Functional TASK-3-Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells.

Ca2+ drives aldosterone synthesis in the cytosolic and mitochondrial compartments of the adrenal zona glomerulosa cell. Membrane potential across each of these compartments regulates the amplitude of the Ca2+ signal; yet, only plasma membrane ion channels and their role in regulating cell membrane potential have garnered investigative attention as pathological causes of human hyperaldosteronism. Previously, we reported that genetic deletion of TASK-3 channels (tandem pore domain acid-sensitive K+ channels) from mice produces aldosterone excess in the absence of a change in the cell membrane potential of zona glomerulosa cells. Here, we report using yeast 2-hybrid, immunoprecipitation, and electron microscopic analyses that TASK-3 channels are resident in mitochondria, where they regulate mitochondrial morphology, mitochondrial membrane potential, and aldosterone production. This study provides proof of principle that mitochondrial K+ channels, by modulating inner mitochondrial membrane morphology and mitochondrial membrane potential, have the ability to play a pathological role in aldosterone dysregulation in steroidogenic cells.

Discovery of Chromane Containing Hepatitis C Virus (HCV) NS5A Inhibitors with Improved Potency against Resistance-Associated Variants.

The discovery of potent and pan-genotypic HCV NS5A inhibitors faces many challenges including the significant diversity among genotypes, substantial potency shift conferred on some key resistance-associated variants, inconsistent SARs between different genotypes and mutants, and the lacking of models of inhibitor/protein complexes for rational inhibitor design. As part of ongoing efforts on HCV NS5A inhibition at Merck, we now describe the discovery of a novel series of chromane containing NS5A inhibitors. SAR studies around the "Z" group of the tetracyclic indole scaffold explored fused bicyclic rings as alternates to the phenyl group of elbasvir (1, MK-8742) and identified novel chromane and 2,3-dihydrobenzofuran derivatives as "Z" group replacements offered good potency across all genotypes. This effort, incorporating the C-1 fluoro substitution at the tetracyclic indole core, led to the discovery of a new series of NS5A inhibitors, such as compounds 14 and 25-28, with significantly improved potency against resistance-associated variants, such as GT2b, GT1a Y93H, and GT1a L31V. Compound 14 also showed reasonable PK exposures in preclinical species (rat and dog).

Alternative core development around the tetracyclic indole class of HCV NS5A inhibitors.

Herein, we describe our research efforts to develop unique cores in molecules which function as HCV nonstructural protein 5A (NS5A) inhibitors. In particular, various fused tetracyclic cores were identified which showed genotype and mutant activities comparable to the indole-based tetracyclic core.

Structure-activity relationships of proline modifications around the tetracyclic-indole class of NS5A inhibitors.

We describe the impact of proline modifications, in our tetracyclic-indole based series of nonstructural protein 5A (NS5A) inhibitors, to their replicon profiles. This work identified NS5A inhibitors with an improved and flattened resistance profiles.

Substituted tetracyclic indole core derivatives of HCV NS5A inhibitor MK-8742.

As part of an ongoing effort in NS5A inhibition at Merck we now describe our efforts for introducing substitution around the tetracyclic indole core of MK-8742. Fluoro substitution on the core combined with the fluoro substitutions on the proline ring improved the potency against GT1a Y93H significantly. However, no improvement on GT2b potency was achieved. Limiting the fluoro substitution to C-1 of the tetracyclic indole core had a positive impact on the potency against the resistance associated variants, such as GT1a Y93H and GT2b, and the PK profile as well. Compounds, such as 62, with reduced potency shifts between wild type GT1a to GT2b, GT1a Y93H, and GT1a L31V were identified.

Matched and mixed cap derivatives in the tetracyclic indole class of HCV NS5A inhibitors.

A matched and mixed capping SAR study was conducted on the tetracyclic indole class of HCV NS5A inhibitors to examine the influence of modifications of this region on the overall HCV virologic resistance profiles.

Aryl or heteroaryl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

Herein we describe our research efforts around the aryl and heteroaryl substitutions at the aminal carbon of the tetracyclic indole-based HCV NS5A inhibitor MK-8742. A series of potent NS5A inhibitors are described, such as compounds 45-47, 54, 56, and 65, which showed improved potency against clinically relevant and resistance associated HCV variants. The improved potency profiles of these compounds demonstrated an SAR that can improve the potency against GT2b, GT1a Y93H, and GT1a L31V altogether, which was unprecedented in our previous efforts in NS5A inhibition.

Alkyl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein we describe our continued research efforts around the alkyl "Z group" modification of the tetracyclic indole-based NS5A inhibitor MK-8742, which led to the discovery of a series of potent NS5A inhibitors. Compounds 10 and 19 are of particular interests since they are as potent as our previous leads and have much improved rat pharmacokinetic profiles.

Discovery of potent macrocyclic HCV NS5A inhibitors.

HCV NS5A inhibitors have demonstrated impressive in vitro virologic profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed-dose combination (FDC) regimen for the treatment of HCV infection. Merck's effort in this area identified MK-4882 and MK-8325 as early development leads. Herein, we describe the discovery of potent macrocyclic NS5A inhibitors bearing the MK-8325 or MK-4882 core structure.

Discovery of fused tricyclic core containing HCV NS5A inhibitors with pan-genotype activity.

HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein, we describe research efforts that led to the discovery of a series of fused tricyclic core containing HCV NS5A inhibitors such as 24, 39, 40, 43, and 44 which have pan-genotype activity and are orally bioavailable in the rat.

Methyl-substitution of an iminohydantoin spiropiperidine ß-secretase (BACE-1) inhibitor has a profound effect on its potency.

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-fold from 11 µM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand. A strategically placed methyl group not only maintained the required pKa of the piperidine nitrogen and filled a small hydrophobic pocket, but more importantly, stabilized the conformation best suited for optimized binding to the receptor.

Discovery of MK-8742: an HCV NS5A inhibitor with broad genotype activity.

The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-pyrrolidin-2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-imidazole]. While preclinical proof-of-concept studies in HCV-infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single- and multiple-dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK-4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK-8742, a tetracyclic indole-based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all-oral, interferon-free regimen for the treatment of HCV infection.

Discovery of a pharmacologically active antagonist of the two-pore-domain potassium channel K2P9.1 (TASK-3).

TWIK-related acid-sensitive K(+) (K(2P) 9.1, TASK-3) ion channels have the capacity to regulate the activity of neuronal pathways by influencing the resting membrane potential of neurons on which they are expressed. The central nervous system (CNS) expression of these channels suggests potential roles in neurologic disorders, and it is believed that the development of TASK-3 antagonists could lead to the therapeutic treatment of a number of neurological conditions. While a therapeutic potential for TASK-3 channel modulation exists, there are only a few documented examples of potent and selective small-molecule channel blockers. Herein, we describe the discovery and lead optimization efforts for a novel series of TASK-3 channel antagonists based on a 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine high-throughput screening lead from which a subseries of potent and selective inhibitors were identified. One compound was profiled in detail with respect to its physical properties and demonstrated pharmacological target engagement as indicated by its ability to modulate sleep architecture in rodent electroencephalogram (EEG) telemetry models.

Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors.

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-L-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.

Assessment of the susceptibility of mutant HIV-1 to antiviral agents.

Traditional phenotypic assays used to assess the susceptibility of mutant human immunodeficiency virus type-1 (HIV-1) obtained from infected patients or from resistance selection to antiviral agents in cell culture are rather tedious and time consuming. To improve the efficiency of this process, a novel method was developed in which mutant viruses are captured with magnetic nano-beads and used to infect gag-GFP reporter cells to evaluate the extent of resistance conferred by the mutant viruses against antiviral agents. The optimal timing for measuring the inhibitory potencies of antiviral agents was found to be day 3 post-infection for integrase strand transfer inhibitors and protease inhibitors and day 4 for non-nucleoside reverse transcriptase inhibitors. Comparable EC(50) values were obtained when bead-captured breakthrough virus from in vitro resistance selection experiments and its matched site-directed mutagenesis virus were tested side by side in this assay. This assay protocol was also employed to evaluate the inhibitor susceptibility of breakthrough viruses collected from resistance selections that were conducted in the presence of increasing concentrations of an HIV-1 protease inhibitor. Taken together, these findings suggest that a rapid, sensitive, non-invasive, and homogeneous phenotypic assay has been developed for assessing the antiviral agent susceptibility of mutant viruses that emerge from in vitro resistance selection studies.

Discovery of aminoheterocycles as a novel beta-secretase inhibitor class: pH dependence on binding activity part 1.

We have developed a novel series of heteroaromatic BACE-1 inhibitors. These inhibitors interact with the enzyme in a unique fashion that allows for potent binding in a non-traditional paradigm. In addition to the elucidation of their binding profile, we have discovered a pH dependent effect on the binding affinity as a result of the intrinsic pK(a) of these inhibitors and the pH of the BACE-1 enzyme binding assay.

PL-100, a novel HIV-1 protease inhibitor displaying a high genetic barrier to resistance: an in vitro selection study.

The development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi-resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug-resistant viruses has produced PL-100, a novel potent protease inhibitor (PI) that incorporates a lysine-based scaffold. A selection for resistance against PL-100 in cord blood mononuclear cells was performed, using the laboratory-adapted IIIb strain of HIV-1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site-directed mutagenesis revealed that all four mutations were required to develop low-level resistance to PL-100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross-resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X-ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations.

Evaluating scoring functions for docking and designing beta-secretase inhibitors.

Several simple scoring methods were examined for 2 series of beta-secretase (BACE-1) inhibitors to identify a docking/scoring protocol which could be used to design BACE-1 inhibitors in a drug discovery program. Both the PLP1 score and MMFFs interaction energy (E(inter)) performed as well or better than more computationally intensive methods for a set of substrate-based inhibitors, while the latter performed well for both sets of inhibitors.

Macrocyclic inhibitors of beta-secretase: functional activity in an animal model.

A macrocyclic inhibitor of beta-secretase was designed by covalently cross-linking the P1 and P3 side chains of an isophthalamide-based inhibitor. Macrocyclization resulted in significantly improved potency and physical properties when compared to the initial lead structures. More importantly, these macrocyclic inhibitors also displayed in vivo amyloid lowering when dosed in a murine model.

BACE-1 inhibition by a series of psi[CH2NH] reduced amide isosteres.

A series of beta-site amyloid precursor protein cleaving enzyme (BACE-1) inhibitors containing a psi(CH2NH) reduced amide bond were synthesized. Incorporation of this reduced amide isostere as a non-cleavable peptide surrogate afforded inhibitors possessing low nanomolar potencies in both an enzymatic and cell-based assay.