Searching for bioactive conformations of drug-like ligands with current force fields: how good are we?

Drug-like ligands obtained from protein–ligand complexes deposited in the Protein Databank were subjected to conformational searching using various force fields and solvation settings. For each ligand, the resulting conformer pool was examined for the presence of the bioactive (crystal pose-like) conformation. Similarity of conformers toward the crystal-pose was quantified as the best achievable root mean squared deviation (RMSD, heavy atoms only). Analyzing the conformer pools generated by various force fields revealed only small differences in the likelihood of finding a crystal pose-like conformation. However, employing different solvents in the conformational search was found to be very important for achieving RMSDs below 1.0 Å. The best statistical values of likelihood were observed with a recently released force field covering a large portion of dihedral angles occurring in drug-like compounds in combination with the water as solvent. In order to enable computational chemists and modelers to efficiently use available software tools, we have additionally performed several focused analyses on ligands, grouped according to descriptors most relevant for the rational drug design

In Silico Pharmacogenetics CYP2D6 Study Focused on the Pharmacovigilance of Herbal Antidepressants

The annual increase in depression worldwide together with an upward trend in the use of alternative medicine as treatment asks for developing reliable safety profiles of herbal based medicine. A considerable risk on adverse reactions exists when herbal remedies are combined with prescription medication. Around 25% of the drugs, including many antidepressants, depend on the activity of CYP2D6 for their metabolism and corresponding efficacy. Therefore, probing CYP2D6 inhibition by the active substances in herbal based medicine within the wild-type enzyme and clinically relevant allelic variants is crucial to avoid toxicity issues. In this in silico study several compounds with herbal origin suggested to have antidepressant activity were analyzed on their CYP2D6 wild-type and CYP2D6*53 inhibition potential using molecular docking. In addition, several pharmacokinetic properties were evaluated to assess their probability to cross the blood brain barrier and subsequently reach sufficient brain bioavailability for the modulation of central nervous system targets as well as characteristics which may hint toward potential safety issues

Species-specific differences in the inhibition of 11β-hydroxysteroid dehydrogenase 2 by itraconazole and posaconazole

11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) converts active 11β-hydroxyglucocorticoids to their inactive 11-keto forms, thereby preventing inappropriate mineralocorticoid receptor activation by glucocorticoids. Disruption of 11β-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypokalemia, hypernatremia and hypertension. Recently, the azole antifungals itraconazole and posaconazole were identified to potently inhibit human 11β-HSD2, and several case studies described patients with acquired AME. To begin to understand why this adverse drug effect was missed during preclinical investigations, the inhibitory potential of itraconazole, its main metabolite hydroxyitraconazole (OHI) and posaconazole against 11β-HSD2 from human and three commonly used experimental animals was assessed. Whilst human 11β-HSD2 was potently inhibited by all three compounds (IC; 50; values in the nanomolar range), the rat enzyme was moderately inhibited (1.5- to 6-fold higher IC; 50; values compared to human), and mouse and zebrafish 11β-HSD2 were very weakly inhibited (IC; 50; values above 7 μM). Sequence alignment and application of newly generated homology models for human and mouse 11β-HSD2 revealed significant differences in the C-terminal region and the substrate binding pocket. Exchange of the C-terminus and substitution of residues Leu170,Ile172 in mouse 11β-HSD2 by the corresponding residues His170,Glu172 of the human enzyme resulted in a gain of sensitivity to itraconazole and posaconazole, resembling human 11β-HSD2. The results provide an explanation for the observed species-specific 11β-HSD2 inhibition by the studied azole antifungals. The obtained structure-activity relationship information should facilitate future assessments of 11β-HSD2 inhibitors and aid choosing adequate animal models for efficacy and safety studies

What contributes to an effective mannose recognition domain?

In general, carbohydrate-lectin interactions are characterized by high specificity but also low affinity. The main reason for the low affinities are desolvation costs, due to the numerous hydroxy groups present on the ligand, together with the typically polar surface of the binding sites. Nonetheless, nature has evolved strategies to overcome this hurdle, most prominently in relation to carbohydrate-lectin interactions of the innate immune system but also in bacterial adhesion, a process key for the bacterium's survival. In an effort to better understand the particular characteristics, which contribute to a successful carbohydrate recognition domain, the mannose-binding sites of six C-type lectins and of three bacterial adhesins were analyzed. One important finding is that the high enthalpic penalties caused by desolvation can only be compensated for by the number and quality of hydrogen bonds formed by each of the polar hydroxy groups engaged in the binding process. In addition, since mammalian mannose-binding sites are in general flat and solvent exposed, the half-lives of carbohydrate-lectin complexes are rather short since water molecules can easily access and displace the ligand from the binding site. In contrast, the bacterial lectin FimH benefits from a deep mannose-binding site, leading to a substantial improvement in the off-rate. Together with both a catch-bond mechanism (i.e., improvement of affinity under shear stress) and multivalency, two methods commonly utilized by pathogens, the affinity of the carbohydrate-FimH interaction can be further improved. Including those just described, the various approaches explored by nature to optimize selectivity and affinity of carbohydrate-lectin interactions offer interesting therapeutic perspectives for the development of carbohydrate-based drugs

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

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 $M^{pro}$. 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 $M^{pro}$ 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 $M^{pro}$ inhibition is rather negligible

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

Spontaneous Ligand Access Events to Membrane-Bound Cytochrome P450 2D6 Sampled at Atomic Resolution

The membrane-anchored enzyme Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of around 25% of marketed drugs and its metabolic performance shows a high interindividual variation. While it was suggested that ligands access the buried active site of the enzyme from the membrane, no proof from unbiased simulations has been provided to support this hypothesis. Laboratory experiments fail to capture the access process which is suspected to influence binding kinetics. Here, we applied unbiased molecular dynamics (MD) simulations to investigate the access of ligands to wild-type CYP2D6, as well as the allelic variant CYP2D6*53. In multiple simulations, substrates accessed the active site of the enzyme from the protein-membrane interface to ultimately adopt a conformation that would allow a metabolic reaction. We propose the necessary steps for ligand access and the results suggest that the increased metabolic activity of CYP2D6*53 might be caused by a facilitated ligand uptake

Allosteric Binding Sites On Nuclear Receptors: Focus On Drug Efficacy and Selectivity

Nuclear receptors (NRs) are highly relevant drug targets in major indications such as oncologic, metabolic, reproductive, and immunologic diseases. However, currently, marketed drugs designed towards the orthosteric binding site of NRs often suffer from resistance mechanisms and poor selectivity. The identification of two superficial allosteric sites, activation function-2 (AF-2) and binding function-3 (BF-3), as novel drug targets sparked the development of inhibitors, while selectivity concerns due to a high conservation degree remained. To determine important pharmacophores and hydration sites among AF-2 and BF-3 of eight hormonal NRs, we systematically analyzed over 10 μ s of molecular dynamics simulations including simulations in explicit water and solvent mixtures. In addition, a library of over 300 allosteric inhibitors was evaluated by molecular docking. Based on our results, we suggest the BF-3 site to offer a higher potential for drug selectivity as opposed to the AF-2 site that is more conserved among the selected receptors. Detected similarities among the AF-2 sites of various NRs urge for a broader selectivity assessment in future studies. In combination with the Supplementary Material, this work provides a foundation to improve both selectivity and potency of allosteric inhibitors in a rational manner and increase the therapeutic applicability of this promising compound class

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