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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.

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).)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

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).)

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.

The protein-ligand residence time, τ, influences molecular function in biological networks and has been recognized as an important determinant of drug efficacy. To predict τ, computational methods must overcome the problem that τ often exceeds the timescales accessible to conventional molecular dynamics (MD) simulation. Here, we apply the τ-Random Acceleration Molecular Dynamics (τRAMD) method to a set of kinetically characterized complexes of T4 lysozyme mutants with small, engineered binding cavities. τRAMD yields relative ligand dissociation rates in good accordance with experiments across this diverse set of complexes that differ with regard to measurement temperature, ligand identity, protein mutation and binding cavity. τRAMD thereby allows a comprehensive characterization of the ligand egress routes and determinants of τ. Although ligand dissociation by multiple egress routes is observed, we find that egress via the predominant route determines the value of τ. We also find that the presence of a greater number of metastable states along egress pathways leads to slower protein-ligand dissociation. These physical insights could be exploited in the rational optimization of the kinetic properties of drug candidates., 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., (© 2021 The Author(s).)

The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ∼30 000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ∼200 virtual screenings of compound libraries on selected protein structures, we redefine the protein's druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Cytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP-CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP-CPR ET rates are low compared to those of soluble bacterial CYPs.

Rare variants in the beta-glucocerebrosidase gene (GBA1) are common genetic risk factors for alpha synucleinopathy, which often manifests clinically as GBA-associated Parkinson's disease (GBA-PD). Clinically, GBA-PD closely mimics idiopathic PD, but it may present at a younger age and often aggregates in families. Most carriers of GBA variants are, however, asymptomatic. Moreover, symptomatic PD patients without GBA variant have been reported in families with seemingly GBA-PD. These observations obscure the link between GBA variants and PD pathogenesis and point towards a role for unidentified additional genetic and/or environmental risk factors or second hits in GBA-PD. In this study, we explored whether rare genetic variants may be additional risk factors for PD in two families segregating the PD-associated GBA1 variants c.115+1G>A (ClinVar ID: 93445) and p.L444P (ClinVar ID: 4288). Our analysis identified rare genetic variants of the HSP70 co-chaperone DnaJ homolog subfamily B member 6 (DNAJB6) and lysosomal protein prosaposin (PSAP) as additional factors possibly influencing PD risk in the two families. In comparison to the wild-type proteins, variant DNAJB6 and PSAP proteins show altered functions in the context of cellular alpha-synuclein homeostasis when expressed in reporter cells. Furthermore, the segregation pattern of the rare variants in the genes encoding DNAJB6 and PSAP indicated a possible association with PD in the respective families. The occurrence of second hits or additional PD cosegregating rare variants has important implications for genetic counseling in PD families with GBA1 variant carriers and for the selection of PD patients for GBA targeted treatments.

The virtual screening of large numbers of compounds against target protein binding sites has become an integral component of drug discovery workflows. This screening is often done by computationally docking ligands into a protein binding site of interest, but this has the drawback of a large number of poses that must be evaluated to obtain accurate estimates of protein-ligand binding affinity. We here introduce a fast pre-filtering method for ligand prioritization that is based on a set of machine learning models and uses simple pose-invariant physicochemical descriptors of the ligands and the protein binding pocket. Our method, Rapid Screening with Physicochemical Descriptors + machine learning (RASPD+), is trained on PDBbind data and achieves a regression performance that is better than that of the original RASPD method and traditional scoring functions on a range of different test sets without the need for generating ligand poses. Additionally, we use RASPD+ to identify molecular features important for binding affinity and assess the ability of RASPD+ to enrich active molecules from decoys., 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 © 2020 Holderbach, Adam, Jayaram, Wade and Mukherjee.)

Dimerization of the small GTPase Arf is prerequisite for the scission of COPI-coated transport vesicles. Here, we quantify the monomer/dimer equilibrium of Arf within the membrane and show that after membrane scission, Arf dimers are restricted to donor membranes. By hydrogen exchange mass spectrometry, we define the interface of activated dimeric Arf within its switch II region. Single amino acid exchanges in this region reduce the propensity of Arf to dimerize. We suggest a mechanism of membrane scission by which the dimeric form of Arf is segregated to the donor membrane. Our data are consistent with the previously reported absence of dimerized Arf in COPI vesicles and could explain the presence of one single scar-like noncoated region in each COPI vesicle., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor drugable by agonists approved for treatment of type 2 diabetes, but also inhibits carcinogenesis and cell proliferation in vivo. Activating mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene mitigate these beneficial effects by promoting a negative feedback-loop comprising extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated kinase kinase 1/2 (MEK1/2)-dependent inactivation of PPARγ. To overcome this inhibitory mechanism, we searched for novel post-translational regulators of PPARγ. Phosphoinositide phosphatase Myotubularin-Related-Protein-7 (MTMR7) was identified as cytosolic interaction partner of PPARγ. Synthetic peptides were designed resembling the regulatory coiled-coil (CC) domain of MTMR7, and their activities studied in human cancer cell lines and C57BL6/J mice. MTMR7 formed a complex with PPARγ and increased its transcriptional activity by inhibiting ERK1/2-dependent phosphorylation of PPARγ. MTMR7-CC peptides mimicked PPARγ-activation in vitro and in vivo due to LXXLL motifs in the CC domain. Molecular dynamics simulations and docking predicted that peptides interact with the steroid receptor coactivator 1 (SRC1)-binding site of PPARγ. Thus, MTMR7 is a positive regulator of PPARγ, and its mimicry by synthetic peptides overcomes inhibitory mechanisms active in cancer cells possibly contributing to the failure of clinical studies targeting PPARγ.

The simulation of membrane proteins requires compatible protein and lipid force fields that reproduce the properties of both the protein and the lipid bilayer. Cytochrome P450 enzymes are bitopic membrane proteins with a transmembrane helical anchor and a large cytosolic globular domain that dips into the membrane. As such, they are representative and challenging examples of membrane proteins for simulations, displaying features of both peripheral and integral membrane proteins. We performed molecular dynamics simulations of three cytochrome P450 isoforms (2C9, 2C19 and 1A1) in a 2-oleoyl-1-palmitoyl-sn-glycerol-3-phosphocholine bilayer using two AMBER force field combinations: GAFF-LIPID with ff99SB for the protein, and LIPID14 with ff14SB for the protein. Comparison of the structural and dynamic properties of the proteins, the lipids and the protein-membrane interactions shows differing sensitivity of the cytochrome P450 isoforms to the choice of force field, with generally better agreement with experiment for the LIPID14 + ff14SB combination.

Chromatosomes are fundamental units of chromatin structure that are formed when a linker histone protein binds to a nucleosome. The positioning of the linker histone on the nucleosome influences the packing of chromatin. Recent simulations and experiments have shown that chromatosomes adopt an ensemble of structures that differ in the geometry of the linker histone-nucleosome interaction. In this article we review the application of Brownian, Monte Carlo, and molecular dynamics simulations to predict the structure of linker histone-nucleosome complexes, to study the binding mechanisms involved, and to predict how this binding affects chromatin fiber structure. These simulations have revealed the sensitivityof the chromatosome structure to variations in DNA and linker histone sequence, as well as to posttranslational modifications, thereby explaining the structural variability observed in experiments. We propose that a concerted application of experimental and computational approaches will reveal the determinants of chromatosome structural variability and how it impacts chromatin packing.

During transmission of malaria-causing parasites from mosquito to mammal, Plasmodium sporozoites migrate at high speed within the skin to access the bloodstream and infect the liver. This unusual gliding motility is based on retrograde flow of membrane proteins and highly dynamic actin filaments that provide short tracks for a myosin motor. Using laser tweezers and parasite mutants, we previously suggested that actin filaments form macromolecular complexes with plasma membrane-spanning adhesins to generate force during migration. Mutations in the actin-binding region of profilin, a near ubiquitous actin-binding protein, revealed that loss of actin binding also correlates with loss of force production and motility. Here, we show that different mutations in profilin, that do not affect actin binding in vitro , still generate lower force during Plasmodium sporozoite migration. Lower force generation inversely correlates with increased retrograde flow suggesting that, like in mammalian cells, the slow down of flow to generate force is the key underlying principle governing Plasmodium gliding motility., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

[This corrects the article DOI: 10.1186/2046-1682-7-4.]., (© The Author(s) 2020.)

Long-term potentiation and depression of synaptic activity in response to stimuli is a key factor in reinforcement learning. Strengthening of the corticostriatal synapses depends on the second messenger cAMP, whose synthesis is catalysed by the enzyme adenylyl cyclase 5 (AC5), which is itself regulated by the stimulatory Gαolf and inhibitory Gαi proteins. AC isoforms have been suggested to act as coincidence detectors, promoting cellular responses only when convergent regulatory signals occur close in time. However, the mechanism for this is currently unclear, and seems to lie in their diverse regulation patterns. Despite attempts to isolate the ternary complex, it is not known if Gαolf and Gαi can bind to AC5 simultaneously, nor what activity the complex would have. Using protein structure-based molecular dynamics simulations, we show that this complex is stable and inactive. These simulations, along with Brownian dynamics simulations to estimate protein association rates constants, constrain a kinetic model that shows that the presence of this ternary inactive complex is crucial for AC5's ability to detect coincident signals, producing a synergistic increase in cAMP. These results reveal some of the prerequisites for corticostriatal synaptic plasticity, and explain recent experimental data on cAMP concentrations following receptor activation. Moreover, they provide insights into the regulatory mechanisms that control signal processing by different AC isoforms., Competing Interests: The authors have declared that no competing interests exist.

The human cytochrome P450 (CYP) 2C9 and 2C19 enzymes are two highly similar isoforms with key roles in drug metabolism. They are anchored to the endoplasmic reticulum membrane by their N-terminal transmembrane helix and interactions of their cytoplasmic globular domain with the membrane. However, their crystal structures were determined after N-terminal truncation and mutating residues in the globular domain that contact the membrane. Therefore, the CYP-membrane interactions are not structurally well-characterized and their dynamics and the influence of membrane interactions on CYP function are not well understood. We describe herein the modeling and simulation of CYP 2C9 and CYP 2C19 in a phospholipid bilayer. The simulations revealed that, despite high sequence conservation, the small sequence and structural differences between the two isoforms altered the interactions and orientations of the CYPs in the membrane bilayer. We identified residues (including K72, P73, and I99 in CYP 2C9 and E72, R73, and H99 in CYP 2C19) at the protein-membrane interface that contribute not only to the differing orientations adopted by the two isoforms in the membrane, but also to their differing substrate specificities by affecting the substrate access tunnels. Our findings provide a mechanistic interpretation of experimentally observed effects of mutagenesis on substrate selectivity.

Drug-target residence times can impact drug efficacy and safety, and are therefore increasingly being considered during lead optimization. For this purpose, computational methods to predict residence times, τ, for drug-like compounds and to derive structure-kinetic relationships are desirable. A challenge for approaches based on molecular dynamics (MD) simulation is the fact that drug residence times are typically orders of magnitude longer than computationally feasible simulation times. Therefore, enhanced sampling methods are required. We recently reported one such approach: the τRAMD procedure for estimating relative residence times by performing a large number of random acceleration MD (RAMD) simulations in which ligand dissociation occurs in times of about a nanosecond due to the application of an additional randomly oriented force to the ligand. The length of the RAMD simulations is used to deduce τ. The RAMD simulations also provide information on ligand egress pathways and dissociation mechanisms. Here, we describe a machine learning approach to systematically analyze protein-ligand binding contacts in the RAMD trajectories in order to derive regression models for estimating τ and to decipher the molecular features leading to longer τ values. We demonstrate that the regression models built on the protein-ligand interaction fingerprints of the dissociation trajectories result in robust estimates of τ for a set of 94 drug-like inhibitors of heat shock protein 90 (HSP90), even for the compounds for which the length of the RAMD trajectories does not provide a good estimation of τ. Thus, we find that machine learning helps to overcome inaccuracies in the modeling of protein-ligand complexes due to incomplete sampling or force field deficiencies. Moreover, the approach facilitates the identification of features important for residence time. In particular, we observed that interactions of the ligand with the sidechain of F138, which is located on the border between the ATP binding pocket and a hydrophobic transient sub-pocket, play a key role in slowing compound dissociation. We expect that the combination of the τRAMD simulation procedure with machine learning analysis will be generally applicable as an aid to target-based lead optimization.

Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Reducing diagnostic delays in cancer has been a major interest worldwide; however, the literature on diagnostic delays in lymphoma remains scarce. Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's lymphoma. We aimed to determine whether certain structural factors predicted diagnostic delays in DLBCL and whether diagnostic delays impacted overall survival (OS)., Methods: Data were extracted via a retrospective cohort design from a single academic tertiary care referral center. A total of 104 patients were included. Time from first symptoms to diagnosis of <3 months was defined as "early diagnosis" and ≥3 months as "delayed diagnosis". Analysis was performed with student's t-test, chi-square testing, binomial logistic regression, and Kaplan-Meier log-rank testing., Results: "Delayed diagnosis" was more likely with lower stage, lower international prognostic index (IPI), and further distance from referral center (OR 0.66, CI 0.46-0.95; OR 0.69, CI 0.51-0.94; OR 1.008, CI 1.001-1.015). Patients of "other" ethnicity and without medical insurance were more likely to have significant diagnostic delays and worse overall survival (P = 0.002 and P = 0.007, respectively). Diagnostic delays of ≥3 months did not predict worse OS. However, delays of >6 months did predict worse OS., Conclusion: Our data suggest that excessive diagnostic delays of more than 6 months, ethnic minority status, and uninsured status in DLBCL may lead to worse outcomes. Efforts should be undertaken to reduce excessive diagnostic delays. More investigation needs to be done on the impacts of diagnostic delays in both DLBCL and other aggressive lymphomas., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.

Human cytochrome P450 (CYP) enzymes play an important role in the metabolism of drugs, steroids, fatty acids, and xenobiotics. Microsomal CYPs are anchored in the endoplasmic reticulum membrane by an N-terminal transmembrane (TM) helix that is connected to the globular catalytic domain by a flexible linker sequence. However, the structural and functional importance of the TM-helix is unclear because it has been shown that CYPs can still associate with the membrane and have enzymatic activity in reconstituted systems after truncation or modification of the N-terminal sequence. Here, we investigated the effect of mutations in the N-terminal TM-helix residues of two human steroidogenic enzymes, CYP 17A1 and CYP 19A1, that are major drug targets for cancer therapy. These mutations were originally introduced to increase the expression of the proteins in Escherichia coli. To investigate the effect of the mutations on protein-membrane interactions and function, we carried out coarse-grained and all-atom molecular dynamics simulations of the CYPs in a phospholipid bilayer. We confirmed the orientations of the globular domain in the membrane observed in the simulations by linear dichroism measurements in a Nanodisc. Whereas the behavior of CYP 19A1 was rather insensitive to truncation of the TM-helix, mutations in the TM-helix of CYP 17A1, especially W2A and E3L, led to a gradual drifting of the TM-helix out of the hydrophobic core of the membrane. This instability of the TM-helix could affect interactions with the allosteric redox partner, cytochrome b5, required for CYP 17A1's lyase activity. Furthermore, the simulations showed that the mutant TM-helix influenced the membrane interactions of the CYP 17A1 globular domain. In some simulations, the mutated TM-helix obstructed the substrate access tunnel from the membrane to the CYP active site, indicating a possible effect on enzyme function., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

This Special Issue contains thirteen articles that provide a vivid snapshot of the state-of-the-art of molecular modeling in drug design, illustrating recent advances and critically discussing important challenges [...].

A growing consensus is emerging that optimizing the drug-target affinity alone under equilibrium conditions does not necessarily translate into higher potency in vivo and that instead binding kinetic parameters should be optimized to ensure better efficacy. Therefore, in silico methods are needed to predict the kinetic parameters and the mechanistic determinants of drug-protein binding. Here we demonstrate the application of COMparative BINding Energy (COMBINE) analysis to derive quantitative structure-kinetics relationships (QSKRs) for the dissociation rate constants ( k off ) of inhibitors of heat shock protein 90 (HSP90) and HIV-1 protease. We derived protein-specific scoring functions by correlating k off rate constants with a subset of weighted interaction energy components determined from the energy-minimized structures of drug-protein complexes. As the QSKRs derived for these sets of chemically diverse compounds have good predictive ability and provide insights into important drug-protein interactions for optimizing k off , COMBINE analysis offers a promising approach for binding kinetics-guided lead optimization., Competing Interests: The authors declare no competing financial interest.

Kinetoplastid parasites-trypanosomes and leishmanias-infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Yvonne Freund was employed by Anacor Pharmaceuticals. Benzoxaboroles were supplied by Anacor Pharmaceuticals (YF) which was later taken over by Pfizer. This does not alter our adherence to all the PLOS Pathogens policies on sharing data and materials.

There is renewed interest in linker histone (LH)-nucleosome binding and how LHs influence eukaryotic DNA compaction. For a long time, the goal was to uncover "the structure of the chromatosome," but recent studies of LH-nucleosome complexes have revealed an ensemble of structures. Notably, the reconstituted LH-nucleosome complexes used in experiments rarely correspond to the sequence combinations present in organisms. For a full understanding of the determinants of the distribution of the chromatosome structural ensemble, studies must include a complete description of the sequences and experimental conditions used, and be designed to enable systematic evaluation of sequence and environmental effects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself. Here we probe the interactions of actin subunits within divergent actin filaments using a comparative dynamic molecular model and explore their functions using Plasmodium, the protozoan causing malaria, and mouse melanoma derived B16-F1 cells as model systems. Parasite actin tagged to a fluorescent protein (FP) did not incorporate into mammalian actin filaments, and rabbit actin-FP did not incorporate into parasite actin filaments. However, exchanging the most divergent region of actin subdomain 3 allowed such reciprocal incorporation. The exchange of a single amino acid residue in subdomain 2 (N41H) of Plasmodium actin markedly improved incorporation into mammalian filaments. In the parasite, modification of most subunit-subunit interaction sites was lethal, whereas changes in actin subdomains 1 and 4 reduced efficient parasite motility and hence mosquito organ penetration. The strong penetration defects could be rescued by overexpression of the actin filament regulator coronin. Through these comparative approaches we identified an essential and common contributor, subdomain 3, which drives the differential dynamic behaviour of two highly divergent eukaryotic actins in motile cells., Competing Interests: The authors have declared that no competing interests exist.

Aims: High levels of glucose and reactive carbonyl intermediates of its degradation pathway such as methylglyoxal (MG) may contribute to diabetic complications partly via increased generation of reactive oxygen species (ROS). This study focused on glutathione peroxidase-1 (GPx1) expression and the impact of carbonylation as an oxidative protein modification on GPx1 abundance and activity in human umbilical vein endothelial cells (HUVEC) under conditions of mild to moderate oxidative stress., Results: High extracellular glucose and MG enhanced intracellular ROS formation in HUVECs. Protein carbonylation was only transiently augmented pointing to an effective antioxidant defense in these cells. Nitric oxide synthase expression was decreased under hyperglycemic conditions but increased upon exposure to MG, whereas superoxide dismutase expression was not significantly affected. Increased glutathione peroxidase (GPx) activity seemed to compensate for a decrease in GPx1 protein due to enhanced degradation via the proteasome. Mass spectrometry analysis identified Lys-114 as a possible carbonylation target which provides a vestibule for the substrate H 2 O 2 and thus enhances the enzymatic reaction., Innovation: Oxidative protein carbonylation has so far been associated with functional inactivation of modified target proteins mainly contributing to aging and age-related diseases. Here, we demonstrate that mild oxidative stress and subsequent carbonylation seem to activate protective cellular redox signaling pathways whereas severe oxidative stress overwhelms the cellular antioxidant defense leading to cell damage., Conclusions: This study may contribute to a better understanding of redox homeostasis and its role in the development of diabetes and related vascular complications., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)