Background:Competitors of LEDGF binding to HIV-1 integrase could prevent targeted integration to chromatin. Results: LEDGF competitors like tBPQAs were also found to inhibit integrase enzyme activity by preventing proper integraseviral DNA assembly. Conclusion: tBPQAs are allosteric inhibitors of integrase with a dual mode of action. Significance: Interference with two distinct steps of integration through the same binding site represents a new antiviral paradigm.

Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The overall virologic profile of BIC supports its ongoing clinical investigation in combination with other antiretroviral agents for both treatment-naive and -experienced HIV-infected patients.

The use of nonhuman primate (NHP) models to study persistent residual virus and viral eradication strategies in combination antiretroviral therapy (cART)-treated individuals requires regimens that effectively suppress SIV replication to clinically relevant levels in macaques. We developed and evaluated two novel cART regimens in SIVmac239-infected rhesus macaques: (1) a "triple regimen" containing the nucleo(s/t)ide reverse transcriptase inhibitors emtricitabine (FTC) and tenofovir disoproxil fumarate [TDF, prodrug of tenofovir (TFV, PMPA)] with the integrase strand transfer inhibitor dolutegravir (DTG) (n = 3), or (2) a "quad regimen" containing the same three drugs plus the protease inhibitor darunavir (DRV) (n = 3), with each regimen coformulated for convenient administration by a single daily subcutaneous injection. Plasma drug concentrations were consistent across animals within the triple and quad regimen-treated groups, although DTG levels were lower in the quad regimen animals. Time to achieve plasma viral loads stably <30 viral RNA copies/ml ranged from 12 to 20 weeks of treatment between animals, and viral loads <30 viral RNA copies/ml plasma were maintained through 40 weeks of follow-up on cART. Notably, although we show virologic suppression and development of viral resistance in a separate cohort of SIV-infected animals treated with oral DRV monotherapy, the addition of DRV in the quad regimen did not confer an apparent virologic benefit during early treatment, hence the quad regimen-treated animals were switched to the triple regimen after 4 weeks. This coformulated triple cART regimen can be safely, conveniently, and sustainably administered to durably suppress SIV replication to clinically relevant levels in rhesus macaques.

The interaction between lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF) and human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for HIV-1 replication. Homogeneous time-resolved fluorescence resonance energy transfer assays were developed to characterize HIV-1 integrase dimerization and the interaction between LEDGF and IN dimers. Using these assays in an equilibrium end point dose-response format with mathematical modeling, we determined the dissociation constants of IN dimers (K dimer ‫؍‬ 67.8 pM) and of LEDGF from IN dimers (K d ‫؍‬ 10.9 nM). When used in a kinetic format, the assays allowed the determination of the on-and off-rate constants for these same interactions. Targeting the viral integration process with small molecules as a strategy to inhibit HIV-1 2 replication has recently yielded an important new class of antiviral drugs. One integrase strand transfer inhibitor (INSTI), raltegravir (MK-0518), has been approved by the Food and Drug Administration for treatment of HIV-1 infection, and a second drug, elvitegravir (GS-9137), is in late stage clinical development. Based on binding experiments (1) and molecular modeling (2), strand transfer inhibitors are thought to interact with a pocket in the active site of integrase that is formed after 3Ј-processing of the viral DNA ends. INSTIs thus prevent the integrase-viral DNA complex from engaging host target DNA. More recently, a second site on integrase that represents the binding site for the cellular cofactor LEDGF was demonstrated to be a viable and attractive target for antiviral drug discovery (3-5). A small molecule inhibitor targeting this second site may retain activity against viral mutants resistant to INSTIs and complement the antiviral activity of INSTIs, akin to non-nucleoside reverse transcriptase inhibitors with nucleoside reverse transcriptase inhibitors. Hence, compounds that interact with the LEDGF binding pocket on IN could be used in combination with INSTIs to decrease the likelihood of resistance emergence.The cellular cofactor LEDGF has been identified as the dominant binding partner of HIV-1 integrase in human cells (6 -10). LEDGF interacts with integrase primarily through an ϳ80-amino acid domain termed the integrase binding domain (IBD) (11). A solution structure of the IBD has been derived (12). In addition, several co-crystal structures involving IBD and IN domains have been solved and include the following: IBD bound to a dimer of HIV-1 integrase catalytic core domain (CCD), IBD bound to an HIV-2 two-domain integrase (N-terminal domain (NTD) and catalytic core domain), and IBD bound to a maedi-visna virus two-domain (NTD ϩ CCD) integrase (13-15). Several lines of evidence point to the requirement of LEDGF for HIV-1 replication as a viral cofactor as follows. 1) Mutations in integrase that preserve the catalytic activity of the enzyme but cause defects in viral replication also disrupt integrase interaction with LEDGF (16 -18). 2) Suppression of LEDGF expression mediated by smal...

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