Structural Insights into Endostatin-Heparan Sulfate Interactions Using Modeling Approaches.

Glycosaminoglycans (GAGs) play a key role in a variety of biological processes in the extracellular matrix (ECM) via interactions with their protein targets. Due to their high flexibility, periodicity and electrostatics-driven interactions, GAG-containing complexes are very challenging to characterize both experimentally and in silico. In this study, we, for the first time, systematically analyzed the interactions of endostatin, a proteolytic fragment of collagen XVIII known to be anti-angiogenic and anti-tumoral, with heparin (HP) and representative heparan sulfate (HS) oligosaccharides of various lengths, sequences and sulfation patterns. We first used conventional molecular docking and a docking approach based on a repulsive scaling-replica exchange molecular dynamics technique, as well as unbiased molecular dynamic simulations, to obtain dynamically stable GAG binding poses. Then, the corresponding free energies of binding were calculated and the amino acid residues that contribute the most to GAG binding were identified. We also investigated the potential influence of Zn2+ on endostatin-HP complexes using computational approaches. These data provide new atomistic details of the molecular mechanism of HP's binding to endostatin, which will contribute to a better understanding of its interplay with proteoglycans at the cell surface and in the extracellular matrix.

Rational design of glycosaminoglycan binding cyclic peptides using cPEPmatch.

Cyclic peptides present a robust platform for drug design, offering high specificity and stability due to their conformationally constrained structures. In this study, we introduce an updated version of the Cyclic Peptide Matching program (cPEPmatch) tailored for the identification of cyclic peptides capable of mimicking protein-glycosaminoglycan (GAG) binding sites. We focused on engineering cyclic peptides to replicate the GAG-binding affinity of antithrombin III (ATIII), a protein that plays a crucial role in modulating anticoagulation through interaction with the GAG heparin. By integrating computational and experimental methods, we successfully identified a cyclic peptide binder with promising potential for future optimization. MD simulations and MM-GBSA calculations were used to assess binding efficacy, supplemented by umbrella sampling to approximate free energy landscapes. The binding specificity was further validated through NMR and ITC experiments. Our findings demonstrate that the computationally designed cyclic peptides effectively target GAGs, suggesting their potential as novel therapeutic agents. This study advances our understanding of peptide-GAG interactions and lays the groundwork for future development of cyclic peptide-based therapeutics.

Why Do Ionic Surfactants Significantly Alter the Chemiluminogenic Properties of Acridinium Salt?

Acridinium esters, due to their capability for chemiluminescence (CL), are employed as indicators and labels in biomedical diagnostics and other fields. In this work, the influence of ionic surfactants, hexadecyltrimethylammonium chloride and bromide (CTAC and CTAB, cationic) and sodium dodecyl sulphate (SDS, anionic) on the CL parameters and mechanism of representative emitter, 10-methyl-9-[(2-methylphenoxy)carbonyl]acridinium trifluoromethanesulphonate (2MeX) in a H2O2/NaOH environment, is studied. Our investigations revealed that the type of surfactant and its form in solution have an impact on the CL kinetic constants and integral efficiencies, while changes in those emission properties resulting from the type of ion (Cl- vs. Br-) are negligible. The major changes were recorded for systems containing surfactants at concentrations higher than the critical micelle concentration. The cationic surfactants (CTAC, CTAB) cause a substantial increase in CL emission kinetics and a moderate increase in its integral efficiency. At the same time, the opposite effect is observed in the case of SDS. Molecular dynamics simulations suggest that changes in emission parameters are likely due to differences in the binding strength of 2MeX substrate with surfactant molecules, which is higher for SDS than for CTAC. The results can help in rational designing of optimal acridinium CL systems and demonstrate their usefulness in distinguishing the pre- and post-micellar environment and the charge of surfactants.

In silico approaches for better understanding cysteine cathepsin-glycosaminoglycan interactions.

Cysteine cathepsins constitute the largest cathepsin family, with 11 proteases in human that are present primarily within acidic endosomal and lysosomal compartments. They are involved in the turnover of intracellular and extracellular proteins. They are synthesized as inactive procathepsins that are converted to mature active forms. Cathepsins play important roles in physiological and pathological processes and, therefore, receive increasing attention as potential therapeutic targets. Their maturation and activity can be regulated by glycosaminoglycans (GAGs), long linear negatively charged polysaccharides composed of recurring dimeric units. In this review, we summarize recent computational progress in the field of (pro)cathepsin-GAG complexes analyses.

Role for Carboxylic Acid Moiety in NSAIDs: Favoring the Binding at Site II of Bovine Serum Albumin.

The molecular structures of nonsteroidal anti-inflammatory drugs (NSAIDs) vary, but most contain a carboxylic acid functional group (RCOOH). This functional group is known to be related to the mechanism of cyclooxygenase inhibition and also causes side effects, such as gastrointestinal bleeding. This study proposes a new role for RCOOH in NSAIDs: facilitating the interaction at the binding site II of serum albumins. We used bovine serum albumin (BSA) as a model to investigate the interactions with ligands at site II. Using dansyl-proline (DP) as a fluorescent site II marker, we demonstrated that only negatively charged NSAIDs such as ibuprofen (IBP), naproxen (NPX), diflunisal (DFS), and ketoprofen (KTP) can efficiently displace DP from the albumin binding site. We confirmed the importance of RCOO by neutralizing IBP and NPX through esterification, which reduced the displacement of DP. The competition was also monitored by stopped-flow experiments. While IBP and NPX displaced DP in less than 1 s, the ester derivatives were ineffective. We also observed a higher affinity of negatively charged NSAIDs using DFS as a probe and ultrafiltration experiments. Molecular docking simulations showed an essential salt bridge between the positively charged residues Arg409 and Lys413 with RCOO-, consistent with the experimental findings. We performed a ligand dissociation pathway and corresponding energy analysis by applying molecular dynamics. The dissociation of NPX showed a higher free energy barrier than its ester. Apart from BSA, we conducted some experimental studies with human serum albumin, and similar results were obtained, suggesting a general effect for other mammalian serum albumins. Our findings support that the RCOOH moiety affects not only the mechanism of action and side effects but also the pharmacokinetics of NSAIDs.

Elucidation of binding mechanisms of bovine serum albumin and 1-alkylsulfonates with different hydrophobic chain lengths.

In this article, the binding interactions between bovine serum albumin (BSA) and three 1-alkylsulfonates, namely sodium 1-dodecanesulfonate, sodium 1-decanesulfonate, and sodium 1-octanesulfonate, have been thoroughly investigated. The study employed various experimental techniques such as isothermal titration calorimetry (ITC), steady-state fluorescence spectroscopy (SF), circular dichroism spectroscopy (CD), and molecular dynamics-based simulations. The objective was to understand the influence of the alkyl chain length of the investigated ligands on several aspects, including the strength of the interaction, the stoichiometry of the resulting complexes, the number of BSA binding sites, and the underlying mechanisms of binding. Notably, the study also demonstrated that sodium dodecyl sulfate (S12S) can serve as an effective site marker for BSA when studying ligands with similar structural and topological features. These findings may have significant implications for enhancing our understanding of the interactions between small amphiphilic molecules and proteins.

Benchmarking Water Models in Molecular Dynamics of Protein-Glycosaminoglycan Complexes.

Glycosaminoglycans (GAGs) made of repeating disaccharide units intricately engage with proteins, playing a crucial role in the spatial organization of the extracellular matrix (ECM) and the transduction of biological signals in cells to modulate a number of biochemical processes. Exploring protein-GAG interactions reveals several challenges for their analysis, namely, the highly charged and periodic nature of GAGs, their multipose binding, and the abundance of the interfacial water molecules in the protein-GAG complexes. Most of the studies on protein-GAG interactions are conducted using the TIP3P water model, and there are no data on the effect of various water models on the results obtained in molecular dynamics (MD) simulations of protein-GAG complexes. Hence, it is essential to perform a systematic analysis of different water models in MD simulations for these systems. In this work, we aim to evaluate the properties of the protein-GAG complexes in MD simulations using different explicit: TIP3P, SPC/E, TIP4P, TIP4PEw, OPC, and TIP5P and implicit: IGB = 1, 2, 5, 7, and 8 water models to find out which of them are best suited to study the dynamics of protein-GAG complexes. The FF14SB and GLYCAM06 force fields were used for the proteins and GAGs, respectively. The interactions of several GAG types, such as heparin, chondroitin sulfate, and hyaluronic acid with basic fibroblast growth factor, cathepsin K, and CD44 receptor, respectively, are investigated. The observed variations in different descriptors used to study the binding in these complexes emphasize the relevance of the choice of water models for the MD simulation of these complexes.

Detecting Protein-Ligand Interactions with Nitroxide Based Paramagnetic Cosolutes.

NMR spectroscopy techniques can provide important information about protein-ligand interactions. Here we tested an NMR approach which relies on the measurement of paramagnetic relaxation enhancements (PREs) arising from analogous cationic, anionic or neutral soluble nitroxide molecules, which distribute around the protein-ligand complex depending on near-surface electrostatic potentials. We applied this approach to two protein-ligand systems, interleukin-8 interacting with highly charged glycosaminoglycans and the SH2 domain of Grb2 interacting with less charged phospho-tyrosine tripeptides. The electrostatic potential around interleukin-8 and its changes upon binding of glycosaminoglycans could be derived from the PRE data and confirmed by theoretical predictions from Poisson-Boltzmann calculations. The ligand influence on the PREs and NMR-derived electrostatic potentials of Grb2 SH2 was localized to a narrow protein region which allowed the localization of the peptide binding pocket. Our analysis suggests that experiments with nitroxide cosolutes can be useful for investigating protein-ligand electrostatic interactions and mapping ligand binding sites.

Ligand binding of interleukin-8: a comparison of glycosaminoglycans and acidic peptides.

Recognition and binding of regulatory proteins to glycosaminoglycans (GAGs) from the extracellular matrix is a process of high biological importance. The interaction between negatively charged sulfate or carboxyl groups of the GAGs and clusters of basic amino acids on the protein is crucial in this binding process and it is believed that electrostatics represent the key factor for this interaction. However, given the rather undirected nature of electrostatics, it is important to achieve a clear understanding of its role in protein-GAG interactions and how specificity and selectivity in these systems can be achieved, when the classical key-lock binding motif is not applicable. Here, we compare protein binding of a highly charged heparin (HP) hexasaccharide with four de novo designed decapeptides of varying negative net charge. The charge density of these peptides was comparable to typical GAGs of the extracellular matrix. We used the regulatory protein interleukin-8 (IL-8) because its interactions with GAGs are well described. All four peptide ligands bind to the same epitope of IL-8 but show much weaker binding affinity as revealed in 1H-15N HSQC NMR titration experiments. Complementary molecular docking and molecular dynamics simulations revealed further atomistic details of the interaction mode of GAG versus peptide ligands. Overall, similar contributions to the binding energy and hydrogen bond formation are determined for HP and the highly charged peptides, suggesting that the entropic loss of the peptides upon binding likely account for the remarkably different affinity of GAG versus peptide ligands to IL-8.

Extension of the SUGRES-1P Coarse-Grained Model of Polysaccharides to Heparin.

Heparin is an unbranched periodic polysaccharide composed of negatively charged monomers and involved in key biological processes, including anticoagulation, angiogenesis, and inflammation. Its structure and dynamics have been studied extensively using experimental as well as theoretical approaches. The conventional approach of computational chemistry applied to the analysis of biomolecules is all-atom molecular dynamics, which captures the interactions of individual atoms by solving Newton's equation of motion. An alternative is molecular dynamics simulations using coarse-grained models of biomacromolecules, which offer a reduction of the representation and consequently enable us to extend the time and size scale of simulations by orders of magnitude. In this work, we extend the UNIfied COarse-gRaiNed (UNICORN) model of biological macromolecules developed in our laboratory to heparin. We carried out extensive tests to estimate the optimal weights of energy terms of the effective energy function as well as the optimal Debye-Hückel screening factor for electrostatic interactions. We applied the model to study unbound heparin molecules of polymerization degree ranging from 6 to 68 residues. We compare the obtained coarse-grained heparin conformations with models obtained from X-ray diffraction studies of heparin. The SUGRES-1P force field was able to accurately predict the general shape and global characteristics of heparin molecules.

Insights into the interaction of human serum albumin with ionic liquids - Thermodynamic, spectroscopic and molecular modelling studies.

Human serum albumin (HSA) effectively binds different types of low-molecular-weight compounds and thus enables their distribution in living organisms. Recently, it has been reported that the protein-ligand interactions play a crucial role in bioaccumulation processes and provide an important sorption phase, especially for ionogenic compounds. Therefore, the binding interactions of such compounds with proteins are the subject of an ongoing interest in environmental and life sciences. In this paper, the influence of some counter-ions, namely [B(CN)4]- and [C(CN)3]- on the affinity of the [IM1-12]+ towards HSA has been investigated and discussed based on experimental methods (isothermal titration calorimetry and steady-state fluorescence spectroscopy) and molecular dynamics-based computational approaches. Furthermore, the thermal stability of the resulting HSA/ligand complexes was assessed using DSC and CD spectroscopy. As an outcome of the work, it has been ascertained that the protein is able to bind simultaneously the ligands under study but in different regions of HSA. Thus, the presence in the system of [IM1-12]+ does not disturb the binding of [C(CN)3]- and [B(CN)4]-. The presented results provide important information on the presence of globular proteins and some ionogenic compounds in the distribution and bioaccumulation of ILs in the environment and living organisms.

Cyclic Peptidic Furin Inhibitors Developed by Combinatorial Chemistry.

Furin is a human serine protease responsible for activating numerous physiologically relevant cell substrates and is also involved in the development of various pathological conditions, including inflammatory diseases, cancers, and viral and bacterial infections. Therefore, compounds with the ability to inhibit furin's proteolytic action are regarded as potential therapeutics. Here we took the combinatorial chemistry approach (library consisting of 2000 peptides) to obtain new, strong, and stable peptide furin inhibitors. The extensively studied trypsin inhibitor SFTI-1 was used as a leading structure. A selected monocylic inhibitor was further modified to finally yield five mono- or bicyclic furin inhibitors with values of K i in the subnanomolar range. Inhibitor 5 was the most active (K i = 0.21 nM) and significantly more proteolytically resistant than the reference furin inhibitor described in the literature. Moreover, it reduced furin-like activity in PANC-1 cell lysate. Detailed analysis of furin-inhibitor complexes using molecular dynamics simulations is also reported.

Glycosaminoglycans: What Remains To Be Deciphered?

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics.

Computational modeling of the molecular basis for the calcium-dependence of the mannuronan C-5 epimerase AvAlgE6 from Azotobacter vinelandii.

The mannuronan C-5 epimerases catalyze epimerization of ß-d-mannuronic acid to α-l-guluronic acid in alginate polymers. The seven extracellular Azotobacter vinelandii epimerases (AvAlgE1-7) are calcium-dependent, and calcium is essential for the structural integrity of their carbohydrate binding R-modules. Ca2+ is also found in the crystal structures of the A-modules, where it is suggested to play a structural role. In this study, the structure of the catalytic A-module of the A. vinelandii mannuronan C-5 epimerase AvAlgE6 is used to investigate the role of this Ca2+. Molecular dynamics (MD) simulations with and without calcium reveal the possible importance of the bound Ca2+ in the hydrophobic packing of ß-sheets. In addition, a putative calcium binding site is found in the active site, indicating a potential direct role of this calcium in the catalysis. According to the literature, two of the residues coordinating calcium in this site are essential for the activity. MD simulations of the interaction with bound substrate indicate that the presence of a calcium ion in this binding site increases the binding strength. Further, explicit calculations of the substrate dissociation pathways with umbrella sampling simulations show and energetically higher dissociation barrier when calcium is present. The present study eludes to a putative catalytic role of calcium in the charge neutralizing first step of the enzymatic reaction. In addition to the importance for understanding these enzymes' molecular mechanisms, this could have implications for engineering strategies of the epimerases in industrial alginate processing.

Solvent Model Benchmark for Molecular Dynamics of Glycosaminoglycans.

In computational studies of glycosaminoglycans (GAGs), a group of anionic, periodic linear polysaccharides, so far there has been very little discussion about the role of solvent models in the molecular dynamics simulations of these molecules. Predominantly, the TIP3P water model is commonly used as one of the most popular explicit water models in general. However, there are numerous alternative explicit and implicit water models that are neglected in the computational research of GAGs. Since solvent-mediated interactions are particularly important for GAG dynamic and structural properties, it would be of great interest for the GAG community to establish the solvent model that is suited the best in terms of the quality of theoretically obtained GAG parameters and, at the same time, would be reasonably demanding in terms of computational resources required. In this study, heparin (HP) was simulated using five implicit and six explicit solvent models with the aim to find out how different solvent models influence HP's molecular descriptors in the molecular dynamics simulations. Here, we initiate the search for the most appropriate solvent representation for GAG systems and we hope to encourage other groups to contribute to this highly relevant subject.

Molecular Dynamics-Based Comparative Analysis of Chondroitin and Dermatan Sulfates.

Glycosaminoglycans (GAGs) are a class of linear anionic periodic polysaccharides containing disaccharide repetitive units. These molecules interact with a variety of proteins in the extracellular matrix and so participate in biochemically crucial processes such as cell signalling affecting tissue regeneration as well as the onset of cancer, Alzheimer's or Parkinson's diseases. Due to their flexibility, periodicity and chemical heterogeneity, often termed "sulfation code", GAGs are challenging molecules both for experiments and computation. One of the key questions in the GAG research is the specificity of their intermolecular interactions. In this study, we make a step forward to deciphering the "sulfation code" of chondroitin sulfates-4,6 (CS4, CS6, where the numbers correspond to the position of sulfation in NAcGal residue) and dermatan sulfate (DS), which is different from CSs by the presence of IdoA acid instead of GlcA. We rigorously investigate two sets of these GAGs in dimeric, tetrameric and hexameric forms with molecular dynamics-based descriptors. Our data clearly suggest that CS4, CS6 and DS are substantially different in terms of their structural, conformational and dynamic properties, which contributes to the understanding of how these molecules can be different when they bind proteins, which could have practical implications for the GAG-based drug design strategies in the regenerative medicine.

Simultaneous approximation of a smooth function and its derivatives by deep neural networks with piecewise-polynomial activations.

This paper investigates the approximation properties of deep neural networks with piecewise-polynomial activation functions. We derive the required depth, width, and sparsity of a deep neural network to approximate any Hölder smooth function up to a given approximation error in Hölder norms in such a way that all weights of this neural network are bounded by 1. The latter feature is essential to control generalization errors in many statistical and machine learning applications.

Investigation of hexacyanoferrate(II)/(III) charge-dependent interactions with bovine and human serum albumins.

In the present paper, the binding interactions of highly negative-charged ions, namely hexacyanoferrates(II/III), i.e. [Fe(CN)6]4- and [Fe(CN)6]3- with bovine and human serum albumins (BSA and HSA, respectively) have been studied for the first time in an aqueous solution (10 mM cacodylate buffer of pH 7.0) using steady-state fluorescence spectroscopy, isothermal titration calorimetry, and CD spectroscopy supported by molecular dynamics-based computational approaches. The Stern-Volmer equation as well as its modifications suggested that hexacyanoferrates(II/III) effectively quenched the intrinsic fluorescence of the albumins through a static mechanism. The proteins under study possess only one binding site on the surface capable of binding one mole of hexacyanoferrates(II/III) ions per one mole of albumin (HSA or BSA). The formation of albumin complexes is an enthalpy-driven process (|ΔHITC| > |TΔSITC|). The strength of the interactions depends mainly on the type of albumin, and changes as follows: BSA-K3[Fe(CN)6] âˆ¼ BSA-K4[Fe(CN)6] > HSA-K3[Fe(CN)6] âˆ¼ HSA-K4[Fe(CN)6]. Finally, potential binding sites of bovine and human serum albumins have been investigated and discussed based on a competitive fluorescence displacement assay (with warfarin and ibuprofen as site markers) and molecular dynamics simulations.

In silico insights into procathepsin S maturation mediated by glycosaminoglycans.

Procathepsins, inactive precursors of cathepsins are present in the extracellular matrix (ECM) and in lysosomes. Their active forms are involved in a number of biologically relevant processes, including bone resorption, intracellular proteolysis and regulation of programmed cell death. These processes might be mediated by glycosaminoglycans (GAGs), long unbranched periodic negatively charged polysaccharides. GAGs are also present in ECM and play important role in anticoagulation, angiogenesis and tissue regeneration. GAGs not only mediate the enzymatic activity of cathepsins but can also regulate the process of procathepsin maturation, as it was shown for procathepsin B and S. In this study, we propose the molecular mechanism underlying the biological role of GAGs in procathepsin S maturation and compare our findings with computational data obtained for procathepsin B. We rigorously analyse procathepsin S-GAG complexes in terms of their dynamics, free energy and potential allosteric regulation. We conclude that the GAG binding region might have an effect on the dynamics of procathepsin S structure and so affect its maturation by two different mechanisms.

Repulsive Scaling Replica Exchange Molecular Dynamics in Modeling Protein-Glycosaminoglycan Complexes.

Glycosaminoglycans are long linear periodic anionic polysaccharides consisting of disaccharide units exhibiting different sulfation patterns forming a highly heterogeneous group of molecules. Due to their flexibility, length, high charge, and periodicity, they are challenging for computational approaches. Despite their biological significance in terms of the important role in various diseases (e.g., Alzheimer, cancer, SARS-CoV-2) and proper cell functioning (e.g., proliferation, maturation), there is a lack of effective molecular docking tools designed specifically for glycosaminoglycans due to their challenging physical-chemical nature. In this chapter we present protocols for the Repulsive Scaling Replica Exchange Molecular Dynamics (RS-REMD) methods to dock glycosaminoglycans with both implicit and explicit solvent models implemented. This novel molecular dynamics-based replica exchange technique should help to elevate our current knowledge on the complexes and interactions between glycosaminoglycans and their protein receptors.