Preparative Biocatalytic Synthesis of α-Ketomethylselenobutyrate-A Putative Agent for Cancer Therapy.

Biomedical studies of the role of organic selenium compounds indicate that the amino acid derivative of L-selenomethionine, α-ketomethylselenobutyrate (KMSB), can be considered a potential anticancer therapeutic agent. It was noted that, in addition to a direct effect on redox signaling molecules, α-ketoacid metabolites of organoselenium compounds are able to change the status of histone acetylation and suppress the activity of histone deacetylases in cancer cells. However, the wide use of KMSB in biomedical research is hindered not only by its commercial unavailability, but also by the fact that there is no detailed information in the literature on possible methods for the synthesis of this compound. This paper describes in detail the procedure for obtaining a high-purity KMSB preparation (purity ≥ 99.3%) with a yield of the target product of more than 67%. L-amino acid oxidase obtained from C. adamanteus was used as a catalyst for the conversion of L-selenomethionine to KMSB. If necessary, this method can be used as a basis both for scaling up the synthesis of KMSB and for developing cost-effective biocatalytic technologies for obtaining other highly purified drugs.

Natural Guanine Derivatives Exert PARP-Inhibitory and Cytoprotective Effects in a Model of Cardiomyocyte Damage under Oxidative Stress.

Inhibitors of human poly(ADP-ribose) polymerase (PARP) are considered as promising agents for treatment of cardiovascular, neurological, and other diseases accompanied by inflammation and oxidative stress. Previously, the ability of natural compounds 7-methylguanine (7mGua) and 8-hydroxy-7-methylguanine (8h7mGua) to suppress activity of the recombinant PARP protein was demonstrated. In the present work, we have investigated the possibility of PARP-inhibitory and cytoprotective action of 7mGua and 8h7mGua against the rat cardiomyoblast cultures (undifferentiated and differentiated H9c2). It was found that 7mGua and 8h7mGua rapidly penetrate into the cells and effectively suppress the H2O2-stimulated PARP activation (IC50 = 270 and 55 µM, respectively). The pronounced cytoprotective effects of 7mGua and 8h7mGua were shown in a cellular model of oxidative stress, and effectiveness of 8h7mGua exceeded the classic PARP inhibitor 3-aminobenzamide. The obtained data indicate promise for the development of PARP inhibitors based on guanine derivatives and their testing using the models of ischemia-reperfusion tissue damage.

Conformational transitions induced by NADH binding promote reduction half-reaction in 2-hydroxybiphenyl-3-monooxygenase catalytic cycle.

2-Hydroxybiphenyl-3-monoxygenase from Pseudomonas azelaica is an effective catalyst of the regiospecific conversions of various aromatic compounds. A comprehensive understanding of the complete catalytic cycle, including the as yet unclear details of NADH binding, NADH/FAD interaction as well as related conformational changes could facilitate the rational design of improved enzyme variants for practical applications. Induced fit formation of a specific pocket for the nicotinamide ring at NADH binding has been revealed using advanced molecular simulation methods including metadynamics and QM/MM modeling. The resulting triple stacking interaction of the nicotinamide as well as isoalloxazine rings and evolutionarily correlated amino acid residues of the active site greatly contributes to the stabilization of the charge-transfer complex and determines the Pro-S stereospecificity of the hydride transfer and the low energy barrier 11 kcal/mol. Then the resulting FADH- anion undergoes the consequent conformational transition of the FAD isoalloxazine ring from the open out to the closed in position which is followed by the binding of an oxygen molecule what is crucial for the next step of substrate oxidation and the completion of the catalytic cycle.

Inhibitory Effects of 7-Methylguanine and Its Metabolite 8-Hydroxy-7-Methylguanine on Human Poly(ADP-Ribose) Polymerase 1.

Previously, we have found that a nucleic acid metabolite, 7-methylguanine (7mGua), produced in the body can have an inhibitory effect on the poly(ADP-ribose) polymerase 1 (PARP1) enzyme, an important pharmacological target in anticancer therapy. In this work, using an original method of analysis of PARP1 activity based on monitoring fluorescence anisotropy, we studied inhibitory properties of 7mGua and its metabolite, 8-hydroxy-7-methylguanine (8h7mGua). Both compounds inhibited PARP1 enzymatic activity in a dose-dependent manner, however, 8h7mGua was shown to be a stronger inhibitor. The IC50 values for 8h7mGua at different concentrations of the NAD+ substrate were found to be 4 times lower, on average, than those for 7mGua. The more efficient binding of 8h7mGua in the PARP1 active site is explained by the presence of an additional hydrogen bond with the Glu988 catalytic residue. Experimental and computational studies did not reveal the effect of 7mGua and 8h7mGua on the activity of other DNA repair enzymes, indicating selectivity of their inhibitory action.

Toxicological Properties of 7-Methylguanine, and Preliminary Data on its Anticancer Activity.

7-Methylguanine (7-MG) competitively inhibits the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) and RNA-modifying enzyme tRNA-guanine transglycosylase (TGT) and represents a potential anticancer drug candidate. Furthermore, as a natural compound, it could escape the serious side effects characteristic for approved synthetic PARP inhibitors. Here we present a comprehensive study of toxicological and carcinogenic properties of 7-MG. It was demonstrated that 7-MG does not induce mutations or structural chromosomal abnormalities, and has no blastomogenic activity. A treatment regimen with 7-MG has been established in mice (50 mg/kg per os, 3 times per week), exerting no adverse effects or changes in morphology. Preliminary data on the 7-MG anticancer activity obtained on transplantable tumor models support our conclusions that 7-MG can become a promising new component of chemotherapy.

Modeling the Structure of Human tRNA-Guanine Transglycosylase in Complex with 7-Methylguanine and Revealing the Factors that Determine the Enzyme Interaction with Inhibitors.

tRNA-guanine transglycosylase, an enzyme catalyzing replacement of guanine with queuine in human tRNA and participating in the translation mechanism, is involved in the development of cancer. However, information on the small-molecule inhibitors that can suppress activity of this enzyme is very limited. Molecular dynamics simulations were used to determine the amino acid residues that provide efficient binding of inhibitors in the active site of tRNA-guanine transglycosylase. It was demonstrated using 7-methylguanine molecule as a probe that the ability of the inhibitor to adopt a charged state in the environment of hydrogen bond acceptors Asp105 and Asp159 plays a key role in complex formation. Formation of the hydrogen bonds and hydrophobic contacts with Gln202, Gly229, Phe109, and Met259 residues are also important. It has been predicted that introduction of the substituents would have a different effect on the ability to inhibit tRNA-guanine transglycosylase, as well as the DNA repair protein poly(ADP-ribose) polymerase 1, which can contribute to the development of more efficient and selective compounds.

Soft Tissue Sarcoma Study: Association of Genetic Alterations in the Apoptosis Pathways with Chemoresistance to Doxorubicin.

Soft tissue sarcomas (STS) are heterogeneous cancers with more than 100 histological subtypes, different in molecular alterations, which make its personalized therapy very complex. Gold standard of chemotherapy for advanced STS includes combinations of Doxorubicin and Ifosfamide or Gemcitabine and Docetaxel. Chemotherapy is efficient for less than 50% of patients and it is followed by a fast development of drug resistance. Our study was directed to the search of genetic alterations in cancer cells associated with chemoresistance of undifferentiated pleomorphic and synovial sarcomas to the abovementioned genotoxic drugs. We analyzed chemoresistance of cancer cells in vitro using primary STS cultures and performed genetic analysis for the components of apoptotic signaling. In 27% of tumors, we revealed alterations in TP53, ATM, PIK3CB, PIK3R1, NTRK1, and CSF2RB. Cells from STS specimens with found genetic alterations were resistant to Dox, excluding the only one case when TP53 mutation resulted in the substitution Leu344Arg associated with partial oligomerization loss and did not cause total loss of TP53 function. Significant association between alterations in the components of apoptosis signaling and chemoresistance to Dox was found. Our data are important to elaborate further the therapeutic strategy for STS patients with alterations in apoptotic signaling.

Guide tree optimization with genetic algorithm to improve multiple protein 3D-structure alignment.

MOTIVATION: With the increasing availability of 3D-data, the focus of comparative bioinformatic analysis is shifting from protein sequence alignments toward more content-rich 3D-alignments. This raises the need for new ways to improve the accuracy of 3D-superimposition. RESULTS: We proposed guide tree optimization with genetic algorithm (GA) as a universal tool to improve the alignment quality of multiple protein 3D-structures systematically. As a proof of concept, we implemented the suggested GA-based approach in popular Matt and Caretta multiple protein 3D-structure alignment (M3DSA) algorithms, leading to a statistically significant improvement of the TM-score quality indicator by up to 220-1523% on 'SABmark Superfamilies' (in 49-77% of cases) and 'SABmark Twilight' (in 59-80% of cases) datasets. The observed improvement in collections of distant homologies highlights the potentials of GA to optimize 3D-alignments of diverse protein superfamilies as one plausible tool to study the structure-function relationship. AVAILABILITY AND IMPLEMENTATION: The source codes of patched gaCaretta and gaMatt programs are available open-access at https://github.com/n-canter/gamaps. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Bifunctional Inhibitors of Influenza Virus Neuraminidase: Molecular Design of a Sulfonamide Linker.

The growing resistance of the influenza virus to widely used competitive neuraminidase inhibitors occupying the active site of the enzyme requires the development of bifunctional compounds that can simultaneously interact with other regulatory sites on the protein surface. When developing such an inhibitor and combining structural fragments that could be located in the sialic acid cavity of the active site and the adjacent 430-cavity, it is necessary to select a suitable linker not only for connecting the fragments, but also to ensure effective interactions with the unique arginine triad Arg118-Arg292-Arg371 of neuraminidase. Using molecular modeling, we have demonstrated the usefulness of the sulfonamide group in the linker design and the potential advantage of this functional group over other isosteric analogues.

Preparative Biocatalytic Synthesis of α-Ketoglutaramate.

α-Ketoglutaramate (KGM) is an underexamined metabolite of L-glutamine in the metabolic pathway of glutaminase II of α-ketoglutarate formation. Presumably, KGM may be a biomarker of hepatic encephalopathy and other hyperammonemic diseases. This metabolite is a substrate for the ω-amidase enzyme and is used to determine its activity in the study of the biochemistry of various types of cancer. However, the commercial unavailability of KGM hinders its widespread use. Methods for the preparative synthesis of KGM are known, but they either do not provide the proper yield or proper purity of the target product. In this work, a detailed description of the procedures is given that allows the production of KGM with a purity above 97% and a yield of the target product above 75% using L-amino acid oxidase from C. adamanteus as a catalyst of L-glutamine conversion. KGM can be obtained both in the form of a highly concentrated aqueous solution and in the form of crystals of sodium salt. The developed methods can be used both for scaling up the synthesis of KGM and for creating economical biocatalytic technologies for the production of other highly purified preparations.

Prospects of Using Biocatalysis for the Synthesis and Modification of Polymers.

Trends in the dynamically developing application of biocatalysis for the synthesis and modification of polymers over the past 5 years are considered, with an emphasis on the production of biodegradable, biocompatible and functional polymeric materials oriented to medical applications. The possibilities of using enzymes not only as catalysts for polymerization but also for the preparation of monomers for polymerization or oligomers for block copolymerization are considered. Special attention is paid to the prospects and existing limitations of biocatalytic production of new synthetic biopolymers based on natural compounds and monomers from biomass, which can lead to a huge variety of functional biomaterials. The existing experience and perspectives for the integration of bio- and chemocatalysis in this area are discussed.

Design, Synthesis, and Molecular Docking Study of New Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Inhibitors Combining Resin Acids and Adamantane Moieties.

In this paper, a series of novel abietyl and dehydroabietyl ureas, thioureas, amides, and thioamides bearing adamantane moieties were designed, synthesized, and evaluated for their inhibitory activities against tyrosil-DNA-phosphodiesterase 1 (TDP1). The synthesized compounds were able to inhibit TDP1 at micromolar concentrations (0.19-2.3 µM) and demonstrated low cytotoxicity in the T98G glioma cell line. The effect of the terpene fragment, the linker structure, and the adamantane residue on the biological properties of the new compounds was investigated. Based on molecular docking results, we suppose that adamantane derivatives of resin acids bind to the TDP1 covalent intermediate, forming a hydrogen bond with Ser463 and hydrophobic contacts with the Phe259 and Trp590 residues and the oligonucleotide fragment of the substrate.

Bioinformatic Analysis of the Nicotinamide Binding Site in Poly(ADP-Ribose) Polymerase Family Proteins.

The PARP family consists of 17 members with diverse functions, including those related to cancer cells' viability. Several PARP inhibitors are of great interest as innovative anticancer drugs, but they have low selectivity towards distinct PARP family members and exert serious adverse effects. We describe a family-wide study of the nicotinamide (NA) binding site, an important functional region in the PARP structure, using comparative bioinformatic analysis and molecular modeling. Mutations in the NA site and D-loop mobility around the NA site were identified as factors that can guide the design of selective PARP inhibitors. Our findings are of particular importance for the development of novel tankyrase (PARPs 5a and 5b) inhibitors for cancer therapy.

Bioinformatic analysis of subfamily-specific regions in 3D-structures of homologs to study functional diversity and conformational plasticity in protein superfamilies.

Local 3D-structural differences in homologous proteins contribute to functional diversity observed in a superfamily, but so far received little attention as bioinformatic analysis was usually carried out at the level of amino acid sequences. We have developed Zebra3D - the first-of-its-kind bioinformatic software for systematic analysis of 3D-alignments of protein families using machine learning. The new tool identifies subfamily-specific regions (SSRs) - patterns of local 3D-structure (i.e. single residues, loops, or secondary structure fragments) that are spatially equivalent within families/subfamilies, but are different among them, and thus can be associated with functional diversity and function-related conformational plasticity. Bioinformatic analysis of protein superfamilies by Zebra3D can be used to study 3D-determinants of catalytic activity and specific accommodation of ligands, help to prepare focused libraries for directed evolution or assist development of chimeric enzymes with novel properties by exchange of equivalent regions between homologs, and to characterize plasticity in binding sites. A companion Mustguseal web-server is available to automatically construct a 3D-alignment of functionally diverse proteins, thus reducing the minimal input required to operate Zebra3D to a single PDB code. The Zebra3D + Mustguseal combined approach provides the opportunity to systematically explore the value of SSRs in superfamilies and to use this information for protein design and drug discovery. The software is available open-access at https://biokinet.belozersky.msu.ru/Zebra3D.

Catalytic and lectin domains in neuraminidase A from Streptococcus pneumoniae are capable of an intermolecular assembly: Implications for biofilm formation.

Neuraminidase A from Streptococcus pneumoniae (NanA) is a cell wall-bound modular enzyme containing one lectin and one catalytic domain. Unlike homologous NanB and NanC expressed by the same bacterium, the two domains within one NanA molecule do not form a stable interaction and are spatially separated by a 16-amino acid-long flexible linker. In this work, the ability of NanA to form intermolecular assemblies was characterized using the methods of molecular modeling and bioinformatic analysis based on crystallographic data and by bringing together previously published experimental data. It was concluded that two catalytic domains, as well as one catalytic and one lectin domain, originating from two cell wall-bound NanA molecules, can interact through a previously uncharacterized interdomain interface to form complexes stabilized by a network of intermolecular hydrogen bonds and salt bridges. Supercomputer modeling strongly indicated that artocarpin, an earlier experimentally discovered inhibitor of the pneumococcal biofilm formation, is able to bind to a site located in the catalytic domain of one NanA entity and prevent its interaction with the lectin or catalytic domain of another NanA entity, thus directly precluding the generation of intermolecular assemblies. The revealed structural adaptation is discussed as one plausible mechanism of noncatalytic participation of this potentially key pathogenicity enzyme in pneumococcal biofilm formation.

Mustguseal and Sister Web-Methods: A Practical Guide to Bioinformatic Analysis of Protein Superfamilies.

Bioinformatic analysis of functionally diverse superfamilies can help to study the structure-function relationship in proteins, but represents a methodological challenge. The Mustguseal web-server can build large structure-guided sequence alignments of thousands of homologs that cover all currently available sequence variants within a common structural fold. The input to the method is a PDB code of the query protein, which represents the protein superfamily of interest. The collection and subsequent alignment of protein sequences and structures is fully automated and driven by the particular choice of parameters. Four integrated sister web-methods-the Zebra, pocketZebra, visualCMAT, and Yosshi-are available to further analyze the resulting superimposition and identify conserved, subfamily-specific, and co-evolving residues, as well as to classify and study disulfide bonds in protein superfamilies. The integration of these web-based bioinformatic tools provides an out-of-the-box easy-to-use solution, first of its kind, to study protein function and regulation and design improved enzyme variants for practical applications and selective ligands to modulate their functional properties. In this chapter, we provide a step-by-step protocol for a comprehensive bioinformatic analysis of a protein superfamily using a web-browser as the main tool and notes on selecting the appropriate values for the key algorithm parameters depending on your research objective. The web-servers are freely available to all users at https://biokinet.belozersky.msu.ru/m-platform with no login requirement.

vsFilt: A Tool to Improve Virtual Screening by Structural Filtration of Docking Poses.

The ability of ligands to form crucial interactions with a protein target, characteristic for the substrate and/or inhibitors, could be considered a structural criterion for identifying potent binders among docked compounds. Structural filtration of predicted poses improves the performance of virtual screening and helps in recovering specifically bound ligands. Here, we present vsFilt-a highly automated and easy-to-use Web server for postdocking structural filtration. The new tool can detect various types of interactions that are known to be involved in the molecular recognition, including hydrogen and halogen bonds, ionic interactions, hydrophobic contacts, π-stacking, and cation-π interactions. A case study for poly(ADP-ribose) polymerase 1 ligands illustrates the utility of the software. The Web server is freely available at https://biokinet.belozersky.msu.ru/vsfilt.

EasyAmber: A comprehensive toolbox to automate the molecular dynamics simulation of proteins.

Conformational plasticity of the functionally important regions and binding sites in protein/enzyme structures is one of the key factors affecting their function and interaction with substrates/ligands. Molecular dynamics (MD) can address the challenge of accounting for protein flexibility by predicting the time-dependent behavior of a molecular system. It has a potential of becoming a particularly important tool in protein engineering and drug discovery, but requires specialized training and skills, what impedes practical use by many investigators. We have developed the easyAmber - a comprehensive set of programs to automate the molecular dynamics routines implemented in the Amber package. The toolbox can address a wide set of tasks in computational biology struggling to account for protein flexibility. The automated workflow includes a complete set of steps from the initial "static" molecular model to the MD "production run": the full-atom model building, optimization/equilibration of the molecular system, classical/conventional and accelerated molecular dynamics simulations. The easyAmber implements advanced MD protocols, but is highly automated and easy-to-operate to attract a broad audience. The toolbox can be used on a personal desktop station equipped with a compatible gaming GPU-accelerator, as well as help to manage huge workloads on a powerful supercomputer. The software provides an opportunity to operate multiple simulations of different proteins at the same time, thus significantly increasing work efficiency. The easyAmber takes the molecular dynamics to the next level in terms of usability for complex processing of large volumes of data, thus supporting the recent trend away from inefficient "static" approaches in biology toward a deeper understanding of the dynamics in protein structures. The software is freely available for download at https://biokinet.belozersky.msu.ru/easyAmber, no login required.

Zebra2: advanced and easy-to-use web-server for bioinformatic analysis of subfamily-specific and conserved positions in diverse protein superfamilies.

Zebra2 is a highly automated web-tool to search for subfamily-specific and conserved positions (i.e. the determinants of functional diversity as well as the key catalytic and structural residues) in protein superfamilies. The bioinformatic analysis is facilitated by Mustguseal-a companion web-server to automatically collect and superimpose a large representative set of functionally diverse homologs with high structure similarity but low sequence identity to the selected query protein. The results are automatically prioritized and provided at four information levels to facilitate the knowledge-driven expert selection of the most promising positions on-line: as a sequence similarity network; interfaces to sequence-based and 3D-structure-based analysis of conservation and variability; and accompanied by the detailed annotation of proteins accumulated from the integrated databases with links to the external resources. The integration of Zebra2 and Mustguseal web-tools provides the first of its kind out-of-the-box open-access solution to conduct a systematic analysis of evolutionarily related proteins implementing different functions within a shared 3D-structure of the superfamily, determine common and specific patterns of function-associated local structural elements, assist to select hot-spots for rational design and to prepare focused libraries for directed evolution. The web-servers are free and open to all users at https://biokinet.belozersky.msu.ru/zebra2, no login required.

Molecular Mechanisms of PARP-1 Inhibitor 7-Methylguanine.

7-Methylguanine (7-MG), a natural compound that inhibits DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1), can be considered as a potential anticancer drug candidate. Here we describe a study of 7-MG inhibition mechanism using molecular dynamics, fluorescence anisotropy and single-particle Förster resonance energy transfer (spFRET) microscopy approaches to elucidate intermolecular interactions between 7-MG, PARP-1 and nucleosomal DNA. It is shown that 7-MG competes with substrate NAD+ and its binding in the PARP-1 active site is mediated by hydrogen bonds and nonpolar interactions with the Gly863, Ala898, Ser904, and Tyr907 residues. 7-MG promotes formation of the PARP-1-nucleosome complexes and suppresses DNA-dependent PARP-1 automodification. This results in nonproductive trapping of PARP-1 on nucleosomes and likely prevents the removal of genotoxic DNA lesions.