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...
In quite a few diseases, drug resistance due to target variability poses a serious problem in pharmacotherapy. This is certainly true for HIV, and hence, it is often unknown which drug is best to use or to develop against an individual HIV strain. In this work we applied ‘proteochemometric’ modeling of HIV Non-Nucleoside Reverse Transcriptase (NNRTI) inhibitors to support preclinical development by predicting compound performance on multiple mutants in the lead selection stage. Proteochemometric models are based on both small molecule and target properties and can thus capture multi-target activity relationships simultaneously, the targets in this case being a set of 14 HIV Reverse Transcriptase (RT) mutants. We validated our model by experimentally confirming model predictions for 317 untested compound – mutant pairs, with a prediction error comparable with assay variability (RMSE 0.62). Furthermore, dependent on the similarity of a new mutant to the training set, we could predict with high accuracy which compound will be most effective on a sequence with a previously unknown genotype. Hence, our models allow the evaluation of compound performance on untested sequences and the selection of the most promising leads for further preclinical research. The modeling concept is likely to be applicable also to other target families with genetic variability like other viruses or bacteria, or with similar orthologs like GPCRs.
Fusion of host cell and human immunodeficiency virus type 1 (HIV-1) membranes is mediated by the 2 "heptad-repeat" regions of the viral gp41 protein. The collapse of the C-terminal heptad-repeat regions into the hydrophobic grooves of a coiled-coil formed by the corresponding homotrimeric N-terminal heptad-repeat regions generates a stable 6-helix bundle. This brings viral and cell membranes together for membrane fusion, facilitating viral entry. The authors developed an assay based on soluble peptides derived from the gp41 N-terminal heptad-repeat region (IQN36) as well as from the C-terminal region (C34). Both peptides were labeled with fluorophores, IQN36 with allophycocyanin (APC) and C34 with the lanthanide europium (Eu
Human immunodeficiency virus type 1 (HIV-1) integrase is, in addition to reverse transcriptase and protease, an important enzymatic target for antiretroviral drug development. Integrase plays a critical role in the HIV-1 life cycle coordinating the integration of the reverse-transcribed viral DNA into the host genome. This integration step is the net result of two consecutive integrase-related processes. First, integrase removes a dinucleotide from the 3' viral DNA ends in a process called 3'-processing. Next, in a process called strand transfer, the viral DNA is integrated into the host genomic DNA. Early on, biochemical assays have played a critical role in understanding the function of HIV-1 integrase and the discovery of small-molecule inhibitors. In this chapter we describe two biochemical assays to identify inhibitors of the 3'-processing and strand transfer process of HIV-1 integrase.
The human immunodeficiency virus type 1 (HIV-1) initiates infection through sequential interactions with CD4 and chemokine coreceptors unmasking the gp41 subunit of the viral envelope protein. Consequently, the N-terminal heptad repeats of gp41 form a trimeric coiled-coil groove in which the C-terminal heptad repeats collapse, generating a stable six-helix bundle. This brings the viral and cell membrane in close proximity enabling fusion and the release of viral genome in the cytosol of the host cell. In this chapter, we describe a homogeneous time-resolved fluorescence assay to identify inhibitors of HIV-1 fusion, based on the ability of soluble peptides, derived from the N- and C-terminal domains of gp41, to form a stable six-helix bundle in vitro. Labeling of the peptides with allophycocyanin and the lanthanide europium results in a Föster resonance energy transfer (FRET) signal upon formation of the six-helix bundle. Compounds interfering with the six-helix bundle formation inhibit the HIV-1 fusion process and suppress the FRET signal.
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