Discovery of Clinical Candidate NT-0796, a Brain-Penetrant and Highly Potent NLRP3 Inflammasome Inhibitor for Neuroinflammatory Disorders.

The NLRP3 inflammasome is a component of the innate immune system involved in the production of proinflammatory cytokines. Neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis, have been shown to have a component driven by NLRP3 inflammasome activation. Diseases such as these with large unmet medical needs have resulted in an interest in inhibiting the NLRP3 inflammasome as a potential pharmacological treatment, but to date, no marketed drugs specifically targeting NLRP3 have been approved. Furthermore, the requirement for CNS-penetrant molecules adds additional complexity to the search for NLRP3 inflammasome inhibitors suitable for clinical investigation of neuroinflammatory disorders. We designed a series of ester-substituted carbamate compounds as selective NLRP3 inflammasome inhibitors, leading to NT-0796, an isopropyl ester that undergoes intracellular conversion to NDT-19795, the carboxylic acid active species. NT-0796 was shown to be a potent and selective NLRP3 inflammasome inhibitor with demonstrated in vivo brain penetration.

Epilogue to the Gerald Maggiora Festschrift: a tribute to an exemplary mentor, colleague, collaborator, and innovator.

In May 2022, JCAMD published a Special Issue in honor of Gerald (Gerry) Maggiora, whose scientific leadership over many decades advanced the fields of computational chemistry and chemoinformatics for drug discovery. Along the way, he has impacted many researchers in both academia and the pharmaceutical industry. In this Epilogue, we explain the origins of the Festschrift and present a series of first-hand vignettes, in approximate chronological sequence, that together paint a picture of this remarkable man. Whether they highlight Gerry's endless curiosity about molecular life sciences or his willingness to challenge conventional wisdom or his generous support of junior colleagues and peers, these colleagues and collaborators are united in their appreciation of his positive influence. These tributes also reflect key trends and themes during the evolution of modern drug discovery, seen through the lens of people who worked with a visionary leader. Junior scientists will find an inspiring roadmap for creative collegiality and collaboration.

Discovery and Optimization of Triazolopyrimidinone Derivatives as Selective NLRP3 Inflammasome Inhibitors.

The NLRP3 inflammasome is a multiprotein complex that facilitates activation and release of the proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 in response to infection or endogenous stimuli. It can be inappropriately activated by a range of danger signals resulting in chronic, low-grade inflammation underlying a multitude of diseases, such as Alzheimer's disease, Parkinson's disease, osteoarthritis, and gout. The discovery of potent and specific NLRP3 inhibitors could reduce the burden of several common morbidities. In this study, we identified a weakly potent triazolopyrimidone hit (1) following an in silico modeling exercise. This was optimized to furnish potent and selective small molecule NLRP3 inflammasome inhibitors. Compounds such as NDT-30805 could be useful tool molecules for a scaffold-hopping or pharmacophore generation project or used as leads toward the development of clinical candidates.

Discovery of Ethyl Ketone-Based Highly Selective HDACs 1, 2, 3 Inhibitors for HIV Latency Reactivation with Minimum Cellular Potency Serum Shift and Reduced hERG Activity.

We describe the discovery of histone deacetylase (HDACs) 1, 2, and 3 inhibitors with ethyl ketone as the zinc-binding group. These HDACs 1, 2, and 3 inhibitors have good enzymatic and cellular activity. Their serum shift in cellular potency has been minimized, and selectivity against hERG has been improved. They are also highly selective over HDACs 6 and 8. These inhibitors contain a variety of substituted heterocycles on the imidazole or oxazole scaffold. Compounds 31 and 48 stand out due to their good potency, high selectivity over HDACs 6 and 8, reduced hERG activity, optimized serum shift in cellular potency, and good rat and dog PK profiles.

Redefining the Histone Deacetylase Inhibitor Pharmacophore: High Potency with No Zinc Cofactor Interaction.

A novel series of histone deacetylase (HDAC) inhibitors lacking a zinc-binding moiety has been developed and described herein. HDAC isozyme profiling and kinetic studies indicate that these inhibitors display a selectivity preference for HDACs 1, 2, 3, 10, and 11 via a rapid equilibrium mechanism, and crystal structures with HDAC2 confirm that these inhibitors do not interact with the catalytic zinc. The compounds are nonmutagenic and devoid of electrophilic and mutagenic structural elements and exhibit off-target profiles that are promising for further optimization. The efficacy of this new class in biochemical and cell-based assays is comparable to the marketed HDAC inhibitors belinostat and vorinostat. These results demonstrate that the long-standing pharmacophore model of HDAC inhibitors requiring a metal binding motif should be revised and offers a distinct class of HDAC inhibitors.

Discovery of an Anion-Dependent Farnesyltransferase Inhibitor from a Phenotypic Screen.

By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.

Discovery of Highly Selective and Potent HDAC3 Inhibitors Based on a 2-Substituted Benzamide Zinc Binding Group.

The selectivity of histone deacetylase inhibitors (HDACis) is greatly impacted by the zinc binding groups. In an effort to search for novel zinc binding groups, we applied a parallel medicinal chemistry (PMC) strategy to quickly synthesize substituted benzamide libraries. We discovered a series containing 2-substituted benzamides as the zinc binding group which afforded highly selective and potent HDAC3 inhibitors, exemplified by compound 16 with a 2-methylthiobenzamide. Compound 16 inhibited HDAC3 with an IC50 of 30 nM and with unprecedented selectivity of >300-fold over all other HDAC isoforms. Interestingly, a subtle change of the 2-methylthio to a 2-hydroxy benzamide in 20 retains HDAC3 potency but loses all selectivity over HDAC 1 and 2. This significant difference in selectivity was rationalized by X-ray crystal structures of HDACis 16 and 20 bound to HDAC2, revealing different binding modes to the catalytic zinc ion. This series of HDAC3 selective inhibitors served as tool compounds for investigating the minimal set of HDAC isoforms that must be inhibited for the HIV latency activation in a Jurkat 2C4 cell model and potentially as leads for selective HDAC3 inhibitors for other indications.

Selective Class I HDAC Inhibitors Based on Aryl Ketone Zinc Binding Induce HIV-1 Protein for Clearance.

HIV persistence in latently infected, resting CD4+ T cells is broadly considered a barrier to eradicate HIV. Activation of the provirus using latency-reversing agents (LRAs) followed by immune-mediated clearance to purge reservoirs has been touted as a promising therapeutic approach. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) control the acetylation level of lysine residues in histones to regulate the gene transcription. Several clinical HDAC inhibitors had been examined as LRAs, which induced HIV activation in vitro and in vivo. Here we report the discovery of a series of selective and potent class I HDAC inhibitors based on aryl ketones as a zinc binding group, which reversed HIV latency using a Jurkat model of HIV latency in 2C4 cells. The SAR led to the discovery of a highly selective class I HDAC inhibitor 10 with excellent potency. HDACi 10 induces the HIV gag P24 protein in patient latent CD4+ T cells.

Prediction of the Favorable Hydration Sites in a Protein Binding Pocket and Its Application to Scoring Function Formulation.

The important role of water molecules in protein-ligand binding energetics has attracted wide attention in recent years. A range of computational methods has been developed to predict the favorable locations of water molecules in a protein binding pocket. Most of the current methods are based on extensive molecular dynamics or Monte Carlo simulations. They are time-consuming and thus cannot be applied to high-throughput tasks. To overcome this difficulty, we have developed an empirical method, called HydraMap, to predict the favorable hydration sites in the binding pocket of a protein molecule. This method uses statistical potentials to quantify the interactions between protein atoms and water molecules. Such statistical potentials were derived from 10,987 crystal structures selected from the Protein Data Bank. The probability of placing a water probe at each spot in the binding pocket was evaluated to derive a density map. The density map was then deduced into explicit hydration sites through a clustering process. HydraMap was validated on two external test sets, where it produced comparable results as 3D-RISM and WATsite but was 30-1000 times faster. In addition, we have attempted to estimate the desolvation energy associated with water molecule replacement upon ligand binding based on the outcomes of HydraMap. This desolvation term, called DEWED, was incorporated into the framework of four scoring functions, i.e., ASP, ChemPLP, GoldScore, and X-Score. The derivative scoring functions were tested in terms of scoring power, docking power, and screening power on a range of data sets. It was observed that X-Score exhibited the most obvious improvement in accuracy after adding the DEWED terms. Moreover, all scoring functions augmented with the DEWED terms exhibited improved or comparable performance on most data sets as the corresponding ones augmented with the GB/SA terms. Our study has demonstrated the potential application of HydraMap and DEWED to the formulation of new scoring functions. A beta-version of the HydraMap software is freely available from our Web site (http://www.sioc-ccbg.ac.cn/software/hydramap/) for testing.

Computational Chemistry: A Rising Tide of Women.

The authors were inspired to explore the topic of gender diversity in computational chemistry on the basis of similar recent publications in the related fields of medicinal chemistry ( Huryn , D. M. ; et al. ACS Med. Chem. Lett. 2017 , 8 , 900 ) and computational biology ( Bonham , K. S. ; Stefan , M. I. PLoS Comput. Biol. 2017 , 13 , e1005134 ). To do so, we examined historical demographics in two different professional settings, i.e., attendance/participation at the Gordon Research Conferences on Computer-Aided Drug Design and Computational Chemistry and membership in the Computers in Chemistry Division of the American Chemical Society. We conclude that female representation in computational chemistry has risen steadily over the last 40 years and likely stands at around 25%, which appears to slightly exceed that of the neighboring fields of computer science and medicinal chemistry. In accordance with the old slogan that "a rising tide lifts all boats", here a rising tide of women scientists is having an impact on the field of computational chemistry. Tactics to ensure that this number continues to improve are highlighted.

Design and Synthesis of Piperazine Sulfonamide Cores Leading to Highly Potent HIV-1 Protease Inhibitors.

Using the HIV-1 protease binding mode of MK-8718 and PL-100 as inspiration, a novel aspartate binding bicyclic piperazine sulfonamide core was designed and synthesized. The resulting HIV-1 protease inhibitor containing this core showed an 60-fold increase in enzyme binding affinity and a 10-fold increase in antiviral activity relative to MK-8718.

Development of a New Structural Class of Broadly Acting HCV Non-Nucleoside Inhibitors Leading to the Discovery of MK-8876.

Studies directed at developing a broadly acting non-nucleoside inhibitor of HCV NS5B led to the discovery of a novel structural class of 5-aryl benzofurans that simultaneously interact with both the palm I and palm II binding regions. An initial candidate was potent in vitro against HCV GT1a and GT1b replicons, and induced multi-log reductions in HCV viral load when orally dosed to chronic GT1 infected chimpanzees. However, in vitro potency losses against clinically relevant GT1a variants prompted a further effort to develop compounds with sustained potency across a broader array of HCV genotypes and mutants. Ultimately, a biology and medicinal chemistry collaboration led to the discovery of the development candidate MK-8876. MK-8876 demonstrated a pan-genotypic potency profile and maintained potency against clinically relevant mutants. It demonstrated moderate bioavailability in rats and dogs, but showed low plasma clearance characteristics consistent with once-daily dosing. Herein we describe the efforts which led to the discovery of MK-8876, which advanced into Phase 1 monotherapy studies for evaluation and characterization as a component of an all-oral direct-acting drug regimen for the treatment of chronic HCV infection.

Molecular Simulations Identify Binding Poses and Approximate Affinities of Stapled α-Helical Peptides to MDM2 and MDMX.

Traditionally, computing the binding affinities of proteins to even relatively small and rigid ligands by free-energy methods has been challenging due to large computational costs and significant errors. Here, we apply a new molecular simulation acceleration method called MELD (Modeling by Employing Limited Data) to study the binding of stapled α-helical peptides to the MDM2 and MDMX proteins. We employ free-energy-based molecular dynamics simulations (MELD-MD) to identify binding poses and calculate binding affinities. Even though stapled peptides are larger and more complex than most protein ligands, the MELD-MD simulations can identify relevant binding poses and compute relative binding affinities. MELD-MD appears to be a promising method for computing the binding properties of peptide ligands with proteins.

The evolution of drug design at Merck Research Laboratories.

On October 5, 1981, Fortune magazine published a cover article entitled the "Next Industrial Revolution: Designing Drugs by Computer at Merck". With a 40+ year investment, we have been in the drug design business longer than most. During its history, the Merck drug design group has had several names, but it has always been in the "design" business, with the ultimate goal to provide an actionable hypothesis that could be tested experimentally. Often the result was a small molecule but it could just as easily be a peptide, biologic, predictive model, reaction, process, etc. To this end, the concept of design is now front and center in all aspects of discovery, safety assessment and early clinical development. At present, the Merck design group includes computational chemistry, protein structure determination, and cheminformatics. By bringing these groups together under one umbrella, we were able to align activities and capabilities across multiple research sites and departments. This alignment from 2010 to 2016 resulted in an 80% expansion in the size of the department, reflecting the increase in impact due to a significant emphasis across the organization to "design first" along the entire drug discovery path from lead identification (LID) to first in human (FIH) dosing. One of the major advantages of this alignment has been the ability to access all of the data and create an adaptive approach to the overall LID to FIH pathway for any modality, significantly increasing the quality of candidates and their probability of success. In this perspective, we will discuss how we crafted a new strategy, defined the appropriate phenotype for group members, developed the right skillsets, and identified metrics for success in order to drive continuous improvement. We will not focus on the tactical implementation, only giving specific examples as appropriate.

Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

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.

High-Throughput Screen of GluK1 Receptor Identifies Selective Inhibitors with a Variety of Kinetic Profiles Using Fluorescence and Electrophysiology Assays.

GluK1, a kainate subtype of ionotropic glutamate receptors, exhibits an expression pattern in the CNS consistent with involvement in pain processing and migraine. Antagonists of GluK1 have been shown to reduce pain signaling in the spinal cord and trigeminal nerve, and are predicted to provide pain and migraine relief. We developed an ultra-high-throughput small-molecule screen to identify antagonists of GluK1. Using the calcium indicator dye fluo-4, a multimillion-member small-molecule library was screened in 1536-well plate format on the FLIPR (Fluorescent Imaging Plate Reader) Tetra against cells expressing a calcium-permeable GluK1. Following confirmation in the primary assay and subsequent counter-screen against the endogenous Par-1 receptor, 6100 compounds were selected for dose titration to assess potency and selectivity. Final triage of 1000 compounds demonstrating dose-dependent inhibition with IC50 values of less than 12 µM was performed in an automated whole-cell patch clamp electrophysiology assay. Although a weak correlation between electrophysiologically active and calcium-imaging active compounds was observed, the identification of electrophysiologically active compounds with a range of kinetic profiles revealed a broad spectrum of mechanisms of action.

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.