Strengthening an Intramolecular Non-Classical Hydrogen Bond to Get in Shape for Binding.

In this research article, we report on the strengthening of a non-classical hydrogen bond (C-H⋅⋅⋅O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E-selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre-organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre-organization, the stabilization by a non-classical hydrogen bond between the H-C5 of l-fucose and the ring oxygen O5 of the neighboring d-galactose moiety is essential and yields 7.4 kJ mol-1. This effect could be further strengthened by replacing l-fucose by 6,6,6-trifluoro-l-fucose leading to an improved non-classical H-bond of 14.9 kJ mol-1, i.e., an improved pre-organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR-shifts of the H-C5Fuc, by X-ray diffraction analysis of glycomimetics co-crystallized with E-selectin as well as by isothermal titration calorimetry. Furthermore, the electron-withdrawing character of the CF3-group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid-stability, a prerequisite for gastrointestinal stability, could be substantially improved.

Analogues of the pan-selectin antagonist rivipansel (GMI-1070).

The selectin family consisting of E-, P- and L-selectin plays dominant roles in atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, and metastatic spreading of some cancers. An early goal in selectin-targeted drug discovery campaigns was to identify ligands binding to all three selectins, so-called pan-selectin antagonists. The physiological epitope, tetrasaccharide sialyl Lewisx (sLex, 1) binds to all selectins, albeit with very different affinities. Whereas P- and L-selectin require additional interactions contributed by sulfate groups for high binding affinity, E-selectin can functionally bind sLex-modified glycolipids and glycoproteins. Rivipansel (3) marked the first pan-selectin antagonist, which simultaneously interacted with both the sLex and the sulfate binding site. The aim of this contribution was to improve the pan-selectin affinity of rivipansel (3) by leveraging a new class of sLex mimetics in combination with an optimized linker length to the sulfate bearing group. As a result, the pan-selectin antagonist 11b exhibits an approximatively 5-fold improved affinity for E-, as well as P-selectin.

In silico and in vitro assessment of drugs potentially causing adverse effects by inhibiting CYP17A1.

Cytochrome P450 enzymes (CYPs) play a crucial role in the metabolism and synthesis of various compound classes. While drug-metabolizing CYP enzymes are frequently investigated as anti-targets, the inhibition of CYP enzymes involved in adrenal steroidogenesis is not well studied. The steroidogenic enzyme CYP17A1 is a dual-function enzyme catalyzing hydroxylase and lyase reactions relevant for the biosynthesis of adrenal glucocorticoids and androgens. Inhibition of CYP17A1-hydroxylase leads to pseudohyperaldosteronism with subsequent excessive mineralocorticoid receptor activation, hypertension and hypokalemia. In contrast, specific inhibition of the lyase function might be beneficial for the treatment of prostate cancer by decreasing adrenal androgen levels. This study combined in silico and in vitro methods to identify drugs inhibiting CYP17A1. The most potent CYP17A1 inhibitors identified are serdemetan, mocetinostat, nolatrexed, liarozole, and talarozole. While some of these drugs are currently under investigation for the treatment of various cancers, their potential for the treatment of prostate cancer is yet to be explored. The DrugBank database was screened for CYP17A1 inhibitors, to increase the awareness for the risk of drug-induced pseudohyperaldosteronism and to highlight drugs so far unknown for their potential to cause side effects resulting from CYP17A1 inhibition.

A battery of in silico models application for pesticides exerting reproductive health effects: Assessment of performance and prioritization of mechanistic studies.

Given the high attention to endocrine disrupting chemicals (EDC), there is an urgent need for the development of rapid and reliable approaches for the screening of large numbers of chemicals with respect to their endocrine disruption potential. This study aimed at the assessment of the correlation between the predicted results of a battery of in silico tools and the reported observed adverse effects from in vivo reproductive toxicity studies. We used VirtualToxLab (VTL) software and the EndocrineDisruptome (ED) online tool to evaluate the binding affinities to nuclear receptors of 17 pesticides, 7 of which were classified as reprotoxic substances under Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP). Then, we aligned the results of the in silico modelling with data from ToxCast assays and in vivo reproductive toxicity studies. We combined results from different in silico tools in two different ways to improve the characteristics of their predictive performance. Reproductive toxicity can be caused by various mechanisms; however, in this study, we demonstrated that the use of a battery of in silico tools for assessing the binding to nuclear receptors can be useful for identifying hazardous compounds and for prioritizing further studies.

St. John's Wort Formulations Induce Rat CYP3A23-3A1 Independent of Their Hyperforin Content.

The pregnane X receptor (PXR) is a ligand-activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John's wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant was used as it contains a high hyperforin content and Rebalance because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant and Rebalance transactivated the CYP3A reporter 3.8-fold and 2.8-fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared with control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant, we observed 1.8 times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount, and a 1.6-fold increase in activity compared with controls. For Rebalance we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared with control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR. SIGNIFICANCE STATEMENT: Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the pregnane X receptor-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro. Importantly, CYP3A induction mimics findings in humans, but our results suggest that another so far unknown constituent of SJW is responsible for the expression- and function-modifying effects in rat liver.

The action of physiological and synthetic steroids on the calcium channel CatSper in human sperm.

The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca2+ concentration ([Ca2+]i) and plays an essential role in sperm function. It is mainly activated by the steroid progesterone (P4) but is also promiscuously activated by a wide range of synthetic and physiological compounds. These compounds include diverse steroids whose action on the channel is so far still controversial. To investigate the effect of these compounds on CatSper and sperm function, we developed a high-throughput screening (HTS) assay to measure changes in [Ca2+]i in human sperm and screened 1,280 approved and off-patent drugs including 90 steroids from the Prestwick chemical library. More than half of the steroids tested (53%) induced an increase in [Ca2+]i and reduced the P4-induced Ca2+ influx in human sperm in a dose-dependent manner. Ten of the most potent steroids (activating and P4-inhibiting) were selected for a detailed analysis of their action on CatSper and their ability to act on sperm acrosome reaction (AR) and penetration in viscous media. We found that these steroids show an inhibitory effect on P4 but not on prostaglandin E1-induced CatSper activation, suggesting that they compete for the same binding site as P4. Pregnenolone, dydrogesterone, epiandrosterone, nandrolone, and dehydroepiandrosterone acetate (DHEA) were found to activate CatSper at physiologically relevant concentrations within the nanomolar range. Like P4, most tested steroids did not significantly affect the AR while stanozolol and estropipate slightly increased sperm penetration into viscous medium. Furthermore, using a hybrid approach integrating pharmacophore analysis and statistical modelling, we were able to screen in silico for steroids that can activate the channel and define the physicochemical and structural properties required for a steroid to exhibit agonist activity against CatSper. Overall, our results indicate that not only physiological but also synthetic steroids can modulate the activity of CatSper with varying potency and if bound to CatSper prior to P4, could impair the timely CatSper activation necessary for proper fertilization to occur.

Assessment of the inhibitory potential of anabolic steroids towards human AKR1D1 by computational methods and in vitro evaluation.

Exposure to xenobiotics can adversely affect biochemical reactions, including hepatic bile acid synthesis. Bile acids are essential for dissolving lipophilic compounds in the hydrophilic environment of the gastrointestinal tract. The critical micellar concentration of bile acids depends on the Δ4-reduction stereochemistry, with the 3-oxo-5ß-steroid-Δ4-dehydrogenase (AKR1D1) introducing the cis ring A/B conformation. Loss-of-function mutations in AKR1D1 cause hepatic cholestasis, which, if left untreated can progress into steatosis and liver cirrhosis. Furthermore, AKR1D1 is involved in clearing steroids with an A-ring Δ4-double bond. Here, we tested whether anabolic-androgenic steroids (AAS), often taken off-label at high doses, might inhibit AKR1D1, thereby potentially causing hepatotoxicity. A computational molecular model was established and used for virtual screening of the DrugBank database consisting of 2740 molecules, yielding mainly steroidal hits. Fourteen AAS were selected for in vitro evaluation, as such compounds can reach high hepatic concentrations in an abuse situation. Nandrolone, clostebol, methasterone, drostanolone, and methenolone inhibited to various extent the AKR1D1-mediated reduction of testosterone. Molecular modeling suggests that 9 out of 14 investigated AAS are competitive inhibitors. Moreover quantum mechanical calculations show that nadrolone and clostebol are substrates of AKR1D1 with different activation energy barriers for the hydrogen transfer from cofactor to the C5 position affecting their turnover. In this multidisciplinary approach, we established a molecular model of AKR1D1, identified several AAS as inhibitors, and described their binding mode. This approach may be applied to study other classes of inhibitors including non-steroidal compounds.

Virtual screening and biological evaluation to identify pharmaceuticals potentially causing hypertension and hypokalemia by inhibiting steroid 11ß-hydroxylase.

Several drugs were found after their market approval to unexpectedly inhibit adrenal 11ß-hydroxylase (CYP11B1)-dependent cortisol synthesis. Known side-effects of CYP11B1 inhibition include hypertension and hypokalemia, due to a feedback activation of adrenal steroidogenesis, leading to supraphysiological concentrations of 11-deoxycortisol and 11-deoxycorticosterone that can activate the mineralocorticoid receptor. This results in potassium excretion and sodium and water retention, ultimately causing hypertension. With the risk known but usually not addressed in preclinical evaluation, this study aimed to identify drugs and drug candidates inhibiting CYP11B1. Two conceptually different virtual screening methods were combined, a pharmacophore based and an induced fit docking approach. Cell-free and cell-based CYP11B1 activity measurements revealed several inhibitors with IC50 values in the nanomolar range. Inhibitors include retinoic acid metabolism blocking agents (RAMBAs), azole antifungals, α2-adrenoceptor ligands, and a farnesyltransferase inhibitor. The active compounds share a nitrogen atom embedded in an aromatic ring system. Structure activity analysis identified the free electron pair of the nitrogen atom as a prerequisite for the drug-enzyme interaction, with its pKa value as an indicator of inhibitory potency. Another important parameter is drug lipophilicity, exemplified by etomidate. Changing its ethyl ester moiety to a more hydrophilic carboxylic acid group dramatically decreased the inhibitory potential, most likely due to less efficient cellular uptake. The presented work successfully combined different in silico and in vitro methods to identify several previously unknown CYP11B1 inhibitors. This workflow facilitates the identification of compounds that inhibit CYP11B1 and therefore pose a risk for inducing hypertension and hypokalemia.

Immunological evaluation of herbal extracts commonly used for treatment of mental diseases during pregnancy.

Nonpsychotic mental diseases (NMDs) affect approximately 15% of pregnant women in the US. Herbal preparations are perceived a safe alternative to placenta-crossing antidepressants or benzodiazepines in the treatment of nonpsychotic mental diseases. But are these drugs really safe for mother and foetus? This question is of great relevance to physicians and patients. Therefore, this study investigates the influence of St. John's wort, valerian, hops, lavender, and California poppy and their compounds hyperforin and hypericin, protopine, valerenic acid, and valtrate, as well as linalool, on immune modulating effects in vitro. For this purpose a variety of methods was applied to assess the effects on viability and function of human primary lymphocytes. Viability was assessed via spectrometric assessment, flow cytometric detection of cell death markers and comet assay for possible genotoxicity. Functional assessment was conducted via flow cytometric assessment of proliferation, cell cycle and immunophenotyping. For California poppy, lavender, hops, and the compounds protopine and linalool, and valerenic acid, no effect was found on the viability, proliferation, and function of primary human lymphocytes. However, St. John's wort and valerian inhibited the proliferation of primary human lymphocytes. Hyperforin, hypericin, and valtrate inhibited viability, induced apoptosis, and inhibited cell division. Calculated maximum concentration of compounds in the body fluid, as well as calculated concentrations based on pharmacokinetic data from the literature, were low and supported that the observed effects in vitro would probably have no relevance on patients. In-silico analyses comparing the structure of studied substances with the structure of relevant control substances and known immunosuppressants revealed structural similarities of hyperforin and valerenic acid to the glucocorticoids. Valtrate showed structural similarities to the T cells signaling modulating drugs.

Evaluating the food safety and risk assessment evidence-base of polyethylene terephthalate oligomers: A systematic evidence map.

BACKGROUND: The presence of polyethylene terephthalate (PET) oligomers in food contact materials (FCMs) is well-documented. Consumers are exposed through their migration into foods and beverages; however, there is no specific guidance for their safety evaluation. OBJECTIVES: This systematic evidence map (SEM) aims to identify and organize existing knowledge and associated gaps in hazard and exposure information on 34 PET oligomers to support regulatory decision-making. METHODS: The methodology for this SEM was recently registered. A systematic search in bibliographic and gray literature sources was conducted and studies evaluated for inclusion according to the Populations, Exposures, Comparators, Outcomes, and Study type (PECOS) framework. Inclusion criteria were designed to record hazard and exposure information for all 34 PET oligomers and coded into the following evidence streams: human, animal, organism (non-animal), ex vivo, in vitro, in silico, migration, hydrolysis, and absorption, distribution, metabolism, excretion/toxicokinetics/pharmacokinetics (ADME/TK/PK) studies. Relevant information was extracted from eligible studies and synthesized according to the protocol. RESULTS: Literature searches yielded 7445 unique records, of which 96 were included. Data comprised migration (560 entries), ADME/TK/PK-related (253 entries), health/bioactivity (98 entries) and very few hydrolysis studies (7 entries). Cyclic oligomers were studied more frequently than linear PET oligomers. In vitro results indicated that hydrolysis of cyclic oligomers generated a mixture of linear oligomers, but not monomers, potentially allowing their absorption in the gastrointestinal tract. Cyclic dimers, linear trimers and the respective smaller oligomers exhibit physico-chemical properties making oral absorption more likely. Information on health/bioactivity effects of oligomers was almost non-existent, except for limited data on mutagenicity. CONCLUSIONS: This SEM revealed substantial deficiencies in the available evidence on ADME/TK/PK, hydrolysis, and health/bioactivity effects of PET oligomers, currently preventing appropriate risk assessment. It is essential to develop more systematic and tiered approaches to address the identified research needs and assess the risks of PET oligomers.

A Structural-Reporter Group to Determine the Core Conformation of Sialyl Lewisx Mimetics.

The d-GlcNAc moiety in sialyl Lewisx (sLex, 1) acts predominantly as a linker to position the d-Gal and the l-Fuc moieties in the bioactive spatial orientation. The hypothesis has been made that the NHAc group of GlcNAc pushes the fucose underneath the galactose and, thus, contributes to the stabilization of the bioactive conformation of the core of sLex (1). To test this hypothesis, GlcNAc mimetics consisting of (R,R)-1,2-cyclohexanediols substituted with alkyl and aryl substituents adjacent to the linking position of the fucose moiety were synthesized. To explore a broad range of extended and spatially demanding R-groups, an enzymatic approach for the synthesis of 3-alkyl/aryl-1,2-cyclohexanediols (3b-n) was applied. These cyclohexanediol derivatives were incorporated into the sLex mimetics 2b-n. For analyzing the relationship of affinity and core conformation, a 1H NMR structural-reporter-group concept was applied. Thus, the chemical shift of H-C5Fuc proved to be a sensitive indicator for the degree of pre-organization of the core of this class of sLex mimetics and therefore could be used to quantify the contribution of the R-groups.

Ligand pathways in estrogen-related receptors.

The three subtypes of estrogen-related receptors ERRα, ERRß, and ERRγ are nuclear receptors mediating metabolic processes in various tissues such as the skeletal muscle, fat tissue, bone, and liver. Although the knowledge on their physiological ligands is limited, they have been implicated as drug targets for important indications including diabetes, cardiovascular diseases, and osteoporosis. As in other nuclear receptors, their ligand binding pocket is buried within the core of the receptor and connected to its surrounding by ligand pathways. Here, we investigated these pathways with conventional molecular dynamics as well as metadynamics simulations to reveal their distribution and their capability to facilitate ligand translocation. Dependent on the ERR subtype and the conformational state of the receptor, we could detect different pathways to be favored. Overall, the results suggested pathways IIIa and IIIb to be favored in the agonistic conformation, while antagonists preferred pathways I, II, and V. Along the pathways, the ligands passed different gating mechanisms of the receptor, including groups of protein residues as well as whole secondary structure elements, to leave the binding site. Even though these pathways are suggested to influence ligand specificity of the receptors and their elucidation might advance rational drug design, they have not yet been studied in ERRs.Communicated by Ramaswamy H. Sarma.

A Dimerosesquiterpene and Sesquiterpene Lactones from Artemisia argyi Inhibiting Oncogenic PI3K/AKT Signaling in Melanoma Cells.

A library of more than 2500 plant extracts was screened for activity on oncogenic signaling in melanoma cells. The ethyl acetate extract from the aerial parts of Artemisia argyi displayed pronounced inhibition of the PI3K/AKT pathway. Active compounds were tracked with the aid of HPLC-based activity profiling, and altogether 21 active compounds were isolated, including one novel dimerosequiterpenoid (1), one new disesquiterpenoid (2), three new guaianolides (3-5), 12 known sesquiterpenoids (6-17), and four known flavonoids (19-22). A new eudesmanolide derivative (13b) was isolated as an artifact formed by methanolysis. Compound 1 is the first adduct comprising a sesquiterpene lactone and a methyl jasmonate moiety. The absolute configurations of compounds 1 and 3-18 were established by comparison of their experimental and calculated ECD spectra. The absolute configuration for 2 was determined by X-ray diffraction analysis. Guaianolide 8 was the most potent sesquiterpene lactone, inhibiting the PI3K/AKT pathway with an IC50 value of 8.9 ± 0.9 µM.

Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors.

With the addition of the compstatin-based complement C3 inhibitor pegcetacoplan, another class of complement targeted therapeutics have recently been approved. Moreover, compstatin derivatives with enhanced pharmacodynamic and pharmacokinetic profiles are in clinical development (e.g., Cp40/AMY-101). Despite this progress, the target binding and inhibitory modes of the compstatin family remain incompletely described. Here, we present the crystal structure of Cp40 complexed with its target C3b at 2.0-Å resolution. Structure-activity-relationship studies rationalize the picomolar affinity and long target residence achieved by lead optimization, and reveal a role for structural water in inhibitor binding. We provide explanations for the narrow species specificity of this drug class and demonstrate distinct target selection modes between clinical compstatin derivatives. Functional studies provide further insight into physiological complement activation and corroborate the mechanism of its compstatin-mediated inhibition. Our study may thereby guide the application of existing and development of next-generation compstatin analogs.

Evaluating the food safety and risk assessment evidence-base of polyethylene terephthalate oligomers: Protocol for a systematic evidence map.

BACKGROUND: Polyethylene terephthalate (PET) oligomers are ubiquitous in PET used in food contact applications. Consumer exposure by migration of PET oligomers into food and beverages is documented. However, no specific risk assessment framework or guidance for the safety evaluating of PET oligomers exist to date. AIM: The aim of this systematic evidence map (SEM) is to identify and organize existing knowledge clusters and associated gaps in hazard and exposure information of PET oligomers. Research needs will be identified as an input for chemical risk assessment, and to support future toxicity testing strategies of PET oligomers and regulatory decision-making. SEARCH STRATEGY AND ELIGIBILITY CRITERIA: Multiple bibliographic databases (incl. Embase, Medline, Scopus, and Web of Science Core Collection), chemistry databases (SciFinder-n, Reaxys), and gray literature sources will be searched, and the search results will be supplemented by backward and forward citation tracking on eligible records. The search will be based on a single-concept PET oligomer-focused strategy to ensure sensitive and unbiased coverage of all evidence related to hazard and exposure in a data-poor environment. A scoping exercise conducted during planning identified 34 relevant PET oligomers. Eligible work of any study type must include primary research data on at least one relevant PET oligomer with regard to exposure, health, or toxicological outcomes. STUDY SELECTION: For indexed scientific literature, title and abstract screening will be performed by one reviewer. Selected studies will be screened in full-text by two independent reviewers. Gray literature will be screened by two independent reviewers for inclusion and exclusion. STUDY QUALITY ASSESSMENT: Risk of bias analysis will not be conducted as part of this SEM. DATA EXTRACTION AND CODING: Will be performed by one reviewer and peer-checked by a second reviewer for indexed scientific literature or by two independent reviewers for gray literature. SYNTHESIS AND VISUALIZATION: The extracted and coded information will be synthesized in different formats, including narrative synthesis, tables, and heat maps. SYSTEMATIC MAP PROTOCOL REGISTRY AND REGISTRATION NUMBER: Zenodo: https://doi.org/10.5281/zenodo.6224302.

Activation of retinoic acid-related orphan receptor γ(t) by parabens and benzophenone UV-filters.

Retinoic acid-related orphan receptor γt (RORγt) regulates immune responses and its impaired function contributes to inflammatory and autoimmune diseases and may promote skin cancer. Synthetic inverse RORγt agonists block the production of Th17-associated cytokines including interleukin (IL)-17A and IL-22 and are under investigation for treatment of such pathologies. Unintentional RORγt activation in skin, following exposure to environmental chemicals, may promote inflammatory skin disease. Parabens and UV-filters, frequently used as additives in cosmetics and body care products, are intensively inspected for endocrine disrupting properties. This study assessed whether such compounds can interfere with RORγ activity using a previously established tetracycline-inducible reporter gene assay in CHO cells. These transactivation experiments revealed hexylparaben, benzylparaben and benzophenone-10 as RORγ agonists (EC50 values: 144 ± 97 nM, 3.39 ± 1.74 µM and 1.67 ± 1.04 µM, respectively), and they could restore RORγ activity after suppression by an inverse agonist. Furthermore, they enhanced RORγt-dependent transcription of the pro-inflammatory IL-17A and/or IL-22 genes in the murine T-cell model EL4. Virtual screening of a cosmetics database for structurally similar chemicals and in vitro testing of the most promising hits revealed benzylbenzoate, benzylsalicylate and 4-methylphenylbenzoate as RORγ agonists (low micromolar EC50 values). Moreover, an analysis of mixtures of the newly identified RORγ agonists suggested additive effects. This study presents novel RORγ(t) agonistic structural scaffolds. By activating RORγ(t) the identified parabens and UV-filters may potentially aggravate pathophysiological conditions, especially skin diseases where highest exposure of such chemicals can be expected. Follow-up studies should assess whether such compounds, either alone or as mixtures, can reach relevant concentrations in tissues and target cells to activate RORγ(t) in vivo.

Evaluation of Xa inhibitors as potential inhibitors of the SARS-CoV-2 Mpro protease.

Based on previous large-scale in silico screening several factor Xa inhibitors were proposed to potentially inhibit SARS-CoV-2 Mpro. In addition to their known anticoagulants activity this potential inhibition could have an additional therapeutic effect on patients with COVID-19 disease. In this study we examined the binding of the Apixaban, Betrixaban and Rivaroxaban to the SARS-CoV-2 Mpro with the use of the MicroScale Thermophoresis technique. Our results indicate that the experimentally measured binding affinity is weak and the therapeutic effect due to the SARS-CoV-2 Mpro inhibition is rather negligible.

A Conserved Allosteric Site on Drug-Metabolizing CYPs: A Systematic Computational Assessment.

Cytochrome P450 enzymes (CYPs) are the largest group of enzymes involved in human drug metabolism. Ligand tunnels connect their active site buried at the core of the membrane-anchored protein to the surrounding solvent environment. Recently, evidence of a superficial allosteric site, here denoted as hotspot 1 (H1), involved in the regulation of ligand access in a soluble prokaryotic CYP emerged. Here, we applied multi-scale computational modeling techniques to study the conservation and functionality of this allosteric site in the nine most relevant mammalian CYPs responsible for approximately 70% of drug metabolism. In total, we systematically analyzed over 44 µs of trajectories from conventional MD, cosolvent MD, and metadynamics simulations. Our bioinformatic analysis and simulations with organic probe molecules revealed the site to be well conserved in the CYP2 family with the exception of CYP2E1. In the presence of a ligand bound to the H1 site, we could observe an enlargement of a ligand tunnel in several members of the CYP2 family. Further, we could detect the facilitation of ligand translocation by H1 interactions with statistical significance in CYP2C8 and CYP2D6, even though all other enzymes except for CYP2C19, CYP2E1, and CYP3A4 presented a similar trend. As the detailed comprehension of ligand access and egress phenomena remains one of the most relevant challenges in the field, this work contributes to its elucidation and ultimately helps in estimating the selectivity of metabolic transformations using computational techniques.

Investigation of Thiocarbamates as Potential Inhibitors of the SARS-CoV-2 Mpro.

In the present study we tested, using the microscale thermophoresis technique, a small library of thionocarbamates, thiolocarbamates, sulfide and disulfide as potential lead compounds for SARS-CoV-2 Mpro drug design. The successfully identified binder is a representative of the thionocarbamates group with a high potential for future modifications aiming for higher affinity and solubility. The experimental analysis was extended by computational studies that show insufficient accuracy of the simplest and widely applied approaches and underline the necessity of applying more advanced methods to properly evaluate the affinity of potential SARS-CoV-2 Mpro binders.

Computational Selectivity Assessment of Protease Inhibitors against SARS-CoV-2.

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious global health threat. Since no specific therapeutics are available, researchers around the world screened compounds to inhibit various molecular targets of SARS-CoV-2 including its main protease (Mpro) essential for viral replication. Due to the high urgency of these discovery efforts, off-target binding, which is one of the major reasons for drug-induced toxicity and safety-related drug attrition, was neglected. Here, we used molecular docking, toxicity profiling, and multiple molecular dynamics (MD) protocols to assess the selectivity of 33 reported non-covalent inhibitors of SARS-CoV-2 Mpro against eight proteases and 16 anti-targets. The panel of proteases included SARS-CoV Mpro, cathepsin G, caspase-3, ubiquitin carboxy-terminal hydrolase L1 (UCHL1), thrombin, factor Xa, chymase, and prostasin. Several of the assessed compounds presented considerable off-target binding towards the panel of proteases, as well as the selected anti-targets. Our results further suggest a high risk of off-target binding to chymase and cathepsin G. Thus, in future discovery projects, experimental selectivity assessment should be directed toward these proteases. A systematic selectivity assessment of SARS-CoV-2 Mpro inhibitors, as we report it, was not previously conducted.