ABSTRACT
Viral infections have a major impact in human health. Ongoing viral transmission and escalating selective pressure have the potential to favor the emergence of vaccine- and antiviral drug-resistant viruses. Target-based approaches for the design of antiviral drugs can play a pivotal role in combating drug-resistant challenges. Drug design computational tools facilitate the discovery of novel drugs. This review provides a comprehensive overview of current drug design strategies employed in the field of antiviral drug resistance, illustrated through the description of a series of successful applications. These strategies include technologies that enhance compound-target affinity while minimizing interactions with mutated binding pockets. Furthermore, emerging approaches such as virtual screening, targeted protein/RNA degradation, and resistance analysis during drug design have been harnessed to curtail the emergence of drug resistance. Additionally, host targeting antiviral drugs offer a promising avenue for circumventing viral mutation. The widespread adoption of these refined drug design strategies will effectively address the prevailing challenge posed by antiviral drug resistance.
Subject(s)
Antiviral Agents , Drug Design , Humans , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Antiviral Agents/metabolism , Drug Resistance, Viral/genetics , MutationABSTRACT
The human immunodeficiency virus type 1 (HIV-1) capsid is a protein core formed by multiple copies of the viral capsid (CA) protein. Inside the capsid, HIV-1 harbours all the viral components required for replication, including the genomic RNA and viral enzymes reverse transcriptase (RT) and integrase (IN). Upon infection, the RT transforms the genomic RNA into a double-stranded DNA molecule that is subsequently integrated into the host chromosome by IN. For this to happen, the viral capsid must open and release the viral DNA, in a process known as uncoating. Capsid plays a key role during the initial stages of HIV-1 replication; therefore, its stability is intimately related to infection efficiency, and untimely uncoating results in reverse transcription defects. How and where uncoating takes place and its relationship with reverse transcription is not fully understood, but the recent development of novel biochemical and cellular approaches has provided unprecedented detail on these processes. In this review, we present the latest findings on the intricate link between capsid stability, reverse transcription and uncoating, the different models proposed over the years for capsid uncoating, and the role played by other cellular factors on these processes.
Subject(s)
Capsid Proteins , Capsid , HIV-1 , Reverse Transcription , Virus Uncoating , HIV-1/genetics , HIV-1/physiology , Humans , Capsid/metabolism , Capsid Proteins/metabolism , Capsid Proteins/genetics , Virus Replication , HIV Infections/virology , HIV Infections/metabolism , RNA, Viral/metabolism , RNA, Viral/genetics , HIV Reverse Transcriptase/metabolism , HIV Reverse Transcriptase/geneticsABSTRACT
Molnupiravir, a prodrug of the nucleoside derivative ß-D-N4-hydroxycytidine (NHC), is currently in clinical trials for COVID-19 therapy. However, the biochemical mechanisms involved in molnupiravir-induced mutagenesis had not been explored. In a recent study, Gordon et al. demonstrated that NHC can be incorporated into viral RNA and subsequently extended and used as template for RNA-dependent RNA synthesis, proposing a mutagenesis model consistent with available virological evidence. Their study uncovers molecular mechanisms by which molnupiravir drives SARS-CoV-2 into error catastrophe.
Subject(s)
Antiviral Agents/pharmacology , COVID-19/virology , Cytidine/analogs & derivatives , Hydroxylamines/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Cytidine/pharmacology , Humans , Point Mutation/drug effects , RNA, Viral/genetics , SARS-CoV-2/metabolismABSTRACT
During the last forty years we have witnessed impressive advances in the field of antiviral drug discovery culminating with the introduction of therapies able to stop human immunodeficiency virus (HIV) replication, or cure hepatitis C virus infections in people suffering from liver disease. However, there are important viral diseases without effective treatments, and the emergence of drug resistance threatens the efficacy of successful therapies used today. In this review, we discuss strategies to discover antiviral compounds specifically designed to combat drug resistance. Currently, efforts in this field are focused on targeted proteins (e.g. multi-target drug design strategies), but also on drug conformation (either improving drug positioning in the binding pocket or introducing conformational constraints), in the introduction or exploitation of new binding sites, or in strengthening interaction forces through the introduction of multiple hydrogen bonds, covalent binding, halogen bonds, additional van der Waals forces or multivalent binding. Among the new developments, proteolysis targeting chimeras (PROTACs) have emerged as a valid approach taking advantage of intracellular mechanisms involving protein degradation by the ubiquitin-proteasome system. Finally, several molecules targeting host factors (e.g. human dihydroorotate dehydrogenase and DEAD-box polypeptide 3) have been identified as broad-spectrum antiviral compounds. Implementation of herein described medicinal chemistry strategies are expected to contribute to the discovery of new drugs effective against current and future threats due to emerging and re-emerging viral pandemics.
Subject(s)
Antiviral Agents/pharmacology , Chemistry, Pharmaceutical , Drug Discovery , Viruses/drug effects , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Drug Resistance, Viral/drug effects , Microbial Sensitivity TestsABSTRACT
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are widely used in combination therapies against HIV-1. However, emergent and transmitted drug resistance compromise their efficacy in the clinical setting. Y181C is selected in patients receiving nevirapine, etravirine and rilpivirine, and together with K103N is the most prevalent NNRTI-associated mutation in HIV-infected patients. Herein, we report on the design, synthesis and biological evaluation of a novel series of indolylarylsulfones bearing acrylamide or ethylene sulfonamide reactive groups as warheads to inactivate Cys181-containing HIV-1 RT via a Michael addition reaction. Compounds I-7 and I-9 demonstrated higher selectivity towards the Y181C mutant than against the wild-type RT, in nucleotide incorporation inhibition assays. The larger size of the NNRTI binding pocket in the mutant enzyme facilitates a better fit for the active compounds, while stacking interactions with Phe227 and Pro236 contribute to inhibitor binding. Mass spectrometry data were consistent with the covalent modification of the RT, although off-target reactivity constitutes a major limitation for further development of the described inhibitors.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/drug effects , Indoles/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Sulfones/pharmacology , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , Dose-Response Relationship, Drug , Drug Resistance, Viral/drug effects , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Indoles/chemical synthesis , Indoles/chemistry , Molecular Structure , Mutation , Reverse Transcriptase Inhibitors/chemical synthesis , Reverse Transcriptase Inhibitors/chemistry , Structure-Activity Relationship , Sulfones/chemical synthesis , Sulfones/chemistryABSTRACT
Human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS) still claim many lives across the world. However, research efforts during the last 40 years have led to the approval of over 30 antiretroviral drugs and the introduction of combination therapies that have turned HIV infection into a chronic but manageable disease. In this chapter, we provide an update on current available drugs and treatments, as well as future prospects towards reducing pill burden and developing long-acting drugs and novel antiretroviral therapies. In addition, we summarize efforts to cure HIV, including pharmaceutical strategies focused on the elimination of the virus.
Subject(s)
Acquired Immunodeficiency Syndrome , Anti-HIV Agents , HIV Infections , Anti-HIV Agents/therapeutic use , HIV Infections/drug therapy , HumansABSTRACT
Recent coronavirus outbreaks of SARS-CoV-1 (2002-2003), MERS-CoV (since 2012), and SARS-CoV-2 (since the end of 2019) are examples of how viruses can damage health care and generate havoc all over the world. Coronavirus can spread quickly from person to person causing high morbidity and mortality. Unfortunately, the antiviral armamentarium is insufficient to fight these infections. In this chapter, we provide a detailed summary of the current situation in the development of drugs directed against pandemic human coronaviruses. Apart from the recently licensed remdesivir, other antiviral agents discussed in this review include molecules targeting viral components (e.g., RNA polymerase inhibitors, entry inhibitors, or protease inhibitors), compounds interfering with virus-host interactions, and drugs identified in large screening assays, effective against coronavirus replication, but with an uncertain mechanism of action.
Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Humans , Pandemics , SARS-CoV-2ABSTRACT
In HIV-1, development of resistance to AZT (3'-azido-3'-deoxythymidine) is mediated by the acquisition of thymidine analogue resistance mutations (TAMs) (i.e., M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q) in the viral reverse transcriptase (RT). Clinically relevant combinations of TAMs, such as M41L/T215Y or D67N/K70R/T215F/K219Q, enhance the ATP-mediated excision of AZT monophosphate (AZTMP) from the 3' end of the primer, allowing DNA synthesis to continue. Additionally, during HIV-1 maturation, the Gag polyprotein is cleaved to release a mature nucleocapsid protein (NCp7) and two intermediate precursors (NCp9 and NCp15). NC proteins interact with the viral genome and facilitate the reverse transcription process. Using wild-type and TAM-containing RTs, we showed that both NCp9 and NCp15 inhibited ATP-mediated rescue of AZTMP-terminated primers annealed to RNA templates but not DNA templates, while NCp7 had no effect on rescue activity. RNase H inactivation by introducing the active-site mutation E478Q led to the loss of the inhibitory effect shown by NCp9. NCp15 had a stimulatory effect on the RT's RNase H activity not observed with NCp7 and NCp9. However, analysis of RNase H cleavage patterns revealed that in the presence of NCp9, RNA/DNA complexes containing duplexes of 12 bp had reduced stability in comparison with those obtained in the absence of NC or with NCp7 or NCp15. These effects are expected to have a strong influence on the inhibitory action of NCp9 and NCp15 by affecting the efficiency of RNA-dependent DNA polymerization after unblocking DNA primers terminated with AZTMP and other nucleotide analogues.
Subject(s)
Anti-HIV Agents , Zidovudine , Adenosine Triphosphate , Anti-HIV Agents/pharmacology , HIV Reverse Transcriptase/genetics , Mutation , Protein Precursors , Reverse Transcriptase Inhibitors/pharmacology , Zidovudine/pharmacologyABSTRACT
During reverse transcription of the HIV-1 genome, two strand-transfer events occur. Both events rely on the RNase H cleavage activity of reverse transcriptases (RTs) and template homology. Using a panel of mutants of HIV-1BH10 (group M/subtype B) and HIV-1ESP49 (group O) RTs and in vitro assays, we demonstrate that there is a strong correlation between RT minus-strand transfer efficiency and template-primer binding affinity. The highest strand transfer efficiencies were obtained with HIV-1ESP49 RT mutants containing the substitutions K358R/A359G/S360A, alone or in combination with V148I or T355A/Q357M. These HIV-1ESP49 RT mutants had been previously engineered to increase their DNA polymerase activity at high temperatures. Now, we found that RTs containing RNase H-inactivating mutations (D443N or E478Q) were devoid of strand transfer activity, whereas enzymes containing F61A or L92P had very low strand transfer activity. The strand transfer defect produced by L92P was attributed to a loss of template-primer binding affinity and, more specifically, to the higher dissociation rate constants (koff) shown by RTs bearing this substitution. Although L92P also deleteriously affected the RT's nontemplated nucleotide addition activity, neither nontemplated nucleotide addition activity nor the RT's clamp activities contributed to increased template switching when all tested mutant and WT RTs were considered. Interestingly, our results also revealed an association between efficient strand transfer and the generation of secondary cleavages in the donor RNA, consistent with the creation of invasion sites. Exposure of the elongated DNA at these sites facilitate acceptor (RNA or DNA) binding and promote template switching.
Subject(s)
DNA, Viral/metabolism , HIV Reverse Transcriptase/metabolism , HIV-1/metabolism , Ribonuclease H/metabolism , HIV Reverse Transcriptase/genetics , HIV-1/genetics , Humans , Point Mutation , Protein Binding , RNA, Viral/metabolism , Templates, GeneticABSTRACT
Nucleoside reverse transcriptase (RT) inhibitors (NRTIs) are the backbone of current antiretroviral treatments. However, the emergence of viral resistance against NRTIs is a major threat to their therapeutic effectiveness. In HIV-1, NRTI resistance-associated mutations either reduce RT-mediated incorporation of NRTI triphosphates (discrimination mechanism) or confer an ATP-mediated nucleotide excision activity that removes the inhibitor from the 3' terminus of DNA primers, enabling further primer elongation (excision mechanism). In HIV-2, resistance to zidovudine (3'-azido-3'-deoxythymidine (AZT)) and other NRTIs is conferred by mutations affecting nucleotide discrimination. Mutations of the excision pathway such as M41L, D67N, K70R, or S215Y (known as thymidine-analogue resistance mutations (TAMs)) are rare in the virus from HIV-2-infected individuals. Here, we demonstrate that mutant M41L/D67N/K70R/S215Y HIV-2 RT lacks ATP-dependent excision activity, and recombinant virus containing this RT remains susceptible to AZT inhibition. Mutant HIV-2 RTs were tested for their ability to unblock and extend DNA primers terminated with AZT and other NRTIs, when complexed with RNA or DNA templates. Our results show that Met73 and, to a lesser extent, Ile75 suppress excision activity when TAMs are present in the HIV-2 RT. Interestingly, recombinant HIV-2 carrying a mutant D67N/K70R/M73K RT showed 10-fold decreased AZT susceptibility and increased rescue efficiency on AZT- or tenofovir-terminated primers, as compared with the double-mutant D67N/K70R. Molecular dynamics simulations reveal that Met73influences ß3-ß4 hairpin loop conformation, whereas its substitution affects hydrogen bond interactions at position 70, required for NRTI excision. Our work highlights critical HIV-2 RT residues impeding the development of excision-mediated NRTI resistance.
Subject(s)
Drug Resistance, Viral , HIV Infections/virology , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/metabolism , HIV-2/enzymology , Nucleosides/pharmacology , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Anti-HIV Agents/pharmacology , DNA Repair/drug effects , HIV Reverse Transcriptase/genetics , HIV-2/chemistry , HIV-2/drug effects , HIV-2/genetics , Humans , Mutation, Missense/drug effects , Reverse Transcriptase Inhibitors/pharmacologyABSTRACT
A novel series of 3-hydroxyquinazoline-2,4(1H,3H)-diones derivatives has been designed and synthesized. Their biochemical characterization revealed that most of the compounds were effective inhibitors of HIV-1 RNase H activity at sub to low micromolar concentrations. Among them, II-4 was the most potent in enzymatic assays, showing an IC50 value of 0.41⯱â¯0.13⯵M, almost five times lower than the IC50 obtained with ß-thujaplicinol. In addition, II-4 was also effective in inhibiting HIV-1 IN strand transfer activity (IC50â¯=â¯0.85⯱â¯0.18⯵M) but less potent than raltegravir (IC50â¯=â¯71⯱â¯14â¯nM). Despite its relatively low cytotoxicity, the efficiency of II-4 in cell culture was limited by its poor membrane permeability. Nevertheless, structure-activity relationships and molecular modeling studies confirmed the importance of tested 3-hydroxyquinazoline-2,4(1H,3H)-diones as useful leads for further optimization.
Subject(s)
Anti-HIV Agents/pharmacology , Drug Design , Enzyme Inhibitors/pharmacology , HIV Integrase/metabolism , HIV Reverse Transcriptase/antagonists & inhibitors , Quinazolinones/pharmacology , Ribonuclease H, Human Immunodeficiency Virus/antagonists & inhibitors , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , Caco-2 Cells , Cell Line , Cell Membrane Permeability/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , HIV Reverse Transcriptase/metabolism , HIV-1/drug effects , HIV-2/drug effects , Humans , Models, Molecular , Molecular Structure , Quinazolinones/chemical synthesis , Quinazolinones/chemistry , Ribonuclease H, Human Immunodeficiency Virus/metabolism , Structure-Activity RelationshipABSTRACT
UNLABELLED: Previous studies have shown that a major block to HIV-1 replication in common marmosets operates at the level of viral entry and that this block can be overcome by adaptation of the virus in tissue-cultured cells. However, our current studies indicate that HIV-1 encounters additional postentry blocks in common marmoset peripheral blood mononuclear cells. Here, we show that the common marmoset APOBEC3G (A3G) and BST2 proteins block HIV-1 in cell cultures. Using a directed-evolution method that takes advantage of the natural ability of HIV-1 to mutate during replication, we have been able to overcome these blocks in tissue-cultured cells. In the adapted viruses, specific changes were observed in gag, vif, env, and nef. The contribution of these changes to virus replication in the presence of the A3G and BST2 restriction factors was studied. We found that certain amino acid changes in Vif and Env that arise during adaptation to marmoset A3G and BST2 allow the virus to replicate in the presence of these restriction factors. The changes in Vif reduce expression levels and encapsidation of marmoset APOBEC3G, while the changes in Env increase viral fitness and discretely favor cell-to-cell transmission of the virus, allowing viral escape from these restriction factors. IMPORTANCE: HIV-1 can infect only humans and chimpanzees. The main reason for this narrow tropism is the presence in many species of dominant-acting factors, known as restriction factors, that block viral replication in a species-specific way. We have been exploring the blocks to HIV-1 in common marmosets, with the ultimate goal of developing a new animal model of HIV-1 infection in these monkeys. In this study, we observed that common marmoset APOBEC3G and BST2, two known restriction factors, are able to block HIV-1 in cell cultures. We have adapted HIV-1 to replicate in the presence of these restriction factors and have characterized the mechanisms of escape. These studies can help in the development of a novel animal model for in vivo infection of marmosets with HIV-1-like viruses.
Subject(s)
Adaptation, Biological , Antigens, CD/metabolism , Cytidine Deaminase/metabolism , HIV-1/immunology , HIV-1/physiology , Virus Replication , Amino Acid Substitution , Animals , Antigens, CD/genetics , Callithrix , Cell Line , Cytidine Deaminase/genetics , HIV-1/genetics , Human Immunodeficiency Virus Proteins/genetics , Humans , Mutation , Virus CultivationABSTRACT
A small library containing 3-hydroxyquinazoline-2,4(1H,3H)-dione and 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one scaffolds was obtained via the copper(I)-catalyzed azidealkyne cycloaddition (CuAAC) reaction and evaluated for their anti-HIV activity in MT-4 cells. Among the synthesized compounds, several 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one derivatives showed remarkable anti-HIV potency with EC50 values ranging from 0.92 to 26.85µM. The most active one, IIA-2, also showed remarkable and selective potency against HIV type 1 integrase (IN). To the best of our knowledge, this is the first report showing that 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-ones are selective HIV IN inhibitors. Preliminary structure-activity relationship (SAR) studies suggested that the divalent metal ion chelators and the nature and position of substituents around the core are important for antiviral potency. Molecular modeling has been used to predict the binding site of the pyrido[2,3-d]pyrimidin-2(1H)-one core in HIV type 1 IN and suggestions are made for improvement of its inhibitory activity.
Subject(s)
HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/pharmacology , Pyrimidinones/chemical synthesis , Pyrimidinones/pharmacology , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Binding Sites , Biological Assay , Crystallography, X-Ray , Enzyme Activation/drug effects , HIV/drug effects , HIV/enzymology , HIV Integrase Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Models, Biological , Molecular Docking Simulation , Molecular Structure , Pyrimidinones/chemistry , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity RelationshipABSTRACT
HIV-1 reverse transcriptase (RT) connection subdomain mutations at positions 348, 369 and 376 have been associated with resistance to non-nucleoside RT inhibitors (NNRTIs). N348I may interfere with the initiation of (+)-strand DNA synthesis by reducing polypurine tract (PPT) removal in the presence of nevirapine. The effect of NNRTIs on the RNase H-mediated cleavage of PPT-containing template-primers has been studied with wild-type HIV-1 RT and mutants N348I, T369I, T369V, T376S and N348I/T369I. In the presence of NNRTIs, all RTs were able to stimulate PPT cleavage after primer elongation. The enhancing effects of nevirapine and efavirenz were reduced in RTs carrying mutation N348I, and specially N348I/T369I. However, those mutations had no effect on rilpivirine-mediated cleavage. Prior to elongation, the PPT remains resilient to cleavage, although efavirenz and rilpivirine facilitate RNase H-mediated trimming of its 3'-end. The integrity of the 3'-end is essential for the initiation of (+)-strand DNA synthesis. In the presence of dNTPs, rilpivirine was the most effective inhibitor of (+)-strand DNA synthesis blocking nucleotide incorporation and preventing usage of available PPT primers. The N348I/T369I RT showed reduced ability to generate short RNA products revealing a cleavage window defect. Its lower RNase H activity could be attributed to enhanced rigidity compared to the wild-type enzyme.
Subject(s)
Anti-HIV Agents/pharmacology , DNA/biosynthesis , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , Mutation , Reverse Transcriptase Inhibitors/pharmacology , Drug Resistance, Viral/genetics , HIV Reverse Transcriptase/chemistry , Nitriles/pharmacology , Protein Structure, Tertiary , Purines/metabolism , Pyridazines/pharmacology , Pyrimidines/pharmacology , RNA , Ribonuclease H/metabolism , Rilpivirine , Templates, GeneticABSTRACT
Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain-containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67(+)) primary CD4(+) T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability.
Subject(s)
Cell Cycle Checkpoints/immunology , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase 6/metabolism , HIV Infections/immunology , Monomeric GTP-Binding Proteins/metabolism , Benzylamines , CD4-Positive T-Lymphocytes/immunology , Cell Cycle/immunology , Cells, Cultured , Cyclams , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/genetics , HEK293 Cells , HIV Infections/virology , HIV-1/immunology , Heterocyclic Compounds/pharmacology , Humans , Lymphocyte Activation/immunology , Lymphocytes/immunology , Macrophages/immunology , Myeloid Cells/immunology , Phosphorylation/drug effects , Phosphorylation/genetics , RNA Interference , RNA, Small Interfering , Receptors, CXCR4/antagonists & inhibitors , SAM Domain and HD Domain-Containing Protein 1ABSTRACT
Asp(443) and Glu(478) are essential active site residues in the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). We have investigated the effects of substituting Asn for Asp(443) or Gln for Glu(478) on the fidelity of DNA-dependent DNA synthesis of phylogenetically diverse HIV-1 RTs. In M13mp2 lacZα-based forward mutation assays, HIV-1 group M (BH10) and group O RTs bearing substitutions D443N, E478Q, V75I/D443N or V75I/E478Q showed 2.0- to 6.6-fold increased accuracy in comparison with the corresponding wild-type enzymes. This was a consequence of their lower base substitution error rates. One-nucleotide deletions and insertions represented between 30 and 68% of all errors identified in the mutational spectra of RNase H-deficient HIV-1 group O RTs. In comparison with the wild-type RT, these enzymes showed higher frameshift error rates and higher dissociation rate constants (koff) for DNA/DNA template-primers. The effects on frameshift fidelity were similar to those reported for mutation E89G and suggest that in HIV-1 group O RT, RNase H inactivation could affect template/primer slippage. Our results support a role for the RNase H domain during plus-strand DNA polymerization and suggest that mutations affecting RNase H function could also contribute to retrovirus variability during the later steps of reverse transcription.
Subject(s)
DNA/biosynthesis , HIV Reverse Transcriptase/metabolism , Ribonuclease H, Human Immunodeficiency Virus/genetics , Amino Acid Substitution , DNA Primers/metabolism , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/genetics , Mutation , Protein Structure, Tertiary , Ribonuclease H, Human Immunodeficiency Virus/chemistry , Templates, GeneticABSTRACT
We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by which those mutations increase the thermal stability of the RT. The three single-mutants (E286R, E302K, and L435R), an RNase H-deficient MMLV RT (carrying the RNase H-inactivating mutation D524A), a quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) and the wild-type enzyme (WT) were produced in Escherichia coli. All RTs exhibited similar dissociation constants (Kd) for heteropolymeric DNA/DNA (2.9-6.5 nM) and RNA/DNA complexes (1.2-2.9 nM). Unlike the WT, mutant enzymes (E286R, E302K, L435R, D524A, and MM4) were devoid of RNase H activity, and were not able to degrade RNA in RNA/DNA complexes. These results suggest that the mutations, E286R, E302K, and L435R increase the thermostability of MMLV RT not by increasing its affinity for T/P but by abolishing its RNase H activity.
Subject(s)
Amino Acid Substitution , Moloney murine leukemia virus/enzymology , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolism , Ribonuclease H/metabolism , Base Sequence , Catalytic Domain , DNA/analysis , DNA/metabolism , Escherichia coli/genetics , Moloney murine leukemia virus/chemistry , Moloney murine leukemia virus/genetics , Mutagenesis, Site-Directed , Protein Stability , RNA/analysis , RNA/metabolism , RNA-Directed DNA Polymerase/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TemperatureABSTRACT
During the last 30 years, significant progress has been made in the development of novel antiviral drugs, mainly crystallizing in the establishment of potent antiretroviral therapies and the approval of drugs inhibiting hepatitis C virus replication. Although major targets of antiviral intervention involve intracellular processes required for the synthesis of viral proteins and nucleic acids, a number of inhibitors blocking virus assembly, budding, maturation, entry or uncoating act on virions or viral capsids. In this review, we focus on the drug discovery process while presenting the currently used methodologies to identify novel antiviral drugs by using a computer-based approach. We provide examples illustrating structure-based antiviral drug development, specifically neuraminidase inhibitors against influenza virus (e.g. oseltamivir and zanamivir) and human immunodeficiency virus type 1 protease inhibitors (i.e. the development of darunavir from early peptidomimetic compounds such as saquinavir). A number of drugs in preclinical development acting against picornaviruses, hepatitis B virus and human immunodeficiency virus and their mechanism of action are presented to show how viral capsids can be exploited as targets of antiviral therapy.
Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Design , Animals , HumansABSTRACT
At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³58-Gly³59-Ala³6° triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³58-Ala³59-Ser³6° triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³58, Gly³59, and Ala³6° in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.
Subject(s)
DNA, Complementary/biosynthesis , DNA/metabolism , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Models, Molecular , RNA, Viral/metabolism , Amino Acid Motifs , Amino Acid Substitution , Binding Sites , Computer Simulation , DNA/chemistry , Databases, Protein , Enzyme Stability , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/genetics , HIV-1/metabolism , Hot Temperature , Kinetics , Molecular Conformation , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/metabolism , RNA, Viral/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribonuclease H, Human Immunodeficiency Virus/chemistry , Ribonuclease H, Human Immunodeficiency Virus/genetics , Ribonuclease H, Human Immunodeficiency Virus/metabolism , Thymine Nucleotides/chemistry , Thymine Nucleotides/metabolismABSTRACT
Influenza is an acute respiratory infection caused by influenza viruses (IFV), According to the World Health Organization (WHO), seasonal IFV epidemics result in approximately 3-5 million cases of severe illness, leading to about half a million deaths worldwide, along with severe economic losses and social burdens. Unfortunately, frequent mutations in IFV lead to a certain lag in vaccine development as well as resistance to existing antiviral drugs. Therefore, it is of great importance to develop anti-IFV drugs with high efficiency against wild-type and resistant strains, needed in the fight against current and future outbreaks caused by different IFV strains. In this review, we summarize general strategies used for the discovery and development of antiviral agents targeting multiple IFV strains (including those resistant to available drugs). Structure-based drug design, mechanism-based drug design, multivalent interaction-based drug design and drug repurposing are amongst the most relevant strategies that provide a framework for the development of antiviral drugs targeting IFV.