Biochemical and cellular insights into the Baz2B protein, a non-catalytic subunit of the chromatin remodeling complex.

Baz2B is a regulatory subunit of the ATP-dependent chromatin remodeling complexes BRF1 and BRF5, which control access to DNA during DNA-templated processes. Baz2B has been implicated in several diseases and also in unhealthy ageing, however limited information is available on the domains and cellular roles of Baz2B. To gain more insight into the Baz2B function, we biochemically characterized the TAM (Tip5/ARBP/MBD) domain with the auxiliary AT-hook motifs and the bromodomain (BRD). We observed alterations in histone code recognition in bromodomains carrying cancer-associated point mutations, suggesting their potential involvement in disease. Furthermore, the depletion of Baz2B in the Hap1 cell line resulted in altered cell morphology, reduced colony formation and perturbed transcriptional profiles. Despite that, super-resolution microscopy images revealed no changes in the overall chromatin structure in the absence of Baz2B. These findings provide insights into the biological function of Baz2B.

Enhanced nucleosome assembly at CpG sites containing an extended 5-methylcytosine analogue.

Methylation of cytosine to 5-methylcytosine (mC) at CpG sites is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the attached methyl groups can alter local structure of DNA and chromatin as well as binding of dedicated proteins. Nucleosome assembly on methylated DNA has been studied extensively, however little is known how the chromatin structure is affected by larger chemical variations in the major groove of DNA. Here, we studied the nucleosome formation in vitro on DNA containing an extended 5mC analog, 5-(6-azidohex-2-ynyl)cytosine (ahyC) installed at biological relevant CpG sites. We found that multiple ahyC residues on 80-Widom and Hsp70 promoter DNA fragments proved compatible with nucleosome assembly. Moreover, unlike mC, ahyC increases the affinity of histones to the DNA, partially altering nucleosome positioning, stability, and the action of chromatin remodelers. Based on molecular dynamics calculations, we suggest that these new features are due to increased DNA flexibility at ahyC-modified sites. Our findings provide new insights into the biophysical behavior of modified DNA and open new ways for directed design of synthetic nucleosomes.

Polyester Dendrimers Based on Bis-MPA for Doxorubicin Delivery.

Although doxorubicin (DOX) is one of the most used chemotherapeutic drugs due to its efficacy against a wide group of cancer types, it presents severe side effects. As such, intensive research is being carried out to find new nanoscale systems that can help to overcome this problem. Polyester dendrimers based on the monomer 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) are very promising systems for biomedical applications due to their biodegradability properties. In this study, bis-MPA-based dendrimers were, for the first time, evaluated as DOX delivery vehicles. Generations 4 and 5 of bis-MPA-based dendrimers with hydroxyl groups at the surface were used (B-G4-OH and B-G5-OH), together with dendrimers partially functionalized with amine groups (B-G4-NH2/OH and B-G5-NH2/OH). Partial functionalization was chosen because the main purpose was to compare the effect of different functional groups on dendrimers' drug delivery behavior without compromising cell viability, which is often affected by dendrimers' cationic charge. Results revealed that bis-MPA-based dendrimers were cytocompatible, independently of the chemical groups that were present at their surface. The B-G4-NH2/OH and B-G5-NH2/OH dendrimers were able to retain a higher number of DOX molecules, but the in vitro release of the drug was faster. On the contrary, the hydroxyl-terminated dendrimers exhibited a lower loading capacity but were able to deliver the drug in a more sustained manner. These results were in accordance with the cytotoxicity studies performed in several models of cancer cell lines and human mesenchymal stem cells. Overall, the results confirmed that it is possible to tune the drug delivery properties of bis-MPA-based dendrimers by modifying surface functionalization. Moreover, molecular modeling studies provided insights into the nature of the interactions established between the drug and the bis-MPA-based dendrimers─DOX molecules attach to their surface rather than being physically encapsulated.

Structure and mechanism of secondary sulfonamide binding to carbonic anhydrases.

Zinc-containing metalloenzyme carbonic anhydrase (CA) binds primary sulfonamides with extremely high, up to picomolar, affinity by forming a coordination bond between the negatively charged amino group and the zinc ion and making hydrogen bonds and hydrophobic contacts with other parts of the inhibitor molecule. However, N-methyl-substituted, secondary or tertiary sulfonamides bind CA with much lower affinity. In search for an explanation for this diminished affinity, a series of secondary sulfonamides were synthesized and, together with analogous primary sulfonamides, the affinities for 12 recombinant catalytically active human CA isoforms were determined by the fluorescent thermal shift assay, stopped-flow assay of the inhibition of enzymatic activity and isothermal titration calorimetry. The binding profile of secondary sulfonamides as a function of pH showed the same U-shape dependence seen for primary sulfonamides. This dependence demonstrated that there were protein binding-linked protonation reactions that should be dissected for the estimation of the intrinsic binding constants to perform structure-thermodynamics analysis. X-ray crystallographic structures of secondary sulfonamides and computational modeling dissected the atomic contributions to the binding energetics. Secondary sulfonamides bind to carbonic anhydrases via coordination bond between the negatively charged nitrogen of alkylated amino group and Zn(II) in the active site of CA. The binding reaction is linked to deprotonation of the amino group and protonation of the Zn(II)-bound hydroxide. To perform the structure-thermodynamics analysis, contributions of these linked reactions must be subtracted to determine the intrinsic energetics. In this aspect, the secondary sulfonamides are similar to primary sulfonamides as CA inhibitors.

Biphenyl substituted lysine derivatives as recognition elements for the matrix metalloproteinases MMP-2 and MMP-9.

Matrix metalloproteinases (MMPs) are an important factor in cancer progression and metastasis, especially gelatinases MMP-2 and MMP-9. A simple methodology for their detection and monitoring is highly desirable. Molecular probes have been very widely and successfully applied to study the activity of MMPs in cellular processes in vitro. We thus synthesized a small compound library of MMP-2 and MMP-9 binding probes based on drug molecules and endowed with free amine groups for the functionalization of transducer surfaces. In this study, we combined experimental results obtained by a kinetic fluorogenic peptide substrate cleavage assay with molecular modeling studies in order to assess the ability of the probe to bind to their target enzymes. The synthesized biphenyl substituted lysine derivatives showed IC50-values in the low nanomolar concentration range against MMP-2 (ligands 3a-d: 3 nM to 8 µM, ligands 4a-d: 45 nM to 350 µM) and low micromolar range against MMP-9 (ligands 3a-d: 350 nM to 60 µM, ligands 4a-d: 5 µM to 600 µM), with a selectivity up to more than 160-fold for MMP-2. The experimental results correlated well with molecular modelling with FleXAID and X-score functions. We showed that in our compound series, the side chain remained far away from the S1' cavity and the ligand for all the docked minima. Ligands 4a-d with their free amine group on the side chain may thus be bound to transducer surfaces for the fabrication of sensors, while retaining their activity against their target enzymes.

Isoform-Selective Enzyme Inhibitors by Exploring Pocket Size According to the Lock-and-Key Principle.

In the design of high-affinity and enzyme isoform-selective inhibitors, we applied an approach of augmenting the substituents attached to the benzenesulfonamide scaffold in three ways, namely, substitutions at the 3,5- or 2,4,6-positions or expansion of the condensed ring system. The increased size of the substituents determined the spatial limitations of the active sites of the 12 catalytically active human carbonic anhydrase (CA) isoforms until no binding was observed because of the inability of the compounds to fit in the active site. This approach led to the discovery of high-affinity and high-selectivity compounds for the anticancer target CA IX and antiobesity target CA VB. The x-ray crystallographic structures of compounds bound to CA IX showed the positions of the bound compounds, whereas computational modeling confirmed that steric clashes prevent the binding of these compounds to other isoforms and thus avoid undesired side effects. Such an approach, based on the Lock-and-Key principle, could be used for the development of enzyme-specific drug candidate compounds.

Template-based modeling of diverse protein interactions in CAPRI rounds 38-45.

Structures of proteins complexed with other proteins, peptides, or ligands are essential for investigation of molecular mechanisms. However, the experimental structures of protein complexes of interest are often not available. Therefore, computational methods are widely used to predict these structures, and, of those methods, template-based modeling is the most successful. In the rounds 38-45 of the Critical Assessment of PRediction of Interactions (CAPRI), we applied template-based modeling for 9 of 11 protein-protein and protein-peptide interaction targets, resulting in medium and high-quality models for six targets. For the protein-oligosaccharide docking targets, we used constraints derived from template structures, and generated models of at least acceptable quality for most of the targets. Apparently, high flexibility of oligosaccharide molecules was the main cause preventing us from obtaining models of higher quality. We also participated in the CAPRI scoring challenge, the goal of which was to identify the highest quality models from a large pool of decoys. In this experiment, we tested VoroMQA, a scoring method based on interatomic contact areas. The results showed VoroMQA to be quite effective in scoring strongly binding and obligatory protein complexes, but less successful in the case of transient interactions. We extensively used manual intervention in both CAPRI modeling and scoring experiments. This oftentimes allowed us to select the correct templates from available alternatives and to limit the search space during the model scoring.

Mycobacterium tuberculosis ß-Carbonic Anhydrases: Novel Targets for Developing Antituberculosis Drugs.

The genome of Mycobacterium tuberculosis (Mtb) encodes three ß-carbonic anhydrases (CAs, EC 4.2.1.1) that are crucial for the life cycle of the bacterium. The Mtb ß-CAs have been cloned and characterized, and the catalytic activities of the enzymes have been studied. The crystal structures of two of the enzymes have been resolved. In vitro inhibition studies have been conducted using different classes of carbonic anhydrase inhibitors (CAIs). In vivo inhibition studies of pathogenic bacteria containing ß-CAs showed that ß-CA inhibitors effectively inhibited the growth of pathogenic bacteria. The in vitro and in vivo studies clearly demonstrated that ß-CAs of not only mycobacterial species, but also other pathogenic bacteria, can be targeted for developing novel antimycobacterial agents for treating tuberculosis and other microbial infections that are resistant to existing drugs. In this review, we present the molecular and structural data on three ß-CAs of Mtb that will give us better insights into the roles of these enzymes in pathogenic bacterial species. We also present data from both in vitro inhibition studies using different classes of chemical compounds and in vivo inhibition studies focusing on M. marinum, a model organism and close relative of Mtb.

Benzimidazole design, synthesis, and docking to build selective carbonic anhydrase VA inhibitors.

The similarity of human carbonic anhydrase (CA) active sites makes it difficult to design selective inhibitors for one or several CA isoforms that are drug targets. Here we synthesize a series of compounds that are based on 5-[2-(benzimidazol-1-yl)acetyl]-2-chloro-benzenesulfonamide (1a) which demonstrated picomolar binding affinity and significant selectivity for CA isoform five A (VA), and explain the structural influence of inhibitor functional groups to the binding affinity and selectivity. A series of chloro-substituted benzenesulfonamides bearing a heterocyclic tail, together with molecular docking, was used to build inhibitors that explore substituent influence on the binding affinity to the CA VA isoform.

Fluorinated benzenesulfonamide anticancer inhibitors of carbonic anhydrase IX exhibit lower toxic effects on zebrafish embryonic development than ethoxzolamide.

The toxic effects of two recently discovered inhibitors (VD12-09 and VD11-4-2) that selectively and with extraordinary strong, picomolar binding affinity to human carbonic anhydrase (CA) isoform IX were investigated on zebrafish embryonic development. CA IX has been recently introduced as an anticancer target since it is highly overexpressed in numerous human cancers but nearly absent in normal tissues. Morphological changes in zebrafish treated by the compounds were studied by light-field microscopy and histological analysis. Homology models of zebrafish CA II and CA IX were built to identify the conserved amino acid residues in the active site of zebrafish CAs. The toxicity studies here showed that the LC50 values at 120 hours post-fertilization (hpf) were 13 µM for VD12-09, 120 µM for VD11-4-2, and 9 µM for ethoxzolamide (EZA), a non-selective CA inhibitor commonly used as a drug in clinics. Thus, EZA was the most toxic of the three compounds. The zebrafish embryos exposed to LC50 doses of VD12-09 and VD11-4-2 showed fewer phenotypic abnormalities compared with the embryos exposed to the corresponding dose of EZA. Histochemical studies did not show any gross morphological changes in the embryos treated with VD12-09 and VD11-4-2 unlike EZA. The results of our study indicate that the compounds exhibited 10-fold lower toxicity and induced fewer side effects in zebrafish than EZA. Therefore, the exposure to VD11-4-2 and VD12-09 at concentrations below LC50 did not lead to deleterious effects on the zebrafish embryonic development and thus both inhibitors may be further developed as drugs.

Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity.

Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.

Comprehensive analysis of resorcinyl-imidazole Hsp90 inhibitor design.

Human Hsp90 chaperones are implicated in various aspects of cancer. Due to this, Hsp90 has been explored as potential target in cancer treatment. Initial attempts to use Hsp90 inhibitors in drug trials failed due to toxicity and inefficacy. The next generation of drugs were less toxic but still insufficiently effective in a clinical setting. Recently, a lot of effort is being put into understanding the consequences of Hsp90 isoform selective inhibition, expecting that this might hold the key in targeting Hsp90 for disease treatment. Here we investigate a series of compounds containing the aryl-resorcinol scaffold with a 5-membered ring as a promising class of new human Hsp90 inhibitors, reaching nanomolar affinity. We compare how the replacement of 5-membered ring, from thiadiazole to imidazole, as well as a variety of their substituents, influences the potency of these inhibitors for Hsp90 alpha and beta isoforms. To further elucidate the dissimilarity in ligand selectivity between the isoforms, a mutant protein was constructed and tested against the ligand library. In addition, we performed a series of molecular dynamics (MD) and docking simulations to further explain our experimental findings as well as evaluated key compounds in cell assays. Our results deepen the understanding of Hsp90 isoform ligand selectivity and serve as an informative base for further Hsp90 inhibitor optimization.

Recent advances in computational and experimental protein-ligand affinity determination techniques.

INTRODUCTION: Modern drug discovery revolves around designing ligands that target the chosen biomolecule, typically proteins. For this, the evaluation of affinities of putative ligands is crucial. This has given rise to a multitude of dedicated computational and experimental methods that are constantly being developed and improved. AREAS COVERED: In this review, the authors reassess both the industry mainstays and the newest trends among the methods for protein - small-molecule affinity determination. They discuss both computational affinity predictions and experimental techniques, describing their basic principles, main limitations, and advantages. Together, this serves as initial guide to the currently most popular and cutting-edge ligand-binding assays employed in rational drug design. EXPERT OPINION: The affinity determination methods continue to develop toward miniaturization, high-throughput, and in-cell application. Moreover, the availability of data analysis tools has been constantly increasing. Nevertheless, cross-verification of data using at least two different techniques and careful result interpretation remain of utmost importance.

Aortic disease and cardiomyopathy in patients with a novel DNMT3A gene variant causing Tatton-Brown-Rahman syndrome.

Tatton-Brown-Rahman syndrome (TBRS) is a rare congenital genetic disorder caused by autosomal dominant pathogenic variants in the DNA methyltransferase DNMT3A gene. Typical TBRS clinical features are overgrowth, intellectual disability, and minor facial anomalies. However, since the syndrome was first described in 2014, a widening spectrum of abnormalities is being described. Cardiovascular abnormalities are less commonly reported but can be a major complication of the syndrome. This article describes a family of three individuals diagnosed with TBRS in adulthood and highlights the variable expression of cardiovascular features. A 34-year-old proband presented with progressive aortic dilatation, mitral valve (MV) regurgitation, left ventricular (LV) dilatation, and ventricular arrhythmias. The affected family members (mother and brother) were diagnosed with MV regurgitation, LV dilatation, and arrhythmias. Exome sequencing and computational protein analysis suggested that the novel familial DNMT3A mutation Ser775Tyr is located in the methyltransferase domain, however, distant from the active site or DNA-binding loops. Nevertheless, this bulky substitution may have a significant effect on DNMT3A protein structure, dynamics, and function. Analysis of peripheral blood cfDNA and transcriptome showed shortened mononucleosome fragments and altered gene expression in a number of genes related to cardiovascular health and of yet undescribed function, including several lncRNAs. This highlights the importance of epigenetic regulation by DNMT3A on cardiovascular system development and function. From the clinical perspective, we suggest that new patients diagnosed with congenital DNMT3A variants and TBRS require close examination and follow-up for aortic dilatation and valvular disease because these conditions can progress rapidly. Moreover, personalized treatments, based on the specific DNMT3A variants and the different pathways of their function loss, can be envisioned in the future.

Understanding the activity of glucose oxidase after exposure to organic solvents.

Long-term stability of enzymes in organic solvents is one of the most challenging problems in modern biotechnology and chemical industries. However, the resistance of enzymes to organic solvents is not very well understood so far. Herein, the effects of apolar, chlorinated, and polar organic solvents on the activity and structure of glucose oxidase from Aspergillus niger were systemically investigated using spectrophotometric activity assay of this enzyme and absorption and chiroptical spectroscopy. Molecular dynamics simulations and correlation of the activity with properties of the organic solvents were employed to understand the effects of organic solvents on the enzyme. The experimental and theoretical results showed that apolar solvents reduce the enzyme activity because they facilitate its aggregation through inter-enzymatic salt bridges. Moreover, polar solvents strongly coordinate with amino acid residues in the glucose binding pocket and prevent binding of the substrates. We found that this enzyme is stable in pure apolar and chlorinated solvents and these solvents can be used for the functionalization of its residues. This work provides an in depth understanding at the molecular level of the impact of various pure organic solvents on the structure and dynamics of glucose oxidase and the regulation of its catalytic activity.

Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin ß-subunit.

Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGß genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of hCG by applying a combination of in silico (sequence and structure analysis, molecular dynamics) and in vitro (co-immunoprecipitation, immuno- and bioassays) approaches. The carrier status of each mutation was determined for 1086 North-Europeans [655 patients with recurrent miscarriage (RM)/431 healthy controls from Estonia, Finland and Denmark] using PCR-restriction fragment length polymorphism. The mutation CGB5 p.Val56Leu (rs72556325) was identified in a single heterozygous RM patient and caused a structural hindrance in the formation of the hCGα/ß dimer. Although the amount of the mutant hCGß assembled into secreted intact hCG was only 10% compared with the wild-type, a stronger signaling response was triggered upon binding to its receptor, thus compensating the effect of poor dimerization. The mutation CGB8 p.Pro73Arg (rs72556345) was found in five heterozygotes (three RM cases and two control individuals) and was inherited by two of seven studied live born children. The mutation caused ~50% of secreted ß-subunits to acquire an alternative conformation, but did not affect its biological activity. For the CGB8 p.Arg8Trp (rs72556341) substitution, the applied in vitro methods revealed no alterations in the assembly of intact hCG as also supported by an in silico analysis. In summary, the accumulated data indicate that only mutations with neutral or mild functional consequences might be tolerated in the major hCGß genes CGB5 and CGB8.

Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studies.

In the present study, the effect of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto branched poly(ethylenimine) (PEI) with different grafting degree was examined for gene delivery applications. The DMAEMA-grafted-PEI conjugates were characterized and complexed with plasmid DNA (pDNA) at various concentrations, and the physicochemical properties, cell viability, and in vitro transfection efficiency of the complexes were evaluated in HEK 293T cells. Computational techniques were used to analyze the interaction energies and possible binding modes between DNA and conjugates at different grafting degrees. The cytotoxicity analysis and in vitro transfection efficiency of the conjugate/pDNA complexes exhibited a beneficial effect of DMAEMA conjugation when compared to PEI alone. The computational results revealed that the DNA/vector interaction energy decreases with increasing grafting degree, which can be associated to an enhanced release of the pDNA from the carrier once inside cells. The results indicate the significance of DMAEMA conjugation onto PEI as a promising approach for gene delivery applications.

Beta and Gamma Amino Acid-Substituted Benzenesulfonamides as Inhibitors of Human Carbonic Anhydrases.

A series of novel benzenesulfonamide derivatives were synthesized bearing para-N ß,γ-amino acid or para-N ß-amino acid and thiazole moieties and their binding to the human carbonic anhydrase (CA) isozymes determined. These enzymes are involved in various illnesses, such as glaucoma, altitude sickness, epilepsy, obesity, and even cancer. There are numerous compounds that are inhibitors of CA and used as pharmaceuticals. However, most of them bind to most CA isozymes with little selectivity. The design of high affinity and selectivity towards one CA isozyme remains a significant challenge. The beta and gamma amino acid-substituted compound affinities were determined by the fluorescent thermal shift assay and isothermal titration calorimetry for all 12 catalytically active human carbonic anhydrase isozymes, showing the full affinity and selectivity profile. The structures of several compounds were determined by X-ray crystallography, and the binding mode in the active site of CA enzyme was shown.

Evaluating the substrate-envelope hypothesis: structural analysis of novel HIV-1 protease inhibitors designed to be robust against drug resistance.

Drug resistance mutations in HIV-1 protease selectively alter inhibitor binding without significantly affecting substrate recognition and cleavage. This alteration in molecular recognition led us to develop the substrate-envelope hypothesis which predicts that HIV-1 protease inhibitors that fit within the overlapping consensus volume of the substrates are less likely to be susceptible to drug-resistant mutations, as a mutation impacting such inhibitors would simultaneously impact the processing of substrates. To evaluate this hypothesis, over 130 HIV-1 protease inhibitors were designed and synthesized using three different approaches with and without substrate-envelope constraints. A subset of 16 representative inhibitors with binding affinities to wild-type protease ranging from 58 nM to 0.8 pM was chosen for crystallographic analysis. The inhibitor-protease complexes revealed that tightly binding inhibitors (at the picomolar level of affinity) appear to "lock" into the protease active site by forming hydrogen bonds to particular active-site residues. Both this hydrogen bonding pattern and subtle variations in protein-ligand van der Waals interactions distinguish nanomolar from picomolar inhibitors. In general, inhibitors that fit within the substrate envelope, regardless of whether they are picomolar or nanomolar, have flatter profiles with respect to drug-resistant protease variants than inhibitors that protrude beyond the substrate envelope; this provides a strong rationale for incorporating substrate-envelope constraints into structure-based design strategies to develop new HIV-1 protease inhibitors.

The hydrolysis of indoxyl acetate: A versatile reaction to assay carbonic anhydrase activity by high-throughput screening.

The interest in CO2 capture and conversion by biological methodologies into various beneficial products is increased. In nature, there are enzymes, with hydration activity, which catalyze the reversible hydration of the CO2 molecule and, thus, are of high interest for biotechnological applications. Such enzymes are carbonic anhydrases (CAs). Structural, functional and mutational studies have shown, that besides hydratase activity, CAs have exposed hydrolytic, particularly esterase activity, and importantly, both activities follow similar catalytic mechanisms in the same catalytic pocket. CAs activity measurement methods based on electrometric assays for hydration activity and nitrophenyl based esters for hydrolytic activity assays do not fulfill the requirements amenable for enzyme activity screening methods. By this study, we were aiming to develop an indigogenic assay method based on the esterase activity of CAs. The first time use of indoxyl acetate as a substrate for CA has shown promising results to gain simplicity, repeatability, and applicability to implement high-throughput screening methods.