High-Throughput Methods for the Discovery of Small Molecule Modulators of Pancreatic Beta-Cell Function and Regeneration.

The progression of type II diabetes (T2D) is characterized by a complex and highly variable loss of beta-cell mass, resulting in impaired insulin secretion. Many T2D drug discovery efforts aimed at discovering molecules that can protect or restore beta-cell mass and function have been developed using limited beta-cell lines and primary rodent/human pancreatic islets. Various high-throughput screening methods have been used in the context of drug discovery, including luciferase-based reporter assays, glucose-stimulated insulin secretion, and high-content screening. In this context, a cornerstone of small molecule discovery has been the use of immortalized rodent beta-cell lines. Although insightful, this usage has led to a more comprehensive understanding of rodent beta-cell proliferation pathways rather than their human counterparts. Advantages gained in enhanced physiological relevance are offered by three-dimensional (3D) primary islets and pseudoislets in contrast to monolayer cultures, but these approaches have been limited to use in low-throughput experiments. Emerging methods, such as high-throughput 3D islet imaging coupled with machine learning, aim to increase the feasibility of integrating 3D microtissue structures into high-throughput screening. This review explores the current methods used in high-throughput screening for small molecule modulators of beta-cell mass and function, a potentially pivotal strategy for diabetes drug discovery.

Automated high-throughput, high-content 3D imaging of intact pancreatic islets.

Diabetes poses a global health crisis affecting individuals across age groups and backgrounds, with a prevalence estimate of 700 million people worldwide by 2045. Current therapeutic strategies primarily rely on insulin therapy or hypoglycemic agents, which fail to address the root cause of the disease - the loss of pancreatic insulin-producing beta-cells. Therefore, bioassays that recapitulate intact islets are needed to enable drug discovery for beta-cell replenishment, protection from beta-cell loss, and islet-cell interactions. Standard cancer insulinoma beta-cell lines MIN6 and INS-1 have been used to interrogate beta-cell metabolic pathways and function but are not suitable for studying proliferative effects. Screening using primary human/rodent intact islets offers a higher level of physiological relevance to enhance diabetes drug discovery and development. However, the 3-dimensionality of intact islets have presented challenges in developing robust, high-throughput assays to detect beta-cell proliferative effects. Established methods rely on either dissociated islet cells plated in 2D monolayer cultures for imaging or reconstituted pseudo-islets formed in round bottom plates to achieve homogeneity. These approaches have significant limitations due to the islet cell dispersion process. To address these limitations, we have developed a robust, intact ex vivo pancreatic islet bioassay in 384-well format that is capable of detecting diabetes-relevant endpoints including beta-cell proliferation, chemoprotection, and islet spatial morphometrics.

Knockout of murine Lyplal1 confers sex-specific protection against diet-induced obesity.

Human genome-wide association studies found single-nucleotide polymorphisms (SNPs) near LYPLAL1 (Lysophospholipase-like protein 1) that have sex-specific effects on fat distribution and metabolic traits. To determine whether altering LYPLAL1 affects obesity and metabolic disease, we created and characterized a mouse knockout (KO) of Lyplal1. We fed the experimental group of mice a high-fat, high-sucrose (HFHS) diet for 23 weeks, and the controls were fed regular chow diet. Here, we show that CRISPR-Cas9 whole-body Lyplal1 KO mice fed an HFHS diet showed sex-specific differences in weight gain and fat accumulation as compared to chow diet. Female, not male, KO mice weighed less than WT mice, had reduced body fat percentage, had white fat mass, and had adipocyte diameter not accounted for by changes in the metabolic rate. Female, but not male, KO mice had increased serum triglycerides, decreased aspartate, and decreased alanine aminotransferase. Lyplal1 KO mice of both sexes have reduced liver triglycerides and steatosis. These diet-specific effects resemble the effects of SNPs near LYPLAL1 in humans, suggesting that LYPLAL1 has an evolutionary conserved sex-specific effect on adiposity. This murine model can be used to study this novel gene-by-sex-by-diet interaction to elucidate the metabolic effects of LYPLAL1 on human obesity.

In Vitro Evaluation and Mitigation of Niclosamide's Liabilities as a COVID-19 Treatment.

Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including the B.1.1.7 (alpha) variant. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future.

In Vitro Evaluation and Mitigation of Niclosamide's Liabilities as a COVID-19 Treatment.

Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro , generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including B.1.1.7. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future. Importance: There is still an urgent need for effective anti-SARS-CoV-2 therapeutics due to waning vaccine efficacy, the emergence of variants of concern, and limited efficacy of existing antivirals. One potential therapeutic option is niclosamide, an FDA approved anthelmintic compound that has shown promising anti-SARS-CoV-2 activity in cell-based assays. Unfortunately, there are significant barriers for the clinical utility of niclosamide as a COVID-19 therapeutic. Our work emphasizes these limitations by showing that niclosamide has high cytotoxicity at antiviral concentrations, variable potency against variants of concern, and significant polypharmacology as a result of its activity as a nonspecific protonophore. Some of these clinical limitations can be mitigated, however, through structural modifications to the niclosamide scaffold, which we demonstrate through a preliminary structure activity relationship analysis. Overall, we show that niclosamide is not a suitable candidate for the treatment of COVID-19, but that structural analogs with improved drug properties may have higher clinical-translational potential.

Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

Morphological Cell Profiling of SARS-CoV-2 Infection Identifies Drug Repurposing Candidates for COVID-19.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10-15 years from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 FDA-approved compounds and clinical candidates, we identified 17 dose-responsive compounds with in vitro antiviral efficacy in human liver Huh7 cells and confirmed antiviral efficacy in human colon carcinoma Caco-2, human prostate adenocarcinoma LNCaP, and in a physiologic relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein classically found in secretory fluids, including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.