Neutral ceramidase-active site inhibitor chemotypes and binding modes.

Ceramides impact a diverse array of biological functions and have been implicated in disease pathogenesis. The enzyme neutral ceramidase (nCDase) is a zinc-containing hydrolase and mediates the metabolism of ceramide to sphingosine (Sph), both in cells and in the intestinal lumen. nCDase inhibitors based on substrate mimetics, for example C6-urea ceramide, have limited potency, aqueous solubility, and micelle-free fraction. To identify non-ceramide mimetic nCDase inhibitors, hit compounds from an HTS campaign were evaluated in biochemical, cell based and in silico modeling approaches. A majority of small molecule nCDase inhibitors contained pharmacophores capable of zinc interaction but retained specificity for nCDase over zinc-containing acid and alkaline ceramidases, as well as matrix metalloprotease-3 and histone deacetylase-1. nCDase inhibitors were refined by SAR, were shown to be substrate competitive and were active in cellular assays. nCDase inhibitor compounds were modeled by in silico DOCK screening and by molecular simulation. Modeling data supports zinc interaction and a similar compound binding pose with ceramide. nCDase inhibitors were identified with notably improved activity and solubility in comparison with the reference lipid-mimetic C6-urea ceramide.

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.

Stabilization of amyloidogenic immunoglobulin light chains by small molecules.

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration.

SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons.

SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.

Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2.

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. METHODS: We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. RESULTS: All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. CONCLUSION: We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

Synthetic fluorescent MYC probe: Inhibitor binding site elucidation and development of a high-throughput screening assay.

We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.

A survey of core replacements in indole-based HIV-1 attachment inhibitors.

Indole- and azaindole-based glyoxylyl amide derivatives have been described as HIV-1 attachment inhibitors (AIs) that act by blocking the interaction between the viral gp120 coat protein and the human host cell CD4 receptor. As part of an effort to more deeply understand the role of the indole/azaindole heterocycle in the expression of antiviral activity, a survey of potential replacements was conducted using parallel synthesis methodology. The design and optimization was guided by a simple 2-dimensional overlay based on an overall planar topography between the indole/azaindole and C-7 substituents that had been deduced from structure-activity studies leading to the discovery of temsavir (3). 2-Substituted naphthalene- and quinoline-derived chemotypes emerged as the most interesting prototypes, with C-5 and C-6 substituents enhancing antiviral potency. Despite the fact that neither of these chemotypes incorporated a H-bond donor that has been shown to engage the side chain carboxylate of Asp113 in gp120, the antiviral potency of several analogues met or exceeded that of 3, demonstrating that engaging Asp113 is not a prerequisite for potent antiviral activity.

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.

Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and ß.

Zinc metalloproteinases meprin α and meprin ß are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools, we have developed high throughput screening assays to enable discovery of inhibitors of both meprin α and meprin ß and screened a collection of well characterized pharmaceutical agents (library of pharmaceutically active compounds, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin ß. Kinetic studies revealed competitive (PPNDS) and mixed competitive/noncompetitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin ß active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin ß reported to the date. These results demonstrate the ability of meprin α and ß assays to identify selective compounds and discard artifacts of primary screening.

Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors.

National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-ß-lactams (ABLs), as potent (IC(50) values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.

A high-throughput cell-based screening method for Zika virus protease inhibitor discovery.

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

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.

Protocol for high throughput 3D drug screening of patient derived melanoma and renal cell carcinoma.

High Throughput Screening (HTS) with 3D cell models is possible thanks to the recent progress and development in 3D cell culture technologies. Results from multiple studies have demonstrated different drug responses between 2D and 3D cell culture. It is now widely accepted that 3D cell models more accurately represent the physiologic conditions of tumors over 2D cell models. However, there is still a need for more accurate tests that are scalable and better imitate the complex conditions in living tissues. Here, we describe ultrahigh throughput 3D methods of drug response profiling in patient derived primary tumors including melanoma as well as renal cell carcinoma that were tested against the NCI oncologic set of FDA approved drugs. We also tested their autologous patient derived cancer associated fibroblasts, varied the in-vitro conditions using matrix vs matrix free methods and completed this in both 3D vs 2D rendered cancer cells. The result indicates a heterologous response to the drugs based on their genetic background, but not on their maintenance condition. Here, we present the methods and supporting results of the HTS efforts using these 3D of organoids derived from patients. This demonstrated the possibility of using patient derived 3D cells for HTS and expands on our screening capabilities for testing other types of cancer using clinically approved anti-cancer agents to find drugs for potential off label use.

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.

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.

Discovery, design and synthesis of a selective S1P(3) receptor allosteric agonist.

Potent and selective S1P3 receptor (S1P3-R) agonists may represent important proof-of-principle tools used to clarify the receptor biological function and assess the therapeutic potential of the S1P3-R in cardiovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide was identified by a high-throughput screening of MLSMR library as a promising S1P3-R agonist. Rational chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide, a S1P3-R agonist endowed with submicromolar activity and exquisite selectivity over the remaining S1P1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide is an allosteric agonist and binds to the S1P3-R in a manner that does not disrupt the S1P3-R-S1P binding. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the molecular basis of the receptor function, and provides the bases for further rational design of more potent and drug-like S1P3-R allosteric agonists.

Inhibitors of HIV-1 attachment. Part 8: the effect of C7-heteroaryl substitution on the potency, and in vitro and in vivo profiles of indole-based inhibitors.

As part of the SAR profiling of the indole-oxoacetic piperazinyl benzamide class of HIV-1 attachment inhibitors, substitution at the C7 position of the lead 4-fluoroindole 2 with various 5- and 6-membered heteroaryl moieties was explored. Highly potent (picomolar) inhibitors of pseudotyped HIV-1 in a primary, cell-based assay were identified and select examples were shown to possess nanomolar inhibitory activity against M- and T-tropic viruses in cell culture. These C7-heteroaryl-indole analogs maintained the ligand efficiency (LE) of 2 and were also lipophilic efficient as measured by LLE and LELP. Pharmacokinetic studies of this class of inhibitor in rats showed that several possessed substantially improved IV clearance and half-lives compared to 2. Oral exposure in the rat correlated with membrane permeability as measured in a Caco-2 assay where the highly permeable 1,2,4-oxadiazole analog 13 exhibited the highest exposure.

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.

Protocol for 3D screening of lung cancer spheroids using natural products.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for ∼84% of all lung cancer cases. NSCLC remains one of the leading causes of cancer-associated death, with a 5-year survival rate less than 25%. This type of cancer begins with healthy cells that change and start growing out of control, leading to the formation of lesions or tumors. Understanding the dynamics of how the tumor microenvironment promotes cancer initiation and progression that leads to cancer metastasis is crucial to help identify new molecular therapies. 3D primary cell tumor models have received renewed recognition due to their ability to better mimic the complexity of in vivo tumors and as a potential bridge between traditional 2D culture and in vivo studies. Vast improvements in 3D cell culture technologies make them much more cost effective and efficient largely because of the use of a cell-repellent surfaces and a novel angle plate adaptor technology. To exploit this technology, we accessed the Natural Products Library (NPL) at UF Scripps, which consists of crude extracts, partially purified fractions, and pure natural products (NPs). NPs generally are not very well represented in most drug discovery libraries and thus provide new insights to discover leads that could potentially emerge as novel molecular therapies. Herein we describe how we combined these technologies for 3D screening in 1536 well format using a panel of ten NSCLC cells lines (5 wild type and 5 mutant) against ∼1280 selected members of the NPL. After further evaluation, the selected active hits were prioritized to be screened against all 10 NSCLC cell lines as concentration response curves to determine the efficacy and selectivity of the compounds between wild type and mutant 3D cell models. Here, we demonstrate the methods needed for automated 3D screening using microbial NPs, exemplified by crude extracts, partially purified fractions, and pure NPs, that may lead to future use targeting human cancer.

A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer.

Pancreatic cancer is one of the deadliest diseases in human malignancies. Among total pancreatic cancer patients, ~10% of patients are categorized as familial pancreatic cancer (FPC) patients, carrying germline mutations of the genes involved in DNA repair pathways (e.g., BRCA2). Personalized medicine approaches tailored toward patients' mutations would improve patients' outcome. To identify novel vulnerabilities of BRCA2-deficient pancreatic cancer, we generated isogenic Brca2-deficient murine pancreatic cancer cell lines and performed high-throughput drug screens. High-throughput drug screening revealed that Brca2-deficient cells are sensitive to Bromodomain and Extraterminal Motif (BET) inhibitors, suggesting that BET inhibition might be a potential therapeutic approach. We found that BRCA2 deficiency increased autophagic flux, which was further enhanced by BET inhibition in Brca2-deficient pancreatic cancer cells, resulting in autophagy-dependent cell death. Our data suggests that BET inhibition can be a novel therapeutic strategy for BRCA2-deficient pancreatic cancer.