Natural Compounds for Bone Remodeling: A Computational and Experimental Approach Targeting Bone Metabolism-Related Proteins.

Osteoporosis, characterized by reduced bone density and increased fracture risk, affects over 200 million people worldwide, predominantly older adults and postmenopausal women. The disruption of the balance between bone-forming osteoblasts and bone-resorbing osteoclasts underlies osteoporosis pathophysiology. Standard treatment includes lifestyle modifications, calcium and vitamin D supplementation and specific drugs that either inhibit osteoclasts or stimulate osteoblasts. However, these treatments have limitations, including side effects and compliance issues. Natural products have emerged as potential osteoporosis therapeutics, but their mechanisms of action remain poorly understood. In this study, we investigate the efficacy of natural compounds in modulating molecular targets relevant to osteoporosis, focusing on the Mitogen-Activated Protein Kinase (MAPK) pathway and the gut microbiome's influence on bone homeostasis. Using an in silico and in vitro methodology, we have identified quercetin as a promising candidate in modulating MAPK activity, offering a potential therapeutic perspective for osteoporosis treatment.

Structural and Functional Insights into CRF Peptides and Their Receptors.

Corticotropin-releasing factor or hormone (CRF or CRH) and the urocortins regulate a plethora of physiological functions and are involved in many pathophysiological processes. CRF and urocortins belong to the family of CRF peptides (CRF family), which includes sauvagine, urotensin, and many synthetic peptide and non-peptide CRF analogs. Several of the CRF analogs have shown considerable therapeutic potential in the treatment of various diseases. The CRF peptide family act by interacting with two types of plasma membrane proteins, type 1 (CRF1R) and type 2 (CRF2R), which belong to subfamily B1 of the family B G-protein-coupled receptors (GPCRs). This work describes the structure of CRF peptides and their receptors and the activation mechanism of the latter, which is compared with that of other GPCRs. It also discusses recent structural information that rationalizes the selective binding of various ligands to the two CRF receptor types and the activation of receptors by different agonists.

W254 in furin functions as a molecular gate promoting anti-viral drug binding: Elucidation of putative drug tunneling and docking by non-equilibrium molecular dynamics.

Furins are serine endoproteases that process precursor proteins into their biologically active forms, and they play essential roles in normal metabolism and disease presentation, including promoting expression of bacterial virulence factors and viral pathogenesis. Thus, furins represent vital targets for development of antimicrobial and antiviral therapeutics. Recent experimental evidence indicated that dichlorophenyl (DCP)-pyridine "BOS" drugs (e.g., BOS-318) competitively inhibit human furin by an induced-fit mechanism in which tryptophan W254 in the furin catalytic cleft (FCC) functions as a molecular gate, rotating nearly 180o through a steep energy barrier about its chi-1 dihedral to an "open" orientation, exposing a buried (i.e., cryptic) hydrophobic pocket 1. Once exposed, the non-polar DCP group of BOS-318, and similar halo-phenyl groups of analogs, enter the cryptic pocket, stabilizing drug binding. Here, we demonstrate flexible-receptor docking of BOS-318 (and various analogs) was unable to emulate the induced-fit motif, even when tryptophan was replaced with less bulky phenylalanine or glycine. While either substitution allowed access to the hydrophobic pocket for most ligands tested, optimal binding was observed only for W254, inferring a stabilizing effect of the indole sidechain. Furthermore, non-equilibrium steered molecular dynamics (sMD) in which the bound drugs (or their fragments) were extracted from the FCC did not cause closure of the open W254 gate, consistent with the thermodynamic stability of the open or closed W254 orientations. Finally, interactive molecular dynamics (iMD) revealed two putative conduits of drug entry and binding into the FCC, each coupled with W254 dihedral rotation and opening of the cryptic pocket. The iMD simulations further revealed ligand entry and binding in the FCC is likely driven in part by energy fluxes stemming from disruption and re-formation of ligand and protein solvation shells during drug migration from the solution phase into the FCC.

A Robust Machine Learning Framework Built Upon Molecular Representations Predicts CYP450 Inhibition: Toward Precision in Drug Repurposing.

Human cytochrome P450 (CYP450) enzymes play a crucial role in drug metabolism and pharmacokinetics. CYP450 inhibition can lead to toxicity, in particular when drugs are co-administered with other drugs and xenobiotics or in the case of polypharmacy. Predicting CYP450 inhibition is also important for rational drug discovery and development, and precision in drug repurposing. In this overarching context, digital transformation of drug discovery and development, for example, using machine and deep learning approaches, offers prospects for prediction of CYP450 inhibition through computational models. We report here the development of a majority-voting machine learning framework to classify inhibitors and noninhibitors for seven major human liver CYP450 isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). For the machine learning models reported herein, we employed interaction fingerprints that were derived from molecular docking simulations, thus adding an additional layer of information for protein-ligand interactions. The proposed machine learning framework is based on the structure of the binding site of isoforms to produce predictions beyond previously reported approaches. Also, we carried out a comparative analysis so as to identify which representation of test compounds (molecular descriptors, molecular fingerprints, or protein-ligand interaction fingerprints) affects the predictive performance of the models. This work underlines the ways in which the structure of the enzyme catalytic site influences machine learning predictions and the need for robust frameworks toward better-informed predictions.

Molecular Epidemiology of SARS-CoV-2: The Dominant Role of Arginine in Mutations and Infectivity.

Background, Aims, Methods, Results, Conclusions: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global challenge due to its ability to mutate into variants that spread more rapidly than the wild-type virus. The molecular biology of this virus has been extensively studied and computational methods applied are an example paradigm for novel antiviral drug therapies. The rapid evolution of SARS-CoV-2 in the human population is driven, in part, by mutations in the receptor-binding domain (RBD) of the spike (S-) protein, some of which enable tighter binding to angiotensin-converting enzyme (ACE2). More stable RBD-ACE2 association is coupled with accelerated hydrolysis by proteases, such as furin, trypsin, and the Transmembrane Serine Protease 2 (TMPRSS2) that augment infection rates, while inhibition of the 3-chymotrypsin-like protease (3CLpro) can prevent the viral replication. Additionally, non-RBD and non-interfacial mutations may assist the S-protein in adopting thermodynamically favorable conformations for stronger binding. This study aimed to report variant distribution of SARS-CoV-2 across European Union (EU)/European Economic Area (EEA) countries and relate mutations with the driving forces that trigger infections. Variants' distribution data for SARS-CoV-2 across EU/EEA countries were mined from the European Centre for Disease Prevention and Control (ECDC) based on the sequence or genotyping data that are deposited in the Global Science Initiative for providing genomic data (GISAID) and The European Surveillance System (TESSy) databases. Docking studies performed with AutoDock VINA revealed stabilizing interactions of putative antiviral drugs, e.g., selected anionic imidazole biphenyl tetrazoles, with the ACE2 receptor in the RBD-ACE2 complex. The driving forces of key mutations for Alpha, Beta, Gamma, Delta, Epsilon, Kappa, Lambda, and Omicron variants, which stabilize the RBD-ACE2 complex, were investigated by computational approaches. Arginine is the critical amino acid in the polybasic furin cleavage sites S1/S2 (681-PRRARS-686) S2' (814-KRS-816). Critical mutations into arginine residues that were found in the delta variant (L452R, P681R) and may be responsible for the increased transmissibility and morbidity are also present in two widely spreading omicron variants, named BA.4.6 and BQ.1, where mutation R346T in the S-protein potentially contributes to neutralization escape. Arginine binders, such as Angiotensin Receptor Blockers (ARBs), could be a class of novel drugs for treating COVID-19.

Autotaxin facilitates selective LPA receptor signaling.

Autotaxin (ATX; ENPP2) produces the lipid mediator lysophosphatidic acid (LPA) that signals through disparate EDG (LPA1-3) and P2Y (LPA4-6) G protein-coupled receptors. ATX/LPA promotes several (patho)physiological processes, including in pulmonary fibrosis, thus serving as an attractive drug target. However, it remains unclear if clinical outcome depends on how different types of ATX inhibitors modulate the ATX/LPA signaling axis. Here, we show that the ATX "tunnel" is crucial for conferring key aspects of ATX/LPA signaling and dictates cellular responses independent of ATX catalytic activity, with a preference for activation of P2Y LPA receptors. The efficacy of the ATX/LPA signaling responses are abrogated more efficiently by tunnel-binding inhibitors, such as ziritaxestat (GLPG1690), compared with inhibitors that exclusively target the active site, as shown in primary lung fibroblasts and a murine model of radiation-induced pulmonary fibrosis. Our results uncover a receptor-selective signaling mechanism for ATX, implying clinical benefit for tunnel-targeting ATX inhibitors.

Reduction of stress responses in honey bees by synthetic ligands targeting an allatostatin receptor.

Honey bees are of great economic and ecological importance, but are facing multiple stressors that can jeopardize their pollination efficiency and survival. Therefore, understanding the physiological bases of their stress response may help defining treatments to improve their resilience. We took an original approach to design molecules with this objective. We took advantage of the previous identified neuropeptide allatostatin A (ASTA) and its receptor (ASTA-R) as likely mediators of the honey bee response to a biologically relevant stressor, exposure to an alarm pheromone compound. A first series of ASTA-R ligands were identified through in silico screening using a homology 3D model of the receptor and in vitro binding experiments. One of these (A8) proved also efficient in vivo, as it could counteract two behavioral effects of pheromone exposure, albeit only in the millimolar range. This putative antagonist was used as a template for the chemical synthesis of a second generation of potential ligands. Among these, two compounds showed improved efficiency in vivo (in the micromolar range) as compared to A8 despite no major improvement in their affinity for the receptor in vitro. These new ligands are thus promising candidates for alleviating stress in honey bees.

Design and synthesis of a novel non peptide CN-NFATc signaling inhibitor for tumor suppression in triple negative breast cancer.

The Ca2+/calmodulin-mediated phosphatase activity of calcineurin (CN) integrates calcium-mediated signaling with gene expression programs involved in the control of essential cellular processes in health and disease, such as the immune response and the pathogenesis of cancer progression and metastasis. In addition, CN is the target of the immunosuppressive drugs cyclosporine A (CsA) and FK-506 which are the cornerstone of immunosuppressant therapy. Unfortunately, long-term administration of these drugs results in severe side effects. Herein, we describe the design, synthesis and evaluation of new synthetic compounds that are capable of inhibiting NFATc activity in a dose-dependent manner, without interfering on CN phosphatase activity. These compounds were designed using the structure-based pharmacophore model of a peptide-derived PxIxIT sequence binding to calcineurin A subunit. Moreover, these compounds inhibit NFATc-dependent cytokine gene expression, secretion and proliferation of human T CD4+ cells. More importantly, compound 5a reduces tumor weight and shows a tendency to reduce tumor angiogenesis in an orthotopic immunocompetent mouse model of triple negative breast cancer, suggesting that 5a has tumor suppressor activity. These findings validate compound 5a as an agent with therapeutic activity against CN-NFATc and highlight its potential as a tool for drug development with therapeutic purposes.

Amidoxime prodrugs convert to potent cell-active multimodal inhibitors of the dengue virus protease.

The flavivirus genus of the Flaviviridae family comprises Dengue, Zika and West-Nile viruses which constitute unmet medical needs as neither appropriate antivirals nor safe vaccines are available. The dengue NS2BNS3 protease is one of the most promising validated targets for developing a dengue treatment however reported protease inhibitors suffer from toxicity and cellular inefficacy. Here we report SAR on our previously reported Zika-active carbazole scaffold, culminating prodrug compound SP-471P (EC50 1.10 µM, CC50 > 100 µM) that generates SP-471; one of the most potent, non-cytotoxic and cell-active protease inhibitors described in the dengue literature. In cell-based assays, SP-471P leads to inhibition of viral RNA replication and complete abolishment of infective viral particle production even when administered 6 h post-infection. Mechanistically, SP-471 appears to inhibit both normal intermolecular protease processes and intramolecular cleavage events at the NS2BNS3 junction, as well as at NS3 internal sites, all critical for virus replication. These render SP-471 a unique to date multimodal inhibitor of the dengue protease.

A COVID moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.

Identification of compounds that bind the centriolar protein SAS-6 and inhibit its oligomerization.

Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerization of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organization of further organelle components. A dimerization interaction between SAS-6 N-terminal "head" domains was previously shown to be essential for protein oligomerization in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low-molecular-weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerization. We demonstrate using NMR spectroscopy that a ligand from this family binds at the head domain dimerization site of algae, nematode, and human SAS-6 variants, but also that another ligand specifically recognizes human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest that ligand specificity derives from favorable Van der Waals interactions with a hydrophobic cavity at the dimerization site.

Publisher Correction: GPCRmd uncovers the dynamics of the 3D-GPCRome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

GPCRmd uncovers the dynamics of the 3D-GPCRome.

G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.

Challenges in identifying large germline structural variants for clinical use by long read sequencing.

Genomic structural variations, previously considered rare events, are widely recognized as a major source of inter-individual variability and hence, a major hurdle in optimum patient stratification and disease management. Herein, we focus on large complex germline structural variations and present challenges towards target treatment via the synergy of state-of-the-art approaches and information technology tools. A complex structural variation detection remains challenging, as there is no gold standard for identifying such genomic variations with long reads, especially when the chromosomal rearrangement in question is a few Mb in length. A clinical case with a large complex chromosomal rearrangement serves as a paradigm. We feel that functional validation and data interpretation are of outmost importance for information growth to be translated into knowledge growth and hence, new working practices are highlighted.

Combined Computational and Structural Approach into Understanding the Role of Peptide Binding and Activation of Melanocortin Receptor 4.

Melanocortin receptor 4 (MC4R) is expressed predominantly in the central nervous system and regulates food intake and sexual function and is also thought to be responsible for effects on mood and cognition. It belongs to the melanocortin receptor subfamily of G protein-coupled receptors (GPCRs). Here, we have synthesized and structurally characterized three peptides that bind to MC4R, producing different signaling events. AgRP is a naturally occurring antagonist, HLWNRS is the minimal sequence of the N-terminal with partial agonist activity, and aMSH is a full agonistic peptide. By implementing molecular dynamics simulations on the different peptide-receptor complexes, we propose their molecular basis of binding to investigate their differential molecular properties regarding the activation states of the receptor. Our analysis shows that the agonist and partial agonist may induce rotation in transmembrane helix 3, which is known to be involved in the key events occurring during GPCR activation, and this movement is impacted by certain aromatic residues and their positioning in the orthosteric binding site of the receptor.

Novel Hit Compounds as Putative Antifungals: The Case of Aspergillus fumigatus.

The prevalence of invasive fungal infections has been dramatically increased as the size of the immunocompromised population worldwide has grown. Aspergillus fumigatus is characterized as one of the most widespread and ubiquitous fungal pathogens. Among antifungal drugs, azoles have been the most widely used category for the treatment of fungal infections. However, increasingly, azole-resistant strains constitute a major problem to be faced. Towards this direction, our study focused on the identification of compounds bearing novel structural motifs which may evolve as a new class of antifungals. To fulfil this scope, a combination of in silico techniques and in vitro assays were implemented. Specifically, a ligand-based pharmacophore model was created and served as a 3D search query to screen the ZINC chemical database. Additionally, molecular docking and molecular dynamics simulations were used to improve the reliability and accuracy of virtual screening results. In total, eight compounds, bearing completely different chemical scaffolds from the commercially available azoles, were proposed and their antifungal activity was evaluated using in vitro assays. Results indicated that all tested compounds exhibit antifungal activity, especially compounds 1, 2, and 4, which presented the most promising minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values and, therefore, could be subjected to further hit to lead optimization.

Cell-active carbazole derivatives as inhibitors of the zika virus protease.

Zika virus (ZIKV) infection recently resulted in an international health emergency the Americas in and despite its high profile there is currently no approved treatment for ZIKV infection with millions of people being at risk. ZIKV is a member of Flaviviridae family which includes prominent members such as dengue virus (DENV) and West Nile virus (WNV). One of the best validated targets for developing anti-flaviviral treatment for DENV and WNV infection is the NS2B/NS3 protease. However the inhibitors reported to date have shown limited promise for further clinical development largely due to poor cellular activity. Prompted by the conserved nature of the viral NS2B/NS3 protease across flaviviruses, we envisaged that small molecule inhibitors of the ZIKVpro may be developed by applying rational design on previously reported scaffolds with demonstrated activity against other flaviviral proteases. Starting with an earlier WNVpro hit we performed a scaffold hopping exercise and discovered that certain carbazole derivatives bearing amidine groups possessed submicromolar potency and significant cellular activity against ZIKV. We successfully addressed various issues with the synthesis of novel N-substituted carbazole-based amidines thus permitting a targeted SAR campaign. The in vitro biochemical and cell-based inhibitory profiles exhibited by the lead molecule described in this work (ZIKVpro IC50 0.52 µM, EC50 1.25 µM), is among the best reported to date. Furthermore, these molecules possess capacity for further optimization of pharmacokinetics and may evolve to broad spectrum flaviviral protease inhibitors.

Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids.

Neurotensin (NT) (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) exerts a dual function as a neurotransmitter/neuromodulator in the central nervous system and as a hormone/cellular mediator in periphery. This dual function of NT establishes a connection between brain and peripheral tissues that renders this peptide a central player in energy homeostasis. Many biological actions of NT are mediated through its interaction with three types of NT receptors (NTS receptors). Despite its role in energy homeostasis, NT has a short half-life that hampers further determination of the biological actions of this peptide and its receptors in brain and periphery. The short half-life of NT is due to the proteolytic degradation of its C-terminal side by several endopeptidases. Therefore, it is important to synthesize NT analogues with resistant bonds against metabolic deactivation. Based on these findings, we herein report the synthesis of ten linear, two cyclic and two dimeric analogues of NT with modifications in its structure that improve their metabolic stability, while retaining the ability to bind to NTS receptors. Modifications at position 11 (introduction of D-Tyrosine (OEthyl) [D-Tyr(Et)] or D-1-naphtylalanine [D-1-Nal] were combined with introduction of a L-Lysine or a D-Arginine at positions 8 or 9, and 1-[2-(aminophenyl)-2-oxoethyl]-1H-pyrrole-2-carboxylic acid (AOPC) at positions 7 or 8, resulting in compounds NT4-NT21. AOPC is an unnatural amino acid with promise in applications as a building block for the synthesis of peptidomimetic compounds. To biologically evaluate these analogues, we determined their plasma stability and their binding affinities to type 1 NT receptor (NTS1), endogenously expressed in HT-29 cells, Among the fourteen NT analogues, compounds, NT5, NT6, and NT8, which have D-Tyr(Et) at position 11, bound to NTS1 in a dose-response manner and with relatively high affinity but still lower than that of the natural peptide. Despite their lower binding affinities compared to NT, the NT5, NT6, and NT8 exhibited a remarkably higher stability, as a result of their chemistry, which provides protection from enzymatic activity. These results will set the basis for the rational design of novel NT molecules with improved pharmacological properties and enhanced enzymatic stability.

Selective antagonism of CRF1 receptor by a substituted pyrimidine.

The corticotrophin-releasing factor (CRF) and its type 1 receptor (CRF1R) regulate the hypothalamic-pituitary-adrenal axis, as well as other systems, thus playing a crucial role in the maintenance of homeostasis. Non-peptide CRF1R-selective antagonists exert therapeutic effects on experimental animals with abnormal regulation of their homeostatic mechanisms. However, none of them is as yet in clinical use. In an effort to develop novel small non-peptide CRF1R-selective antagonists, we have synthesized a series of substituted pyrimidines described in a previous study. These small molecules bind to CRF1R, with analog 3 having the highest affinity. Characteristic structural features of analog 3 are a N,N-bis(methoxyethyl)amino group at position 6 and a methyl in the alkythiol group at position 5. Based on the binding profile of analog 3, we selected it in the present study for further pharmacological characterization. The results of this study suggest that analog 3 is a potent CRF1R-selective antagonist, blocking the ability of sauvagine, a CRF-related peptide, to stimulate cAMP accumulation in HEK 293 cells via activation of CRF1R, but not via CRF2R. Moreover, analog 3 blocked sauvagine to stimulate the proliferation of macrophages, further supporting its antagonistic properties. We have also constructed molecular models of CRF1R to examine the interactions of this receptor with analog 3 and antalarmin, a prototype CRF1R-selective non-peptide antagonist, which lacks the characteristic structural features of analog 3. Our data facilitate the design of novel non-peptide CRF1R antagonists for clinical use.

Conformational plasticity of the VEEV macro domain is important for binding of ADP-ribose.

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contribution in viral replication is still under investigation. NMR-driven titration experiments of ADPr in solution with the VEEV macro domain (in apo- and complex state) show that it adopts a suitable conformation for ADPr binding. Specific experiments indicate that the flexibility of the loops ß5-α3 and α3-ß6 is critical for formation of the complex and assists a wrapping mechanism for ADPr binding. Furthermore, along with this sequence of events, the VEEV MD undergoes a conformational exchange process between the apo state and a low-populated "dark" conformational state.