We have discovered a novel class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone compounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed "hot spots" for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-specific footprinting experiments revealed that INDOPY-1 traps the complex in the posttranslocational state, preventing binding and incorporation of the next complementary nucleotide. The novel mode of action translates into a unique resistance profile. While INDOPY-1 susceptibility is unaffected by mutations associated with NNRTI or multidrug NRTI resistance, mutations M184V and Y115F are associated with decreased susceptibility, and mutation K65R confers hypersusceptibility to INDOPY-1. This resistance profile provides additional evidence for active site binding. In conclusion, this class of indolopyridones can occupy the nucleotide binding site of HIV RT by forming a stable ternary complex whose stability is mainly dependent on the nature of the primer 3 end.The reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1) remains a major target in antiretroviral therapy, with the current standard of care being the use of two nucleoside or nucleotide analogue reverse transcriptase inhibitors (NRTIs), combined with one nonnucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (32).Upon entry into the cell, the virus is uncoated, and the viral RT enzyme converts its single-stranded RNA genome into double-stranded proviral DNA. Inhibition of this crucial event in the early viral life cycle ultimately precludes the virus from proliferating. Following the intracellular phosphorylation of NRTIs, NRTI-triphosphates compete with natural deoxyribonucleoside triphosphate (dNTP) pools and bind to the RT active site. They act as chain terminators due to the lack of a 3Ј-hydroxyl group (31). In contrast, NNRTIs represent a chemically diverse class of compounds that bind to a pocket in the vicinity of the catalytic site (25,29,30). Binding of these inhibitors is noncompetitive with respect to both dNTPs and template/primer (16).Despite the potency of combinations of NRTIs and NNRTIs, the emergence of mutations conferring resistance remains a major cause for treatment failure. The advent of novel RT inhibitors with a different mechani...

Thymidine analogue-associated mutations (TAMs) in reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) cause resistance to 3-azido-3-deoxythymidine (AZT) through excision of the incorporated monophosphate. Mutations in the connection domain of HIV-1 RT can augment AZT resistance. It has been suggested that these mutations compromise RNase H cleavage, providing more time for AZT excision to occur. However, the underlying mechanism remains elusive. Here, we focused on connection mutations N348I and A360V that are frequently observed in clinical samples of treatment-experienced patients. We show that both N348I and A360V, in combination with TAMs, decrease the efficiency of RNase H cleavage and increase excision of AZT in the presence of the pyrophosphate donor ATP. The TAMs/N348I/A360V mutant accumulates transiently formed, shorter hybrids that can rebind to RT before the template is irreversibly degraded. These hybrids dissociate selectively from the RNase H-competent complex, whereas binding in the polymerase-competent mode is either not affected with N348I or modestly improved with A360V. Both connection domain mutations can compensate for TAM-mediated deficits in processive DNA synthesis, and experiments with RNase H negative mutant enzymes confirm an RNase H-independent contribution to increased levels of resistance to AZT. Moreover, the combination of diminished RNase H cleavage and increased processivity renders the use of both PP i and ATP advantageous, whereas classic TAMs solely enhance the ATP-dependent reaction. Taken together, our findings demonstrate that distinct, complementary mechanisms can contribute to higher levels of excision of AZT, which in turn can amplify resistance to this drug.Human immunodeficiency virus type 1 (HIV-1) 4 replicates using a virally encoded reverse transcriptase (RT), which contains a catalytically active large subunit (p66) and a smaller p66-derived subunit (p51). The p66 subunit comprises the DNA polymerase (residues 1-321), connection (residues 322-440), and RNase H (residues 441-560) domains (1, 2). The RNase H activity, which degrades the RNA of DNA⅐RNA hybrids, is essentially required to convert the single-stranded viral RNA genome into double-stranded proviral DNA (3).Due to its key role in viral replication, HIV-1 RT represents a major therapeutic target (4, 5). Approved RT inhibitors belong to two distinct classes: nucleoside analogue and nonnucleoside analogue RT inhibitors (NRTIs and NNRTIs, respectively). NRTIs are synthetic derivatives of the natural deoxynucleosides. The triphosphate forms of NRTIs and cellular dNTPs serve as substrates for HIV-1 RT. In contrast to natural dNTPs, NRTIs lack the 3Ј-hydroxyl group of the sugar moiety. The incorporated monophosphate (MP) therefore acts as a chain terminator, which prevents phosphodiester bond formation with the next complementary nucleotide (6). NNRTIs are structurally diverse, and members of this family of compounds bind to a hydrophobic pocket (NNRTI-binding pocket) near the polymeras...

High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5, 6,7,8-tetrahydro-4H-cyclohepta[b] thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT).A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor β-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Sitedirected mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or nonnatural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, diva-lent metal-chelating inhibitors that have been reported.Although an absolute requirement for reverse transcriptase (RT)-associated ribonuclease H (RNase H) activity for human immunodeficiency virus (HIV) replication was documented almost two decades ago (1,2), development of potent and selective RNase H inhibitors has been surprisingly slow compared with the nucleoside and non-nucleoside DNA polymerase inhibitors currently in clinical use. Recently, however,4), diketo acids (5,6), and dihydroxytropolones (7) have shown promise by specifically inhibiting RNase H activity of HIV-1 and HIV-2 RT, and in some instances acting synergistically with clinically approved RT inhibitors. The preliminary crystal structure of an N-hydroxyimide bound to the RNase H domain of HIV-1 RT (4) suggests that it sequesters the divalent metal cofactor, laying the foundation for rational design of improved inhibitors. Increasing the diversity of RNase H © 2008 American Chemical Society * Corresponding author, slegrice@ncifcrf.gov.. Supporting Information Available:This material is free of charge via the Internet. In order to examine the NSC727447 binding site, we performed mass spectrometric protein footprinting based on biotin modification of exposed lysine residues in the free protein and the protein-inhibitor complex (8-10). Cys280 and Lys281, located in helix I of the thumb subdomain, were protected from modification by inhibitor binding. Proximity between the p51 thumb subdomain and the p66 RNase H domain implies that inhibitor binding adjacent to the catalytic center affects either divalent metal coordination or positioning of the nucleic acid substrate in the active site. Although DNA polymerase activity was less sensi...

Tremendous progress has been made over the last 2 decades to discover and develop approaches to control hepatitis B virus (HBV) infections and to prevent the development of hepatocellular carcinoma using various interferons and small molecules as antiviral agents. However, none of these agents have significant impact on eliminating HBV from infected cells. Currently the emphasis is on silencing or eliminating cccDNA, which could lead to a cure for HBV. Various approaches are being developed including the development of capsid effectors, CRISPR/Cas9, TALENS, siRNA, entry and secretion inhibitors, as well as immunological approaches. It is very likely that a combination of these modalities will need to be employed to successfully eliminate HBV or prevent virus rebound on discontinuation of therapy. In the next 5 years clinical data will emerge which will provide insight on the safety and feasibility of these approaches and if they can be applied to eradicate HBV infections globally. In this review, we summarize current treatments and we highlight and examine recent therapeutic strategies that are currently being evaluated at the preclinical and clinical stage. K E Y W O R D Santiviral agents, cccDNA, HBV cure, immunotherapy

Chutes and Ladders is an exciting up-and-down-again game in which players race to be the first to the top of the board. Along the way, they will find ladders to help them advance, and chutes that will cause them to move backwards. The development of nucleoside analogs for clinical treatment of hepatitis C presents a similar scenario in which taking shortcuts may help quickly advance a program, but there is always a tremendous risk of being sent backwards as one competes for the finish line. In recent years the treatment options for chronic hepatitis C virus (HCV) infection have expand due to the development of a replicon based in vitro evaluation system, allowing for the identification of multiple drugable viral targets along with a concerted and substantial drug discovery effort. Three major drug targets have reached clinical study for chronic HCV infection: the NS3/4A serine protease, the large phosphoprotein NS5A, and the NS5B RNA-dependent RNA polymerase. Recently, two oral HCV protease inhibitors were approved by the FDA and were the first direct acting anti-HCV agents to result from the substantial research in this area. There are currently many new chemical entities from several different target classes that are being evaluated worldwide in clinical trials for their effectiveness at achieving a sustained virologic response (SVR) (Pham et al., 2004; Radkowski et al., 2005). Clearly the goal is to develop therapies leading to a cure that are safe, widely accessible and available, and effective against all HCV genotypes (GT), and all stages of the disease. Nucleoside analogs that target the HCV NS5B polymerase that have reached human clinical trials is the focus of this review as they have demonstrated significant advantages in the clinic with broader activity against the various HCV GT and a higher barrier to the development of resistant viruses when compared to all other classes of HCV inhibitors.