During the COVID-19 pandemic, drug repurposing represented an effective strategy to obtain quick answers to medical emergencies. Based on previous data on methotrexate (MTX), we evaluated the ...anti-viral activity of several DHFR inhibitors in two cell lines. We observed that this class of compounds showed a significant influence on the virus-induced cytopathic effect (CPE) partly attributed to the intrinsic anti-metabolic activity of these drugs, but also to a specific anti-viral function. To elucidate the molecular mechanisms, we took advantage of our EXSCALATE platform for in-silico molecular modelling and further validated the influence of these inhibitors on nsp13 and viral entry. Interestingly, pralatrexate and trimetrexate showed superior effects in counteracting the viral infection compared to other DHFR inhibitors. Our results indicate that their higher activity is due to their polypharmacological and pleiotropic profile. These compounds can thus potentially give a clinical advantage in the management of SARS-CoV-2 infection in patients already treated with this class of drugs.
Abstract
Worldwide, there are intensive efforts to identify repurposed drugs as potential therapies against SARS-CoV-2 infection and the associated COVID-19 disease. To date, the anti-inflammatory ...drug dexamethasone and (to a lesser extent) the RNA-polymerase inhibitor remdesivir have been shown to be effective in reducing mortality and patient time to recovery, respectively, in patients. Here, we report the results of a phenotypic screening campaign within an EU-funded project (H2020-EXSCALATE4COV) aimed at extending the repertoire of anti-COVID therapeutics through repurposing of available compounds and highlighting compounds with new mechanisms of action against viral infection. We screened 8702 molecules from different repurposing libraries, to reveal 110 compounds with an anti-cytopathic IC
50
< 20 µM. From this group, 18 with a safety index greater than 2 are also marketed drugs, making them suitable for further study as potential therapies against COVID-19. Our result supports the idea that a systematic approach to repurposing is a valid strategy to accelerate the necessary drug discovery process.
Abstract Objectives Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can generate any given cell type in the human body. One challenge for cell-replacement therapy ...is the efficient differentiation and expansion of large quantities of progenitor cells from pluripotent stem cells produced under good manufacturing practice (GMP). FOXA2 and SOX17 double positive definitive endoderm (DE) progenitor cells can give rise to all endoderm-derived cell types in the thymus, thyroid, lung, pancreas, liver, and gastrointestinal tract. FOXA2 is a pioneer transcription factor in DE differentiation that is also expressed and functionally required during pancreas development and islet cell homeostasis. Current differentiation protocols can successfully generate endoderm; however, generation of mature glucose-sensitive and insulin-secreting β-cells is still a challenge. As a result, it is of utmost importance to screen for small molecules that can improve DE and islet cell differentiation for cell-replacement therapy for diabetic patients. Methods The aim of this study was to identify and validate small molecules that can induce DE differentiation and further enhance pancreatic progenitor differentiation. Therefore, we developed a large scale, high-content screen for testing a chemical library of 23,406 small molecules to identify compounds that induce FoxA2 in mouse embryonic stem cells (mESCs). Results Based on our high-content screen algorithm, we selected 84 compounds that directed differentiation of mESCs towards the FoxA2 lineage. Strikingly, we identified ROCK inhibition (ROCKi) as a novel mechanism of endoderm induction in mESCs and hESCs. DE induced by the ROCK inhibitor Fasudil efficiently gives rise to PDX1+ pancreatic progenitors from hESCs. Conclusion Taken together, DE induction by ROCKi can simplify and improve current endoderm and pancreatic differentiation protocols towards a GMP-grade cell product for β-cell replacement.
The drug discovery community faces high costs in bringing safe and effective medicines to market, in part due to the rising volume and complexity of data which must be generated during the research ...and development process. Fully utilising these expensively created experimental and computational data resources has become a key aim of scientists due to the clear imperative to leverage the power of artificial intelligence (AI) and machine learning-based analyses to solve the complex problems inherent in drug discovery. In turn, AI methods heavily rely on the quantity, quality, consistency, and scope of underlying training data. While pre-existing preclinical and clinical data cannot fully replace the need for
de novo
data generation in a project, having access to relevant historical data represents a valuable asset, as its reuse can reduce the need to perform similar experiments, therefore avoiding a “reinventing the wheel” scenario. Unfortunately, most suitable data resources are often archived within institutes, companies, or individual research groups and hence unavailable to the wider community. Hence, enabling the data to be Findable, Accessible, Interoperable, and Reusable (FAIR) is crucial for the wider community of drug discovery and development scientists to learn from the work performed and utilise the findings to enhance comprehension of their own research outcomes. In this mini-review, we elucidate the utility of FAIR data management across the drug discovery pipeline and assess the impact such FAIR data has made on the drug development process.
The pharmacodynamics of drug-candidates is often optimized by metrics that describe target binding (K
or K
value) or target modulation (IC
). However, these metrics are determined at equilibrium ...conditions, and consequently information regarding the onset and offset of target engagement and modulation is lost. Drug-target residence time is a measure for the lifetime of the drug-target complex, which has recently been receiving considerable interest, as target residence time is shown to have prognostic value for the
efficacy of several drugs. In this study, we have investigated the relation between the increased residence time of antihistamines at the histamine H
receptor (H
R) and the duration of effective target-inhibition by these antagonists. Hela cells, endogenously expressing low levels of the H
R, were incubated with a series of antihistamines and dissociation was initiated by washing away the unbound antihistamines. Using a calcium-sensitive fluorescent dye and a label free, dynamic mass redistribution based assay, functional recovery of the H
R responsiveness was measured by stimulating the cells with histamine over time, and the recovery was quantified as the
. Using these assays, we determined that the receptor recovery time for a set of antihistamines differed more than 40-fold and was highly correlated to their H
R residence times, as determined with competitive radioligand binding experiments to the H
R in a cell homogenate. Thus, the receptor recovery time is proposed as a cell-based and physiologically relevant metric for the lead optimization of G protein-coupled receptor antagonists, like the H
R antagonists. Both, label-free or real-time, classical signaling assays allow an efficient and physiologically relevant determination of kinetic properties of drug molecules.
Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that possesses an epoxide hydrolase and lipid phosphatase activity (sEH-P) at two distinct catalytic domains. While the physiological role of ...the epoxide hydrolase domain is well understood, the consequences of the phosphatase activity remain unclear. Herein we describe the bacterial expression of the recombinant N-terminal domain of sEH-P and the development of a high-throughput screening protocol using a sensitive and commercially available substrate fluorescein diphosphate. The usability of the assay system was demonstrated and novel inhibitors of sEH-P were identified.
The COVID-19 pandemic has highlighted the need for FAIR (Findable, Accessible, Interoperable, and Reusable) data more than any other scientific challenge to date. We developed a flexible, ...multi-level, domain-agnostic FAIRification framework, providing practical guidance to improve the FAIRness for both existing and future clinical and molecular datasets. We validated the framework in collaboration with several major public-private partnership projects, demonstrating and delivering improvements across all aspects of FAIR and across a variety of datasets and their contexts. We therefore managed to establish the reproducibility and far-reaching applicability of our approach to FAIRification tasks.
The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link ...module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA8 and HA10 oligosaccharides, respectively. Equilibrium experiments showed that the binding affinity of Link_TSG6 to HA was maximal at pH 6.0, and reduced dramatically above and below this pH. In contrast, G1 had maximum binding at pH 7.0–8.0 and moderate to strong binding affinity over a much broader pH range (5.5–8.0). The KD determined for Link_TSG6 binding to HA showed a 100-fold increase in binding affinity between pH 7.4 and 6.0, whereas G1 showed a 75-fold decrease in binding affinity over the same pH range. The sharp difference observed in their pH binding suggests that pH controls the physiological function of TSG-6, with a low affinity for HA at neutral pH, but with increased affinity as the pH falls below pH 7. TSG-6 and aggrecan interact with HA through structurally homologous domains and the difference in pH-dependent binding can be understood in terms of differences in the presence and topographical distribution of key regulatory amino acids in Link_TSG6 and in the related tandem Link domains in aggrecan G1.
Around 20% of the American population have chronic pain and estimates in other Western countries report similar numbers. This represents a major challenge for global health care systems. Additional ...problems for the treatment of chronic and persistent pain are the comparably low efficacy of existing therapies, the failure to translate effects observed in preclinical pain models to human patients and related setbacks in clinical trials from previous attempts to develop novel analgesics. Drug repurposing offers an alternative approach to identify novel analgesics as it can bypass various steps of classical drug development. In recent years, several approved drugs were attributed analgesic properties. Here, we review available data and discuss recent findings suggesting that the approved drugs minocycline, fingolimod, pioglitazone, nilotinib, telmisartan, and others, which were originally developed for the treatment of different pathologies, can have analgesic, antihyperalgesic, or neuroprotective effects in preclinical and clinical models of inflammatory or neuropathic pain. For our analysis, we subdivide the drugs into substances that can target neuroinflammation or substances that can act on peripheral sensory neurons, and highlight the proposed mechanisms. Finally, we discuss the merits and challenges of drug repurposing for the development of novel analgesics.
Blood-pressure-lowering drugs are proposed to foster SARS-CoV-2 infection by pharmacological upregulation of angiotensin-converting enzyme 2 (ACE2), the binding partner of the virus spike (S) ...protein, located on the surface of the host cells. Conversely, it is postulated that angiotensin–renin system antagonists may prevent lung damage caused by SARS-CoV-2 infection, by reducing angiotensin II levels, which can induce permeability of lung endothelial barrier via its interaction with the AT1 receptor (AT1R). Methods: We have investigated the influence of the ACE inhibitors (lisinopril, captopril) and the AT1 antagonists (telmisartan, olmesartan) on the level of ACE2 mRNA and protein expression as well as their influence on the cytopathic effect of SARS-CoV-2 and on the cell barrier integrity in a Caco-2 cell model. Results: The drugs revealed no effect on ACE2 mRNA and protein expression. ACE inhibitors and AT1R antagonist olmesartan did not influence the infection rate of SARS-CoV-2 and were unable to prevent the SARS-CoV-2-induced cell barrier disturbance. A concentration of 25 µg/mL telmisartan significantly reduced the virus replication rate. Conclusion: ACE inhibitors and AT1R antagonist showed neither beneficial nor detrimental effects on SARS-CoV-2-infection and cell barrier integrity in vitro at pharmacologically relevant concentrations.
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