From Linear to Nets: Multiconfiguration Polymer Structure Generation with PolyFlin.

Molecular modeling and simulations are essential tools in polymer science and engineering, enabling researchers to predict and understand the properties of macromolecules, including their structure, dynamics, thermodynamics, and overall material characteristics. However, one of the key challenges in polymer simulation and modeling lies in the initial topology design, as existing programs often lack the capability to generate all types of polymer forms. In this study, we present PolyFlin, a powerful Python module that addresses this limitation by allowing the generation of a wide range of polymer structures, from simple homopolymers to complex copolymers, including grafts, cyclic, star, dendrimers, and nets. PolyFlin offers a versatile and efficient tool for exploring and creating diverse polymer architectures, facilitating advancements in various fields that require precise polymer modeling and simulation.

A promising synthetic citric crosslinked ß-cyclodextrin derivative for antifungal drugs: Solubilization, cytotoxicity, and antifungal activity.

Effective antifungal therapy for the treatment of fungal keratitis requires a high drug concentration at the corneal surface. However, the use of natural ß-cyclodextrin (ßCD) in the preparation of aqueous eye drop formulations for treating fungal keratitis is limited by its low aqueous solubility. Here, we synthesized water-soluble anionic ßCD derivatives capable of forming water-soluble complexes and evaluated the solubility, cytotoxicity, and antifungal efficacy of drug prepared using the ßCD derivative. To achieve this, a citric acid crosslinked ßCD (polyCTR-ßCD) was successfully synthesized, and the aqueous solubilities of selected antifungal drugs, including voriconazole, miconazole (MCZ), itraconazole, and amphotericin B, in polyCTR-ßCD and analogous ßCD solutions were evaluated. Among the drugs tested, complexation of MCZ with polyCTR-ßCD (MCZ/polyCTR-ßCD) increased MCZ aqueous solubility by 95-fold compared with that of MCZ/ßCD. The inclusion complex formation of MCZ/ßCD and MCZ/polyCTR-ßCD was confirmed by spectroscopic techniques. Additionally, the nanoaggregates of saturated MCZ/polyCTR-ßCD and MCZ/ßCD solutions were observed using dynamic light scattering and transmission electron microscopy. Moreover, MCZ/polyCTR-ßCD solution exhibited good mucoadhesion, sustained drug release, and high drug permeation of porcine cornea ex vivo. Hen's Egg test-chorioallantoic membrane assay and cell viability study using Statens Seruminstitut Rabbit Cornea cell line showed that both MCZ/polyCTR-ßCD and MCZ/ßCD exhibited no sign of irritation and non-toxic to cell line. Additionally, antifungal activity evaluation demonstrated that all isolated fungi, including Candida albicans, Aspergillus flavus, and Fusarium solani, were susceptible to MCZ/polyCTR-ßCD. Overall, the results showed that polyCTR-ßCD could be a promising nanocarrier for the ocular delivery of MCZ.

CHO-produced RBD-Fc subunit vaccines with alternative adjuvants generate immune responses against SARS-CoV-2.

Subunit vaccines feature critical advantages over other vaccine platforms such as stability, price, and minimal adverse effects. To maximize immunological protection of subunit vaccines, adjuvants are considered as main components that are formulated within the subunit vaccine. They can modulate adverse effects and enhance immune outcomes. However, the most suitable formulation providing the best immunological outcomes and safety are still under investigation. In this report, we combined recombinant RBD with human IgG1 Fc to create an RBD dimer. This fusion protein was expressed in CHO and formulated with alternative adjuvants with different immune activation including Montanide ISA51, Poly (I:C), and MPLA/Quil-A® as potential vaccine candidate formulations. Using the murine model, a potent induction of anti-RBD IgG antibodies in immunized mice sera were observed. IgG subclass analyses (IgG1/IgG2a) illustrated that all adjuvanted formulations could stimulate both Th1 and Th2-type immune responses in particular Poly (I:C) and MPLA/Quil-A®, eliciting greater balance. In addition, Montanide ISA51-formulated RBD-Fc vaccination provided a promising level of neutralizing antibodies against live wild-type SARS-CoV-2 in vitro followed by Poly (I:C) and MPLA/Quil-A®, respectively. Also, mice sera from adjuvanted formulations could strongly inhibit RBD:ACE2 interaction. This study offers immunogenicity profiles, forecasted safety based on Vaccine-associated enhanced disease (VAED) caused by Th1-skewed immunity, and neutralizing antibody analysis of candidates of RBD-Fc-based subunit vaccine formulations to obtain an alternative subunit vaccine formulation against SARS-CoV-2.

Structure-based computational screening of 470 natural quercetin derivatives for identification of SARS-CoV-2 Mpro inhibitor.

Coronavirus disease 2019 (COVID-19) is a global pandemic infecting the respiratory system through a notorious virus known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to viral mutations and the risk of drug resistance, it is crucial to identify new molecules having potential prophylactic or therapeutic effect against SARS-CoV-2 infection. In the present study, we aimed to identify a potential inhibitor of SARS-CoV-2 through virtual screening of a compound library of 470 quercetin derivatives by targeting the main protease-Mpro (PDB ID: 6LU7). The study was carried out with computational techniques such as molecular docking simulation studies (MDSS), molecular dynamics (MD) simulations, and molecular mechanics generalized Born surface area (MMGBSA) techniques. Among the natural derivatives, compound 382 (PubChem CID 65604) showed the best binding affinity to Mpro (-11.1 kcal/mol). Compound 382 interacted with LYS5, TYR126, GLN127, LYS137, ASP289, PHE291, ARG131, SER139, GLU288, and GLU290 of the Mpro protein. The SARS-CoV-2 Mpro-382 complex showed acceptable stability during the 100 ns MD simulations. The SARS-CoV-2 Mpro-382 complex also showed an MM-GBSA binding free energy value of -54.0 kcal/mol. The binding affinity, stability, and free energy results for 382 and Mpro were better than those of the native ligand and the standard inhibitors ledipasvir and cobicistat. The conclusion of our study was that compound 382 has the potential to inhibit SARS-Cov-2 Mpro. However, further investigations such as in-vitro assays are recommended to confirm its in-silico potency.

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry.

Characterizing a protein's higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein's structure by MS, specific chemical reactions are performed in solution that encode a protein's structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as ß2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

Utilization of Hydrophobic Microenvironment Sensitivity in Diethylpyrocarbonate Labeling for Protein Structure Prediction.

Diethylpyrocarbonate (DEPC) labeling analyzed with mass spectrometry can provide important insights into higher order protein structures. It has been previously shown that neighboring hydrophobic residues promote a local increase in DEPC concentration such that serine, threonine, and tyrosine residues are more likely to be labeled despite low solvent exposure. In this work, we developed a Rosetta algorithm that used the knowledge of labeled and unlabeled serine, threonine, and tyrosine residues and assessed their local hydrophobic environment to improve protein structure prediction. Additionally, DEPC-labeled histidine and lysine residues with higher relative solvent accessible surface area values (i.e., more exposed) were scored favorably. Application of our score term led to reductions of the root-mean-square deviations (RMSDs) of the lowest scoring models. Additionally, models that scored well tended to have lower RMSDs. A detailed tutorial describing our protocol and required command lines is included. Our work demonstrated the considerable potential of DEPC covalent labeling data to be used for accurate higher order structure determination.

Complementary Structural Information for Stressed Antibodies from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry.

Identifying changes in the higher-order structure (HOS) of therapeutic monoclonal antibodies upon storage, stress, or mishandling is important for ensuring efficacy and avoiding adverse effects. Here, we demonstrate diethylpyrocarbonate (DEPC)-based covalent labeling (CL) mass spectrometry (MS) and hydrogen-deuterium exchange (HDX)/MS can be used together to provide site-specific information about subtle conformational changes that are undetectable by traditional techniques. Using heat-stressed rituximab as a model protein, we demonstrate that CL/MS is more sensitive than HDX/MS to subtle HOS structural changes under low stress conditions (e.g., 45 and 55 °C for 4 h). At higher heat stress (65 °C for 4 h), we find CL/MS and HDX/MS provide complementary information, as CL/MS reports on changes in side chain orientation while HDX/MS reveals changes in backbone dynamics. More interestingly, we demonstrate that the two techniques work synergistically to identify likely aggregation sites in the heat-stressed protein. In particular, the CH3 and CL domains experience decreases in deuterium uptake after heat stress, while only the CH3 domain experiences decreases in DEPC labeling extent as well, suggesting the CH3 domain is a likely site of aggregation and the CL domain only undergoes a decrease in backbone dynamics. The combination of DEPC-CL/MS and HDX/MS provides valuable structural information, and the two techniques should be employed together when investigating the HOS of protein therapeutics.

Potential drug-drug interactions of antiretrovirals and antimicrobials detected by three databases.

Standard treatment for HIV infection involves a combination of antiretrovirals. Additionally, opportunistic infections in HIV infected patients require further antimicrobial medications that might cause drug-drug interactions (DDIs). The objective of this study was to to compare the recognition of DDIs between antiretrovirals and antimicrobials by three proprietary databases and evaluate their concordance. 114 items of antiretrovirals and antimicrobials from the National List of Essential Medicines of Thailand 2018 were used in the study. However, 21 items were not recognised by Micromedex, Drugs.com, and Liverpool HIV interactions. Only 93 items were available for the detection of potential DDIs by the three databases. Potential DDIs detected from the three databases included 292 pairs. Liverpool showed the highest number of DDIs with 285 pairs compared with 259 pairs by drugs.com and 133 pairs by Micromedex. Regarding the severity classifications, Liverpool reported 10% Contraindicated; Micromedex reported 14% contraindicated and 59% major; Drugs.com reported 21% major. The Fleiss' kappa agreements were fair to poor among the three databases, higher agreement was observed for DDIs classified as severe. This study highlights the need to harmonize the evaluation and interpretation of DDI risk in order to produce standardized information to support prescribers.

Covalent Labeling/Mass Spectrometry of Monoclonal Antibodies with Diethylpyrocarbonate: Reaction Kinetics for Ensuring Protein Structural Integrity.

Diethylpyrocarbonate (DEPC)-based covalent labeling together with mass spectrometry is a promising tool for the higher-order structural analysis of antibody therapeutics. Reliable information about antibody higher-order structure can be obtained, though, only when the protein's structural integrity is preserved during labeling. In this work, we have evaluated the applicability of DEPC reaction kinetics for ensuring the structural integrity of monoclonal antibodies (mAbs) during labeling. By monitoring the modification extent of selected proteolytic fragments as a function of DEPC concentration, we find that a common DEPC concentration can be used for different monoclonal antibodies in formulated samples without perturbing their higher-order structure. Under these labeling conditions, we find that the antibodies can accommodate up to four DEPC modifications without being structurally perturbed, indicating that multidomain proteins can withstand more than one label, which contrasts to previously studied single-domain proteins. This more extensive labeling provides a more sensitive measure of structure, making DEPC-based covalent labeling-mass spectrometry suitable for the higher-order structural analyses of mAbs.

Covalent Labeling with an α,ß-Unsaturated Carbonyl Scaffold for Studying Protein Structure and Interactions by Mass Spectrometry.

A new covalent labeling (CL) reagent based on an α,ß-unsaturated carbonyl scaffold has been developed for studying protein structure and protein-protein interactions when coupled with mass spectrometry. We show that this new reagent scaffold can react with up to 13 different types of residues on protein surfaces, thereby providing excellent structural resolution. To illustrate the value of this reagent scaffold, it is used to identify the residues involved in the protein-protein interface that is formed upon Zn(II) binding to the protein ß-2-microglobulin. The modular design of the α,ß-unsaturated carbonyl scaffold allows facile variation of the functional groups, enabling labeling kinetics and selectivity to be tuned. Moreover, by introducing isotopically enriched functional groups into the reagent structure, labeling sites can be more easily identified by MS and MS/MS. Overall, this reagent scaffold should be a valuable CL reagent for protein higher order structure characterization by MS.

Higher-Order Structure Influences the Kinetics of Diethylpyrocarbonate Covalent Labeling of Proteins.

The combination of covalent labeling (CL) and mass spectrometry (MS) has emerged as a useful tool for studying protein structure due to its good structural coverage, the ability to study proteins in mixtures, and its high sensitivity. Diethylpyrocarbonate (DEPC) is an effective CL reagent that can label N-termini and the side chains of several nucleophilic residues, providing information for about 30% of the residues in the average protein. For DEPC to provide accurate structural information, the extent of labeling must be controlled to minimize label-induced structural perturbations. In this work, we establish a quantitative correlation between general protein structural factors and DEPC reaction rates by measuring the reaction rate coefficients for several model proteins. Using principal component and regression analyses, we find that the solvent accessible surface areas of histidine and lysine residues in proteins are the primary factors that determine a protein's reactivity toward DEPC, despite the fact that other more abundant residues, such as tyrosine, threonine, and serine, are also labeled by DEPC. From the statistical analysis, a model emerges that can be used to predict the reactivity of a protein based on its structure and sequence, allowing the optimal DEPC concentration to be chosen for a given protein. The resulting model is supported by cross-validation studies and by accurately predicting of the reactivity of five test proteins. Overall, our model reveals interesting insight into the reactivity of proteins with DEPC, and it will facilitate identification of optimal DEPC labeling conditions for proteins.

Synergistic Structural Information from Covalent Labeling and Hydrogen-Deuterium Exchange Mass Spectrometry for Protein-Ligand Interactions.

Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) and covalent labeling (CL) MS are typically considered to be complementary methods for protein structural analysis, because one probes the protein backbone, while the other probes side chains. For protein-ligand interactions, we demonstrate in this work that the two labeling techniques can provide synergistic structural information about protein-ligand binding when reagents like diethylpyrocarbonate (DEPC) are used for CL because of the differences in the reaction rates of DEPC and HDX. Using three model protein-ligand systems, we show that the slower time scale for DEPC labeling makes it only sensitive to changes in solvent accessibility and insensitive to changes in protein structural fluctuations, whereas HDX is sensitive to changes in both solvent accessibility and structural fluctuations. When used together, the two methods more clearly reveal binding sites and ligand-induced changes to structural fluctuations that are distant from the binding site, which is more comprehensive information than either technique alone can provide. We predict that these two methods will find widespread usage together for more deeply understanding protein-ligand interactions.

Covalent Labeling with Diethylpyrocarbonate: Sensitive to the Residue Microenvironment, Providing Improved Analysis of Protein Higher Order Structure by Mass Spectrometry.

Covalent labeling with mass spectrometry is increasingly being used for the structural analysis of proteins. Diethylpyrocarbonate (DEPC) is a simple to use, commercially available covalent labeling reagent that can readily react with a range of nucleophilic residues in proteins. We find that in intact proteins weakly nucleophilic side chains (Ser, Thr, and Tyr) can be modified by DEPC in addition to other residues such as His, Lys, and Cys, providing very good structural resolution. We hypothesize that the microenvironment around these side chains, as formed by a protein's higher order structure, tunes their reactivity such that they can be labeled. To test this hypothesis, we compare DEPC labeling reactivity of Ser, Thr, and Tyr residues in intact proteins with peptide fragments from the same proteins. Results indicate that these residues almost never react with DEPC in free peptides, supporting the hypothesis that a protein's local microenvironment tunes the reactivity of these residues. From a close examination of the structural features near the reactive residues, we find that nearby hydrophobic residues are essential, suggesting that the enhanced reactivity of certain Ser, Thr, and Tyr residues occurs due to higher local concentrations of DEPC.

Covalent labeling and mass spectrometry reveal subtle higher order structural changes for antibody therapeutics.

Monoclonal antibodies are among the fastest growing therapeutics in the pharmaceutical industry. Detecting higher-order structure changes of antibodies upon storage or mishandling, however, is a challenging problem. In this study, we describe the use of diethylpyrocarbonate (DEPC)-based covalent labeling (CL) - mass spectrometry (MS) to detect conformational changes caused by heat stress, using rituximab as a model system. The structural resolution obtained from DEPC CL-MS is high enough to probe subtle conformation changes that are not detectable by common biophysical techniques. Results demonstrate that DEPC CL-MS can detect and identify sites of conformational changes at the temperatures below the antibody melting temperature (e.g., 55 á´¼C). The observed labeling changes at lower temperatures are validated by activity assays that indicate changes in the Fab region. At higher temperatures (e.g., 65 á´¼C), conformational changes and aggregation sites are identified from changes in CL levels, and these results are confirmed by complementary biophysical and activity measurements. Given the sensitivity and simplicity of DEPC CL-MS, this method should be amenable to the structural investigations of other antibody therapeutics.

Covalent labeling-mass spectrometry with non-specific reagents for studying protein structure and interactions.

Using mass spectrometry (MS) to obtain information about a higher order structure of protein requires that a protein's structural properties are encoded into the mass of that protein. Covalent labeling (CL) with reagents that can irreversibly modify solvent accessible amino acid side chains is an effective way to encode structural information into the mass of a protein, as this information can be read-out in a straightforward manner using standard MS-based proteomics techniques. The differential reactivity of proteins under two or more conditions can be used to distinguish protein topologies, conformations, and/or binding sites. CL-MS methods have been effectively used for the structural analysis of proteins and protein complexes, particularly for systems that are difficult to study by other more traditional biochemical techniques. This review provides an overview of the non-specific CL approaches that have been combined with MS with a particular emphasis on the reagents that are commonly used, including hydroxyl radicals, carbenes, and diethylpyrocarbonate. We describe the reagent and protein factors that affect the reactivity of amino acid side chains. We also include details about experimental design and workflow, data analysis, recent applications, and some future prospects of CL-MS methods.

Disruption of the open conductance in the ß-tongue mutants of Cytolysin A.

Cytolysin A (ClyA) is a water-soluble alpha pore-forming toxin that assembles to form an oligomeric pore on host cell membranes. The ClyA monomer possesses an α-helical bundle with a ß-sheet subdomain (the ß-tongue) previously believed to be critical for pore assembly and/or insertion. Oligomerization of ClyA pores transforms the ß-tongue into a helix-turn-helix that embeds into the lipid bilayer. Here, we show that mutations of the ß-tongue did not prevent oligomerization or transmembrane insertion. Instead, ß-tongue substitution mutants yielded pores with decreased conductance while a deletion mutation resulted in pores that rapidly closed following membrane association. Our results suggest that the ß-tongue may play an essential structural role in stabilizing the open conformation of the transmembrane domain.

Simultaneous determination of curcumin diethyl disuccinate and its active metabolite curcumin in rat plasma by LC-MS/MS: Application of esterase inhibitors in the stabilization of an ester-containing prodrug.

Four esterase inhibitors, ethylenediamine tetraacetic acid disodium (Na2EDTA), sodium fluoride (NaF), bis(4-nitrophenyl) phosphate (BNPP) and phenylmethanesulfonyl fluoride (PMSF), were evaluated for their inhibitory effects on enzymatic hydrolysis of labile phenolate esters in curcumin diethyl disuccinate (CDD), a prodrug of curcumin (CUR), in rat plasma. BNPP and PMSF at 10mM exhibited stabilization by preventing degradation of CDD. BNPP at a final concentration of 10mM was subsequently selected to prevent ex vivo metabolism of CDD throughout LC-MS/MS analysis of CDD and CUR in rat plasma. A simple protein precipitation technique using acetonitrile as a precipitating agent was used to extract CDD, CUR and dimethylcurcumin (DMC), an internal standard, from rat plasma. Chromatographic separation was performed on a Halo C8 column (4.6×50mm, 2.7µm) using an isocratic mobile phase containing acetonitrile-0.2% formic acid in water (73:27v/v) with a flow rate of 0.4mLmin(-1). An AB SCIEX QTRAP(®) 6500 mass spectrometer was operated using a positive ion electrospray mode for ionization and detection of analytes and internal standard. Calibration curves for CDD and CUR were established using 50µL of rat plasma over the concentration range of 1-500ngmL(-1). The developed method was fully validated according to US Food and Drug Administration (FDA) guidelines for selectivity, sensitivity, linearity, accuracy, precision, dilution integrity, recovery, matrix effect, and stability. The validated method was applied to evaluate the pharmacokinetics of CDD and CUR in rats after a single intravenous dose of 40mgkg(-1). The method using BNPP as an esterase inhibitor was successful in determining the remaining CDD in rat plasma. The pharmacokinetic results indicate that CDD in rats is converted instantaneously to CUR after intravenous administration and a higher CUR plasma concentration at 5min is achieved in comparison with direct intravenous injection of CUR.