Substitution models of protein evolution with selection on enzymatic activity

Substitution models of evolution are necessary for diverse evolutionary analyses including phylogenetic tree and ancestral sequence reconstructions. At the protein level, empirical substitution models are traditionally used due to their simplicity, but they ignore the variability of substitution patterns among protein sites. Next, in order to improve the realism of the modeling of protein evolution, a series of structurally constrained substitution models were presented, but still they usually ignore constraints on the protein activity. Here, we present a substitution model of protein evolution with selection on both protein structure and enzymatic activity, and that can be applied to phylogenetics. In particular, the model considers the binding affinity of the enzyme–substrate complex as well as structural constraints that include the flexibility of structural flaps, hydrogen bonds, amino acids backbone radius of gyration, and solvent-accessible surface area that are quantified through molecular dynamics simulations. We applied the model to the HIV-1 protease and evaluated it by phylogenetic likelihood in comparison with the best-fitting empirical substitution model and a structurally constrained substitution model that ignores the enzymatic activity. We found that accounting for selection on the protein activity improves the fitting of the modeled functional regions with the real observations, especially in data with high molecular identity, which recommends considering constraints on the protein activity in the development of substitution models of evolution.Agencia Estatal de Investigación | Ref. PID2019-107931GA-I00Xunta de Galicia | Ref. ED481A-2020/192Fundação para a Ciência e a TecnologiaUniversidade de Vigo/CISU

The evolution of the HIV-1 protease folding stability

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGThe evolution of structural proteins is generally constrained by the folding stability. However, little is known about the particular capacity of viral proteins to accommodate mutations that can potentially affect the protein stability and, in general, the evolution of the protein stability over time. As an illustrative model case, here, we investigated the evolution of the stability of the human immunodeficiency virus (HIV-1) protease (PR), which is a common HIV-1 drug target, under diverse evolutionary scenarios that include (1) intra-host virus evolution in a cohort of seventy-five patients sampled over time, (2) intra-host virus evolution sampled before and after specific PR-based treatments, and (3) inter-host evolution considering extant and ancestral (reconstructed) PR sequences from diverse HIV-1 subtypes. We also investigated the specific influence of currently known HIV-1 PR resistance mutations on the PR folding stability. We found that the HIV-1 PR stability fluctuated over time within a constant and wide range in any studied evolutionary scenario, accommodating multiple mutations that partially affected the stability while maintaining activity. We did not identify relationships between change of PR stability and diverse clinical parameters such as viral load, CD4+ T-cell counts, and a surrogate of time from infection. Counterintuitively, we predicted that nearly half of the studied HIV-1 PR resistance mutations do not significantly decrease stability, which, together with compensatory mutations, would allow the protein to adapt without requiring dramatic stability changes. We conclude that the HIV-1 PR presents a wide structural plasticity to acquire molecular adaptations without affecting the overall evolution of stability.Ministerio de Economía y Competitividad | Ref. RYC-2015-18241Xunta de Galicia | Ref. ED431F 2018/08Xunta de Galicia | Ref. ED481A-2020/19

Substitution models of protein evolution with selection on enzymatic activity

<p>Substitution models of evolution are required for diverse evolutionary analyses including phylogenetic tree and ancestral sequence reconstructions. At the protein level, empirical substitution models have been commonly used in phylogenetics due to their simplicity, but they assume the same substitution patterns for all the protein sites. In order to improve the realism of the modeling of protein evolution, a series of substitution models that account for selection on the protein structure were presented but they usually ignore constraints on the protein activity, which can also be informative for modeling protein evolution. Here we present a substitution model of protein evolution that considers selection on both, the protein structure and enzymatic activity, and that can be applied to phylogenetics. In particular, the model considers the binding affinity of the enzyme-substrate complex and diverse structural constraints such as flexibility of structural flaps, hydrogen bonds, amino acids backbone radius of gyration and solvent-accessible surface area that are quantified through molecular dynamics simulations. We applied the model to the HIV-1 protease and evaluate it by phylogenetic likelihood in comparison with the best-fitting empirical substitution model and a structurally constrained substitution model that ignores the enzymatic activity. We found that accounting for selection on the enzymatic activity improves the fitting of the modeling in sites related with the enzymatic activity and especially for data with high molecular similarity.</p&gt

Substitution models of protein evolution with selection on enzymatic activity

Substitution models of evolution are required for diverse evolutionary analyses including phylogenetic tree and ancestral sequence reconstructions. At the protein level, empirical substitution models have been commonly used in phylogenetics due to their simplicity, but they assume the same substitution patterns for all the protein sites. In order to improve the realism of the modeling of protein evolution, a series of substitution models that account for selection on the protein structure were presented but they usually ignore constraints on the protein activity, which can also be informative for modeling protein evolution. Here we present a substitution model of protein evolution that considers selection on both, the protein structure and enzymatic activity, and that can be applied to phylogenetics. In particular, the model considers the binding affinity of the enzyme-substrate complex and diverse structural constraints such as flexibility of structural flaps, hydrogen bonds, amino acids backbone radius of gyration and solvent-accessible surface area that are quantified through molecular dynamics simulations. We applied the model to the HIV-1 protease and evaluate it by phylogenetic likelihood in comparison with the best-fitting empirical substitution model and a structurally constrained substitution model that ignores the enzymatic activity. We found that accounting for selection on the enzymatic activity improves the fitting of the modeling in sites related with the enzymatic activity and especially for data with high molecular similarity

Substitution models of protein evolution with selection on enzymatic activity

<p>Substitution models of evolution are required for diverse evolutionary analyses including phylogenetic tree and ancestral sequence reconstructions. At the protein level, empirical substitution models have been commonly used in phylogenetics due to their simplicity, but they assume the same substitution patterns for all the protein sites. In order to improve the realism of the modeling of protein evolution, a series of substitution models that account for selection on the protein structure were presented but they usually ignore constraints on the protein activity, which can also be informative for modeling protein evolution. Here we present a substitution model of protein evolution that considers selection on both, the protein structure and enzymatic activity, and that can be applied to phylogenetics. In particular, the model considers the binding affinity of the enzyme-substrate complex and diverse structural constraints such as flexibility of structural flaps, hydrogen bonds, amino acids backbone radius of gyration and solvent-accessible surface area that are quantified through molecular dynamics simulations. We applied the model to the HIV-1 protease and evaluate it by phylogenetic likelihood in comparison with the best-fitting empirical substitution model and a structurally constrained substitution model that ignores the enzymatic activity. We found that accounting for selection on the enzymatic activity improves the fitting of the modeling in sites related with the enzymatic activity and especially for data with high molecular similarity.</p&gt

Virtual Screening, Synthesis and Biological Evaluation of Streptococcus mutans Mediated Biofilm Inhibitors

Dental caries, a global oral health concern, is a biofilm-mediated disease. Streptococcus mutans, the most prevalent oral microbiota, produces extracellular enzymes, including glycosyltransferases responsible for sucrose polymerization. In bacterial communities, the biofilm matrix confers resistance to host immune responses and antibiotics. Thus, in cases of chronic dental caries, inhibiting bacterial biofilm assembly should prevent demineralization of tooth enamel, thereby preventing tooth decay. A high throughput screening was performed in the present study to identify small molecule inhibitors of S. mutans glycosyltransferases. Multiple pharmacophore models were developed, validated with multiple datasets, and used for virtual screening against large chemical databases. Over 3000 drug-like hits were obtained that were analyzed to explore their binding mode. Finally, six compounds that showed good binding affinities were further analyzed for ADME (absorption, distribution, metabolism, and excretion) properties. The obtained in silico hits were evaluated for in vitro biofilm formation. The compounds displayed excellent antibiofilm activities with minimum inhibitory concentration (MIC) values of 15.26–250 µg/mL

Voriconazole Cyclodextrin Based Polymeric Nanobeads for Enhanced Solubility and Activity: In Vitro/In Vivo and Molecular Simulation Approach

Hydroxypropyl β-cyclodextrin (HPβCD) based polymeric nanobeads containing voriconazole (VRC) were fabricated by free radical polymerization using N, N′-methylene bisacrylamide (MBA) as a cross-linker, 2-acrylamide-2-methylpropane sulfonic acid (AMPS) as monomer and ammonium persulfate (APS) as reaction promoter. Optimized formulation (CDN5) had a particle size of 320 nm with a zeta potential of −35.5 mV and 87% EE. Scanning electron microscopy (SEM) depicted porous and non-spherical shaped beads. No evidence of chemical interaction was evident in FT-IR studies, whereas distinctive high-intensity VRC peaks were found superimposed in XRD. A stable polymeric network formation was evident in DSC studies owing to a lower breakdown in VRC loaded HPβCD in comparison to blank HPβCD. In vitro release studies showed 91 and 92% drug release for optimized formulation at pH 1.2 and 6.8, respectively, with first-order kinetics as the best-fit model and non-Fickian diffusion as the release mechanism. No evidence of toxicity was observed upon oral administration of HPβCD loaded VRC polymeric nanobeads owing to with cellular morphology of vital organs as observed in histopathology. Molecular docking indicates the amalgamation of the compounds highlighting the hydrophobic patching mediated by nanogel formulation. It can be concluded that the development of polymeric nanobeads can be a promising tool to enhance the solubility and efficacy of hydrophobic drugs such as VRC besides decreased toxicity and for effective management of fungal infections

Voriconazole Cyclodextrin Based Polymeric Nanobeads for Enhanced Solubility and Activity: In Vitro/In Vivo and Molecular Simulation Approach

Hydroxypropyl β-cyclodextrin (HPβCD) based polymeric nanobeads containing voriconazole (VRC) were fabricated by free radical polymerization using N, N′-methylene bisacrylamide (MBA) as a cross-linker, 2-acrylamide-2-methylpropane sulfonic acid (AMPS) as monomer and ammonium persulfate (APS) as reaction promoter. Optimized formulation (CDN5) had a particle size of 320 nm with a zeta potential of −35.5 mV and 87% EE. Scanning electron microscopy (SEM) depicted porous and non-spherical shaped beads. No evidence of chemical interaction was evident in FT-IR studies, whereas distinctive high-intensity VRC peaks were found superimposed in XRD. A stable polymeric network formation was evident in DSC studies owing to a lower breakdown in VRC loaded HPβCD in comparison to blank HPβCD. In vitro release studies showed 91 and 92% drug release for optimized formulation at pH 1.2 and 6.8, respectively, with first-order kinetics as the best-fit model and non-Fickian diffusion as the release mechanism. No evidence of toxicity was observed upon oral administration of HPβCD loaded VRC polymeric nanobeads owing to with cellular morphology of vital organs as observed in histopathology. Molecular docking indicates the amalgamation of the compounds highlighting the hydrophobic patching mediated by nanogel formulation. It can be concluded that the development of polymeric nanobeads can be a promising tool to enhance the solubility and efficacy of hydrophobic drugs such as VRC besides decreased toxicity and for effective management of fungal infections