The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC 50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started.

Background & Aims. The chimeric tyrosine kinase BcrAbl triggers malignant transformation of myeloid cells via phosphorylation of a number of substrates including the CrkL adaptor protein. Pharmacological inhibition of Bcr-Abl mediated signaling is a major strategy in treatment of patients with chronic myeloid leukemia (CML). A new specific Bcr-Abl inhibitor (PF-114) was designed using a molecular modeling approach. The paper defines the cytotoxicity of PF-114 against CML cells and its effect on the CrkL phosphorylation. Methods. The cytotoxicity was determined using the MTT assay. The total intracellular CrKL pool (phosphorylated and non-phosphorylated forms) was determined by means of flow cytometry. Results. Exposure of Bcr-Abl-positive, K562 cell line to PF114 blocked intracellular CrkL phosphorylation and caused cell death. In contrast, virtually no phosphorylated CrkL was detectable in Bcr-Abl-negative HL60, U937 and Jurkat leukemia cell lines. Conclusion. Absence of CrkL phosphorylation in Bcr-Ablnegative cells (HL60, U937 and Jurkat) and death of HL60 cells under the effect of PF-114 at concentrations exceeding those required to kill K562 cells supports the emergence of PF-114 as a promising drug candidate for CML.

A protein kinase inhibitor of Formula I ** Formula ** or a tautomer, a stereoisomer or a mixture of stereoisomers, or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein: X1 is N or CRt1; X2 is N or CRt2; X3 is N or CRt3; X4 is N or CH; where X1, X2, X3 and X4 are independently selected; Rt1 is selected from -H, halo, -COOH, -CN, -CH2OH, C1-C4 alkyl, -O (C1-C3 alkyl); Rt2 is selected from -H, halo, -CH3, -CH2CH3.-OH, -OCH3, and -NH2; Rt3 is selected from -H, halo, -S (O) rR4, -CN and C (O) YR4; ring A is aryl or a 5- or 6-membered heteroaryl ring, wherein the heteroaryl forming ring A contains 1-2 heteroatoms selected from N, S and O, and wherein ring A is optionally substituted with 1-4 Ra groups; ring B is a 5- or 6-membered phenyl or heteroaryl ring, wherein ring B heteroaryl contains 1-2 ring heteroatoms selected from N or S and where ring B is optionally substituted with 1-5 Rb groups; Ra and Rb are each independently selected from -H, halo, -CN, -R6, -OR4, -NR4R5, -C (O) YR4, -S (O) rR4, - SO2NR4R5, -NR4SO2NR4R5; alternatively, an Rb substituent on ring B may be ring C, where ring C is a 5- or 6-membered heteroaryl or heterocyclyl ring comprising carbon atoms and 1-3 heteroatoms independently selected from O, N and S (O) r , and wherein ring C is optionally substituted with 1 to Rc substituents; each Rc is independently selected from -H, halo, and -R6; alternatively, one of the Rb substituents may have a -L2-D structure, where D is a ring, L2 is (CH2) z, and z is 1, 2, 3, or 4; or L2 is (OCH2) x, where x is 0, 1, 2 or 3, and wherein ring D is a 5- or 6-membered heteroaryl or heterocyclyl ring comprising carbon atoms and 1-3 heteroatoms independently selected from O , N and S (O) r, and wherein ring D is optionally substituted with 1 to Rd substituents; each Rd is independently selected from -H, halo, R6, -OR4 or -NR4R5; L1 represents an NR3C (O) or C (O) NR3; each Y is independently selected from a chemical bond, -O-, -S-, and -NR5; each R3, each R4 and each R5 is independently selected from H and C1-C6 alkyl, where alternatively an NR4R5 group may represent a 5- or 6-membered ring, saturated or unsaturated; each R6 is independently selected from C1-C6 alkyl or C2-C6 alkenyl; and r is 0, 1 or 2.

We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca2+-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.

A search for poly(ADP-ribose) polymerase-1 inhibitors by virtual screening of a chemical compound database and a subsequent experimental verification of their activities have been performed. It was shown that the most efficient method to predict inhibitory properties implies a combinatorial approach joining molecular docking capabilities with structural filtration. Among more than 300000 low molecular chemical compounds, 9 PARP1 inhibitors were revealed; the most active ones, namely, STK031481, STK056130, and STK265022, displayed biological effect at a micromolar concentration (IC50 = 2.0, 1.0, and 2.6 μM, respectively).

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.

Modeling of the catalytic mechanism of penicillin acylase, a member of the N-terminal nucleophile hydrolase superfamily, is for the first time conducted at ab initio quantum chemistry level. The uniqueness of this family of enzymes is that their active site lacks His and Asp (Glu) residues, comprising together with a serine residue the classical catalytic triad. The current investigation confirms that the amino group of the N-terminal serine residue in N-terminal hydrolases is capable of activating its own hydroxyl group. Using the MP2/RHF method with the 6−31+G** basis set, stationary points on the potential energy surface of the considered molecular system were located, corresponding to local minima (complexes of reagents, products, intermediate) and to saddle points (transition states). It turned out that the stage of acyl-serine formation proceeds via two transition states; the first one, which separates reagents from the so-called tetrahedral intermediate, has the highest relative energy (30 kcal/mol). In contrast to recently proposed empiric suggestions, we have found that participation of a bridging water molecule in proton shuttling is not necessary for the catalysis. The quantum chemical calculations showed a crucial role of a specific solvation in decreasing the activation barrier of the reaction by approximately 10 kcal/mol.

A complete set of AMBER force field parameters for 6-aminopenicillanic acid, crucial moiety of β-lactam antibiotics, has been developed. Equilibrium geometry was derived from RHF/6-31G* calculations, stretching constants were computed from quantum mechanical hessian, partial atomic charges were assigned according to restrained electrostatic potential fit methodology. Torsional parameters were also derived from quantum mechanical calculations. It was shown that unusual chemical structure of 6-aminopenicillanic acid with two merged four- and five-member rings has found its reflection in the set of molecular mechanical parameters: a number of bonds and angles appeared to be much more rigid than those described by classic AMBER field. The quality of derived set parameters was attested by computing molecular geometry, spectroscopic and thermodynamic properties.

A new strategy for the biocatalytic resolution of (R,S)-phenylglycinonitrile, a crucial intermediate in the antibiotic industry, has been developed. While former techniques exploit nitrilases or combinations of nitrile hydratases and amidases, manipulating with nitrile functionality, the current approach is based on a highly efficient and enantioselective acylation of the α-amino group with phenylacetic acid catalyzed by a well known enzyme, penicillin acylase from E. coli, in slightly acidic aqueous medium. It is shown that since the condensation product is poorly soluble, removal of (S)-phenylglycinonitrile from the reaction sphere is almost complete and irreversible, favoring kinetics of the process and making high conversion possible. The proposed approach is characterized by high space-time yield and extends the scope of enzymatic synthesis in aqueous medium.

The application of the two-phase "aqueous solution – water-immiscible organic solvent" system is suggested not for effective biocatalytic synthesis, but for hydrolytic purposes. Enzymatic hydrolysis of benzylpenicillin and N-phenylacetamidodesacetoxycephalosporanic acid to corresponding antibiotic nuclei 6-aminopenicillanic and 7-aminodesacetoxycephalosporanic acids in a two-phase water–butylacetate system at pH 3–4 is proposed as an alternative to the biocatalytic hydrolysis in an alkaline medium. An experimental study has been performed and a model has been developed, which describes the influence of pH, phase volume ratio, thermodynamic constants, and initial antibiotic concentration on the effectiveness of their hydrolysis in a two-phase "aqueous solution – water-immiscible organic solvent" system. The thermodynamic evaluation of penicillin G and 7-phenylacetamidodesacetoxycephalosporanic acid hydrolysis at low pH in a two-phase aqueous solution – water-immiscible organic solvent system has demonstrated high practical potential. The suggested approach allows for the exclusion of several technological steps during the transformation of natural β-lactam antibiotics to their semi-synthetic analogues: alkaline extraction of the biosynthetic antibiotic from butylacetate followed by its enzymatic hydrolysis at pH 7.5–8.0 and further acidification of the reaction mixture, which results in the precipitation of the antibiotic nucleus. Experimental observations also revealed a specific feature of this process: the kinetic supersaturation of the antibiotic nucleus slows down the attainment of the equilibrium, which should be taken into account when further developing this approach.Key words: enzymatic hydrolysis, β-lactam antibiotic nuclei, two-phase systems, supersaturation, penicillin acylase.

Nucleophile reactivity of two most known nuclei of penicillins and cephalosporins, 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids, was quantitatively characterized. In penicillin acylase (PA)-catalyzed acyl transfer reactions the relative reactivity of the added nucleophile compared to the water (i.e. nucleophile reactivity) is defined by two complex kinetic parameters β0 and γ, and depends on the nucleophile concentration. In turn, parameters β0 and γ were shown to be dependent on the structure of both reactants involved: nucleophile and acyl donor. Analysis of the kinetic scheme revealed that nucleophile reactivity is one of a few key parameters controlling efficiency of PA-catalyzed acyl transfer to the added nucleophile in an aqueous medium. Computation of the maximum nucleophile conversion to the product using determined nucleophile reactivity parameters in the synthesis of three different antibiotics, ampicillin, amoxicillin and cephalexin, showed good correlation with the results of corresponding synthetic experiments. Suggested approach can be extended to the quantitative description and optimization of PA-catalyzed acyl transfer reactions in a wide range of experimental conditions.

Inhibition of penicillin acylases from Escherichia coli and Alcaligenes faecalis by aliphatic and aromatic alcohols was studied. It was shown that the inhibition of both enzymes has competitive nature and they bind the alcohols at the acyl group binding site of the enzyme active center. The free energy of alcohol sorption was shown to be linearly dependent on the hydrophobicity of the inhibitor with slopes of 1.6 and 1.7, demonstrating extremely effective hydrophobic interactions. To rationalize the observed distinctions in the inhibiting properties of aromatic and aliphatic alcohols beginning with butanol, it was suggested that the loss of entropy occurring on the interaction of the ligand with the tightly restricted hydrophobic pocket of the active center makes an essential contribution to the overall energetics of complex formation.