Expanding the ligandable proteome by paralog hopping with covalent probes.

More than half of the ~20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.

Translational PK-PD for targeted protein degradation.

Targeted protein degradation has emerged from the chemical biology toolbox as one of the most exciting areas for novel therapeutic development across the pharmaceutical industry. The ability to induce the degradation, and not just inhibition, of target proteins of interest (POIs) with high potency and selectivity is a particularly attractive property for a protein degrader therapeutic. However, the physicochemical properties and mechanism of action for protein degraders can lead to unique pharmacokinetic (PK) and pharmacodynamic (PD) properties relative to traditional small molecule drugs, requiring a shift in perspective for translational pharmacology. In this review, we provide practical insights for building the PK-PD understanding of protein degraders in the context of translational drug development through the use of quantitative mathematical frameworks and standard experimental assays. Published datasets describing protein degrader pharmacology are used to illustrate the applicability of these insights. The learnings are consolidated into a translational PK-PD roadmap for targeted protein degradation that can enable a systematic, rational design workflow for protein degrader therapeutics.

REL-1017 (Esmethadone) as Adjunctive Treatment in Patients With Major Depressive Disorder: A Phase 2a Randomized Double-Blind Trial.

Objective: The purpose of this study was to examine the effects of REL-1017 (esmethadone), a novel N-methyl-d-aspartate receptor (NMDAR) channel blocker, in patients with major depressive disorder who failed to benefit from one to three standard antidepressant treatments in their current major depressive episode. Methods: A 7-day phase 2 multicenter randomized double-blind placebo-controlled trial, comprising three arms, was conducted to assess the safety, tolerability, pharmacokinetics, and efficacy of two dosages of REL-1017 (25 mg or 50 mg orally once a day). Patients were randomly assigned in a 1:1:1 ratio to placebo (N=22), REL-1017 25 mg/day (N=19), or REL-1017 50 mg/day (N=21). Safety scales included the 4-item Positive Symptom Rating Scale for psychotomimetic symptoms, the Clinician-Administered Dissociative States Scale for dissociative symptoms, the Clinical Opiate Withdrawal Scale for withdrawal signs and symptoms, and the Columbia-Suicide Severity Rating Scale for suicidality. The primary efficacy endpoint was the Montgomery-Åsberg Depression Scale (MADRS) score. All 62 randomly assigned patients were included in the full analysis set population analysis. Results: Patients experienced mild or moderate transient adverse events and no evidence of dissociative or psychotomimetic effects, opioid effects, or withdrawal signs and symptoms. The improvement in MADRS score shown on day 4 in both of the REL-1017 dosage groups was sustained through day 7 (last dose) and day 14 (7 days after the last dose), with effect sizes from 0.7 to 1.0. Conclusions: This trial showed favorable safety, tolerability, and pharmacokinetic profiles and suggests that REL-1017 may have rapid and sustained antidepressant effects compared with placebo in patients with inadequate responses to antidepressant treatments. These results will need confirmation in larger and longer trials.

Intramolecular Ring-Opening Decomposition of Aryl Azetidines.

The ring strain present in azetidines can lead to undesired stability issues. Herein, we described a series of N-substituted azetidines which undergo an acid-mediated intramolecular ring-opening decomposition via nucleophilic attack of a pendant amide group. Studies were conducted to understand the decomposition mechanism enabling the design of stable analogues.

Snapshots and ensembles of BTK and cIAP1 protein degrader ternary complexes.

Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton's tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.

A kinetic proofreading model for bispecific protein degraders.

Bispecific protein degraders (BPDs) engage the ubiquitin-proteasome system (UPS) to catalytically degrade intracellular proteins through the formation of ternary complexes with the target protein and E3 ubiquitin ligases. Here, we describe the development of a mechanistic modeling framework for BPDs that includes the reaction network governing ternary complex formation and degradation via the UPS. A critical element of the model framework is a multi-step process that results in a time delay between ternary complex formation and protein degradation, thereby balancing ternary complex stability against UPS degradation rates akin to the kinetic proofreading concept that has been proposed to explain the accuracy and specificity of biological processes including protein translation and T cell receptor signal transduction. Kinetic proofreading likely plays a central role in the cell's ability to regulate substrate recognition and degradation by the UPS, and the model presented here applies this concept in the context of a quantitative pharmacokinetic (PK)-pharmacodynamic (PD) framework to inform the design of potent and selective BPDs.

Delineating the role of cooperativity in the design of potent PROTACs for BTK.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

Inducing protein-protein interactions with molecular glues.

The drugable proteome is limited by the number of functional binding sites that can bind small molecules and respond with a therapeutic effect. Orthosteric and allosteric modulators of enzyme function or receptor signaling are well-established mechanisms of drug action. Drugs that perturb protein-protein interactions have only recently been launched. This approach is more difficult due to the extensive contact surfaces that must be perturbed antagonistically. Compounds that promote novel protein-protein interactions promise to dramatically expand opportunities for therapeutic intervention. This approach is precedented with natural products (rapamycin, FK506, sanglifehrin A), synthetic small molecules (thalidomide and IMiD derivatives) and indisulam analogues.

Discovery of Trifluoromethyl Glycol Carbamates as Potent and Selective Covalent Monoacylglycerol Lipase (MAGL) Inhibitors for Treatment of Neuroinflammation.

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.

Structure-Based Approach To Identify 5-[4-Hydroxyphenyl]pyrrole-2-carbonitrile Derivatives as Potent and Tissue Selective Androgen Receptor Modulators.

In an effort to find new and safer treatments for osteoporosis and frailty, we describe a novel series of selective androgen receptor modulators (SARMs). Using a structure-based approach, we identified compound 7, a potent AR (ARE EC50 = 0.34 nM) and selective (N/C interaction EC50 = 1206 nM) modulator. In vivo data, an AR LBD X-ray structure of 7, and further insights from modeling studies of ligand receptor interactions are also presented.

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1-((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1-yl)prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans.

Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of 11 (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor 11 led to its evaluation in several human clinical studies.

Clearance Prediction of Targeted Covalent Inhibitors by In Vitro-In Vivo Extrapolation of Hepatic and Extrahepatic Clearance Mechanisms.

The concept of target-specific covalent enzyme inhibitors appears attractive from both an efficacy and a selectivity viewpoint considering the potential for enhanced biochemical efficiency associated with an irreversible mechanism. Aside from potential safety concerns, clearance prediction of covalent inhibitors represents a unique challenge due to the inclusion of nontraditional metabolic pathways of direct conjugation with glutathione (GSH) or via GSH S-transferase-mediated processes. In this article, a novel pharmacokinetic algorithm was developed using a series of Pfizer kinase selective acrylamide covalent inhibitors based on their in vitro-in vivo extrapolation of systemic clearance in rats. The algorithm encompasses the use of hepatocytes as an in vitro model for hepatic clearance due to oxidative metabolism and GSH conjugation, and the use of whole blood as an in vitro surrogate for GSH conjugation in extrahepatic tissues. Initial evaluations with clinical covalent inhibitors suggested that the scaling algorithm developed from rats may also be useful for human clearance prediction when species-specific parameters, such as hepatocyte and blood stability and blood binding, were considered. With careful consideration of clearance mechanisms, the described in vitro-in vivo extrapolation approach may be useful to facilitate candidate optimization, selection, and prediction of human pharmacokinetic clearance during the discovery and development of targeted covalent inhibitors.

Chiral Sulfoxide-Induced Single Turn Peptide α-Helicity.

Inducing α-helicity through side-chain cross-linking is a strategy that has been pursued to improve peptide conformational rigidity and bio-availability. Here we describe the preparation of small peptides tethered to chiral sulfoxide-containing macrocyclic rings. Furthermore, a study of structure-activity relationships (SARs) disclosed properties with respect to ring size, sulfur position, oxidation state, and stereochemistry that show a propensity to induce α-helicity. Supporting data include circular dichroism spectroscopy (CD), NMR spectroscopy, and a single crystal X-ray structure for one such stabilized peptide. Finally, theoretical studies are presented to elucidate the effect of chiral sulfoxides in inducing backbone α-helicity.

Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition.

PF-06651600, a newly discovered potent JAK3-selective inhibitor, is highly efficacious at inhibiting γc cytokine signaling, which is dependent on both JAK1 and JAK3. PF-06651600 allowed the comparison of JAK3-selective inhibition to pan-JAK or JAK1-selective inhibition, in relevant immune cells to a level that could not be achieved previously without such potency and selectivity. In vitro, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and in vivo it reduces disease pathology in rat adjuvant-induced arthritis as well as in mouse experimental autoimmune encephalomyelitis models. Importantly, by sparing JAK1 function, PF-06651600 selectively targets γc cytokine pathways while preserving JAK1-dependent anti-inflammatory signaling such as the IL-10 suppressive functions following LPS treatment in macrophages and the suppression of TNFα and IL-1ß production in IL-27-primed macrophages. Thus, JAK3-selective inhibition differentiates from pan-JAK or JAK1 inhibition in various immune cellular responses, which could potentially translate to advantageous clinical outcomes in inflammatory and autoimmune diseases.

Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism.

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.

Optimization of a Dicarboxylic Series for in Vivo Inhibition of Citrate Transport by the Solute Carrier 13 (SLC13) Family.

Inhibition of the sodium-coupled citrate transporter (NaCT or SLC13A5) has been proposed as a new therapeutic approach for prevention and treatment of metabolic diseases. In a previous report, we discovered dicarboxylate 1a (PF-06649298) which inhibits the transport of citrate in in vitro and in vivo settings via a specific interaction with NaCT. Herein, we report the optimization of this series leading to 4a (PF-06761281), a more potent inhibitor with suitable in vivo pharmacokinetic profile for assessment of in vivo pharmacodynamics. Compound 4a was used to demonstrate dose-dependent inhibition of radioactive [(14)C]citrate uptake in liver and kidney in vivo, resulting in modest reductions in plasma glucose concentrations.

Developing predictive assays: the phenotypic screening "rule of 3".

Phenotypic drug discovery approaches can positively affect the translation of preclinical findings to patients. However, not all phenotypic assays are created equal. A critical question then follows: What are the characteristics of the optimal assays? We analyze this question and propose three specific criteria related to the disease relevance of the assay-system, stimulus, and end point-to help design the most predictive phenotypic assays.

Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors.

Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.