Identification of Src as a Therapeutic Target in Oesophageal Adenocarcinoma through Functional Genomic and High-Throughput Drug Screening Approaches.

Drug resistance limits the effectiveness of oesophageal adenocarcinoma (OAC) chemotherapies, leading to a poor prognosis for this disease. Elucidation of the underlying resistance mechanisms is key to enabling the identification of more effective treatments. This study, therefore, aims to identify novel therapeutic and/or chemotherapy sensitising drug targets in OAC. Transcriptional data from a cohort of 273 pre-treatment OAC biopsies, from patients who received neoadjuvant chemotherapy followed by surgical resection, were analysed using gene set enrichment analysis (GSEA) to determine differential gene expression between responding and non-responding OAC tumours. From this, 80 genes were selected for high-throughput siRNA screening in OAC cell lines with or without standard chemotherapy treatment. In parallel, cell viability assays were performed using a panel of FDA-approved drugs and combination index (CI) values were calculated to evaluate drug synergy with standard chemotherapy. Mechanisms of synergy were investigated using western blot, propidium iodide flow cytometry, and proliferation assays. Taken together, the screens identified that targeting Src, using either siRNA or the small molecule inhibitor dasatinib, enhanced the efficacy of chemotherapy in OAC cells. Further in vitro functional analysis confirmed Src inhibition to be synergistic with standard OAC chemotherapies, 5-fluorouracil (5-FU), and cisplatin (CDDP). In conclusion, a compound screen together with a functional genomic approach identified Src as a potential chemosensitising target in OAC, which could be assessed in a clinical study for poor prognosis OAC patients.

Persister state-directed transitioning and vulnerability in melanoma.

Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination.

Mapping Chromone-3-Phenylcarboxamide Pharmacophore: Quid Est Veritas?

Chromone-3-phenylcarboxamides (Crom-1 and Crom-2) were identified as potent, selective, and reversible inhibitors of human monoamine oxidase B (hMAO-B). Since they exhibit some absorption, distribution, metabolism, and excretion (ADME)-toxicity liabilities, new derivatives were synthesized to map the chemical structural features that compose the pharmacophore, a process vital for lead optimization. Structure-activity relationship data, supported by molecular docking studies, provided a rationale for the contribution of the heterocycle's rigidity, the carbonyl group, and the benzopyran heteroatom for hMAO-B inhibitory activity. From the study, N-(3-chlorophenyl)-4H-thiochromone-3-carboxamide (31) (hMAO-B IC50 = 1.52 ± 0.15 nM) emerged as a reversible tight binding inhibitor with an improved pharmacological profile. In in vitro ADME-toxicity studies, compound 31 showed a safe cytotoxicity profile in Caco-2, SH-SY5Y, HUVEC, HEK-293, and MCF-7 cells, did not present cardiotoxic effects, and did not affect P-gp transport activity. Compound 31 also protected SH-SY5Y cells from iron(III)-induced damage. Collectively, these studies highlighted compound 31 as the first-in-class and a suitable candidate for in vivo preclinical investigation.

A SARS-CoV-2 cytopathicity dataset generated by high-content screening of a large drug repurposing collection.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic, in which acute respiratory infections are associated with high socio-economic burden. We applied high-content screening to a well-defined collection of 5632 compounds including 3488 that have undergone previous clinical investigations across 600 indications. The compounds were screened by microscopy for their ability to inhibit SARS-CoV-2 cytopathicity in the human epithelial colorectal adenocarcinoma cell line, Caco-2. The primary screen identified 258 hits that inhibited cytopathicity by more than 75%, most of which were not previously known to be active against SARS-CoV-2 in vitro. These compounds were tested in an eight-point dose response screen using the same image-based cytopathicity readout. For the 67 most active molecules, cytotoxicity data were generated to confirm activity against SARS-CoV-2. We verified the ability of known inhibitors camostat, nafamostat, lopinavir, mefloquine, papaverine and cetylpyridinium to reduce the cytopathic effects of SARS-CoV-2, providing confidence in the validity of the assay. The high-content screening data are suitable for reanalysis across numerous drug classes and indications and may yield additional insights into SARS-CoV-2 mechanisms and potential therapeutic strategies.

Nelfinavir and Its Active Metabolite M8 Are Partial Agonists and Competitive Antagonists of the Human Pregnane X Receptor.

The HIV protease inhibitor nelfinavir is currently being analyzed for repurposing as an anticancer drug for many different cancers because it exerts manifold off-target protein interactions, finally resulting in cancer cell death. Xenosensing pregnane X receptor (PXR), which also participates in the control of cancer cell proliferation and apoptosis, was previously shown to be activated by nelfinavir; however, the exact molecular mechanism is still unknown. The present study addresses the effects of nelfinavir and its major and pharmacologically active metabolite nelfinavir hydroxy-tert-butylamide (M8) on PXR to elucidate the underlying molecular mechanism. Molecular docking suggested direct binding to the PXR ligand-binding domain, which was confirmed experimentally by limited proteolytic digestion and competitive ligand-binding assays. Concentration-response analyses using cellular transactivation assays identified nelfinavir and M8 as partial agonists with EC50 values of 0.9 and 7.3 µM and competitive antagonists of rifampin-dependent induction with IC50 values of 7.5 and 25.3 µM, respectively. Antagonism exclusively resulted from binding into the PXR ligand-binding pocket. Impaired coactivator recruitment by nelfinavir as compared with the full agonist rifampin proved to be the underlying mechanism of both effects on PXR. Physiologic relevance of nelfinavir-dependent modulation of PXR activity was investigated in respectively treated primary human hepatocytes, which showed differential induction of PXR target genes and antagonism of rifampin-induced ABCB1 and CYP3A4 gene expression. In conclusion, we elucidate here the molecular mechanism of nelfinavir interaction with PXR. It is hypothesized that modulation of PXR activity may impact the anticancer effects of nelfinavir. SIGNIFICANCE STATEMENT: Nelfinavir, which is being investigated for repurposing as an anticancer medication, is shown here to directly bind to human pregnane X receptor (PXR) and thereby act as a partial agonist and competitive antagonist. Its major metabolite nelfinavir hydroxy-tert-butylamide exerts the same effects, which are based on impaired coactivator recruitment. Nelfinavir anticancer activity may involve modulation of PXR, which itself is discussed as a therapeutic target in cancer therapy and for the reversal of chemoresistance.

An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds.

The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting. Graphical abstract •The embryonic stem cell test to predict teratogenicity was made automation-compatible. •Several key improvements to the assay procedure have been introduced to increase performance. •The workflow was adapted to human iPS cells and isogenic fibroblast donor cells.

A tiered high-throughput screening approach for evaluation of estrogen and androgen receptor modulation by environmentally relevant bisphenol A substitutes.

Bisphenol A (BPA) is a high production volume chemical with a broad application spectrum. As an endocrine disrupting chemical, mainly by modulation of nuclear receptors (NRs), BPA has an adverse impact on organisms and is identified as a substance of very high concern under the European REACH regulation. Various BPA substitution candidates have been developed in recent years, however, information concerning the endocrine disrupting potential of these substances is still incomplete or missing. In this study, we intended to investigate the endocrine potential of BPA substitution candidates used in environmentally relevant applications such as thermal paper or epoxy resins. Based on an extensive literature and patent search, 33 environmentally relevant BPA substitution candidates were identified. In order to evaluate the endocrine potential of the BPA replacements, a screening cascade consisting of biochemical and cell-based assays was employed to investigate substance binding to the NRs estrogen receptor α and ß, as well as androgen receptor, co-activator recruitment and NR-mediated reporter gene activation. In addition, a computational docking approach for retrospective prediction of receptor binding was carried out. Our results show that some BPA substitution candidates, for which so far no or only very few data were available, possess a substantial endocrine disrupting potential (TDP, BPZ), while several substances (BPS, D-8, DD70, DMP-OH, TBSA, D4, CBDO, ISO, VITC, DPA, and DOPO) did not reveal any NR binding.

Mutations in PMPCB Encoding the Catalytic Subunit of the Mitochondrial Presequence Protease Cause Neurodegeneration in Early Childhood.

Mitochondrial disorders causing neurodegeneration in childhood are genetically heterogeneous, and the underlying genetic etiology remains unknown in many affected individuals. We identified biallelic variants in PMPCB in individuals of four families including one family with two affected siblings with neurodegeneration and cerebellar atrophy. PMPCB encodes the catalytic subunit of the essential mitochondrial processing protease (MPP), which is required for maturation of the majority of mitochondrial precursor proteins. Mitochondria isolated from two fibroblast cell lines and induced pluripotent stem cells derived from one affected individual and differentiated neuroepithelial stem cells showed reduced PMPCB levels and accumulation of the processing intermediate of frataxin, a sensitive substrate for MPP dysfunction. Introduction of the identified PMPCB variants into the homologous S. cerevisiae Mas1 protein resulted in a severe growth and MPP processing defect leading to the accumulation of mitochondrial precursor proteins and early impairment of the biogenesis of iron-sulfur clusters, which are indispensable for a broad range of crucial cellular functions. Analysis of biopsy materials of an affected individual revealed changes and decreased activity in iron-sulfur cluster-containing respiratory chain complexes and dysfunction of mitochondrial and cytosolic Fe-S cluster-dependent enzymes. We conclude that biallelic mutations in PMPCB cause defects in MPP proteolytic activity leading to dysregulation of iron-sulfur cluster biogenesis and triggering a complex neurological phenotype of neurodegeneration in early childhood.

Identification of approved drugs as potent inhibitors of pregnane X receptor activation with differential receptor interaction profiles.

Activation of pregnane X receptor (PXR) results in the induction of first-pass metabolism and drug efflux. Hereby, PXR may cause adverse drug reactions or therapeutic failure of drugs. PXR inhibition is thus an attractive option to minimise adverse effects or to improve therapeutic efficiencies; however, only a limited number of antagonists have been identified so far. We performed a cell-based high-throughput screen to identify PXR antagonists, using a library of approved and investigational drugs. Two approved drugs, pimecrolimus and pazopanib, emerged as novel potent antagonists of PXR activation, with IC50 values of 1.2 and 4.1 µM, respectively. We further characterised these with respect to receptor specificity, assembly of the PXR ligand-binding domain (LBD) and interactions with co-factors. In vitro and in silico assays were carried out to identify the site(s) of interaction with the PXR LBD. Primary human hepatocytes were used to investigate antagonism of the induction of endogenous PXR target genes. Pimecrolimus and pazopanib did not affect the transcriptional activity of other nuclear receptors. Both induced the release of co-repressor from PXR and likewise interfered with agonist-induced recruitment of co-activator. Cumulative evidence from cellular and in vitro assays, as well as molecular docking, suggested additional or exclusive binding outside the PXR ligand-binding pocket for both. The compounds differentially antagonised the induction of PXR-regulated genes by rifampicin in primary human hepatocytes. In conclusion, we here have identified two approved drugs as novel potent PXR inhibitors with differential receptor interaction profiles and gene selectivity in primary human hepatocytes.

Effects of exercise on Irisin, BDNF and IL-6 serum levels in patients with progressive multiple sclerosis.

BACKGROUND: Clinical studies have suggested beneficial effects of exercise on cognitive function in ageing adults and neurodegenerative diseases such as dementia. Recent work indicates the same for progressive multiple sclerosis (MS), an inflammatory and degenerative disease of the central nervous system (CNS). The biological pathways associated with these effects are however not well understood. OBJECTIVE: In this randomized controlled study, we explored serum levels of the myokine Irisin, the neurotrophin brain-derived neurotrophic factor (BDNF) and Interleukin-6 (IL-6) during acute endurance exercise and over the course of a 9-weeks endurance exercise training period in n=42 patients with progressive MS. RESULTS: We detected a significant increase of BDNF levels in progressive MS patients after 30min of bicycling (p<0.001). However, there were no significant changes for baseline levels after 22 sessions of training. No significant effects of acute or prolonged exercise could be found for Irisin or Interleukin-6. CONCLUSION: Our results indicate that BDNF is strongly induced during acute exercise even in patients with progressive MS and advanced physical disability. Long-term effects of exercise programs on biological parameters (Irisin, BDNF, IL-6) were much less pronounced. Given the hypothesis-driven selection of a limited set of biological markers in this pilot study, future studies should use unbiased approaches in larger samples to obtain a comprehensive picture of the networks involved in exercise effects on neurological diseases.

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs.

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 µM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 µM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

Novel MK2 inhibitors by fragment screening.

Inhibitors of MAPKAP kinase 2 (MK2) are expected to attenuate the p38alpha signal transduction pathway in macrophages in a similar way to p38alpha inhibitors and to have a lower propensity for toxic side effects that have slowed the clinical development of the latter. Therefore, novel MK2 inhibitors may find therapeutic application in acute and chronic, TNFalpha-mediated inflammatory conditions like rheumatoid arthritis and others. Herein we have applied fragment screening, using physiologically relevant bioassays and fragment binding mode mapping by protein-observed NMR spectroscopy to the discovery of novel efficient chemical starting points for MK2.