The discovery of novel and potent indazole NLRP3 inhibitors enabled by DNA-encoded library screening.

NLRP3 is an intracellular sensor protein that detects a broad range of danger signals and environmental insults. Its activation results in a protective pro-inflammatory response designed to impair pathogens and repair tissue damage via the formation of the NLRP3 inflammasome. Assembly of the NLRP3 inflammasome leads to caspase 1-dependent secretory release of the pro-inflammatory cytokines IL-1ß and IL-18 as well as to gasdermin d-mediated pyroptotic cell death. Herein, we describe the discovery of a novel indazole series of high affinity, reversible inhibitors of NLRP3 activation through screening of DNA-encoded libraries and the potent lead compound 3 (BAL-0028, IC50 = 25 nM) that was identified directly from the screen. SPR studies showed that compound 3 binds tightly (KD range 104-123 nM) to the NACHT domain of NLRP3. A CADD analysis of the interaction of compound 3 with the NLRP3 NACHT domain proposes a binding site that is distinct from those of ADP and MCC950 and includes specific site interactions. We anticipate that compound 3 (BAL-0028) and other members of this novel indazole class of neutral inhibitors will demonstrate significantly different physical, biochemical, and biological properties compared to NLRP3 inhibitors previously identified.

Diazepinone HBV capsid assembly modulators.

The HBV capsid core protein serves a number of important functions in the viral life cycle enabling chronic HBV infection to persist, and therefore is a promising drug target. Interfering with capsid assembly has shown efficacy in clinical trials with small molecule capsid assembly modulators (CAMs). Herein is described the further optimization of a progressive series of diazepinone HBV CAMs.

Oxadiazepinone HBV capsid assembly modulators.

The HBV core protein serves multiple essential functions in the viral life cycle that enable chronic HBV infection to persist, and as such, represents a promising drug target. Modulation of the HBV capsid assembly has shown efficacy in early clinical trials through use of small molecule capsid assembly modulators (CAMs). Herein is described the evolution and SAR of a novel pyrazolo piperidine lead series into advanced oxadiazepinone HBV CAMs.

Identification of a new class of HBV capsid assembly modulator.

The HBV core protein is a druggable target of interest due to the multiple essential functions in the HBV life cycle to enable chronic HBV infection. The core protein oligomerizes to form the viral capsid, and modulation of the HBV capsid assembly has shown efficacy in clinical trials. Herein is described the identification and hit to lead SAR of a novel series of pyrazolo piperidine HBV capsid assembly modulators.

P450s under Restriction (PURE) Screen Using HepaRG and Primary Human Hepatocytes for Discovery of Novel HBV Antivirals.

Herein is reported a novel screening paradigm PURE (P450s under restriction) for the identification and optimization of hits as part of a hepatitis B virus (HBV) antiviral discovery program. To closely represent in vivo hepatocytes, differentiated HepaRG cells (dHRGs) and primary human hepatocytes (PHHs) were used as the basis for an HBV infection system. However, a significant challenge arose during potency evaluation in using cultured dHRGs and PHHs as screening platforms because, as with hepatocytes in vivo, these cells express active cytochrome P450 enzymes and thus can metabolize test compounds. The observed antiviral effects may be the cumulative result of a dynamic pool of parent compound and metabolites thus confounding structure activity relationship (SAR) interpretation and subsequent optimization design initiatives. We show here that PURE methodology restricts metabolism of HBV-infected dHRGs and PHHs and thus provides highly informative potency data for decision-making on key representative antiviral compounds.

SAR studies in the sulfonyl carboxamide class of HBV capsid assembly modulators.

The HBV core protein has multiple essential functions in the HBV life cycle to enable chronic HBV infection. The core protein oligomerizes to form the viral capsid, and modulation of the HBV capsid assembly process has shown clinical efficacy in early clinical trials. Herein is described the SAR exploration of NVR 3-778, the first clinical compound in the sulfonyl carboxamide class.

Antiviral Activity, Safety, and Pharmacokinetics of Capsid Assembly Modulator NVR 3-778 in Patients with Chronic HBV Infection.

BACKGROUND & AIMS: NVR 3-778 is a first-in-class hepatitis B virus (HBV) capsid assembly modulator that can inhibit HBV replication. We performed a proof-of-concept study to examine the safety, pharmacokinetics, and antiviral activity of NVR 3-778 in patients with chronic HBV infection. METHODS: We performed a phase 1 study in 73 hepatitis B envelope antigen (HBeAg)-positive patients with chronic HBV infection without cirrhosis. In a 2-part study (part 1 in New Zealand and part 2 in Hong Kong, Singapore, Taiwan, Korea, and the United States), patients were randomly assigned to groups that were given oral NVR 3-778 (100 mg, 200 mg, or 400 mg daily or 600 mg or 1000 mg twice daily) or placebo for 4 weeks. Additional groups received combination treatment with pegylated interferon (pegIFN) and NVR 3-778 (600 mg twice daily) or pegIFN with placebo. RESULTS: Reductions in serum levels of HBV DNA and HBV RNA were observed in patients receiving ≥1200 mg/d NVR 3-778. The largest mean reduction in HBV DNA was observed in the group given NVR 3-778 plus pegIFN (1.97 log10 IU/mL), compared with the groups given NVR 3-778 or pegIFN alone (1.43 log10 IU/mL and 1.06 log10 IU/mL, respectively). The mean reduction in HBV RNA was also greatest in the group given NVR 3-778 plus pegIFN (2.09 log10 copies/mL), compared with the groups given NVR 3-778 or pegIFN alone (1.42 log10 copies/mL and 0.89 log10 copies/mL, respectively). There was no significant mean reduction in HBsAg during the 4-week treatment period. There were no discontinuations and no pattern of dose-related adverse effects with NVR 3-778. CONCLUSIONS: In a phase 1 study of HBeAg-positive patients with chronic HBV infection without cirrhosis, NVR 3-778 was well tolerated and demonstrated antiviral activity. The agent reduced serum levels of HBV DNA and HBV RNA, to the greatest extent in combination with pegIFN. The observed reductions in HBV RNA confirmed the novel mechanism of NVR 3-778. Clinicaltrials.gov no. NCT02112799 (single-center) and NCT02401737 (multicenter).

Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus.

NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.

MK-7622: A First-in-Class M1 Positive Allosteric Modulator Development Candidate.

Identification of ligands that selectively activate the M1 muscarinic signaling pathway has been sought for decades to treat a range of neurological and cognitive disorders. Herein, we describe the optimization efforts focused on addressing key physicochemical and safety properties, ultimately leading to the clinical candidate MK-7622, a highly selective positive allosteric modulator of the M1 muscarinic receptor that has entered Phase II studies in patients with Alzheimer's disease.

Efficacy of NVR 3-778, Alone and In Combination With Pegylated Interferon, vs Entecavir In uPA/SCID Mice With Humanized Livers and HBV Infection.

BACKGROUND & AIMS: NVR3-778 is a capsid assembly modulator in clinical development. We determined the in vivo antiviral efficacy and effects on innate and endoplasmic reticulum (ER) stress responses of NVR3-778 alone or in combination with pegylated interferon alpha (peg-IFN) and compared with entecavir. METHODS: We performed 2 studies, with a total of 61 uPA/SCID mice with humanized livers. Mice were infected with a hepatitis B virus (HBV) genotype C preparation; we waited 8 weeks for persistent infection of the human hepatocytes in livers of mice. Mice were then randomly assigned to groups (5 or 6 per group) given vehicle (control), NVR3-778, entecavir, peg-IFN, NVR3-778 + entecavir, or NVR3-778 + peg-IFN for 6 weeks. We measured levels of HB surface antigen, HB e antigen, HBV RNA, alanine aminotransferase, and human serum albumin at different time points. Livers were collected and analyzed by immunohistochemistry; levels of HBV DNA, covalently closed circular DNA, and HBV RNA, along with markers of ER stress and IFN response, were quantified. RESULTS: Mice given NVR3-778 or entecavir alone for 6 weeks had reduced serum levels of HBV DNA compared with controls or mice given peg-IFN. The largest reduction was observed in mice given NVR3-778 + peg-IFN; in all mice in this group, the serum level of HBV DNA was below the limit of quantification. NVR3-778 and peg-IFN, but not entecavir, also reduced serum level of HBV RNA. The largest effect was obtained in the NVR3-778 + peg-IFN group, in which serum level of HBV RNA was below the limit of quantification. Levels of HB surface antigen and HB e antigen were reduced significantly in only the groups that received peg-IFN. Levels of covalently closed circular DNA did not differ significantly among groups. NVR3-778 was not associated with any significant changes in level of alanine aminotransferase, the ER stress response, or IFN-stimulated genes. CONCLUSIONS: NVR3-778 has high antiviral activity in mice with humanized livers and stable HBV infection, reducing levels of serum HBV DNA and HBV RNA. Entecavir reduced levels of serum HBV DNA, but had no effect on HBV RNA. The combination of NVR3-778 and peg-IFN prevented viral replication and HBV RNA particle production to a greater extent than each compound alone or entecavir.

Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants.

The hepatitis B virus (HBV) core protein serves multiple essential functions in the viral life cycle, and antiviral agents that target the core protein are being developed. Capsid assembly modulators (CAMs) are compounds that target core and misdirect capsid assembly, resulting in the suppression of HBV replication and virion production. Besides HBV DNA, circulating HBV RNA has been detected in patient serum and can be associated with the treatment response. Here we studied the effect of HBV CAMs on the production of extracellular HBV RNA using infected HepaRG cells and primary human hepatocytes. Representative compounds from the sulfonamide carboxamide and heteroaryldihydropyrimidine series of CAMs were evaluated and compared to nucleos(t)ide analogs as inhibitors of the viral polymerase. The results showed that CAMs blocked extracellular HBV RNA with efficiencies similar to those with which they blocked pregenomic RNA (pgRNA) encapsidation, HBV DNA replication, and Dane particle production. Nucleos(t)ide analogs inhibited viral replication and virion production but not encapsidation or production of extracellular HBV RNA. Profiling of HBV RNA from both culture supernatants and patient serum showed that extracellular viral RNA consisted of pgRNA and spliced pgRNA variants with an internal deletion(s) but still retained the sequences at both the 5' and 3' ends. Similar variants were detected in the supernatants of infected cells with and without nucleos(t)ide analog treatment. Overall, our data demonstrate that HBV CAMs represent direct antiviral agents with a profile differentiated from that of nucleos(t)ide analogs, including the inhibition of extracellular pgRNA and spliced pgRNA.

Discovery of MK-1832, a Kv1.5 inhibitor with improved selectivity and pharmacokinetics.

Selective inhibition of Kv1.5, which underlies the ultra-rapid delayed rectifier current, IKur, has been pursued as a treatment for atrial fibrillation. Here we describe the discovery of MK-1832, a Kv1.5 inhibitor with improved selectivity versus the off-target current IKs, whose inhibition has been associated with ventricular proarrhythmia. MK-1832 exhibits improved selectivity for IKur over IKs (>3000-fold versus 70-fold for MK-0448), consistent with an observed larger window between atrial and ventricular effects in vivo (>1800-fold versus 210-fold for MK-0448). MK-1832 also exhibits an improved preclinical pharmacokinetic profile consistent with projected once daily dosing in humans.

High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein.

The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010-001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010-001-E2 binds at the dimer-dimer interface of the core proteins, forms a new interaction surface promoting protein-protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010-001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein-protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties.

Discovery of MK-3697: a selective orexin 2 receptor antagonist (2-SORA) for the treatment of insomnia.

Orexin receptor antagonists have demonstrated clinical utility for the treatment of insomnia. The majority of clinical efforts to date have focused on the development of dual orexin receptor antagonists (DORAs), small molecules that antagonize both the orexin 1 and orexin 2 receptors. Our group has recently disclosed medicinal chemistry efforts to identify highly potent, orally bioavailable selective orexin 2 receptor antagonists (2-SORAs) that possess acceptable profiles for clinical development. Herein we report additional SAR studies within the 'triaryl' amide 2-SORA series focused on improvements in compound stability in acidic media and time-dependent inhibition of CYP3A4. These studies resulted in the discovery of 2,5-disubstituted isonicotinamide 2-SORAs such as compound 24 that demonstrated improved stability and TDI profiles as well as excellent sleep efficacy across species.

Discovery of naphthyl-fused 5-membered lactams as a new class of m1 positive allosteric modulators.

Selective activation of the M1 muscarinic receptor via positive allosteric modulation represents an original approach to treat the cognitive decline in patients with Alzheimer's disease. A series of naphthyl-fused 5-membered lactams were identified as a new class of M1 positive allosteric modulators and were found to possess good potency and in vivo efficacy.

Adenosine analogue inhibitors of S-adenosylhomocysteine hydrolase.

Elevated plasma homocysteine (Hcy) levels are an independent risk factor for the onset and progression of Alzheimer's disease. Reduction of Hcy to normal levels therefore presents a new approach for disease modification. Hcy is produced by the cytosolic enzyme S-adenosylhomocysteine hydrolase (AHCY), which converts S-adenosylhomocysteine (SAH) to Hcy and adenosine. Herein we describe the design and characterization of novel, substrate-based S-adenosylhomocysteine hydrolase inhibitors with low nanomolar potency in vitro and robust activity in vivo.

Identification of a methoxynaphthalene scaffold as a core replacement in quinolizidinone amide M(1) positive allosteric modulators.

A series of methoxynaphthalene amides were prepared and evaluated as alternatives to quinolizidinone amide M1 positive allosteric modulators. A methoxy group was optimal for M1 activity and addressed key P-gp issues present in the aforementioned quinolizidinone amide series.

Benzimidazole CB2 agonists: design, synthesis and SAR.

A new series of CB2-selective agonists containing a benzimidazole core is reported. Design, synthesis, SAR and pharmacokinetic data for selected compounds are described.

Discovery of 5''-chloro-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2,2':5',3''-terpyridine-3'-carboxamide (MK-1064): a selective orexin 2 receptor antagonist (2-SORA) for the treatment of insomnia.

The field of small-molecule orexin antagonist research has evolved rapidly in the last 15 years from the discovery of the orexin peptides to clinical proof-of-concept for the treatment of insomnia. Clinical programs have focused on the development of antagonists that reversibly block the action of endogenous peptides at both the orexin 1 and orexin 2 receptors (OX1 R and OX2 R), termed dual orexin receptor antagonists (DORAs), affording late-stage development candidates including Merck's suvorexant (new drug application filed 2012). Full characterization of the pharmacology associated with antagonism of either OX1 R or OX2 R alone has been hampered by the dearth of suitable subtype-selective, orally bioavailable ligands. Herein, we report the development of a selective orexin 2 antagonist (2-SORA) series to afford a potent, orally bioavailable 2-SORA ligand. Several challenging medicinal chemistry issues were identified and overcome during the development of these 2,5-disubstituted nicotinamides, including reversible CYP inhibition, physiochemical properties, P-glycoprotein efflux and bioactivation. This article highlights structural modifications the team utilized to drive compound design, as well as in vivo characterization of our 2-SORA clinical candidate, 5''-chloro-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2,2':5',3''-terpyridine-3'-carboxamide (MK-1064), in mouse, rat, dog, and rhesus sleep models.

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs).

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX1R/OX2R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.