Enhancing the Small-Scale Screenable Biological Space beyond Known Chemogenomics Libraries with Gray Chemical Matter─Compounds with Novel Mechanisms from High-Throughput Screening Profiles.

Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.

DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance.

Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.

Activation of human STING by a molecular glue-like compound.

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

DRUG-seq Provides Unbiased Biological Activity Readouts for Neuroscience Drug Discovery.

Unbiased transcriptomic RNA-seq data has provided deep insights into biological processes. However, its impact in drug discovery has been narrow given high costs and low throughput. Proof-of-concept studies with Digital RNA with pertUrbation of Genes (DRUG)-seq demonstrated the potential to address this gap. We extended the DRUG-seq platform by subjecting it to rigorous testing and by adding an open-source analysis pipeline. The results demonstrate high reproducibility and ability to resolve the mechanism(s) of action for a diverse set of compounds. Furthermore, we demonstrate how this data can be incorporated into a drug discovery project aiming to develop therapeutics for schizophrenia using human stem cell-derived neurons. We identified both an on-target activation signature, induced by a set of chemically distinct positive allosteric modulators of the N-methyl-d-aspartate (NMDA) receptor, and independent off-target effects. Overall, the protocol and open-source analysis pipeline are a step toward industrializing RNA-seq for high-complexity transcriptomics studies performed at a saturating scale.

An IMiD-inducible degron provides reversible regulation for chimeric antigen receptor expression and activity.

The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 ß-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated in vivo when dosing a US Food and Drug Administration-approved drug, lenalidomide.

Author Correction: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.

DRUG-seq for miniaturized high-throughput transcriptome profiling in drug discovery.

Here we report Digital RNA with pertUrbation of Genes (DRUG-seq), a high-throughput platform for drug discovery. Pharmaceutical discovery relies on high-throughput screening, yet current platforms have limited readouts. RNA-seq is a powerful tool to investigate drug effects using transcriptome changes as a proxy, yet standard library construction is costly. DRUG-seq captures transcriptional changes detected in standard RNA-seq at 1/100th the cost. In proof-of-concept experiments profiling 433 compounds across 8 doses, transcription profiles generated from DRUG-seq successfully grouped compounds into functional clusters by mechanism of actions (MoAs) based on their intended targets. Perturbation differences reflected in transcriptome changes were detected for compounds engaging the same target, demonstrating the value of using DRUG-seq for understanding on and off-target activities. We demonstrate DRUG-seq captures common mechanisms, as well as differences between compound treatment and CRISPR on the same target. DRUG-seq provides a powerful tool for comprehensive transcriptome readout in a high-throughput screening environment.

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

Resorufin butyrate as a soluble and monomeric high-throughput substrate for a triglyceride lipase.

Triglyceride lipases such as lipoprotein lipase, endothelial lipase, and hepatic lipase play key roles in controlling the levels of plasma lipoprotein. Accordingly, small-molecule modulation of these species could alter patient lipid profiles with corresponding health effects. Screening of these enzymes for small-molecule therapeutics has historically involved the use of lipid-based particles to mimic native substrates. However, particle-based artifacts can complicate the discovery of therapeutic molecules. As a simplifying solution, the authors sought to develop an approach involving a soluble and monomeric lipase substrate. Using purified bovine lipoprotein lipase as a model system, they show that the hydrolysis of resorufin butyrate can be fluorescently monitored to give a robust assay (Z' > 0.8). Critically, using parallel approaches, they show that resorufin butyrate is soluble and monomeric under assay conditions. The presented assay should be useful as a simple and inexpensive primary or secondary screen for the discovery of therapeutic lipase modulators.

Discovery of novel P2Y14 agonist and antagonist using conventional and nonconventional methods.

P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.

Applying the pro-drug approach to afford highly bioavailable antagonists of P2Y(14).

Our series of competitive antagonists against the G-protein coupled receptor P2Y(14) were found to be highly shifted in the presence of serum (>99% protein bound). A binding assay using 2% human serum albumin (HSA) was developed to guide further SAR studies and led to the identification of the zwitterion 2, which is substantially less shifted (18-fold) than our previous lead compound 1 (323-fold). However, as the bioavailability of 2 was low, a library of ester pro-drugs was prepared (7a-7j) and assessed in vitro. The most interesting candidates were then profiled in vivo and led to the identification of the pro-drug 7j, which possesses a substantially improved pharmacokinetic profile.

The identification of 4,7-disubstituted naphthoic acid derivatives as UDP-competitive antagonists of P2Y14.

A weak, UDP-competitive antagonist of the pyrimidinergic receptor P2RY(14) with a naphthoic acid core was identified through high-throughput screening. Optimization provided compounds with improved potency but poor pharmacokinetics. Acylglucuronidation was determined to be the major route of metabolism. Increasing the electron-withdrawing nature of the substituents markedly reduced glucuronidation and improved the pharmacokinetic profile. Additional optimization led to the identification of compound 38 which is an 8 nM UDP-competitive antagonist of P2Y(14) with a good pharmacokinetic profile.

Synthesis and SAR of pyrimidine-based, non-nucleotide P2Y14 receptor antagonists.

A weak antagonist of the pyrimidinergic receptor P2Y(14) containing a dihydropyridopyrimidine core was identified through high-throughput screening. Subsequent optimization led to potent, non-UTP competitive antagonists and represent the first reported non-nucleotide antagonists of this receptor. Compound 18q was identified as a 10 nM P2Y(14) antagonist with good oral bioavailability and provided sufficient exposure in mice to be used as a tool for future in vivo studies.

An activity-based probe for high-throughput measurements of triacylglycerol lipases.

Modulating the activity of lipases involved in the metabolism of plasma lipoproteins is an attractive approach for developing lipid raising/lowering therapies to treat cardiovascular disease. Identifying small molecule inhibitors for these membrane-active enzymes, however, is complicated by difficulties associated with measuring lipase activity and inhibition at the water-membrane interface; substrate and compound dynamics at the particle interface have the potential to confound data interpretation. Here, we describe a novel ELISA-based lipase activity assay that employs as "bait" a biotinylated active-site probe that irreversibly binds to the catalytic active-site serine of members of the triacylglycerol lipase family (hepatic lipase, lipoprotein lipase, and endothelial lipase) in solution with high affinity. Detection of "captured" (probe-enzyme) complexes on streptavidin-coated plates using labeled secondary antibodies to specific primary antibodies offers several advantages over conventional assays, including the ability to eliminate enzyme-particle and compound-particle effects; specifically measure lipase activity in complex mixtures in vitro; preferentially identify active-site-directed inhibitors; and distinguish between reversible and irreversible inhibitors through a simple assay modification. Using EL as an exemplar, we demonstrate the versatility of this assay both for high-throughput screening and for compound mechanism-of-action studies.

Tricyclic imidazole antagonists of the Neuropeptide S Receptor.

A new structural class of potent antagonists of the Neuropeptide S Receptor (NPSR) is reported. High-throughput screening identified a tricyclic imidazole antagonist of NPSR, and medicinal chemistry optimization of this structure was undertaken to improve potency against the receptor as well as CNS penetration. Detailed herein are synthetic and medicinal chemistry studies that led to the identification of antagonists 15 and NPSR-PI1, which demonstrate potent in vitro NPSR antagonism and central exposure in vivo.

Synthesis and evaluation of a new series of Neuropeptide S receptor antagonists.

Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood-brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited (125)I-NPS binding in the CNS when administered to rats.

Design, synthesis, and evaluation of novel 3-amino-4-hydrazine-cyclobut-3-ene-1,2-diones as potent and selective CXCR2 chemokine receptor antagonists.

We describe herein a novel series of 3-amino-4-hydrazine-cyclobut-3-ene-1,2-diones as potent and selective inhibitors against the CXCR2 chemokine receptor and IL-8-mediated chemotaxis of a CXCR2-expressing cell line. Furthermore, these alkyl-hydrazine series inhibitors such as 5b demonstrated acceptable metabolic stability when incubated in human and rat microsomes.

Stimulation of toll-like receptor 2 with bleomycin results in cellular activation and secretion of pro-inflammatory cytokines and chemokines.

The clinical use of bleomycin results in systemic and pulmonary inflammatory syndromes that are mediated by the production of cytokines and chemokines. In this study, we demonstrate that cell activation is initiated upon the recognition of bleomycin as a pathogen-associated molecular pattern by toll-like receptor (TLR) 2. The THP1 human monocytic cell line, which constitutively expresses high levels of TLR2, secretes interleukin (IL)-1beta, IL-8, and tumor necrosis factor (TNF)-alpha during bleomycin exposure. The TLR2-dependent nature of cell activation and cytokine secretion is supported by (1) the inability of TLR2-deficient human embryonic kidney (HEK) 293 cells to exhibit nuclear factor-kappa B (NF-kappaB) activation and secrete IL-8 in response to bleomycin; (2) the acquired ability of HEK293 to exhibit NF-kappaB activation and secrete IL-8 upon experimental expression of TLR2; and (3) the inhibition of cell activation in TLR2-expressing HEK293 and THP1 by anti-TLR2 monoclonal antibody. Collectively, these observations identify TLR2 activation as a critical event that triggers NF-kappaB activation and secretion of cytokines and chemokines during bleomycin exposure. Our in vitro findings could serve as a molecular mechanism underlying the pro-inflammatory toxicity associated with bleomycin. Whether bleomycin engages with other cellular receptors that results in activation of alternate signaling pathways and whether the TLR2-agonist activity of bleomycin contribute to its anti-neoplastic property deserve further study.