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Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure–activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

At a molecular level, insects utilize members of several highly divergent and unrelated families of cell-surface chemosensory receptors for detection of volatile odorants. Most odors are detected via a family of odorant receptors (ORs), which form heteromeric complexes consisting of a well-conserved OR co-receptor (Orco) ion channel and a non-conserved tuning OR that provides coding specificity to each complex. Orco functions as a non-selective cation channel and is expressed in the majority of olfactory receptor neurons (ORNs). As the destructive behaviors of many insects are principally driven by olfaction, Orco represents a novel target for behavior-based control strategies. While many natural and synthetic odorants have been shown to agonize Orco/Or complexes, only a single direct Orco modulator, VUAA1, has been described. In an effort to identify additional Orco modulators, we have investigated the structure/activity relationships around VUAA1. A search of our compound library identified several VUAA1 analogs that were selected for evaluation against HEK cells expressing Orco from the malaria vector Anopheles gambiae (AgOrco). While the majority of compounds displayed no activity, many of these analogs possess no intrinsic efficacy, but instead, act as competitive VUAA1 antagonists. Using calcium mobilization assays, patch clamp electrophysiology, and single sensillum in vivo recording, we demonstrate that one such candidate, VU0183254, is a specific allosteric modulator of OR signaling, capable of broadly inhibiting odor-mediated OR complex activation. We have described and characterized the first Orco antagonist, that is capable of non-competitively inhibiting odorant-evoked activation of OR complexes, thereby providing additional insight into the structure/function of this unique family of ligand-gated ion channels. While Orco antagonists are likely to have limited utility in insect control programs, they represent important pharmacological tools that will facilitate the investigation of the molecular mechanisms underlying insect olfactory signal transduction.

Plasma membrane organization profoundly impacts cellular functionality. A well-known mechanism underlying this organization is through nanoscopic clustering of distinct lipids and proteins in membrane rafts. Despite their physiological importance, rafts remain a difficult-to-study aspect of membrane organization, in part because of the paucity of chemical tools to experimentally modulate their properties. Methods to selectively target rafts for therapeutic purposes are also currently lacking. To tackle these problems, we developed a high-throughput screen and an accompanying image analysis pipeline to identify small molecules that enhance or inhibit raft formation. Cell-derived giant plasma membrane vesicles were used as the experimental platform. A proof-of-principle screen using a bioactive lipid library demonstrates that this method is robust and capable of validating established raft modulators including C6- and C8-ceramide, miltefosine, and epigallocatechin gallate as well as identifying new ones. The platform we describe here represents a powerful tool to discover new chemical approaches to manipulate rafts and their components.

A Xenopus laevis two-reporter screen identifies the antihelminthic drug pyrvinium as an inhibitor of the Wnt/β-catenin signaling pathway that works by activating CK1α, which is likely working at the level of Pygopus, a core transcriptional component of the Wnt pathway. Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC 50 of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.

Building on the role of human intuition in small molecule drug design, we explored whether crowdsourcing could recruit citizen scientists to this task while in parallel building awareness for this scientific process. Here, we introduce Drugit ( https://drugit.org ), the small molecule design mode of the online citizen science game Foldit. We demonstrate its utility by identifying distinct binders to the von Hippel Lindau E3 ligase. Several thousand molecules were suggested by players in a series of ten puzzle rounds. The proposed molecules were further evaluated in silico and manually by an expert panel. Selected candidates were synthesized and tested. One of these molecules shows dose-dependent shift perturbations in protein-observed NMR experiments. The co-crystal structure in complex with the E3 ligase reveals that the observed binding mode matches the player’s original idea. The completion of one full design cycle is a proof of concept for the Drugit approach and highlights the potential of involving citizen scientists in early drug discovery. Citizen science taps the efforts of non-experts. Here, authors describe Drugit, an extension of the crowdsourcing game Foldit, and its use in designing a non-peptide binder of Von Hippel Lindau E3 ligase for use with proteolysis targeting chimeras.

Analysis of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678-1020) with a structurally related propargylic amine (8). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure-activity relationship understanding of the covalent modifying potential and biological activity of a series of acetylenic thienopyrimidine compounds with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.

Currently, there is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and its analog mundulone acetate (MA) as inhibitors of intracellular Ca -regulated myometrial contractility. In this study, we probed the tocolytic and therapeutic potential of these small molecules using myometrial cells and tissues obtained from patients receiving cesarean deliveries, as well as a mouse model of PL resulting in preterm birth. In a phenotypic assay, mundulone displayed greater efficacy in the inhibition of intracellular-Ca from myometrial cells; however, MA showed greater potency and uterine-selectivity, based IC and E values between myometrial cells compared to aorta vascular smooth muscle cells, a major maternal off-target site of current tocolytics. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted concentration-dependent inhibition of myometrial contractions and that neither mundulone or MA affected vasoreactivity of ductus arteriosus, a major fetal off-target of current tocolytics. A high-throughput combination screen of intracellular Ca -mobilization identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these synergistic combinations, mundulone + atosiban demonstrated a favorable therapeutic index (TI)=10, a substantial improvement compared to TI=0.8 for mundulone alone. The and synergism of mundulone and atosiban was substantiated, yielding greater tocolytic efficacy and potency on isolated mouse and human myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone 5hrs after mifepristone administration (and PL induction) dose-dependently delayed the timing of delivery. Importantly, mundulone in combination with atosiban (FR 3.7:1, 6.5mg/kg + 1.75mg/kg) permitted long-term management of PL after induction with 30 μg mifepristone, allowing 71% dams to deliver viable pups at term (> day 19, 4-5 days post-mifepristone exposure) without any visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the future development of mundulone as a stand-alone single- and/or combination-tocolytic therapy for management of PL.

Congenital long QT syndrome (LQTS) promotes risk for life-threatening cardiac arrhythmia and sudden death in children and young adults. Pathogenic variants in the voltage-gated potassium channel KCNQ1 are the most frequently discovered genetic cause. Most LQTS-associated KCNQ1 variants cause loss-of-function secondary to impaired trafficking of the channel to the plasma membrane. There are currently no therapeutic approaches that address this underlying molecular defect. Using a high-throughput screening paradigm, we identified VU0494372, a small molecule that increases total and cell surface levels and trafficking efficiency of WT KCNQ1 as well as three LQTS-associated variants. Additionally, 16-hour treatment of cells with VU0494372 increased IKs (KCNQ1-KCNE1 current) for WT KCNQ1 and the LQTS-associated variant V207M in cells co-expressing KCNE1. VU0494372 had no impact on KCNQ1 transcription, degradation, or thermal stability, and increased the rate of KCNQ1 reaching the cell surface. We identified a potential direct interaction site with KCNQ1 at or near the binding site of the KCNQ1 potentiator ML277. Together, these findings demonstrate that small molecules can increase the expression levels and cell surface trafficking efficiency of KCNQ1 and introduce a potential new pharmacological approach for treating LQTS.

N-acyl-phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase that hydrolyzes N-acyl-phosphatidylethanolamine (NAPEs) to form N-acyl-ethanolamides (NAEs) and phosphatidic acid. Several lines of evidence suggest that reduced NAPE-PLD activity could contribute to cardiometabolic diseases. For instance, NAPEPLD expression is reduced in human coronary arteries with unstable atherosclerotic lesions, defective efferocytosis is implicated in the enlargement of necrotic cores of these lesions, and NAPE-PLD products such as palmitoylethanolamide and oleoylethanolamide have been shown to enhance efferocytosis. Thus, enzyme activation mediated by a small molecule may serve as a therapeutic treatment for cardiometabolic diseases. As a proof-of-concept study, we sought to identify small molecule activators of NAPE-PLD. High-throughput screening followed by hit validation and primary lead optimization studies identified a series of benzothiazole phenylsulfonyl-piperidine carboxamides that variably increased activity of both mouse and human NAPE-PLD. From this set of small molecules, two NAPE-PLD activators (VU534 and VU533) were shown to increase efferocytosis by bone-marrow derived macrophages isolated from wild-type mice, while efferocytosis was significantly reduced in Napepld-/- BMDM or after Nape-pld inhibition. Together these studies demonstrate an essential role for NAPE-PLD in the regulation of efferocytosis and the potential value of NAPE-PLD activators as a strategy to treat cardiometabolic diseases.Competing Interest StatementJEZ, KK, AWG, AMD, GAS, and SSD are named as inventors on patent application for the use of benzothiazole phenylsulfonyl-piperidine carboxamides as small molecule NAPE-PLD activators. This work was funded in part by grants from the NIH/NHLBI awarded to SSD and AMD P01HL116263 and by American Heart Association fellowship 835504 awarded to JEZ.

Wnt/b-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote b-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC50 of ~10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1a (CK1a) kinase activity. CK1a knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and b-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or b-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1a as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.