A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells.

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

Establishment of intracellular tenofovir-diphosphate as the key determinant for in vitro-in vivo translation of antiviral efficacy.

In vitro evaluation of tenofovir disproxil fumarate (TDF) and tenofovir alafenamide (TAF) revealed comparable antiviral effects with respect to the tenofovir-diphosphate (TFV-DP) level in human peripheral blood mononuclear cells (PBMCs), despite the EC50 values determined based on prodrug concentrations were nearly two orders of magnitude apart. In vivo EC50 obtained from meta-analyses were in good agreement with the in vitro results, indicating intracellular TFV-DP can be employed for in vitro-in vivo translation of viral inhibition for tenofovir prodrugs. Current analysis indicated that the intracellular concentrations of TFV-DP achieving maximal antiviral effect in vitro can be directly translatable in the clinic to accomplish maximal viral load suppression attainable by tenofovir-prodrugs.

P2-quinazolinones and bis-macrocycles as new templates for next-generation hepatitis C virus NS3/4a protease inhibitors: discovery of MK-2748 and MK-6325.

With the goal of identifying inhibitors of hepatitis C virus (HCV) NS3/4a protease that are potent against a wide range of genotypes and clinically relevant mutant viruses, several subseries of macrocycles were investigated based on observations made during the discovery of MK-5172. Quinazolinone-containing macrocycles were identified as promising leads, and optimization for superior cross-genotype and mutant enzyme potency as well as rat liver and plasma concentrations following oral dosing, led to the development of MK-2748. Additional investigation of a series of bis-macrocycles containing a fused 18- and 15-membered ring system were also optimized for the same properties, leading to the discovery of MK-6325. Both compounds display the broad genotype and mutant potency necessary for clinical development as next-generation HCV NS3/4a protease inhibitors.

Emergence of resistance-associated variants after failed triple therapy with vaniprevir in treatment-experienced non-cirrhotic patients with hepatitis C-genotype 1 infection: a population and clonal analysis.

BACKGROUND: Vaniprevir with P/R improved SVR rates over P/R alone in treatment-experienced patients with chronic HCV-genotype 1 infection, but treatment failure presents therapeutic challenges. We identified RAVs from non-cirrhotic patients failing to achieve SVR on vaniprevir-containing regimens from a dose/duration-ranging trial of triple-combination therapy. METHODS: Using population analysis, resistance sequencing was performed on all baseline samples and on samples at virologic failure in the vaniprevir arms. Longitudinal clonal analyses were performed on viral isolates from six vaniprevir recipients experiencing breakthrough viremia. RESULTS: Baseline RAVs were detected in two patients subsequently experiencing virologic failure. At virologic failure, the majority of RAVs had substitutions at R155, A156, or D168. Clonal analyses identified novel double/triple variants emerging with continuing vaniprevir dosing. CONCLUSIONS: RAVs were predominantly observed at R155, A156, and/or D168 during virologic failure on vaniprevir/P/R. Double/triple RAVs were identified in patients remaining viremic on triple therapy, suggesting evolution of resistance under selective pressure.

Development of macrocyclic inhibitors of HCV NS3/4A protease with cyclic constrained P2-P4 linkers.

A series of macrocyclic compounds containing a cyclic constraint in the P2-P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K, A156T, A156V, and D168V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ~20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2-P4 linker.

Development of potent macrocyclic inhibitors of genotype 3a HCV NS3/4A protease.

A series of macrocyclic compounds containing 2-substituted-quinoline moieties have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K mutant activity while maintaining the high rat liver exposure. Cyclization of the 2-substituted quinoline substituent led to a series of tricyclic P2 compounds which also display superb gt3a potency.

MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants.

HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.

Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor.

A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.

A time-resolved, internally quenched fluorescence assay to characterize inhibition of hepatitis C virus nonstructural protein 3-4A protease at low enzyme concentrations.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) with its cofactor NS4A is a pivotal enzyme for the replication of HCV. Inhibition of NS3-4A protease activity has been validated as an antiviral target in clinical studies of inhibitors of the enzyme. We have developed a sensitive time-resolved fluorescence (TRF) assay capable of detecting very low NS3-4A concentrations. A depsipeptide substrate is used that contains a europium-cryptate moiety and an efficient quenching group, QSY-7. The TRF assay is at least 30-fold more sensitive than a fluorescence energy transfer (FRET) assay and allows evaluation of NS3 protease inhibitors in reactions catalyzed by low enzyme concentrations (30 pM). Use of low enzyme concentrations allows for accurate measurement of inhibition by compounds with subnanomolar inhibition constants. The inhibitory potency of the potent protease inhibitor, BILN-2061, is significantly greater than previously reported. The ability to accurately determine inhibitory potency in reactions with low picomolar concentrations of NS3-4A is crucially important to allow valid comparisons between potent inhibitors. Studies of the interaction of NS3 with its NS4A cofactor at low enzyme concentration also reveal that the protease activity is salt dependent. This salt dependence of the enzyme activity is not present when high enzyme concentrations are used in the FRET assay.