Manuel Tsiang

New Class of HIV-1 Integrase (IN) Inhibitors with a Dual Mode of Action.

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 integrase-viral 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. 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.

Preclinical Evaluation of GS-9160, a Novel Inhibitor of Human Immunodeficiency Virus Type 1 Integrase

ABSTRACT 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 (EC50) of ∼2 nM, with a selectivity index (50% cytotoxic concentration/EC50) of ∼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.

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

ABSTRACT 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.

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 (Kdimer = 67.8 pm) and of LEDGF from IN dimers (Kd = 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 kon of 0.1247 nm−1·min−1 and a koff of 0.0080 min−1 resulting in a Kdimer of 64.5 pm. LEDGF binding to IN dimers had a kon of 0.0285 nm−1·min−1 and a koff of 0.2340 min−1 resulting in a Kd 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 Ki 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 Ki 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.

Biochemical characterization of rhinovirus RNA-dependent RNA polymerase

Human rhinoviruses (HRV) represent the single most important causative agent of the common cold. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) designated 3D polymerase that is required for replication of the HRV RNA genome. We have expressed and purified recombinant HRV-16 3D polymerase to near homogeneity from Escherichia coli transformed with an expression plasmid containing the full-length 460 amino acid HRV-16 3D sequence with a methionine at the N-terminus and a glycine-serine linker followed by a 6-histidine affinity tag at the C-terminus. The purified recombinant protein has rifampicin-resistant activity in a poly(A)-dependent poly(U) polymerase assay while corresponding fractions similarly purified from E. coli transformed with an expression plasmid without the HRV-16 3D sequence showed no activity. The optimal conditions for temperature, pH, divalent cations Mg(2+) and Mn(2+), and KCl were determined. The recombinant protein has RNA polymerase activity on homopolymeric templates poly(A) and poly(C) and heteropolymeric RNA templates primed with either RNA or DNA oligonucleotide primers or self-primed by a copy-back mechanism. A unique, secondary structureless heteropolymeric RNA template that is an efficient substrate was developed to facilitate kinetic characterizations of the enzyme. In the presence of Mg(2+), the enzyme displayed strong base and sugar specificity. However, when Mg(2+) was replaced by Mn(2+) specificity for ribonucleotides was lost, utilization of deoxynucleotides became possible and primer-independent activity was observed on the poly(C) template. Zn(2+) was found to inhibit HRV-16 3D polymerase with an IC(50) as low as 0.6 microM by a mechanism distinct from the magnesium ion stimulation. The activity of this 6His-tagged HRV-16 3D polymerase was compared with that of a recombinant HRV-16 3D polymerase expressed without the 6His-tag and was found to be identical. The availability of recombinant rhinovirus RdRp in a purified form will facilitate the structure-function analysis of this enzyme as well as the identification of specific inhibitors to the rhinovirus 3D polymerase that have therapeutic value in the treatment of the common cold.

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.

Analysis of hepatitis B virus dynamics and its impact on antiviral development.

Viral dynamics is the study of the population dynamics of viral infection within the body of an infected individual. It describes how viruses spread from cell to cell, with the aim of revealing the basic laws that govern the spread of the virus within the host, their interaction with the immune system, and their response to therapy. For viruses that produce a viremia, changes in the size of the virus population can be measured and followed from blood samples obtained by phlebotomy at various time intervals using very sensitive polymerase chain reaction (PCR)-based assays to quantify viral nucleic acids accurately over a dynamic range of six orders of magnitude. To analyze these data, mathematical models based on a firm understanding of the biology of the virus and its interaction with the host have been developed. Such models can often provide nonintuitive insights into the dynamics of viral infection. The advent of potent antiviral drugs for the treatment of viral diseases in recent years has facilitated the mathematical analysis of the dynamics of viral infections by providing a means for perturbing the steady state that commonly exists during chronic infection. Such studies have yielded not only insight into our understanding of the establishment, maintenance, and clearance of viral infection, but also predictors of response and principles useful in the design of more effective treatment strategies for human immunodeficiency virus (HIV) (1–4) and hepatitis C virus (HCV) (5,6) and promises to do the same for hepatitis B virus (HBV) as well. Importantly, viral dynamics analyses now offer a more sophisticated way of analyzing the response to therapy, particularly in Phase II clinical studies of short duration. Viral dynamics analysis maximizes the use of readily available data and allows different drugs, dosages and regimens to be compared more precisely. In this chapter, the basic model that has been formulated for HBV dynamics is explained and its application to the analysis of response to antiviral therapy is

A Cell-Based Strategy To Assess Intrinsic Inhibition Efficiencies of HIV-1 Reverse Transcriptase Inhibitors

ABSTRACT During HIV-1 reverse transcription, there are increasing opportunities for nucleos(t)ide (NRTI) or nonnucleoside (NNRTI) reverse transcriptase (RT) inhibitors to stop elongation of the nascent viral DNA (vDNA). In addition, RT inhibitors appear to influence the kinetics of vDNA synthesis differently. While cell-free kinetic inhibition constants have provided detailed mechanistic insight, these assays are dependent on experimental conditions that may not mimic the cellular milieu. Here we describe a novel cell-based strategy to provide a measure of the intrinsic inhibition efficiencies of clinically relevant RT inhibitors on a per-stop-site basis. To better compare inhibition efficiencies among HIV-1 RT inhibitors that can stop reverse transcription at any number of different stop sites, their basic probability, p, of getting stopped at any potential stop site was determined. A relationship between qPCR-derived 50% effective inhibitory concentrations (EC50s) and this basic probability enabled determination of p by successive approximation. On a per-stop-site basis, tenofovir (TFV) exhibited 1.4-fold-greater inhibition efficiency than emtricitabine (FTC), and as a class, both NRTIs exhibited an 8- to 11-fold greater efficiency than efavirenz (EFV). However, as more potential stops sites were considered, the probability of reverse transcription failing to reach the end of the template approached equivalence between both classes of RT inhibitors. Overall, this novel strategy provides a quantitative measure of the intrinsic inhibition efficiencies of RT inhibitors in the natural cellular milieu and thus may further understanding of drug efficacy. This approach also has applicability for understanding the impact of viral polymerase-based inhibitors (alone or in combination) in other virus systems.