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BackgroundPulmonary endothelial damage has been shown to precede the development of emphysema in animals, and vascular changes in humans have been observed in COPD and emphysema.Research questionIs intraparenchymal vascular pruning associated with longitudinal progression of emphysema on CT imaging or decline in lung function over 5 years?Study design and methodsThe Genetic Epidemiology of COPD Study enrolled ever smokers with and without COPD from 2008 through 2011. The percentage of emphysema-like lung, or "percent emphysema," was assessed at baseline and after 5 years on noncontrast CT imaging as the percentage of lung voxels < -950 Hounsfield units. An automated CT imaging-based tool assessed and classified intrapulmonary arteries and veins. Spirometry measures are postbronchodilator. Pulmonary arterial pruning was defined as a lower ratio of small artery volume (< 5 mm2 cross-sectional area) to total lung artery volume. Mixed linear models included demographics, anthropomorphics, smoking, and COPD, with emphysema models also adjusting for CT imaging scanner and lung function models adjusting for clinical center and baseline percent emphysema.ResultsAt baseline, the 4,227 participants were 60 ± 9 years of age, 50% were women, 28% were Black, 47% were current smokers, and 41% had COPD. Median percent emphysema was 2.1 (interquartile range, 0.6-6.3) and progressed 0.24 percentage points/y (95% CI, 0.22-0.26 percentage points/y) over 5.6 years. Mean FEV1 to FVC ratio was 68.5 ± 14.2% and declined 0.26%/y (95% CI, -0.30 to -0.23%/y). Greater pulmonary arterial pruning was associated with more rapid progression of percent emphysema (0.11 percentage points/y per 1-SD increase in arterial pruning; 95% CI, 0.09-0.16 percentage points/y), including after adjusting for baseline percent emphysema and FEV1. Arterial pruning also was associated with a faster decline in FEV1 to FVC ratio (-0.04%/y per 1-SD increase in arterial pruning; 95% CI, -0.008 to -0.001%/y).InterpretationPulmonary arterial pruning was associated with faster progression of percent emphysema and more rapid decline in FEV1 to FVC ratio over 5 years in ever smokers, suggesting that pulmonary vascular differences may be relevant in disease progression.Trial registryClinicalTrials.gov; No.: NCT00608764; URL: www.clinicaltrials.gov.

Background: The coronary artery calcium score (CACS) and ratio of the pulmonary artery to aorta diameters (PA:A ratio) measured from chest CT scans have been established as predictors of cardiovascular events and COPD exacerbations, respectively. However, little is known about the reciprocal relationship between these predictors and outcomes. Furthermore, the prognostic implications of COPD subtypes on clinical outcomes remain insufficiently characterized., Research Question: How can these two chest CT scan-derived parameters predict subsequent cardiovascular events and COPD exacerbations in different COPD subtypes?, Study Design and Methods: Using COPDGene study data, we assessed prospective cardiovascular disease (CVD) and COPD exacerbation risk in participants with COPD (Global Initiative for Chronic Obstructive Lung Disease spirometric grades 2-4), focusing on CACS and PA:A ratio at study enrollment, with logistic regression models. These outcomes were analyzed in three COPD subtypes: 1,042 participants with non-emphysema-predominant COPD (NEPD; low attenuation area at -950 Hounsfield units [LAA-950] < 5%), 1,324 participants with emphysema-predominant COPD (EPD; LAA-950 ≥ 10%), and 465 participants with intermediate emphysema COPD (IE; 5% ≤ LAA-950 < 10%)., Results: Our study indicated significantly higher overall risk for cardiovascular events in participants with higher CACS (≥ median; OR, 1.61; 95% CI, 1.30-2.00) and increased COPD exacerbations in those with higher PA:A ratios (≥ 1; OR, 1.80; 95% CI, 1.46-2.23). Notably, participants with NEPD showed a stronger association between these indicators and clinical events compared to EPD (with CACS/CVD, NEPD vs EPD: OR, 2.02 vs 1.41; with PA:A ratio/COPD exacerbation, NEPD vs EPD: OR, 2.50 vs 1.65); the difference in ORs between COPD subtypes was statistically significant for CACS/CVD., Interpretation: Two chest CT scan parameters, CACS and PA:A ratio, hold distinct predictive values for cardiovascular events and COPD exacerbations that are influenced by specific COPD subtypes., Trial Registration: ClinicalTrials.gov; No.: NCT00608764; URL: www., Clinicaltrials: gov., Competing Interests: Financial/Nonfinancial Disclosures The authors have reported to CHEST the following: In the past 3 years, E. K. S. has received grant support from Bayer and Northpond Laboratories. J. M. W. has received grant support from ARCUS-Med, Mereo BioPharma, Medscape, Verona Pharma, Grifols, Alpha-1 Foundation, and InhibrX. P. J. C. has received grant support from Bayer and Sanofi. C. P. H. has received grant support from Alpha-1 Foundation, Bayer, Boehringer-Ingelheim, and Vertex. None declared (H.-M. Y., M. H. R., V. J. C., K. Y., G. L. K., M. T. D., R. C. W., M. J. B.)., (Copyright © 2024 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Background: The EF-hand Ca 2+ sensor protein S100A1 has been identified as a molecular regulator and enhancer of cardiac performance. The ability of S100A1 to recognize and modulate the activity of targets such as SERCA2a (sarcoplasmic reticulum Ca 2+ ATPase) and RyR2 (ryanodine receptor 2) in cardiomyocytes has mostly been ascribed to its hydrophobic C-terminal α-helix (residues 75-94). We hypothesized that a synthetic peptide consisting of residues 75 through 94 of S100A1 and an N-terminal solubilization tag (S100A1ct) could mimic the performance-enhancing effects of S100A1 and may be suitable as a peptide therapeutic to improve the function of diseased hearts., Methods: We applied an integrative translational research pipeline ranging from in silico computational molecular modeling and in vitro biochemical molecular assays as well as isolated rodent and human cardiomyocyte performance assessments to in vivo safety and efficacy studies in small and large animal cardiac disease models., Results: We characterize S100A1ct as a cell-penetrating peptide with positive inotropic and antiarrhythmic properties in normal and failing myocardium in vitro and in vivo. This activity translates into improved contractile performance and survival in preclinical heart failure models with reduced ejection fraction after S100A1ct systemic administration. S100A1ct exerts a fast and sustained dose-dependent enhancement of cardiomyocyte Ca 2+ cycling and prevents β-adrenergic receptor-triggered Ca 2+ imbalances by targeting SERCA2a and RyR2 activity. In line with the S100A1ct-mediated enhancement of SERCA2a activity, modeling suggests an interaction of the peptide with the transmembrane segments of the sarcoplasmic Ca 2+ pump. Incorporation of a cardiomyocyte-targeting peptide tag into S100A1ct (cor-S100A1ct) further enhanced its biological and therapeutic potency in vitro and in vivo., Conclusions: S100A1ct is a promising lead for the development of novel peptide-based therapeutics against heart failure with reduced ejection fraction.

Background: COPD has high mortality, compounded by comorbid cardiovascular disease. We investigated two electrocardiogram (ECG) markers, Cardiac Infarction Injury Score (CIIS) and P pulmonale, as prognostic tools for adverse cardiopulmonary events in COPD., Methods: Post hoc analysis of the IMPACT trial. Outcomes included odds (odds ratio [95% confidence intervals]) of adverse cardiopulmonary events stratified by CIIS threshold (<20/≥20) and P pulmonale (baseline). Events included all-cause death, hospitalisation/death, cardiovascular adverse event of special interest (CVAESI), severe COPD exacerbations, and moderate/severe COPD exacerbations. Effects of fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) versus FF/VI or UMEC/VI based on CIIS and P pulmonale were also assessed., Results: We included 9448 patients. Patients with CIIS ≥20 had greater odds of all-cause death (1.73[1.27-2.37]; p <0.001), hospitalisation/death (1.33[1.17-1.50]; p <0.001), CVAESI (1.27[1.08-1.48]; p <0.005), severe COPD exacerbations (1.41[1.21-1.64]; p <0.001) and moderate/severe COPD exacerbations (1.25[1.13-1.40]; p <0.001) versus CIIS <20. Patients with P pulmonale ( versus without) had greater odds of all-cause death (2.25[1.54-3.29]; p <0.001), hospitalisation/death (1.51[1.28-1.79]; p <0.001), severe COPD exacerbations (2.00[1.65-2.41]; p <0.001) and moderate/severe COPD exacerbations (1.25[1.08-1.46]; p <0.001). A combined model demonstrated patients with CIIS ≥20 and P pulmonale had increased risk of all-cause death (3.38[1.23-9.30]; p=0.019), hospitalisation/death (1.61[1.14-2.22]; p=0.004), and rate of severe COPD exacerbations (1.89[1.22-2.91]; p=0.004) and moderate/severe COPD exacerbations (1.25[1.00-1.56]; p=0.046). The risk of all-cause death and CVAESI was reduced with FF/UMEC/VI versus UMEC/VI in patients with CIIS ≥20, but not CIIS <20., Conclusions: These findings suggest potential clinical relevance of CIIS and P pulmonale as risk indicators for adverse cardiopulmonary events in COPD., (Copyright ©The authors 2024.)

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2' functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential., Competing Interests: Competing interests statement:The authors declare no competing interest.

Cytochrome P450 2B4 (CYP 2B4) is one of the best-characterized CYPs and serves as a key model system for understanding the mechanisms of microsomal class II CYPs, which metabolize most known drugs. The highly flexible nature of CYP 2B4 is apparent from crystal structures that show the active site with either a wide open or a closed heme binding cavity. Here, we investigated the conformational ensemble of the full-length CYP 2B4 in a phospholipid bilayer, using multiresolution molecular dynamics (MD) simulations. Coarse-grained MD simulations revealed two predominant orientations of CYP 2B4's globular domain with respect to the bilayer. Their refinement by atomistic resolution MD showed adaptation of the enzyme's interaction with the lipid bilayer, leading to open configurations that facilitate ligand access to the heme binding cavity. CAVER analysis of enzyme tunnels, AquaDuct analysis of water routes, and Random Acceleration Molecular Dynamics simulations of ligand dissociation support the conformation-dependent passage of molecules between the active site and the protein surroundings. Furthermore, simulation of the re-entry of the inhibitor bifonazole into the open conformation of CYP 2B4 resulted in binding at a transient hydrophobic pocket within the active site cavity that may play a role in substrate binding or allosteric regulation. Together, these results show how the open conformation of CYP 2B4 facilitates the binding of substrates from and release of products to the membrane, whereas the closed conformation prolongs the residence time of substrates or inhibitors and selectively allows the passage of smaller reactants via the solvent and water channels., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The dissociation rate, or its reciprocal, the residence time (τ), is a crucial parameter for understanding the duration and biological impact of biomolecular interactions. Accurate prediction of τ is essential for understanding protein-protein interactions (PPIs) and identifying potential drug targets or modulators for tackling diseases. Conventional molecular dynamics simulation techniques are inherently constrained by their limited timescales, making it challenging to estimate residence times, which typically range from minutes to hours. Building upon its successful application in protein-small molecule systems, τ-Random Acceleration Molecular Dynamics (τRAMD) is here investigated for estimating dissociation rates of protein-protein complexes. τRAMD enables the observation of unbinding events on the nanosecond timescale, facilitating rapid and efficient computation of relative residence times. We tested this methodology for three protein-protein complexes and their extensive mutant datasets, achieving good agreement between computed and experimental data. By combining τRAMD with MD-IFP (Interaction Fingerprint) analysis, dissociation mechanisms were characterized and their sensitivity to mutations investigated, enabling the identification of molecular hotspots for selective modulation of dissociation kinetics. In conclusion, our findings underscore the versatility of τRAMD as a simple and computationally efficient approach for computing relative protein-protein dissociation rates and investigating dissociation mechanisms, thereby aiding the design of PPI modulators., (© 2024. The Author(s).)

Background: Neural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimer's disease (AD) pathology. Addressing these impairments by using neurotrophic factors is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations., Methods: Here, we use a combination of mouse and human stem cell models for comprehensive in-vitro characterization of a novel neurogenic compound, focusing on the brain-derived neurotrophic factor (BDNF) pathway. The ability of ENT-A011, a steroidal dehydroepiandrosterone derivative, to activate the tyrosine receptor kinase B (TrkB) receptor was tested through western blotting in NIH-3T3 cells and its neurogenic and neuroprotective action were assessed through proliferation, cell death and Amyloid-β (Aβ) toxicity assays in mouse primary adult hippocampal NSCs, mouse embryonic cortical NSCs and neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. RNA-seq profiling was used to assess if the compound acts through the same gene network as BDNF in human NPCs., Results: ENT-A011 was able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Aβ-induced cell death, acting selectively through TrkB activation. The compound was able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from Αβ toxicity and prevent synapse loss after Aβ treatment. ENT-A011 successfully induces proliferation and prevents cell death after Aβ toxicity in human NPCs, acting through a core gene network shared with BDNF as shown through RNA-seq., Conclusions: Our work characterizes a novel BDNF mimetic with preferable pharmacological properties and neurogenic and neuroprotective actions in Alzheimer's disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing., (© 2024. The Author(s).)

Inhibition of K-RAS effectors like B-RAF or MEK1/2 is accompanied by treatment resistance in cancer patients via re-activation of PI3K and Wnt signaling. We hypothesized that myotubularin-related-protein-7 (MTMR7), which inhibits PI3K and ERK1/2 signaling downstream of RAS, directly targets RAS and thereby prevents resistance. Using cell and structural biology combined with animal studies, we show that MTMR7 binds and inhibits RAS at cellular membranes. Overexpression of MTMR7 reduced RAS GTPase activities and protein levels, ERK1/2 phosphorylation, c-FOS transcription and cancer cell proliferation in vitro. We located the RAS-inhibitory activity of MTMR7 to its charged coiled coil (CC) region and demonstrate direct interaction with the gastrointestinal cancer-relevant K-RAS G12V mutant, favouring its GDP-bound state. In mouse models of gastric and intestinal cancer, a cell-permeable MTMR7-CC mimicry peptide decreased tumour growth, Ki67 proliferation index and ERK1/2 nuclear positivity. Thus, MTMR7 mimicry peptide(s) could provide a novel strategy for targeting mutant K-RAS in cancers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Krabbe disease is a sphingolipidosis characterized by the genetic deficiency of the acid hydrolase β-galactosylceramidase (GALC). Most of the studies concerning the biological role of GALC performed on Krabbe patients and Galc -deficient twitcher mice (an authentic animal model of the disease) indicate that the pathogenesis of this disorder is the consequence of the accumulation of the neurotoxic GALC substrate β-galactosylsphingosine (psychosine), ignoring the possibility that this enzyme may exert a wider biological impact. Indeed, limited information is available about the effect of GALC downregulation on the cell lipidome in adult and developing organisms. The teleost zebrafish ( Danio rerio ) has emerged as a useful platform to model human genetic diseases, including sphingolipidoses, and two GALC co-orthologs have been identified in zebrafish ( galca and galcb ). Here, we investigated the effect of the competitive and irreversible GALC inhibitor β-galactose-cyclophellitol (GCP) on the lipid profile of zebrafish embryos. Molecular modelling indicates that GCP can be sequestered in the catalytic site of the enzyme and covalently binds human GALC, and the zebrafish Galca and Galcb proteins in a similar manner. Accordingly, GCP inhibits the β-galactosylceramide hydrolase activity of zebrafish in vitro and in vivo , leading to significant alterations of the lipidome of zebrafish embryos. These results indicate that the lack of GALC activity deeply affects the lipidome during the early stages of embryonic development, and thereby provide insights into the pathogenesis of Krabbe disease., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Introduction: In 2019, the Beta-Blockers for the Prevention of Acute Exacerbations of Chronic Obstructive Pulmonary Disease study (BLOCK-COPD) evaluated the effect of metoprolol on exacerbation risk and mortality in a COPD population without indications for beta-blocker use. We hypothesized that an imaging metric of coronary artery disease (CAD), the coronary artery calcium (CAC) score, would predict exacerbation risk and identify a differential response to metoprolol treatment., Methods: The study population includes participants in the BLOCK-COPD study from multiple study sites. Participants underwent clinically indicated thoracic computed tomography (CT) scans ± 12 months from enrollment. The Weston scoring system quantified CAC. Adjusted Cox proportional hazards models evaluated for associations between CAC and time to exacerbation., Results: Data is included for 109 participants. The mean CAC score was 5.1±3.7, and 92 participants (84%) had CAC scores greater than 0. Over a median (interquartile range) follow-up time of 350 (280 to 352) days, there were 61 mild exacerbations and 19 severe/very severe exacerbations. No associations were found between exacerbations of any severity and CAC>0 or total CAC. Associations were observed between total CAC and CAC>0 in the left circumflex (LCx) and time to exacerbation of any severity (adjusted hazard ratio [aHR]=1.39, confidence interval [CI]: 1.08-1.79, p =0.01) and (aHR=1.96, 95% CI: 1.04-3.70, p =0.04), respectively., Conclusions: CAD is a prevalent comorbidity in COPD accounting for significant mortality. Our study confirms the high prevalence of CAD using the CAC score; however, we did not discover an association between CAC and exacerbation risk. We did find novel associations between CAC in the LCx and exacerbation risk which warrant further investigation in larger cohorts., (JCOPDF © 2024.)

The COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the "Billion molecules against COVID-19 challenge", to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 molecules, which were subsequently ranked to find 'consensus compounds'. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for biological activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (only the Nsp12 domain), and (alpha) spike protein S. Overall, 27 compounds with weak inhibition/binding were experimentally identified by binding-, cleavage-, and/or viral suppression assays and are presented here. Open science approaches such as the one presented here contribute to the knowledge base of future drug discovery efforts in finding better SARS-CoV-2 treatments., (© 2023 The Authors. Molecular Informatics published by Wiley-VCH GmbH.)

Rationale: Pulmonary hypertension (PH) in COPD confers increased risk of exacerbations (ECOPD). Electrocardiogram (ECG) indicators of PH are prognostic both in PH and COPD. In the Beta-Blockers for the Prevention of Acute Exacerbations of COPD (BLOCK-COPD) trial, metoprolol increased risk of severe ECOPD through unclear mechanisms., Objective: We evaluated whether an ECG indicator of PH, P-pulmonale, would be associated with ECOPD and whether participants with P-pulmonale randomized to metoprolol were at higher risk of ECOPD and worsened respiratory symptoms given the potential detrimental effects of beta-blockers in PH., Methods: ECGs of 501 participants were analyzed for P-pulmonale (P wave enlargement in lead II). Cox proportional hazards models evaluated for associations between P-pulmonale and time to ECOPD (all and severe) for all participants and by treatment assignment (metoprolol vs. placebo). Linear mixed-effects models evaluated the association between treatment assignment and P-pulmonale on change in symptom scores (measured by CAT and SOBQ)., Results: We identified no association between P-pulmonale and risk of any ECOPD or severe ECOPD. However, in individuals with P-pulmonale, metoprolol was associated with increased risk for ECOPD (aHR 2.92, 95% CI: 1.45-5.85). There was no association between metoprolol and ECOPD in individuals without P-pulmonale (aHR 1.01, 95% CI: 0.77-1.31). Individuals with P-pulmonale assigned to metoprolol experienced worsening symptoms (mean increase of 3.95, 95% CI: 1.32-6.58) whereas those assigned to placebo experienced a mean improvement in CAT score of -2.45 (95% CI: -0.30- -4.61)., Conclusions: In individuals with P-pulmonale, metoprolol was associated with increased exacerbation risk and worsened symptoms. These findings may explain the findings observed in BLOCK-COPD., (© 2023. The Author(s).)

The propensity of poorly water-soluble drugs to aggregate at supersaturation impedes their bioavailability. Supersaturated amorphous drug-salt-polymer systems provide an emergent approach to this problem. However, the effects of polymers on drug-drug interactions in aqueous phase are largely unexplored and it is unclear how to choose an optimal salt-polymer combination for a particular drug. Here, we describe a comparative experimental and computational characterization of amorphous solid dispersions containing the drug celecoxib, and a polymer, polyvinylpyrrolidone vinyl acetate (PVP-VA) or hydroxypropyl methylcellulose acetate succinate, with or without Na + /K + salts. Classical models for drug-polymer interactions fail to identify the best drug-salt-polymer combination. In contrast, more stable drug-polymer interaction energies computed from molecular dynamics simulations correlate with prolonged stability of supersaturated amorphous drug-salt-polymer systems, along with better dissolution and pharmacokinetic profiles. The celecoxib-salt-PVP-VA formulations exhibit excellent biopharmaceutical performance, offering the prospect of a low-dosage regimen for this widely used anti-inflammatory, thereby increasing cost-effectiveness, and reducing side-effects., (© 2023. Springer Nature Limited.)

A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC 50 ) against THP-1 macrophages being >100 μM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Human ecto-5'-nucleotidase (h-ecto-5'-NT, CD73) is a homodimeric Zn 2+ -binding metallophosphoesterase that hydrolyzes adenosine 5'-monophosphate (5'-AMP) to adenosine and phosphate. h-Ecto-5'-NT is a key enzyme in purinergic signaling pathways and has been recognized as a promising biological target for several diseases, including cancer and inflammatory, infectious, and autoimmune diseases. Despite its importance as a biological target, little is known about h-ecto-5'-NT dynamics, which poses a considerable challenge to the design of inhibitors of this target enzyme. Here, to explore h-ecto-5'-NT flexibility, all-atom unbiased molecular dynamics (MD) simulations were performed. Remarkable differences in the dynamics of the open (catalytically inactive) and closed (catalytically active) conformations of the apo-h-ecto-5'-NT were observed during the simulations, and the nucleotide analogue inhibitor AMPCP was shown to stabilize the protein structure in the closed conformation. Our results suggest that the large and complex domain motion that enables the h-ecto-5'-NT open/closed conformational switch is slow, and therefore, it could not be completely captured within the time scale of our simulations. Nonetheless, we were able to explore the faster dynamics of the h-ecto-5'-NT substrate binding site, which is mainly located at the C-terminal domain and well conserved among the protein's open and closed conformations. Using the TRAPP ("Transient Pockets in Proteins") approach, we identified transient subpockets close to the substrate binding site. Finally, conformational states of the substrate binding site with higher druggability scores than the crystal structure were identified. In summary, our study provides valuable insights into h-ecto-5'-NT structural flexibility, which can guide the structure-based design of novel h-ecto-5'-NT inhibitors.

The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gomez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Martini 3, the latest version of the widely used Martini force field for coarse-grained molecular dynamics simulations, is a promising tool to investigate proteins in phospholipid bilayers. However, simulating other lipid environments, such as detergent micelles, presents challenges due to the absence of validated parameters for their constituent molecules. Here, we propose parameters for the micelle-forming surfactant, dodecylphosphocholine (DPC). These result in micelle assembly with aggregation numbers in agreement with the experimental values. However, we identified a lack of hydrophobic interactions between transmembrane helix protein dimers and the tails of DPC molecules, preventing insertion and stabilization of the protein in the micelles. This problem was also observed for protein insertion by self-assembling 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) or dipalmitoylphosphatidylcholine (DPPC) bilayers. We propose the reduction of the nonbonded interactions between protein and water beads by 10% as a simple and effective solution to this problem that enables protein encapsulation in phospholipid micelles and bilayers without altering protein dimerization or the bilayer structure.

Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory function. MtFAS generates the octanoic acid precursor for lipoic acid synthesis, but the role of longer fatty acid products has remained unclear. The structurally well-characterized component of mtFAS, human 2E-enoyl-ACP reductase (MECR) rescues respiratory growth and lipoylation defects of a Saccharomyces cerevisiae Δetr1 strain lacking native mtFAS enoyl reductase. To address the role of longer products of mtFAS, we employed in silico molecular simulations to design a MECR variant with a shortened substrate binding cavity. Our in vitro and in vivo analyses indicate that the MECR G165Q variant allows synthesis of octanoyl groups but not long chain fatty acids, confirming the validity of our computational approach to engineer substrate length specificity. Furthermore, our data imply that restoring lipoylation in mtFAS deficient yeast strains is not sufficient to support respiration and that long chain acyl-ACPs generated by mtFAS are required for mitochondrial function., (© 2023. The Author(s).)

Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7 , we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers., Competing Interests: LC, SF, MS, OS, EC, SF, AL, CP, MT, GG, PS, GC, LL, DC, AV, AQ, PL, LT, MM, RG, SP, RL, FG, SH, SA, NS, Ad, EG, GP, PP, AR, GC, RS, RW, SM, GM, DD, GP, MC No competing interests declared, (© 2022, Costantino et al.)

The therapeutic targeting of the immune system, for example in vaccinology and cancer treatment, is a challenging task and the subject of active research. Several in silico tools used for predicting immunogenicity are based on the analysis of peptide sequences binding to the Major Histocompatibility Complex (pMHC). However, few of these bioinformatics tools take into account the pMHC three-dimensional structure. Here, we describe a new bioinformatics tool, MatchTope, developed for predicting peptide similarity, which can trigger cross-reactivity events, by computing and analyzing the electrostatic potentials of pMHC complexes. We validated MatchTope by using previously published data from in vitro assays. We thereby demonstrate the strength of MatchTope for similarity prediction between targets derived from several pathogens as well as for indicating possible cross responses between self and tumor peptides. Our results suggest that MatchTope can enhance and speed up future studies in the fields of vaccinology and cancer immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mendes, de Souza Bragatte, Vianna, de Freitas, Pöhner, Richter, Wade, Salzano and Vieira.)

Background: With the expansion of animal production, parasitic helminths are gaining increasing economic importance. However, application of several established deworming agents can harm treated hosts and environment due to their low specificity. Furthermore, the number of parasite strains showing resistance is growing, while hardly any new anthelminthics are being developed. Here, we present a bioinformatics workflow designed to reduce the time and cost in the development of new strategies against parasites. The workflow includes quantitative transcriptomics and proteomics, 3D structure modeling, binding site prediction, and virtual ligand screening. Its use is demonstrated for Acanthocephala (thorny-headed worms) which are an emerging pest in fish aquaculture. We included three acanthocephalans (Pomphorhynchus laevis, Neoechinorhynchus agilis, Neoechinorhynchus buttnerae) from four fish species (common barbel, European eel, thinlip mullet, tambaqui)., Results: The workflow led to eleven highly specific candidate targets in acanthocephalans. The candidate targets showed constant and elevated transcript abundances across definitive and accidental hosts, suggestive of constitutive expression and functional importance. Hence, the impairment of the corresponding proteins should enable specific and effective killing of acanthocephalans. Candidate targets were also highly abundant in the acanthocephalan body wall, through which these gutless parasites take up nutrients. Thus, the candidate targets are likely to be accessible to compounds that are orally administered to fish. Virtual ligand screening led to ten compounds, of which five appeared to be especially promising according to ADMET, GHS, and RO5 criteria: tadalafil, pranazepide, piketoprofen, heliomycin, and the nematicide derquantel., Conclusions: The combination of genomics, transcriptomics, and proteomics led to a broadly applicable procedure for the cost- and time-saving identification of candidate target proteins in parasites. The ligands predicted to bind can now be further evaluated for their suitability in the control of acanthocephalans. The workflow has been deposited at the Galaxy workflow server under the URL tinyurl.com/yx72rda7 ., (© 2022. The Author(s).)

Crowded environments are known to affect the diffusion of macromolecules, but their effects on the diffusion of small molecules are largely uncharacterized. We investigate how three protein crowders, bovine serum albumin (BSA), hen egg-white lysozyme, and myoglobin, influence the diffusion rates and interactions of four small molecules: fluorescein, and three drugs, doxorubicin, glycogen synthase kinase-3 inhibitor SB216763, and quinacrine. Using Line-FRAP measurements, Brownian dynamics simulations, and molecular docking, we find that the diffusion rates of the small molecules are highly affected by self-aggregation, interactions with the proteins, and surface adsorption. The diffusion of fluorescein is decreased because of its interactions with the protein crowders and their surface adsorption. Protein crowders increase the diffusion rates of doxorubicin and SB216763 by reducing surface interactions and self-aggregation, respectively. Quinacrine diffusion was not affected by protein crowders. The mechanistic insights gained here may assist in optimization of compounds for higher mobility in complex macromolecular environments., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Introduction: Trypanosomatidic parasitic infections in humans and animals caused by Trypanosoma brucei, Trypanosoma cruzi , and Leishmania species pose a significant health and economic burden in developing countries. There are few effective and accessible treatments for these diseases, and the existing therapies suffer from problems, such as parasite resistance and side effects. Structure-based drug design (SBDD) is one of the strategies that has been applied to discover new compounds targeting trypanosomatid-borne diseases., Areas Covered: We review the current literature (mostly over the last 5 years, searched in the PubMed database on 11 November 2021) on the application of structure-based drug design approaches to identify new anti-trypanosomatidic compounds that interfere with a validated target biochemical pathway, the trypanosomatid folate pathway., Expert Opinion: The application of structure-based drug design approaches to perturb the trypanosomatid folate pathway has successfully provided many new inhibitors with good selectivity profiles, most of which are natural products or their derivatives or have scaffolds of known drugs. However, the inhibitory effect against the target protein(s) often does not translate to anti-parasitic activity. Further progress is hampered by our incomplete understanding of parasite biology and biochemistry, which is necessary to complement SBDD in a multiparameter optimization approach to discovering selective anti-parasitic drugs.

The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure-activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC 50 values against T. brucei brucei while retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

Modeling in neuroscience occurs at the intersection of different points of view and approaches. Typically, hypothesis-driven modeling brings a question into focus so that a model is constructed to investigate a specific hypothesis about how the system works or why certain phenomena are observed. Data-driven modeling, on the other hand, follows a more unbiased approach, with model construction informed by the computationally intensive use of data. At the same time, researchers employ models at different biological scales and at different levels of abstraction. Combining these models while validating them against experimental data increases understanding of the multiscale brain. However, a lack of interoperability, transparency, and reusability of both models and the workflows used to construct them creates barriers for the integration of models representing different biological scales and built using different modeling philosophies. We argue that the same imperatives that drive resources and policy for data - such as the FAIR (Findable, Accessible, Interoperable, Reusable) principles - also support the integration of different modeling approaches. The FAIR principles require that data be shared in formats that are Findable, Accessible, Interoperable, and Reusable. Applying these principles to models and modeling workflows, as well as the data used to constrain and validate them, would allow researchers to find, reuse, question, validate, and extend published models, regardless of whether they are implemented phenomenologically or mechanistically, as a few equations or as a multiscale, hierarchical system. To illustrate these ideas, we use a classical synaptic plasticity model, the Bienenstock-Cooper-Munro rule, as an example due to its long history, different levels of abstraction, and implementation at many scales., Competing Interests: OE, KB, SC, DK, AK, ML, AS, RW, JH No competing interests declared, UB Reviewing Editor, eLife, (© 2022, Eriksson et al.)

Angiogenesis, the formation of new blood vessels from preexisting ones, is crucial for tumor growth and metastatization, and is considered a promising therapeutic target. Unfortunately, drugs directed against a specific proangiogenic growth factor or receptor turned out to be of limited benefit for oncology patients, likely due to the high biochemical redundancy of the neovascularization process. In this scenario, multitarget compounds that are able to simultaneously tackle different proangiogenic pathways are eagerly awaited. UniPR1331 is a 3β-hydroxy-Δ 5 -cholenic acid derivative, which is already known to inhibit Eph-ephrin interaction. Here, we employed an analysis pipeline consisting of molecular modeling and simulation, surface plasmon resonance spectrometry, biochemical assays, and endothelial cell models to demonstrate that UniPR1331 directly interacts with the vascular endothelial growth factor receptor 2 (VEGFR2) too. The binding of UniPR1331 to VEGFR2 prevents its interaction with the natural ligand vascular endothelial growth factor and subsequent autophosphorylation, signal transduction, and in vitro proangiogenic activation of endothelial cells. In vivo, UniPR1331 inhibits tumor cell-driven angiogenesis in zebrafish. Taken together, these data shed light on the pleiotropic pharmacological effect of UniPR1331, and point to Δ 5 -cholenic acid as a promising molecular scaffold for the development of multitarget antiangiogenic compounds., (© 2021. The Author(s).)

Biological Field-Effect Transistors (BioFETs) have already demonstrated enormous potential for detecting minute amounts of ions and molecules. The use of two-dimensional (2D) materials has been shown to boost their performance and to enable the design of new applications. This combination deserves special interest in the current pandemic caused by the SARS-CoV-2 virus which demands fast, reliable and cheap detection methods. However, in spite of the experimental advances, there is a lack of a comprehensive and in-depth computational approach to capture the mechanisms underlying the sensor behaviour. Here, we present a multiscale platform that combines detailed atomic models of the molecules with mesoscopic device-level simulations. The fine-level description exploited in this approach accounts for the charge distribution of the receptor, its reconfiguration when the target binds to it, and the consequences in terms of sensitivity on the transduction mechanism. The results encourage the further exploration of improved sensor designs and 2D materials combined with diverse receptors selected to achieve the desired specificity., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Neurotrophins are growth factors that exert important neuroprotective effects by preventing neuronal death and synaptic loss. Nerve Growth Factor (NGF) acts through the activation of its high-affinity, pro-survival TrkA and low-affinity, pro-apoptotic p75 NTR receptors. NGF has been shown to slow or prevent neurodegenerative signals in Alzheimer's Disease (AD) progression. However, its low bioavailability and its blood-brain-barrier impermeability limit the use of NGF as a potential therapeutic agent against AD. Based on our previous findings on synthetic dehydroepiandrosterone derivatives, we identified a novel NGF mimetic, named ENT-A013, which selectively activates TrkA and exerts neuroprotective, anti-amyloid-β actions. We now report the chemical synthesis, in silico modelling, metabolic stability, CYP-mediated reaction phenotyping and biological characterization of ENT-A013 under physiological and neurodegenerative conditions. We show that ENT-A013 selectively activates the TrkA receptor and its downstream kinases Akt and Erk1/2 in PC12 cells, protecting these cells from serum deprivation-induced cell death. Moreover, ENT-A013 promotes survival of primary Dorsal Root Ganglion (DRG) neurons upon NGF withdrawal and protects hippocampal neurons against Amyloid β-induced apoptosis and synaptic loss. Furthermore, this neurotrophin mimetic partially restores LTP impairment. In conclusion, ENT-A013 represents a promising new lead molecule for developing therapeutics against neurodegenerative disorders, such as Alzheimer's Disease, selectively targeting TrkA-mediated pro-survival signals.

The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes . GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro . As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Eisenberg, Albert, Teuffel, Zitzow, Michaelis, Jarick, Sehlke, Große, Bader, Nunes-Alves, Kreikemeyer, Schindelin, Wade and Fiedler.)

Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor-binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan coreceptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

We develop an approach to characterize the effects of gating by a multiconformation protein consisting of macrostate conformations that are either accessible or inaccessible to ligand binding. We first construct a Markov state model of the apo-protein from atomistic molecular dynamics simulations from which we identify macrostates and their conformations, compute their relative macrostate populations and interchange kinetics, and structurally characterize them in terms of ligand accessibility. We insert the calculated first-order rate constants for conformational transitions into a multistate gating theory from which we derive a gating factor γ that quantifies the degree of conformational gating. Applied to HIV-1 protease, our approach yields a kinetic network of three accessible (semi-open, open, and wide-open) and two inaccessible (closed and a newly identified, "parted") macrostate conformations. The parted conformation sterically partitions the active site, suggesting a possible role in product release. We find that the binding kinetics of drugs and drug-like inhibitors to HIV-1 protease falls in the slow gating regime. However, because γ = 0.75, conformational gating only modestly slows ligand binding. Brownian dynamics simulations of the diffusional association of eight inhibitors to the protease─having a wide range of experimental association constants (∼10 4 -10 10 M -1 s -1 )─yields gated rate constants in the range of ∼0.5-5.7 × 10 8 M -1 s -1 . This indicates that, whereas the association rate of some inhibitors could be described by the model, for many inhibitors either subsequent conformational transitions or alternate binding mechanisms may be rate-limiting. For systems known to be modulated by conformational gating, the approach could be scaled computationally efficiently to screen association kinetics for a large number of ligands.

There is a growing appreciation of the importance of drug-target binding kinetics for lead optimization. For G protein-coupled receptors (GPCRs), which mediate signaling over a wide range of time scales, the drug dissociation rate is often a better predictor of in vivo efficacy than binding affinity, although it is more challenging to compute. Here, we assess the ability of the τ-Random Acceleration Molecular Dynamics (τRAMD) approach to reproduce relative residence times and reveal dissociation mechanisms and the effects of allosteric modulation for two important membrane-embedded drug targets: the β2-adrenergic receptor and the muscarinic acetylcholine receptor M2. The dissociation mechanisms observed in the relatively short RAMD simulations (in which molecular dynamics (MD) simulations are performed using an additional force with an adaptively assigned random orientation applied to the ligand) are in general agreement with much more computationally intensive conventional MD and metadynamics simulations. Remarkably, although decreasing the magnitude of the random force generally reduces the number of egress routes observed, the ranking of ligands by dissociation rate is hardly affected and agrees well with experiment. The simulations also reproduce changes in residence time due to allosteric modulation and reveal associated changes in ligand dissociation pathways.

The binding kinetic properties of potential drugs may significantly influence their subsequent clinical efficacy. Predictions of these properties based on computer simulations provide a useful alternative to their expensive and time-consuming experimental counterparts, even at an early drug discovery stage. Herein, we perform scaled molecular dynamics (ScaledMD) simulations on a set of 27 ligands of HSP90 belonging to more than seven chemical series to estimate their relative residence times. We introduce two new techniques for the analysis and the classification of the simulated unbinding trajectories. The first technique, which helps in estimating the limits of the free energy well around the bound state, and the second one, based on a new contact map fingerprint, allow the description and the comparison of the paths that lead to unbinding. Using these analyses, we find that ScaledMD's relative residence time generally enables the identification of the slowest unbinders. We propose an explanation for the underestimation of the residence times of a subset of compounds, and we investigate how the biasing in ScaledMD can affect the mechanistic insights that can be gained from the simulations.

Linker histones (LHs) bind to nucleosomes with their globular domain (gH) positioned in either an on- or an off-dyad binding mode. Here, we study the effect of the linker DNA (L-DNA) sequence on the binding of a full-length LH, Xenopus laevis H1.0b, to a Widom 601 nucleosome core particle (NCP) flanked by two 40 bp long L-DNA arms, by single-pair FRET spectroscopy. We varied the sequence of the 11 bp of L-DNA adjoining the NCP on either side, making the sequence either A-tract, purely GC, or mixed with 64% AT. The labeled gH consistently exhibited higher FRET efficiency with the labeled L-DNA containing the A-tract than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA when compared to the pure-GC stretch. We explain our observations with a model that shows that the gH binds on dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove. To investigate whether this on-dyad minor groove-based recognition was distinct from previously identified off-dyad major groove-based recognition, a nucleosome was designed with A-tracts on both the L-DNA arms. One A-tract was complementary to thymine and the other to deoxyuridine. The major groove of the thymine-tract was lined with methyl groups that were absent from the major groove of the deoxyuridine tract. The gH exhibited similar FRET for both these A-tracts, suggesting that it does not interact with the thymine methyl groups exposed on the major groove. Our observations thus complement previous studies that suggest that different LH isoforms may employ different ways of recognizing AT-rich DNA and A-tracts. This adaptability may enable the LH to universally compact scaffold-associated regions and constitutive heterochromatin, which are rich in such sequences., (Copyright © 2021. Published by Elsevier Inc.)

There is growing consensus that the optimization of the kinetic parameters for drug-protein binding leads to improved drug efficacy. Therefore, computational methods have been developed to predict kinetic rates and to derive quantitative structure-kinetic relationships (QSKRs). Many of these methods are based on crystal structures of ligand-protein complexes. However, a drawback is that each ligand-protein complex is usually treated as having a single structure. Here, we present a modification of COMparative BINding Energy (COMBINE) analysis, which uses the structures of ligand-protein complexes to predict binding parameters. We introduce the option of using multiple structures to describe each ligand-protein complex in COMBINE analysis and apply this to study the effects of protein flexibility on the derivation of dissociation rate constants ( k off ) for inhibitors of p38 mitogen-activated protein (MAP) kinase, which has a flexible binding site. Multiple structures were obtained for each ligand-protein complex by performing docking to an ensemble of protein configurations obtained from molecular dynamics simulations. Coefficients to scale ligand-protein interaction energies determined from energy-minimized structures of ligand-protein complexes were obtained by partial least squares regression, and they allowed for the computation of k off values. The QSKR model obtained using single, energy-minimized crystal structures for each ligand-protein complex had higher predictive power than the QSKR model obtained with multiple structures from ensemble docking. However, incorporation of ligand-protein flexibility helped to highlight additional ligand-protein interactions that lead to longer residence times, such as interactions with residues Arg67 and Asp168, which are close to the ligand in many crystal structures. These results show that COMBINE analysis is a promising method to guide the design of compounds that bind to flexible proteins with improved binding kinetics.

Background: Secondary polycythemia is associated with cigarette smoking and chronic obstructive pulmonary disease (COPD). However, the prevalence of polycythemia in COPD and the contributing risk factors for polycythemia in COPD have not been extensively studied., Methods: We analyzed the presence of secondary polycythemia in current and former smokers with moderate to very severe COPD at the five-year follow-up visit in the observational COPDGene study. We used logistic regression to evaluate the association of polycythemia with age, sex, race, altitude, current smoking status, spirometry, diffusing capacity for carbon monoxide (DLCO), quantitative chest CT measurements (including emphysema, airway wall thickness, and pulmonary artery to aorta diameter ratio), resting hypoxemia, exercise-induced hypoxemia, and long-term oxygen therapy., Results: In a total of 1928 COPDGene participants with moderate to very severe COPD, secondary polycythemia was found in 97 (9.2%) male and 31 (3.5%) female participants. In a multivariable logistic model, severe resting hypoxemia (OR 3.50, 95% CI 1.41-8.66), impaired DLCO (OR 1.28 for each 10-percent decrease in DLCO % predicted, CI 1.09-1.49), male sex (OR 3.60, CI 2.20-5.90), non-Hispanic white race (OR 3.33, CI 1.71-6.50), current smoking (OR 2.55, CI 1.49-4.38), and enrollment in the Denver clinical center (OR 4.42, CI 2.38-8.21) were associated with higher risk for polycythemia. In addition, continuous (OR 0.13, CI 0.05-0.35) and nocturnal (OR 0.46, CI 0.21-0.97) supplemental oxygen were associated with lower risk for polycythemia. Results were similar after excluding participants with anemia and participants enrolled at the Denver clinical center., Conclusions: In a large cohort of individuals with moderate to very severe COPD, male sex, current smoking, enrollment at the Denver clinical center, impaired DLCO, and severe hypoxemia were associated with increased risk for secondary polycythemia. Continuous or nocturnal supplemental oxygen use were associated with decreased risk for polycythemia., (© 2021. The Author(s).)

Simulations of macromolecular diffusion and adsorption in confined environments can offer valuable mechanistic insights into numerous biophysical processes. In order to model solutes at atomic detail on relevant time scales, Brownian dynamics simulations can be carried out with the approximation of rigid body solutes moving through a continuum solvent. This allows the precomputation of interaction potential grids for the solutes, thereby allowing the computationally efficient calculation of forces. However, hydrodynamic and long-range electrostatic interactions cannot be fully treated with grid-based approaches alone. Here, we develop a treatment of both hydrodynamic and electrostatic interactions to include the presence of surfaces by modeling grid-based and long-range interactions. We describe its application to simulate the self-association and many-molecule adsorption of the well-characterized protein hen egg-white lysozyme to mica-like and silica-like surfaces. We find that the computational model can recover a number of experimental observables of the adsorption process and provide insights into their determinants. The computational model is implemented in the Simulation of Diffusional Association (SDA) software package.

There is increasing evidence of a significant correlation between prolonged drug-target residence time and increased drug efficacy. Here, we report a structural rationale for kinetic selectivity between two closely related kinases: focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2). We found that slowly dissociating FAK inhibitors induce helical structure at the DFG motif of FAK but not PYK2. Binding kinetic data, high-resolution structures and mutagenesis data support the role of hydrophobic interactions of inhibitors with the DFG-helical region, providing a structural rationale for slow dissociation rates from FAK and kinetic selectivity over PYK2. Our experimental data correlate well with computed relative residence times from molecular simulations, supporting a feasible strategy for rationally optimizing ligand residence times. We suggest that the interplay between the protein structural mobility and ligand-induced effects is a key regulator of the kinetic selectivity of inhibitors of FAK versus PYK2., Competing Interests: Declaration of interests M.F., J.B., and T.H. are employees of Merck KGaA, Darmstadt, Germany. M.A. was an employee of Merck KGaA, Darmstadt, Germany at the time of experimental procedures and is now an employee of Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany. Other authors have no conflict of interest to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Glycosaminoglycans (GAGs) are linear polysaccharides. In proteoglycans (PGs), they are attached to a core protein. GAGs and PGs can be found as free molecules, associated with the extracellular matrix or expressed on the cell membrane. They play a role in the regulation of a wide array of physiological and pathological processes by binding to different proteins, thus modulating their structure and function, and their concentration and availability in the microenvironment. Unfortunately, the enormous structural diversity of GAGs/PGs has hampered the development of dedicated analytical technologies and experimental models. Similarly, computational approaches (in particular, molecular modeling, docking and dynamics simulations) have not been fully exploited in glycobiology, despite their potential to demystify the complexity of GAGs/PGs at a structural and functional level. Here, we review the state-of-the art of computational approaches to studying GAGs/PGs with the aim of pointing out the "bitter" and "sweet" aspects of this field of research. Furthermore, we attempt to bridge the gap between bioinformatics and glycobiology, which have so far been kept apart by conceptual and technical differences. For this purpose, we provide computational scientists and glycobiologists with the fundamentals of these two fields of research, with the aim of creating opportunities for their combined exploitation, and thereby contributing to a substantial improvement in scientific knowledge.

The S100A1ct peptide, consisting of the C-terminal 20 residues of the S100A1 protein fused to an N-terminal 6-residue hydrophilic tag, has been found to exert a positive inotropic effect, resulting in improved contractile performance of failing cardiac and skeletal muscle without arrhythmic side-effects. The S100A1ct peptide thus has high potential for the treatment of acute heart failure. As a step toward understanding its molecular mechanism of action, and to provide a basis for peptidomimetic design to optimize its properties, we here describe de novo structure predictions and molecular dynamics simulations to characterize the conformational landscape of S100A1ct in aqueous environment. In S100A1, the C-terminal 20 residues form an α-helix, but de novo peptide structure predictions indicate that other conformations are also possible. Conventional molecular dynamics simulations in implicit and explicit solvent corroborated this finding. To ensure adequate sampling, we performed simulations of a tagged 10-residue segment of S100A1ct, and we carried out Gaussian accelerated molecular dynamics simulations of the peptides. These simulations showed that although the helical conformation of S100A1ct was the most energetically stable, the peptide can adopt a range of kinked conformations, suggesting that its activity may be related to its ability to act as a conformational switch.