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...
Integration of viral DNA into the host chromosome is an essential step in the life cycle of retroviruses and is facilitated by the viral integrase enzyme. The first generation of integrase inhibitors recently approved or currently in late-stage clinical trials shows great promise for the treatment of human immunodeficiency virus (HIV) infection, but virus is expected to develop resistance to these drugs. Therefore, we used a novel resistance selection protocol to follow the emergence of resistant HIV in the presence of the integrase inhibitor elvitegravir (GS-9137). We find the primary resistance-conferring mutations to be Q148R, E92Q, and T66I and demonstrate that they confer a reduction in susceptibility not only to elvitegravir but also to raltegravir (MK-0518) and other integrase inhibitors. The locations of the mutations are highlighted in the catalytic sites of integrase, and we correlate the mutations with expected drug-protein contacts. In addition, mutations that do not confer reduced susceptibility when present alone (H114Y, L74M, R20K, A128T, E138K, and S230R) are also discussed in relation to their position in the catalytic core domain and their proximity to known structural features of integrase. These data broaden the understanding of antiviral resistance against integrase inhibitors and may give insight facilitating the discovery of second-generation compounds.Integration of retroviral DNA is an essential step in the life cycle of human immunodeficiency virus (HIV) (29). The integration process is facilitated by the viral integrase (IN) enzyme which catalyzes the insertion of the viral DNA into the host genome in a multistep process involving viral and host proteins. HIV IN recognizes and binds specific sequences in the long terminal repeats (LTRs) of the viral retrotranscribed DNA in the cytoplasm. After DNA binding, IN cleaves GT dinucleotides from the 3Ј termini of the linear cDNA in a process called 3Ј processing (2). The processed viral DNA, as part of the preintegration complex, is then translocated into the nucleus, where IN inserts the viral DNA into the host chromosome by a process called strand transfer (2,12,13). There are few cellular enzymes with comparable function to HIV integrase (24), apart from the V(D)J polynucleotide recombinase RAG1 (34). Therefore, the IN enzyme has been considered an attractive target for antiretroviral therapy for the last decade (27, 36). Recent progress has resulted in two IN inhibitors (5, 30), with one drug in late-stage clinical trials and one currently approved for use in treatment-experienced patients (18). For all currently targeted retroviral proteins, inhibition with antiretroviral drugs has led to emergence of resistance in treated patients, often leading to treatment failure and requiring changes in the composition of the highly active antiretroviral therapy (HAART) drug regimen (6). Nevertheless, the emergence of new classes of drugs will enable new combinations of inhibitors to be used and will offer more treatment options to HIV-infected pati...
Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure–activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.
Six-helix bundle (6HB) formation is an essential step for many viruses that rely on a class I fusion protein to enter a target cell and initiate replication. Because the binding modes of small molecule inhibitors of 6HB formation are largely unknown, precisely how they disrupt 6HB formation remains unclear, and structure-based design of improved inhibitors is thus seriously hampered. Here we present the high resolution crystal structure of TMC353121, a potent inhibitor of respiratory syncytial virus (RSV), bound at a hydrophobic pocket of the 6HB formed by amino acid residues from both HR1 and HR2 heptad-repeats. Binding of TMC353121 stabilizes the interaction of HR1 and HR2 in an alternate conformation of the 6HB, in which direct binding interactions are formed between TMC353121 and both HR1 and HR2. Rather than completely preventing 6HB formation, our data indicate that TMC353121 inhibits fusion by causing a local disturbance of the natural 6HB conformation.cocrystal structure | respiratory syncytial virus | TMC353121 | viral fusion T o allow the deposition of their nucleic acid genome into a host cell, and to initiate their replication cycle, enveloped viruses have evolved complex membrane fusion machinery that includes a fusion protein (1, 2). Based on structural similarity, the viral fusion proteins from different viruses have been grouped into three distinct classes: I, II, and III (3, 4). Prototypic trimeric class I fusion proteins include HIV-1 gp41, influenza hemagglutinin and the fusion proteins from paramyxoviruses. The fusion protein (F) of respiratory syncytial virus (RSV), a paramyxovirus belonging to the pneumovirinae subfamily, assembles into a homotrimer that is cleaved at two proximal furin cleavage sites during biosynthesis, priming the protein for membrane fusion. Proteolytic cleavage of the fusion protein precursor (F 0 ) yields two polypeptides, F 1 and F 2 , joined by a disulfide bridge (Fig. 1). F 1 consists of an N-terminal hydrophobic fusion peptide, followed by a first heptad-repeat (HR1), an intervening globular domain, and a second heptadrepeat (HR2), which itself is N-terminal to the viral transmembrane and cytoplasmic regions (3). Once fusion is triggered, dramatic refolding of the prefusion conformation of the viral fusion protein occurs. Functional and structural studies have provided evidence that a folding intermediate is formed that contains a coiled-coil structure of three HR1 heptad repeats (5-8). This intermediate allows the fusion peptide to be inserted into the plasma membrane of a target cell. In the final stage of membrane fusion, the HR1-CTC structure irreversibly refolds into a 6HB complex with three HR2 heptad-repeats, resulting in membrane merger and stable fusion pore formation (5-14). In many viruses that rely on class I fusion proteins, the central HR1 trimeric coiled-coil (HR1-CTC) contains a hydrophobic pocket in each of its three grooves that has been proposed as a potential drug binding site (9, 10).The therapeutic value of inhibiting 6HB formation was establ...
Emergence of resistance to raltegravir reduces its treatment efficacy in HIV-1-infected patients. To delineate the effect of resistance mutations on viral susceptibility to integrase inhibitors, in vitro resistance selections with raltegravir and with MK-2048, an integrase inhibitor with a second-generation-like resistance profile, were performed. Mutation Q148R arose in four out of six raltegravir-selected resistant viruses. In addition, mutations Q148K and N155H were selected. In the same time frame, no mutations were selected with MK-2048. Q148H/K/R and N155H conferred resistance to raltegravir, but only minor changes in susceptibility to MK-2048. V54I, a previously unreported mutation, selected with raltegravir, was identified as a possible compensation mutation. Mechanisms by which N155H, Q148H/K/R, Y143R and E92Q confer resistance are proposed based on a structural model of integrase. These data improve the understanding of resistance against raltegravir and cross-resistance to MK-2048 and other integrase inhibitors, which will aid in the discovery of second-generation integrase inhibitors.
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