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1.
Biochemistry ; 60(24): 1896-1908, 2021 06 22.
Article in English | MEDLINE | ID: mdl-34096272

ABSTRACT

Feline immunodeficiency virus (FIV) is a veterinary infective agent for which there is currently no efficient drug available. Drugs targeting the lentivirus capsid are currently under development for the treatment of human immunodeficiency virus 1 (HIV-1). Here we describe a lead compound that interacts with the FIV capsid. This compound, 696, modulates the in vitro assembly of and stabilizes the assembled capsid protein. To decipher the mechanism of binding of this compound to the protein, we performed the first nuclear magnetic resonance (NMR) assignment of the FIV p24 capsid protein. Experimental NMR chemical shift perturbations (CSPs) observed after the addition of 696 enabled the characterization of a specific binding site for 696 on p24. This site was further analyzed by molecular modeling of the protein:compound interaction, demonstrating a strong similarity with the binding sites of existing drugs targeting the HIV-1 capsid protein. Taken together, we characterized a promising capsid-interacting compound with a low cost of synthesis, for which derivatives could lead to the development of efficient treatments for FIV infection. More generally, our strategy combining the NMR assignment of FIV p24 with NMR CSPs and molecular modeling will be useful for the analysis of future compounds targeting p24 in the quest to identify an efficient treatment for FIV.


Subject(s)
Antiviral Agents/pharmacology , Benzimidazoles/pharmacology , Gene Products, gag/antagonists & inhibitors , Immunodeficiency Virus, Feline/drug effects , Animals , Binding Sites , Capsid/metabolism , Capsid Proteins/antagonists & inhibitors , Capsid Proteins/metabolism , Cats , Gene Products, gag/metabolism , Immunodeficiency Virus, Feline/metabolism , Lead/pharmacology , Protein Domains
2.
Mar Drugs ; 19(5)2021 Apr 28.
Article in English | MEDLINE | ID: mdl-33925208

ABSTRACT

Walleye dermal sarcoma virus (WDSV) is a type of retrovirus, which affects most of the adult walleye fishes during the spawning time. The virus causes multiple epithelial tumors on the fish's skin and fins that are liable for more than 50% of the mortality rate of fish around the world. Till now, no effective antiviral drug or vaccine candidates have been developed that can block the progression of the disease caused by the pathogen. It was found that the 582-amino-acid (aa) residues long internal structural gag polyprotein of the virus plays an important role in virus budding and virion maturation outside of the cell. Inhibition of the protein can block the budding and virion maturation process and can be developed as an antiviral drug candidate against the virus. Therefore, the study aimed to identify potential natural antiviral drug candidates from the tropical mangrove marine plant Avicennia alba, which will be able to block the budding and virion maturation process by inhibiting the activity of the gag protein of the virus. Initially, a homology modeling approach was applied to identify the 3D structure, followed by refinement and validation of the protein. The refined protein structures were then utilized for molecular docking simulation. Eleven phytochemical compounds have been isolated from the marine plant and docked against the virus gag polyprotein. Three compounds, namely Friedlein (CID244297), Phytosterols (CID12303662), and 1-Triacontanol (CID68972) have been selected based on their docking score -8.5 kcal/mol, -8.0 kcal/mol and -7.9 kcal/mol, respectively, and were evaluated through ADME (Absorption, Distribution, Metabolism and Excretion), and toxicity properties. Finally, molecular dynamics (MD) simulation was applied to confirm the binding stability of the protein-ligands complex structure. The ADME and toxicity analysis reveal the efficacy and non-toxic properties of the compounds, where MD simulation confirmed the binding stability of the selected three compounds with the targeted protein. This computational study revealed the virtuous value of the selected three compounds against the targeted gag polyprotein and will be effective and promising antiviral candidates against the pathogen in a significant and worthwhile manner. Although in vitro and in vivo study is required for further evaluation of the compounds against the targeted protein.


Subject(s)
Antiviral Agents/pharmacology , Avicennia/chemistry , Epsilonretrovirus/drug effects , Fish Diseases/prevention & control , Plant Extracts/pharmacology , Retroviridae Infections/veterinary , Tumor Virus Infections/veterinary , Animals , Antiviral Agents/isolation & purification , Epsilonretrovirus/metabolism , Epsilonretrovirus/pathogenicity , Fish Diseases/virology , Gene Products, gag/antagonists & inhibitors , Gene Products, gag/metabolism , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Plant Extracts/isolation & purification , Protein Conformation , Retroviridae Infections/prevention & control , Retroviridae Infections/virology , Structure-Activity Relationship , Tumor Virus Infections/prevention & control , Tumor Virus Infections/virology , Virus Release/drug effects
3.
Curr Top Microbiol Immunol ; 389: 171-201, 2015.
Article in English | MEDLINE | ID: mdl-25731773

ABSTRACT

The advances made in the treatment of HIV-1 infection represent a major success of modern biomedical research, prolonging healthy life and reducing virus transmission. There remain, however, many challenges relating primarily to side effects of long-term therapy and the ever-present danger of the emergence of drug-resistant strains. To counter these threats, there is a continuing need for new and better drugs, ideally targeting multiple independent steps in the HIV-1 replication cycle. The most successful current drugs target the viral enzymes: protease (PR), reverse transcriptase (RT), and integrase (IN). In this review, we outline the advances made in targeting the Gag protein and its mature products, particularly capsid and nucleocapsid, and highlight possible targets for future pharmacological intervention.


Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Anti-HIV Agents/pharmacology , Gene Products, gag/antagonists & inhibitors , HIV-1 , Capsid Proteins/antagonists & inhibitors , Humans , Nucleocapsid/antagonists & inhibitors , Viral Matrix Proteins/antagonists & inhibitors , Virus Replication/drug effects
4.
J Virol ; 86(12): 6643-55, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22496222

ABSTRACT

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.


Subject(s)
Anti-HIV Agents/pharmacology , Capsid/drug effects , Gene Products, gag/antagonists & inhibitors , HIV Infections/virology , HIV-1/drug effects , Benzimidazoles/pharmacology , Benzodiazepines/pharmacology , Capsid/metabolism , Cell Line , Gene Products, gag/chemistry , Gene Products, gag/genetics , Gene Products, gag/metabolism , HIV Infections/drug therapy , HIV-1/chemistry , HIV-1/genetics , HIV-1/physiology , Humans , Protein Structure, Tertiary , Virus Assembly/drug effects
5.
J Virol Methods ; 173(1): 92-8, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21277903

ABSTRACT

Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection.


Subject(s)
Gene Products, env/antagonists & inhibitors , Gene Products, gag/antagonists & inhibitors , Gene Silencing , Human T-lymphotropic virus 1/genetics , RNA, Small Interfering/genetics , Artificial Gene Fusion , Gene Products, env/genetics , Gene Products, gag/genetics , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans
6.
Biochemistry ; 49(19): 4006-17, 2010 May 18.
Article in English | MEDLINE | ID: mdl-20387899

ABSTRACT

Assembly of retrovirus particles is promoted by interaction of the Gag polyprotein with RNA. Nonspecific RNA association with the nucleocapsid domain (NC) of Gag induces the dimerization of Gag through protein-protein contacts in the capsid domain (CA), followed by higher order assembly to form the immature virus particle. NMR relaxation studies were conducted to investigate the initial steps of Rous sarcoma virus (RSV) assembly by examining the association with nucleic acid of a fragment of Gag comprising the C-terminal domain of CA (CTD) postulated to mediate Gag dimerization, the spacer region between CA and NC (SP), and NC. This fragment, CTD-SP-NC (residues 394-577), spans the critical SP region and allows assessment of this key Gag-nucleic acid interaction in the context of the Gag polyprotein rather than the isolated domains. Main-chain amide relaxation of CTD-SP-NC was measured in the absence and presence of (GT)(4), an 8-mer DNA oligonucleotide that binds tightly to the polyprotein but is too short to promote Gag dimerization. The results show that the CTD and NC domains tumble independently. In contrast, the two zinc finger domains within NC are rotationally coupled in both the unbound and bound states, even though only the first zinc finger appears to make direct contact with (GT)(4). In addition, the NMR data indicate that SP and flanking residues undergo a conformational exchange process that is slowed in the presence of (GT)(4). This region around SP where relaxation is strongly affected by (GT)(4) binding is nearly identical to the assembly domain defined previously by mutagenesis studies. Other changes in relaxation induced by (GT)(4) implicate conformational perturbations of helices 1 and 4 in CTD. On the basis of the combined data, we propose a model for the promotion of Gag dimerization by RNA association in which NC-RNA binding disrupts an assembly inhibitory, intramolecular interaction involving SP and CTD. Disruption of this intramolecular interaction is proposed to enhance the accessibility of the Gag dimer contact surface and release the assembly domain to promote intermolecular oligomerization.


Subject(s)
Gene Products, gag/chemistry , Gene Products, gag/metabolism , RNA, Viral/chemistry , Rous sarcoma virus/metabolism , Base Sequence , Binding Sites , Gene Products, gag/antagonists & inhibitors , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Nucleocapsid/chemistry , Nucleocapsid/metabolism , Protein Structure, Secondary , RNA, Viral/metabolism
7.
J Mol Biol ; 378(3): 565-80, 2008 May 02.
Article in English | MEDLINE | ID: mdl-18374356

ABSTRACT

The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer alpha-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.7 A. Using this structural information, we have utilized a structure-based rational design approach to stabilize the alpha-helical structure of CAI and convert it to a cell-penetrating peptide (CPP). The modified peptide (NYAD-1) showed enhanced alpha-helicity. Experiments with laser scanning confocal microscopy indicated that NYAD-1 penetrated cells and colocalized with the Gag polyprotein during its trafficking to the plasma membrane where virus assembly takes place. NYAD-1 disrupted the assembly of both immature- and mature-like virus particles in cell-free and cell-based in vitro systems. NMR chemical shift perturbation analysis mapped the binding site of NYAD-1 to residues 169-191 of the C-terminal domain of HIV-1 capsid encompassing the hydrophobic cavity and the critical dimerization domain with an improved binding affinity over CAI. Furthermore, experimental data indicate that NYAD-1 most likely targets capsid at a post-entry stage. Most significantly, NYAD-1 inhibited a large panel of HIV-1 isolates in cell culture at low micromolar potency. Our study demonstrates how a structure-based rational design strategy can be used to convert a cell-impermeable peptide to a cell-permeable peptide that displays activity in cell-based assays without compromising its mechanism of action. This proof-of-concept cell-penetrating peptide may aid validation of capsid as an anti-HIV-1 drug target and may help in designing peptidomimetics and small molecule drugs targeted to this protein.


Subject(s)
Anti-HIV Agents/chemistry , HIV-1/drug effects , Peptides, Cyclic/chemistry , Peptides/chemistry , Anti-HIV Agents/pharmacology , Circular Dichroism , Gene Products, gag/antagonists & inhibitors , Gene Products, gag/metabolism , HIV-1/metabolism , Humans , Magnetic Resonance Spectroscopy , Microscopy, Electron , Peptides/metabolism , Peptides/pharmacology , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacology , Permeability/drug effects , Protein Structure, Secondary , Transfection , Virion/ultrastructure , Virus Assembly/drug effects
8.
Nat Med ; 13(5): 631-5, 2007 May.
Article in English | MEDLINE | ID: mdl-17435772

ABSTRACT

Mammalian cells have developed diverse strategies to restrict retroviral infection. Retroviruses have therefore evolved to counteract such restriction factors, in order to colonize their hosts. Tripartite motif-containing 5 isoform-alpha (TRIM5alpha) protein from rhesus monkey (TRIM5alpharh) restricts human immunodeficiency virus type 1 (HIV-1) infection at a postentry, preintegration stage in the viral life cycle, by recognizing the incoming capsid and promoting its premature disassembly. TRIM5alpha comprises an RBCC (RING, B-box 2 and coiled-coil motifs) domain and a B30.2(SPRY) domain. Sequences in the B30.2(SPRY) domain dictate the potency and specificity of the restriction. As TRIM5alpharh targets incoming mature HIV-1 capsid, but not precursor Gag, it was assumed that TRIM5alpharh did not affect HIV-1 production. Here we provide evidence that TRIM5alpharh, but not its human ortholog (TRIM5alphahu), blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. The specificity for this restriction is determined by sequences in the RBCC domain. Our observations suggest that TRIM5alpharh interacts with HIV-1 Gag during or before Gag assembly through a mechanism distinct from the well-characterized postentry restriction. This finding demonstrates a cellular factor blocking HIV-1 production by actively degrading a viral protein. Further understanding of this previously unknown restriction mechanism may reveal new targets for future anti-HIV-1 therapy.


Subject(s)
Gene Products, gag/antagonists & inhibitors , HIV Infections/prevention & control , Proteins/physiology , Virus Replication , Animals , Cell Line , Cells, Cultured , Gene Products, gag/metabolism , HIV-1/physiology , Humans , Kidney , Macaca mulatta , Proteins/genetics , Transfection , Ubiquitin-Protein Ligases
9.
Curr Opin Investig Drugs ; 7(8): 759-65, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16955688

ABSTRACT

Panacos Pharmaceuticals Inc is developing the HIV Gag protein and viral maturation inhibitor bevirimat for the potential oral treatment of HIV infection. Phase II clinical trials are underway and phase III trials expected to commence in 2007.


Subject(s)
Gene Products, gag/antagonists & inhibitors , HIV Infections/drug therapy , HIV-1/metabolism , Succinates/pharmacology , Triterpenes/pharmacology , Animals , Clinical Trials, Phase II as Topic , Drug Evaluation, Preclinical , HIV Infections/metabolism , Humans , Succinates/adverse effects , Succinates/pharmacokinetics , Succinates/therapeutic use , Triterpenes/adverse effects , Triterpenes/pharmacokinetics , Triterpenes/therapeutic use
10.
Curr HIV Res ; 4(3): 379-86, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16842089

ABSTRACT

There is a continued need to develop inexpensive and effective drugs specific for novel targets of human immunodeficiency virus type 1 (HIV-1). The HIV-1 nucleocapsid p7 (NCp7) protein plays a critical role in early and late stages of the virus life cycle and possesses two highly conserved retroviral zinc fingers that are essential for its function. We have previously shown that zinc finger inhibitors (ZFI) based on the S-acyl 2-mercaptobenzamide thioester (SAMT) chemotype specifically target HIV NCp7 and are effective at reducing levels of infectious virus in an HIV-1-transgenic mouse model. Here, we did an initial proof-of-concept study to test the potential of a lead SAMT compound to reduce virus infectivity in the simian immunodeficiency virus (SIV) nonhuman primate model. SAMT-19 had potent antiviral and virucidal effects against the primary pathogenic isolate SIV/DeltaB670 and was non-cytotoxic in vitro. Cynomolgus macaques were infected intrarectally with SIV/DeltaB670 and treated with a low dose of SAMT-19 by continuous infusion from day 8 to day 28 post infection. Monkeys in the treatment group had significantly lower levels of infectious virus in peripheral blood mononuclear cells during the course of therapy as compared to monkeys in the control group, although therapy had no demonstrable effect on virus load. SAMT-19 therapy did not alter liver, kidney or immunologic function and was well tolerated by all treated monkeys. These data demonstrate that SAMT-19 is safe and virucidal in the nonhuman primate model. Further studies directed at optimizing SAMT bioavailability and pharmacokinetics likely will result in enhanced therapeutic efficacy of this promising HIV therapeutic.


Subject(s)
Anti-HIV Agents/therapeutic use , Capsid Proteins/antagonists & inhibitors , Gene Products, gag/antagonists & inhibitors , Simian Acquired Immunodeficiency Syndrome/drug therapy , Sulfhydryl Compounds/therapeutic use , Viral Proteins/antagonists & inhibitors , Zinc Fingers/drug effects , Animals , Leukocytes, Mononuclear/virology , Macaca fascicularis , Male , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , T-Lymphocytes/immunology , gag Gene Products, Human Immunodeficiency Virus
11.
J Virol ; 80(12): 5716-22, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16731910

ABSTRACT

3-O-(3',3'-Dimethysuccinyl) betulinic acid (PA-457) has been shown to potently inhibit human immunodeficiency virus (HIV) replication in culture. In contrast to inhibitors that act upon the viral proteinase, PA-457 appears to block only the final maturational cleavage of p25CA-p2 to p24CA. However, attempts to replicate this effect in vitro using recombinant Gag have failed, leading to the hypothesis that activity is dependent upon the assembly state of Gag. Using a synthesis/assembly system for chimeric HIV type 1 Gag proteins, we have replicated the activity of PA-457 in vitro. The processing of assembled chimeric Gag can be inhibited by the addition of drug with only the final cleavage of p25CA-p2 to p24CA blocked. Consistent with our hypothesis and with previous findings, inhibition appears specific to Gag assembled into an immature capsid-like structure, since synthetic Gag that remains unassembled is properly processed in the presence of the compound. To further analyze the authenticity of the assay, PA-457 was tested in parallel with its inactive parental compound, betulinic acid. Betulinic acid had no effect upon p25 processing in this system. Analysis of a PA-457-resistant mutant, A1V, in this system pointed to more rapid cleavage as a possible mechanism for resistance. However, characterization of additional mutations at the cleavage site and in p2 suggests that resistance does not strictly correlate with the rate of cleavage. With the establishment of an in vitro assay for the detection of PA-457 activity, a more detailed characterization of its mechanism of action will be possible.


Subject(s)
Gene Products, gag/antagonists & inhibitors , HIV-1/chemistry , Succinates/pharmacology , Triterpenes/pharmacology , Capsid Proteins/antagonists & inhibitors , Hydrolysis , Kinetics , Protein Precursors/antagonists & inhibitors , Virus Replication/drug effects , gag Gene Products, Human Immunodeficiency Virus
12.
J Med Chem ; 48(8): 2847-58, 2005 Apr 21.
Article in English | MEDLINE | ID: mdl-15828823

ABSTRACT

The HIV-1 nucleocapsid protein (NCp7) is a small basic protein with two CysCysHisCys zinc-binding domains that specifically recognizes the Psi-site of the viral RNA. NCp7 plays a number of crucial roles in the viral lifecycle, including reverse transcription and RNA encapsidation. Several classes of potential anti-HIV compounds have been designed to inactivate NCp7 through zinc ejection, including a special class of thioester compounds. We have investigated the mechanism of action of two N-substituted-S-acyl-2-mercaptobenzamide compounds (compounds 1 and 2) that target NCp7. UV/Visible spectroscopy studies demonstrated that both thioesters were able to eject metal from NCp7. NMR and mass spectroscopy studies showed that the thioester compounds specifically ejected zinc from the carboxyl-terminal zinc-binding domain of NCp7 by covalent modification of Cys(39). Exposure of NCp7 to compounds 1 and 2 destroyed its ability to specifically bind RNA, whereas NCp7 already bound to RNA was protected from zinc ejection by the thioesters. The thiol component of the thioesters (compound 3, 2-mercaptobenzoyl-beta-alaninamide) did not eject zinc from NCp7, but when compound 3 was incubated with acetyl CoA prior to incubation with NCp7, we observed extensive metal ejection. Thus, the thiol released by the reaction of compounds 1 and 2 could be re-acylated in vivo by acyl CoA to form a new thioester compound that is able to react with NCp7. These studies provide a better understanding of the mechanism of action of thioester compounds, which is important for future design of anti-HIV-1 compounds that target NCp7.


Subject(s)
Anti-HIV Agents/chemistry , Benzamides/chemistry , Capsid Proteins/antagonists & inhibitors , Capsid Proteins/chemistry , Gene Products, gag/antagonists & inhibitors , Gene Products, gag/chemistry , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , beta-Alanine/analogs & derivatives , beta-Alanine/chemistry , Amino Acid Sequence , Anti-HIV Agents/chemical synthesis , Benzamides/chemical synthesis , Cysteine/chemistry , Electrophoretic Mobility Shift Assay , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence Data , Spectrophotometry/methods , Sulfhydryl Compounds/chemistry , Zinc/chemistry , beta-Alanine/chemical synthesis , gag Gene Products, Human Immunodeficiency Virus
13.
J Neuroimmunol ; 159(1-2): 196-202, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15652420

ABSTRACT

We analyzed the relationship between the expression of interferon (IFN)-gamma and HTLV-I p19 antigen and activation of p38 mitogen-activated protein kinase (p38 MAPK) in two HTLV-I-infected T cell lines derived from two patients (HCT-1 and HCT-4) with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and three HTLV-I-infected T cell lines derived from three patients with adult T cell leukemia (ATL). Expression of phosphorylated (activated)-p38 MAPK was markedly increased concomitant with high levels of both IFN-gamma and HTLV-I p19 antigen expression in both HCT-1 and HCT-4 compared with cell lines derived from ATL patients. Treatment with SB203580, a specific inhibitor of p38 MAPK, suppressed IFN-gamma and HTLV-I p19 antigen expression levels in HCT-1, HCT-4 and peripheral blood CD4(+) T cells of HAM/TSP patients. These findings strongly suggest that activation of p38 MAPK signaling pathway is involved in the up-regulation of IFN-gamma expression with high HTLV-I proviral load in HAM/TSP patients.


Subject(s)
Gene Products, gag/biosynthesis , HTLV-I Antigens/biosynthesis , Human T-lymphotropic virus 1/immunology , Interferon-gamma/biosynthesis , MAP Kinase Signaling System/immunology , Paraparesis, Tropical Spastic/immunology , Retroviridae Proteins, Oncogenic/biosynthesis , p38 Mitogen-Activated Protein Kinases/physiology , Adult , Aged , Antiviral Agents/pharmacology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Cell Line , Female , Gene Products, gag/antagonists & inhibitors , Humans , Imidazoles/pharmacology , Interferon-gamma/antagonists & inhibitors , Male , Middle Aged , Paraparesis, Tropical Spastic/enzymology , Paraparesis, Tropical Spastic/virology , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Proviruses/immunology , Pyridines/pharmacology , Retroviridae Proteins, Oncogenic/antagonists & inhibitors , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , T-Lymphocytes/virology , gag Gene Products, Human Immunodeficiency Virus , p38 Mitogen-Activated Protein Kinases/biosynthesis , p38 Mitogen-Activated Protein Kinases/metabolism
15.
J Virol ; 77(22): 12132-9, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14581550

ABSTRACT

The presence of porcine endogenous retroviruses presents a potential risk of transmission of infectious diseases (xenozoonosis) if tissues and organs from genetically modified pigs are to be used in xenotransplantation. Here, we report that intracellular expression of a llama single-domain antibody against p15, the matrix domain protein of the porcine endogenous retrovirus Gag polyprotein, blocks retrovirus production, providing the possibility of eliminating the risk of infection in xenotransplantation.


Subject(s)
Antibodies, Viral/physiology , Endogenous Retroviruses/physiology , Gene Products, gag/antagonists & inhibitors , Retroviridae Infections/prevention & control , Swine/virology , Transplantation, Heterologous/adverse effects , Viral Matrix Proteins/antagonists & inhibitors , Amino Acid Sequence , Animals , Antibody Affinity , Epitope Mapping , Gene Products, gag/immunology , Humans , Immune Sera/immunology , Immunoglobulin Heavy Chains/physiology , Immunoglobulin Variable Region/physiology , Molecular Sequence Data , Viral Matrix Proteins/immunology , Virus Replication
16.
Proc Natl Acad Sci U S A ; 100(23): 13555-60, 2003 Nov 11.
Article in English | MEDLINE | ID: mdl-14573704

ABSTRACT

New HIV therapies are urgently needed to address the growing problem of drug resistance. In this article, we characterize the anti-HIV drug candidate 3-O-(3',3'-dimethylsuccinyl) betulinic acid (PA-457). We show that PA-457 potently inhibits replication of both WT and drug-resistant HIV-1 isolates and demonstrate that the compound acts by disrupting a late step in Gag processing involving conversion of the capsid precursor (p25) to mature capsid protein (p24). We find that virions from PA-457-treated cultures are noninfectious and exhibit an aberrant particle morphology characterized by a spherical, acentric core and a crescent-shaped, electron-dense shell lying just inside the viral membrane. To identify the determinants of compound activity we selected for PA-457-resistant virus in vitro. Consistent with the effect on Gag processing, we found that mutations conferring resistance to PA-457 map to the p25 to p24 cleavage site. PA-457 represents a unique class of anti-HIV compounds termed maturation inhibitors that exploit a previously unidentified viral target, providing additional opportunities for HIV drug discovery.


Subject(s)
Anti-HIV Agents/pharmacology , Gene Products, gag/chemistry , Succinates/pharmacology , Triterpenes/pharmacology , Binding Sites , Chromobox Protein Homolog 5 , Drug Design , Gene Products, gag/antagonists & inhibitors , Genotype , HIV Core Protein p24/metabolism , HeLa Cells , Humans , Inhibitory Concentration 50 , Microscopy, Electron , Models, Chemical , Models, Genetic , Mutation , Plasmids/metabolism , Precipitin Tests , Succinates/chemistry , Triterpenes/chemistry , gag Gene Products, Human Immunodeficiency Virus
17.
Bioorg Med Chem ; 11(14): 3215-9, 2003 Jul 17.
Article in English | MEDLINE | ID: mdl-12818684

ABSTRACT

The anti-HIV activity of (+/-)-cis-4,5-dihydroxy-1,2-dithiane 1,1-dioxide [(+/-)-cis-1,1-dioxo-[1,2]-dithiane-4,5-diol, NSC-624151] and its attack on the zinc finger domain of the HIV-1 nucleocapsid p7 (NCp7) protein has been established [Rice, W. G.; Baker, D. C.; Schaeffer, C. A.; Graham, L.; Bu, M.; Terpening, S.; Clanton, D.; Schultz, R.; Bader, J. P.; Buckheit, R. W.; Field, L.; Singh, P. K. Turpin, J. A. Antimicrob. Agents Chemother. 1997, 41, 419]. In order to determine which enantiomer of NSC-624151 is the more active component, the compound was resolved via its bis-'Mosher ester', which was prepared via its reaction with two equiv of (-)-(R)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride. The diastereoisomeric esters were separated, and each ester was hydrolyzed to yield enantiomers with (D)(21) +151 degrees (c 0.5, MeOH) and (D)(21) -146 degrees (c 0.5, MeOH). Single-crystal X-ray analysis of the (-)-bis-'Mosher ester' showed that the (-)-enantiomer is the (4S, 5R)-compound. The (-)-enantiomer (NSC 693195) was ca. twice as active (EC(50) 8.8+/-0.2 microM) as its (+)-counterpart (NSC 693194) (EC(50) 16.2+/-2.4 microM) in the XTT assay against HIV-1. All three compounds were found to be approximately equally effective in promoting Zn ejection from the NCp7 zinc finger. As the more anti-HIV active enantiomer is only slightly more active than the racemic form, it appears to offer no advantages over the racemic form.


Subject(s)
Anti-HIV Agents/chemical synthesis , Capsid Proteins/antagonists & inhibitors , Gene Products, gag/antagonists & inhibitors , HIV-1/drug effects , Sulfones/chemical synthesis , Viral Proteins , Anti-HIV Agents/pharmacology , HIV-1/metabolism , Models, Molecular , Stereoisomerism , Structure-Activity Relationship , Sulfones/pharmacology , Zinc Fingers , gag Gene Products, Human Immunodeficiency Virus
18.
AIDS Res Hum Retroviruses ; 19(2): 91-101, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12639244

ABSTRACT

Control of human immunodeficiency virus through the use of inexpensive chemotherapeutics, with minimal side effects and decreased potential for engendering resistant virus, is a long-term therapeutic goal. In principle, this goal can be accomplished if viral replication in reservoirs of chronically and latently infected cells is addressed. As a first step, we have developed novel antiviral compounds based on a 2-mercaptobenzamide thioester chemotype, including the pyridinioalkanoyl thioesters, which specifically target the zinc fingers of the human immunodeficiency virus nucleocapsid protein (NCp7). Using these compounds in a murine transgenic model, in which infectious human immunodeficiency virus is induced from an integrated provirus, we show inhibition of transgenic spleen cell p24 expression with potencies comparable to acute infection assays using human peripheral blood lymphocytes. More importantly, transgenic mice treated in vivo with two 2-mercaptobenzamide thioesters expressed significantly lower plasma p24, and splenocytes from these animals produced fewer infectious virions. Thus, these thioesters may provide an effective means for inhibiting the expression of human immunodeficiency virus from integrated viral reservoirs.


Subject(s)
Anti-HIV Agents/pharmacology , Benzamides/pharmacology , Capsid Proteins , Esters/pharmacology , Gene Products, gag/antagonists & inhibitors , HIV-1/drug effects , Sulfhydryl Compounds/pharmacology , Viral Proteins , Zinc Fingers/drug effects , Animals , Benzamides/chemistry , Capsid/chemistry , Disease Models, Animal , Esters/chemistry , Gene Products, gag/chemistry , HIV Infections/drug therapy , HIV Infections/virology , HIV-1/genetics , Humans , Mice , Microbial Sensitivity Tests/methods , Pyridinium Compounds/chemistry , Pyridinium Compounds/pharmacology , Sulfhydryl Compounds/chemistry , gag Gene Products, Human Immunodeficiency Virus
19.
Expert Rev Anti Infect Ther ; 1(1): 97-128, 2003 Jun.
Article in English | MEDLINE | ID: mdl-15482105

ABSTRACT

There are presently 42 million people worldwide living with HIV/AIDS, the majority of which have limited access to antiretrovirals. Even if worldwide penetration was possible, our current chemotherapeutic strategies still suffer from issues of cost, patient compliance, deleterious acute and chronic side effects, emerging single and multidrug resistance, and generalized treatment and economic issues. Even our best antiretroviral therapeutic strategy, highly active antiretroviral therapy (HAART), falls short of completely suppressing HIV replication. Therefore, expansion of current therapeutic options by discovering new antiretrovirals and targets will be critical in the coming years. This review addresses the current status of reverse transcriptase and protease inhibitor development, and summarizes the progress in emerging classes of HIV inhibitors, including entry (T-20, T-1249), coreceptor (SCH-C, SCH-D), integrase (beta-Diketos) and p7 nucleocapsid Zn finger inhibitors (thioesters and PATEs). In addition, the processes of virus entry, PIC transport to the nucleus, HIV interaction with nuclear pores, Tat function, Rev function and virus budding (Tsg101 and ubiquitination) are examined, and proof of concept inhibitors and potential antiviral targets discussed.


Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Anti-HIV Agents/pharmacology , Anti-HIV Agents/therapeutic use , Antiretroviral Therapy, Highly Active , Capsid Proteins/antagonists & inhibitors , Gene Products, gag/antagonists & inhibitors , HIV Integrase Inhibitors/pharmacology , Humans , Membrane Fusion/drug effects , Receptors, HIV/antagonists & inhibitors , Reverse Transcriptase Inhibitors/pharmacology , Viral Proteins/antagonists & inhibitors , Virus Assembly/drug effects , gag Gene Products, Human Immunodeficiency Virus
20.
J Med Chem ; 45(24): 5249-59, 2002 Nov 21.
Article in English | MEDLINE | ID: mdl-12431052

ABSTRACT

The interaction of a series gag peptide analogues with human cyclophilin A (hCypA) have been studied employing molecular docking and 3D-QSAR approaches. The Lamarckian Genetic Algorithm (LGA) and divide-and-conquer methods were applied to locate the binding orientations and conformations of the inhibitors interacting with hCypA. Good correlations between the calculated interaction free energies and experimental inhibitory activities suggest that the binding conformations of these inhibitors are reasonable. A novel interaction model was identified for inhibitors 11, 15, and 17 whose N-termini were modified by addition of the deaminovaline (Dav) group and the C-termini of 15 and 17 were modified by addition of a benzyl group. Accordingly, two new binding sites (sites A and D in Figure 1) were revealed, which show a strong correlation with inhibitor potency and thus can be used as a starting point for new inhibitor design. In addition, two predictive 3D-QSAR models were obtained by CoMFA and CoMSIA analyses based on the binding conformations derived from the molecular docking calculations. The reasonable r(cross)(2) (cross-validated) values 0.738 and 0.762 were obtained for CoMFA and CoMSIA models, respectively. The predictive ability of these models was validated by four peptide analogues test set. The CoMFA and CoMSIA field distributions are in general agreement with the structural characteristics of the binding groove of hCypA. This indicates the reasonableness of the binding model of the inhibitors with hCypA. Considering all these results together with the valuable clues of binding from references published recently, reasonable pharmacophore elements have been suggested, demonstrating that the 3D-QSAR models about peptide analogue inhibitors are expected to be further employed in predicting activities of the novel compounds for inhibiting hCypA.


Subject(s)
Cyclophilin A/chemistry , Gene Products, gag/chemistry , Oligopeptides/chemistry , Binding Sites , Gene Products, gag/antagonists & inhibitors , Humans , Molecular Conformation , Oligopeptides/antagonists & inhibitors , Quantitative Structure-Activity Relationship , Thermodynamics
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