U.S. patent application number 11/150485 was filed with the patent office on 2006-01-19 for compounds and methods of use to treat infectious diseases.
Invention is credited to Bradley Berger, Michael I. Bukrinsky, Anthony Cerami, Peter Ulrich.
Application Number | 20060014760 11/150485 |
Document ID | / |
Family ID | 24944447 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060014760 |
Kind Code |
A1 |
Bukrinsky; Michael I. ; et
al. |
January 19, 2006 |
Compounds and methods of use to treat infectious diseases
Abstract
The present invention concerns alkyl aryl carbonyl compounds
that possess anti-infective activity. The compounds of the
invention can be used to target specific nuclear localization
signal, thereby blocking importation of specific proteins or
molecular complex into the nucleus of a cell. The invention
encompasses methods of use of such compounds for treatment or
prevention of infectious diseases, such as parasitic and viral
diseases, including, for example, malaria and acquired
immunodeficiency syndrome. The use of the compounds to detect
certain specific protein structures which are present in nuclear
localization sequences is also taught.
Inventors: |
Bukrinsky; Michael I.;
(Potomac, MD) ; Cerami; Anthony; (Sleepy Hollow,
NY) ; Ulrich; Peter; (Portland, OR) ; Berger;
Bradley; (Medicine Hat, CA) |
Correspondence
Address: |
DLA PIPER RUDNICK GRAY CARY US LLP;Patent Group
1200 Nineteenth Street, N.W.
Washington
DC
20036-2412
US
|
Family ID: |
24944447 |
Appl. No.: |
11/150485 |
Filed: |
June 13, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10660748 |
Sep 12, 2003 |
6906076 |
|
|
11150485 |
Jun 13, 2005 |
|
|
|
09887020 |
Jun 25, 2001 |
6649797 |
|
|
10660748 |
Sep 12, 2003 |
|
|
|
08732653 |
Oct 15, 1996 |
6297253 |
|
|
09887020 |
Jun 25, 2001 |
|
|
|
Current U.S.
Class: |
514/256 ;
435/455; 514/1.2; 514/3.8; 514/4.4 |
Current CPC
Class: |
A61P 33/06 20180101;
C07D 239/48 20130101; Y10T 436/173845 20150115; A61K 31/505
20130101; Y02A 50/402 20180101; A61K 31/53 20130101; Y02A 50/30
20180101; A61P 31/12 20180101; Y02A 50/423 20180101; Y02A 50/411
20180101; Y10T 436/200833 20150115 |
Class at
Publication: |
514/256 ;
435/455; 514/012 |
International
Class: |
A61K 31/505 20060101
A61K031/505; C12N 15/85 20060101 C12N015/85 |
Claims
1. A method for inactivating a nuclear localization signal of a
protein comprising contacting the protein with a compound that is
capable of stable reversible binding with basic amino acid residues
of the nuclear localization signal of the protein.
2-25. (canceled)
Description
1 FIELD OF THE INVENTION
[0001] The field of the present invention concerns compounds that
react with specific sequences in proteins. The present invention
more particularly concerns a class of compounds that react, under
physiologic conditions, with proteins having adjacent or
neighboring basic amino acid sequences. The compounds of the
invention can be used to label specifically such proteins for
research purposes and to disrupt their function for pharmacologic
purposes. The compounds of the invention can be used for targeted
inactivation of nuclear localization signal in specific proteins or
molecular complexes. The compounds of the invention can also be
used to treat infectious diseases such as HIV infection and
malaria.
2 BACKGROUND TO THE INVENTION
2.1 The Derivation of Proteins
[0002] Those skilled in the art will appreciate that there are many
compounds that can react with specific amino-acid residues in
proteins, e.g., with sulfhydryl, amino, carboxyl moieties. These
reagents are substrate specific, in the sense that each reacts only
with one or a few specific amino acids wherever they occur within a
protein's sequence. However, the reactivity of such reagents is not
affected by the adjacent or neighboring amino acids that form the
environment of the reactive moiety. Thus, the reactivity of such
compounds is not context or neighborhood specific.
2.2 Nuclear Importation
[0003] The function of an intracellular protein is usually the
result of the overall three dimensional (tertiary) structure of the
protein. However, nuclear importation is determined by the simple
presence of a short sequence, called a nuclear localization signal
(NLS), which functions relatively independently of its position
relative to the remainder of the structure of object that is
imported. In eukaryotic cells all proteins are made in the
cytoplasm, which is outside of the nucleus. In general, those
proteins larger than 40 kD that are specifically localized in the
nucleus of the cell must be actively imported into the nucleus
through the nuclear membrane from the cytoplasm via an
ATP-dependent mechanism that is independent of cell division. The
proteins, and other objects, that are imported have a nuclear
localization signal (NLS), usually located within the NH.sub.2
terminal segment of the protein. Several such sequences are known:
[0004] a. PKKKRKV from large T antigen of SV40 and other
papillomaviruses such as JC, see Kalderon, D., et al., 1984, Cell
39:499-509; [0005] b. [AV]KRPAATKKAGQAKKKK[LD] from nucleoplasmin,
in which only one of the two bracketed sequences is required,
Dingwall, C., et al., 1988, J. Cell Biol. 107:841-49; [0006] c.
PRRRRSQS from hepatitis B HbcAg- Yeh, C. T., 1990, J. Virol. [0007]
d. KRSAEGGNPPKPLKKLR from the retinoblastoma gene product
p110.sup.rb1-Zacksenhaus E. et al., 1993, Mol. Cell. Biol. 13:4588
[0008] e. KIRLPRGGKKKYKLK from the matrix protein of HIV-1,
Bukrinsky, M. I., et al., 1993, Nature 365:666.
[0009] Other viruses that contain NLS sequences include influenza
virus (NP, PA, PB1, PB2 proteins which have lysine-rich NLS similar
to SV40), hepatitis delta virus (HDAg, which has the sequence
PKKKXKK), parvoviruses such as RA1 (NS, VP proteins which have
lysine-rich NLS similar to SV40), Herpes simplex and measles virus.
The recognition of an NLS sequence is largely independent of the
detailed structure of the object which includes it and of its site
of attachment. Goldfarb, D. S. et al., 1986, Nature 332:641-44;
Lanford, R. E., 1986, Cell 46:575. Mere juxtaposition of the amino
acids of the NLS is not sufficient for function, for example NLS
function is generally not conferred by the peptide having the same
sequence of amino acids in the opposite order as the NLS sequence.
Adam, S. A. et al., 1989, Nature 337:276-79.
[0010] The primary structure, i.e., the linear sequence, of the NLS
most frequently contains consecutive lysines, the N.sup..epsilon.
moieties of which presumably closely approach one another, i.e.,
they are neighbors. However, certain functional NLS peptides lack
consecutive lysines. Robbins, J., et al., 1991, Cell 64:615-23.
Presumably the secondary and tertiary structure of these so called
"bipartite" NLS peptides gives rise to neighboring N.sup..epsilon.
moieties, which may be important for their activity.
[0011] Docking and subsequent movement of proteins across the
nuclear pore complex require transport factors. Import of
NLS-containing proteins across the nuclear pore complex is mediated
by karyopherin .alpha..beta. heterodimers (also termed NLS
receptor/importin) which bind NLS-containing proteins in the
cytosol and target them to the nucleus (Gorlich, D., et al., 1995,
Curr. Biol. 5:383-392; Radu, A., et al., 1995, Proc. Natl. Acad.
Sci. 92:1765-1773). Karyopherin .alpha. binds the NLS (Adam and
Gerace, 1991, Cell 66:837-847) whereas karyopherin .beta. enhances
the affinity of .alpha. for the NLS (Rexach and Blobel, 1995, Cell
83:683-692) and mediates docking of karyopherin-NLS protein
complexes to nucleoporins (a collective term for nuclear pore
complex proteins) that contain FXFG peptide repeats. The GTPase Ran
and its interacting protein p10 (also termed NTF2) (Moore and
Blobel, 1994, Proc. Natl. Acad. Sci. 91:10212-10216) impart
mobility to the translocation process by catalyzing the disruption
of karyopherin .alpha..beta. heterodimers that have docked to a
nucleoporin (Nerhbass and Blobel, 1996, Science 272:120-122).
Partial reactions of the nuclear import can be reproduced in vitro
using solution binding assays and recombinant karyopherins (Rexach
and Blobel, 1995, supra).
[0012] Two inhibitors of the nuclear localization process have been
described. Nuclear localization has been inhibited by lectins
(e.g., wheat germ agglutinin (WGA)) that bind to the O-linked
glycoproteins associated with nuclear localization. Dabauvalle, M.
-C., 1988, Exp. Cell Res. 174:291-96; Sterne-Marr R., et al., 1992,
J. Cell Biol. 116:271. The nuclear localization process, which also
depends upon the hydrolysis of GTP, is blocked by a
non-hydrolyzable analog of GTP, e.g., (.gamma.-S)GTP, Melchior, F.,
1993, J. Cell Biol. 123:1649.
[0013] However, neither (.gamma.-S)GTP nor WGA can be used as
pharmaceuticals. Proteins, such as WGA, can be introduced into the
interior of a cell only with considerable difficulty. The same
limitation applies to thiotriphospates such as [.gamma.-S] GTP.
Further, GTPases are involved in a multitude of cell processes and
intercellular signaling, thus, the use of a general inhibitor of
GTPases would likely lead to unacceptable side effects.
2.3 The Significance of Nuclear Importation in HIV-1 Infections
[0014] Although HIV-1 is a retrovirus, it and other lentiviruses
must be distinguished from viruses of the onco-retrovirus group,
which are not associated with progressive fatal infection. For
example, lentiviruses replicate in non-proliferating cells, e.g.,
terminally differentiated macrophages, Weinberg, J. B., 1991,
J.Exp. Med. 172:1477-82, while onco-retroviruses, do not.
Humphries, E. H., & Temin, H. M., 1974, J. Virol. 14:531-46.
Secondly, lentiviruses are able to maintain themselves in a
non-integrated, extrachromosomal form in resting T-cells.
Stevenson, M., et al., 1990, EMBO J. 9:1551-60; Bukrinsky, M. I.,
et al., 1991, Science 254:423; Zack, J. L., et al., 1992, J. Virol.
66:1717-25. However, it is unclear whether this phenomenon is
related to the presence of latently infected peripheral blood
lymphocytes (PBL) in HIV-1 infected subjects, wherein the virus is
present in a provirus form. Schnittman, S. M., 1989, Science
245:305; Brinchmann, J. E., et al., 1991, J. Virol. 65:2019;
Chapel, A., et al., 1992 J. Virol. 66:3966.
[0015] The productive infection of a cell by a retroviruses
involves the steps of penetration into the cell, synthesis of a DNA
genome from the RNA genetic material in the virion and insertion of
the DNA genome into a chromosome of the host, thereby forming a
provirus. Both lenti- and oncoretroviruses gain access to the host
cell's nucleus during mitosis when the nuclear membrane dissolves.
However, the lentiviruses are also able to cross the nuclear
membrane because viral proteins containing nuclear localization
sequences are associated with the viral nucleoprotein complex.
[0016] The productive infection of terminally differentiated
macrophages located in the central nervous system is thought to be
responsible for the dementia associated with AIDS. Keonig, S., et
al., 1986, Science 233:1089; Wiley, C. A. et al., 1986, Proc. Natl.
Acad. Sci. 83:7089-93; Price, R. W., et al., 1988, Science
239:586-92. The infection of terminally differentiated macrophages
in the lymphoid system is known to cause aberrant cytokine
production. Guilian, D., et al., 1990, Science 250:1593; Fauci, A.
S., et al., 1991, Ann. Int. Med. 114:678. Thus, the wasting
syndrome associated with HIV-1, also known as "slim" disease, is
believed to be a pathological process that is independent of the
loss of CD4-T-cells. Rather the pathobiology of the wasting is
closely related to the pathobiology of cachexia in chronic
inflammatory and malignant diseases. Weiss, R. A., 1993, Science
260:1273. For these reasons, the inhibition on HIV-1 infection of
macrophages and other non-dividing cells is understood to represent
a highly desired modality in the treatment of HIV-1 infection,
especially for patients wherein dementia or cachexia dominate the
clinical picture.
[0017] Macrophages play an important role in the transmission of
HIV as well. During early stages of the infection, macrophages and
cells of the macrophage lineage (i.e. dendritic cells) may be the
primary reservoir of HIV-1 in the body, supporting infection of T
cells by antigen presentation activities, Pantaleo, G., et al.,
1993, Nature 362:355-358, as well as via the release of free virus.
Direct cell-to-cell transmission of the virus may constitute the
major route by which infection spreads during the early-stages of
the disease, after resolution of the initial viremia.
[0018] It is noteworthy, in this regard, that macrophage-tropic
strains of HIV-1 predominate in the early stages of infection.
Thus, it appears that the infection of macrophages is particularly
important during the development of a chronic infective state of
the host in a newly infected subject. Secondly, macrophages are the
HIV-susceptible cell type most readily passed during sexual
intercourse from an HIV-infected individual into the circulation of
an uninfected individual.
[0019] Finally, infection of quiescent T cells by HIV-1 has been
shown to take place in vitro, Stevenson, M., et al., 1990, EMBO J.
9:1551-1560; Zack, J. A., 1990, Cell 61:213-222, and probably
constitutes an important pathway for the spread of infection in
vivo at various stages of the disease. Bukrinsky, M. I., et al.,
1991, Science 254:423-427. Although HIV-1 does not establish
productive replication in quiescent T cells, the extrachromosomal
retroviral DNA can persist in the cytoplasm of such cells for a
considerable period of time, and initiate replication upon
activation of the host cell. Stevenson, M., et al., 1990, EMBO J.
9:1551-1560; Spina, C. A., et al., 1994, J. Exp. Med. 179:115-123;
Miller, M. D., et al., 1994, J. Exp. Med. 179:101-113. A recent
report suggests that the duration of viral persistence in the
quiescent T cell depends on the presence of a functional NLS. von
Schwedler, U., et al., 1994, Proc. Natl. Acad. Sci. 91:6992-6996.
Thus, physicians recognize the desirability of preventing the
infection of macrophages by HIV and understand that substantial
benefits would be obtained from the use of a pharmacologic agent
that prevents HIV infection in this cell type.
[0020] The mechanism whereby HIV, but not oncoretroviruses, infect
non-dividing cells is now understood in broad outline. It is
established that the function of the pre-integration complex of
retrovirus in this regard does not depend upon the cellular
mechanisms of mitosis or DNA replication, per se. Rather the
integration complex must merely gain access to nucleus. Brown, P.
O., et al., 1987, Cell 49:347. Onco-retroviruses gain access to the
nucleus upon the dissolution of the nuclear membrane in mitosis. By
contrast, lentiviruses contain two distinct proteins that mediate
nuclear access through the nuclear pore complex in the absence of
cellular division. For the first of these, the matrix protein (MA
or p17), nuclear importation activity is clearly due to the
presence of a trilysyl-containing NLS sequence. Bukrinsky, M. I.,
et al., 1993, Nature 365:666; von Schwedler, U., et al., 1994,
Proc. Natl. Acad. Sci. 91:6992. A second protein subserving the
function of nuclear entry, the vpr protein, does not contain an
identifiable NLS consensus sequence. Emerman, M., et al., 1994,
Nature 369:108; Heinzinger, N. K. et al., 1994, Proc. Natl. Acad.
Sci. 91:7311.
[0021] The significance of the NLS sequence in the importation of
HIV-1 into the nucleus of non-dividing cells has been illustrated
in experiments wherein the presence in the medium of a high
concentration (0.1 M) of the peptide having the sequence of the
SV40 T-antigen NLS blocked the importation of HIV-1 into the
nucleus of aphidicolin-arrested CD4.sup.+ MT4 cells. Gulizia, J.,
et al., 1994, J. Virol. 68:2021-25.
2.4 Infectious Diseases and Its Treatment
[0022] Treatment of an infectious disease with chemicals involves
killing or inhibition of growth of the infectious agent, which may
include free-living and parasitic organisms. Parasitic diseases are
widespread in the animal world where a parasitic organism lives at
the expense of a host organism, and causes damage, or kills its
host. Humans, domestic pets and livestocks are hosts to a variety
of parasites. Parasites do not comprise a single taxonomic group,
but are found within the protozoans and metazoans, among other
groups. In many ways, infectious parasitic diseases resemble
infectious diseases caused by microbiologicals such as fungi,
bacteria and viruses.
[0023] Malaria remains one of the major health problems in the
tropics. It is estimated that 300 million people a year are
infected with malaria (World Health Organization, 1990, Malaria pp.
15-27. In Tropical Diseases, Progress in Research 1989-1990,
Geneva). Malaria is transmitted by Anopheles mosquitos in endemic
areas, and often by blood transfusion in eradicated areas.
[0024] Malaria in humans is caused by at least four protozoan
species of Plasmodium: P. falciparum, P. vivax, P. ovale and P.
malariae. The asexual erythrocytic parasite, merozoite, is the
stage in the life cycle that causes the pathology of malaria with a
characteristic pattern of fever, chills and sweats. Anemia, acute
renal failure and disturbances in consciousness are often
associated with malarial infection. P. falciparum can produce a
large number of parasites in blood rapidly, and causes the most
morbidity and mortality.
[0025] The most important treatment of malaria to date is
chemotherapy using a number of natural and synthetic drugs.
Antifolates, such as pyrimethamine, inhibit the parasite's
dihydrofolate reductase, whereas the aminoquinolines, such as
chloroquine (4-aminoquinoline) have the digestive vacuoles as their
major site of action. Prior to the introduction of chloroquine in
the 1940's, quinine was the only effective drug for treatment of
malaria. Chloroquine is commonly used to treat acute infections
with all four species, but has no effect on relapses of infection
by P. vivax or P. ovale. Chloroquine (500 mg weekly) may also be
used to prevent malaria by suppressing the stages that multiply in
the erythrocytes and cause the symptoms.
[0026] However, the use of these drugs in certain areas and in the
future will be seriously hampered by the emergence of drug
resistant parasites. Chloroquine resistance is widespread and will
continue to appear in new areas. Due to the possibility of
resistance, the presence of parasites in blood (i.e., parasitemia)
is followed closely during treatment, and alternative drugs
instituted if indicated. The decision on drug regimen will depend
on the origin of the infection. Combination therapy, such as
quinine and Fansidar (pyrimethamine and sulfadoxine), is applied to
treat chloroquine-resistant P. falciparum. Because of the presence
of multidrug resistant P. falciparum in many parts of the world,
prevention of malaria by chemoprophylaxis with currently available
drugs is not always effective.
[0027] In the last 20 years, only several drugs, such as
mefloquine, halofantrine and artemisinin derivatives, have been
developed to treat P. falciparum (Nosten et al., 1995, Drug Saf.
12:264-73). In view of the continuing spread of multidrug resistant
P. falciparum, it is apparent that novel effective chemotherapeutic
agents are needed for use against malaria.
3 SUMMARY OF THE INVENTION
[0028] The present invention encompasses a class of alkyl aryl
carbonyl compounds that forms stable binding interactions,
preferably through formation of reversible covalent bonds, with one
or more basic amino acid residues, wherein such basic amino acid
residues are a part of a nuclear localization signal (NLS). The
stable binding interaction results in the inhibition or
neutralization of the nuclear localization activity of the NLS. The
binding interaction is mediated by one functional component of the
compound, i.e., the reactive group, whereas another functional
component of the compound, i.e. the targetting group, determines
the specificity of the compound for different NLS.
[0029] This targetting function occurs by interaction of the
targetting group with a docking site that is positioned proximately
to the susceptible basic residues of the target NLS, such that
docking of the compound places it in a favorable configuration to
form a stable interaction with basic amino acid residues of the
target NLS. The docking site is located either on the same
NLS-bearing protein, or on another component of a larger molecular
complex that includes the NLS-bearing protein.
[0030] Preferred compounds of the invention provide divalent aryl
carbonyl moieties as the reactive group, particularly aryl
bis(ketone), aryl bis(.alpha.-diketone) or aryl
bis(.beta.-diketone), linked to a targetting group, preferably to a
nitrogen-containing heterocyclic functionality via an N-linkage.
Particularly preferred compounds provided are bis acetyl,
propanoyl, glyoxyloyl, pyruvoyl, 2-oxobutanoyl, acetoacetyl,
3-oxopentanoyl, 3-oxo-2,2-dimethylbutanoyl or
3-oxo-2,2-dimethylpentanoyl substituted aniline moieties N-linked
to a pyrimidinium, pyrimidine or triazine moiety.
[0031] The invention further encompasses methods of using the
compounds of the invention to form tandem binding interactions with
proteins having neighboring basic residues. As used, herein,
neighboring basic residues are two basic amino acid residues of a
protein, particularly lysine and arginine residues, the side chain
amino or guanidino functions of which approach each other as
closely as the bis carbonyl functions of the arylene bis(carbonyl)
compounds of the invention, when the protein is in its natured
conformation. As used herein neighboring, adjacent and juxtaposed
are equivalent terms in reference to amino or guanidino moieties,
and refer to the physical locations of the amino or guanidino
moieties in the structure of the native protein and not to the
positions of the basic amino acid residues themselves in the linear
sequence.
[0032] In one embodiment of the invention, the compounds of the
invention can be used to react with neighboring nitrogenous
moieties in lysine and arginine residues of nuclear localization
sequence (NLS) of a protein, thereby inactivating the NLS. The
invention also encompasses uses of compounds of the invention for
specifically inhibiting importation of a NLS-containing protein or
molecular complex comprising a NLS-containing protein, into the
nucleus of a cell. The carbonyl group(s) of the compounds of the
invention inactivates the NLS of a protein which is essential for
nuclear translocation of the complex. The compounds of the
invention are targeted to the complex by the specific interaction
between the targetting group, preferrably a nitrogen-containing
heterocyclic targetting group, and a docking site on a molecule in
the complex that is distinct from the NLS. The invention further
encompasses methods of screening for alkyl aryl bis(carbonyl) and
bis(dicarbonyl) compounds that are capable of inhibiting
importation of a specific NLS-containing protein or molecular
complex comprising a NLS-containing protein into the nucleus of a
cell. The screening assays of the present invention allow a
compound of the present invention to be identified and selected
without advance knowledge of the specific docking site that confers
specificity on the NLS inhibitory activity of the compound.
[0033] The invention further encompasses methods of inhibiting
productive infection by HIV-1 of terminally differentiated
(non-dividing cells), particularly macrophages, by inhibition of
the importation of the cytoplasmic HIV-1 complex into the nucleus
of cell. Particularly the invention concerns the administration of
the compound effective to block such importation to a cell. Thus,
in one embodiment, the invention encompasses methods of using the
above-described compounds to prevent productive infection of
terminally differentiated macrophages and resting T-cells in HIV-1
infected subjects.
[0034] Without limitation as to theory, the compounds of the
invention is believed to block HIV-1 replication by binding to
reverse transcriptase and formation of tandem Schiff bases with
neighboring N.sup..epsilon. moieties of lysines in the nuclear
localization signal of HIV matrix antigen. As a result, the matrix
antigen is unable to interact with karyopherin .alpha. of the host
cell and the viral nucleoprotein complex does not pass across the
nuclear membrane via interaction with the nuclear pore transport
complex and/or other cellular components.
[0035] Moreover, compounds of the present invention are also useful
for inhibiting viral infection or nuclear translocation of viral
proteins in proliferating cell populations, to the extent that such
occurs in some of the cells in the population during periods of the
cell cycle in which the nuclear membrane is intact. Such infection
or nuclear translocation of proteins in a proliferating population
of cells is susceptible to treatment with the compounds of the
present invention on the same basis as non-dividing or quiescent
populations would be susceptible.
[0036] The invention further encompasses methods of using the
compounds of the invention in treating or preventing infectious
diseases such as those caused by parasites, particularly Plasmodium
species that cause malaria.
4 BRIEF DESCRIPTION OF THE FIGURES
[0037] FIG. 1A-C. The structures of exemplary Compounds No. 2, 11
and 13 are, respectively, FIGS. 1A, 1B, 1C.
[0038] FIG. 2A-C. The effect of various concentrations of Compound
No. 2 on RT activity in the supernatant of HIV-1-infected
monocytes. FIG. 2A: Multiplicity of Infection (MOI) 1 ng
p24/10.sup.6 monocytes, cultured in presence of M-CSF. FIG. 2B: MOI
8 ng p24/10.sup.6 monocytes, cultured in absence of M-CSF. FIG. 2C:
MOI 0.8 ng p24/10.sup.6 monocytes, cultured in absence of
M-CSF.
[0039] FIG. 3. The effect of various concentrations of Compound No.
2 on RT activity in the supernatant of HIV-1-infected
mitogen-stimulated peripheral blood leukocytes at infected at 10
and 1.0 ng p24/10.sup.6 cells, FIGS. 3A and 3B, respectively.
[0040] FIG. 4A-F. The structures of the compounds used in Example 7
are shown respectively in FIGS. 4A-4F. FIG. 4A:
2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium
chloride (CNI-0294). FIG. 4B:
2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine (CNI-1194).
FIG. 4C: 2-amino-4-(3-acetylphenyl)amino-6-methylpyrimidine
(CNI-1594). FIG. 4D:
2-amino-4-(4-acetylphenyl)amino-6-methylpyrimidine (CNI-1794). FIG.
E: 3.5-diacetylaniline (CNI-1894). FIG. 4F:
4-phenylamino-2-amino-6-methylpyrimidine (CNI-4594).
[0041] FIG. 5. Representative plasma concentrations over time in
mice treated with CNI-1194. Female ND4 Swiss-Webster mice were
given a single 50 mg/kg injection intraperitoneally (circles) or
orally (squares). The calculated plasma concentrations, in
.mu.g/ml, was then plotted against the time of sampling.
[0042] FIGS. 6A-6B. Chromatograms of plasma extracts from animals
treated with CNI-0294 or CNI-1594. Female ND4 Swiss-Webster mice
were given a single i.p. injection of 50 mg/kg CNI-0294 (A) or 20
mg/kg CNI-1594 (B). The chromatogram shown for CNI-0294 was from
the 2 hr time point, and that for CNI-1594 for the 1 hr time point.
The peaks labeled "2" and "15" are the parent peaks for CNI-0294
and CNI-1594 respectively. The other peaks in the chromatogram
represent possible metabolites (labeled "x") and endogenous plasma
peaks.
[0043] FIGS. 7A-7D. The in vitro metabolism of the CNI compounds.
The drugs were incubated with mouse liver post-mitochondrial
supernatants and NADPH for various lengths of time. The
chromatograms shown are from the 60 min time point for (A)
CNI-0294, (B) CNI-1194, (C) CNI-1594, and (D) CNI-1894. The peaks
labeled "2, 11, 15, 18" refer to the parent compound peaks, and
those labeled "a-n" to putative metabolite peaks that increased
over time and were not present in control incubations. All
off-scale peaks were single peaks, and the scale was chosen to
allow presentation of trace metabolite peaks.
[0044] FIGS. 8A-8D. The in vivo metabolism of the CNI compounds.
Female ND4 Swiss Webster mice received a single intraperitoneal
dose of (A) 50 mg/kg CNI-0294, (B) 50 mg/kg CNI-1194, (C) 20 mg/kg
CNI-1594, or (D) 50 mg/kg CNI-1894. In all four graphs, the open
bar represents the peak area of the parent compound and the black
bars the apparent metabolite peaks. The metabolite peaks shown are
(from left to right in each graph): (a) peak "d" (see FIG. 7 for
letter-designated peaks), peak "a", peak "c", and a peak eluting at
13 minutes; (b) peak "h", peak "e", peak "f", peak "g", a peak
eluting at 14 minutes, and a peak eluting at 23 minutes; (C) peak
"j", peak "i", peak "l", and a peak eluting at 14 minutes; (D) peak
"m", peak "n", and a peak eluting at 11 minutes. The peak area
units are arbitrary and calculated by the HPLC operating
system.
[0045] FIG. 9. The activity of CNI-0294 against Plasmodium berghei
infected mice. Female ND4 Swiss Webster mice were infected with
infected erythrocytes and then treated once daily, for four days,
with 50 mg/kg CNI-0294, or with distilled water. Six hours after
the last dose, thin blood smears were made from each of the animals
and the parasitemia was determined. The bars represent the median
parasitemia (n=4 for controls and n=5 for treated).
[0046] FIG. 10A-10C. Binding of HIV-1 nucleoprotein complexes to
karyopherin .alpha..
[0047] A. Binding of HIV-1 to karyopherin .alpha. is mediated by
both MA and Vpr.
[0048] Cytoplasmic extracts prepared 4 hours after infection of H9
cells with equivalent amounts (100 ng of p24 per 10.sup.6 cells) of
wild-type HIV-1.sub.NLHX, or variants carrying inactivating
mutations in MA NLS or/and Vpr were divided in two aliquots. DNA
was extracted from one aliquot and quantified by PCR using primers
specific for the HIV-1 pol gene (bottom panel). The obtained signal
represented the total amount of the HIV-1 DNA in the cytoplasm. The
second aliquot was incubated with GST-karyopherin .alpha.
immobilized on Sepharose beads. HIV-1 DNA was extracted from the
beads and analyzed by PCR using pol-specific primers. The obtained
signal represented the amount of HIV-1 pre-integration complexes
bound to karyopherin .alpha..
[0049] B. Binding of HIV-1 pseudovirion nucleoprotein complexes to
karyopherin .alpha. is mediated by MA NLS.
[0050] H9 cells were inoculated with HIV-like gag-env pseudovirions
that contain gag RNA; equal amounts of wild-type (wt) and mutant
pseudovirions that carry amino acid substitutions in the MA NLS
(.DELTA.MA NLS) were used. Cytoplasmic extracts prepared from
infected cells were incubated with polyclonal anti-MA serum
(.DELTA.MA, lane 2), pre-immune serum (NSS, lane 3), or nothing
(lanes 1, 4, and 5). Samples were then mixed with GST-karyopherin
.alpha. immobilized on glutathione Sepharose beads for 30 min at
25.degree. C. Nucleic acids were extracted from Sepharose beads and
quantified by RT-PCR using primers specific for HIV-1 gag gene. To
control for possible differences in cell entry of wild-type vs.
mutant agents, HIV-specific nucleic acids were extracted directly
from cytoplasmic extracts and assayed by RT-PCR (lanes 4 and
5).
[0051] C. CNI-H0294 inhibits interactions of karyopherin .alpha.
with HIV-1 pre-integration complexes, but not with
pseudovirion-derived nucleoprotein complexes.
[0052] Cytoplasmic lysates of H9 cells infected with HIV-1RF (upper
panel) or HIV-like pseudovirions (bottom panel) were treated for 2
hours with various concentrations of CNI-H0294, and were then mixed
with GST-karyopherin .alpha. immobilized on glutathione Sepharose
beads. HIV-1 DNA or RNA that co-precipitated with karyopherin
.alpha. was quantified as in A and B.
[0053] FIG. 11. CNI-H0294 binds to recombinant RT in solution.
[0054] Twenty nmol of [.sup.14C]-CNI-H0294 were mixed with 0.28
nmol of recombinant MA or RT (a p51/p66 heterodimer) in 40 .mu.l of
binding buffer. Samples were incubated for 2 h at 37.degree. C. in
the presence or absence of 200 nmol of unlabeled CNI-H0294. MA and
RT proteins were immunoprecipitated using protein G agarose and
sheep polyclonal anti-MA (.alpha.MA) or rabbit anti-RT (.alpha.RT)
sera, respectively. Pre-immune sera (PI) was used as control. Bound
material was eluted from protein G using 0.1 M glycine buffer [pH
2.8] and the radioactivity in the eluate was quantified in a
scintillation counter.
[0055] FIG. 12A-12C. CNI-H0294 interacts with RT to produce the
anti-HIV effect.
[0056] A. CNI-H3094 competes with CNI-H0294 for binding to RT.
[0057] Recombinant RT (0.2 .mu.M) was incubated with 3.3 .mu.M of
[.sup.14C]-labeled CNI-H0294 and increasing concentrations of
unlabeled (cold) CNI-3094. The amount of [.sup.14C]-CNI-H0294 that
bound to RT was measured as in FIG. 11.
[0058] B. CNI-H3094 reduces CNI-H0294-mediated inhibition of
HIV-karyopherin .alpha. interaction.
[0059] Cytoplasmic lysates prepared from H9 cells infected with
HIV-1RF were treated with 10 .mu.M CNI-H3094 (lane 1) or with 1
.mu.M CNI-H0294 and 10 .mu.M (lane 5), 5 .mu.M (lane 4), 1 .mu.M
(lane 3), or no CNI-H3094 (lane 2). The amount of pre-integration
complexes available for interaction with karyopherin .alpha. was
quantified as in FIG. 10.
[0060] C. CNI-H3094 inhibits anti-HIV activity of CNI-H0294 in
monocyte cultures.
[0061] Monocytes infected with HIV-1.sub.ADA were cultured in the
presence of CNI-H0294 and CNI-H3094 in various concentrations. Nine
days after infection, RT activity in culture supernatants was
quantified. The results are presented as percent of total RT
activity in untreated cultures (control). Three independent samples
were assayed for each drug concentration, and the standard
deviation was less than 15%.
[0062] FIG. 13A-13B. Analysis of CNI-H0294 interactions with the
HIV-1 pre-integration complex.
[0063] A. CNI-H0294 does not disrupt the interaction between MA and
HIV-1 cDNA.
[0064] Cytoplasmic extracts prepared from H9 cells infected with
HIV-1RF were treated with 10 .mu.M CNI-H0294 (lanes 2, 3, and 4) or
left untreated (lane 1). Samples were divided into two aliquots.
One aliquot was mixed with GST-karyopherin .alpha. immobilized on
Sepharose beads and the HIV-1 DNA that bound was quantified by PCR.
The second aliquot was immunoprecipitated (IP) with anti-MA serum
(.alpha.MA, lane 3) or with pre-immune serum (NSS, lane 4) as
control.
[0065] B. CNI-H0294 reacts with MA, but only when MA is associated
with the HIV-1 pre-integration complex.
[0066] Cytoplasmic extracts from HIV-1RF-infected H9 cells were
treated with [.sup.14C]CNI-H0294 (10 .mu.M). After borohydride
reduction the extracts were immunoprecipitated with anti-MA
(.alpha.MA), anti-IN (.alpha.IN), or pre-immune serum (PI). As
control, similar reactions were performed-using lysates of
pseudovirion-infected cells which lack RT and thus do not bind
CNI-H0294. Immunoprecipitated radioactivity was quantified in a
scintillation counter.
[0067] FIG. 14A-14B. CNI-H0294 inhibits MA-, but not Vpr-mediated
binding of HIV-1 pre-integration complexes to karyopherin
.alpha..
[0068] H9 cells were infected with equal amounts of wild-type
HIV-1.sub.NLHX or with mutant HIV-1.sub.NLHXthat lack Vpr
(Vpr.sup.-) or carry a mutation that inactivates the MA NLS (MA
NLS.sup.-). Infected cells were washed and incubated for 4 h. An
aliquot of each sample was used to quantify the total HIV-1 DNA
(FIG. 14A) and the rest was used to prepare cytoplasmic extracts.
Extracts were incubated with 1 .mu.M CNI-H0294 (FIG. 14B, lanes 2,
4, 6, 8) or were left untreated (lanes 1, 3, 5, 7). The amount of
pre-integration complexes available for binding to karyopherin
.alpha. was determined as in FIG. 10.
[0069] FIG. 15. Proposed mechanism of CNI-H0294 action.
[0070] CNI-H0294 binds to HIV-1 pre-integration complexes via RT,
and then reacts with an adjacent MA NLS. This interaction prevents
binding of karyopherin .alpha. to MA NLS; this nearly abolishes
nuclear import of the pre-integration complexes. Note that Vpr
binds to karyopherin .alpha. even in the presence of CNI-H0294;
this explains the low level of import detected in the presence of
the drug.
5 DETAILED DESCRIPTION OF THE INVENTION
5.1 The Compounds and Methods of Their Synthesis
[0071] The present invention encompasses a class of alkyl aryl
carbonyl compounds that forms stable, but preferably reversible,
and most preferably reversible covalent interactions with one or
more basic amino acid residues, wherein such basic amino acid
residues are a part of a nuclear localization signal (NLS). The
stable covalent interaction results in the inhibition or
neutralization of the nuclear localization activity of the NLS.
[0072] Two structural features are involved in conferring such
capabilities of a compound of the invention: (a) a moiety
comprising at least one carbonyl group that reacts with the side
chains of basic amino acid residues in the target protein, i.e.,
the reactive group; and (b) a second moiety, i.e., the targetting
group, that interacts with a specific docking site and determines
the specificity of the compound for different NLS.
[0073] Although the reactive group(s) of the compounds of the
invention can react reversibly with any susceptible basic side
chains in a protein, e.g., arginines and lysines, the interaction
between the targetting group and the docking site confers
specificity to the activity of the carbonyl group(s), such that the
carbonyl group(s) react only with particular target residue(s) in a
protein. This targetting function occurs by interaction of the
targetting group with a docking site that is located proximately to
the susceptible side chains of basic amino acid residues of the
target NLS, such that docking of the compound places it in a
favorable configuration to form a stable interaction with the side
chains of the basic amino acid residues of the target NLS. It is to
be understood that the docking site is located either on the same
NLS-bearing protein, or on another component of a larger molecular
complex that includes the NLS-bearing protein.
[0074] Preferred compounds of the invention provide divalent aryl
carbonyl moieties as the reactive group, particularly aryl
bis(ketone) or aryl bis(.alpha.-diketone), aryl
bis(.beta.-diketone), linked to a targetting group, preferably to a
nitrogen-containing heterocyclic functionality via an N-linkage.
Particularly preferred compounds provided are bis acetyl,
propanoyl, glyoxyloyl, pyruvoyl, 2-oxobutanoyl, acetoacetyl,
3-oxopentanoyl, 3-oxo-2,2-dimethylbutanoyl or
3-oxo-2,2-dimethylpentanoyl substituted aniline moieties N-linked
to a pyrimidinium, pyrimidine or triazine moiety.
[0075] The compounds of the present invention form reversible
adducts with a target NLS containing protein. In particular, the
compounds form Schiff bases with adjacent lysine residues, and
other reversible adducts with adjacent arginine residues. Thus, the
compounds of the invention, e.g., aryl bis(ketone), can
advantageously be used for inactivation of an NLS where the NLS
comprises lysine residues. On the other hand, where the NLS
contains arginine residues, aryl bis(diketone) compounds,
particularly dimethyl-substituted compounds, i.e., those lacking a
methylene hydrogen between the ketones, can be used
advantageously.
[0076] In one particular embodiment, the compounds of the invention
are capable of forming Schiff bases with lysine residues of a
target NLS containing protein in a molecular complex, and
interacting with a specific docking site on a molecule in the
molecular complex, said docking site being positioned proximately
to the lysine residues in the protein. Because of the proximity of
the target lysine residues to the docking site, the interaction of
the targetting group with the docking site localizes the compound
of the invention to the vicinity of the target lysine residues,
thereby facilitating the reaction of the carbonyl group(s) of the
compound with the target N.sup..epsilon. group of lysine residue(s)
in the NLS, and resulting in the formation of stable Schiff bases
and inactivation of the NLS.
[0077] Specific compounds of the invention, e.g., compound No. 2 or
CNI-H0294, or
2-Amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium salts,
and their synthesis are described in section 6 et seq, and are
disclosed in earlier filed U.S. patent application Ser. Nos.
08/369,830, 08/463,405, 08/471,797, 08/470,103, and 08/584,857 each
of which is hereby incorporated by reference.
[0078] According to the invention, the compounds of the invention
are alkyl aryl carbonyl compounds of formula (I): ##STR1## wherein
A, independently, .dbd.CH.sub.3, CH.sub.2CH.sub.3, COH, COCH.sub.3,
COCH.sub.2CH.sub.3, CH.sub.2COCH.sub.3, CH.sub.2COCH.sub.2CH.sub.3,
C(CH.sub.3).sub.2COCH.sub.3, C(CH.sub.3).sub.2COCH.sub.2CH.sub.3 or
the like to yield an acetyl, propanoyl, glyoxyloyl, pyruvoyl,
2-oxobutanoyl, acetoacetyl, 3-oxopentanoyl,
3-oxo-2,2-dimethylbutanoyl or 3-oxo-2,2-dimethylpentanoyl
substituted aniline; P=1 or 2; L is a linker group containing an S,
O, N or C atom, e.g., --SO.sub.2--, --O--, --NH--, --N.dbd.,
--CH.sub.2-- or --CH.dbd.; K is 0 or a positive integer, preferably
K=1; and wherein J represents (i) a saturated or unsaturated,
substitued or unsubstituted, straight or branched acyclic
hydrocarbon group; (ii) a saturated or unsaturated, substitued or
unsubstituted, straight or branched acyclic group containing hetero
atoms such as nitrogen, sulfur or oxygen; (iii) a substituted or
unsubstituted, saturated or aromatic, mono- or poly-cyclic group
having 3 to 20 carbon atoms; or (iv) a substituted or
unsubstituted, saturated or aromatic, mono- or poly-heterocyclic
group having 3 to 20 atoms, at least one of which is a nitrogen,
sulfur or oxygen. The compounds of the invention can contain one or
more linker groups (L), however, if J contains a linker group as
defined above, K can be 0.
[0079] The acyclic and cyclic groups defined above may be saturated
or unsaturated and may, if desired, bear one or more substituents.
Exemplary of such substituents are alkyl, alkoxy, phenoxy, alkenyl,
alkynyl, phenylalkyl, hydroxyalkyl, haloalkyl, aryl, arylalkyl,
alkyloxy, alkylthio, alkenylthio, phenylalkylthio,
hydroxyalkyl-thio, alkylthiocarbamylthio, phenyl, cyclohexyl,
pyridyl, piperidinyl, alkylamino, amino, nitro, mercapto, cyano,
hydroxyl or a halogen atom.
[0080] For example, J can be a substituted or unsubstituted five or
six membered ring having 1-4 hetero ring atoms, at least one of
which is nitrogen, the remainder of which are selected from
nitrogen, oxygen or sulfur, e.g., pyridine, pyrrole, imidazole,
thiazole, isothiazole, isoxazole, furazan, pyrrolidine, piperidine,
imidazolidine, piperazine, oxazole, tetrazole, pyrazole, triazole,
oxadiazole, thiodiazole. Alternatively, J can be a substituted or
unsubstituted polycyclic group having 1 to 4 hetero ring atoms, one
of which is nitrogen and the remainder of which are nitrogen,
oxygen or sulfur, e.g., indole, quinoxaline, quinazoline,
quinoline, isoquinoline, purine.
[0081] By the term "alkyl" as used herein is meant a straight or
branched chain saturated hydrocarbon group having from 1 to 20
carbons such as methyl, ethyl, isopropyl, n-butyl, s-butyl,
t-butyl, n-amyl, isoamyl, n-hexyl, n-octyl and n-decyl. The terms
"alkenyl" and "alkynyl" are used to mean straight or branched chain
hydrocarbon groups having from 2 to 10 carbons and unsaturated by a
double or triple bond respectively, such as vinyl, allyl,
propargyl, 1-methylvinyl, but-1-enyl, but-2-enyl, but-2-ynyl, 1
methylbut-2-enyl, pent-1-enyl, pent-3-enyl, 3-methylbut-1-ynyl,
1,1-dimethylallyl, hex-2-enyl and 1-methyl-1-ethylallyl. The term
"phenylalkyl" means the aforementioned alkyl groups substituted by
a phenyl group such as benzyl, phenethyl, phenopropyl,
1-benzylethyl, phenobutyl and 2-benzylpropyl. The term "aryl" as
used herein is meant to include a monocyclic, bicyclic, tricyclic
or other polycyclic compounds, wherein at least one ring is
aromatic including aromatic hydrocarbons or hetero-aromatic
hydrocarbons having heteroaromatic atoms such as nitrogen, sulfur
and oxygen. The term "hydroxy-alkyl" means the aforementioned alkyl
groups substituted by a single hydroxyl group such as
2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 4-hydroxybutyl,
1-hydroxybutyl and 6-hydroxyhexyl. The terms "alkylthio,
alkenylthio, alkynylthio, hydroxy-alkylthio and phenyl-alkylthio"
as used herein mean the aforementioned alkyl, alkenyl, alkynyl,
hydroxy-alkyl and phenyl-alkyl groups is linked through a sulfur
atom.
[0082] The term "substituted" as used herein means that the group
in question, e.g., alkyl group, aryl group, etc., may bear one or
more substituents including but not limited to halogen, hydroxy,
cyano amino, nitro, mercapto, carboxy and other substituents known
to those skilled in the art.
[0083] The terms "saturated" as used herein means an organic
compound with neither double or triple bonds. The term
"unsaturated" as used herein means an organic compound containing
either double or triple bonds.
[0084] In another embodiment, the compounds of the invention are
formed according to formula (II): ##STR2## wherein A,
independently, .dbd.CH.sub.3 or CH.sub.2CH.sub.3 and P=1 or 2; and
##STR3## wherein X.dbd.NH.sub.2, CH.sub.3 or CH.sub.2CH.sub.3;
X'.dbd.CH.sub.3 or CH.sub.2CH.sub.3; Y.dbd.NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, 1-pyrrolidino or 1-piperidino; and Z.dbd.H,
CH.sub.3 or CH.sub.2CH.sub.3; or ##STR4## wherein Y' and Z',
independently, .dbd.H, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2 or
N.sup.+ (CH.sub.3).sub.3, 1-pyrrolidino or 1-piperidino; Q is N or
CH; and salts thereof.
[0085] In a preferred embodiment the compounds of the invention are
bis ketone arylene compounds having a third nitrogenous
substituent. The nitrogenous substituent can be further substituted
with an aromatic nitrogen-containing heterocyclic compound.
[0086] More precisely the bis ketone arylene compounds of the
invention are formed according to the formula (III): ##STR5##
wherein A.dbd.CH.sub.3 or CH.sub.2CH.sub.3 and ##STR6## wherein
X.dbd.NH.sub.2, CH.sub.3 or CH.sub.2CH.sub.3; X'.dbd.CH.sub.3 or
CH.sub.2CH.sub.3; Y.dbd.NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2,
1-pyrrolidino or 1-piperidino; and Z.dbd.H, CH.sub.3 or
CH.sub.2CH.sub.3; or ##STR7## wherein Y' and Z', independently,
.dbd.H, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2 or N.sup.+
(CH.sub.3).sub.3, 1-pyrrolidino or 1-piperidino; Q is N or CH; and
salts thereof.
[0087] In yet another embodiment of the invention, the compounds of
the invention are compounds formed according to formula (I),
wherein J is not R; L is not --NH--; and wherein ##STR8## and
wherein, independently, X.dbd.NH.sub.2, CH.sub.3 or
CH.sub.2CH.sub.3; X'.dbd.CH.sub.3 or CH.sub.2CH.sub.3;
Y.dbd.NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2; and Z.dbd.H,
CH.sub.3 or CH.sub.2CH.sub.3; or ##STR9## wherein Y' and Z',
independently, .dbd.H, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2 or
N.sup.+ (CH.sub.3).sub.3; and salts thereof.
[0088] The compounds of the present invention can be synthesized by
reacting aniline--to form a compound of formula (II), wherein P is
0--or an acetyl or propanoyl derivative of aniline--to form a
compound of formula (II), wherein P is 1--or a diacetyl or
dipropanoyl derivative of aniline--to form a compound of formula
(II) or formula (III) wherein P is 2.revreaction.with a chloro
derivative of purine, aminomethylpyrimidine, diamino-triazine, or
with a cyanoguanidine. The reaction can be performed at
90-100.degree. C. in an aqueous solvent in the presence of a
mineral acid to yield the corresponding aminophenyl pyridine or
triazine. The pyrimidinium can be synthesized from the pyrimidine
by reaction with an excess methyl iodide at 40-45.degree. C. under
reflux conditions in 1:1 acetonitrile/tetrahydrofuran or in a 1:1:2
mixture of dichloromethane/acetonitrile/tetrahydrofuran.
[0089] For synthesis of aryl bis(diketone) compounds, the acyl
groups attached to the benzene ring, for example in
3,5-diacetylaniline, may be converted to 2-oxoacyl groups by
reaction with selenium dioxide or selenious acid in wet dioxane or
other suitable medium (Rabjohn, N., 1976, Org. React. 24:261-415).
Acyl groups attached to benzene rings may be converted to
higher-chain 3-oxoacyl groups by treatment with strong base such as
sodium hydride or sodium metal in the presence of an aliphatic
ester such as ethyl acetate, when the acyl groups are of the
structure X--CH.sub.2--CO-- (March, J., 1992, Advanced Organic
Chemistry, 4th ed., Wiley Interscience, New York, pp 491-493;
Fenton, D. E. et al., 1980, Inorg. Chim. Acta 44:L105-L106)
[0090] Any chemistries for generating chemical libraries known in
the art can be used to form the J group in the compound of formula
(I), including but not limited to combinatorial chemistries, in
which interchangeable chemical building blocks are systematically
assembled to provide diverse structures. See generally Gordon, E.
M. et al., 1994, J. Med. Chem. 37:1385; Chen, C. et al., 1994, J.
Amer. Chem. Soc. 116:2661; Cho, C. Y. et al., 1993, Science
261:1303. The modification and adaptation of the various
chemistries for generating diversities for the formation of the J
group in the compound of formula (I) will be apparent to persons of
skill in the art. Moreover, automated synthesis systems can be used
to generate the desired chemical diversities including, for
example, workstations and robots made by Takeda Chemical Industries
Ltd, Osaka, Japan; Zymark Corporation, Hopkinton, Mass.; and
Hewlett Packard, Palo Alto, Calif.
5.2 The Inhibition of HIV-1 Importation into the Nucleus of
Non-Dividing Cells
[0091] A quantitative measurement of the activity of the compounds
of the invention to block the replication of HIV-1 in non-dividing
cells can be determined by culture of a macrophage-tropic strain of
HIV-1 on peripheral blood-derived macrophages. The cells are
cultured for 5-6 days prior to infection in a medium consisting of
DMEM supplemented with 10% type A/B human serum and 200 U/ml
Macrophage Colony Stimulating Factor, with half the medium changed
after 3 days, to reach a density of about 10.sup.6 cells per 5 ml
well. A macrophage-tropic viral stock may be grown on these cells.
The concentration of infectious particles in the stock is estimated
by measurement of p24 antigen concentration.
[0092] To test the effect of compounds of the invention on HIV-1
infection in the above-described culture system, the medium is
removed and replaced with medium containing HIV-1 at a
concentration of 1 ng of p24 (10.sup.4 TCID.sub.50/ml (TCID=tissue
culture infectious doses)) and a known concentration of the
compound of the invention (the inhibitor). After 24 hours, the
cultures are washed to remove non-adherent virus and the culture is
re-fed with medium containing the inhibitor at the desired
concentration. The amount of replication of HIV-1 is estimated by
an assay of the reverse transcriptase activity or by an assay of
the concentration of p24 antigen in the culture medium every 2-3
days throughout the post-infection period. In a preferred
embodiment the anti-HIV potency of the candidate drug is measured
by comparison of the concentration of reverse transcriptase (RT) or
of p24 antigen in the medium of the treated and control cultures at
the time of the peak of these values in non-treated control
cultures, that is about day 5 or 6 post-infection. Repetition at
various levels of inhibitor allows for the calculation of the
concentration of inhibitor that achieves 50% inhibition of viral
growth, IC.sub.50. Table I discloses the IC.sub.50 of various
inhibitors. TABLE-US-00001 TABLE I Compound IC.sub.50
2-amino-4-(3,5-diacetylphenyl)amino-1,6- 1 nM dimethylpyrimidinium
iodide (Compound No. 2) 2-amino-4-(3-acetylphenyl)amino-1,6- 10 nM
dimethylpyrimidinium iodide (Compound No. 14)
2-amino-4-(3,5-diacetylphenyl)amino-6- 50 nM methylpyrimidine
(Compound No. 11) 4-(3-acetylphenyl)amino-2-amino-6- 15 nM
methylpyrimidine (Compound No. 15)
[0093] Alternatively, the compounds may all be compared for
inhibition of HIV replication at a fixed concentration. Presented
in Table II are compounds that were used at a concentration of 100
nM to inhibit the production of HIV-1 in cultured monocytes
infected with HIV-1 10 days prior to assay (10 ng of p24/10.sup.6
cells). The production of HIV-1 in each treated culture is reported
as percentage of untreated control. TABLE-US-00002 TABLE II Viral
Compound Production N-(3,5-diacetylphenyl)biguanide hydrochloride
12% (Compound No. 12)
2-(3,5-diacetylphenyl)amino-4,6-diamino-1,3,5-triazine 14%
(Compound No. 13)
4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine 20% (Compound
No. 17) 3,5-diacetylaniline 20% N,N-dimethyl-3,5-diacetylaniline
25% 2,6-diacetylaniline 28% 3,5-diacetylpyridine 58%
[0094] FIG. 2A presents further results of the use of the most
active of the compounds of Table I, Compound No. 2, to block the
replication of HIV-1 in purified monocytes, cultured in medium
supplemented with monocyte-colony stimulating factor (M-CSF). The
cultures were treated with none or between 10.sup.-12 and 10.sup.-6
M Compound No. 2 and, simultaneously with the beginning of
treatment, the cells were exposed to the monocyte-tropic strain
HIV-1.sub.ADA at about 0.01 TCID.sub.50/cell (1 ng p24/10.sup.6
cells) for 2 hours. Samples were withdrawn at days 3, 6, 10, 14 and
17 after infection and assayed for reverse transcription activity.
Compound No. 2 does not inhibit reverse transcriptase, data not
shown. The results show that under these conditions the IC.sub.50
concentrations is between 0.1 and 1.0 nM and that a concentration
of between 0.1 .mu.M and 1.0 .mu.M completely inhibits the
replication of the virus.
[0095] FIGS. 2B and 2C show the effects of various concentrations
of Compound No. 2 on the production of HIV-1 in monocyte cultures
not supplemented with M-CSF. In these studies MOI, as determined by
concentration of p24 antigen was; FIG. 2B (8 ng/10.sup.6 cells) and
FIG. 2C (0.8 ng/10.sup.6 cells). These experiments showed
IC.sub.50s of about 10 nM and of less than 1.0 nM respectively.
[0096] The inhibition of the replication of HIV-1 is not due to
general cytotoxic effects of the compound. Concentrations of
Compound No. 2 as high as 10 .mu.M were without toxic effects on
the monocyte cultures as determined by lactate dehydrogenase
release and trypan blue exclusion. Further evidence of the
specificity of the inhibition due to Compound No. 2 is provided by
the data presented in FIGS. 3A and 3B wherein mitogen-stimulated
peripheral blood leukocytes were cultured in IL-2-supplemented
medium and were exposed to the HIV-1.sub.ADA at p24 concentrations
of 10 and 1 ng/10.sup.6 cells, respectively. in this experiment up
to 10 .mu.M Compound No. 2 had only a marginal effect on viral
production at the higher MOI. At the lower MOI, 1 and 10 .mu.M of
Compound No. 2 caused an approximate 2-fold reduction in viral
output.
[0097] The inhibition of HIV-1 importation into the nucleus of
non-dividing cells can also be directly measured. One suitable
method to determine directly the activity of compounds of the
invention utilizes a cell line that is susceptible to HIV-1
infection, e.g., MT-4 cells, that is growth arrested by treatment
with aphidicolin and exposed to HIV-1. PCR amplification is used to
detect double-stranded closed circular HIV-1 genomes, which are
formed only after nuclear importation, by selecting primers that
bridge the junction point of the genome. For greater detail see
Bukrinsky, M. I., et al., 1992, Proc. Natl. Acad. Sci.
89:6580-84.
5.3 The Treatment of HIV Infection
[0098] The present invention provides a method of treatment of
HIV-1 infection by administering to an HIV-1-infected subject a
pharmaceutical composition having, as an active ingredient, an
effective amount of a compound of formula (I) and (III), and
particularly a compound of formula (III). In one embodiment the
compound to be administered is Compound No. 2. Pharmaceutical
compositions suitable for oral, intraperitoneal, and intravenous
administration can be used in the practice of the invention. Such
pharmaceutical compositions include, by way of non-limiting
examples, aqueous solutions of the chloride, bicarbonate, phosphate
and acetate salts of Compound No. 2 and pH-buffered mixtures
thereof. The chloride salt of compound 2 is herein referred to as
CNI-0294. Compound 11, Compound 14 and Compound 15 are also known
as CNI-1194, CNI-H1494 and CNI-1594, respectively.
[0099] The effective dose of the active ingredient can be
determined by methods well known to those skilled in medicinal
chemistry and pharmacology. An effective dose is the dose that
achieves in the subject's plasma a concentration of the active
ingredient that is sufficient to inhibit the replication of HIV-1
in monocyte cultures as described in Section 5.4, supra, but does
not lead to cytopathic effects in such cultures.
[0100] The daily dose and dosing schedule to be given a subject can
be determined by those skilled in the art, using the
pharmacokinetic constants set forth in Table III below, to achieve
a target plasma concentration. The target plasma concentration can
be selected by routine pharmacological and clinical investigation
methods well-known to those skilled in the art, and can be based on
a range of concentrations which encompass the IC.sub.50 calculated
for each particular compound. For example, the dose can be adjusted
to achieve a range of target plasma concentrations that included
the IC.sub.50 for the compounds as shown in Table I above.
TABLE-US-00003 TABLE III Pharmacokinetic parameters of the CNI
compounds. CNI- CNI- CNI- CNI- CNI- CNI- CNI- 0294 0294 0294 1194
1194 1594 1894 Route of i.p. i.p. oral i.p. oral i.p. i.p.
Injection Dose (mg/kg) 50 50 50 50 50 20 50 Vehicle DP* W* DP W W W
W AUC (.mu. * hr/ml) 9.15 8.83 0.56 3.93 0.57 0.82 20.20 C.sub.max
(.mu.g/ml) 18.76 18.93 0.41 5.70 0.35 1.93 13.43 t.sub.max (min) 5
5 60 15 15 15 5 .alpha. (hr.sup.-1) 1.12 1.74 -- 1.83 -- 2.14 1.19
.beta. (hr.sup.-1) 0.15 0.19 -- 0.19 -- 0.04 0.03 A (.mu.g/ml)
14.00 16.07 -- 5.22 -- 1.10 14.93 B (.mu.g/ml) 0.07 0.05 -- 0.14 --
0.01 0.15 t.sub.1/2.alpha. (hr) 0.62 0.40 -- 0.38 -- 0.32 0.58
t.sub.1/2.beta. (hr) 4.62 3.65 -- 3.65 -- 17.33 23.10 V.sub.D (L)
14.14 19.80 -- 5.21 -- 39.60 6.60 Cl.sub.tot (ml/min) 35.35 62.70
-- 16.50 -- 26.40 3.30 Bioavailability -- -- 0.06 -- 0.15 -- -- *DP
= DMSO/peanut oil, W = water
[0101] For example, using the foregoing pharmacokinetic constants,
particularly, the clearance rate, the daily dose and dosing
schedule needed to obtain a given target average plasma
concentration can be calculated. The results of such calculations
for Compound Nos. 2, 11 and 15 are presented in Table IV. The
calculated doses of Compound Nos. 2 and 15 are considerably below
the toxic levels, as measured by the LD.sub.50, of these compounds.
See, Section 6.4 below. TABLE-US-00004 TABLE IV Target Compound
serum Clearance.dagger-dbl. Dose No. M.W. conc. (ml/min) (mg/Kg
day) 2* 334 10 nM 35.35 6.80 11 280 50 nM 16.50 13.3 15 250 15 nM
26.40 5.70 .dagger-dbl.measured in a 25 gr mouse *Chloride salt
(CNI-0294)
[0102] Using such methods, a dose can be calculated to achieve a
predetermined target plasma concentration. A practicable target
plasma concentration of Compound No. 2 ranges from 0.5 nM to 10 nM;
for Compound No. 11, a practicable target range is from 25 nM to
100 nM; for Compound No. 15, a practicable target range is from 7.5
nM to 50 nM.
[0103] Subjects who can benefit from the administration of the
compounds of the invention according to this method include all
persons infected by HIV-1. More particularly, firstly, those who
benefit include those subjects who have or are at risk to develop
CNS signs of HIV-1 infection and/or subjects that have developed
significant weight loss. Secondly, those who benefit include those
who have been recently exposed to HIV-1, but who do not yet have an
established chronic infection.
5.4 Pharmaceutical Formulations
[0104] Because of their pharmacological properties, the compounds
of the present invention can be used especially as agents to treat
patients suffering from HIV and can be used as agents to treat
patients suffering from other viral infections or chronic diseases
that are dependent upon nuclear localization as part of the
pathogenic process. The compounds of the invention can also be used
to treat or prevent other infectious diseases such as parasitic
diseases, and in particular malaria. Such a compound can be
administered to a patient either by itself, or in pharmaceutical
compositions where it is mixed with suitable carriers or
excipient(s).
[0105] Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present invention,
in particular, those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well-known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated.
[0106] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein.
[0107] In addition to the active ingredients these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions.
[0108] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levitating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0109] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0110] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0111] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0112] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added.
5.5 Use of the Compounds of the Invention to Derivatize
Proteins
[0113] The compounds of the present invention of formula (III),
wherein P is 1 or 2, can be used to derivatize a target protein and
thereby determine the presence of adjacent
N.sup..epsilon.-moieties. The test reaction can be conducted in
aqueous buffer at mild to moderate alkaline pH, between about 7.2
and 8.0. Specific derivatization of the target protein can be
detected by any means that separates protein-bound and free
derivatizing compound. The derivatizing compound optionally can be
detected by radiolabeling it. In one embodiment, the compound can
be synthesized using .sup.14C-methyliodide in place of
methyliodide. Alternatively, use can be made of the strong UV
absorption or fluorescence of the derivatizing compounds. Compound
No. 2, for example has a absorption peak of 16,000 M.sup.-1
cm.sup.-1 at .lamda.=298 nm. In a preferred embodiment the target
protein is derivatized by a compound of the invention, irreversibly
reduced with sodium borohydride or cyanoborohydride and fragmented
into peptides by trypsin or the like. The resultant peptides can be
compared with the peptides obtained from an unreacted sample of the
protein by analysis using any chromatographic or electrophoretic
technique that resolves peptides, e.g., reverse phase High
Performance Liquid Chromatography (HPLC). When the peptides are
resolved by any high resolution chromatography procedure, the
derivatized peptides can be readily detected by their altered
elution time and the absorbance at .lamda.=298 nm.
[0114] In a preferred embodiment the practitioner will conduct the
reaction at various pH points to determine whether a positive
result can be obtained at any point within the expected range. A
positive result, i.e., a result that indicates the presence of
adjacent N.sup..epsilon.-moieties, is one in which a large fraction
of each of a limited number, i.e., between 1-4, of peptides of the
target protein are derivatized and negligible amounts of other
peptides are affected.
[0115] The above-described protein derivatization technique can be
used to determine whether a candidate compound can be used,
according to the invention to prevent productive HIV-1 infection of
macrophages. A comparison of the activity of a candidate compound
and that of Compound No. 2 as derivatizing agents specific for
nuclear localization sequences can be made. A compound that
derivatizes the same peptides to the same extent as Compound No. 2
can be used to practice the invention.
5.6 The Treatment of Infectious Diseases
[0116] The compounds of the present invention can be used to
prevent or treat infectious diseases in animals, including mammals
and preferably humans, and these compounds are. particularly suited
to treatment of parasitic diseases, more particularly, malaria. The
invention described herein provides methods for treatment of
infection, including and without limitation, infection with
parasites, and methods of preventing diseases associated with such
infection. The compounds can reduce parasitemia when administered
to an animal infected with a parasite.
[0117] Infectious diseases may include without limitation:
protozoal diseases such as those caused by Kinetoplastida such as
Trypanosoma and Leishmania, by Diplomonadina such as Giardia, by
Trichomonadida such as Dientamoeba and Trichomonas, by Gymnamoebia
such as Naegleria and the Amoebida such as Entamoeba and
Acanthamoeba, by Sporozoasida such as Babesia and the Coccidiasina
such as Isospora, Toxoplasma, Cryptosporidium, Eimeria, Thelleria,
and Plasmodium; metazoal diseases such as those caused by the
Nematoda (roundworms) such as Ascaris, Toxocara, the hookworms,
Strongyloides, the whipworms, the pinworms, Dracunculus,
Trichinella, and the filarial worms, and by the Platyhelminthes
(flatworms) such as the Trematoda such as Schistosoma, the blood
flukes, liver flukes, intestinal flukes, and lung flukes, and the
Cestoda such as the tapeworms; viral and chlamydial diseases
including for instance those caused by the Poxviridae,
Iridoviridae, Herpesviridae, Adenoviridae, Papovaviridae,
Hepadnaviridae, Parvoviridae, Reoviridae, Birnaviridae,
Togaviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae,
Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae,
Retroviridae, Picornaviridae, Calciviridae and by Chlamydia;
bacterial diseases; mycobacterial diseases; spirochetal diseases;
rickettsial diseases; and fungal diseases.
[0118] In one embodiment, the compounds of the invention having
anti-infective activity are formed according to formula (I), (II)
and (III) as described in section 5.1.
[0119] In another embodiment, the compounds of the invention may be
used therapeutically against infections with Plasmodium species
such as P. falciparum, P. vivax, P. ovale and P. malariae, that
cause acute and recurrent malaria in humans. The compounds of the
invention are also active against infection by other Plasmodium
species, which include P. berghei, P. knowlesi, P. simium, P.
cynomolgi bastianelli and P. brasilianum.
[0120] In yet another embodiment of the invention, the compounds
may be useful in providing chemoprophylaxis for individuals at risk
of infection, such as when travelling in endemic areas. By
maintaining in circulation an effective concentration of a compound
of the invention, malaria can be prevented by suppressing the
pathological stages of infection with Plasmodium species. Without
being bound by any theory, the compounds of the invention can be
effective against various stages of the life cycle of the parasite,
including sporozoites and merozoites, as well as dormant, asexual
and sexual stages. The compounds of the invention may be active in
the blood stream, in erythrocytes, in the liver, or in other
tissues where the malaria parasite may reside.
[0121] In a specific embodiment of the invention, the compound of
the invention can be used to prevent malaria, or to treat malaria,
or to treat infection with Plasmodium species that are resistant to
antimalarial drugs, such as, but not limited to, chloroquine and
pyrimethamine. The antimalarial properties of the compounds are not
diminished against P. falciparum known to be resistant to
chloroquine or pyrimethamine (see section 8 infra). Although not
wishing to be bound by any theory of mechanism of the compounds, it
is contemplated that the compounds interact with biochemical
targets that are different and independent from those affected by
these two classic antimalarial drugs. Thus, the compounds of the
invention may be used preferentially to treat malarial infections
arising out of areas that are known or suspected to harbor
drug-resistant Plasmodium species.
[0122] In a further embodiment, the compounds may contain a single
acyl group, i.e., P=1, on the arylene ring or the acyl group can be
absent therefrom, i.e., P=0, and/or the heterocyclic substituent,
i.e., R, can be uncharged. In the embodiment of the invention
wherein there are two acyl groups, i.e., P=2, on the arylene ring,
it is preferred that such acyl groups are not in an ortho
arrangement relative to each other. In another preferred embodiment
of the invention, the compounds that possess potent antimalarial
activity are arylene bis(methylketone) compounds that contain a
charged heterocylic ring such as a pyrimidinium, as in CNI-0294
(see FIG. 4A).
[0123] The antimalarial properties of the compounds of the
invention can be analyzed by techniques, assays and experimental
animal models well known in the art. For example, the inhibition of
growth of Plasmodium falciparum in vitro by the compounds may be
assessed by the hypoxanthine-incorporation method (Desjardins et
al., 1979, Antimicrob. Ag. Chemother. 16:710-718). The in vitro
antiparasitic activities of several exemplary compounds of the
invention were assessed by this method, and the results are
described in Section 8. The in vivo efficacy of the compounds can
also be tested in mouse models in which parasitemia is enumerated
following administration of the compound (Ager, A. L. 1984, Rodent
malaria models, pp 225-264. In Handbook of Experimental
Pharmacology vol. 68, Antimalarial Drugs, Peters and Richards eds,
Springer-Verlag, Berlin). The in vivo activity of several exemplary
compounds have been evaluated in a four-day suppression model in
mouse, and the results are provided in Section 8.
[0124] The present invention also provides pharmaceutical
compositions. Such pharmaceutical compositions comprises a
prophylactically or therapeutically effective amount of the
compound and a pharmaceutical carrier, such as those described in
section 5.4. More specifically, an effective amount means an amount
effective to prevent development of or to alleviate the existing
symptoms of the subject being treated. Determination of the
effective amounts is well within the capability of those skilled in
the art, especially in light of the detailed disclosure provided
herein. For any compound used in the method of the invention, the
effective dose can be estimated initially from in vitro assays. A
dose can be formulated in animal models to achieve a circulating
range that includes the ICS.sub.50 (i.e., the concentration of
compound which achieves a half-maximal inhibition of growth of
parasite) as determined in the in vitro assay. Such information can
be used to more accurately determine useful doses in subjects, for
example, humans. The dosage may vary within this range depending
upon the dosage form employed and the route of administration.
Various delivery systems are known and can be used for
administration of the compound, e.g., encapsulation in liposomes.
Other methods of administration include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal and oral routes.
[0125] In another embodiment, the invention provides a method of
preventing or treating malaria by administering to a subject in
need thereof an effective amount of a compound of the invention. In
a further aspect there is provided a method of preventing or
treating malaria, especially malaria caused by drug resistant
Plasmodium species in humans, which method comprise administering
to the individual in need thereof an effective amount of a compound
of the present invention and an effective amount of an antimalarial
drug. The invention also provides the use of a compound of the
invention and an antimalarial drug in the manufacture of a
medicament for the prevention or treatment of malaria. Such
antimalarial drugs may include but are not limited to quinine,
aminoquinolines (chloroquine and primaquine), pyrimethamine,
mefloquine, halofantrine, and artemisinins.
[0126] The "adjunct administration" of a compound of the invention
and an antimalarial drug means that the two are administered either
as a mixture or sequentially. When administered sequentially, the
compound may be administered before or after the antimalarial drug,
so long as the first administered agent is still providing
antimalarial activity in the animal when the second agent is
administered. Any of the above-described modes of administration
may be used in combination to deliver the compound and the
antimalarial drug.
[0127] The present invention is to be understood as embracing all
such regimens and the term "adjunct administration" is to be
interpreted accordingly. When a compound of the invention and an
antimalarial drug are administered adjunctively as a mixture, they
are preferably given in the form of a pharmaceutical composition
comprising both agents. Thus, in a further embodiment of the
invention, it is provided a pharmaceutical composition comprising a
compound of the invention and an antimalarial drug, together with a
pharmaceutically acceptable carrier.
5.7 Compounds and Assays for Compounds that Target Specific NLS
[0128] The present invention also provides methods of use of the
alkyl aryl carbonyl compounds as described in section 5.1. The
reactive group(s) of the compounds of the invention are capable of
forming stable but reversible covalent interactions with the side
chain of basic amino acid residue(s) of a protein. In certain
embodiments of the invention, the divalency of the compound ensures
that the compound will form especially stable associations with
sequences, such as nuclear localization signals (NLS), that
comprise multiple basic amino acid residues, such as lysines and
arginines. Nuclear localization signals, in general, and the HIV
matrix antigen (MA) p17 NLS in particular are characterized by a
stretch of basic, and often positively charged, amino acids
typically including one or more lysines. Such NLS-containing
proteins are often associated with other molecules in a
complex.
[0129] The interaction of basic side chain(s) with the reactive
carbonyl group(s) of the compound is facilitated by the prior
interaction of the targetting group of the compound with a specific
docking site, said docking site being positioned proximately to the
side chain of the basic amino acid residue(s) in the target NLS.
The docking site may be located on the NLS-containing protein or
another molecule in a complex comprising the NLS-containing
protein. As a result of the formation of stable but reversible
covalent interactions with the compound, the NLS-containing protein
or the molecular complex comprising NLS-containing protein is
prevented from interacting with cellular receptors for the NLS. The
protein or molecular complex is thus blocked from importation into
the nucleus.
[0130] Although the carbonyl group(s) of the compounds of the
invention can react with alternative susceptible side chains in a
protein, the interaction between the targetting group and the
docking site confers specificity to the activity of the carbonyl
group(s), such that the carbonyl group(s) react only with
particular target basic amino acid residues in a protein. The
specific recognition and binding of the compound to a docking site
determines which basic amino acid residue(s) in a protein will
become inactivated by the carbonyl group(s) of the compound.
Because of the proximity of the basic residues in the target NLS to
the docking site, the interaction of the compound with the docking
site localizes the compound to the vicinity of the target NLS,
thereby facilitating the reaction of the carbonyl group(s) of the
compound with the target NLS. It is to be understood that the
docking site can be on the NLS-containing protein or another
molecule in a complex comprising the NLS-containing protein. Thus,
in one embodiment, the compounds of the invention can be used to
target a specific nuclear localization signal, thereby blocking
importation of specific proteins or molecular complexes into the
nucleus.
[0131] As demonstrated by the experiments in section 9, the
compounds of the invention, particularly Compound No. 2 or
CNI-H0294, are capable of inhibiting nuclear translocation of HIV-1
pre-integration complexes. The inhibitory effect is caused by the
inactivation of the NLS of HIV MA in a reaction that requires the
presence of HIV reverse transcriptase (RT), i.e. the target
nucleoprotein complex. The carbonyl groups of Compound No. 2 or
CNI-H0294 react with the NLS of MA by formation of Schiff base
adducts, and prevent the binding of MA to karyopherin .alpha., the
cellular receptor for NLS, and that blocks translocation of
proteins into the nucleus. The inventors further showed that the
interaction between Compound No. 2 or CNI-H0294 via its pyrimidine
moiety with RT that contains a docking site determines the
specificity of the compound towards HIV pre-integration complex,
and its low cytotoxicity to the host cell. Thus, the alkyl aryl
carbonyl compounds of the invention can be used for targeted
inactivation of NLS in a protein or a molecular complex, such as
the HIV-1 or other viral pre-integration complex, comprising
contacting the protein or molecular complex with a compound of the
invention. Furthermore, variations and modifications of the
targetting group provides for altered binding specificities, and
can serve to target the alkyl aryl carbonyl compound to a different
NLS-containing protein or molecular complex. Thus, it is envisioned
that the alkyl aryl carbonyl compounds of the invention, can be
selected to inactivate specifically various different
NLS-containing proteins or molecular complexes.
[0132] Accordingly, it is contemplated that a molecular complex
comprises at least one NLS-containing protein and can include other
proteins, nucleic acids, carbohydrates, lipids and other biological
molecules. The docking site to which the targeting group of the
compound binds may reside on any molecule in the molecular complex.
In a preferred embodiment, the target of the compound of the
present invention is a viral nucleoprotein complex or a viral
protein. Alternatively, a target may be a cellular protein such as
a transcription factor. An exemplary, non-limiting list of such
targets may include, viruses which translocate both viral proteins
and nucleic acids into the infected cell nucleus, such as human
immunodeficiency virus and other retroviruses, influenza virus,
hepatitis B and hepatitis delta virus, papillomaviruses and
parvoviruses; viruses which translocate only viral protein into the
infected cell nucleus such as adenoviruses, measles and other
paramyxoviruses, herpes viruses, and rabies and other bunyaviruses;
and single protein, such as NF-.kappa.B and other transcription
factors.
[0133] In another embodiment of the invention, screening assays are
provided for identification of alkyl aryl carbonyl compounds of the
invention that can inactivate the NLS of a specific NLS-containing
protein or NLS-containing molecular complex. The assays of the
invention involves monitoring the binding of the NLS-containing
protein or molecular complexes to at least one protein or fragment
thereof that interacts with the NLS in the course of nuclear
translocation, i.e., the cellular receptor, in the presence of an
alkyl aryl carbonyl test compound of the present invention, and a
comparison of such binding in the absence of the test compound. It
is anticipated that the desired compound binds specifically to a
docking site in the NLS-containing protein or molecular-complex,
and inactivates the NLS. As a result, the binding of the NLS
protein or molecular complex to the cellular receptor is reduced or
abolished.
[0134] Any cellular protein(s) that interact with the NLS, and
functionally effect or contribute to the nuclear translocation of
the NLS-containing protein or complex can be used in the assays of
the invention. Fragments of such cellular proteins that bind NLS
can also be used in the assays of the invention. Such cellular
proteins and fragments thereof, collectively referred herein to as
the cellular receptor moiety, may include but are not limited to,
karyopherin .alpha..beta. heterodimer and fragments thereof, or
fusion proteins, such as karyopherin .alpha.-glutathione
S-transferase (GST-karyopherin .alpha.).
[0135] The assays are performed in vitro in which the cellular
receptor moiety is immobilized directly or indirectly onto a solid
support. The NLS-containing protein or molecular complex, purified
or in a cell extract, is contacted with the immobilized cellular
receptor moiety in the presence of test compound. As a control, the
NLS-containing protein or molecular complex is contacted with the
immobilized cellular receptor moiety under the same condition, but
in the absence of the test compound. After an interval sufficient
for binding reactions to occur among the components in the assay,
the solid support is washed to remove any unbound molecules. A
detection procedure is performed with the solid support to quantify
the binding of NLS-containing protein or molecular complex to the
immobilized cellular receptor moiety as compared to binding
reactions in the absence of test compound. The absence of bound
NLS-containing protein or molecular complex, or a reduction in the
binding of the NLS-containing protein or molecular complex to the
solid support, in the presence of a test compound, indicates that
the test compound can be useful in inhibiting the importation of
the specific NLS-containing protein or molecular complex into the
nucleus.
[0136] The detection procedure may employ an antibody or a ligand
that recognizes and binds the NLS-containing protein or a component
of the molecular complex. Alternatively, if the molecular complex
comprises nucleic acids, such as the case of HIV-1 pre-integration
complex, polymerase chain reaction may be employed to detect the
presence of specific nucleic acid sequences on the solid support.
Any appropriate isotopic and nonisotopic labels can be used in
conjunction with the detection procedure. Detection or measurement
of the antibody, ligand or amplified nucleic acids is accomplished
by standard techniques well known in the art. Those skilled in the
art will be able to determine operative and optimal conditions for
the above-described techniques by employing routine
experimentation.
[0137] For example, a screening assay for alkyl aryl carbonyl
compounds that inactivate the NLS of HIV matrix antigen can be set
up as follows. A glutathione S-transferase-karyopherin .alpha.
fusion protein (GST-karyopherin .alpha.) that binds NLS is used as
the cellular receptor moiety. The fusion-protein is immobilized
onto glutathione Sepharose (Pharmacia) by incubation for 30-60
minutes at room temperature. Cytoplasmic extracts from cells that
have been infected by HIV may be prepared by mechanically lysing
infected cells in the presence of protease inhibitors, and removing
the nuclei by centrifugation.
[0138] Alternatively, HIV MA and RT, and possibly other accessory
proteins, such as Vpr, that are present in the pre-integration
complex can be produced by recombinant DNA methods, and
reconstituted in vitro into a complex for use in the assay. The
cytoplasmic extract or the reconstituted MA/RT complex is incubated
at room temperature with the GST-karyopherin .alpha. Sepharose
beads in the presence of various concentrations of compounds of the
invention, for an interval sufficient for binding interactions to
occur, for example, 30 minutes. At the end of the incubation, the
beads are washed to remove any unbound material. To quantitate the
binding of pre-integration complex to the beads, HIV-1 DNA was
isolated from the beads by SDS-proteinase K treatment with
subsequent phenol-chloroform extraction, and analyzed by polymerase
chain reaction using primers specific for the pol gene, as
described in Bukrinsky, M. I., et al., 1995, J. Exp. Med.
181:735-745. Alternatively, the amount of MA or RT bound to the
beads can be determined by an immunoassay using anti-MA or anti-RT
sera. Compounds of the invention that can be used to block nuclear
importation of HIV are indicated by a reduction or inhibition of
binding to the beads by the HIV-1 preintegration complex or
reconstituted MA/RT complex as compared to the control.
6 EXAMPLES
6.1 Synthesis of Specific Compounds
[0139] Compound No. 2, FIG. 1A: A suspension of Compound No. 11
(2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine) (0.284 g),
was suspended in 1:1 acetonitrile-tetrahydrofuran was treated with
methyl iodide (2 mL) and heated at 40-45.degree. C. under a reflux
condenser for 18 hr. Cooling and filtration gave 0.35 g of
2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium
iodide, mp 292.degree. C.
[0140]
2-Amino-4-(3,5-diacetylphenyl)imino-1,4-dihydro-1,6-dimethylpyrimi-
dine. A suspension of 21 g (49.3 mmole) of
2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium iodide
(compound No. 2, synthesized as described in section 6.1) in 1:1
methanol/water (750 mL) at 60.degree. C. was treated with excess 2N
NaOH with cooling to maintain about 60.degree. C. An additional 200
mL of water was added and the mixture was cooled in ice and
filtered to give 14.69 g
2-amino-4-(3,5-diacetylphenyl)imino-1,4-dihydro-1,6-dimethylpyrim-
idine as yellow crystals, mp 219-220.degree..
[0141] 2-Amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium
chloride (CNI-0294). CNI-0294 is the chloride salt of compound No.
2. The base
2-amino-4-(3,5-diacetylphenyl)imino-1,4-dihydro-1,6-dimethylpyrimidi-
ne (14.35 g, 48 mmole) was dissolved in 500 mL of methanol and
treated with HCl gas until precipitation appeared complete.
Filtration gave 12.8 g of white crystals with a faint yellowish
tinge, mp 306.5-307.5.degree..
[0142] Compound No. 11 (CNI-1194): A suspension of
3,5-diacetylaniline (0.885 g) in water (18 mL) was treated with
2-amino-4-chloro-6-methylpyrimidine (0.718 g) and concentrated HCl
(0.42 mL) and heated at 90-100.degree. C. for 30 min. After cooling
the mixture was treated with 10 mL of aqueous 1N KOH. The mixture
was stirred for 10 min and the solid was filtered out, washed with
water, and dried, to give 1.332 g of tan crystals.
Recrystallization from ethyl acetate-2-methoxyethanol gave 1.175 g
of 2-amino-4-(3,5-diacetyl-phenyl)amino-6-methylpyrimidine as light
buff crystals, mp 240-241.degree. C.
[0143] Compound No. 12. A suspension of 3,5-diacetylaniline (0.531
g) in water (8 mL) was treated with cyanoguanidine (0.285 g) and
conc. HCl (0.25 mL) and heated at reflux. After 6 hr the mixture
was cooled and concentrated and 0.248 g of off-white solid was
filtered out and dried to give N-(3,5-diacetylphenyl)biguanide
hydrochloride, mp 260-70.degree. C. (dec).
[0144] Compound No. 13: A suspension of 3,5-diacetylaniline (1.95
g) in water (10 mL) was treated with
2-chloro-4,6-diamino-1,3,5-triazine (1.455 g) and concentrated HCl
(0.1 mL) and heated at reflux for 20 min. After cooling the
hydrochloride of Compound No. 13 separated as a white powder. This
was filtered out, dissolved in 60 mL of boiling aqueous 75%
methanol and treated with triethylamine (1.5 mL). On cooling,
off-white flakes separated. Filtration and drying gave 1.79 g of
2-(3,5-diacetylphenyl)amino-4,6-diamino-1,3,5-triazine, mp
271-2.degree. C.
[0145] Compound No. 14 (CNI-H1494):
4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine, Compound No.
15, (0.968 g) was suspended in acetone (5 mL) containing methyl
iodide (2 mL) was heated at reflux for 48 hr. Filtration after
cooling gave 0.657 g of
4-(3-acetylphenyl)amino-2-amino-1,6-dimethylpyrimidinium iodide as
a white powder, mp 238-40.degree. C.
[0146] Compound No. 15 (CNI-1594): A suspension of
m-aminoacetophenone (2.7 g) and
2-amino-4-chloro-6-methyl-pyrimidine (2.87 g) in 40 mL water was
treated with 1.7 mL concentrated HCl and heated at reflux for 1
hour. Addition of 40 mL 1N KOH gave a light buff solid, which was
filtered out and dried to give 3.8 g
4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine, mp
196-98.degree. C.
[0147] Compound No. 16: A suspension of 3,5-diacetylaniline (0.531
g) in water (10 mL) was treated with 6-chloropurine (0.464 g) and
concentrated HCl (0.25 mL) and heated at reflux for 30 min. After
cooling the mixture was treated with 6 mL of aqueous 1N KOH. The
mixture was stirred for 10 min and the solid was filtered out,
washed with water, and dried, to give 0.80 g of
6-[(3,5-diacetylphenyl)amino]purine, mp dec 340-350.degree. C.
[0148] Compound NO. 17 (CNI-1794): A suspension of
p-aminoacetophenone (1.35 g) and
2-amino-4-chloro-6-methyl-pyrimidine (1.435 g) in 20 mL water was
treated with 0.85 mL conc HCl and heated at reflux for 1 hr.
Addition of 20 mL 1N KOH gave a light buff solid, which was
filtered out and dried to give 2.28 g
4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine, mp
194-196.degree. C. Of this, 1.21 g was treated with methyl iodide
(3 mL) in dimethylformamide (15 mL) at room temperature for 42 hr.
Dilution with ethyl acetate and filtration gave 1.11 g
4-(4-acetylphenyl)amino-2-amino-1,6-dimethylpyrimidinium iodide as
a white powder, mp 302-3.degree. C.
[0149] Compound No. 45. (CNI-4594) A mixture of aniline (0.93 g)
and 2-amino-4-chloro-6-methylpyrimidine (1.44 g) in 36 mL water was
treated with 0.84 mL conc HCl and heated at reflux for 1 hr.
Addition of 20 mL 1N KOH gave a light buff solid, which was
filtered out, dried, and recrystallized from ethyl
acetate/2-methoxyethanol and ethyl acetate/hexane to give 0.69 g
4-phenylamino-2-amino-6-methylpyrimidine, mp 179-180.degree. C.
[0150] Compound No. 46. A suspension of
4-phenylamino-2-amino-6-methylpyrimidine, Compound No. 45, (0.25 g)
in ethanol (4 mL) was treated with methyl methanesulfonate (0.090
g) and heated at reflux for 5 days. Additional methyl
methanesulfonate (0.090 g) was added and the mixture refluxed
another 2 days. Concentration and recrystallization from a mixture
of methanol, ethyl acetate, and tert-butyl ethyl ether gave 0.10 g
of 4-phenylamino-2-amino-1,6-dimethylpyrimidinium
methanesulfonate.
[0151] 3,5-diacetylaniline (CNI-1894) was synthesized as per Ulrich
et al. (1983, J Med Chem 27:35-40). Diacetylanilines substituted in
other positions can be synthesized according to Ulrich et al. supra
or McKinnon et al. (1971, Can J Chem 49:2019-2022). All other
starting materials were obtained from the Aldrich Chemical Co.
Nuclear magnetic resonance spectra and elemental analysis for all
the compounds agreed with expected values.
6.2 The Use Of Compound No. 2 to Inhibit HIV Replication in Primary
Macrophage Lines
6.2.1 Materials and Methods
[0152] Primary human monocytes were obtained from peripheral blood
by Ficoll-Hypaque centrifugation and adherence to plastic as
described previously. Gartner S. P., et al., 1986, Science 233:215.
Briefly, after Ficoll-Hypaque (Pharmacia) separation, PBMCs were
washed 4 times with DMEM (the last wash was done at 800 rpm to
remove platelets) and resuspended in monocyte culture medium [DMEM
supplemented with 1 mM glutamine, 10% heat-inactivated human serum,
1% penicillin+streptomycin mixture (Sigma)] at a density of
6.times.10.sup.6 cells/ml. Cells were seeded in 24-well plates (1
ml per well) and incubated for 2 h at 37.degree. C., 5% CO.sub.2.
Following incubation, cells were washed 3 times with DMEM to remove
non-adherent cells and incubation was continued in monocyte culture
medium supplemented with 250 U/ml human M-CSF (Sigma). Cells were
allowed to mature for 7 days prior to infection with the
monocyte-tropic strain, HIV-1.sub.ADA. Nuovo, G. J., et al., 1992,
Diagn. Mol. Pathol. 1:98. Two hours after infection, cells were
washed with medium and cultured in RPMI supplemented with 10% human
serum. In experiments where PCR analysis was performed, virus was
pretreated with RNAse-free DNAse (Boehinger-Mannheim) for 2 h at
room temperature and then filtered though a 0.2 .mu.m pore
nitrocellulose filter prior to infection.
[0153] PBMCs were purified by Ficoll-Hypaque centrifugation and
activated by 10 .mu.g/ml PHA-P (Sigma) and 20 U/ml recombinant
human IL-2 (rhIL-2) in RPMI 1640 supplemented with 10% FBS
(HyClone). After 24 h incubation, cells were washed and inoculated
with HIV-1.sub.ADA in RPMI 1640 supplemented with 10% FBS. After a
2 h adsorption, free virus was washed away and cells were cultured
in RPMI 1640 supplemented with 10% FBS and 20 U/ml rhIL-2.
[0154] Virus stock and infection. Macrophage-tropic strain
HIV-1.sub.ADA was amplified in primary human monocytes and
concentrated to produce stock with TCID.sub.50 of about
10.sup.5/ml. The concentration of HIV-1 was determined by
immunoassay of viral p24, concentration; using a conversion factor
of 1 ng/200 HIV-1 particles.
6.2.2 p24 and RT Assay
[0155] For p24 assay, sequential 1:9 dilutions of culture
supernatant were prepared and analyzed by ELISA as suggested by the
manufacturer (Cellular Products, Buffalo, N.Y.). For the reverse
transcriptase (RT) assay, 10 .mu.l of culture supernatant was added
to 40 .mu.l of reaction mixture (final composition was 50 mM
Tris-HCl, pH 7.8; 20 mM KCl; 5 mM MgCl.sub.2; 1 mM DTT; 0.1% Triton
X-100; 0.2 OD/ml polyA; 0.2 OD/ml oligo(dT).sub.12-18; and 40
.mu.Ci/ml .sup.3H-dTTP (76 Ci/mmol, DuPont) and incubated 2 hr at
37.degree. C. 5 .mu.l of the reaction mixture was then spotted onto
the DE 81 (Whatman) paper. Paper was air dried and washed 5 times
with 5% Na.sub.2HPO.sub.4, followed by rinsing with distilled
water. After air drying, paper was put on a Flexi Filter plate
(Packard), covered with scintillation fluid and counted in a Top
Count Microplate Counter (Packard). Results are expressed as counts
per minute in 1 ml of supernatant (cpm/ml).
6.2.3 Results Dividing and Quiescent Cells
[0156] The cytotoxicity of Compound No. 2 was tested in monocyte
cultures by trypan blue exclusion assay or lactate dehydrogenase
(LDH) release. By both assays, no cytotoxic effect was observed
with concentrations of the compound up to 10 .mu.M (data not
shown). Results presented in FIG. 2 show the effect of various
concentrations of Compound 2 on HIV-1 replication in monocytes.
From this experiment, we estimate the IC.sub.50 for this compound
between 0.1 and 1 nM. Similar and higher concentrations of the
compound were also tested on activated PBLs. The anti-viral effect
of this compound was much less expressed in these actively dividing
cell populations (FIG. 3). No anti-viral effect was detected when
cultures of replicating cells were infected at the multiplicity of
infection used to infect monocytes.
6.2.4 AZT and Compound No. 2 in Combination
[0157] AZT is a drug that is routinely used to treat HIV-1 infected
persons. However, two factors are known to diminish the
effectiveness of AZT: its toxicity and the emergence of resistant
mutant strains of HIV-1. The effects of both of these factors can
be reduced by administering a second, synergistic HIV-1-inhibitory
drug with AZT.
[0158] In view of these premises, the effects on HIV-1 replication
in human monocyte cultures of the various concentrations of AZT,
alone or in combination with 100 nM Compound No. 2, were tested
using the protocols of Sections 6.2.1 and 6.2.2. Drugs were added
to the monocyte cultures together with HIV-1 at about 10.sup.5
TCID/ml. The concentration of drugs was maintained on refeeding.
HIV-1 replication was assessed by assay of the supernatant for
reverse transcriptase activity. The results are expressed as
mean.+-.std. dev. (cpmx10.sup.-3) in Table V. TABLE-US-00005 TABLE
V Effects of Combined AZT/Compound No. 2 on HIV-1 infected Monocyte
Cultures day-7 day-11 [AZT] (-) No. 2 (+) No. 2 (-) No. 2 (+) No. 2
0 1.46 .+-. 0.43 0.37 .+-. 0.07 1.81 .+-. 0.75 0.72 .+-. 0.30 10 pM
0.92 .+-. 0.21 0.15 .+-. 0.05 1.63 .+-. 0.81 0.18 .+-. 0.06 100 pM
0.79 .+-. 0.14 0.13 .+-. 0.04 1.34 .+-. 0.59 0.15 .+-. 0.06 1 nM
0.60 .+-. 0.28 0.04 .+-. 0.02 1.07 .+-. 0.49 0.09 .+-. 0.03 10 nM
0.05 .+-. 0.02 0.03 .+-. 0.02 0.08 .+-. 0.03 0.07 .+-. 0.03
[0159] These results demonstrate that there is synergy between the
AZT and Compound No. 2. The synergistic effects are most pronounced
at the lower doses of AZT on day 11. For example, 10 pM AZT alone
produces an about 20% reduction in RT activity on day-11, 100 nM
Compound No. 2 alone produces about a 60% reduction. Without
synergy, the combination should produce a 70% reduction
(100.times.(1-(0.8.times.0.4)). Instead the observed reduction was
90%.
6.3 The Compounds of the Invention do not Block the Nuclear
Importation of Essential Proteins in Cells
6.3.1 Direct Demonstration of the Inhibition of HIV-1 Nuclear
Importation by Compound No. 2
[0160] The effects of Compound No. 2 on the nuclear importation of
HIV-1 preintegration complexes can be directly measured by
detecting the presence of circularized duplex HIV-1 genomic DNA.
These duplex circles can be readily detected by PCR amplification
using primers which span the junction of the circularized HIV-1
genome. Bukrinsky, M. I., et al., 1992, Proc. Natl. Acad. Sci.
89:6580-84.
[0161] Briefly, the efficiency of nuclear translocation was
estimated by the ratio between the 2-LTR- and pol-specific PCR
products, which reflects the portion of 2-LTR circle DNA molecules
as a fraction of the entire pool of intracellular HIV-1 DNA. Viral
2-LTR circle DNA is formed exclusively within the nucleus of
infected cells and thus is a convenient marker of successful
nuclear translocation. Bukrinsky, M. I., 1992, Procd. Natl. Acad.
Sci. 89:6580-84; Bukrinsky, M. I., 1993, Nature 365:666-669.
[0162] PCR analysis of HIV-1 DNA: Total DNA was extracted from
HIV-1-infected cells using the IsoQuick extraction kit (Microprobe
Corp., Garden Grove, Calif.). DNA was then analyzed by PCR using
primer pairs that amplify the following sequences: a fragment of
HIV-1 (LTR/gag) that is the last one to be synthesized during
reverse transcription and therefore represents the pool of
full-length viral DNA molecules; a fragment of polymerase gene
(pol); a 2-LTR junction region found only in HIV-1 2-LTR circle DNA
molecules; or a fragment of the cellular a-tubulin gene. Dilutions
of 8E5 cells (containing 1 integrated copy of HIV-1 DNA per genome)
into CEM cells were used as standards. Amplification products were
transferred to nylon membrane filters and hybridized to
.sup.32P-labeled oligonucleotides corresponding to internal
sequences specific for each PCR amplification fragment, followed by
exposure to Kodak XAR-5 film or a phosphor screen.
[0163] Quantitation of PCR Reactions: Bands of correct size
revealed after hybridization were quantitated with a PhosphorImager
(Molecular Dynamics) by measuring the total density (integrated
volume) of rectangles enclosing the corresponding product band.
Efficiency of nuclear translocation of HIV-1 DNA was estimated by
measurement of the amount of 2-LTR circle DNA (N.sub.2-LTR)
relative to total viral DNA (N.sub.tot) in each culture, indexed to
the same ratio of appropriate control cultures. Thus, Translocation
Index=(N.sub.2-LTR/N.sub.tot)/(C.sub.2-LTR/C.sub.tot).times.100.
[0164] Results: Primary human monocytes were infected with
HIV-1.sub.ADA in the presence of 100 nM concentration of Compound
No. 2 or without drugs (control). Half the medium was changed every
3 days, and drugs were present throughout the whole experiment.
Cell samples were taken at 48 and 96 hours post infection and the
Translocation Index, relative to the drug free control was
determined. At both time points the Translocation Index was less
than 10, indicating there was greater than 90% inhibition of
nuclear importation.
7 PHARMACOKINETIC AND TOXICOLOGICAL STUDIES
[0165] This section describes in detail the techniques that were
used to study the toxicological and pharmacological properties of
the compounds of the invention.
7.1 Drug Analysis
[0166] Standard addition curves for each test compound were
constructed by adding increased amounts of drug to mouse or human
A.sup.+ plasma (Long Island Blood Services; Melville, N.Y.). An
equal volume of 10 mM tetramethylammonium chloride/10 mM heptane
sulfonate/4.2 mM H.sub.3PO.sub.4 (Buffer A) was added to the plasma
sample, which was then loaded onto a washed 1 g cyanopropylsilane
(or octadecylsilane for CNI-1894) solid-phase extraction column
(Fisher Scientific). The column was washed with 1.0 ml of water and
then eluted with 1.0 ml of 10 mM tetramethylammonium chloride/10 mM
heptane sulfonate/4.2 mM H.sub.3PO.sub.4/95% CH.sub.3CN/5% H.sub.2O
(Buffer C). The eluted sample was reduced to dryness in a rotary
evaporator and resuspended in 1.0 ml Buffer A.
[0167] Two hundred .mu.l of the resuspended sample was injected
onto a Hewlett-Packard model 1090 high performance liquid
chromatography system (HPLC) (Wilmington, Del.) equipped with a
photodiode array ultraviolet/visible spectrophotometric detector,
autosampler, and Chemstation operating software. The column used
was a 250.times.4.6 mm Zorbax RX-C8 column (Mac-Mod Analyticals.;
Chadd's Ford, Pa.) kept at room temperature and run at 1.5 ml/min.
The mobile phase used was Buffer A and 10 mM tetramethylammonium
chloride/10 mM heptane sulfonate/4.2 mM H.sub.3PO.sub.4/75%
CH.sub.3CN/25% H.sub.2O (Buffer B), with all runs initiated at 10%
Buffer B. A linear 30 min gradient to 60% Buffer B was then
performed, followed by a 4 min reverse gradient to initial
conditions. Compounds CNI-0294, -1194, -1594, and -1794 were
detected by ultraviolet absorbance at 300 nm, CNI-1894at 240 nm,
and pentamidine at 265 nm. In this assay system, the CNI test
compounds have a linear response and are detectable down to at
least 19.5 ng per injection.
7.2 Toxicity Studies
7.2.1 Method
[0168] The doses of compounds of the invention found to be lethal
to 50% of the mice (LD.sub.50) were determined by intraperitoneal
injection of groups of five animals with increasing doses of each
compound. CNI-0294 was administered from 0, 2, 10, 20, 40, 80, 160,
320, 640, 1280 mg/kg in 0.5 ml of water/HCl; CNI-1594 at 0, 2.4, 5,
10, 20, 40, 80 mg/kg in 0.5 ml of water/HCl; CNI-1794 at 0, 20, 50,
80 mg/kg in 0.5 ml of water/HCl; and CNI-1894 at 0, 10, 20, 40, 80,
240, 480, 960 mg/kg in water/HCl. All animals were observed for
visible signs of acute or long-term toxicity. The percentage of
animals in each group which died were utilized to calculate the
LD.sub.50 by non-linear curve fitting with the Enzfit software
(Elsevier Bioscience; Cambridge, UK) programmed with the Chou
equation (Chou 1976, J Theor Biol 39:253-276)).
7.2.2 Results
[0169] The compounds (FIG. 4A-E), were screened for toxicity via a
modified LD.sub.50 assay procedure as described above in an outbred
strain of mice. The results are shown in Table VII as follows:
TABLE-US-00006 TABLE VI The toxicity of the CNI compounds, as
measured by the median lethal dose determined as described above.
LD.sub.50 .+-. standard deviation Compound (mg/kg) 0294 587.77 .+-.
65.79 1194 >160* 1594 49.04 .+-. 0.08 1794 48.93 .+-. 0.12 1894
258.64 .+-. 1.37 *Higher doses were not tested due to limiting
amounts of the compound.
[0170] CNI-0294 was found to be very well tolerated (see Table VI),
with no overt signs of toxicity detectable at doses approaching the
LD.sub.50. The other compounds in the CNI series were designed to
allow for structure-function relationships with respect to activity
and toxicity. CNI-1194, which differs from CNI-0294 only by the
lack of a methyl group on the heterocyclic nitrogen, was also well
tolerated, with a high LD.sub.50 (Table VI). However, CNI-1594,
which is similar to CNI-1194 plus the omission of one of the acetyl
groups on the benzene rings, was appreciably more lethal (Table
VI). This toxicity was immediate, with death occurring in minutes
and the animals displaying signs of acute neurotoxicity. CNI-1794,
which is identical to CNI-1594 except that the single acetyl group
is moved para to the heterocyclic substituent, had an LD.sub.50
identical to that for CNI-1594 (Table VI). CNI-1894, which is
similar to CNI-0294 and -1194 but lacks the heterocyclic ring, was
also reasonably well tolerated. Animals dosed with large amounts of
CNI-1894 died 2-3 days post injection, and showed no sign of any
immediate toxicity. Based on the above observation, it is concluded
that the presence of the heterocyclic ring in the compounds of the
invention plays only a small role in determining toxicity, while
the presence of two acetyl groups on the benzene ring is very
important. Therefore, a preferred compound of the invention showing
low toxicity contains two acetyl groups on the benzene ring.
7.3 Pharmacokinetic Studies
7.3.1 Methods
[0171] Female ND4 Swiss Webster mice (21-24 g) were obtained from
Harlan Sprague Dawley (Indianapolis, Ind.) and randomly placed in
groups of five in cages with free access to food and water. Each
group of animals received 50 mg/kg of CNI-0294, -1194, or -1894, or
20 mg/kg of CNI-1594in a volume of 0.5 ml. Compound CNI-0294 was
administered intraperitoneally or by oral gavage as a solution in
water or a suspension in 10% DMSO/peanut oil. The other CNI
compounds were administered intraperitoneally or by oral gavage as
a solution in water titrated with sufficient HCl to dissolve the
drug. At various time points, ranging from 5 min to 4 days, a
single group of animals was euthanized by carbon dioxide inhalation
and bled by cardiac puncture using heparin as an anticoagulant. The
blood from the five mice in the group was pooled and centrifuged at
14000.times.g for 10 min. The volume of plasma was measured, and
equal volume of Buffer A added, and the mixture extracted and
analyzed as described above, except that the dried eluates were
resuspended in 200 .mu.l Buffer A and 100 .mu.l was injected onto
the high performance liquid chromatography (HPLC) system.
[0172] As inspection of the blood concentration-time curves for a
single i.p. injection showed a typical biphasic appearance,
standard methods of pharmacokinetic measurement were employed
(1982, Gibaldi et al., Pharmacokinetics. Marcel Dekker, New York).
The area under the plasma concentration-time curve (AUC) was
determined, and bioavailability was measured as
AUC.sub.oral/AUC.sub.i.p. A and B represent the zero time intercept
of the distribution and elimination phases respectively, and
.alpha. and .beta. the respective slopes of the phases multiplied
by 2.303. The t.sub.1/2.alpha. and t.sub.1/2.beta. are calculated
half-lives of the drug in each phase (0.693/.alpha. and
0.693/.beta. respectively). The volume of distribution (V.sub.D)
was calculated as dose/B, and the total clearance rate (Cl.sub.tot)
calculated as .beta.*V.sub.D. C.sub.max and t.sub.max are the
maximal plasma concentration and time of this measurement,
respectively.
7.3.2 Results
[0173] As judged by the plasma concentration-time curves from a
single intraperitoneal injection, each compound in the CNI series
had similar pharmacokinetic properties despite the structural
differences. The kinetic parameters are summarized in Table III and
a typical pattern is shown for CNI-1194 in FIG. 5. The drugs were
rapidly absorbed, with the maximal plasma concentration reached in
5-15 min, and also had a rapid distribution phase, with a
t.sub.1/2.alpha. of 0.32-0.62 hr. Differences were found to occur
in the maximal plasma concentration and parameters related to the
elimination phase. CNI-0294 achieved the highest maximal plasma
level for a single 50 mg/kg i.p. injection, with 18.76 .mu.g/ml,
and CNI-1894 was very similar with a value of 13.43 .mu.g/ml. As
CNI-1194 had an appreciably lower maximal plasma level and a slower
t.sub.max when compared with CNI-0294, it appears that the presence
of the methyl substituent on the heterocyclic nitrogen enhances
drug absorption from the peritoneum. A comparison of CNI-1194 and
CNI-1594 implied that the number of acetyl groups had little effect
on drug absorption. The values relating to elimination (.beta., B,
t.sub.1/2.beta., V.sub.d, Cl.sub.tot) were found to vary, but no
clear structural relationship could be discerned. All the
compounds, except CNI-1894, were undetectable in plasma after 24 hr
and approached the limit of detection after 5-6 hr. Therefore, as a
general property, the compounds of the invention are absorbed and
eliminated rapidly. A preferred compound of the invention has a
methyl substituent on the heterocyclic ring nitrogen at position 1
and possesses enhanced absorption from the peritoneum.
[0174] Experiments were also performed with CNI-0294 and -1194 to
evaluate relative bioavailability. By comparing the AUC.sub.oral
against the AUC.sub.i.p. for a single 50 mg/kg dose, CNI-0294 was
found to have 6% relative bioavailability and CNI-1194 15%. The
maximal plasma level was 0.4 .mu.g/ml for CNI-0294 and 0.35
.mu.g/ml for CNI-1194, and the drugs were detectable in plasma for
at least 6 hr (see FIG. 5).
7.4 Metabolic Studies
[0175] During the analysis of the plasma samples for the
pharmacokinetic parameters, a number of additional HPLC peaks were
detected which increased and decreased over time. Extra peaks of
this nature were seen in samples from each of the CNI series as
shown in FIGS. 8A-8D. As it was possible that these peaks
represented metabolites of the CNI compounds, the compounds of the
invention were screened in a simple model of primary
metabolism.
7.4.1 Method
[0176] Several female ND4 Swiss Webster mice were euthanized by
carbon dioxide inhalation and the livers excised and rinsed with
ice cold phosphate buffered saline (pH 7.4). The livers were
minced, gently homogenized in 50 mM phosphate buffer (pH 7.4) with
a Dounce homogenizer, and centrifuged at 9600.times.g for 20 min.
The post-mitochondrial supernatant was kept, glycerol added to 20%,
and frozen at -70.degree. C. in 1.0 ml aliquots until used. For
each incubation, 1.0 ml of a 1.0 mg/ml drug solution was added to
3.0 ml of 50 mM phosphate buffer (pH 7.4), 1.0 ml of 2 mg/ml NADPH
in 50 mM phosphate (pH 7.4), and 1.0 ml of the post-mitochondrial
supernatant. Five hundred .mu.l of each incubate was then
immediately transferred to an ice-cold tube to provide the
zero-time sample, and addition 500 .mu.l aliquots removed to
ice-cold tubes at 8, 15, 30, and 60 min. The samples were then
extracted, and analyzed by HPLC as described in section 7.1.
Control incubations were also performed where drug or
post-mitochondrial supernatant was omitted. An incubation using
pentamidine was performed to confirm microsomal activity (Berger et
al., 1992, Antimicrob. Ag. Chemother. 36:1825-1831). Peaks in the
CNI compound incubations which increased over time, and were not
present in control samples lacking the enzyme preparation were
treated as putative metabolites.
7.4.2 Results
[0177] Using post-mitochondrial supernatants of homogenized mouse
livers as a source of enzyme, the drugs were incubated in the
presence of NADPH. As described in Berger et al. supra, pentamidine
was used as a positive control, and the seven, expected, primary
metabolites were detectable, confirming the activity of the enzyme
preparation. Extraction and analysis of the CNI incubates showed
the presence of numerous, putative metabolite peaks that were not
present in negative control incubations (FIG. 6). Incubation of
CNI-0294, -1594, or -1194 was found to produce three minor and one
major metabolite and CNI-1894 had one minor and one major
metabolite. The major metabolite was found to elute 0.9-1.2 min
closer to the solvent front for CNI-0294, -1194, and -1594,
suggesting that the same position was being altered in each of
these compounds. The metabolic conversion in the post-mitochondrial
supernatant system was considerable, with 43.5% of CNI-0294, 65.19%
of CNI-1194, 11.74% of CNI-1594, and 17.28% of CNI-1894 altered
during the course of a 60 min incubation (as judged by peak area).
These results indicated that appreciable metabolism of the
compounds of the invention should occur in vivo.
[0178] Re-examination of the plasma samples confirmed that the
several of the unknown plasma peaks seen in FIGS. 6A and 6B
corresponded to the putative metabolites in FIGS. 7A-7D. However,
the metabolic model system did not produce all the unknown peaks
seen in the plasma samples. In particular, a plasma peak eluting at
11-14 min was seen with all the compounds in vivo, but not seen at
all in the in vitro test system. As was evident from the plasma
time-course samples, there appeared to be a large amount of
metabolic conversion in vivo of all of the compounds, regardless of
the route of administration.
7.5 Conclusions
[0179] The toxicity, pharmacokinetics, and metabolism of the novel
arylene bis(methylketone) compounds of the invention, and several
novel analogues thereof likewise of the invention were examined in
mice. With a median lethal dose of 587.77 mg/kg, CNI-0294 was well
tolerated when administered intraperitoneally. Analogues which also
had two acetyl groups on the phenyl moiety were also well
tolerated, with median lethal doses exceeding 160 mg/kg i.p. All
visible toxic reactions appeared to be rather delayed (generally
2-3 days post injection). While no biopsy samples were taken, such
a delay would be consistent with organ damage by very high doses
these compounds. Compounds which had only one acetyl group were
found to be more toxic, with median lethal doses of 48.93-49.04
mg/kg i.p. While the visible symptoms following injection of
CNI-1594 or -1794 suggested a lethal neurotoxicity, the structural
differences between the two drugs indicate that antagonism of an
endogenous neurotransmitter is unlikely.
[0180] In test animals, all of the compounds possessed very rapid
pharmacokinetic properties, with the plasma maximal concentration,
for intraperitoneal injection, being reached in 5-15 min, and 15-60
min for oral dosing. For CNI-0294, a plasma maximal concentration
of 18.76-18.93 .mu.g/ml was reached after injection of 50 mg/kg
i.p. The other compounds tested achieved lower maximal plasma
levels (1.9-13.43 .mu.g/ml). The half-life of the distribution
phase (t.sub.1/2.alpha.) was 0.32-0.62 hours, and that for the
elimination phase (t.sub.1/2.beta.) was 3.65-23.10 hours. All of
the kinetic parameters are consistent with drugs that are very
rapidly cleared from the plasma and are not retained in tissues for
a long period of time. Both CNI-0294 and -1194 were orally
absorbed, with a relative bioavailability of 6 and 15 percent
respectively. This latter feature is very favorable for continued
development of these compounds as anti-infective agents,
particularly as antiviral and antiparasitic agents, and more
particularly as anti-retroviral and anti-protozoal agents, and yet
particularly as anti-HIV agents and antimalarials. The toxicity,
kinetic, and bioavailability data suggest that frequent, high, oral
doses of the CNI-0294 can safely maintain therapeutically effective
plasma concentration.
[0181] Metabolism of the drugs was assessed in a mouse liver
post-mitochondrial supernatant system, and extensive metabolism was
discovered (11.74-65.19% metabolized during, a 60 minute
incubation). Examination of plasma samples showed that there was
considerable in vivo metabolism, with at least 4-6 metabolites
easily detected during the first 3 hours following i.p.
administration of the test compounds. The levels of metabolite
rapidly exceeded plasma concentrations of the parent compound. The
HPLC retention times indicated that the compounds were likely
altered in the same positions. In addition, the metabolites, like
the parent compounds, appeared to have very rapid plasma
kinetics.
8 EXAMPLE: DEMONSTRATION OF ANTI-MALARIAL ACTIVITY
8.1 The Compounds have Anti-Malarial Activity in Vitro
8.1.1 Method
[0182] The antimalarial activity of the compounds was determined
essentially as described in Desjardins et al. supra. Fifty .mu.l of
various concentrations of a compound of the invention, chloroquine,
or pyrimethamine were added to the wells of microtiter plates,
followed by 200 .mu.l of ring-stage, synchronized, P.
falciparum-infected erythrocytes (final hematocrit=1.5% , final
parasitemia=1-5%). The plates were incubated for 24 hr in a candle
jar kept at 37.degree. C., and then 25 .mu.l of
[.sup.3H]-hypoxanthine (Amersham, Arlington Heights, Ill.; 2.5
.mu.l Ci/well) was added. The plates were then incubated for a
further 24 hr, before harvesting onto Unifilter-96 GF/C
filter-microplates (Packard; Meriden, Conn.). Twenty-five .mu.l of
Microscint scintillation fluid (Packard) was added to each well of
the filter-microplate, which was subsequently counted in a
Top-count microplate scintillation counter (Packard). The percent
of [.sup.3H]-hypoxanthine uptake relative to control
infect-erythrocytes was used to determine the IC.sub.50 value for
the compounds by non-linear regression for LD.sub.50
determination.
8.1.2 Results
[0183] Using the hypoxanthine-incorporation method for assessing
Plasmodium falciparum growth in vitro as described above, CNI-0294
was found to have considerable anti-malarial activity (Table VII).
TABLE-US-00007 TABLE VII The antimalarial activity of CNI- 0294,
chloroquine, and pyrimethamine in vitro against several Plasmodium
falciparum clones. The median inhibitory concentration was
determined as described above. Pyrimethamine IC.sub.50 CNI-0294
IC.sub.50 Clone Chloroquine IC.sub.50 (.mu.M) (.mu.M) D10 26.99
.+-. 2.42* 170.70 .+-. 24.60 4.00 .+-. 0.41 Dd2 122.54 .+-. 7.26
103.70 .+-. 9.79 3.52 .+-. 0.10 FCR-3 104.68 .+-. 9.98 0.04 .+-.
0.01 3.09 .+-. 0.30 HB3 6.73 .+-. 0.16 8.97 .+-. 2.75 1.79 .+-.
0.27 W2mef 143.79 .+-. 13.30 17.81 .+-. 13.46 2.29 .+-. 0.22 *Each
value is .+-. standard deviation (n = 4 for chloroquine and
CNI-0294, and n = 2 for pyrimethamine).
[0184] The median inhibitory concentration (IC.sub.50) for CNI-0294
was calculated to be 1.79-4.00 .mu.M for a series of cloned
parasites which have different sensitivities to chloroquine or
pyrimethamine (Table VII).
[0185] The Dd2 clone of P. falciparum, which was both chloroquine
and pyrimethamine resistant, was utilized to compare the
antimalarial activity of the remaining CNI compounds (Table VIII).
TABLE-US-00008 TABLE VIII The antimalarial activities of the CNI
compounds against the chloroquine- and pyrimethamine-resistant P.
falciparum clone Dd2. The median inhibitory concentration was
determined as described above. Compound IC.sub.50 .+-. standard
deviation (.mu.M) 0294 3.67 .+-. 0.57* 1194 20.27 .+-. 1.62 1594
23.73 .+-. 0.59 1894 >200** 4594 25.11 .+-. 0.72 *n = 4 for all.
The CNI-0294 replicates were independent of those shown in Table
VII. **Highest concentration tested.
[0186] In independent measurements, CNI-0294 agreed well with the
results in Table VII, and CNI-1194 was found to be approximately
5-fold less active. This difference suggested that the heterocyclic
methyl group is required for maximal activity. CNI-1594 had an
IC.sub.50 equal to that for CNI-1194 or CNI-4594 demonstrating that
loss of one or both of the acetyl groups can have little effect on
the antimalarial activity. CNI-1894, however, was inactive at the
highest concentration tested.
8.2 The Compounds have Anti-Malarial Activity in Vivo
8.2.1 Method
[0187] The antimalarial activity of CNI-0294 in vivo was assessed
by infecting female ND4 Swiss Webster mice with 100 .mu.l of
Plasmodium berghei NYU-2 infected mouse erythrocytes (50%
parasitemia) by intraperitoneal injection. The animals were
subsequently injected intraperitoneally once per day on days 1-4 of
the infection with 0.5 ml water or 0.5 ml of 50 mg/kg CNI-0294 in
water. Four hours after the final injection, small blood samples
were taken from the tail, and thin smears stained with Dif-Quick
(Baxter, Miami, Fla.). The parasitemia of control and treated
animals was enumerated by inspection of at least 1000 erythrocytes
in each animal.
8.2.2 Results
[0188] As the CNI-0294 IC.sub.50 for P. falciparum was in the range
achieved for approximately one hr following a single i.p. injection
of 50 mg/kg in mice, the compound was also screened in vivo in mice
infected with Plasmodium berghei. Utilizing the four day
suppression test, where parasitemia is enumerated following four
daily injections of the test compound (in this case 50 mg/kg i.p.),
CNI-0294 was found to significantly (P.ltoreq.0.01) lower the
parasitemia by 10-fold (FIG. 9).
8.3 Conclusions
[0189] As indicated in Table VII, CNI-0294 was effective against
various clones of P. falciparum. The consistency in CNI-0294
IC.sub.50 over such a range of chloroquine and pyrimethamine
IC.sub.50's suggested that CNI-0294 had a different mechanism of
action than either of these established antimalarials.
[0190] While daily 50 mg/kg injections i.p., for 4 days, were found
to strongly suppress P. berghei infection in mice, these animals
were not completely cured during this course of treatment. The
difference between these in vivo results and the more striking P.
falciparum in vitro results are likely due to the kinetic and
metabolic properties of the compound. In vitro, the parasites are
exposed to a constant level of the drug for 48 hr, with no source
of host metabolizing enzymes. In the case in vivo, the single,
daily i.p. injection only provides therapeutic plasma
concentrations for approximately one hour and there is considerable
metabolism to compounds which may have reduced anti-plasmodial
activity. In light of these observations, one of ordinary skill in
the art would be able to further optimize the dosing regimens.
9 EXAMPLE: MECHANISM OF INHIBITION OF HIV-1 NUCLEAR TRANSLOCATION
BY COMPOUND NO. 2
[0191] The following experiments demonstrate the inhibitory
mechanism of compound No. 2 (also known as CNI-0294 or CNI-H0294)
on HIV nuclear localization, which is based on the inactivation of
the nuclear localization sequence (NLS) of the HIV matrix antigen
(MA) in the presence of HIV reverse transcriptase (RT). The results
described herein provide a basis for the development of a novel
class of antiviral compound that inhibit nuclear localization and
that are selective against specific NLS-containing proteins or
molecular complexes comprising NLS-containing protein.
9.1 Materials and Methods
[0192] Infection with Mutant HIV-1 or HIV-Like Pseudovirions H9
cells were infected with HIV-1 or HIV-like pseudovirions (Haffar,
et al., 1990, J. Virol., 64:2653-2659) at a multiplicity adjusted.
according to p24 content (50 ng p24 per 106 cells). The MA
NLS.sup.- virus contains substitutions of isoleucine residues for
lysines in positions 26 and 27 of MA in an NLHX backbone
(Westervelt., et al., 1992, J. Virol. 66:2577-2582), thus
inactivating the NLS. The Vpr.sup.- virus has the initiating ATG of
the vpr gene changed to GTG, thus abolishing expression of this
gene. The .DELTA.MA NLS pseudovirions have leucine substituting for
lysine in position 28 of MA. This mutation abrogates nuclear
translocation of HIV-1 gag RNA in growth-arrested H9 cells infected
with pseudovirions. After a 1 hour absorption, excess viruses or
pseudovirions were washed away, and cells were incubated for an
additional 2-3 hour period at 37.degree. C. prior to analysis.
Preparation of Cytoplasmic Lysates
[0193] Cytoplasmic extracts were prepared by lysing cells in cold
extraction buffer (10 mM KCl, 10 mM Tris-HCl [pH 7.6], 0.5 mM
MgCl.sub.2, 1 .mu.g/ml each of leupeptin and aprotinin, and 1 mM
phenylmethylsulfonyl fluoride [PMSF]) by 20-30 strokes of a Dounce
homogenizer under the control of phase-contrast microscopy. After
removal of nuclei, cytoplasmic extract was cleared by
centrifugation at 15,000 g for 10 min.
Analysis of Binding of Nucleoprotein Complexes to Karyopherin
.alpha.
[0194] Cytoplasmic extracts prepared from HIV-1- or
pseudovirion-infected H9 cells were adjusted to 0.14 M NaCl, 0.1%
Tween 20 and precleared with glutathione-Sepharose beads for 30
min. at room temperature. Karyopherin .alpha. was expressed as a
fusion protein with glutathione S-transferase (GST) which can bind
glutathione beads in solution. GST-karyopherin .alpha. immobilized
on Sepharose beads was then added (about 50 .mu.g of immobilized
karyopherin .alpha. per extract from 108 infected cells) and the
mixture was incubated at room temperature for another 30 min. Beads
were then pelleted by centrifugation and washed 3 times with PBS
supplemented with 0.1% Tween 20, 1 .mu.g/ml each of leupeptin and
aprotinin, and 1 mM PMSF. HIV-1 DNA was isolated from the beads by
SDS-proteinase K treatment with subsequent phenol-chloroform
extraction, while pseudovirion gag RNA was isolated by RNazol
(Biotecx Laboratories Inc.).
Analysis of CNI-H0294 Interaction with HIV-1 Proteins in
Solution
[0195] 0.28 nanomoles of recombinant MA or RT [p66/p51 dimer were
mixed with 20 nmol of [.sup.14C]CNI-H0294 (specific activity
5.times.10.sup.4 cpm/nmol) and incubated 2 hr at room temperature
in 40 .mu.l of binding buffer (PBS supplemented with 1% BSA, 0.1%
Tween 20, 1 .mu.g/ml leupeptin, 1 .mu.g/ml aprotinin, 1 mM PMSF).
Sheep anti-MA or rabbit anti-RT sera (both obtained from NIH AIDS
Research and Reference Reagent Program) or pre-immune control sera
were then added (at 1:100 dilution) and incubation continued for
another 1 hr at room temperature. Immune complexes were
precipitated with protein G-agarose, washed, and then eluted with
0.1 M glycine, pH 2.8. Radioactivity of the eluate was measured in
a scintillation counter.
Analysis of CNI-H0294 Interaction with HIV-1 Pre-Integration
Complexes
[0196] Cytoplasmic lysates prepared from HIV-1-infected cells were
treated with 10 .mu.M of [.sup.14C]-labeled CNI-H0294 (specific
activity 5.times.10.sup.4 cpm/nmol) in 1 ml extraction buffer
adjusted to 0.14 M NaCl. Sodium borohydride was then added to a
final concentration 10 mM and samples were incubated 1 hr at room
temperature prior to immunoprecipitation to reduce double bonds of
Schiff bases to an irreversible secondary amine.
Immunoprecipitation was performed as described above, but beads
were stringently washed three times with PBS supplemented with 0.1%
SDS, 1% sodium deoxycholate, 1 .mu.g/ml each of aprotinin and
leupeptin, and 1 mM PMSF.
9.2 Results
[0197] CNI-H0294 reacts with adjacent lysines in the NLS, thus
making it capable of neutralizing NLSs on many different proteins.
Interestingly, CNI-H0294 exhibited remarkably low cytotoxicity in
monocyte and T lymphocyte cultures in vitro (50% toxic dose >1
mM) and in vivo in mice (LD50=590 mg/kg, see Table VI). These
results suggest that the molecular mechanism of MA NLS inactivation
by CNI-H0294 is very specific. Indeed, this compound, did not block
nuclear import of nucleoplasmin-coated gold particles, nor of BSA
with conjugated NLS peptides that mimic the NLS of SV40 large T
antigen.
CNI-H0294 Inhibits Interaction Between HIV-1 Pre-Integration
Complexes and Karyopherin .alpha., but does not Affect Binding of
Karyopherin .alpha. to Pseudovirion-Derived Nucleoprotein
Complexes.
[0198] The initial step in the process of nuclear import is binding
of karyopherin .alpha. (also termed NLS-receptor/importin) to an
NLS. Results presented in FIG. 10A demonstrate that wild-type HIV-1
pre-integration complexes bound to GST-karyopherin .alpha.
immobilized on glutathione-Sepharose beads (lane 1). Mutant
pre-integration complexes that lack Vpr (MA NLS.sup.+Vpr.sup.-,
lane 3) bound with reduced-efficiency, while binding of the
complexes with mutated MA NLS (MA NLS.sup.-Vpr.sup.+, lane 2) was
even more impaired. Pre-integration complexes that lack Vpr and are
mutant in MA NLS (MA NLS.sup.-Vpr.sup.-) did not bind to
karyopherin .alpha. (lane 4). These results are consistent with the
analysis of MA and Vpr binding to karyopherin .alpha. which
demonstrated that while Vpr can bind weakly to karyopherin .alpha.,
its main role is to enhance the MA NLS-karyopherin .alpha.
interaction.
[0199] To facilitate analysis of HIV-1 nuclear translocation and of
the mechanism of drug effects on this process, a simplified model
of the HIV-1 pre-integration complex was used which comprises a
minimal number of non-essential proteins. This model employs
gag-env pseudovirions which exhibit an HIV-like core but are
composed exclusively of Gag (MA, CA, NC, p6) and Env (gp41 and
gp120) proteins. These pseudovirions package HIV-1 gag RNA and
translocate this RNA into the nucleus of an infected cell in a
manner similar to the behavior of HIV-1 pre-integration complexes.
Results presented in FIG. 10B demonstrate that karyopherin .alpha.
binds nucleoprotein complexes formed in pseudovirion-infected CD4+
T cells (lane 3). Binding required a functional MA NLS as mutation
of the NLS (FIG. 10A, lane 1) or pre-treatment of nucleoprotein
complexes with polyclonal anti-MA antibodies (lane 2) greatly
diminished binding to karyopherin .alpha.. Thus, it is concluded
that pseudovirion-derived nucleoprotein complexes interact with
karyopherin .alpha. in a manner similar to HIV-1 pre-integration
complexes.
[0200] The effect of CNI-H0294 on the interaction between
karyopherin .alpha. and HIV-1 versus pseudovirion nucleoprotein
complexes was examined. It was found that CNI-H0294 inhibited in a
dose-dependent manner binding of karyopherin .alpha. to HIV-1
pre-integration complexes (FIG. 10C, top panel). Quantitation on a
Phosphorimager demonstrated that 0.1 .mu.M and 1 .mu.M of CNI-H0294
reduced karyopherin .alpha./HIV-1 binding 8- and 25-fold,
respectively. These results explain the inhibition of HIV-1 nuclear
import by the compound and correlate well with the dose response
curve obtained when HIV-1-infected monocyte cultures were treated
with CNI-H0294. Surprisingly, CNI-H0294 did not inhibit binding of
karyopherin .alpha. to pseudovirion-derived nucleoprotein complexes
(FIG. 10C, bottom panel) or to purified recombinant MA. These
results suggest that the mechanism of CNI-0294 inhibition requires
a factor(s) present in the HIV-1 pre-integration complex but absent
from pseudovirion-derived complexes.
Structure-Activity Relationships Within the CNI-H Group of
Compounds
[0201] To further investigate the mechanism of action of CNI-H0294,
the structure-activity relationships within compounds of the
invention were examined (Table IX).
Table IX Structure-Function Analysis of Anti-HIV Activity of CNI
Compounds
[0202] CNI compounds were added at various concentrations (10 pM to
10 nM) to cultures of primary human monocytes together with
HIV-1.sub.ADA and were present throughout the entire experiment. A
50% inhibitory concentration (IC.sub.50) was determined at day 9
after infection. Some compounds did not achieve 50% inhibition at
maximal concentration yet exhibited anti-HIV activity; in these
cases the results are present as >10 .mu.M. TABLE-US-00009
Compound Structure IC.sub.50 CNI-H0294 ##STR10## 50 nM CNI-H1894
##STR11## >10 .mu.M CNI-H1494 ##STR12## 1 .mu.M CNI-H3094
##STR13## >10 .mu.M
[0203] Absence of the reactive carbonyl groups (compounds CNI-H1494
and CNI-H3094) or the pyrimidine side chain (compound CNI-H1894)
resulted in a dramatic decrease of the drug's potency. As the
carbonyl groups were designed to react with lysine residues within
MA NLS, it was not surprising that their absence decreased the
drug's activity. In contrast, a role for the pyrimidine side chain
was unexpected, and suggested that this side group may be involved
in binding CNI-H0294 to the pre-integration complex.
CNI-H0294 Binds to RT
[0204] Binding of CNI-H0294 to RT or MA was tested in vitro using
[.sup.14C]-labeled CNI-H0294 and recombinant RT and MA proteins
(FIG. 11). Specific immunoprecipitation was used to quantify the
amount of bound CNI-H0294. Preliminary experiments showed that both
anti-RT and anti-MA reagents specifically recognized and
immunoprecipitated RT and MA, respectively. As shown in FIG. 11,
about 17,000 cpm, or 0.34 nmol of CNI-H0294 (specific activity
50,000 cpm/nmol) were immunoprecipitable from incubations of drug
with 0.28 nmol RT, suggesting that CNI-H0294 binds to RT in a 1:1
molar ratio. The specificity of this interaction was further
confirmed by immunoprecipitation experiments using cold CNI-H0294
to compete out precipitable counts associated with labeled drug
(FIG. 11). No binding was observed with recombinant MA, and no
radioactivity was precipitated by immune sera if the recombinant
protein was omitted from the reaction mixture. In similar
experiments, the binding of CNI-H0294 to Vpr nor to integrase, two
other proteins known to be present within the HIV-1 pre-integration
complex, were detected. These experiments established that
CNI-H0294 bound directly to RT, but not to other proteins of the
HIV-1 pre-integration complex. Of interest, CNI-H0294 did not
significantly inhibit reverse transcription of HIV-1 in infected
cells, nor did it block in concentrations up to 50 .mu.M the
enzymatic activity of HIV-1 RT in vitro, suggesting that an effect
on RT activity cannot account for the anti-viral action of the
compound.
Binding to RT is Critical for the Anti-HIV Activity of
CNI-H0294
[0205] The role of CNI-H0294/RT interaction in the drug's activity
was analyzed in experiments with compound CNI-H3094. As CNI-H3094
does not have reactive carbonyl groups but contains the active
pyrimidine side chain (see Table IX), it could effectively compete
with CNI-H0294 for binding to the same site on the HIV-1
pre-integration complex, albeit it did not inhibit nuclear import
of HIV-1. FIG. 12A shows that unlabeled CNI-H3094 inhibits binding
of [.sup.14C]-labeled CNI-H0294 to RT in a dose-dependent manner.
Likewise, CNI-H3094 restored binding of HIV-1 pre-integration
complexes to karyopherin .alpha. in the presence CNI-H0294 (FIG.
12B). A 5-fold excess of CNI-H3094 (FIG. 12B, lane 4) reduced
significantly the inhibitory effect of CNI-H0294 on binding of
HIV-1 pre-integration complexes to karyopherin .alpha., and a
10-fold excess (lane 5) completely eliminated the inhibitory
effect. In a control experiment, CNI-H3094 did not inhibit binding
of HIV-1 pre-integration complexes to karyopherin .alpha. (FIG.
12B, lane 1); this correlates with the compound's lack of anti-HIV
activity (Table IX). Finally, CNI-H3094 eliminated the inhibitory
effect of CNI-H0294 on HIV-1 replication in monocyte cultures (FIG.
12C). These results confirm the critical role of CNI-H0294/RT
interaction in the drug's mechanism of action and also show a
direct correlation between the drug's binding to RT, inhibition of
HIV-1/karyopherin .alpha. interaction, and repression of viral
replication.
CNI-H0294 Inactivates the NLS of MA Without Disrupting MA
Association with the HIV-1 Genome
[0206] The results presented herein indicate a direct role for RT
in the anti-HIV effect of CNI-H0294 and provide a molecular
explanation for the high specificity of the compound. However,
these results do not explain how CNI-H0294 prevents binding of
HIV-1 pre-integration complexes to karyopherin .alpha., as RT does
not bind to directly to karyopherin .alpha.. One possibility was
that binding of CNI-H0294 to RT disrupts the pre-integration
complex and causes dissociation of MA from HIV-1 cDNA.
[0207] To test this hypothesis, cytoplasmic lysates of
HIV-1-infected H9 cells were treated with CNI-H0294 and mixed with
immobilized karyopherin .alpha. or subjected to immunoprecipitation
with antibodies that bind MA. Although CNI-H0294 blocked the
interaction of the pre-integration complexes with karyopherin
.alpha. (FIG. 13A, lane 2), it did not prevent immunoprecipitation
of viral DNA with anti-MA serum (FIG. 13A, lane 3); thus MA was
still associated with HIV-1 cDNA but lost its ability to bind
karyopherin .alpha.. As binding of pre-integration complexes to
karyopherin .alpha. is controlled mainly by the MA NLS (FIGS. 10A
and 10B), these results indicate that CNI-H0294 neutralizes the NLS
activity of MA, either directly (through chemical modification) or
indirectly (e.g. by steric hindrance).
[0208] To discriminate between these two possibilities, cytoplasmic
extracts of HIV-1-infected cells were treated with
[.sup.14C]-CNI-H0294 and then with sodium borohydride, to reduce
the reversible Schiff bases hypothesized to form between the
compound and the lysines of MA NLS and convert the attached drug
molecules to irreversible adducts. MA was then immunoprecipitated
with specific serum in a buffer containing of 0.1% SDS and 1%
sodium deoxycholate which disrupts weak protein-drug and
protein-protein interactions in the pre-integration complex without
disrupting covalent bonds. Under these conditions, a significant
amount of radioactivity was immunoprecipitated by anti-MA serum
(FIG. 13B), in contrast to results obtained with recombinant MA
(FIG. 11). These results corroborated requirement for RT for the
drug's effect and suggested that the CNI-H0294 had been covalently
linked to MA by borohydride treatment. Without borohydride
treatment, no radioactivity was immunoprecipitated with MA. In
control experiments, no radioactivity was precipitated by anti-IN
serum, or by anti-MA serum from the cytoplasmic extract of cells
infected with pseudovirions (FIG. 13B) which lack RT and thus do
not bind CNI-H0294 (FIG. 11).
CNI-H0294 Inhibits MA NLS.sup.-, but not Vpr-Mediated Binding of
HIV-1 Pre-Integration Complexes to Karyopherin .alpha.
[0209] Because of the role for MA NLS and Vpr in HIV-1 nuclear
import, the effects of CNI-H0294 on the interaction between
karyopherin .alpha. and pre-integration complexes derived from
viruses that carry mutations in Vpr (Vpr.sup.-), MA NLS (MA
NLS.sup.-), or both were investigated (FIG. 11). These viruses
(except for MA NLS.sup.- Vpr.sup.- double mutant which was slightly
attenuated) entered target cells and reverse transcribed their
genome with similar efficiencies (FIG. 14A). The presence of
CNI-H0294 diminished binding of karyopherin .alpha. to wild-type
(wt) (FIG. 14B, lanes 1, 2) and Vpr.sup.- complexes (lanes 3, 4) by
95% but had no effect on binding to MA NLS.sup.- complexes (lanes 5
and 6) which is only 1-5% of that observed with MA NLS+ complexes.
The inability of CNI-H0294 to block binding of MA NLS.sup.-
complexes to karyopherin .alpha. can be explained by the lack of a
consensus NLS in vpr and that Vpr binds to karyopherin .alpha. in
an NLS-independent manner.
[0210] Results presented hereinabove reveal the molecular mechanism
of action of CNI-H0294 that specifically target nuclear import of
HIV-1. The mechanism by which CNI-H0294 can inactivate the MA NLS
and thus prevent nuclear import of HIV-1 is depicted in FIG. 15.
According to the invention, the compound first binds to HIV-1
pre-integration complexes via RT and then forms reversible Schiff
bases with contiguous lysines in an adjacent MA NLS. This
interaction prevents binding of karyopherin .alpha. to the MA NLS
and significantly inhibits nuclear translocation of the HIV-1
pre-integration complex. The results with pseudovirion-derived
nucleoprotein complexes indicate that formation of the functional
complex capable of binding karyopherin .alpha. and translocating
into the nucleus resides entirely within the HIV-1 Gag proteins.
Although other proteins present in the HIV-1 pre-integration
complex (e.g. Vpr, IN, RT) may enhance nuclear translocation, they
are not necessary for this process. The results indicate that RT
and MA NLS are positioned in close proximity within the HIV-1
pre-integration complex as CNI-H0294 is very small yet seems to
bind RT and the MA NLS simultaneously. As MA is made from the Gag
precursor and RT is made from the Gag-Pol precursor, the ratio of
RT to MA in the virion is expected to be 1:50 because the
translational frameshift that leads to synthesis of the Gag-Pol
precursor (rather than the Gag precursor) occurs about 2% of the
time. Interestingly, about 1-2% of the virion MA protein is
phosphorylated and only these molecules are incorporated into the
HIV-1 pre-integration complex. This leads to an important
conclusion that there is roughly an equivalent number of RT and MA
molecules per HIV-1 pre-integration complex. Given the efficient
inactivation of MA NLS by CNI-H0294, most if not all RT and MA
molecules are in close proximity in the pre-integration
complex.
[0211] The present invention is not to be limited in scope by the
specific embodiments described which were intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components were within the
scope of the invention. Indeed, various modifications of the
invention, in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
Sequence CWU 1
1
6 1 7 PRT Simian virus 40 1 Pro Lys Lys Lys Arg Lys Val 1 5 2 18
PRT Xenopus laevis MOD_RES (1) Ala or Val 2 Xaa Lys Arg Pro Ala Ala
Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys 1 5 10 15 Lys Xaa 3 8 PRT
Hepatitis B virus 3 Pro Arg Arg Arg Arg Ser Gln Ser 1 5 4 17 PRT
Unknown Organism Description of Unknown Organism Unknown
retinoblastoma gene peptide 4 Lys Arg Ser Ala Glu Gly Gly Asn Pro
Pro Lys Pro Leu Lys Lys Leu 1 5 10 15 Arg 5 15 PRT Human
immunodeficiency virus type 1 5 Lys Ile Arg Leu Pro Arg Gly Gly Lys
Lys Lys Tyr Lys Leu Lys 1 5 10 15 6 7 PRT Hepatitis delta virus
MOD_RES (5) Variable amino acid 6 Pro Lys Lys Lys Xaa Lys Lys 1
5
* * * * *