The NLRP3 inflammasome is an intracellular protein complex that causes inflammation via the release of IL-1β and pyroptosis. NLRP3 activation is associated with many age-related inflammatory diseases, and NLRP3 inhibition is a promising therapeutic strategy. We previously performed a DNA-encoded library screen to identify novel NLRP3-binding molecules. Herein we describe the characterization of BAL-0028 as a potent and specific inhibitor of NLRP3 signaling. Notably, BAL-0028 is a poor inhibitor of mouse NLRP3 but inhibits human and primate NLRP3 with nanomolar potency. Using cellular and biochemical analyses, we demonstrate that BAL-0028 binds to the NLRP3 NACHT domain at a site that is distinct from the MCC950-binding pocket. Using humanized NLRP3 mice, we show that a derivative of BAL-0028, BAL-0598, inhibits NLRP3 activation in vivo in a peritonitis model. Finally, we demonstrate that both BAL-0028 and BAL-0598 inhibit select hyperactive NLRP3 mutations associated with autoinflammatory diseases more potently than MCC950. BAL-0028 and BAL-0598 thus represent a new modality for NLRP3 inhibition in inflammatory diseases.

The NLRP3 inflammasome is an intracellular protein complex that causes inflammation via the release of IL-1β and pyroptosis. NLRP3 activation is associated with many age-related inflammatory diseases, and NLRP3 inhibition is a promising therapeutic strategy. We previously performed a DNA encoded library screen to identify novel NLRP3 binding molecules. Herein we describe the characterization of BAL-0028 as a potent and specific inhibitor of NLRP3 signaling. Notably, BAL-0028 is a poor inhibitor of mouse NLRP3 but inhibits human and primate NLRP3 with nanomolar potency. Using cellular and biochemical analyses we demonstrate that BAL-0028 binds to the NLRP3 NACHT domain at a site that is distinct from the MCC950 binding pocket. Using humanized NLRP3 mice we show that a derivative of BAL-0028 inhibits NLRP3 activation in vivo in a peritonitis model. Finally, we demonstrate that BAL-0028 inhibits select hyperactive NLRP3 mutations associated with autoinflammatory diseases more potently than does MCC950. BAL-0028 thus represents a new modality for NLRP3 inhibition in inflammatory diseases. ### Competing Interest Statement K.W., A.D., S.T., M.M., M.B., M.C., S.C., K.F., G.H., R.H., R.M., C.P., P.R., Y.W., S.Y. are employees of BIOAGE Labs. R.C.C. is a co-inventor on patent applications for NLRP3 inhibitors that have been licensed to Inflazome Ltd, a company acquired by Roche. R.C.C. and M.G. are consultants to BioAge Labs, USA (since 2020). R.C.C. serves on the Scientific Advisory Board of Viva In Vitro diagnostics, Spain (since 2024). All other authors have no competing interests.

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-1 beta 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 (K 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.

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.

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.

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.

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).

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.

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 mu M in HepG2.2.15 cells. The anti-viral 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 mu M against HBV DNA and between 3.7 and 4.8 mu M 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 mu M 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.

ABSTRACT 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.

BACKGROUND & AIMSNVR3-778 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.METHODSWe 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.RESULTSMice 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.CONCLUSIONSNVR3-778 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.

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.

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.