Target-based discovery of a broad-spectrum flukicide.

Diseases caused by parasitic flatworms impart a considerable healthcare burden worldwide. Many of these diseases-for example, the parasitic blood fluke infection schistosomiasis-are treated with the drug praziquantel (PZQ). However, PZQ is ineffective against disease caused by liver flukes from the genus Fasciola because of a single amino acid change within the target of PZQ, a transient receptor potential ion channel in the melastatin family (TRPMPZQ), in Fasciola species. Here, we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and tegumental damage to these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad-spectrum activity manifests as BZQ adopts a pose within the binding pocket of TRPMPZQ that is dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad-spectrum flukicide.

Protocol for kinetic mode potassium channel assays on common plate readers and microscopes.

Fluorescence-based potassium channel assays are typically run on expensive, hard to obtain, fluorescence imaging kinetic plate readers that are uncommon in most laboratories. Here we describe the use of the Brilliant Thallium Snapshot assay to conduct an endpoint potassium channel assay, so that it can be used across multiple plate reader platforms that are more common in many labs. These methods will allow users to identify modulators of potassium channels. For this work, we have taken a kinetic mode Molecular Devices FLIPR based protocol and adapted it to be utilized on endpoint plate readers, such as the BMG Labtech PHERAstar, to identify activators of GIRK channels in CHO cells. We demonstrate that both plate readers are functionally competent at generating excellent Z' values which makes them ideally suited to finding corollary hits from the Sigma LOPAC 1,280 screening collection. Importantly, this assay has also been validated using a high content reader, demonstrating the possibility of spatially resolving signals from individual cells within a mixed cell population. The compendium of these results shows the flexibility, accessibility and functionality of endpoint-compatible potassium channel assay readouts on more common plate readers.

Rebamipide and Derivatives are Potent, Selective Inhibitors of Histidine Phosphatase Activity of the Suppressor of T Cell Receptor Signaling Proteins.

The suppressor of T cell receptor signaling (Sts) proteins are negative regulators of immune signaling. Genetic inactivation of these proteins leads to significant resistance to infection. From a 590,000 compound high-throughput screen, we identified the 2-(1H)-quinolinone derivative, rebamipide, as a putative inhibitor of Sts phosphatase activity. Rebamipide, and a small library of derivatives, are competitive, selective inhibitors of Sts-1 with IC50 values from low to submicromolar. SAR analysis indicates that the quinolinone, the acid, and the amide moieties are all essential for activity. A crystal structure confirmed the SAR and reveals key interactions between this class of compound and the protein. Although rebamipide has poor cell permeability, we demonstrated that a liposomal preparation can inactivate the phosphatase activity of Sts-1 in cells. These studies demonstrate that Sts-1 enzyme activity can be pharmacologically inactivated and provide foundational tools and insights for the development of immune-enhancing therapies that target the Sts proteins.

Target-based discovery of a broad spectrum flukicide.

Diseases caused by parasitic flatworms impart a considerable healthcare burden worldwide. Many of these diseases - for example, the parasitic blood fluke infection, schistosomiasis - are treated with the drug praziquantel (PZQ). However, PZQ is ineffective against disease caused by liver flukes from the genus Fasciola. This is due to a single amino acid change within the target of PZQ, a transient receptor potential ion channel (TRPMPZQ), in Fasciola species. Here we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and damage to the protective tegument of these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad spectrum activity was manifest as BZQ adopts a pose within the binding pocket of TRPMPZQ dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad spectrum flukicide.

High throughput screening for drugs that inhibit 3C-like protease in SARS-CoV-2.

The SARS coronavirus 2 (SARS-CoV-2) pandemic remains a major problem in many parts of the world and infection rates remain at extremely high levels. This high prevalence drives the continued emergence of new variants, and possibly ones that are more vaccine-resistant and that can drive infections even in highly vaccinated populations. The high rate of variant evolution makes clear the need for new therapeutics that can be clinically applied to minimize or eliminate the effects of COVID-19. With a hurdle of 10 years, on average, for first in class small molecule therapeutics to achieve FDA approval, the fastest way to identify therapeutics is by drug repurposing. To this end, we developed a high throughput cell-based screen that incorporates the essential viral 3C-like protease and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or FDA-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Medicinal chemistry efforts to optimize the hits identified Tranilast as a potential lead. Here, we report the rapid screening and identification of potentially relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral 3C-like protease.

High throughput screening for compounds to the orphan nuclear receptor NR2F6.

NR2F6 is considered an orphan nuclear receptor since its endogenous ligand has yet to be identified. Recently, NR2F6 has emerged as a novel cancer therapeutic target. NR2F6 has been demonstrated to be upregulated or overexpressed in several cancers. Importantly, Nr2f6-/- mice spontaneously reject tumors and develop host-protective immunological memory, a consequence of NR2F6 acting as an immune checkpoint in effector T cells. Collectively, these data suggest that modulation of NR2F6 activity may have important clinical applications in the fight against cancer. The nuclear receptor superfamily of ligand-regulated transcription factors has proven to be an excellent source of targets for therapeutic intervention of a broad range of diseases. Approximately 15% of FDA approved drugs target NRs, demonstrating their clinical efficacy. To identify small molecule regulators of NR2F6 activity, with the overall goal of immuno-oncology, we developed and initiated a high-throughput cell-based assay that specifically measures the transcriptional activity of NR2F6. We completed automated screening of approximately 666,000 compounds and identified 5,008 initial hits. Further screening efforts, including counterscreening assays, confirmed 128 of these hits, most of which had IC50s of equal to or less than 5µM potencies. Here, we report, for the first time, the identification of several small molecule compounds to the orphan nuclear receptor, NR2F6.

Identification of novel modulators of a schistosome transient receptor potential channel targeted by praziquantel.

Given the worldwide burden of neglected tropical diseases, there is ongoing need to develop novel anthelmintic agents to strengthen the pipeline of drugs to combat these burdensome infections. Many diseases caused by parasitic flatworms are treated using the anthelmintic drug praziquantel (PZQ), employed for decades as the key clinical agent to treat schistosomiasis. PZQ activates a flatworm transient receptor potential (TRP) channel within the melastatin family (TRPMPZQ) to mediate sustained Ca2+ influx and worm paralysis. As a druggable target present in many parasitic flatworms, TRPMPZQ is a promising target for a target-based screening campaign with the goal of discovering novel regulators of this channel complex. Here, we have optimized methods to miniaturize a Ca2+-based reporter assay for Schistosoma mansoni TRPMPZQ (Sm.TRPMPZQ) activity enabling a high throughput screening (HTS) approach. This methodology will enable further HTS efforts against Sm.TRPMPZQ as well as other flatworm ion channels. A pilot screen of ~16,000 compounds yielded a novel activator of Sm.TRPMPZQ, and numerous potential blockers. The new activator of Sm.TRPMPZQ represented a distinct chemotype to PZQ, but is a known chemical entity previously identified by phenotypic screening. The fact that a compound prioritized from a phenotypic screening campaign is revealed to act, like PZQ, as an Sm.TRPMPZQ agonist underscores the validity of TRPMPZQ as a druggable target for antischistosomal ligands.

Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR.

Cystic fibrosis (CF) is caused by a mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which disrupts an ion channel involved in hydration maintenance via anion homeostasis. Nearly 5% of CF patients possess one or more copies of the G542X allele, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small-molecule modulators of mutant CFTR biosynthesis that affect the readthrough of this and other premature termination codons to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large-scale screening of a homogeneous, 1536-well functional G542X-CFTR readthrough assay. The assay uses HEK 293 cells engineered to overexpress the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR to the cell surface. Compounds that allow translational readthrough and synthesis of functional CFTR chloride channels are reflected by changes in membrane potential in response to cAMP stimulation with forskolin and CFTR channel potentiation with genistein. Assay statistics yielded Z' values of 0.69 ± 0.06. As further evidence of its suitability for high-throughput screening, we completed automated screening of approximately 666,000 compounds, identifying 7761 initial hits. Following secondary and tertiary assays, we identified 188 confirmed hit compounds with low and submicromolar potencies. Thus, this approach takes advantage of a phenotypic screen with high-throughput scalability to identify new small-molecule G542X-CFTR readthrough modulators.

High-Throughput Screening for Drugs That Inhibit Papain-Like Protease in SARS-CoV-2.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 has triggered an ongoing global pandemic whereby infection may result in a lethal severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, millions of confirmed cases and hundreds of thousands of deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. The purported development of a vaccine could require at least 1-4 years, while the typical timeline from hit finding to drug registration of an antiviral is >10 years. Thus, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we developed and initiated a high-throughput cell-based screen that incorporates the essential viral papain-like protease (PLpro) and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or US Food and Drug Administration (FDA)-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Here, we report the identification of four clinically relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral PLpro.

Rescue of mutant gonadotropin-releasing hormone receptor function independent of cognate receptor activity.

Molecules that correct the folding of protein mutants, restoring their functional trafficking, are called pharmacoperones. Most are clinically irrelevant and possess intrinsic antagonist or agonist activity. Here, we identify compounds capable of rescuing the activity of mutant gonadotropin-releasing hormone receptor or GnRHR which, is sequestered within the cell and if dysfunctional leads to Hypogonadotropic Hypogonadism. To do this we screened the E90K GnRHR mutant vs. a library of 645,000 compounds using a cell-based calcium detection system. Ultimately, we identified 399 compounds with EC50 ≤ 5 µM with no effect in counterscreen assays. Medicinal chemistry efforts confirmed activity of 70 pure samples and mode of action studies, including radioligand binding, inositol phosphate, and toxicity assays, proved that we have a series of tractable compounds that can be categorized into structural clusters. These early lead molecules rescue mutant GnRHR function and are neither agonist nor antagonists of the GnRHR cognate receptor, a feature required for potential clinical utility.

Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.

Activation of the IRE1/XBP1s signaling arm of the unfolded protein response (UPR) is a promising strategy to correct defects in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. However, no pharmacologic activators of this pathway identified to date are suitable for ER proteostasis remodeling through selective activation of IRE1/XBP1s signaling. Here, we use high-throughput screening to identify non-toxic compounds that induce ER proteostasis remodeling through IRE1/XBP1s activation. We employ transcriptional profiling to stringently confirm that our prioritized compounds selectively activate IRE1/XBP1s signaling without activating other cellular stress-responsive signaling pathways. Furthermore, we demonstrate that our compounds improve ER proteostasis of destabilized variants of amyloid precursor protein (APP) through an IRE1-dependent mechanism and reduce APP-associated mitochondrial toxicity in cellular models. These results establish highly selective IRE1/XBP1s activating compounds that can be widely employed to define the functional importance of IRE1/XBP1s activity for ER proteostasis regulation in the context of health and disease.

Discovery and Characterization of Two Classes of Selective Inhibitors of the Suppressor of the TCR Signaling Family of Proteins.

The suppressor of T-cell receptor signaling (Sts) proteins, Sts-1, has recently emerged as a potential immunostimulatory target for drug development. Genetic inactivation of the Sts proteins dramatically increases host survival of systemic infection and leads to improved pathogen clearance. The protein tyrosine phosphatase (PTP) activity of these proteins arises from a C-terminal 2-histidine phosphatase (HP) domain. To identify new inhibitors of the HP activity of Sts-1, we miniaturized a phosphatase assay to a 1536-well format and conducted a 20 580 compound screen. Among the hits were two classes of structurally related compounds, tetracycline variants and sulfonated azo dyes. These hits had low micromolar to nanomolar IC50 values. Orthogonal screening confirmed the validity of these inhibitors and demonstrated that both act competitively on Sts-1 phosphatase activity. When tested on other PTPs, PTP1B and SHP1, it was found that the tetracycline PTP1B, SHP1, the tetracycline variant (doxycycline), and the sulfonated azo dye (Congo red) are selective inhibitors of Sts-1HP, with selectivity indices ranging from 19 to as high as 200. The planar polyaromatic moieties present in both classes of compounds suggested a common binding mode. The mutation of either tryptophan 494 or tyrosine 596, located near the active site of the protein, reduced the Ki of the inhibitors from 3- to 18-fold, indicating that these residues may help to promote the binding of substrates with aromatic groups. This work provides new insights into substrate selectivity mechanisms and describes two classes of compounds that can serve as probes of function or as a basis for future drug discovery.

Chemical validation and optimization of pharmacoperones targeting vasopressin type 2 receptor mutant.

A series of compounds formerly identified by high-throughput screening was studied for their ability to serve as pharmacoperones for the vasopressin type 2 receptor (V2R) mutant L83Q, which is known to cause nephrogenic diabetes insipidus (NDI). Three compounds were particularly effective in rerouting the mutant receptor in a concentration-dependent manner, were neither agonists nor antagonists, and displayed low cellular toxicity. Compound 1 was most effective and can be used as a molecular probe for future studies of how small molecules may affect NDI caused by mutant V2R. These compounds, however, failed to rescue the V2R Y128S mutant, indicating that the compounds described may not work in the rescue of all known mutants of V2R. Taken collectively, the present studies have now identified a promising lead compound that could function as a pharmacoperone to correct the trafficking defect of the NDI-associated mutant V2R L83Q and thus has the therapeutic potential for the treatment of NDI.

Whole-genome sequencing of Atacama skeleton shows novel mutations linked with dysplasia.

Over a decade ago, the Atacama humanoid skeleton (Ata) was discovered in the Atacama region of Chile. The Ata specimen carried a strange phenotype-6-in stature, fewer than expected ribs, elongated cranium, and accelerated bone age-leading to speculation that this was a preserved nonhuman primate, human fetus harboring genetic mutations, or even an extraterrestrial. We previously reported that it was human by DNA analysis with an estimated bone age of about 6-8 yr at the time of demise. To determine the possible genetic drivers of the observed morphology, DNA from the specimen was subjected to whole-genome sequencing using the Illumina HiSeq platform with an average 11.5× coverage of 101-bp, paired-end reads. In total, 3,356,569 single nucleotide variations (SNVs) were found as compared to the human reference genome, 518,365 insertions and deletions (indels), and 1047 structural variations (SVs) were detected. Here, we present the detailed whole-genome analysis showing that Ata is a female of human origin, likely of Chilean descent, and its genome harbors mutations in genes (COL1A1, COL2A1, KMT2D, FLNB, ATR, TRIP11, PCNT) previously linked with diseases of small stature, rib anomalies, cranial malformations, premature joint fusion, and osteochondrodysplasia (also known as skeletal dysplasia). Together, these findings provide a molecular characterization of Ata's peculiar phenotype, which likely results from multiple known and novel putative gene mutations affecting bone development and ossification.

A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel.

Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. In this study, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1,536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a cystic fibrosis bronchial epithelial (CFBE41o-) cell line, which was engineered to report CFTR-mediated intracellular flux of iodide by a halide-sensitive yellow fluorescence protein (YFP) reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z' value interval of 0.61 ± 0.05. As further evidence of high-throughput screen suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays, we identified 187 potential CFTR modulators, which EC50's < 5 µM. Thus, the assay has integrated the advantages of a phenotypic screen with high-throughput scalability to discover new small-molecule CFTR modulators.

Discovery and SAR of a novel series of potent, CNS penetrant M4 PAMs based on a non-enolizable ketone core: Challenges in disposition.

This Letter describes the chemical optimization of a novel series of M4 PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided analogs for which M4 PAM potency and CNS exposure were maintained; yet, clearance remained high. Metabolite identification studies demonstrated that this series was subject to rapid, and near quantitative, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M4 PAM activity.

Receptor antagonism/agonism can be uncoupled from pharmacoperone activity.

Pharmacoperones rescue misrouted mutants of the vasopressin receptor type 2 (V2R) and enable them to traffic to the correct biological locus where they function. Previously, a library of nearly 645,000 structures was interrogated with a high throughput screen; pharmacoperones were identified for V2R mutants with a view toward correcting the underlying mutational defects in nephrogenic diabetes insipidus. In the present study, an orthologous assay was used to evaluate hits from the earlier study. We found no consistent relation between antagonism or agonism and pharmacoperone activity. Active pharmacoperones were identified which had minimal antagonistic activity. This increases the therapeutic reach of these drugs, since virtually all pharmacoperone drugs reported to date were selected from peptidomimetic antagonists. Such mixed-activity drugs have a complex pharmacology limiting their therapeutic utility and requiring their removal prior to stimulation of the receptor with agonist.

Identification of Potential Pharmacoperones Capable of Rescuing the Functionality of Misfolded Vasopressin 2 Receptor Involved in Nephrogenic Diabetes Insipidus.

Pharmacoperones correct the folding of otherwise misfolded protein mutants, restoring function (i.e., providing "rescue") by correcting their trafficking. Currently, most pharmacoperones possess intrinsic antagonist activity because they were identified using methods initially aimed at discovering such functions. Here, we describe an ultra-high-throughput homogeneous cell-based assay with a cAMP detection system, a method specifically designed to identify pharmacoperones of the vasopressin type 2 receptor (V2R), a GPCR that, when mutated, is associated with nephrogenic diabetes insipidus. Previously developed methods to identify compounds capable of altering cellular trafficking of V2R were modified and used to screen a 645,000 compound collection by measuring the ability of library compounds to rescue a mutant hV2R [L83Q], using a cell-based luminescent detection system. The campaign initially identified 3734 positive modulators of cAMP. The confirmation and counterscreen identified only 147 of the active compounds with an EC50 of ≤5 µM. Of these, 83 were reconfirmed as active through independently obtained pure samples and were also inactive in a relevant counterscreen. Active and tractable compounds within this set can be categorized into three predominant structural clusters, described here, in the first report detailing the results of a large-scale pharmacoperone high-throughput screening campaign.

Discovery and optimization of a novel series of highly CNS penetrant M4 PAMs based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core.

This Letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma Kp=0.74), within the original core after several rounds of optimization; however, the thieno[2,3-d]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma Kp>10). Moreover, this campaign provided fundamentally distinct M4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target.

An Integrated Approach for Screening and Identification of Positive Allosteric Modulators of N-Methyl-D-Aspartate Receptors.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia.