MEK1/2 activity modulates TREM2 cell surface recruitment.

Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer's disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface. Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that noncanonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 were required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.

High-Throughput Label-Free Biochemical Assays Using Infrared Matrix-Assisted Desorption Electrospray Ionization Mass Spectrometry.

Mass spectrometry (MS) can provide high sensitivity and specificity for biochemical assays without the requirement of labels, eliminating the risk of assay interference. However, its use had been limited to lower-throughput assays due to the need for chromatography to overcome ion suppression from the sample matrix. Direct analysis without chromatography has the potential for high throughput if sensitivity is sufficient despite the presence of a matrix. Here, we report and demonstrate a novel direct analysis high-throughput MS system based on infrared matrix-assisted desorption electrospray ionization (IR-MALDESI) that has a potential acquisition rate of 33 spectra/s. We show the development of biochemical assays in standard buffers for wild-type isocitrate dehydrogenase 1 (IDH1), diacylglycerol kinase zeta (DGKζ), and p300 histone acetyltransferase (P300) to demonstrate the suitability of this system for a broad range of high-throughput lead discovery assays. A proof-of-concept pilot screen of ∼3k compounds is also shown for IDH1 and compared to a previously reported fluorescence-based assay. We were able to obtain reliable data at a speed amenable for high-throughput screening of large-scale compound libraries.

Fragment-based discovery of a potent NAMPT inhibitor.

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor.

Off-target pharmacological activity at various kinases: Potential functional and pathological side effects.

In drug discovery, during the lead optimization and candidate characterization stages, novel small molecules are frequently evaluated in a battery of in vitro pharmacology assays to identify potential unintended, off-target interactions with various receptors, transporters, ion channels, and enzymes, including kinases. Furthermore, these screening panels may also provide utility at later stages of development to provide a mechanistic understanding of unexpected safety findings. Here, we present a compendium of the most likely functional and pathological outcomes associated with interaction(s) to a panel of 95 kinases based on an extensive curation of the scientific literature. This panel of kinases was designed by AbbVie based on safety-related data extracted from the literature, as well as from over 20 years of institutional knowledge generated from discovery efforts. For each kinase, the scientific literature was reviewed using online databases and the most often reported functional and pathological effects were summarized. This work should serve as a practical guide for small molecule drug discovery scientists and clinical investigators to predict and/or interpret adverse effects related to pharmacological interactions with these kinases.

New Platform for Label-Free, Proximal Cellular Pharmacodynamic Assays: Identification of Glutaminase Inhibitors Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.

Cellular pharmacodynamic assays are crucial aspects of lead optimization programs in drug discovery. These assays are sometimes difficult to develop, oftentimes distal from the target and frequently low throughput, which necessitates their incorporation in the drug discovery funnel later than desired. The earlier direct pharmacodynamic modulation of a target can be established, the fewer resources are wasted on compounds that are acting via an off-target mechanism. Mass spectrometry is a versatile tool that is often used for direct, proximal cellular pharmacodynamic assay analysis, but liquid chromatography-mass spectrometry methods are low throughput and are unable to fully support structure-activity relationship efforts in early medicinal chemistry programs. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization method amenable to high-throughput cellular assays, capable of diverse analyte detection, ambient and rapid laser sampling processes, and low cross-contamination. Here, we demonstrate the capability of IR-MALDESI for the detection of diverse analytes directly from cells and report the development of a high-throughput, label-free, proximal cellular pharmacodynamic assay using IR-MALDESI for the discovery of glutaminase inhibitors and a biochemical assay for hit confirmation. We demonstrate the throughput with a ∼100,000-compound cellular screen. Hits from the screening were confirmed by retesting in dose-response with mass spectrometry-based cellular and biochemical assays. A similar workflow can be applied to other targets with minimal modifications, which will speed up the discovery of cell active lead series and minimize wasted chemistry resources on off-target mechanisms.

Screen for Modulation of Nucleocapsid Protein Condensation Identifies Small Molecules with Anti-Coronavirus Activity.

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase-separated condensates that are hypothesized to play critical roles in viral replication, transcription, and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43, and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

Discovery of a Potent Chloroacetamide GPX4 Inhibitor with Bioavailability to Enable Target Engagement in Mice, a Potential Tool Compound for Inducing Ferroptosis In Vivo.

Compounds that inhibit glutathione peroxidase 4 (GPX4) hold promise as cancer therapeutics in their ability to induce a form of nonapoptotic cell death called ferroptosis. Our research identified 24, a structural analog of the potent GPX4 inhibitor RSL3, that has much better plasma stability (t1/2 > 5 h in mouse plasma). The bioavailability of 24 provided efficacious plasma drug concentrations with IP dosing, thus enabling in vivo studies to assess tolerability and efficacy. An efficacy study in mouse using a GPX4-sensitive tumor model found that doses of 24 up to 50 mg/kg were tolerated for 20 days but had no effect on tumor growth, although partial target engagement was observed in tumor homogenate.

1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) induces phosphorylation of eukaryotic elongation factor-2 (eEF2): a cautionary note on the anticancer mechanism of an eEF2 kinase inhibitor.

Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.

The HPK1 Inhibitor A-745 Verifies the Potential of Modulating T Cell Kinase Signaling for Immunotherapy.

Hematopoietic progenitor kinase 1 (HPK1) is an MAP4K family member within the Ste20-like serine/threonine branch of the kinome. HPK1 expression is limited to hematopoietic cells and has a predominant role as a negative regulator of T cell function. Because of the central/dominant role in negatively regulating T cell function, HPK1 has long been in the center of interest as a potential pharmacological target for immune therapy. The development of a small molecule HPK1 inhibitor remains challenging because of the need for high specificity relative to other kinases, including additional MAP4K family members, that are required for efficient immune cell activation. Here, we report the identification of the selective and potent HPK1 chemical probe, A-745. In unbiased cellular kinase-binding assays, A-745 demonstrates an excellent cellular selectivity binding profile within pharmacologically relevant concentrations. This HPK1 selectivity translates to an in vitro immune cell activation phenotype reminiscent of Hpk1-deficient and Hpk1-kinase-dead T cells, including augmented proliferation and cytokine production. The results from this work give a path forward for further developmental efforts to generate additional selective and potent small molecule HPK1 inhibitors with the pharmacological properties for immunotherapy.

Identification and structure-based drug design of cell-active inhibitors of interleukin 17A at a novel C-terminal site.

Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein-protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (< 50 nM) and showed functional inhibition of IL17A-induced cellular signaling (IC50~1 µM). We also describe a fluorescence-based probe molecule suitable to discern/screen for additional molecules binding in this C-terminal site.

Small Molecule Phenotypic Screen Identifies Novel Regulators of LDLR Expression.

Alzheimer's Disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia. The current treatment options for AD are limited to ameliorating cognitive decline temporarily and not reversing or preventing the progression of dementia. Hence, more effective therapeutic strategies are needed to combat this devastating disease. The low-density lipoprotein receptor has been shown to modulate the neuronal metabolism of cholesterol and apolipoprotein E, a major genetic risk factor for AD. LDLR overexpression in mice has been shown to increase amyloid-ß clearance and reduce amyloid deposition. We conducted a phenotypic screen to identify novel signaling pathways and targets that regulate LDLR expression in glial cells using an annotated compound library of approximately 29 000 compounds. The screen identified novel targets such as polo like kinase 1 (PLK1), activin receptor like kinase 5 (ALK5), and serotonin transporter (SERT). We used genetic, chemical biology and pathway analysis to confirm the target hypothesis. This work highlights that phenotypic screening is a promising strategy to identify novel mechanisms and targets for therapeutic intervention of complex neurodegenerative disorders.

Small Molecule IL-36γ Antagonist as a Novel Therapeutic Approach for Plaque Psoriasis.

IL-36 cytokines are pro-inflammatory members of the IL-1 family that are upregulated in inflammatory disorders. Specifically, IL-36γ is highly expressed in active psoriatic lesions and can drive pro-inflammatory processes in 3D human skin equivalents supporting a role for this target in skin inflammation. Small molecule antagonists of interleukins have been historically challenging to generate. Nevertheless, we performed a small molecule high-throughput screen to identify IL-36 antagonists using a novel TR-FRET binding assay. Several compounds, including 2-oxypyrimidine containing structural analogs of the marketed endothelin receptor A antagonist Ambrisentan, were identified as hits from the screen. A-552 was identified as a the most potent antagonist of human IL-36γ, but not the closely related family member IL-36α, was capable of attenuating IL-36γ induced responses in mouse and human disease models. Additionally, x-ray crystallography studies identified key amino acid residues in the binding pocket present in human IL-36γ that are absent in human IL-36α. A-552 represents a first-in-class small molecule antagonist of IL-36 signaling that could be used as a chemical tool to further investigate the role of this pathway in inflammatory skin diseases such as psoriasis.

Role of Rho kinase pathway in chondroitin sulfate proteoglycan-mediated inhibition of neurite outgrowth in PC12 cells.

Activation of the Rho kinase (ROCK) pathway has been associated with inhibition of neurite regeneration and outgrowth in spinal cord injury. Growth-inhibitory substances present in the glial scar such as chondroitin sulfate proteoglycans (CSPGs) have been shown to create a nonpermissive environment for axon regeneration that results in growth cone collapse. In this study, an in vitro model was developed in nerve growth factor-differentiated PC12 cells where the Rho/ROCK pathway was modulated by CSPG. CSPG elicited concentration-dependent inhibition of neurite outgrowth in PC12 cells, which was reversed by ROCK inhibitors such as fasudil, dimethylfasudil, and Y27632. Further studies on the interactions of CSPG with ROCK inhibitors revealed that the modulation of ROCK by CSPG is noncompetitive in nature. It was also observed that ROCK inhibitors increased neurite outgrowth in undifferentiated PC12 cells, indicating constitutive ROCK activity in the cells. Analysis of signaling pathways demonstrated that the effect of CSPG increases the phosphorylation of myosin phosphatase, a substrate immediately downstream of ROCK activation. Fasudil, dimethylfasudil, and Y27632 inhibited the phosphorylation of myosin phosphatase induced by CSPG with rank order potencies comparable to those observed in the neurite outgrowth assay. In addition, ROCK inhibitors reversed cofilin phosphorylation induced by CSPG with similar rank order potencies. Taken together, our data demonstrate that the interaction of CSPG with the ROCK pathway involves downstream effectors of ROCK such as myosin phosphatase and cofilin.

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315.

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,ß-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.

Identification of Selective Dual ROCK1 and ROCK2 Inhibitors Using Structure-Based Drug Design.

A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.

Implementation of a human podocyte injury model of chronic kidney disease for profiling of renoprotective compounds.

Degradation of podocyte structural integrity and function are hallmarks of proteinuric chronic kidney disease. In vivo, injury of podocytes manifests itself in the form of disruption of foot process morphology and associated cytoskeletal architecture, de-differentiation, and loss of adhesion to the glomerular basement membrane. Given the critical role played by this highly specialized cell type in maintaining glomerular filtration, there is a need for improved physiologically relevant cellular models that enable detection of disease-relevant indicators of podocyte perturbation. We have addressed this need by evaluating a subclone of conditionally immortalized human podocytes through quantitative benchmarking against freshly isolated primary human podocytes. Benchmarking was performed by measuring key phenotypic parameters, expression of podocyte specific proteins and multiparametric responses to stressors that model different aspects of podocyte perturbation. We subsequently employed the subcloned cells to profile the protective activity of structurally distinct adenosine kinase inhibitors. Our results support the translatability of our cellular model and set the stage for broader screening of renoprotective compounds with a view to eventually treat proteinuric kidney disease.

An offline-addition format for identifying GPCR modulators by screening 384-well mixed compounds in the FLIPR.

Although fluorescence imaging plate reader (FLIPR)-based assays have been widely used in high-throughput screening, improved efficiencies in throughput and fidelity continue to be investigated. This study presents an offline compound addition protocol coupled with a testing strategy using mixtures of compounds in a 384-well format to identify antagonists of the neurokinin-1 receptor expressed in the human astrocytoma cell line (U373 MG). Substance P evoked a concentration-dependent increase in intracellular cellular Ca(2+) with an EC(50) value of 0.30 +/- 0.17 nM, which was inhibited by neurokinin-1 (NK1) antagonists L-733,060 and L-703,606. Test compounds, as mixtures of 10 compounds/well, were added to the cells offline using an automated dispensing unit and incubated prior to performing the assay in the FLIPR. Using the offline protocol, a higher through put of ~200,000 compounds was achieved in an 8-h working day, and several novel structural classes of compounds were identified as antagonists for the NK1 receptor. These studies demonstrate that the offline compound addition format using a mixture of compounds in a 384-well FLIPR assay provides an efficient platform for screening and identifying modulators for G-protein-coupled receptors.

Application of Micro Arrayed Compound Screening (microARCS) to identify inhibitors of caspase-3.

Micro Arrayed Compound Screening (microARCS) is a miniaturized ultra-high-throughput screening platform developed at Abbott Laboratories. In this format, 8,640 discrete compounds are spotted and dried onto a polystyrene sheet, which has the same footprint as a 96-well plate. A homogeneous time-resolved fluorescence assay format (LANCE) was applied to identify the inhibitors of caspase-3 using a peptide substrate labeled with a fluorescent europium chelate and a dabcyl quencher. The caspase-3 enzyme was cast into a thin agarose gel, which was placed on a sheet containing test compounds. A second gel containing caspase substrate was then laid above the enzyme gel to initiate the reaction. Caspase-3 cleaves the substrate and separates the europium from the quencher, giving rise to a time-resolved fluorescent signal, which was detected using a ViewLux charge-coupled device imaging system. Potential inhibitors of caspase-3 appeared as dark spots on a bright fluorescent background. Results from the microARCS assay format were compared to those from a conventional 96-well plate-screening format.

Development and validation of a fluorescence-based HTS assay for the identification of P/Q-type calcium channel blockers.

Dysfunction of P/Q-type calcium channels is thought to underlie a variety of neurological diseases. There is evidence that migraine, Alzheimer's disease, and epilepsy involve a gain-of-function of the channel, leading to abnormal presynaptic vesicle release. P/Q-channel blockers may normalize current flow and consequently lead to an alleviation of disease symptoms. Although the medical need is high, there are no such compounds on the market. Here we describe a high throughput screen (HTS) for P/Q-type calcium channel blockers and the confirmation of hits by automated electrophysiology. We generated a HEK293 cell line stably expressing the α1A subunit of the P/Q-type calcium channel under control of a tetracycline (Tet) promoter. The accessory ß1.1 and α2δ1 subunits were co-expressed constitutively. The cell line was pharmacologically characterized by ion channel specific modulators, and revealed functional P/Q-type calcium currents. Using a fluorescence imaging plate reader (FLIPR), an assay for P/Q-type calcium channels was established based on a calcium sensitive dye. HTS of a 150,000 compound-containing sub-library led to the identification of 3262 hits that inhibited the fluorescence signal with potencies below 10 µM. Hit-to-lead (HTL) efforts identified 12,400 analogues. Compounds were clustered into 37 series, and 8 series of interest were prioritized. An electrophysiological secondary screen, providing a more direct measure of channel function, was implemented into the HTL process. 27 selected exemplars of different chemotypes were validated by automated whole-cell patch clamp analysis at inactivated channel state. The discovery of P/Q-channel blockers may foster the development of new therapeutics for a variety of neurological diseases.

Functional characterization and high-throughput screening of positive allosteric modulators of α7 nicotinic acetylcholine receptors in IMR-32 neuroblastoma cells.

α7 nicotinic acetylcholine receptors (nAChRs) are characterized by relatively low ACh sensitivity, rapid activation, and fast desensitization kinetics. ACh/agonist evoked currents at the α7 nAChR are transient, and, typically, calcium flux responses are difficult to detect using conventional fluorometric assay techniques. One approach to study interactions of agonists with the α7 nAChR is by utilizing positive allosteric modulators (PAMs). In this study, we demonstrate that inclusion of type II PAMs such as PNU-120596, but not type I, can enable detection of endogenous α7 nAChR-mediated calcium responses in human neuroblastoma (IMR-32) cells. Using this approach, we characterized the pharmacological profile of nicotine, epibatidine, choline, and other nAChR agonists such as PNU-282987, SSR-180711, GTS-21, OH-GTS21, tropisetron, NS6784, and A-582941. The rank order potency of agonists well correlated with α7 nAChR binding affinities measured in brain membranes. Inhibition of calcium response by methyllycaconitine in the presence of increasing concentrations of PNU-282987 or PNU-120596 revealed that the IC(50) value of methyllycaconitine was sensitive to varying concentrations of the agonist, but not that of the PAM. This format demonstrated the feasibility of this approach for high-throughput screening to identify small molecule, PAMs, which were further confirmed in electrophysiological assays of human α7 nAChR expressed in oocytes.