TLR7 Agonist GS-9620 Is a Potent Inhibitor of Acute HIV-1 Infection in Human Peripheral Blood Mononuclear Cells.

GS-9620 is a potent and selective oral Toll-like receptor 7 (TLR7) agonist that directly activates plasmacytoid dendritic cells (pDCs). GS-9620 suppressed hepatitis B virus (HBV) in animal models of chronic infection and transiently activated HIV expression ex vivo in latently infected peripheral blood mononuclear cells (PBMCs) from virally suppressed patients. Currently, GS-9620 is under clinical evaluation for treating chronic HBV infection and for reducing latent reservoirs in virally suppressed HIV-infected patients. Here, we investigated the in vitro anti-HIV-1 activity of GS-9620. GS-9620 potently inhibited viral replication in PBMCs, particularly when it was added 24 to 48 h prior to HIV infection (50% effective concentration = 27 nM). Depletion of pDCs but not other immune cell subsets from PBMC cultures suppressed GS-9620 antiviral activity. Although GS-9620 was inactive against HIV in purified CD4+ T cells and macrophages, HIV replication was potently inhibited by conditioned medium derived from GS-9620-treated pDC cultures when added to CD4+ T cells prior to infection. This suggests that GS-9620-mediated stimulation of PBMCs induced the production of a soluble factor(s) inhibiting HIV replication in trans GS-9620-treated PBMCs primarily showed increased production of interferon alpha (IFN-α), and cotreatment with IFN-α-blocking antibodies reversed the HIV-1-inhibitory effect of GS-9620. Additional studies demonstrated that GS-9620 inhibited a postentry event in HIV replication at a step coincident with or prior to reverse transcription. The simultaneous activation of HIV-1 expression and inhibition of HIV-1 replication are important considerations for the clinical evaluation of GS-9620 since these antiviral effects may help restrict potential local HIV spread upon in vivo latency reversal.

Antiviral Activity of Bictegravir (GS-9883), a Novel Potent HIV-1 Integrase Strand Transfer Inhibitor with an Improved Resistance Profile.

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.

Short Communication: Comparative Evaluation of Coformulated Injectable Combination Antiretroviral Therapy Regimens in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

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.

Abyssomicin 2 reactivates latent HIV-1 by a PKC- and HDAC-independent mechanism.

Screening of a marine natural products library afforded three new analogues of the tetronic acid containing polyketide abyssomicin family and identified abyssomicin 2 as a selective reactivator of latent HIV virus. Examination of the mode of action of this new latent HIV reactivating agent demonstrated that it functions via a distinct mechanism compared to that of existing reactivating agents and is effective at reactivating latent virus in a subset of primary patient cell lines.

Study of marine natural products including resorcyclic acid lactones from Humicola fuscoatra that reactivate latent HIV-1 expression in an in vitro model of central memory CD4+ T cells.

An extract of Humicola fuscoatra (UCSC strain no. 108111A) was shown to reactivate latent HIV-1 expression in an in vitro model of central memory CD4+ T cells. We report the bioassay-guided isolation and structure determination of several resorcyclic acid lactones, including four known compounds, radicicol (1, aka. monorden) and pochonins B (2), C (3), and N (4), and three new analogues, radicicols B-D (5-7). Compounds 1-3 and 5 showed moderate activities in the memory T cell model of HIV-1 latency. Radicicol (1) displayed lower potency in reactivating latent HIV-1 (EC50 = 9.1 µM) relative to the HDAC inhibitors apicidin (EC50 = 0.3 µM), romidepsin (EC50 = 0.003 µM), and SAHA (EC50 = 0.6 µM); however, it achieved equivalent maximum efficacy relative to the positive control compounds (98% of SAHA and romidepsin).

New class of HIV-1 integrase (IN) inhibitors with a dual mode of action.

tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.

In vitro resistance selections using elvitegravir, raltegravir, and two metabolites of elvitegravir M1 and M4.

Elvitegravir is a strand transfer inhibitor of HIV-1 integrase that is currently undergoing phase 3 clinical testing. The two predominant metabolites of elvitegravir, M1 and M4 (elvitegravir hydroxide and elvitegravir glucuronide), have been shown to inhibit HIV-1 integrase in vitro. While they are markedly less potent than elvitegravir and present only at low levels in plasma clinically, we investigated their potential to select for elvitegravir resistance in vitro. Resistance selection experiments using metabolites M1 and M4 led to the development of the previously reported elvitegravir integrase resistance mutations H51Y, T66A, E92G, and S147G, as well as a novel S153F substitution. Additional resistance selection experiments using elvitegravir led to the development of previously reported integrase inhibitor resistance mutations (T66I, F121Y, and S153Y) as well as a novel R263K integrase mutation. Phenotypic analyses of site-directed mutants with these mutations demonstrated broad cross-resistance between elvitegravir and its M1 and M4 metabolites with more limited cross-resistance to the integrase inhibitor raltegravir. Overall, our in vitro studies demonstrate that the resistance profile of the M1 and M4 metabolites of elvitegravir overlaps with that of the parent molecule elvitegravir; as such, their presence at low levels is not considered clinically relevant.

Dithiothreitol causes HIV-1 integrase dimer dissociation while agents interacting with the integrase dimer interface promote dimer formation.

We have developed a homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based assay that detects the formation of HIV-1 integrase (IN) dimers. The assay utilizes IN monomers that express two different epitope tags that are recognized by their respective antibodies, coupled to distinct fluorophores. Surprisingly, we found that dithiothreitol (DTT), a reducing agent essential for in vitro enzymatic activity of IN, weakened the interaction between IN monomers. This effect of DTT on IN is dependent on its thiol groups, since the related chemical threitol, which contains hydroxyls in place of thiols, had no effect on IN dimer formation. By studying mutants of IN, we determined that cysteines in IN appear to be dispensable for the dimer dissociation effect of DTT. Peptides derived from the IN binding domain (IBD) of lens epithelium derived growth factor/transcriptional coactivator p75 (LEDGF), a cellular cofactor that interacts with the IN dimer interface, were tested in this IN dimerization assay. These peptides, which compete with LEDGF for binding to IN, displayed an intriguing equilibrium binding dose-response curve characterized by a plateau rising to a peak, then descending to a second plateau. Mathematical modeling of this binding system revealed that these LEDGF-derived peptides promote IN dimerization and block subunit exchange between IN dimers. This dose-response behavior was also observed with a small molecule that interacts with the IN dimer interface and inhibits LEDGF binding to IN. In conclusion, this novel IN dimerization assay revealed that peptide and small molecule inhibitors of the IN-LEDGF interaction also stabilize IN dimers and promote their formation.

Affinities between the binding partners of the HIV-1 integrase dimer-lens epithelium-derived growth factor (IN dimer-LEDGF) complex.

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. Integrase dimerization had a k(on) of 0.1247 nm(-1) x min(-1) and a k(off) of 0.0080 min(-1) resulting in a K(dimer) of 64.5 pm. LEDGF binding to IN dimers had a k(on) of 0.0285 nm(-1).min(-1) and a k(off) of 0.2340 min(-1) resulting in a K(d) of 8.2 nm. These binding assays can also be used in an equilibrium end point competition format. In this format, the IN catalytic core domain produced a K(i) of 15.2 nm while competing for integrase dimerization, confirming the very tight interaction of IN with itself. In the same format, LEDGF produced a K(i) value of 35 nm when competing for LEDGF binding to IN dimers. In summary, this study describes a methodology combining homogeneous time-resolved fluorescence resonance energy transfer and mathematical modeling to derive the affinities between IN monomers and between LEDGF and IN dimers. This study revealed the significantly tighter nature of the IN-IN dimer compared with the IN-LEDGF interaction.

Preclinical evaluation of GS-9160, a novel inhibitor of human immunodeficiency virus type 1 integrase.

GS-9160 is a novel and potent inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN) that specifically targets the process of strand transfer. It is an authentic inhibitor of HIV-1 integration, since treatment of infected cells results in an elevation of two-long terminal repeat circles and a decrease of integration junctions. GS-9160 has potent and selective antiviral activity in primary human T lymphocytes producing a 50% effective concentration (EC(50)) of approximately 2 nM, with a selectivity index (50% cytotoxic concentration/EC(50)) of approximately 2,000. The antiviral potency of GS-9160 decreased by 6- to 10-fold in the presence of human serum. The antiviral activity of GS-9160 is synergistic in combination with representatives from three different classes of antiviral drugs, namely HIV-1 protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and nucleotide reverse transcriptase inhibitors. Viral resistance selections performed with GS-9160 yielded a novel pattern of mutations within the catalytic core domain of IN; E92V emerged initially, followed by L74M. While E92V as a single mutant conferred 12-fold resistance against GS-9160, L74M had no effect as a single mutant. Together, these mutations conferred 67-fold resistance to GS-9160, indicating that L74M may potentiate the resistance caused by E92V. The pharmacokinetic profile of GS-9160 in healthy human volunteers revealed that once-daily dosing was not likely to achieve antiviral efficacy; hence, the clinical development of this compound was discontinued.

HIV-1 integrase preassembled on donor DNA is refractory to activity stimulation by LEDGF/p75.

LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed. Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, but displayed 3- to 7-fold higher IC50 values. Our analyses indicate that, in the presence of 150 nM LEDGF/p75, active integrase/donor DNA complexes were increased by 5.3-fold during the 3'-processing step. In addition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNA during the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant of catalysis of the strand transfer step when 150 nM LEDGF/p75 was present during the 3'-processing step. In contrast, when LEDGF/p75 was added at the beginning of the strand transfer step, no increase in either the concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysis was observed. This observation suggested that the integrase/donor DNA formed in the absence of LEDGF/p75 became refractory to the stimulatory effect of LEDGF/p75. Instead, this LEDGF/p75 added at the start of the strand transfer step was able to promote the formation of a new cohort of active integrase/donor DNA complexes which became functional with a delay of 45 min after LEDGF/p75 addition. We propose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereas donor DNA can engage either free or LEDGF/p75-bound integrase.

Ethnic differences in cardiac potassium channel variants: implications for genetic susceptibility to sudden cardiac death and genetic testing for congenital long QT syndrome.

OBJECTIVE: To determine the spectrum, frequency, and ethnic-specificity of channel variants in the potassium channel genes implicated in congenital long QT syndrome (LQTS) among healthy subjects. SUBJECTS AND METHODS: Genomic DNA from 744 apparently healthy individuals-305 black, 187 white, 134 Asian, and 118 Hispanic--was subject to a comprehensive mutational analysis of the 4 LQTS-causing potassium channel genes: KCNQ1 (LQT1), KCNH2 (LQT2), KCNE1 (LQT5), and KCNE2 (LQT6). RESULTS: Overall, 49 distinct amino acid-altering variants (36 novel) were identified: KCNQ1 (n = 16), KCNH2 (n = 25),KCNE1 (n = 5), and KCNE2 (n = 3). More than half of these variants (26/49) were found exclusively in black subjects. The known K897T-HERG and the G38S-min K common polymorphisms were identified in all 4 ethnic groups. Excluding these 2 common polymorphisms, 25% of black subjects had at least 1 nonsynonymous potassium channel variant compared with 14% of white subjects (P < .01). CONCLUSIONS: To our knowledge, this study represents the first comprehensive determination of the frequency and spectrum of cardiac channel variants found among healthy subjects from 4 major ethnic groups. Defining the population burden of genetic variants in these critical cardiac ion channels is crucial for proper interpretation of genetic test results of individuals at risk for LQTS. This compendium provides a resource for epidemiological and functional investigation of variant effects on the repolarization properties of cardiac tissues, including susceptibility to lethal cardiac arrhythmias.