ABSTRACT
Nucleocapsid p7 (NCp7) proteins of human immunodeficiency virus type 1 (HIV-1) contain two zinc binding domains of the sequence Cys-(X)2-Cys-(X)4-His-(X)4-Cys (CCHC). The spacing pattern and metal-chelating residues (3 Cys, 1 His) of these nucleocapside CCHC zinc fingers are highly conserved among retroviruses. These CCHC domains are required during both the early and late phases of retroviral replication, making them attractive targets for antiviral agents. toward that end, we have identified a number of antiviral chemotypes that electrophilically attack the sulfur atoms of the zinc-coordinating cysteine residues of the domains. Such nucleocapside inhibitors were directly virucidal by preventing the initiation of reverse transcription and blocked formation of infectious virus from cells through modification of CCHC domains within Gag precursors. Herein we report that azodicarbonamide (ADA) represents a new compound that inhibits HIV-1 and a broad range of retroviruses by targeting the the nucleocapsid CCHC domains. Vandevelde et al. also recently disclosed that ADA inhibits HIV-1 infection via an unidentified mechanism and that ADA was introduced into Phase I/II clinical trials in Europe for advanced AIDS. These studies distinguish ADA as the first known nucleocapsid inhibitor to progress to human trials and provide a lead compound for drug optimization.
Subject(s)
Anti-HIV Agents/pharmacology , Azo Compounds/pharmacology , Capsid Proteins , Capsid/drug effects , Gene Products, gag/drug effects , HIV Infections/virology , HIV-1/physiology , Viral Proteins , Virus Replication/drug effects , Binding Sites , Cell Line , HIV-1/drug effects , Humans , gag Gene Products, Human Immunodeficiency VirusABSTRACT
Human neutrophils can initiate the rapid degradation of extracellular matrix macromolecules by localizing the destructive process to sites of cell-substrate contact. Although plasma and its filtrates contain multiple proteinase inhibitors, these inhibitors did not prevent neutrophils from attacking either underlying fibronectin or elastin. However, subjacent substrates could be protected from neutrophils by recombinant secretory leukoprotease inhibitor, a structurally unique serine proteinase inhibitor whose natural counterpart is normally confined to human mucous secretions. The identification of this extravascular proteinase inhibitor as a potent regulator of subjacent proteolysis could lead to the development of a new class of anti-inflammatory therapeutics.
Subject(s)
Extracellular Matrix/metabolism , Neutrophils/metabolism , Proteins , Serine Proteinase Inhibitors/metabolism , Cell Adhesion , Cells, Cultured , Elastin/metabolism , Fibronectins/metabolism , Humans , Proteinase Inhibitory Proteins, Secretory , Recombinant Proteins/metabolism , SolubilityABSTRACT
Strategies for the treatment of human immunodeficiency virus-type 1 (HIV-1) infection must contend with the obstacle of drug resistance. HIV-1 nucleocapsid protein zinc fingers are prime antiviral targets because they are mutationally intolerant and are required both for acute infection and virion assembly. Nontoxic disulfide-substituted benzamides were identified that attack the zinc fingers, inactivate cell-free virions, inhibit acute and chronic infections, and exhibit broad antiretroviral activity. The compounds were highly synergistic with other antiviral agents, and resistant mutants have not been detected. Zinc finger-reactive compounds may offer an anti-HIV strategy that restricts drug-resistance development.
Subject(s)
Antiviral Agents/pharmacology , Benzamides/pharmacology , Capsid Proteins , Capsid/metabolism , Disulfides/pharmacology , Gene Products, gag/antagonists & inhibitors , HIV-1/drug effects , Viral Proteins , Zinc Fingers/drug effects , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Benzamides/chemistry , Benzamides/pharmacokinetics , Biological Availability , Capsid/chemistry , Cell Line , Disulfides/chemistry , Disulfides/pharmacokinetics , Drug Resistance, Microbial , Drug Synergism , Gene Products, gag/chemistry , HIV-1/physiology , Humans , Male , Mice , Molecular Sequence Data , gag Gene Products, Human Immunodeficiency VirusABSTRACT
BACKGROUND: The principal agent in the etiology of cervical cancer, i.e., human papillomavirus (HPV) type 16, encodes three oncoproteins, E5, E6, and E7. Structural and mutational studies have identified two potential zinc-finger domains as critical for E6 protein function. We investigated several assays to identify and characterize compounds that interfere with the binding of zinc to E6. METHODS: Thirty-six compounds were selected on the basis of their structure, which would facilitate their participation in sulfhydryl residue-specific redox reactions, and were tested for their ability to release zinc from E6 protein. The zinc-ejecting compounds were then tested for their ability to inhibit E6 binding to E6-associated protein (E6AP) and E6-binding protein (E6BP), two coactivators of E6-mediated cellular transformation. The binding of E6 to E6BP and E6AP was measured by use of surface plasmon resonance (a technique that monitors molecular interactions by measuring changes in refractive index) and by use of in vitro translation assays. The compounds were also tested for their effects on the viability of HPV-containing cell lines. RESULTS: Nine of the 36 tested compounds ejected zinc from E6. Two of the nine compounds inhibited the interaction of E6 with E6AP and E6BP, and one of these two, 4, 4'-dithiodimorpholine, selectively inhibited cell viability and induced higher levels of p53 protein (associated with the induction of apoptosis [programmed cell death]) in tumorigenic HPV-containing cells. CONCLUSION: We have described assay systems to identify compounds, such as 4,4'-dithiodimorpholine, that can potentially interfere with the biology and pathology of HPV. These assay systems may be useful in the development of drugs against cervical cancer, genital warts, and asymptomatic infections by genital HPVs.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor/methods , Morpholines/pharmacology , Oncogene Proteins, Viral/drug effects , Papillomaviridae , Repressor Proteins , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/virology , Zinc Fingers/drug effects , Zinc/metabolism , Amino Acid Sequence , Apoptosis/drug effects , Blotting, Western , Calcium-Binding Proteins/drug effects , Female , Gene Expression Regulation, Neoplastic , Glutathione/metabolism , Glutathione Transferase/biosynthesis , Humans , Ligases/drug effects , Molecular Sequence Data , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/complications , Protein Binding/drug effects , Protein Biosynthesis , Recombinant Fusion Proteins/metabolism , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , Tumor Virus Infections/complications , Ubiquitin-Protein Ligases , Uterine Cervical Neoplasms/metabolismABSTRACT
All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two zinc fingers, consisting of the sequence C-X2-C-X4-H-X4-C. Rice et al. (Science 270:1194-1197, 1995) have described a series of compounds which inactivate HIV-1 particles and oxidize the sulfur atoms in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. In contrast, the compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV-1 by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses, and the lethality of all known mutations altering the zinc-binding residues, suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.
Subject(s)
Antiviral Agents/pharmacology , Cross-Linking Reagents/pharmacology , HIV-1/physiology , Moloney murine leukemia virus/physiology , Nucleocapsid/chemistry , Spumavirus/physiology , Virus Replication/physiology , Amino Acid Sequence , Animals , Disulfides , Humans , Mice , Moloney murine leukemia virus/drug effects , Mutagenesis , Nucleocapsid/drug effects , Virus Replication/drug effects , Zinc FingersABSTRACT
The intersection of the HIV and the chemokine fields began with the observation that HIV entry into cells could be blocked by certain chemokines. Subsequent work showed that HIV entry is dependent on the presence of specific chemokine receptors. These observations led us to evaluate a series of compounds, ureido analogs of distamycin previously reported to block HIV entry into cells in vitro, for chemokine antagonist activity. One of the distamycin analogs, 2,2'[4,4'-[[aminocarbonyl]amino]bis[N,4'-di[pyrrole-2-carboxamide- 1,1'-dimethyl]]-6,8 napthalenedisulfonic acid] hexasodium salt (NSC 651016), is shown here to inhibit syncytia formation and cell fusion. Mechanistic studies showed that this inhibition was not due to conformational changes in gp120-gp41 induced by target cell CD4 and chemokine co-receptor and was therefore not due to interference with binding of HIV-1. Additional mechanistic studies demonstrated that NSC 651016 inhibited chemokine binding to specific chemokine receptors, induced CXCR4 and CCR5 receptor internalization, and inhibited chemokine-induced chemotaxis by macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and stromal-derived factor-1alpha but not monocyte chemotactic protein-1. Thus, we describe a novel compound that inhibits in vivo replication of HIV-1 by down-regulation of co-receptors. These data lead us to propose that NSC 651016 may have in vivo anti-inflammatory activity.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , HIV-1/physiology , Membrane Fusion/drug effects , Naphthalenesulfonates/pharmacology , Receptors, Chemokine/antagonists & inhibitors , Virus Replication/drug effects , CD4-Positive T-Lymphocytes/virology , Cell Line , Chemokines/metabolism , HeLa Cells , Humans , Receptors, Chemokine/physiologyABSTRACT
The 3-nitrosobenzamide (NOBA) drug abolishes SIV replication sharply at 20 microM concentration when CEM x 174 cells are preincubated for 1 h with the drug prior to viral infection. Treatment of CEM x 174 cells with 20 microM NOBA resulted in the inhibition of the synthesis of the DNA sequence coding for the gag gene, as determined by the PCR technique. Cell viability was directly proportional to the antiviral action of NOBA. Replication of AZT-resistant SIV 23740 in MMU 23740 cells in vitro was suppressed by NOBA in a concentration-dependent manner without significant effects on cell viability. Reverse transcriptase activity of SIVmac239 was unaffected by NOBA up to 800 microM concentration. Preincubation of two SIV strains with NOBA completely abolished their infectivity in human PHA-PBL cells. Replication of two strains of SIV in PHA-PBL cells was also inhibited by NOBA.
Subject(s)
Antiviral Agents/pharmacology , Benzamides/pharmacology , Nitroso Compounds/pharmacology , Simian Immunodeficiency Virus/drug effects , Virus Replication/drug effects , Zidovudine/pharmacology , Animals , Benzamides/administration & dosage , Cell Line , DNA, Viral/biosynthesis , Drug Resistance, Microbial , Genes, gag , Humans , Macaca mulatta , Nitroso Compounds/administration & dosage , Polymerase Chain Reaction , Simian Acquired Immunodeficiency Syndrome/microbiology , Simian Immunodeficiency Virus/physiologyABSTRACT
The highly conserved and mutationally intolerant retroviral zinc finger motif of the HIV-1 nucleocapsid protein (NC) is an attractive target for drug therapy due to its participation in multiple stages of the viral replication cycle. A literature search identified cystamine, thiamine disulfide, and disulfiram as compounds that have been shown to inhibit HIV-1 replication by poorly defined mechanisms and that have electrophilic functional groups that might react with the metal-coordinating sulfur atoms of the retroviral zinc fingers and cause zinc ejection. 1H NMR studies reveal that these compounds readily eject zinc from synthetic peptides with sequences corresponding to the HIV-1 NC zinc fingers, as well as from the intact HIV-1 NC protein. In contrast, the reduced forms of disulfiram and cystamine, diethyl dithiocarbamate and cysteamine, respectively, were found to be ineffective at zinc ejection, although cysteamine formed a transient complex with the zinc fingers. Studies with HIV-1-infected human T-cells and monocyte/macrophage cultures revealed that cystamine and cysteamine possess significant antiviral properties at nontoxic concentrations, which warrant their consideration as therapeutically useful anti-HIV agents.
Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Capsid Proteins , Capsid/metabolism , Cystamine/therapeutic use , Disulfiram/therapeutic use , Gene Products, gag/metabolism , Thiamine/analogs & derivatives , Viral Proteins , Zinc Fingers , Amino Acid Sequence , Anti-HIV Agents/chemistry , Anti-HIV Agents/therapeutic use , Cells, Cultured , Cystamine/chemistry , Disulfiram/chemistry , Ditiocarb/chemistry , Ditiocarb/pharmacology , Ditiocarb/therapeutic use , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Thiamine/chemistry , Thiamine/therapeutic use , gag Gene Products, Human Immunodeficiency VirusABSTRACT
Several molecular modeling techniques were used to generate an all-atom molecular model of a receptor binding site starting only from Ca atom coordinates. The model consists of 48 noncontiguous residues of the non-nucleoside binding site of HIV-1 reverse transcriptase and was generated using a congeneric series of nevirapine analogs as structural probes. On the basis of the receptor-ligand atom contacts, the program HINT was used to develop a 3D quantitative structure activity relationship that predicted the rank order of binding affinities for the series of inhibitors. Electronic profiles of the ligands in their docked conformations were characterized using electrostatic potential maps and frontier orbital calculations. These results led to the development of a 3D stereoelectronic pharmacophore which was used to construct 3D queries for database searches. A search of the National Cancer Institute's open database identified a lead compound that exhibited moderate antiviral activity.
Subject(s)
Antiviral Agents/chemistry , HIV-1/enzymology , RNA-Directed DNA Polymerase/chemistry , Reverse Transcriptase Inhibitors/chemistry , Antiviral Agents/pharmacology , Binding Sites , HIV Reverse Transcriptase , HIV-1/drug effects , Information Systems , Models, Molecular , Reverse Transcriptase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
The human immunodeficiency virus type one integrase (HIV-1 integrase) is required for integration of a double-stranded DNA copy of the viral RNA genome into a host chromosome and for HIV replication. We have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE), tyrphostins, and curcumin confer inhibitory activity against HIV-1 integrase. We have investigated the actions of several recently described protease inhibitors, possessing novel structural features, on HIV-1 integrase. NSC 158393, which contains four 4-hydroxycoumarin residues, was found to exhibit antiviral, antiprotease, and antiintegrase activity. Both the DNA binding and catalytic activities (3'-processing and strand transfer) of integrase were inhibited at micromolar concentrations. Disintegration catalyzed by an integrase mutant containing only the central catalytic domain was also inhibited, indicating that the binding site for these compounds resides in the central 50-212 amino acids of HIV-1 integrase. Binding at or near the integrase catalytic site was also suggested by a global inhibition of the choice of attacking nucleophile in the 3'-processing reaction. NSC 158393 inhibited HIV-2, feline, and simian immunodeficiency virus integrases while eukaryotic topoisomerase I was inhibited at higher concentrations, suggesting selective inhibition of retroviral integrases. Molecular modeling studies revealed that the two hydroxyls and two carbonyl moieties in NSC 158393 may represent essential elements of the pharmacophore. Antiviral efficacy was observed with NSC 158393 derivatives that inhibited both HIV protease and integrase, and the most potent integrase inhibitors also inhibited HIV protease. Hydroxycoumarins may provide lead compounds for development of novel antiviral agents based upon the concurrent inhibition of two viral targets, HIV-1 integrase and protease.
Subject(s)
4-Hydroxycoumarins/pharmacology , Antiviral Agents/pharmacology , DNA Nucleotidyltransferases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , 4-Hydroxycoumarins/chemistry , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Base Sequence , Binding Sites , DNA Nucleotidyltransferases/metabolism , DNA, Viral/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/metabolism , Integrases , Models, Chemical , Molecular Sequence Data , Molecular Structure , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Polydeoxyribonucleotides/metabolism , Quinones/pharmacology , Structure-Activity Relationship , Topoisomerase I Inhibitors , Ultraviolet RaysABSTRACT
Several new analogues of the novel anti-HIV agent cosalane have been synthesized and evaluated as inhibitors of HIV-1 integrase and protease, HIV-1 replication, HIV-1 and HIV-2 cytopathicity, HIV-1- and HIV-2-mediated syncytium formation, and cytopathicity of a variety of human pathogenic viruses. The congeners displayed enhanced potencies relative to cosalane itself as inhibitors of HIV-1 integrase and protease. The two most potent analogues against HIV-1 integrase displayed IC50 values of 2.2 microM, while the three most potent compounds against HIV-1 protease had IC50 values in the 0.35-0.39 microM range. In addition to its activity against HIV-1 and HIV-2 cytopathicity, cosalane inhibited the cytopathic effects of herpes simplex virus-1, herpes simplex virus-2, and human cytomegalovirus at concentrations that were well below the cytotoxic concentrations. Potentially useful antiviral activities were also revealed for some of the new cosalane congeners against influenza virus, Junin virus, and Tacaribe virus.
Subject(s)
Antiviral Agents/pharmacology , Aurintricarboxylic Acid/analogs & derivatives , DNA Nucleotidyltransferases/antagonists & inhibitors , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Amino Acid Sequence , Aurintricarboxylic Acid/chemistry , Aurintricarboxylic Acid/pharmacology , Cell Line , HIV-1/enzymology , HIV-1/physiology , Herpesviridae/drug effects , Humans , Integrases , Microbial Sensitivity Tests , Molecular Sequence Data , Virus Replication/drug effectsABSTRACT
Fifteen novel non-peptide HIV-1 protease inhibitors were identified by flexible 3D database pharmacophore searching of the NCI DIS 3D database. The pharmacophore query used in the search was derived directly from the X-ray determined structures of protease/inhibitor complexes. These 15 inhibitors, belonging to nine different chemical classes, are promising leads for further development. The two best inhibitors found, NSC 32180, a "dimer" of 4-hydroxycoumarin, and NSC 117027, a "tetramer" of 2-hydroxy quinone, had ID50 values of 0.32 and 0.75 microM for HIV-1 protease inhibition, respectively, and two other inhibitors had ID50 values close to 1 microM. Among the potent inhibitors, NSC 158393 not only demonstrated activity against HIV-1 protease (ID50 1.7 microM) but also exhibited promising antiviral activity in HIV-1-infected CEM-SS cells (EC50 = 11.5 microM). Validation of the pharmacophore used in the search was accomplished by conformational analysis. The binding modes of the most potent inhibitor found in our studies, NSC 32180, were predicted employing docking and molecular dynamics techniques.
Subject(s)
HIV Protease Inhibitors/chemistry , Amino Acid Sequence , Binding Sites , Cell Line , Crystallography, X-Ray , HIV Protease Inhibitors/metabolism , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Humans , Information Systems , Models, Molecular , Molecular Sequence DataABSTRACT
Several novel alkenyldiarylmethane (ADAM) non-nucleoside HIV-1 reverse transcriptase inhibitors were synthesized. The most potent of these proved to be 3',3"-dibromo-4',4"-dimethoxy-5'5"-bis(methoxycarbonyl)-1,1-diphenyl-1-+ ++heptene (8) ADAM 8 inhibited the cytopathic effect of HIV-1 in CEM cell culture with an EC50 value of 7.1 microM and was active against an array of laboratory strains of HIV-1 in CEM-SS and MT-4 cells, but was inactive as an inhibitor of HIV-2. In common with the other known non-nucleoside reverse transcriptase inhibitors, ADAM 8 was an effective inhibitor of HIV-1 reverse transcriptase (IC50 1 microM) with poly(rC).oligo(dG), but not with poly(rA).oligo(dT), as the template/primer. ADAM 8 was inactive against HIV-1 reverse transcriptases containing non-nucleoside reverse transcriptase inhibitor resistance mutations at residues 101, 106, 108, 139, 181, 188, and 236, while it remained active against enzymes with mutations at residues 74, 98, 100, 103, and at 103/181. An AZT-resistant virus having four mutations in reverse transcriptase was more sensitive to inhibition by ADAM 8 than the wild-type HIV-1. In addition, ADAM 8 displayed synergistic activity with AZT, but lacked synergy with ddI. ADAM 8 or a structurally related analog may therefore be useful as an antiviral agent in combination with AZT or with other NNRTIs that are made ineffective by mutations at residues which do not confer resistance to ADAM 8.
Subject(s)
Antiviral Agents/chemical synthesis , Benzoates/chemical synthesis , Biphenyl Compounds/chemical synthesis , HIV-1/drug effects , RNA-Directed DNA Polymerase/metabolism , Reverse Transcriptase Inhibitors/chemical synthesis , Amino Acid Sequence , Antiviral Agents/pharmacology , Aurintricarboxylic Acid/analogs & derivatives , Aurintricarboxylic Acid/pharmacology , Benzoates/pharmacology , Binding Sites , Biphenyl Compounds/pharmacology , Cells, Cultured , Didanosine/pharmacology , Drug Resistance, Microbial , HIV Core Protein p24/analysis , HIV Reverse Transcriptase , HIV-1/enzymology , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence Data , Molecular Structure , Mutagenesis, Site-Directed , Reverse Transcriptase Inhibitors/pharmacologyABSTRACT
Twenty-two new alkenyldiarylmethanes (ADAMs) were synthesized and evaluated for inhibition of HIV-1 replication. The most potent compound proved to be methyl 3',3"-dichloro-4',4"-dimethoxy-5', 5"-bis(methoxycarbonyl)-6,6-diphenyl-5-hexenoate (ADAM II), which displayed an EC50 of 13 nM for inhibition of the cytopathic effect of HIV-1RF in CEM-SS cells. ADAM II inhibited HIV-1 reverse transcriptase with an IC50 of 0.3 microM but was inactive as an inhibitor of HIV-1 attachment/fusion to cells, protease, integrase, and the nucleocapsid protein. Molecular target-based and cell-based assays revealed that ADAM II acted biologically as a nonnucleoside reverse transcriptase inhibitor (NNRTI). ADAM II inhibited replication of a wide variety of laboratory, clinical, and clade-representative isolates of HIV-1 in T cell lines and cultures of peripheral blood mononuclear cells or monocyte/macrophages. Mutations that conferred resistance to ADAM II clustered at residues 101, 103, 108, 139, 179, 181, and 188, which line the nonnucleoside binding pocket of HIV-1 reverse transcriptase. However, HIV-1 NL4-3 strain expressing a mutation at residue 100 of reverse transcriptase, and an AZT-resistant virus, displayed increased sensitivity to ADAM II. Thus, ADAM II could serve as an adjunct therapy to AZT and NNRTIs that select for L100I resistance mutations.
Subject(s)
Alkanes , Anti-HIV Agents , Caproates , HIV-1/drug effects , Reverse Transcriptase Inhibitors , Alkanes/chemical synthesis , Alkanes/chemistry , Alkanes/metabolism , Alkanes/pharmacology , Animals , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Anti-HIV Agents/pharmacology , Binding Sites , Caproates/chemical synthesis , Caproates/chemistry , Caproates/metabolism , Caproates/pharmacology , Cell Line , Cytopathogenic Effect, Viral/drug effects , Drug Evaluation, Preclinical , Drug Resistance, Microbial , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , HIV-1/genetics , HIV-1/physiology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/virology , Macrophages/drug effects , Macrophages/virology , Mice , Models, Molecular , Mutation , Reverse Transcriptase Inhibitors/chemical synthesis , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/virology , Virus Replication/drug effects , Zidovudine/pharmacologyABSTRACT
Agents that target the two highly conserved Zn fingers of the human immunodeficiency virus (HIV) nucleocapsid p7 (NCp7) protein are under development as antivirals. These agents covalently modify Zn-coordinating cysteine thiolates of the fingers, causing Zn ejection, loss of native protein structure and nucleic acid binding capacity, and disruption of virus replication. Concentrations of three antiviral agents that promoted in vitro Zn ejection from NCp7 and inhibited HIV replication did not impact the functions of cellular Zn finger proteins, including poly(ADP-ribose) polymerase and the Sp1 and GATA-1 transcription factors, nor did the compounds inhibit HeLa nuclear extract mediated transcription. Selectivity of interactions of these agents with NCp7 was supported by molecular modeling analysis which (1) identified a common saddle-shaped nucleophilic region on the surfaces of both NCp7 Zn fingers, (2) indicated a strong correspondence between computationally docked positions for the agents tested and overlap of frontier orbitals within the nucleophilic loci of the NCp7 Zn fingers, and (3) revealed selective steric exclusion of the agents from the core of the GATA-1 Zn finger. Further modeling analysis suggests that the thiolate of Cys49 in the carboxy-terminal finger is the site most susceptible to electrophilic attack. These data provide the first experimental evidence and rationale for antiviral agents that selectively target retroviral nucleocapsid protein Zn fingers.
Subject(s)
Anti-HIV Agents/pharmacology , Capsid Proteins , Capsid/metabolism , Gene Products, gag/metabolism , HIV-1/drug effects , Viral Proteins , Zinc Fingers , Animals , Anti-HIV Agents/metabolism , Azo Compounds/metabolism , Azo Compounds/pharmacology , Benzamides/metabolism , Benzamides/pharmacology , Binding Sites , Capsid/antagonists & inhibitors , Capsid/chemistry , Cell Line , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Gene Products, gag/antagonists & inhibitors , Gene Products, gag/chemistry , HIV-1/metabolism , HIV-1/physiology , Host Cell Factor C1 , Humans , Ligands , Mice , Models, Molecular , Nitroso Compounds/metabolism , Nitroso Compounds/pharmacology , Octamer Transcription Factor-1 , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Sp1 Transcription Factor/chemistry , Sp1 Transcription Factor/metabolism , Sulfones/metabolism , Sulfones/pharmacology , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Virus Replication/drug effects , gag Gene Products, Human Immunodeficiency VirusABSTRACT
Nucleocapsid p7 protein (NCp7) zinc finger domains of the human immunodeficiency virus type 1 (HIV-1) are being developed as antiviral targets due to their key roles in viral replication and their mutationally nonpermissive nature. On the basis of our experience with symmetrical disulfide benzamides (DIBAs; Rice et al. Science 1995, 270, 1194-1197), we synthesized and evaluated variants of these dimers, including sets of 4,4'- and 3,3'-disubstituted diphenyl sulfones and their monomeric benzisothiazolone derivatives (BITA). BITAs generally exhibited diminished antiviral potency when compared to their disulfide precursors. Novel, monomeric structures were created by linking haloalkanoyl groups to the benzamide ring through -NH-C(=O)- (amide) or -S-C(=O)- (thiolester) bridges. Amide-linked compounds generally lacked antiviral activity, while haloalkanoyl thiolesters and non-halogen-bearing analogues frequently exhibited acceptable antiviral potency, thus establishing thiolester benzamides per se as a new anti-HIV chemotype. Pyridinioalkanoyl thiolesters (PATEs) exhibited superior anti-HIV-1 activity with minimal cellular toxicity and appreciable water solubility. PATEs were shown to preferentially target the NCp7 Zn finger when tested against other molecular targets, thus identifying thiolester benzamides, and PATEs in particular, as novel NCp7 Zn finger inhibitors for in vivo studies.
Subject(s)
Anti-HIV Agents/chemical synthesis , Capsid Proteins , Capsid/antagonists & inhibitors , Gene Products, gag/antagonists & inhibitors , HIV-1/drug effects , Pyridinium Compounds/chemical synthesis , Sulfonamides/chemical synthesis , Sulfones/chemical synthesis , Viral Proteins , Zinc Fingers , Animals , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Cell Line , HIV-1/metabolism , Ligands , Mice , Models, Molecular , Pyridinium Compounds/chemistry , Pyridinium Compounds/pharmacology , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology , Sulfones/chemistry , Sulfones/pharmacology , Virus Replication/drug effects , gag Gene Products, Human Immunodeficiency VirusABSTRACT
Cosalane (3), a novel anti-HIV agent having a disalicylmethane unit linked to C-3 of cholestane by a three-carbon linker, was synthesized from commercially available starting materials by a convergent route. Cosalane proved to be a potent inhibitor of HIV with a broad range of activity against a variety of laboratory, drug-resistant, and clinical HIV-1 isolates, HIV-2, and Rauscher murine leukemia virus. The cytotoxicity of cosalane is relatively low as reflected by an in vitro therapeutic index of > 100. Although cosalane inhibits HIV-1 reverse transcriptase and protease, time of addition experiments indicate that it prevents the cytopathic effect of HIV by acting earlier than reverse transcription in the viral replication cycle. The available evidence indicates that the primary mechanism of action of cosalane involves inhibition of gp120-CD4 binding as well as inhibition of a postattachment event prior to reverse transcription.
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Aurintricarboxylic Acid/analogs & derivatives , HIV-1/drug effects , Virus Replication/drug effects , Aurintricarboxylic Acid/chemical synthesis , Aurintricarboxylic Acid/pharmacology , B-Lymphocytes/microbiology , Cell Fusion , Cells, Cultured , DNA, Viral/biosynthesis , HIV Infections/blood , HIV Infections/drug therapy , HIV-1/physiology , HeLa Cells , Humans , Macrophages/microbiology , Phenotype , T-Lymphocytes/microbiology , Virion/drug effects , Zidovudine/pharmacologyABSTRACT
Conservation of the Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys retroviral zinc finger sequences and their absolute requirement in both the early and late phases of retroviral replication make these chemically reactive structures prime antiviral targets. We recently reported that select 2,2'-dithiobisbenzamides (DIBAs) chemically modify the zinc finger Cys residues, resulting in release of zinc from the fingers and inhibition of HIV replication. In the current study we surveyed 21 categories of disulfide-based compounds from the chemical repository of the National Cancer Institute for their capacity to act as retroviral zinc finger inhibitors. Aromatic disulfides that exerted anti-HIV activity tended to cluster in the substituted aminobenzene, benzoate, and benzenesulfonamide disulfide subclasses. Only one thiuram derivative exerted moderate anti-HIV activity, while a number of nonaromatic thiosulfones and miscellaneous disulfide congeners were moderately antiviral. Two compounds (NSC 20625 and NSC 4493) demonstrated anti-cultures. The two compounds chemically modified the p7NC zinc fingers in two separate in vitro assays, and interatomic surface molecular modeling docked the compounds efficiently but differentially into the zinc finger domains. The combined efforts of rational drug selection, cell-based screening, and molecular target-based screening led to the identification of zinc finger inhibitors that can now be optimized by medicinal chemistry for the development of biopharmaceutically useful anti-HIV agents.
Subject(s)
Anti-HIV Agents/pharmacology , Disulfides/pharmacology , Guanidines/pharmacology , HIV-1/drug effects , Hydrazines/pharmacology , Virus Replication/drug effects , Zinc Fingers , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Binding Sites , Cells, Cultured , Disulfides/chemistry , Guanidines/chemistry , HIV-1/physiology , Humans , Hydrazines/chemistry , Macrophages/virology , Models, Molecular , Molecular Structure , Monocytes/virology , RNA, Messenger/metabolismABSTRACT
The anti-HIV agent cosalane inhibits both the binding of gp120 to CD4 as well as an undefined postattachment event prior to reverse transcription. Several cosalane analogues having an extended polyanionic "pharmacophore" were designed based on a hypothetical model of the binding of cosalane to CD4. The analogues were synthesized, and a number of them displayed anti-HIV activity. One of the new analogues was found to possess enhanced potency as an anti-HIV agent relative to cosalane itself. Although the new analogues inhibited both HIV-1 and HIV-2, they were more potent as inhibitors of HIV-1 than HIV-2. Mechanism of action studies indicated that the most potent of the new analogues inhibited fusion of the viral envelope with the cell membrane at lower concentrations than it inhibited attachment, suggesting inhibition of fusion as the primary mechanism of action.
Subject(s)
Anti-HIV Agents/chemical synthesis , Aurintricarboxylic Acid/analogs & derivatives , Benzoates/chemical synthesis , Cholestanes/chemical synthesis , Animals , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Anti-HIV Agents/pharmacology , Aurintricarboxylic Acid/chemistry , Benzoates/chemistry , Benzoates/metabolism , Benzoates/pharmacology , CD4 Antigens/chemistry , CD4 Antigens/metabolism , Cell Line , Cholestanes/chemistry , Cholestanes/metabolism , Cholestanes/pharmacology , HIV-1/drug effects , HIV-2/drug effects , Humans , Models, Molecular , Protein Binding , Structure-Activity RelationshipABSTRACT
A series of thiazolothiazepines were prepared and tested against purified human immunodeficiency virus type-1 integrase (HIV-1 IN) and viral replication. Structure-activity studies reveal that the compounds possessing the pentatomic moiety SC(O)CNC(O) with two carbonyl groups are in general more potent against purified IN than those containing only one carbonyl group. Substitution with electron-donating or -withdrawing groups did not enhance nor abolish potency against purified IN. By contrast, compounds with a naphthalene ring system showed enhanced potency, suggesting that a hydrophobic pocket in the IN active site might accommodate an aromatic system rather than a halogen. The position of sulfur in the thiazole ring appears important for potency against IN, as its replacement with an oxygen or carbon abolished activity. Further extension of the thiazole ring diminished potency. Compounds 1, 19, and 20 showed antiviral activity and inhibited IN within similar concentrations. These compounds inhibited IN when Mn(2+) or Mg(2+) was used as cofactor. None of these compounds showed detectable activities against HIV-1 reverse transcriptase, protease, virus attachment, or nucleocapsid protein zinc fingers. Therefore, thiazolothiazepines are potentially important lead compounds for development as inhibitors of IN and HIV replication.