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In the fight against heart failure, therapeutics that have the ability to increase the contractile power of the heart are urgently needed. One possible route of action to improve heart contractile power is increasing the calcium sensitivity of the thin filament. From a pharmaceutical standpoint, calcium sensitizers have the distinct advantage of not altering cardiomyocyte calcium levels and thus have lower potential for side-effects. Small chemical molecules have been shown to bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium-sensitizing properties, possibly by stabilizing cTnC in an open conformation. Building on existing structural data of a known calcium sensitizer bound to cardiac troponin, we combined computational structure-based virtual screening drug discovery methods and solution NMR titration assays to identify a novel calcium sensitizer 4-(4-(2,5-dimethylphenyl)-1-piperazinyl)-3-pyridinamine (NSC147866) which binds to cTnC and the cTnC-cTnI147-163 complex. Its presence increases the affinity of switch peptide to cTnC by approximately a factor of two. This action is comparable to that of known levosimendan analogues. In the fight against heart failure, therapeutics that increase the calcium sensitivity of the thin filament are a promising option to improve heart contractile power. Here, we combined computational drug discovery methods and solution NMR titration assays to identify a novel calcium sensitizer which binds to cTnC and the cTnC-cTnI147-163 complex. Its presence increases the affinity of switch peptide to cTnC by approximately a factor of two, making its action comparable to that of known levosimendan analogues.

Net fluxes of gaseous sulphur compounds, water vapour and carbon dioxide to current- and one-year-old shoots of Scots pine were simultaneously measured in the field. The shoots were fumigated in temperature-controlled assimilation chambers with sulphur dioxide at concentrations ranging from 40 to 250 μg m-3 (15-95 ppb). The hypothesis that stomatal opening regulates the uptake of sulphur dioxide was tested. The following conclusions were reached concerning the dry deposition of sulphur dioxide to a dry coniferous shoot. There was a marked diurnal variation in the uptake rate of sulphur dioxide. The net deposition velocity of sulphur was about three times higher during the day than during the night during July-October. Stomatal opening was not the primary factor controlling the dry deposition rate of sulphur dioxide. The net dry deposition rate was significantly smaller than the calculated rate based on stomatal conductances for water vapour. Part of this deviation could be explained by re-emission of reduced sulphur compounds(s) from the needles. The re-emission of sulphur was a light-dependent process and might be of great significance for the sulphur balance calculations. A variable deposition velocity should be used in atmospheric transport models to account for the diurnal and seasonal variation in the surface resistance of a dry canopy. [ABSTRACT FROM AUTHOR]

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most common and aggressive primary brain tumor with less than 10% of patients surviving beyond 5 years after diagnosis. Even when treated early with the current standard of care, GBM recurs in nearly all patients. Therefore, there is an urgent need to develop novel therapies to overcome treatment resistance in GBM, including those that could be combined with the current standard of care to improve patient outcomes. In previous studies, we identified proteasome subunit alpha type-7 (PSMA7) as a potentially druggable target in GBM. Moreover, we discovered small molecules (SMs), predicted in silico to be blood-brain barrier (BBB)-permeable and target PSMA7, with greater toxicity in GBM cells compared to normal cells. In this study, we further investigated the potential of these SMs to be used as novel therapeutics for the treatment of GBM by examining their ability to inhibit PSMA7 expression, directly bind to PSMA7, block the interaction(s) between PSMA7 with its reported binding partners, and/or disrupt phosphorylation of tyrosine residues on PSMA7. In silico molecular docking screenings were performed using the National Cancer Institute Diversity Set VI SM library and regions of interaction on PSMA7 with its reported binding partners (i.e., HIF-1alpha, Parkin, and PAC4) or regions around two tyrosine residues of PSMA7 (i.e., Y106 and Y153) that are potentially phosphorylated by cABL. Top-scoring SMs were screened using SwissADME to select SMs predicted to be BBB-permeable. Western blots (WB) were performed to examine protein expression. Surface Plasmon Resonance (SPR) was performed using a Biacore S200 to determine real-time quantitative binding affinities and kinetics for interactions between PSMA7 with select SMs. Co-immunoprecipitation (Co-IP) was used to evaluate protein-protein interactions and IP/WB for examining changes to protein phosphorylation. Preliminary in vitro screening of primary human GBM cells with eight SMs predicted to target PSMA7 was performed to identify whether they inhibit PSMA7 protein expression. Western blot analysis revealed that two SMs significantly inhibited PSMA7 in both 08-387 and 3359 cells. SPR data analysis found that three SMs had equilibrium dissociation constant (K D) values < 10-6 M, indicating their direct binding with PSMA7. Co-IP/WB and IP/WB studies were conducted to examine the ability of these three SMs to block interactions with their targeted PSMA7 binding partners and/or inhibit phosphorylation on PSMA7Y106 and PSMA7Y153. Our study identified BBB-permeable SMs that inhibit the biological functions of PSMA7, a potential therapeutic target in GBM. These SMs are potential candidates for in vivo preclinical studies and combining these novel targeted molecules with the standard of care may aid in overcoming treatment resistance in GBM. Our group is actively investigating the binding specificity of these SMs for PSMA7. [ABSTRACT FROM AUTHOR]

Native Mass Spectrometry (nMS) is a versatile technique for elucidating protein structure. Surface-Induced Dissociation (SID) is an activation method in tandem MS predominantly employed for determining protein complex stoichiometry alongside information about interface strengths. SID-nMS data can be collected over a range of acceleration energies, yielding Energy Resolved Mass Spectrometry (ERMS) data. Previous work demonstrated that the onset and appearance energy from SID-nMS can be used in integrative computational and experimental modeling to guide multimeric structure determination in some cases. However, the appearance energy is a single data point, while the ERMS data provide a full pattern of interface breakage. We hypothesized that incorporation of ERMS data into multimeric protein structure prediction would significantly outperform appearance energy. To test this hypothesis, we generated models of 20 protein complexes with RosettaDock using subunits generated from AlphaFold2. We simulated the ERMS data for each predicted model and rescored based on its agreement to experimental ERMS data. We demonstrated that more accurately predicted models exhibited simulated ERMS data in better agreement with the experimental data. As part of our ERMS-based rescoring, we matched or improved the RMSD of the best scoring model compared to Rosetta in 16 out of 20 cases, with 4 out of 20 cases improving to become a highly accurate (below 5 Å) structure. Finally, we benchmarked our method against our previously published appearance energy-based rescoring and showed improvement in 14 out of 20 cases, with 6 out of 20 becoming a highly accurate (below 5 Å) model. Our method is freely available through Rosetta Commons, with a usage tutorial and test files provided in the Supporting Information.

Breast cancer (BC) is the most frequent cancer and second-leading cause of cancer deaths in women in the United States. While RAS mutations are infrequent in BC, triple-negative (TN) and HER2-positive (HER2+) BC both exhibit increased RAS activity. Here, we tested the RAS effectors RALA and RALB, which are overexpressed in BC, as tractable molecular targets in these subtypes. While analysis of the breast cancer patient sample data suggests that the RALs are associated with poor outcome in both TNBC and HER2+ BC, our in vivo and in vitro experimental findings revealed the RALs to be essential in only the TNBC cell lines. While testing the response of the BC cell lines to the RAL inhibitors RBC8 and BQU57, we observed no correlation between drug efficacy and cell line dependency on RAL expression for survival, suggesting that these compounds kill via off-target effects. Finally, we report the discovery of a new small molecule inhibitor, OSURALi, which exhibits strong RAL binding, effectively inhibits RAL activation, and is significantly more toxic to RAL-dependent TNBC cells than RAL-independent HER2+ and normal cell lines. These results support the RALs as viable molecular targets in TNBC and the further investigation of OSURALi as a therapeutic agent.

Multidrug-resistant bacterial infections pose an ever-evolving threat to public health. Since the outset of the antibacterial age, bacteria have developed a multitude of diverse resistance mechanisms that suppress the effectiveness of current therapies. New drug entities, such as Novel Bacterial Topoisomerase Inhibitors (NBTIs), can circumvent this major issue. A computational docking model was employed to predict the binding to DNA gyrase of atypical NBTIs with novel pharmacophores. Synthesis of NBTIs based on computational docking and subsequent antibacterial evaluation against both Gram-positive and Gram-negative bacteria yielded congeners with outstanding anti-staphylococcal activity and varying activity against select Gram-negative pathogens., Competing Interests: The authors declare the following competing financial interest(s): MMF is a shareholder of Pfizer., (© 2024 The Authors. Published by American Chemical Society.)

We used a structure-based drug discovery approach to identify novel inhibitors of human dihydroorotate dehydrogenase (DHODH), which is a therapeutic target for treating cancer and autoimmune and inflammatory diseases. In the case of acute myeloid leukemia, no previously discovered DHODH inhibitors have yet succeeded in this clinical application. Thus, there remains a strong need for new inhibitors that could be used as alternatives to the current standard-of-care. Our goal was to identify novel inhibitors of DHODH. We implemented prefiltering steps to omit PAINS and Lipinski violators at the earliest stages of this project. This enriched compounds in the data set that had a higher potential of favorable oral druggability. Guided by Glide SP docking scores, we found 20 structurally unique compounds from the ChemBridge EXPRESS-pick library that inhibited DHODH with IC 50, DHODH values between 91 nM and 2.7 μM. Ten of these compounds reduced MOLM-13 cell viability with IC 50, MOLM-13 values between 2.3 and 50.6 μM. Compound 16 (IC 50, DHODH = 91 nM) inhibited DHODH more potently than the known DHODH inhibitor, teriflunomide (IC 50, DHODH = 130 nM), during biochemical characterizations and presented a promising scaffold for future hit-to-lead optimization efforts. Compound 17 (IC 50, MOLM-13 = 2.3 μM) was most successful at reducing survival in MOLM-13 cell lines compared with our other hits. The discovered compounds represent excellent starting points for the development and optimization of novel DHODH inhibitors.

Ion mobility coupled to mass spectrometry informs on the shape and size of protein structures in the form of a collision cross section (CCSIM). Although there are several computational methods for predicting CCSIM based on protein structures, including our previously developed projection approximation using rough circular shapes (PARCS), the process usually requires prior experience with the command-line interface. To overcome this challenge, here we present a web application on the Rosetta Online Server that Includes Everyone (ROSIE) webserver to predict CCSIM from protein structure using projection approximation with PARCS. In this web interface, the user is only required to provide one or more PDB files as input. Results from our case studies suggest that CCSIM predictions (with ROSIE-PARCS) are highly accurate with an average error of 6.12%. Furthermore, the absolute difference between CCSIM and CCSPARCS can help in distinguishing accurate from inaccurate AlphaFold2 protein structure predictions. ROSIE-PARCS is designed with a user-friendly interface, is available publicly and is free to use. The ROSIE-PARCS web interface is supported by all major web browsers and can be accessed via this link (https://rosie.graylab.jhu.edu)., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Hypertrophic cardiomyopathy (HCM) is one of the most common heritable cardiovascular diseases and variants of TNNT2 (cardiac troponin T) are linked to increased risk of sudden cardiac arrest despite causing limited hypertrophy. In this study, a TNNT2 variant, R278C +/- , was generated in both human cardiac recombinant/reconstituted thin filaments (hcRTF) and human- induced pluripotent stem cells (hiPSCs) to investigate the mechanisms by which the R278C +/- variant affects cardiomyocytes at the proteomic and functional levels. The results of proteomics analysis showed a significant upregulation of markers of cardiac hypertrophy and remodeling in R278C +/- vs. the isogenic control. Functional measurements showed that R278C +/- variant enhances the myofilament sensitivity to Ca 2+ , increases the kinetics of contraction, and causes arrhythmia at frequencies >75 bpm. This study uniquely shows the profound impact of the TNNT2 R278C +/- variant on the cardiomyocyte proteomic profile, cardiac electrical and contractile function in the early stages of cardiac development., Competing Interests: Competing interests. R.J.S. is a member of the Scientific Advisor Board of Cytokinetics, Inc., and a consultant to Myokardia/Bristol- Myers-Squibb.

In contrast to other mammals, the spiny mouse ( Acomys ) regenerates skin and ear tissue, which includes hair follicles, glands, and cartilage, in a scar-free manner. Ear punch regeneration is asymmetric with only the proximal wound side participating in regeneration. Here, we show that cues originating from the proximal side are required for normal regeneration and use spatially resolved transcriptomics (tomo-seq) to understand the molecular and cellular events underlying this process. Analyzing gene expression across the ear and comparing expression modules between proximal and distal wound sides, we identify asymmetric gene expression patterns and pinpoint regenerative processes in space and time. Moreover, using a comparative approach with nonregenerative rodents ( Mus , Meriones ), we strengthen a hypothesis in which particularities in the injury-induced immune response may be one of the crucial determinants for why spiny mice regenerate whereas their relatives do not. Our data are available in SpinyMine, an easy-to-use and expandable web-based tool for exploring Acomys regeneration-associated gene expression., (© 2023 van Beijnum et al.; Published by Cold Spring Harbor Laboratory Press.)

Amadori rearrangement products are stable sugar-amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose-lysine (F-Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F-Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6-phosphofructose-lysine (6-P-F-Lys). FrlB, a deglycase, then converts 6-P-F-Lys to L-lysine and glucose-6-phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8-Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6-P-F-Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine-6-phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB-6-P-F-Lys on GlmS-fructose-6-phosphate structures revealed parallels in their active-site arrangement and guided our selection of seven putative active-site residues in FrlB for site-directed mutagenesis. Activity assays with eight recombinant single-substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface-induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x-ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure-function and mechanistic studies of enzymes., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function. Here, we examined the effect of our previously identified calcium-sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR-derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This integrated structural-biochemical-physiological approach led to the identification of three novel low-affinity binders, which had similar binding affinities to the known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 μM.

The cardiac thin filament comprises F-actin, tropomyosin, and troponin (cTn). cTn is composed of three subunits: troponin C (cTnC), troponin I (cTnI), and troponin T (cTnT). To computationally study the effect of the thin filament on cTn activation events, we employed targeted molecular dynamics followed by umbrella sampling using a model of the thin filament to measure the thermodynamics of cTn transition events. Our simulations revealed that the thin filament causes an increase in the free energy required to open the cTnC hydrophobic patch and causes a more favorable interaction between this region and the cTnI switch peptide. Mutations to the cTn complex can lead to cardiomyopathy, a collection of diseases that present clinically with symptoms of hypertrophy or dilation of the cardiac muscle, leading to impairment of the heart's ability to function normally and ultimately myocardial infarction or heart failure. Upon introduction of cardiomyopathic mutations to R145 of cTnI, we observed a general decrease in the free energy of opening the cTnC hydrophobic patch, which is on par with previous experimental results. These mutations also exhibited a decrease in electrostatic interactions between cTnI-R145 and actin-E334. After introduction of a small molecule to the wild-type cTnI-actin interface to intentionally disrupt intersubunit contacts, we successfully observed similar thermodynamic consequences and disruptions to the same protein-protein contacts as observed with the cardiomyopathic mutations. Computational studies utilizing the cTn complex in isolation would have been unable to observe these effects, highlighting the importance of using a more physiologically relevant thin-filament model to investigate the global consequences of cardiomyopathic mutations to the cTn complex.

Covalent labeling (CL) in combination with mass spectrometry can be used as an analytical tool to study and determine structural properties of protein-protein complexes. However, data from these experiments is sparse and does not unambiguously elucidate protein structure. Thus, computational algorithms are needed to deduce structure from the CL data. In this work, we present a hybrid method that combines models of protein complex subunits generated with AlphaFold with differential CL data via a CL-guided protein-protein docking in Rosetta. In a benchmark set, the RMSD (root-mean-square deviation) of the best-scoring models was below 3.6 Å for 5/5 complexes with inclusion of CL data, whereas the same quality was only achieved for 1/5 complexes without CL data. This study suggests that our integrated approach can successfully use data obtained from CL experiments to distinguish between nativelike and non-nativelike models., (© 2022. The Author(s).)

Calcium-dependent heart muscle contraction is regulated by the cardiac troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). cNTnC contains one calcium binding site (site II), and altered calcium binding in this site has been studied for decades. It has been previously shown that cNTnC mutants, which increase calcium sensitization may have therapeutic benefits, such as restoring cardiac muscle contractility and functionality post-myocardial infarction events. Here, we computationally characterized eight mutations for their potential effects on calcium binding affinity in site II of cNTnC. We utilized two distinct methods to estimate calcium binding: adaptive steered molecular dynamics (ASMD) and thermodynamic integration (TI). We observed a sensitizing trend for all mutations based on the employed ASMD methodology. The TI results showed excellent agreement with experimentally known calcium binding affinities in wild-type cNTnC. Based on the TI results, five mutants were predicted to increase calcium sensitivity in site II. This study presents an interesting comparison of the two computational methods, which have both been shown to be valuable tools in characterizing the impacts of calcium sensitivity in mutant cNTnC systems.

Cardiac troponin C (cTnC) is the critical Ca 2+ -sensing component of the troponin complex. Binding of Ca 2+ to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)-associated TNNC1 variants generally induce a greater degree and duration of Ca 2+ binding, which may underly the hypertrophic phenotype. Regulation of contraction has long been thought to occur exclusively through Ca 2+ binding to site II of cTnC. However, work by several groups including ours suggest that Mg 2+ , which is several orders of magnitude more abundant in the cell than Ca 2+ , may compete for binding to the same cTnC regulatory site. We previously used isothermal titration calorimetry (ITC) to demonstrate that physiological concentrations of Mg 2+ may decrease site II Ca 2+ -binding in both N-terminal and full-length cTnC. Here, we explore the binding of Ca 2+ and Mg 2+ to cTnC harbouring a series of TNNC1 variants thought to be causal in HCM. ITC and thermodynamic integration (TI) simulations show that A8V, L29Q and A31S elevate the affinity for both Ca 2+ and Mg 2+ . Further, L48Q, Q50R and C84Y that are adjacent to the EF hand binding motif of site II have a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg 2+ in modifying the Ca 2+ affinity of cTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg 2+ both at baseline and when elevated on modifying the Ca 2+ binding properties of cTnC and the subsequent conformational changes which precede cardiac contraction., (© 2022 Federation of European Biochemical Societies.)

The cardiac troponin (cTn) complex is an important regulatory protein in heart contraction. Upon binding of Ca 2+ , cTn undergoes a conformational shift that allows the troponin I switch peptide (cTnI SP ) to be released from the actin filament and bind to the troponin C hydrophobic patch (cTnC HP ). Mutations and modifications to this complex can change its sensitivity to Ca 2+ and alter the energetics of the transition from the Ca 2+ -unbound, cTnI SP -unbound form to the Ca 2+ -bound, cTnI SP -bound form. We utilized targeted molecular dynamics (TMD) to obtain a trajectory of this transition pathway, followed by umbrella sampling to estimate the free energy associated with the cTnI SP -cTnC HP binding and the cTnC HP opening events for wild-type (WT) cTn. We were able to reproduce experimental values for the cTnI SP -cTnC HP binding event and obtain cTnC HP opening free energies in agreement with previous computational measurements of smaller cTnC systems. This excellent agreement for WT cTn demonstrated the strength of computational methods in studying the dynamics and energetics of the cTn complex. We then introduced mutations to the cTn complex that cause cardiomyopathy or alter its Ca 2+ sensitivity and observed a general decrease in the free energy of opening the cTnC HP . For these same mutations, we observed no general trend in the effect on the cTnI SP -cTnC HP binding event. Our method sets the stage for future computational studies on this system that predict the consequences of yet uncharacterized mutations on cTn dynamics and energetics.