and/or are capable to form one or more
interactions to the polypeptide in a similar manner as the
co-crystallized ligand. If a compound exhibits one or more of these
features, the compound is identified as a potential inhibitor of
DHODH.
[0105] A functional group or moiety of the compound is said to
"interact" with a subsite of the DHODH ubiquinone binding site if
it participates in an energetically favourable, or stabilizing,
interaction with one or more complementary moieties within the
subsite, as defined above.
[0106] A functional group or moiety of the compound is said to
interact in a "similar" manner as the co-crystallized ligand if one
or more, preferably two or more of its functional groups or
moieties capable of forming the attractive interactions mentioned
above can be superimposed on those functional groups or moieties of
the co-crystallized ligand capable of forming the attractive
interactions. The superposition can be performed based on the
identity of atoms, and/or the identity or similarity of functional
groups, and/or the similarity of molecular shape and/or the
identity or similarity of interaction possibilities. For example,
an --OH group of a compound and an --NH group of the cocrystallized
ligand may interact in the same way, namely as hydrogen bond
donors, with a hydrogen bond acceptor atom suitably positioned in
the enzyme. Therefore, the --OH group and the --NH group are said
to have similar interaction properties, and a molecule containing
an --OH group may be superimposed onto a molecule carrying an --NH
group at the corresponding position.
[0107] Typically, the assessment of interactions between (1) the
test compound and the DHODH ubiquinone binding site and (2) the
superposition of a test compound and the co-crystallized ligand
employ computer-based computational methods, such as those known in
the art, in which, for the first case, possible interactions of a
compound with the protein, as defined by atomic coordinates, are
evaluated with respect to interaction strength by calculating the
interaction energy upon binding the compound to the protein. For
the second case, the superposition of a test compound and the
cocrystallized ligand is performed according to the identity of
atoms, and/or the identity or similarity of functional groups,
and/or the similarity of molecular shape and/or the identity or
similarity of interaction possibilities in a process termed
alignment. Matching atoms/functional groups/shape/interaction
possibilities are evaluated and summarized to an alignment score
enabling the ranking of the tested molecules.
[0108] Compounds which have calculated interaction energies within
a preselected range or which otherwise, in the opinion of the
computational chemist employing the method, have the greatest
potential as DHODH inhibitors, can then be provided, for example,
from a compound library or via synthesis, and assayed for the
ability to inhibit DHODH. The interaction energy for a given
compound generally depends upon the ability of the compound to
interact with one or more subsites within the protein catalytic
domain.
[0109] In one embodiment, the atomic coordinates used in the method
are the atomic coordinates set forth in FIGS. 2, 3 and 4. It is to
be understood that the coordinates set forth in FIGS. 2, 3 and 4
can be transformed, for example, into a different coordinate
system, in ways known to those of skill in the art without
substantially changing the three dimensional structure represented
thereby.
[0110] In certain cases a moiety of the compound can interact with
a subsite via two or more individual interactions. A moiety of the
compound and a subsite can interact if they have complementary
properties and are positioned in sufficient proximity and in a
suitable orientation for a stabilizing interaction to occur. The
possible range of distances for the moiety of the compound and the
subsite depends upon the distance dependence of the interaction, as
known in the art. For example, a hydrogen bond typically occurs
when a hydrogen bond donor atom, which bears a hydrogen atom, and a
hydrogen bond acceptor atom are separated by about 2.5 .ANG. and
about 3.5 .ANG.. Hydrogen bonds are well known in the art.
Generally, the overall interaction, or binding, between the
compound and the ubiquinone binding site will depend upon the
number and strength of these individual interactions.
[0111] The ability of a test compound to interact with one or more
subsites of the ubiquinone binding site can be determined by
computationally evaluating interactions between functional groups,
or moieties, of the test compound and one or more amino acid side
chains and/or backbone atoms in the ubiquinone binding site.
Typically, a compound which is capable of participating in
stabilizing interactions with a preselected number of subsites,
preferably without simultaneously participating in significant
destabilizing interactions, is identified as a potential inhibitor
of DHODH. Such a compound will interact with one or more subsites,
preferably with two or more subsites and, more preferably, with
three or more subsites.
[0112] The invention further provides methods of designing a
compound which is a potential inhibitor of DHODH.
[0113] The first method includes the steps of (1) identifying one
or more functional groups capable of interacting with one or more
subsites of the DHODH ubiquinone binding site; and (2) identifying
a scaffold which presents the functional group or functional groups
identified in step 1 in a suitable orientation for interacting with
one or more subsites of the DHODH ubiquinone binding site. The
compound which results from attachment of the identified functional
groups or moieties to the identified scaffold is a potential
inhibitor of DHODH. The DHODH ubiquinone binding site is,
generally, defined by the atomic coordinates of a polypeptide
comprising the DHODH ubiquinone binding site, for example, the
atomic coordinates set forth in FIGS. 2, 3 and 4.
[0114] The second method comprises the steps of (1) identifying one
or more functional groups or moieties capable of interacting in a
similar way as one or more functional groups or moieties of the
co-crystallized ligand, and (2) identifying a scaffold which
presents the functional group or functional groups identified in
step 1 in a suitable orientation for interacting in a similar way
as one or more functional groups or moieties of the co-crystallized
ligand. The compound which results from attachment of the
identified functional groups or moieties to the identified scaffold
is a potential inhibitor of DHODH. The co-crystallized ligand is,
generally, defined by the atomic coordinates of a ligand complexed
in the polypeptide comprising the DHODH ubiquinone binding site,
for example, the atomic coordinates set forth in FIGS. 2, 3 and
4.
[0115] Suitable methods, as known in the art, can be used to
identify chemical moieties, fragments or functional groups which
are capable of interacting favorably with a particular subsite or
sets of subsites. These methods include, but are not limited to:
interactive molecular graphics; molecular mechanics; conformational
analysis; energy evaluation; docking; database searching; virtual
high-throughput screening (U.S. Pat. No. 4,223,03, DE 10009479, EP
1094415, U.S. Pat. No. 6,937,31, U.S. Pat. No. 8,858,93, U.S. Pat.
No. 8,855,17); structural alignment; functional group alignment;
interaction-point alignment; pharmacophore modeling; de novo
design; property estimation and descriptor-based database
searching. These methods can also be employed to assemble chemical
moieties, fragments or functional groups into a single inhibitor
molecule. These same methods can also be used to determine whether
a given chemical moiety, fragment or functional group is able to
interact favorably with a particular subsite or sets of
subsites.
[0116] In one embodiment, the design of potential DHODH inhibitors
begins from the general perspective of three-dimensional shape and
electrostatic complementarity for the ubiquinone binding site, and
subsequently, interactive molecular modeling techniques can be
applied by one skilled in the art to visually inspect the quality
of the fit of a candidate molecule into the binding site. Suitable
visualization programs include SYBYL (Tripos Inc., St. Louis, Mo.),
MOLOC (Gerber Molecular Design, Basel), RASMOL (Sayle et al. Trends
Biochem. Sci. 20:374-376 (1995)) and MOE (Chemical Computing Group
Inc., Montreal).
[0117] A further embodiment of the present invention utilizes a
database searching program which is capable of scanning a database
of small molecules of known three-dimensional structure for
candidates which fit into the target protein site. Suitable
software programs include 4SCan.RTM. (U.S. Pat. No. 4,223,03, DE
10009479, EP 1094415, U.S. Pat. No. 6,937,31, U.S. Pat. No.
8,858,93, U.S. Pat. No. 8,855,17), FLEXX (Rarey et al., J. Mol.
Biol. 261:470-489 (1996)), and UNITY (Tripos Inc., St. Louis, Mo.).
Especially 4SCan.RTM. was developed to scan/screen large virtual
databases up to several millions of small molecules in a reasonable
time-frame.
[0118] A further embodiment of the present invention utilizes a
database searching program which is capable of scanning a database
of small molecules of known three-dimensional structure for
candidates which align properly with the co-crystallized ligand,
both in shape and interaction properties. Suitable software
programs include 4SCan.RTM. (U.S. Pat. No. 4,223,03, DE 10009479,
EP 1094415, U.S. Pat. No. 6,937,31, U.S. Pat. No. 8,858,93, U.S.
Pat. No. 8,855,17) and FLEXS (Lemmen et al., J. Med. Chem.
41:4502-4520 (1998)). Especially 4SCan.RTM. is capable of aligning
large virtual databases up to several millions of small molecules
in a reasonable time-frame.
[0119] It is not expected that the molecules found in the search
will necessarily be leads themselves, since a complete evaluation
of all interactions will necessarily be made during the initial
search. Rather, it is anticipated that such candidates might act as
the framework for further design, providing molecular skeletons to
which appropriate atomic replacements can be made. Of course, the
chemical complementarity of these molecules can be evaluated, but
it is expected that the scaffold, functional groups, linkers and/or
monomers may be changed to maximize the electrostatic, hydrogen
bonding, and hydrophobic interactions with the enzyme.
[0120] Goodford (Goodford J. Med. Chem. 28:849-857 (1985)) has
produced a computer program, GRID, which seeks to determine regions
of high affinity for different chemical groups (termed probes) on
the molecular surface of the binding site. GRID hence provides a
tool for suggesting modifications to known ligands that might
enhance binding.
[0121] Consequently, virtual combinatorial libraries covering
numerous variations of the addressed scaffold, functional groups,
linkers and/or monomers can be build up using suitable software
programs including LEGION (Tripos Inc., St. Louis, Mo.) or ACCORD
FOR EXCEL (Accelrys Inc., San Diego, Calif.), followed by scanning
or virtual screening or docking of these libraries using suitable
software mentioned above.
[0122] A range of factors, including electrostatic interactions,
hydrogen bonding, hydrophobic interactions, desolvation effects,
conformational strain, ligand flexibility and cooperative motions
of ligand and enzyme, all influence the binding effect and should
be taken into account in attempts to design bioactive
inhibitors.
[0123] Yet another embodiment of a computer-assisted molecular
design method for identifying inhibitors of DHODH comprises
searching for fragments which fit into a binding region subsite and
link to a pre-defined scaffold. The scaffold itself may be
identified in such a manner. A representative program suitable for
the searching of such functional groups and monomers include LUDI
(Boehm, J. Comp. Aid. Mol. Des. 6:61-78 (1992)) and MCSS (Miranker
et al., Proteins 11: 314-328 (1991)).
[0124] Yet another embodiment of a computer-assisted molecular
design method for identifying inhibitors of DHODH comprises the de
novo synthesis of potential inhibitors by algorithmic connection of
small molecular fragments that will exhibit the desired structural
and electrostatic complementarity with the active site of the
enzyme. The methodology employs a large template set of small
molecules which are iteratively pierced together in a model of the
DHODH ubiquinone binding site. Programs suitable for this task
include GROW (Moon et al. Proteins 11:314-328 (1991)) and SPROUT
(Gillet et al. J. Comp. Aid. Mol. Des. 7:127 (1993)).
[0125] In yet another embodiment, the suitability of inhibitor
candidates can be determined using an empirical scoring function,
which can rank the binding affinities for a set of inhibitors. For
examples of such a method see Muegge et al. and references therein
(Muegge et al., J. Med. Chem. 42:791-804 (1999)) and ScoreDock (Tao
et al. J. Comp. Aid. Mol. Des. 15: 429-446 (2001)).
[0126] Other modeling techniques can be used in accordance with
this invention, for example, those described by Stahl (Stahl, in:
Virtual Screening for Bioactive Molecules, Wiley-VCH, Weinheim,
2000, pp. 229-264), Cohen et al. (J. Med. Chem. 33:883-894 (1990));
Navia et al. (Current Opinions in Structural Biology 2:202-210
(1992)); Baldwin et al. (J. Med. Chem. 32:2510-2513 (1989)); Appelt
et al. (J. Med. Chem. 34:1925-1934 (1991)); Ealick et al. (Proc.
Nat. Acad. Sci. USA 88:11540-11544 (1991));
[0127] A compound which is identified by one of the foregoing
methods as a potential inhibitor of DHODH can then be obtained, for
example, by synthesis or from a compound library, and assessed for
the ability to inhibit DHODH in vitro. Such an in vitro assay can
be performed as is known in the art, for example, by contacting
DHODH in solution with the test compound in the presence of the
substrate and cofactor of DHODH and ubiquinone. The rate of
substrate transformation can be determined in the presence of the
test compound and compared with the rate in the absence of the test
compound. Suitable assays for DHODH biological activity are
described below, the teachings of each of which are hereby
incorporated by reference herein in their entity.
[0128] An inhibitor identified or designed by a method of the
present invention can be a competitive inhibitor, an uncompetitive
inhibitor or a noncompetitive inhibitor with respect to
ubiquinone.
[0129] A screen of thousands of compounds using 4Scan as described
above was performed. Hits were ranked according to consensus
score.
[0130] In table 25 the structures of the highest ranking compounds
of the combinatorial library are shown. The consensus score of each
molecule is calculated by the summation of the two predicted
4SCan.RTM. activity scores for the two different structures of the
ubiquinone binding site.
[0131] The compounds of the present invention can be used for a
variety of human and animal diseases, preferably human diseases,
where inhibition of the pyrimidine metabolism is beneficial. Such
diseases are:
[0132] fibrosis, uveitis, rhinitis, asthma or arthropathy, in
particular, arthrosis
[0133] all forms of rheumatism
[0134] acute immunological events and disorders such as sepsis,
septic shock, endotoxic shock, Gram-negative sepsis, toxic shock
syndrome, acute respiratory distress syndrome, stroke, reperfusion
injury, CNS injury, serious forms of allergy, graft versus host and
host versus graft reactions, alzheimer's disease or pyresis,
restenosis, chronic pulmonary inflammatory disease, silicosis,
pulmonary sarcosis, bone resorption disease. These immunological
events also include a desired modulation and suppression of the
immune system;
[0135] all types of autoimmune diseases, in particular rheumatoid
arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis,
multiple sclerosis, insulin dependent diabetes mellitus and
non-insulin dependent diabetes mellitus, and lupus erythematoidis,
ulcerative colitis, Morbus Crohn, inflammatory bowel disease, as
well as other chronic inflammations, chronic diarrhea;
[0136] dermatological disorders such as psoriasis
[0137] progressive retinal atrophy
[0138] all kinds of infections including opportunistic
infections.
[0139] The compounds according to the invention and medicaments
prepared therewith are generally useful for the treatment of cell
proliferation disorders, for the treatment or prophylaxis,
immunological diseases and conditions (as for instance inflammatory
diseases, neuroimmunological diseases, autoimmune diseases or
other).
[0140] The compounds of the present invention are also useful for
the development of immunomodulatory and anti-inflammatory
medicaments or, more generally, for the treatment of diseases where
the inhibition of the pyrimidine biosynthesis is beneficial.
[0141] The compounds of the present invention are also useful for
the treatment of diseases which are caused by malignant cell
proliferation, such as all forms of hematological and solid cancer.
Therefore the compounds according to the invention and medicaments
prepared therewith are generally useful for regulating cell
activation, cell proliferation, cell survival, cell
differentiation, cell cycle, cell maturation and cell death or to
induce systemic changes in metabolism such as changes in sugar,
lipid or protein metabolism. They can also be used to support cell
generation poiesis, including blood cell growth and generation
(prohematopoietic effect) after depletion or destruction of cells,
as caused by, for example, toxic agents, radiation, immunotherapy,
growth defects, malnutrition, malabsorption, immune dysregulation,
anemia and the like or to provide a therapeutic control of tissue
generation and degradation, and therapeutic modification of cell
and tissue maintenance and blood cell homeostasis.
[0142] These diseases and conditions include but are not limited to
cancer as hematological (e.g. leukemia, lymphoma, myeloma) or solid
tumors (for example breast, prostate, liver, bladder, lung,
esophageal, stomach, colorectal, genitourinary, gastrointestinal,
skin, pancreatic, brain, uterine, colon, head and neck, ovarian,
melanoma, astrocytoma, small cell lung cancer, glioma, basal and
squameous cell carcinoma, sarcomas as Kaposi's sarcoma and
osteosarcoma), treatment of disorders involving T-cells such as
aplastic anemia and DiGeorge syndrome, Graves' disease.
[0143] Leflunomide was previously found to inhibit HCMV replication
in cell culture. Ocular herpes is the most common cause of
infectious blindness in the developed world. There are about 50.000
cases per year in the US alone, of which 90% are recurrences of
initial infections. Recurrences are treated with antivirals and
corticosteroids. Cytomegalovirus, another herpes virus, is a common
cause of retinal damage and blindness in patients with aids. The
compounds of the present invention can be used alone or in
combination with other antiviral compounds such as ganciclovir and
foscarnet to treat such diseases.
[0144] The compounds of the present invention can further be used
for diseases that are caused by protozoal infestations in humans
and animals. Such veterinary and human pathogenic protozoas are
preferably intracellular active parasites of the phylum Apicomplexa
or Sarcomastigophora, especially Trypanosoma, Plasmodia,
Leishmania, Babesia and Theileria, Cryptosporidia, Sacrocystida,
Amoebia, Coccidia and Trichomonadia. These active substances or
corresponding drugs are especially suitable for the treatment of
Malaria tropica, caused by Plasmodium falciparum, Malaria tertiana,
caused by Plasmodium vivax or Plasmodium ovale and for the
treatment of Malaria quartana, caused by Plasmodium malariae. They
are also suitable for the treatment of Toxoplasmosis, caused by
Toxoplasma gondii, Coccidiosis, caused for instance by Isospora
belli, intestinal Sarcosporidiosis, caused by Sarcocystis
suihominis, dysentery caused by Entamoeba histolytica,
Cryptosporidiosis, caused by Cryptosporidium parvum, Chargas'
disease, caused by Trypanosoma cruzi, sleeping sickness, caused by
Trypanosoma brucei rhodesiense or gambiense, the cutaneous and
visceral as well as other forms of Leishmaniosis. They are also
suitable for the treatment of animals infected by veterinary
pathogenic protozoa, like Theileria parva, the pathogen causing
bovine East coast fever, Trypanosoma congolense congolense or
Trypanosoma vivax vivax, Trypanosoma brucei brucei, pathogens
causing Nagana cattle disease in Africa, Trypanosoma brucei evansi
causing Surra, Babesia bigemina, the pathogen causing Texas fever
in cattle and buffalos, Babesia bovis, the pathogen causing
european bovine Babesiosis as well as Babesiosis in dogs, cats and
sheep, Sarcocystis ovicanis and ovifelis pathogens causing
Sarcocystiosis in sheep, cattle and pigs, Cryptosporidia, pathogens
causing Cryptosporidioses in cattle and birds, Eimeria and Isospora
species, pathogens causing Coccidiosis in rabbits, cattle, sheep,
goats, pigs and birds, especially in chickens and turkeys. The use
of the compounds of the present invention is preferred in
particular for the treatment of Coccidiosis or Malaria infections,
or for the preparation of a drug or feed stuff for the treatment of
these diseases. This treatment can be prophylactic or curative. In
the treatment of malaria, the compounds of the present invention
may be combined with other anti-malaria agents.
[0145] The compounds of the present invention can further be used
for viral infections or other infections caused for instance by
Pneumocystis carinii.
EXAMPLES
[0146] 1. X-Ray Structure Determination
Expression and Purification
[0147] The cDNA encoding for an N-terminally truncated human
DHODH(Met30-Arg396) was amplified by the polymerase chain reaction
(PCR) from a human liver cDNA bank (Invitrogen, Groningen). The
following primers were used to amplify the DHODH gene form the cDNA
bank: TABLE-US-00003 DHODH-V: 5'-GGA ATT CCA TAT GGC CAC GGG AGA
TGA GCG-3' DHODH-R: 5'-GCG CGG ATC CTC ACC TCC GAT GAT CTG C-3'
[0148] The underlined sequence regions encode for the cutting sites
of the restriction enzymes NdeI (DHODH-V) and BamHI (DHODH-R),
respectively. The primers are designed such that subcloning using
the NdeI and BamHI restriction sites into a pET-19b vector is
possible. The amplified DNA bands were purified and isolated from
an agarose gel (QIAquick PCR purification kit). The band showed the
expected length of 1.2 kb. The isolated PCR fragment was subcloned
into a TOPO vector (Invitrogen, Groningen) according to the
protocol outlined in the TOPT TA Cloning Kit. The TOPO vector
including the ligated PCR fragment was digested with the
restriction enzymes NdeI and BamHI (New England Biolabs Inc.) to
produce sticky ends. Finally, the fragment was cloned into the
NdeI/BamHI sites of a pET-19b vector (Novagen, Madison, Wis.). This
vector produced the human DHODH(Met30-Arg396) as an N-terminal ten
histidine fusion protein (his10-hDHODH(Met30-Arg396)). The vector
was transformed into chemical competent E. coli BL21(DE3) Gold
cells (Stratagene, LaJolla, Calif.). Cells were stored as glycerol
stocks at -80.degree. C. until further use.
[0149] 100 ml LB-medium in 250 ml flasks containing 100 .mu.L
freshly prepared ampicilline were inoculated with BL21(DE3) Gold
cells hosting the pET-19b/hDHODH(Met30-Arg396) construct. Cells
were grown overnight at 25.degree. C. and constantly vortexed with
150 rpm.
[0150] For the expression cultures four 2 L flasks each were filled
with 800 mL rich medium (LB) containing 800 .mu.L ampicilline. The
flasks were inoculated with 40 mL of overnight culture and were
grown to an optical density O.D..sub.600 of 0.6-0.8 at 25.degree.
C. The cells were induced with 80 .mu.L of a 1 M
isopropyl-.beta.-D-thiogalactoside (IPTG) stock solution and grown
for another 20 h at 25.degree. C.
[0151] The cells were harvested by centrifugation for 15 min in a
JA-10 Beckmann rotor at 5000 rpm at 4.degree. C. The cell pellet
was stored until further use at -20.degree. C.
[0152] The pellets of 4.times.800 mL expression were thawed on ice
and resuspended in 100 mL lysisbuffer containing 50 mM HEPES at pH
7.7, 300 mM NaCl, 10% glycerol, 10% bugbuster (Novagen, 10x), two
tablets of protease-inhibitor mix (Complete Tabletes EDTA-free,
Roche) and 1% triton X-100. The cell suspension was incubated under
gentle rocking for 20 min at room temperature.
[0153] Cell lysis was performed via ultra sonification using a
Branson sonotrode. The chosen parameters for sonification were the
following: TABLE-US-00004 amplitude: 60% duration: 3 .times. 3 min
maximal allowed temperature: 37.degree. C. pulse duration: 0.5 sec
duty cycle: 0.1 sec
[0154] The resulting suspension was centrifuged in a JA-25.50 rotor
(Beckmann) at 25.000 rpm for 1 hour at 4.degree. C.
[0155] The supernatant was loaded onto a Ni-NTA-column (resin was
from Quiagen, column adapter from Pharmacia). The column had a bed
volume of 3 mL and was equilibrated with 5 column volumes (CV) of
starting buffer (50 mM HEPES pH 7.7; 300 mM NaCl; 10% glycerol and
10 mM imidazole). The sample was loaded with a flow rate of 1
mL/min at 4.degree. C. using a BioRad Econopump. Then the column
was mounted on a BioRad BioLogic-LP chromatography system and
washed with 5-10 CVs of 50 mM HEPES pH 7.7, 300 mM NaCl, 10%
glycerol, 10 mM imidazole and 10 mM N,N-dimethylundecylamin-N-oxide
(C11DAO) at a rate of 1 mL/min. Another more stringent washing step
was performed by applying step gradients consisting of the above
washing buffer containing 20 mM and 50 mM imidazole, respectively.
At this point, pure DHODH was eluted with 50 mM HEPES pH 7.7, 300
mM NaCl, 10% glycerol, 200 mM imidazole and 10 mM
N,N-dimethylundecylamin-N-oxide. Elution was carried out with a
flow rate of 0.5 mL/min and the eluate was collected in 4 mL
fractions. Fractions containing hDHODH(Met30-Arg396) are
characterized by a bright yellow colour and showed full activity in
an in vitro assay (as described above/below).
[0156] Fractions containing hDHODH were combined (approx. 10 mL)
and dialysed against 3 L of buffer containing 50 mM HEPES pH 7.7,
400 mM NaCl, 30% glycerol, 1 mM EDTA and 10 mM
N,N-dimethylundecylamin-N-oxide overnight at 4.degree. C. The
dialysed protein sample was concentrated to a final concentration
of 20 mg/mL using an Ultrafree 4/YM-30 device from Millipore.
During the concentrating procedure the temperature was kept at
4.degree. C. The protein concentration was determined
spectrometrically. The His-tag was not removed for further
studies.
[0157] Finally, aliquots of 50 .mu.L were flash frozen in liquid
nitrogen and stored at -80.degree. C. until further use.
Crystallization and Data Collection
[0158] Human his10-hDHODH(Met30-Arg396) was co-crystallized with
compound 1 and compound 2 at 20.degree. C. using the hanging-drop
vapour diffusion method. Drops were formed by mixing equal amounts
of 20 mg/ml protein in 50 mM HEPES pH 7.7, 400 mM NaCl, 30%
glycerol, 1 mM EDTA and 10 mM N,N-dimethylundecylamin-N-oxide
(C11DAO) with a precipitant solution of 0.1 M acetate pH 4.6-5.0,
40 mM C11DAO, 20.8 mM N,N-dimethyldecylamine-N-oxide (DDAO), 2 mM
dihydroorotate (DHO), 1.8-2.4 M ammonium sulfate, 1 mM compound 1
or 2. The hanging drops were incubated against 0.5 mL reservoir of
0.1 M acetate pH 4.8, 2.4-2.6 M ammonium sulfate and 30% glycerol.
The crystallization conditions were screened by variation of pH
versus ammonium sulfate concentration using a small grid screen
(see FIG. 5):
[0159] The same procedure was applied to obtain single crystals of
DHODH(Met30-Arg396) in complex with compounds 3, 4, 5, 6, 7, 8, 9
and 10. Compounds 5 and 6 were synthesized as racemic mixtures due
to the presence of a stereo center at the five membered ring. The
racemic mixtures were used for crystallization experiments.
[0160] Crystals usually appeared as small cubes within three days.
They usually reached a full size of 0.2.times.0.2.times.0.2 mm
within three to four weeks. The protein crystallized in the space
group P3.sub.221. Crystals were harvested with pre-mounted loops of
size 0.5 mm (Hampton Research) and were flash frozen directly in
the cryo stream of the measurement device.
[0161] Data were collected at the beamline BW6 at the DESY Hamburg
on a MAR-CCD camera. A total of 120 frames (0.5.degree. each) were
collected from a human DHODH(Met30-Arg396) crystal co-crystallized
with compound 1. For the crystal cocrystallized with compound 2 a
total of 85 frames (1.degree. each) was recorded. The crystals were
maintained at a temperature of 100 K during data collection. The
indexing and integration of the reflection intensities were
performed with the program MOSFLM (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Data were
scaled and merged with SCALA and reduced to structure factor
amplitudes with TRUNCATE, both from the CCP4 program suite
(Collaborative Computational Project, Number 4 (1994). Acta Cryst.
D50, 760-763.). At this stage 5% and 10% (the "test set") of unique
reflections were flagged for cross validation to calculate the free
R-factor (R.sub.free) during the refinement process later on for
compound 1 and compound 2, respectively. The remaining 95% and 90%
of the reflections constituted the "working set" for calculation of
the R-factor (R), respectively. The statistics of data collection
are shown in table 1 and table 2. TABLE-US-00005 TABLE 1 Crystal
& Data collection statistics for compound 1 A. Crystal data
Spacegroup P3.sub.221 Cell dimensions (.ANG.) a = 90.69 b = 90.69 c
= 123.22 Molecules/asymmetric unit 1 Matthews' constant
(V.sub.m)(.ANG..sup.3/Da) 4.1 Maximum resolution (.ANG.) 2.35 B.
Data Collection X-Ray source DESY BW6 Wavelength (.ANG.) 1.05
Total/unique reflections 91431/24977 Completeness (%) 98.2 (99.0)
I/sigma 23.9 (6.5) R.sub.merge (%) 5.7 (20.2)
[0162] TABLE-US-00006 TABLE 2 Crystal & Data collection
statistics for compound 2 A. Crystal data Spacegroup P3.sub.221
Cell dimensions (.ANG.) a = 90.65 b = 90.65 c = 123.07
Molecules/asymmetric unit 1 Matthews' constant
(V.sub.m)(.ANG..sup.3/Da) 4.1 Maximum resolution (.ANG.) 2.4 B.
Data Collection X-Ray source DESY BW6 Wavelength (.ANG.) 1.05
Total/unique reflections 101935/22253 Completeness (%) 95.8 (97.1)
I/sigma 14.6 (3.8) R.sub.merge (%) 9.1 (38.1)
[0163] Datasets for the crystals of human DHODH(Met30-Arg396)
co-crystallized with compounds 3, 4, 6, 7, 8, 9 and 10 were also
collected at the beamline BW6 at the DESY Hamburg on a MAR-CCD
camera. Co-crystals with compound 5 were recorded at an in house
generator using CuK.alpha. radiation and a MAR-dtb image plate.
[0164] A total of 55 frames, 65 frames, 96 frames, 62 frames, 120
frames, 60 frames, 100 frames and 100 frames (1.degree. each) were
collected from human DHODH(Met30-Arg396) crystals co-crystallized
with compound 3, 4, 5, 6, 7, 8, 9 and 10 respectively. The crystals
were maintained at a temperature of 100 K during data collection.
The indexing and integration of the reflection intensities were
performed with the program MOSFLM (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Data were
scaled and merged with SCALA and reduced to structure factor
amplitudes with TRUNCATE, both from the CCP4 program suite
(Collaborative Computational Project, Number 4 (1994). Acta Cryst.
D50, 760-763.). At this stage 5% or 10% (the "test set") of unique
reflections were flagged for cross validation to calculate the free
R-factor (R.sub.free) during the refinement process. The remaining
95% or 90% of the reflections constituted the "working set" for
calculation of the R-factor (R), respectively. The statistics of
data collection are shown in tables 5 to 12, respectively.
TABLE-US-00007 TABLE 5 Crystal & Data collection statistics for
compound 3 A. Crystal data Spacegroup P3.sub.221 Cell dimensions
(.ANG.) a = 90.43 b = 90.43 c = 123.00 Molecules/asymmetric unit 1
Matthews' constant (V.sub.m)(.ANG..sup.3/Da) 4.1 Maximum resolution
(.ANG.) 1.95 B. Data Collection X-Ray source DESY BW6 Wavelength
(.ANG.) 1.05 Total/unique reflections 142628/42908 Completeness (%)
99.8/99.9 I/sigma 12.6/3.4 R.sub.merge (%) 8.2/38.3
[0165] TABLE-US-00008 TABLE 6 Crystal & Data collection
statistics for compound 4 A. Crystal data Spacegroup P3.sub.221
Cell dimensions (.ANG.) a = 90.65 b = 90.65 c = 123.21
Molecules/asymmetric unit 1 Matthews' constant
(V.sub.m)(.ANG..sup.3/Da) 4.1 Maximum resolution (.ANG.) 2.15 B.
Data Collection X-Ray source DESY BW6 Wavelength (.ANG.) 1.05
Total/unique reflections 124056/32175 Completeness (%) 99.2/99.0
I/sigma 14.7/5.7 R.sub.merge (%) 7.1/24.8
[0166] TABLE-US-00009 TABLE 7 Crystal & Data collection
statistics for compound 5 A. Crystal data Spacegroup P3.sub.221
Cell dimensions (.ANG.) a = 90.30 b = 90.30 c = 123.09
Molecules/asymmetric unit 1 Matthews' constant
(V.sub.m)(.ANG..sup.3/Da) 4.1 Maximum resolution (.ANG.) 2.2 B.
Data Collection X-Ray source CuK.alpha. Wavelength (.ANG.) 1.54
Total/unique reflections 171127/30057 Completeness (%) 99.9 (99.9)
I/sigma 4.0/1.9 R.sub.merge (%) 15.4/43.5
[0167] TABLE-US-00010 TABLE 8 Crystal & Data collection
statistics for compound 6 A. Crystal data Spacegroup P3.sub.221
Cell dimensions (.ANG.) a = 90.44 b = 90.44 c = 123.20
Molecules/asymmetric unit 1 Matthews' constant
(V.sub.m)(.ANG..sup.3/Da) 4.1 Maximum resolution (.ANG.) 1.9 B.
Data Collection X-Ray source DESY BW 6 Wavelength (.ANG.) 1.05
Total/unique reflections 173775/46257 Completeness (%) 99.4/99.9
I/sigma 13.8/2.8 R.sub.merge (%) 8.5/46.0
[0168] TABLE-US-00011 TABLE 9 Crystal & Data collection
statistics for compound 7 A. Crystal data Spacegroup P3.sub.221
Cell dimensions (.ANG.) a = 90.74 b = 90.74 c = 122.88
Molecules/asymmetric unit 1 Matthews' constant
(V.sub.m)(.ANG..sup.3/Da) 4.1 Maximum resolution (.ANG.) 1.9 B.
Data Collection X-Ray source DESY BW 6 Wavelength (.ANG.) 1.05
Total/unique reflections 341319/46198 Completeness (%) 98.6/99.7
I/sigma 23.5/5.1 R.sub.merge (%) 8.2/21.8
[0169] TABLE-US-00012 TABLE 10 Crystal & Data collection
statistics for compound 8 A. Crystal data Spacegroup P3.sub.221
Cell dimensions (.ANG.) a = 90.56 b = 90.56 c = 123.06
Molecules/asymmetric unit 1 Matthews' constant (V.sub.m)
(.ANG..sup.3/Da) 4.1 Maximum resolution (.ANG.) 1.8 B. Data
Collection X-Ray source DESY BW 6 Wavelength (.ANG.) 1.05
Total/unique reflections 190208/53993 Completeness (%) 98.8/96.7
I/sigma 16.7/2.9 R.sub.merge (%) 6.3/38.3
[0170] TABLE-US-00013 TABLE 11 Crystal & Data collection
statistics for compound 9 A. Crystal data Spacegroup P3.sub.221
Cell dimensions (.ANG.) a = 90.29 b = 90.29 c = 122.69
Molecules/asymmetric unit 1 Matthews' constant (V.sub.m)
(.ANG..sup.3/Da) 4.1 Maximum resolution (.ANG.) 2.0 B. Data
Collection X-Ray source DESY BW 6 Wavelength (.ANG.) 1.05
Total/unique reflections 103711/39080 Completeness (%) 98.6/99.0
I/sigma 14.1/3.9 R.sub.merge (%) 6.5/24.8
[0171] TABLE-US-00014 TABLE 12 Crystal & Data collection
statistics for compound 10 A. Crystal data Spacegroup P3.sub.221
Cell dimensions (.ANG.) a = 90.75 b = 90.75 c = 122.71
Molecules/asymmetric unit 1 Matthews' constant (V.sub.m)
(.ANG..sup.3/Da) 4.1 Maximum resolution (.ANG.) 1.8 B. Data
Collection X-Ray source DESY BW 6 Wavelength (.ANG.) 1.05
Total/unique reflections 326425/54728 Completeness (%) 99.9/100
I/sigma 27.5/6.0 R.sub.merge (%) 6.0/30.6
Structure Determination and Refinement of DHODH/Compound 1
Complex
[0172] The structure for the human DHODH(Met30-Arg396) in complex
with compound 1 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.5 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 35.6% and
a correlation coefficient of 69.4% for compound 1 complex.
[0173] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard CNX
protocols. Finally, SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of compound 1 could be interpreted
unambiguously.
[0174] A pdb file for compound 1 was created using the program MOE
(Chemical Computing Group Inc., MOE 2002.02). After energy
minimization the compound was built into the electron density
manually. Topology and parameter files for compound 1 were created
using the program Xplo2d (Uppsala Software Factory; Kleywegt, G. M.
(1997) J. Mol. Biol. 273, 371-376). After an additional round of
model building and water picking using CNX another complete round
of refinement was performed. The final model included the
DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide
(FMN), one orotate molecule (ORO), one acetate molecule (ACT), two
sulfate ions (SO4), one molecule of compound 1 (INH) and 153 water
molecules (TIP) (see FIG. 2). The model is well refined and has
very good geometry. The refinement process which included data from
12.0-2.35 .ANG. resulted in an R-factor of 18.5% and a free
R-factor of 21.7%. With the exception of glycine residues, 92.4%
(278) of the residues are located in the most favoured region of
the ramachandran plot and 7.6% (22) cluster in the additional
allowed regions. Table 13 summarizes the refinement statistics for
the inhibitor compound 1 in complex with human DHODH. Values in
parentheses give the R-factor and R.sub.free-factors, respectively,
for the last resolution bin ranging from 2.50 to 2.35.
[0175] The N-terminal His tag could not be detected in the electron
density map. TABLE-US-00015 TABLE 13 Refinement Statistics for
DHODH/compound 1 complex R-factor (%) 18.5 (19.6) R.sub.free 21.7
(24.2) RMS deviation from ideal values bond length (.ANG.) 0.006
Bond angle (.degree.) 1.2 Dihedral angles (.degree.) 21.4 Improper
angles (.degree.) 0.83
Structure Determination and Refinement of DHODH/Compound 2
Complex
[0176] The structure for the human DHODH(Met30-Arg396) in complex
with compound 2 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.5 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 33.8% and
a correlation coefficient of 68.2% for the DHODH/compound 2
complex.
[0177] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard
CNX-protocols. Finally a SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of the inhibitor compound 2 could
be interpreted unambiguously. The electron density around the
five-membered ring carrying the carboxy group clearly showed the
presence of two alternative conformations of compound 2. In one
conformation (conformation A) the carboxy group interacts with
residues Gln 47 and Arg 136, whereas in the second conformation
(conformation B) the interaction involves residues His 56 and Tyr
356 (see above). For each conformation a separate DHODH/compound 2
complex was subjected to refinement.
[0178] Pdb files for the compound 2 in conformation A and B were
created using the program MOE (Chemical Computing Group Inc., MOE
2002.02). Both compounds were energy minimized and built into the
electron density manually. Topology and parameter files for
compound 2 were created using the program Xplo2d (Uppsala Software
Factory; Kleywegt, G. M. (1997) J. Mol. Biol., 273, 371-376). After
an additional round of model building and water picking using CNX,
another complete round of refinement was performed. The final model
included the human DHODH(Met30-Arg396) protein, the cofactor
flavinmononucleotide (FMN), one orotate molecule (ORO), one acetate
molecule (ACT), four sulfate ions (SO4), one molecule of compound 2
(INH) either in conformation A or conformation B and 250 water
molecules (TIP) (see FIGS. 3 and 4). The models are well refined
and show very good geometry. The refinement process which included
data from 12.0-2.4 .ANG. resulted in an R-factor of 17.5% and a
free R-factor of 21.1% for conformation A complex and an R-factor
of 17.6% and a free R-factor of 21.6% for conformation B complex,
respectively. With the exception of glycine residues, 91.7% (276)
of the residues are located in the most favoured region of the
ramachandran plot and 8.3% (24) cluster in the additional allowed
regions. Table 14 summarizes the refinement statistics for compound
2 in complex with human DHODH. Values in parentheses give the
R-factor and R.sub.free-factors, respectively, for the last
resolution bin ranging from 2.55 to 2.40. TABLE-US-00016 TABLE 14
Refinement Statistics for DHODH/compound 2 complex Conformation A
Conformation B R-factor (%) 17.5 (19.6) 17.6 (19.4) R.sub.free 21.1
(23.6) 21.6 (23.2) RMS deviation from ideal values bond length
(.ANG.) 0.005 0.005 Bond angle (.degree.) 1.2 1.2 Dihedral angles
(.degree.) 21.3 21.3 Improper angles (.degree.) 0.81 0.81
[0179] Structure Determination and Refinement of DHODH/Compound 3
Complex
[0180] The structure for the human DHODH(Met30-Arg396) in complex
with compound 3 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.0 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 33.9% and
a correlation coefficient of 72.5 for the DHODH/compound 3
complex.
[0181] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard
CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of the inhibitor compound 3 could
be interpreted unambiguously. The electron density around the
five-membered ring carrying the carboxy group clearly showed the
presence of two alternative conformations of compound 3. In one
conformation (conformation A) the carboxy group interacts with
residues Gln 47 and Arg 136, whereas in the second conformation
(conformation B) the interaction involves residues His 56 and Tyr
356 (see above). For each conformation a separate DHODH/compound 3
complex was subjected to refinement.
[0182] The pdb files for the compound 3 in conformation A and B
were created using the program MOE (Chemical Computing Group Inc.,
MOE 2002.02). Both compounds were energy minimized and built into
the electron density manually. Topology and parameter files for
compound 3 were created using the program Xplo2d (Uppsala Software
Factory; Kleywegt, G. M. (1997) J. Mol. Biol., 273, 371-376). After
an additional round of model building and water picking using CNX,
another complete round of refinement was performed. The final model
included the human DHODH(Met30-Arg396) protein, the cofactor
flavinmononucleotide (FMN), one orotate molecule (ORO), two acetate
molecules (ACT), two sulfate ions (SO4), one molecule of compound 3
(INH) either in conformation A or conformation B and 263 water
molecules (WAT). Residues which are missing the coordinate file due
to very poor electron density are listed in the header of the pdb
files.
[0183] The models are well refined and show very good geometry. The
refinement process which included data from 19.9-1.95 .ANG.
resulted in an R-factor of 18.5% and a free R-factor of 20.3% for
the complex in conformation A and an R-factor of 18.5% and a free
R-factor of 20.3% for the complex in conformation B, respectively.
The almost identical R-factors indicate that non of the conformers
A and B represent a preferred conformation. Except for non-glycine
and non-proline residues 91.6% are located in the most favoured
region of the ramachandran plot and 8% cluster in the additional
allowed regions. There are no residues in the disallowed region.
Table 15 summarizes the refinement statistics for compound 3 in
complex with human DHODH. Values in parentheses give the R-factor
and R.sub.free-factors, respectively, for the last resolution bin
ranging from 2.07 to 1.95. TABLE-US-00017 TABLE 15 Refinement
Statistics for DHODH/compound 3 complex conformation A conformation
B R-factor (%) 18.5 (20.6) 18.5 (20.6) R.sub.free 20.3 (23.5) 20.2
(23.6) RMS deviation from ideal values bond length (.ANG.) 0.005
0.005 Bond angle (.degree.) 1.2 1.2 Dihedral angles (.degree.) 21.2
21.2 Improper angles (.degree.) 0.81 0.81
[0184] Structure Determination and Refinement of DHODH/Compound 4
Complex
[0185] The structure for the human DHODH(Met30-Arg396) in complex
with compound 4 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.0 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 34.6% and
a correlation coefficient of 71.1 for the DHODH/compound 4
complex.
[0186] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard
CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of the inhibitor compound 4 could
be interpreted unambiguously. The electron density around the
five-membered ring carrying the carboxy group clearly showed the
carboxy group in contact with residues His 56 and Tyr 356 in
non-brequinar-like conformation.
[0187] A pdb file for compound 4 was created using the program MOE
(Chemical Computing Group Inc., MOE 2002.02). After energy
minimization the compound was built into the electron density
manually. Topology and parameter files for compound 4 were created
using the program Xplo2d (Uppsala Software Factory; Kleywegt, G. M.
(1997) J. Mol. Biol. 273, 371-376). After an additional round of
model building and water picking using CNX another complete round
of refinement was performed. The final model included the
DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide
(FMN), one orotate molecule (ORO), one acetate molecule (ACT), one
sulfate ion (SO4), one molecule of compound 4 (INH) and 192 water
molecules (TIP).
[0188] The model is well refined and shows very good stereochemical
geometry. The refinement process which included data from 19.9-2.15
.ANG. resulted in an R-factor of 20.1% and a free R-factor of
22.1%. Except for non-glycine and non-proline residues 91.6% of the
residues are located in the most favoured region of the
ramachandran plot and 8% and 0.3% cluster in the additional allowed
or generously allowed regions, respectively. There are no residues
in the disallowed region. Table 16 summarizes the refinement
statistics for compound 4 in complex with human DHODH. Values in
parentheses give the R-factor and R.sub.free-factors, respectively,
for the last resolution bin ranging from 2.28 to 2.15.
TABLE-US-00018 TABLE 16 Refinement Statistics for DHODH/compound 4
complex R-factor (%) 20.1 (19.1) R.sub.free 22.1 (20.9) RMS
deviation from ideal values bond length (.ANG.) 0.005 Bond angle
(.degree.) 1.2 Dihedral angles (.degree.) 21.5 Improper angles
(.degree.) 0.80
[0189] Structure Determination and Refinement of DHODH/Compound 5
Complex
[0190] The structure for the human DHODH(Met30-Arg396) in complex
with compound 5 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.0 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 33.8% and
a correlation coefficient of 71.5 for the DHODH/compound 5
complex.
[0191] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard
CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of the inhibitor compound 5 could
be interpreted unambiguously. The electron density around the
five-membered ring carrying the carboxy group clearly showed the
carboxy group in contact with residues His 56 and Tyr 356 in
non-brequinar-like conformation. Interestingly the protein's active
site discriminates between the S- and R-enantiomere. Inspection of
the corresponding electron density unequivocally shows the
presences of the R-enantiomere only.
[0192] A pdb file for compound 5 was created using the program MOE
(Chemical Computing Group Inc., MOE 2002.02). After energy
minimization the compound was built into the electron density
manually. Topology and parameter files for compound 5 were created
using the program Xplo2d (Uppsala Software Factory; Kleywegt, G. M.
(1997) J. Mol. Biol. 273, 371-376). After an additional round of
model building and water picking using CNX another complete round
of refinement was performed. The final model included the
DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide
(FMN), one orotate molecule (ORO), one acetate molecule (ACT), two
sulfate ions (SO4), one molecule of compound 5 (INH) and 287 water
molecules (TIP).
[0193] The model is well refined and shows very good stereochemical
geometry. The refinement process which included data from 25.5-2.2
.ANG. resulted in an R-factor of 18.3% and a free R-factor of
20.9%. Except for non-glycine and non-proline residues 92.6% of the
residues are located in the most favoured region of the
ramachandran plot and 7% and 0.3% cluster in the additional allowed
or generously allowed regions, respectively. There are no residues
in the disallowed region. Table 17 summarizes the refinement
statistics for compound in complex with human DHODH. Values in
parentheses give the R-factor and R.sub.free-factors, respectively,
for the last resolution bin ranging from 2.34 to 2.2.
TABLE-US-00019 TABLE 17 Refinement Statistics for DHODH/compound 5
complex R-factor (%) 18.3 (19.4) R.sub.free 20.9 (22.0) RMS
deviation from ideal values bond length (.ANG.) 0.005 Bond angle
(.degree.) 1.2 Dihedral angles (.degree.) 21.3 Improper angles
(.degree.) 0.83
[0194] Structure Determination and Refinement of DHODH/Compound 6
Complex
[0195] The structure for the human DHODH(Met30-Arg396) in complex
with compound 6 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.0 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 32.7% and
a correlation coefficient of 74.5 for the DHODH/compound 6
complex.
[0196] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard
CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of the inhibitor compound 6 could
be interpreted unambiguously. The electron density around the
five-membered ring carrying the carboxy group clearly showed that
the inhibitor molecule adopts both the brequinar and non-brequinar
binding mode. The carboxy group is in contact with both anion
binding sites. Interestingly the protein's active site
discriminates between the S- and R-enantiomere. Inspection of the
corresponding electron density unequivocally shows the presences of
the R-enantiomere only.
[0197] A pdb file for compound 6 was created using the program MOE
(Chemical Computing Group Inc., MOE 2002.02). After energy
minimization the compound was built into the electron density
manually. Topology and parameter files for compound 6 were created
using the program Xplo2d (Uppsala Software Factory; Kleywegt, G. M.
(1997) J. Mol. Biol. 273, 371-376). After an additional round of
model building and water picking using CNX another complete round
of refinement was performed. The final model included the
DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide
(FMN), one orotate molecule (ORO), one acetate molecule (ACT), one
sulfate ion (SO4), one molecule of compound 6 (INH) and 312 water
molecules (TIP).
[0198] The models are well refined and show very good geometry. The
refinement process which included data from 19.3-1.9 .ANG. resulted
in an R-factor of 18.5% and a free R-factor of 20.8% for the
complex in conformation A and an R-factor of 18.5% and a free
R-factor of 20.7% for the complex in conformation B, respectively.
The almost identical R-factors indicate that non of the conformers
A and B represent a preferred conformation. Except for non-glycine
and non-proline residues 92.6% are located in the most favoured
region of the ramachandran plot and 7.4% cluster in the additional
allowed regions. There are no residues in the disallowed region.
Table 18 summarizes the refinement statistics for compound 6 in
complex with human DHODH. Values in parentheses give the R-factor
and R.sub.free-factors, respectively, for the last resolution bin
ranging from 2.02 to 1.9. TABLE-US-00020 TABLE 18 Refinement
Statistics for DHODH/compound 6 complex conformation A conformation
B R-factor (%) 18.5 (21.1) 18.5 (21.2) R.sub.free 20.8 (21.5) 20.7
(21.6) RMS deviation from ideal values bond length (.ANG.) 0.005
0.005 Bond angle (.degree.) 1.2 1.2 Dihedral angles (.degree.) 21.3
21.3 Improper angles (.degree.) 0.79 0.79
[0199] Structure Determination and Refinement of DHODH/Compound 7
Complex
[0200] The structure for the human DHODH(Met30-Arg396) in complex
with compound 7 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.0 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 32.7% and
a correlation coefficient of 73.9 for the DHODH/compound 7
complex.
[0201] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard
CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of the inhibitor compound 7 could
be interpreted unambiguously. The electron density around the
five-membered ring carrying the carboxy group clearly showed the
carboxy group in contact with residues His 56 and Tyr 356 in
non-brequinar-like conformation addressing subsite 3. In compound 7
a hydroxy group at 3-position at the five membered ring was
introduced creating a stereo center at this position. The racemic
mixture was used for crystallization experiments. Analysis of the
electron density reveals the presence of both enantiomeres.
Interestingly only the R-enantiomere is able to form additional
contacts to the side chains of residues Gln47 and Arg136 and to a
conserved water molecule. As is clearly shown from experimental
data compound 7 is able to form interactions with both subsite 2
and subsite 3 at the same time. This feature clearly discriminates
this compound class from, for example, compounds 2, 6 and 10 which
can address both binding sites utilizing alternative conformations
but not at the same time.
[0202] A pdb file for compound 7 was created using the program MOE
(Chemical Computing Group Inc., MOE 2002.02). After energy
minimization the compound was built into the electron density
manually. Topology and parameter files for compound 7 were created
using the program Xplo2d (Uppsala Software Factory; Kleywegt, G. M.
(1997) J. Mol. Biol. 273, 371-376). After an additional round of
model building and water picking using CNX another complete round
of refinement was performed. The final model included the
DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide
(FMN), one orotate molecule (ORO), one acetate molecule (ACT), two
sulfate ions (SO4), one molecule of compound 7 (INH) and 229 water
molecules (TIP).
[0203] The model is well refined and shows very good stereochemical
geometry. The refinement process which included data from 17.0-2.0
.ANG. resulted in an R-factor of 17.5% and a free R-factor of 20.4%
for the R-form and S-form. Except for non-glycine and non-proline
residues 92.3% of the residues are located in the most favoured
region of the ramachandran plot and 7.7% cluster in the additional
allowed regions. There are no residues in the disallowed region.
Table 19 summarizes the refinement statistics for compound 7 in
complex with human DHODH. Values in parentheses give the R-factor
and R.sub.free-factors, respectively, for the last resolution bin
ranging from 2.13 to 2.0. TABLE-US-00021 TABLE 19 Refinement
Statistics for DHODH/compound 7 complex R-form S-form R-factor (%)
17.5 (17.3) 17.5 (17.3) R.sub.free 20.4 (21.4) 20.4 (21.4) RMS
deviation from ideal values bond length (.ANG.) 0.005 0.008 Bond
angle (.degree.) 1.2 1.2 Dihedral angles (.degree.) 21.2 21.2
Improper angles (.degree.) 0.82 0.81
[0204] Structure Determination and Refinement of DHODH/Compound 8
Complex
[0205] The structure for the human DHODH(Met30-Arg396) in complex
with compound 8 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.0 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 33.3% and
a correlation coefficient of 73.9 for the DHODH/compound 8
complex.
[0206] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard
CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of the inhibitor compound 8 could
be interpreted unambiguously. The electron density around the
five-membered ring carrying the carboxy group clearly showed the
carboxy group in contact with residues His 56 and Tyr 356 in
non-brequinar-like conformation addressing subsite 3. In compound 8
a hydroxy group at 5-position at the five membered ring was
introduced creating a stereo center at this position. The racemic
mixture was used for crystallization experiments. Analysis of the
electron density reveals that both enantiomeres fit into the
electron density. The R-enantiomere appears to be positioned in a
more favourable position to form interactions with subsite 3
whereas in the S-enantiomere the hydroxy group protrudes into the
direction of subsite 4 (remote hydrophobic pocket) in a less
favourable manner.
[0207] A pdb file for compound 8 was created using the program MOE
(Chemical Computing Group Inc., MOE 2002.02). After energy
minimization the compound was built into the electron density
manually. Topology and parameter files for compound 8 were created
using the program Xplo2d (Uppsala Software Factory; Kleywegt, G. M.
(1997) J. Mol. Biol. 273, 371-376). After an additional round of
model building and water picking using CNX another complete round
of refinement was performed. The final model included the
DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide
(FMN), one orotate molecule (ORO), one acetate molecule (ACT), five
sulfate ions (SO4), one molecule of compound 8 (INH) and 218 water
molecules (TIP).
[0208] The model is well refined and shows very good stereochemical
geometry. The refinement process which included data from 19.0-1.8
.ANG. resulted in an R-factor of 18.2% and a free R-factor of 19.6%
for the R-form and S-form (statistics are given only for R-form).
Except for non-glycine and non-proline residues 91.6% of the
residues are located in the most favoured region of the
ramachandran plot and 8.4% cluster in the additional allowed
regions. There are no residues in the disallowed region. Table 20
summarizes the refinement statistics for compound 8 in complex with
human DHODH. Values in parentheses give the R-factor and
R.sub.free-factors, respectively, for the last resolution bin
ranging from 1.91 to 1.8. TABLE-US-00022 TABLE 20 Refinement
Statistics for DHODH/compound 8 complex R-factor (%) 18.2 (22.1)
R.sub.free 19.6 (24.6) RMS deviation from ideal values bond length
(.ANG.) 0.005 Bond angle (.degree.) 1.2 Dihedral angles (.degree.)
21.2 Improper angles (.degree.) 0.83
[0209] Structure Determination and Refinement of DHODH/Compound 9
Complex
[0210] The structure for the human DHODH(Met30-Arg396) in complex
with compound 9 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.0 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 32.8% and
a correlation coefficient of 73.6 for the DHODH/compound 9
complex.
[0211] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard
CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of the inhibitor compound 9 could
be interpreted unambiguously. The electron density around the
five-membered ring carrying the carboxy group clearly showed the
carboxy group in contact with residues Gln 47 and Arg 136 and a
conserved water molecule in a unique brequinar-like conformation
addressing subsite 2 only. In this conformation the sulfur atom of
the five membered ring comes into close contact to Val 134 and Val
143 which form in part subsite 4 (remote hydrophobic pocket).
[0212] A pdb file for compound 9 was created using the program MOE
(Chemical Computing Group Inc., MOE 2002.02). After energy
minimization the compound was built into the electron density
manually. Topology and parameter files for compound 9 were created
using the program Xplo2d (Uppsala Software Factory; Kleywegt, G. M.
(1997) J. Mol. Biol. 273, 371-376). After an additional round of
model building and water picking using CNX another complete round
of refinement was performed. The final model included the
DHODH(Met30-Arg396) protein, the cofactor flavinmononucleotide
(FMN), one orotate molecule (ORO), one acetate molecule (ACT), five
sulfate ions (SO4), one molecule of compound 9 (INH) and 291 water
molecules (TIP).
[0213] The model is well refined and shows very good stereochemical
geometry. The refinement process which included data from 17.2-2.0
.ANG. resulted in an R-factor of 18.1% and a free R-factor of
20.0%. Except for non-glycine and non-proline residues 92.1% of the
residues are located in the most favoured region of the
ramachandran plot and 7.9% cluster in the additional allowed
regions. There are no residues in the disallowed region. Table 21
summarizes the refinement statistics for compound 9 in complex with
human DHODH. Values in parentheses give the R-factor and
R.sub.free-factors, respectively, for the last resolution bin
ranging from 2.13 to 2.0. TABLE-US-00023 TABLE 21 Refinement
Statistics for DHODH/compound 9 complex R-factor (%) 18.1 (19.7)
R.sub.free 20.0 (22.0) RMS deviation from ideal values bond length
(.ANG.) 0.005 Bond angle (.degree.) 1.2 Dihedral angles (.degree.)
21.2 Improper angles (.degree.) 0.80
[0214] Structure Determination and Refinement of DHODH/Compound 10
Complex
[0215] The structure for the human DHODH(Met30-Arg396) in complex
with compound 10 was solved using the method of molecular
replacement (MR). The free accessible pdb entry 1D3G.pdb was used
as a search model. The ligands brequinar and DDQ as well as all of
the water molecules were removed prior to the MR search. The search
model included the polypeptide chain of hDHODH(Met30-Arg396), one
molecule of orotate, one molecule of the cofactor
flavinmononucleotide (FMN) and one acetate molecule which was
present under the crystallization conditions. A standard rotational
and translational molecular replacement search at 3.0 .ANG. was
performed using the program molrep (Collaborative Computational
Project, Number 4 (1994). Acta Cryst. D50, 760-763.). Solutions for
both the rotational and translational search were well above the
next ranking solutions. The MR resulted in an R-factor of 32.8% and
a correlation coefficient of 74.1 for the DHODH/compound 10
complex.
[0216] In a first round of refinement the MR model was subjected to
rigid body refinement and a slow cooling simulated annealing
protocol using a maximum likelihood target to remove model bias
(Accelrys Inc. CNX program suite, CNX2002). Additionally, an
individual b-factor refinement was carried out using standard
CNX-protocols. Finally SIGMAA weighted 2Fo-Fc and Fo-Fc electron
density maps were calculated and displayed together with the
protein model in the program O (DatOno AB; Jones, T. A., Zou, J.
Y., Cowan, S. W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.). The resulting experimental electron density was so
excellent that the conformation of the inhibitor compound 10 could
be interpreted unambiguously. The electron density around the
five-membered ring carrying the carboxy group clearly showed the
presence of two alternative conformations of compound 10. In one
conformation (conformation A) the carboxy group interacts with
residues Gln 47 and Arg 136, whereas in the second conformation
(conformation B) the interaction involves residues His 56 and Tyr
356 (see above). For each conformation a separate DHODH/compound 10
complex was subjected to refinement.
[0217] The pdb files for the compound 10 in conformation A and B
were created using the program MOE (Chemical Computing Group Inc.,
MOE 2002.02). Both compounds were energy minimized and built into
the electron density manually. Topology and parameter files for
compound 10 were created using the program Xplo2d (Uppsala Software
Factory; Kleywegt, G. M. (1997) J. Mol. Biol., 273, 371-376). After
an additional round of model building and water picking using CNX,
another complete round of refinement was performed. The final model
included the human DHODH(Met30-Arg396) protein, the cofactor
flavinmononucleotide (FMN), one orotate molecule (ORO), two acetate
molecules (ACT), four sulfate ions (SO4), one molecule of compound
10 (INH) either in conformation A or conformation B and 226 water
molecules (TIP). Residues which are missing the coordinate file due
to very poor electron density are listed in the header of the pdb
files.
[0218] The models are well refined and show very good geometry. The
refinement process which included data from 19.5-1.8 .ANG. resulted
in an R-factor of 19.5% and a free R-factor of 20.5% for the
complex in conformation A and for the complex in conformation B,
respectively. The identical R-factors indicate that non of the
conformers A and B represent a preferred conformation. Except for
non-glycine and non-proline residues 91.6% are located in the most
favoured region of the ramachandran plot and 8.4% cluster in the
additional allowed regions. There are no residues in the disallowed
region. Table 22 summarizes the refinement statistics for compound
10 in complex with human DHODH. Values in parentheses give the
R-factor and R.sub.free-factors, respectively, for the last
resolution bin ranging from 1.91 to 1.8. TABLE-US-00024 TABLE 22
Refinement Statistics for DHODH/compound 10 complex conformation A
& B R-factor (%) 19.5 (20.5) R.sub.free 20.5 (22.7) RMS
deviation from ideal values bond length (.ANG.) 0.005 Bond angle
(.degree.) 1.2 Dihedral angles (.degree.) 21.9 Improper angles
(.degree.) 0.82
[0219] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
[0220] The following compounds are preferred:
[0221] 3-(Biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylic acid;
3-(2'-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylic
acid;
3-(3'-Ethoxy-3,5-difluoro-biphenyl-4-yl-carbamoyl)-thiophene-2-carb-
oxylic acid;
3-(3,5-Difluoro-2',4'-dimethoxy-biphenyl-4-yl-carbamoyl)-thiophene-2-carb-
oxylic acid;
3-(2,3,5,6-Tetrafluoro-2'-methoxy-biphenyl-4-yl-carbamoyl)-thiophene-2-ca-
rboxylic acid;
3-(2'-Chloro-3,5-difluoro-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylic
acid;
3-(3,5,2'-Trifluoro-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylic
acid;
3-(2-Chloro-2'-methoxy-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxyl-
ic acid;
3-(2,3,5,6-Tetrafluoro-3'-trifluoromethoxy-biphenyl-4-ylcarbamoyl-
)-thiophene-2-carboxylic acid;
3-(3-Fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylic
acid;
3-(3,5-Difluoro-3'-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-thiophe-
ne-2-carboxylic acid; 3-(Biphenyl-4-ylcarbamoyl)-furan-2-carboxylic
acid; 4-(Biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylic acid;
4-(2-Chloro-2'-methoxy-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylic
acid;
4-(3,5,2'-Trifluoro-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylic
acid;
4-(3'-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-thiophene-3-carbo-
xylic acid;
4-(2'-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylic
acid;
4-(3,5-Difluoro-3'-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-thiophe-
ne-3-carboxylic acid;
4-(3-Fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylic
acid; 4-(Biphenyl-4-ylcarbamoyl)-furan-3-carboxylic acid;
2-(Biphenyl-4-ylcarbamoyl)-thiophene-3-carboxylic acid;
2-(Biphenyl-4-ylcarbamoyl)-furan-3-carboxylic acid;
3-(3-Fluoro-3'-methoxy-biphenyl-4-yl-carbamoyl)-cyclopent-2-ene-1,2-dicar-
boxylic acid;
2-(3-Fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl)-cyclopent-1-ene-1,3-dicarb-
oxylic acid;
2-(3-Fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarboxylic
acid methyl ester; Cyclopent-1-ene-1,2-dicarboxylic acid
1-[(3-fluoro-3'-methoxy-biphenyl-4-yl)-amide]2-hydroxyamide;
3-Hydroxy-2-(2,3,5,6-tetrafluoro-3'-trifluoromethoxy-biphenyl-4-ylcarbamo-
yl)-cyclopent-1-enecarboxylic acid;
5-Hydroxy-2-(2,3,5,6-tetrafluoro-3'-trifluoromethoxy-biphenyl-4-ylcarbamo-
yl)-cyclopent-1-enecarboxylic acid;
2-(3'-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclopent-1-e-
necarboxylic acid;
2-(3'-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-5-hydroxy-cyclo-pent-1--
enecarboxylic acid;
2-(1',3'di-methoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclope-
nt-1-enecarboxylic acid;
2-(1',3'di-methoxy-3,5-difluoro-biphenyl-4-yl-carbamoyl)-5-hydroxy-cyclop-
ent-1-enecarboxylic acid;
3-Hydroxy-2-(3,5,2'-trifluoro-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarb-
oxylic acid;
5-Hydroxy-2-(3,5,2'-trifluoro-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarb-
oxylic acid;
2-(2-Chloro-2'-methoxy-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclopent-1-enec-
arboxylic acid;
2-(2-Chloro-2'-methoxy-biphenyl-4-ylcarbamoyl)-5-hydroxy-cyclopent-1-enec-
arboxylic acid;
2-(2'-Chloro-3,5-difluoro-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclopent-1-e-
necarboxylic acid;
2-(2'-Chloro-3,5-difluoro-biphenyl-4-ylcarbamoyl)-5-hydroxy-cyclopent-1-e-
necarboxylic acid;
2-(3-Fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl)-3-hydroxy-cyclopent-1-enec-
arboxylic acid;
2-(3-Fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl)-5-hydroxy-cyclopent-1-enec-
arboxylic acid; trans
2-(3-Fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl)-cyclopentane
carboxylic acid;
cis-2-(3-Fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl)-cyclopentane
carboxylic acid;
2-(2'-Chloro-3,5-difluoro-biphenyl-4-ylcarbamoyl)-cyclopentane
carboxylic acid;
2-(3,5-Difluoro-2',4'-dimethoxy-biphenyl-4-ylcarbamoyl)-cyclopentan-
e carboxylic acid;
2-(3'-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-cyclopentane
carboxylic acid;
2-(2'-Ethoxy-3,5-difluoro-biphenyl-4-ylcarbamoyl)-cyclopentane
carboxylic acid; 2-(Biphenyl-4-ylcarbamoyl)-cyclopentane carboxylic
acid;
2-(2,3,5,6-Tetrafluoro-3'-trifluoro-methoxy-biphenyl-4-ylcarbamoyl)-cyclo-
pentane carboxylic acid;
2-(3,5-Difluoro-3'-trifluoro-methoxy-biphenyl-4-yl-carbamoyl)-cyclopentan-
e carboxylic acid TABLE-US-00025 TABLE 25 Con- sensus Structure
Score ##STR4## -63.04 ##STR5## -61.85 ##STR6## -60.26 ##STR7##
-59.46 ##STR8## -58.50 ##STR9## -58.21 ##STR10## -58.13 ##STR11##
-58.12 ##STR12## -58.05 ##STR13## -57.99 ##STR14## -57.66 ##STR15##
-57.60 ##STR16## -57.56 ##STR17## -57.55 ##STR18## -57.51 ##STR19##
-57.18 ##STR20## -57.14 ##STR21## -57.00 ##STR22## -56.93 ##STR23##
-56.85 ##STR24## -56.85 ##STR25## -56.72 ##STR26## -56.71 ##STR27##
-56.31 ##STR28## -56.25 ##STR29## -55.96 ##STR30## -55.93 ##STR31##
-55.89 ##STR32## -55.67 ##STR33## -55.64 ##STR34## -55.58 ##STR35##
-55.52 ##STR36## -55.51 ##STR37## -55.29 ##STR38## -55.14 ##STR39##
-55.10 ##STR40## -54.92 ##STR41## -54.72 ##STR42## -54.51 ##STR43##
-54.49 ##STR44## -54.47 ##STR45## -54.47 ##STR46## -54.38 ##STR47##
-54.35 ##STR48## -54.35 ##STR49## -54.29 ##STR50## -54.29 ##STR51##
-54.28 ##STR52## -54.16 ##STR53## -54.10 ##STR54## -54.10 ##STR55##
-54.07 ##STR56## -54.05 ##STR57## -54.04 ##STR58## -53.92 ##STR59##
-53.92 ##STR60## -53.79 ##STR61## -53.75 ##STR62## -53.73 ##STR63##
-53.70 ##STR64## -53.56 ##STR65## -53.54 ##STR66## -53.21 ##STR67##
-53.20 ##STR68## -53.18 ##STR69## -53.15 ##STR70## -53.15 ##STR71##
-53.08 ##STR72## -53.01 ##STR73## -53.01 ##STR74## -53.00 ##STR75##
-52.99 ##STR76## -52.89 ##STR77## -52.74 ##STR78## -52.73 ##STR79##
-52.69 ##STR80## -52.65 ##STR81## -52.60 ##STR82## -52.58 ##STR83##
-52.57 ##STR84## -52.51 ##STR85## -52.49 ##STR86## -52.33 ##STR87##
-52.30
##STR88## -52.12 ##STR89## -52.08 ##STR90## -52.04 ##STR91## -51.98
##STR92## -51.91 ##STR93## -51.86 ##STR94## -51.76 ##STR95## -51.76
##STR96## -51.74 ##STR97## -51.66 ##STR98## -51.65 ##STR99## -51.55
##STR100## -51.54 ##STR101## -51.45 ##STR102## -51.40 ##STR103##
-51.37
[0222]
Sequence CWU 1
1
3 1 367 PRT Human 1 Met Ala Thr Gly Asp Glu Arg Phe Tyr Ala Glu His
Leu Met Pro Thr 1 5 10 15 Leu Gln Gly Leu Leu Asp Pro Glu Ser Ala
His Arg Leu Ala Val Arg 20 25 30 Phe Thr Ser Leu Gly Leu Leu Pro
Arg Ala Arg Phe Gln Asp Ser Asp 35 40 45 Met Leu Glu Val Arg Val
Leu Gly His Lys Phe Arg Asn Pro Val Gly 50 55 60 Ile Ala Ala Gly
Phe Asp Lys His Gly Glu Ala Val Asp Gly Leu Tyr 65 70 75 80 Lys Met
Gly Phe Gly Phe Val Glu Ile Gly Ser Val Thr Pro Lys Pro 85 90 95
Gln Glu Gly Asn Pro Arg Pro Arg Val Phe Arg Leu Pro Glu Asp Gln 100
105 110 Ala Val Ile Asn Arg Tyr Gly Phe Asn Ser His Gly Leu Ser Val
Val 115 120 125 Glu His Arg Leu Arg Ala Arg Gln Gln Lys Gln Ala Lys
Leu Thr Glu 130 135 140 Asp Gly Leu Pro Leu Gly Val Asn Leu Gly Lys
Asn Lys Thr Ser Val 145 150 155 160 Asp Ala Ala Glu Asp Tyr Ala Glu
Gly Val Arg Val Leu Gly Pro Leu 165 170 175 Ala Asp Tyr Leu Val Val
Asn Val Ser Ser Pro Asn Thr Ala Gly Leu 180 185 190 Arg Ser Leu Gln
Gly Lys Ala Glu Leu Arg Arg Leu Leu Thr Lys Val 195 200 205 Leu Gln
Glu Arg Asp Gly Leu Arg Arg Val His Arg Pro Ala Val Leu 210 215 220
Val Lys Ile Ala Pro Asp Leu Thr Ser Gln Asp Lys Glu Asp Ile Ala 225
230 235 240 Ser Val Val Lys Glu Leu Gly Ile Asp Gly Leu Ile Val Thr
Asn Thr 245 250 255 Thr Val Ser Arg Pro Ala Gly Leu Gln Gly Ala Leu
Arg Ser Glu Thr 260 265 270 Gly Gly Leu Ser Gly Lys Pro Leu Arg Asp
Leu Ser Thr Gln Thr Ile 275 280 285 Arg Glu Met Tyr Ala Leu Thr Gln
Gly Arg Val Pro Ile Ile Gly Val 290 295 300 Gly Gly Val Ser Ser Gly
Gln Asp Ala Leu Glu Lys Ile Arg Ala Gly 305 310 315 320 Ala Ser Leu
Val Gln Leu Tyr Thr Ala Leu Thr Phe Trp Gly Pro Pro 325 330 335 Val
Val Gly Lys Val Lys Arg Glu Leu Glu Ala Leu Leu Lys Glu Gln 340 345
350 Gly Phe Gly Gly Val Thr Asp Ala Ile Gly Ala Asp His Arg Arg 355
360 365 2 367 PRT Human 2 Met Ala Thr Gly Asp Glu Arg Phe Tyr Ala
Glu His Leu Met Pro Thr 1 5 10 15 Leu Gln Gly Leu Leu Asp Pro Glu
Ser Ala His Arg Leu Ala Val Arg 20 25 30 Phe Thr Ser Leu Gly Leu
Leu Pro Arg Ala Arg Phe Gln Asp Ser Asp 35 40 45 Met Leu Glu Val
Arg Val Leu Gly His Lys Phe Arg Asn Pro Val Gly 50 55 60 Ile Ala
Ala Gly Phe Asp Lys His Gly Glu Ala Val Asp Gly Leu Tyr 65 70 75 80
Lys Met Gly Phe Gly Phe Val Glu Ile Gly Ser Val Thr Pro Lys Pro 85
90 95 Gln Glu Gly Asn Pro Arg Pro Arg Val Phe Arg Leu Pro Glu Asp
Gln 100 105 110 Ala Val Ile Asn Arg Tyr Gly Phe Asn Ser His Gly Leu
Ser Val Val 115 120 125 Glu His Arg Leu Arg Ala Arg Gln Gln Lys Gln
Ala Lys Leu Thr Glu 130 135 140 Asp Gly Leu Pro Leu Gly Val Asn Leu
Gly Lys Asn Lys Thr Ser Val 145 150 155 160 Asp Ala Ala Glu Asp Tyr
Ala Glu Gly Val Arg Val Leu Gly Pro Leu 165 170 175 Ala Asp Tyr Leu
Val Val Asn Val Ser Ser Pro Asn Thr Ala Gly Leu 180 185 190 Arg Ser
Leu Gln Gly Lys Ala Glu Leu Arg Arg Leu Leu Thr Lys Val 195 200 205
Leu Gln Glu Arg Asp Gly Leu Arg Arg Val His Arg Pro Ala Val Leu 210
215 220 Val Lys Ile Ala Pro Asp Leu Thr Ser Gln Asp Lys Glu Asp Ile
Ala 225 230 235 240 Ser Val Val Lys Glu Leu Gly Ile Asp Gly Leu Ile
Val Thr Asn Thr 245 250 255 Thr Val Ser Arg Pro Ala Gly Leu Gln Gly
Ala Leu Arg Ser Glu Thr 260 265 270 Gly Gly Leu Ser Gly Lys Pro Leu
Arg Asp Leu Ser Thr Gln Thr Ile 275 280 285 Arg Glu Met Tyr Ala Leu
Thr Gln Gly Arg Val Pro Ile Ile Gly Val 290 295 300 Gly Gly Val Ser
Ser Gly Gln Asp Ala Leu Glu Lys Ile Arg Ala Gly 305 310 315 320 Ala
Ser Leu Val Gln Leu Tyr Thr Ala Leu Thr Phe Trp Gly Pro Pro 325 330
335 Val Val Gly Lys Val Lys Arg Glu Leu Glu Ala Leu Leu Lys Glu Gln
340 345 350 Gly Phe Gly Gly Val Thr Asp Ala Ile Gly Ala Asp His Arg
Arg 355 360 365 3 367 PRT Human 3 Met Ala Thr Gly Asp Glu Arg Phe
Tyr Ala Glu His Leu Met Pro Thr 1 5 10 15 Leu Gln Gly Leu Leu Asp
Pro Glu Ser Ala His Arg Leu Ala Val Arg 20 25 30 Phe Thr Ser Leu
Gly Leu Leu Pro Arg Ala Arg Phe Gln Asp Ser Asp 35 40 45 Met Leu
Glu Val Arg Val Leu Gly His Lys Phe Arg Asn Pro Val Gly 50 55 60
Ile Ala Ala Gly Phe Asp Lys His Gly Glu Ala Val Asp Gly Leu Tyr 65
70 75 80 Lys Met Gly Phe Gly Phe Val Glu Ile Gly Ser Val Thr Pro
Lys Pro 85 90 95 Gln Glu Gly Asn Pro Arg Pro Arg Val Phe Arg Leu
Pro Glu Asp Gln 100 105 110 Ala Val Ile Asn Arg Tyr Gly Phe Asn Ser
His Gly Leu Ser Val Val 115 120 125 Glu His Arg Leu Arg Ala Arg Gln
Gln Lys Gln Ala Lys Leu Thr Glu 130 135 140 Asp Gly Leu Pro Leu Gly
Val Asn Leu Gly Lys Asn Lys Thr Ser Val 145 150 155 160 Asp Ala Ala
Glu Asp Tyr Ala Glu Gly Val Arg Val Leu Gly Pro Leu 165 170 175 Ala
Asp Tyr Leu Val Val Asn Val Ser Ser Pro Asn Thr Ala Gly Leu 180 185
190 Arg Ser Leu Gln Gly Lys Ala Glu Leu Arg Arg Leu Leu Thr Lys Val
195 200 205 Leu Gln Glu Arg Asp Gly Leu Arg Arg Val His Arg Pro Ala
Val Leu 210 215 220 Val Lys Ile Ala Pro Asp Leu Thr Ser Gln Asp Lys
Glu Asp Ile Ala 225 230 235 240 Ser Val Val Lys Glu Leu Gly Ile Asp
Gly Leu Ile Val Thr Asn Thr 245 250 255 Thr Val Ser Arg Pro Ala Gly
Leu Gln Gly Ala Leu Arg Ser Glu Thr 260 265 270 Gly Gly Leu Ser Gly
Lys Pro Leu Arg Asp Leu Ser Thr Gln Thr Ile 275 280 285 Arg Glu Met
Tyr Ala Leu Thr Gln Gly Arg Val Pro Ile Ile Gly Val 290 295 300 Gly
Gly Val Ser Ser Gly Gln Asp Ala Leu Glu Lys Ile Arg Ala Gly 305 310
315 320 Ala Ser Leu Val Gln Leu Tyr Thr Ala Leu Thr Phe Trp Gly Pro
Pro 325 330 335 Val Val Gly Lys Val Lys Arg Glu Leu Glu Ala Leu Leu
Lys Glu Gln 340 345 350 Gly Phe Gly Gly Val Thr Asp Ala Ile Gly Ala
Asp His Arg Arg 355 360 365
* * * * *