High-throughput Screening Hit Research Articles

Discovery of 2-Amide-3-methylester Thiophenes that Target SARS-CoV-2 Mac1 and Repress Coronavirus Replication, Validating Mac1 as an Antiviral Target.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed. Here, we describe small-molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation-mediated innate immune responses. Three high-throughput screening hits had the same 2-amide-3-methylester thiophene scaffold. We studied the compound binding mode using X-ray crystallography, allowing us to design analogues. Compound 27 (MDOLL-0229) had an IC50 of 2.1 μM and was selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human proteins. The improved potency allowed testing of its effect on virus replication, and indeed, 27 inhibited replication of both murine hepatitis virus (MHV) prototypes CoV and SARS-CoV-2. Sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1-targeted small molecule demonstrated to inhibit coronavirus replication in a cell model.

Abstract 4584: Novel YAP1/TAZ pathway inhibitors identified through phenotypic screening with potent anti-tumor activity via blockade of GGTase-I and Rho-GTPase signaling

Abstract YAP1/TAZ have been shown to be aberrantly activated oncogenes in several human solid tumors, resulting in enhanced cell proliferation, metastasis and provision of a pro-tumorigenic microenvironment, making YAP1/TAZ targets for novel cancer therapies. Yet, the development of effective inhibitors of these potent oncogenes has been challenging. In this work, we break new ground in this direction through the identification of novel inhibitors of YAP1/TAZ activity. This study describes the identification and target deconvolution of novel small molecule inhibitors of oncogenic YAP1/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. HTS hits that selectively inhibited TEAD-luciferase, but not TK-Renilla-luciferase (n = 3.994) were subsequently assessed regarding their ability to induce translocation of YAP1 from the nucleus to the cytoplasm in MDA-MB-231 cells, which is the physiological mechanism of inactivation. Out of these, 392 hits showed activity to induce translocation of YAP1. Finally, selected hits active in both assays (n = 96) were assessed regarding their effect on endogenous YAP1/TAZ target genes. The small molecule BAY-856 demonstrated the most consistent YAP1/TAZ inhibitory activity of all selected hits, which warranted further exploration of the unknown direct drug target. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex, as the direct target of YAP1/TAZ pathway inhibitor BAY-856. BAY-856 and close analogs with improved in vitro potency blocked the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in several tumor types in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties. BAY-593 demonstrated anti-tumor activity in solid tumor models and blockade of YAP1/TAZ signaling in vivo. BAY-593 is a novel tool compound to explore Rho-GTPase signaling and downstream YAP1/TAZ biology in vitro and in vivo. Citation Format: Keith Graham, Philip Lienau, Benjamin Bader, Stefan Prechtl, Jan Naujoks, Ralf Lesche, Barbara Nicke, Wilhelm Bone, Sven Golfier, Krzysztof Brzezinka, Stefan Kaulfuss, Charlotte Kopitz, Holger Steuber, Nico Braeuer, Katrin Nowak-Reppel, Carlo Stresemann, Patrick Steigemann, Julia Kuehnlenz, Lisette Potze, Francesca Zanconato, Anna Montebaur, Sabine Pilari, Sikander Hayat, Atanas Kamburov, Andreas Steffen, Andreas Schlicker, Philipp Buchgraber, Nuria Aiguabella Font, Tobias Heinrich, Lara Kuhnke, Annette O. Walter, Simona Blotta, Matthias Ocker, Ashley Lakner, Dominik Mumberg, Knut Eis, Stefano Piccolo, Martin Lange. Novel YAP1/TAZ pathway inhibitors identified through phenotypic screening with potent anti-tumor activity via blockade of GGTase-I and Rho-GTPase signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4584.

Discovery of IRAK4 Inhibitors BAY1834845 (Zabedosertib) and BAY1830839.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate inflammatory processes. Here, we describe the discovery of two clinical candidate IRAK4 inhibitors, BAY1834845 (zabedosertib) and BAY1830839, starting from a high-throughput screening hit derived from Bayer's compound library. By exploiting binding site features distinct to IRAK4 using an in-house docking model, liabilities of the original hit could surprisingly be overcome to confer both candidates with a unique combination of good potency and selectivity. Favorable DMPK profiles and activity in animal inflammation models led to the selection of these two compounds for clinical development in patients.

Lies and Liabilities: Computational Assessment of High-Throughput Screening Hits to Identify Artifact Compounds.

Hits from high-throughput screening (HTS) of chemical libraries are often false positives due to their interference with assay detection technology. In response, we generated the largest publicly available library of chemical liabilities and developed "Liability Predictor," a free web tool to predict HTS artifacts. More specifically, we generated, curated, and integrated HTS data sets for thiol reactivity, redox activity, and luciferase (firefly and nano) activity and developed and validated quantitative structure-interference relationship (QSIR) models to predict these nuisance behaviors. The resulting models showed 58-78% external balanced accuracy for 256 external compounds per assay. QSIR models developed and validated herein identify nuisance compounds among experimental hits more reliably than do popular PAINS filters. Both the models and the curated data sets were implemented in "Liability Predictor," publicly available at https://liability.mml.unc.edu/. "Liability Predictor" may be used as part of chemical library design or for triaging HTS hits.

Synthesis and Structure-Activity Relationships of a New Class of Oxadiazoles Targeting DprE1 as Antitubercular Agents.

The continuing prevalence of drug-resistant tuberculosis threatens global TB control programs, highlighting the need to discover new drug candidates to feed the drug development pipeline. In this study, we describe a high-throughput screening hit (4-benzylpiperidin-1-yl)(1-(5-phenyl-1,3,4-oxadiazol-2-yl)piperidin-4-yl)methanone (P1) as a potent antitubercular agent. Structure-activity guided synthesis led to the discovery of several analogs with high in vitro potency. P1 was found to have promising potency against many drug-resistant strains, as well as drug-susceptible clinical isolates. It also showed cidality against Mtb growing in host macrophages. Whole genome sequencing of genomic DNA from resistant mutants raised to P1 revealed mutations in decaprenylphosphoryl-β-d-ribose 2'-oxidase (DprE1). This novel oxadiazole scaffold expands the set of chemical tools for targeting a well-validated pathway to treat tuberculosis.

Design, synthesis, and pharmacological evaluation of N-(3-carbamoyl-1H-pyrazol-4-yl)-1,3-oxazole-4-carboxamide derivatives as interleukin-1 receptor-associated kinase 4 inhibitors with reduced potential for cytochrome P450 1A2 induction

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical molecule in Toll-like receptor/interleukin-1 receptor signaling and an attractive therapeutic target for a wide range of inflammatory and autoimmune diseases as well as cancers. In our search for novel IRAK4 inhibitors, we conducted structural modification of a thiazolecarboxamide derivative 1, a lead compound derived from high-throughput screening hits, to elucidate structure–activity relationship and improve drug metabolism and pharmacokinetic (DMPK) properties. First, conversion of the thiazole ring of 1 to an oxazole ring along with introduction of a methyl group at the 2-position of the pyridine ring aimed at reducing cytochrome P450 (CYP) inhibition were conducted to afford 16. Next, modification of the alkyl substituent at the 1-position of the pyrazole ring of 16 aimed at improving CYP1A2 induction properties revealed that branched alkyl and analogous substituents such as isobutyl (18) and (oxolan-3-yl)methyl (21), as well as six-membered saturated heterocyclic groups such as oxan-4-yl (2), piperidin-4-yl (24, 25), and dioxothian-4-y (26), are effective for reducing induction potential. Representative compound AS2444697 (2) exhibited potent IRAK4 inhibitory activity with an IC50 value of 20 nM and favorable DMPK properties such as low risk of drug–drug interactions mediated by CYPs as well as excellent metabolic stability and oral bioavailability.

Discovery of a Novel Series of Potent, Selective, Orally Available, and Brain-Penetrable C1s Inhibitors for Modulation of the Complement Pathway.

A novel series of non-amidine-based C1s inhibitors have been explored. Starting from high-throughput screening hit 3, isoquinoline was replaced with 1-aminophthalazine to enhance C1s inhibitory activity while exhibiting good selectivity against other serine proteases. We first disclose a crystal structure of a complex of C1s and a small-molecule inhibitor (4e), which guided structure-based optimization around the S2 and S3 sites to further enhance C1s inhibitory activity by over 300-fold. Improvement of membrane permeability by incorporation of fluorine at the 8-position of 1-aminophthalazine led to identification of (R)-8 as a potent, selective, orally available, and brain-penetrable C1s inhibitor. (R)-8 significantly inhibited membrane attack complex formation induced by human serum in a dose-dependent manner in an in vitro assay system, proving that selective C1s inhibition blocked the classical complement pathway effectively. As a result, (R)-8 emerged as a valuable tool compound for both in vitro and in vivo assessment.

Discovery of Novel Substrate-Competitive Lysine Methyltransferase G9a Inhibitors as Anticancer Agents.

Identification of structurally novel inhibitors of lysine methyltransferase G9a has been a subject of intense research in cancer epigenetics. Starting with the high-throughput screening (HTS) hit rac-10a obtained from the chemical library of the University of Tokyo Drug Discovery Initiative, the structure-activity relationship of the unique substrate-competitive inhibitors was established with the help of X-ray crystallography and fragment molecular orbital (FMO) calculations for the ligand-protein interaction. Further optimization of the in vitro characteristics and drug metabolism and pharmacokinetics (DMPK) properties led to the identification of 26j (RK-701), which is a structurally distinct potent inhibitor of G9a/GLP (IC50 = 27/53 nM). Compound 26j exhibited remarkable selectivity against other related methyltransferases, dose-dependent attenuation of cellular H3K9me2 levels, and tumor growth inhibition in MOLT-4 cells in vitro. Moreover, compound 26j showed inhibition of tumor initiation and growth in a carcinogen-induced hepatocellular carcinoma (HCC) in vivo mouse model without overt acute toxicity.

Discovery of a novel, highly potent and orally bioavailable pyrrolidinone indole series of irreversible Myeloperoxidase (MPO) inhibitors

Myeloperoxidase (MPO) is a heme-containing peroxidase from phagocytic cells, which plays an important role in the innate immune response. The primary anti-microbial function of MPO is achieved by catalyzing the oxidation of halides by hydrogen peroxide (H2O2). Upon activation of phagocytes, MPO activity is detectable in both phagosomes and extracellularly, where it can remain or transcytose into interstitial compartments. Activated MPO leads to oxidative stress and tissue damage in many inflammatory states, including cardiovascular disease. Starting from a low molecular weight (LMW) high throughput screening (HTS) hit, here we report the discovery of a novel pyrrolidinone indole (IN-4) as a highly potent MPO inhibitor. This compound displays similar in vitro potency across peroxidation, plasma and NETosis assays. In a dilution/dialysis study, <5% of the original MPO activity was detected post-incubation of MPO with IN-4, suggesting irreversible enzyme inhibition. A fast MPO inactivation rate (kinact/Ki) and low partition ratio (k3/k4) make IN-4 kinetic properties attractive for an MPO inhibitor. This compound also displays significant selectivity over the closely related thyroid peroxidase (TPO), and is selective for extracellular MPO over intracellular (neutrophil) MPO. Moreover, IN-4 shows good exposure, low clearance and high oral bioavailability in mice, rats and dogs. The high in vitro MPO activity and high oral exposure observed with IN-4 result in a dose-dependent inhibition of MPO activity in three mouse models of inflammation. In conclusion, IN-4 is a novel, potent, mechanism-based and selective MPO inhibitor, which may be used as superior therapeutic agent to treat multiple inflammatory conditions, including cardiovascular disease.

BAY-7081: A Potent, Selective, and Orally Bioavailable Cyanopyridone-Based PDE9A Inhibitor.

Despite advances in the treatment of heart failure in recent years, options for patients are still limited and the disease is associated with considerable morbidity and mortality. Modulating cyclic guanosine monophosphate levels within the natriuretic peptide signaling pathway by inhibiting PDE9A has been associated with beneficial effects in preclinical heart failure models. We herein report the identification of BAY-7081, a potent, selective, and orally bioavailable PDE9A inhibitor with very good aqueous solubility starting from a high-throughput screening hit. Key aspect of the optimization was a switch in metabolism of our lead structures from glucuronidation to oxidation. The switch proved being essential for the identification of compounds with improved pharmacokinetic profiles. By studying a tool compound in a transverse aortic constriction mouse model, we were able to substantiate the relevance of PDE9A inhibition in heart diseases.

The discovery of (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile, a potent and selective agonist of human transient receptor potential cation channel subfamily m member 5 (TRPM5) and evaluation of as a potential gastrointestinal prokinetic agent

This publication details the discovery of a series of selective transient receptor potential cation channel subfamily M member 5 (TRPM5) agonists culminating with the identification of the lead compound (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (39). We describe herein our biological rationale for agonism of the target, the examination of the then current literature tool molecules, and finally the process of our discovery starting with a high throughput screening hit through lead development. We also detail the selectivity of the lead compound 39 versus related family members TRPA1, TRPV1, TRPV4, TRPM4 and TRPM8, the drug metabolism and pharmacokinetics (DMPK) profile and in vivo efficacy in a mouse model of gastrointestinal motility.

Structure–activity relationship studies in a new series of 2-amino-N-phenylacetamide inhibitors of Slack potassium channels

In this Letter we describe structure–activity relationship (SAR) studies conducted in five distinct regions of a new 2-amino-N-phenylacetamides series of Slack potassium channel inhibitors exemplified by recently disclosed high-throughput screening (HTS) hit VU0606170 (4). New analogs were screened in a thallium (Tl+) flux assay in HEK-293 cells stably expressing wild-type human (WT) Slack. Selected analogs were screened in Tl+ flux versus A934T Slack and other Slo family members Slick and Maxi-K and evaluated in whole-cell electrophysiology (EP) assays using an automated patch clamp system. Results revealed the series to have flat SAR with significant structural modifications resulting in a loss of Slack activity. More minor changes led to compounds with Slack activity and Slo family selectivity similar to the HTS hit.

Structure-based design, synthesis and evaluation of a novel family of PEX5-PEX14 interaction inhibitors against Trypanosoma

Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic stage of the disease, urgently calling for the identification of new targets and drug candidates.Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzo[b,f][1,4]thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases.

Fragment-based discovery of novel phenyltriazolyl derivatives as allosteric type-I1/2 ALK inhibitors with promising antitumor effects

Based on the high-throughput screening hit BY-1, a series of phenyltriazolyl derivatives were developed. Satisfyingly, most compounds were detected moderate to excellent antitumor effects against Karpas299 and H2228 cells. Among them, 12k bearing 4‑hydroxypiperidinyl group exhibited the optimal activities against tested cells with IC50 values of 51 nM and 175 nM, as well as promising inhibitory effects on ALKWT (3.7 nM) and ALKL1196M (6.8 nM). Unlike the conventional type-I ALK inhibitors, molecular models identified 12k as an allosteric type-I1/2 inhibitor by forming key interactions in both the ATP binding region and the hydrophobic back pocket of ALK. Intriguingly, 12k could dose-dependently induce apoptosis on H2228 cell and inhibit colony formation and tumor cell migration. Taken together, the rationalization of 12k may shed new light on the identification of novel allosteric type-I1/2 ALK inhibitors.

Discovery and In Vivo Evaluation of ACT-660602: A Potent and Selective Antagonist of the Chemokine Receptor CXCR3 for Autoimmune Diseases.

The chemokine receptor CXCR3 is a seven-transmembrane G-protein-coupled receptor (GPCR) involved in various pathologies, in particular autoimmune diseases. It is activated by the three chemokine ligands CXCL9, CXCL10, and CXCL11 and enables the recruitment of immune cell subsets leading to damage of inflamed tissues. Starting from a high-throughput screening hit, we describe the iterative optimization of a chemical series culminating in the discovery of the selective CXCR3 antagonist ACT-660602 (9j). The careful structural modifications during the lead optimization phase led to a compound with high biological potency in inhibiting cell migration together with improvements of the metabolic stability and hERG issue. In a LPS-induced lung inflammation model in mice, ACT-660602 led to significantly reduced recruitment of the CXCR3+ CD8+ T cell in the bronchoalveolar lavage compartment when administered orally at a dose of 30 mg/kg.

Accelerating GPCR Drug Discovery With Conformation-Stabilizing VHHs.

The human genome encodes 850 G protein-coupled receptors (GPCRs), half of which are considered potential drug targets. GPCRs transduce extracellular stimuli into a plethora of vital physiological processes. Consequently, GPCRs are an attractive drug target class. This is underlined by the fact that approximately 40% of marketed drugs modulate GPCRs. Intriguingly 60% of non-olfactory GPCRs have no drugs or candidates in clinical development, highlighting the continued potential of GPCRs as drug targets. The discovery of small molecules targeting these GPCRs by conventional high throughput screening (HTS) campaigns is challenging. Although the definition of success varies per company, the success rate of HTS for GPCRs is low compared to other target families (Fujioka and Omori, 2012; Dragovich et al., 2022). Beyond this, GPCR structure determination can be difficult, which often precludes the application of structure-based drug design approaches to arising HTS hits. GPCR structural studies entail the resource-demanding purification of native receptors, which can be challenging as they are inherently unstable when extracted from the lipid matrix. Moreover, GPCRs are flexible molecules that adopt distinct conformations, some of which need to be stabilized if they are to be structurally resolved. The complexity of targeting distinct therapeutically relevant GPCR conformations during the early discovery stages contributes to the high attrition rates for GPCR drug discovery programs. Multiple strategies have been explored in an attempt to stabilize GPCRs in distinct conformations to better understand their pharmacology. This review will focus on the use of camelid-derived immunoglobulin single variable domains (VHHs) that stabilize disease-relevant pharmacological states (termed ConfoBodies by the authors) of GPCRs, as well as GPCR:signal transducer complexes, to accelerate drug discovery. These VHHs are powerful tools for supporting in vitro screening, deconvolution of complex GPCR pharmacology, and structural biology purposes. In order to demonstrate the potential impact of ConfoBodies on translational research, examples are presented of their role in active state screening campaigns and structure-informed rational design to identify de novo chemical space and, subsequently, how such matter can be elaborated into more potent and selective drug candidates with intended pharmacology.

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 (27b) was identified as a novel M5 orthosteric antagonist with high potency (human M5 IC50 = 36 nM), M5 subtype selectivity (>100-fold selectivity against human M1-4) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

Discovery of EP300/CBP histone acetyltransferase inhibitors through scaffold hopping of 1,4-oxazepane ring

EP300 and its paralog CBP play an important role in post-translational modification as histone acetyltransferases (HATs). EP300/CBP inhibition has been gaining attention as an anticancer treatment target in recent years. Herein, we describe the identification of a novel, highly selective EP300/CBP inhibitor, compound 11 (DS17701585), by scaffold hopping and structure-based optimization of a high-throughput screening hit 1. Compound 11 (DS17701585) shows dose-dependent inhibition of SRY-box transcription factor 2 (SOX2) mRNA expression in a human lung squamous cell carcinoma cell line LK2-xenografted mouse model.

Discovery of 5-Aryl-2,4-diaminopyrimidine Compounds as Potent and Selective IRAK4 Inhibitors.

IRAK4 kinase plays a key role in TLR/IL-1R signaling pathways that regulate innate immune responses, and if uncontrolled, it is responsible for various inflammatory disorders. By high-throughput screening (HTS) and hit-to-lead optimization, compounds with a 5-aryl-2,4-diaminopyrimidine core structure have been identified as potent IRAK4 inhibitors. A cocrystal structure of IRAK4 protein with an early lead molecule helped with understanding the structure-activity relationship and the design of the new compounds. Initial HTS hits from this series of compounds were also found to inhibit TAK1 kinase, which would cause liver toxicity and potentially bone marrow failure. Optimization of this series resulted in improved selectivity over TAK1 kinase. The TAK1 selectivity was found to be closely associated with different sizes and types of substituents at the 5-position of the pyrimidine. The impact of other pyrimidine substituents on the potency and selectivity was also explored. A few representative compounds were evaluated in IL-1β-induced IL-6 inhibition animal model studies and showed modest efficacy.

Thieno[2,3-d]pyrimidine-Based Positive Allosteric Modulators of Human Mas-Related G Protein-Coupled Receptor X1 (MRGPRX1).

Mas-related G protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and potential target for the treatment of pain. Positive allosteric modulators (PAMs) of MRGPRX1 have the potential to preferentially activate the receptors at the central terminals of primary sensory neurons and minimize itch side effects caused by peripheral activation. Using a high-throughput screening (HTS) hit, a series of thieno[2,3-d]pyrimidine-based molecules were synthesized and evaluated as human MRGPRX1 PAMs in HEK293 cells stably transfected with human MrgprX1 gene. An iterative process to improve potency and metabolic stability led to the discovery of orally available 6-(tert-butyl)-5-(3,4-dichlorophenyl)-4-(2-(trifluoromethoxy)phenoxy)thieno[2,3-d]pyrimidine (1t), which can be distributed to the spinal cord, the presumed site of action, following oral administration. In a neuropathic pain model induced by sciatic nerve chronic constriction injury (CCI), compound 1t (100 mg/kg, po) reduced behavioral heat hypersensitivity in humanized MRGPRX1 mice, demonstrating the therapeutic potential of MRGPRX1 PAMs in treating neuropathic pain.