Effect of histidine protonation state on ligand binding at the ATP-binding site of human protein kinase CK2.

Histidine residues contribute to numerous molecular interactions, owing to their structure with the ionizable aromatic side chain with pKa close to the physiological pH. Herein, we studied how the two histidine residues, His115 and His160 of the catalytic subunit of human protein kinase CK2, affect the binding of the halogenated heterocyclic ligands at the ATP-binding site. Thermodynamic studies on the interaction between five variants of hCK2α (WT protein and four histidine mutants) and three ionizable bromo-benzotriazoles and their conditionally non-ionizable benzimidazole counterparts were performed with nanoDSF, MST, and ITC. The results allowed us to identify the contribution of interactions involving the particular histidine residues to ligand binding. We showed that despite the well-documented hydrogen bonding/salt bridge formation dragging the anionic ligands towards Lys68, the protonated His160 also contributes to the binding of such ligands by long-range electrostatic interactions. Simultaneously, His 115 indirectly affects ligand binding, placing the hinge region in open/closed conformations.

5,6-diiodo-1H-benzotriazole: new TBBt analogue that minutely affects mitochondrial activity.

4,5,6,7-Tetrabromo-1H-benzotriazole is widely used as the reference ATP-competitive inhibitor of protein kinase CK2. Herein, we study its new analogs: 5,6-diiodo- and 5,6-diiodo-4,7-dibromo-1H-benzotriazole. We used biophysical (MST, ITC) and biochemical (enzymatic assay) methods to describe the interactions of halogenated benzotriazoles with the catalytic subunit of human protein kinase CK2 (hCK2α). To trace the biological activity, we measured their cytotoxicity against four reference cancer cell lines and the effect on the mitochondrial inner membrane potential. The results obtained lead to the conclusion that iodinated compounds are an attractive alternative to brominated ones. One of them retains the cytotoxicity against selected cancer cell lines of the reference TBBt with a smaller side effect on mitochondrial activity. Both iodinated compounds are candidate leaders in the further development of CK2 inhibitors.

Synthesis of Novel Halogenated Heterocycles Based on o-Phenylenediamine and Their Interactions with the Catalytic Subunit of Protein Kinase CK2.

Protein kinase CK2 is a highly pleiotropic protein kinase capable of phosphorylating hundreds of protein substrates. It is involved in numerous cellular functions, including cell viability, apoptosis, cell proliferation and survival, angiogenesis, or ER-stress response. As CK2 activity is found perturbed in many pathological states, including cancers, it becomes an attractive target for the pharma. A large number of low-mass ATP-competitive inhibitors have already been developed, the majority of them halogenated. We tested the binding of six series of halogenated heterocyclic ligands derived from the commercially available 4,5-dihalo-benzene-1,2-diamines. These ligand series were selected to enable the separation of the scaffold effect from the hydrophobic interactions attributed directly to the presence of halogen atoms. In silico molecular docking was initially applied to test the capability of each ligand for binding at the ATP-binding site of CK2. HPLC-derived ligand hydrophobicity data are compared with the binding affinity assessed by low-volume differential scanning fluorimetry (nanoDSF). We identified three promising ligand scaffolds, two of which have not yet been described as CK2 inhibitors but may lead to potent CK2 kinase inhibitors. The inhibitory activity against CK2α and toxicity against four reference cell lines have been determined for eight compounds identified as the most promising in nanoDSF assay.

Coumarin Derivatives as New Toxic Compounds to Selected K12, R1-R4 E. coli Strains.

Coumarins are natural compounds that were detected in 80 species of plants. They have numerous applications including the medical, food, tobacco, perfumery, and spirit industries. They show anti-swelling and diastolic effects. However, excess consumption of coumarins may adversely affect our health, because they are easily absorbed from the intestines into the lymph and blood, causing cirrhosis of the liver. Peptidomimetics are molecules whose structure and function are similar to those of peptides. They are an important group of compounds with biological, microbiological, anti-inflammatory, and anti-cancer properties. Therefore, studies on new peptidomimetics, which load the effect of native peptides, whose half-life in the body is much longer due to structural modifications, are extremely important. A preliminary study of coumarin analogues and its derivatives as new potential antimicrobial drugs containing carboxylic acid or ester was performed to determine their basic structure related to their biological features against various types of Gram-stained bacteria by lipopolysaccharide (LPS). We hypothesized that the toxicity (antibacterial activity) of coumarin derivatives is dependent on the of LPS in bacteria and nature and position of the substituent which may be carboxylic acid, hydroxyl groups, or esters. In order to verify this hypothesis, we used K12 (smooth) and R1-R4 (rough) Escherichia coli strains which are characterized by differences in the type of LPS, especially in the O-antigen region, the outermost LPS layer. In our work, we synthesized 17 peptidomimetics containing a coumarin scaffold and checked their influence on K12 and R1-R4 E. coli strains possessing smooth and rough LPS. We also measured the damage of plasmid DNA caused by target compounds. The results of our studies clearly support the conclusion that coumarin peptidomimetics are antagonistic compounds to many of the currently used antibiotics. The high biological activity of the selected coumarin peptidomimetic was associated with identification of the so-called magic methyl groups, which substantially change the biochemical properties of target compounds. Investigating the effects of these compounds is particularly important in the era of increasingly common resistance in bacteria.

Thermodynamic contribution of iodine atom to the binding of heterogeneously polyhalogenated benzotriazoles by the catalytic subunit of human protein kinase CK2.

A series of novel benzotriazole derivatives containing iodine atom(s) were synthesized. The binding of these compounds to the catalytic subunit of human protein kinase CK2 was evaluated using differential scanning fluorimetry. The obtained thermodynamic data were compared with those determined previously for the brominated and chlorinated benzotriazole analogues to get a deeper insight into the thermodynamic contribution of iodine substitution to the free energy of ligand binding. We have shown that iodine atom(s) attached to the benzene ring of benzotriazole enhance(s) its binding by the target protein. This effect is the strongest when two iodine atoms are attached at positions peripheral to the triazole ring, which according to the structures deposited in protein data bank may be indicative for the formation of the halogen bond between iodine and carbonyl groups of residues located in the hinge region of the protein. Finally, quantitative structure-activity relationship analysis pointed the solute hydrophobicity as the main factor contributing to the binding affinity.

The influence of the isocyanoesters structure on the course of enzymatic Ugi reactions.

The impact of isocyanoesters structure on enzymatic three-component Ugi reactions course has been determined. The significant promiscuous ability of enzyme in Ugi-type reaction switching between four (U-4CR) and three (U-3CR) components reactions depending on the size of used isocyanoester. The application of short-chain cyanoesters up to isocyanpropionate leading to product of three component reaction exclusively while longer isocyanobutyrate gives only the product of four component reaction. The limitation of studied enzymatic Ugi reaction is a substrate selectivity of lipases.

Multicomponent Reactions Accelerated by Aqueous Micelles.

Multicomponent reactions are powerful synthetic tools for the efficient creation of complex organic molecules in an one-pot one-step fashion. Moreover, the amount of solvents and energy needed for separation and purification of intermediates is significantly reduced what is beneficial from the green chemistry issues point of view. This review highlights the development of multicomponent reactions conducted using aqueous micelles systems during the last two decades.

The sustainable synthesis of peptidomimetics via chemoenzymatic tandem oxidation-Ugi reaction.

A simply and green synthetic protocol based on the selective laccase-oxidation of alcohol to a corresponding aldehyde and a following Ugi reaction in a micellar system made of SDS was developed and is reported herein. Special emphasis was placed on the metal-free chemoenzymatic tandem reaction based on alcohol oxidation strategies using molecular oxygen from air, followed by an Ugi reaction. The reaction was carried out without the use of a transition metal or organic solvents as a reaction medium. The presented protocol offers an efficient and environmentally friendly procedure.

Enzymatic Ugi Reaction with Amines and Cyclic Imines.

The application of the Ugi reaction to the construction of new peptide scaffolds is an important goal of organic chemistry. To date, there are no examples of the Ugi reaction being performed with a cyclic imine and amine simultaneously. The application of 2-substituted cyclic imines in an enzymatic three-component Ugi-type reaction provides an elegant and attractive synthesis of substituted pyrrolidine and piperidine derivatives in up to 60 % yield. Results on studies of the selection of an enzyme, amount of water, and solvent used in a novel three-component Ugi reaction and the limitations thereof are reported herein. The presented methodology exploiting enzyme promiscuity in the multicomponent reaction fulfills the requirements associated with green chemistry. Several methods, such as isotope labeling and enzyme inhibition, were used to probe the possible mechanism of this complex synthesis. This research is the first example of an enzyme-catalyzed Ugi-type reaction with an imine, amine, and isocyanide.

Self-immolative versatile fluorogenic probes for screening of hydrolytic enzyme activity.

Enzyme triggered probes with a self-immolative linker for rapid and sensitive hydrolase detection through a cascade reaction have been reported. Their utility was proved by the preparation of three model compounds and their evaluation as enzyme substrates and demonstration of their applicability as fluorogenic probes for screening lipase, esterase and protease activities. These probes represent a new class of fluorogenic compounds, are stable under aqueous conditions and not susceptible to nonspecific degradation. The utilization of the carbamate cleavable linkage in a probe structure allows moving away of the bulky fluorophore from the enzyme recognition unit and targets different classes of enzymes with the same substrate.

Chemoenzymatic Synthesis of Proxyphylline Enantiomers.

A novel synthetic route for preparation of proxyphylline enantiomers using a kinetic resolution (KR) procedure as the key step is presented. The reactions were catalyzed by immobilized Candida antarctica lipase B in acetonitrile. Three types of reactions were examined: (i) enantioselective transesterification of racemic proxyphylline with vinyl acetate as well as (ii) hydrolysis and (iii) methanolysis of its esters. The influence of reaction conditions on the substrate conversion and enantiomeric purity of the products were investigated. Studies on analytical scale reactions revealed that the titled API enantiomers could be successfully obtained with excellent enantiomeric excess (up to >99% ee). The process was easily conducted on a 5 g scale at 100 g/L. In a preparative-scale reaction, unreacted (S)-(+)-butanoate (97% ee) and (R)-(-)-alcohol (96% ee) were obtained after 2 days in yields of 45% and 46%, respectively. When the reaction time was extended to 6 days, (S)-(+)-butanoate was isolated in >99% ee and acceptable high enantioselectivity (E = 90). Importantly, the KR's products could be conveniently isolated by exploiting varying solubility of the ester/alcohol in acetonitrile at room temperature. In addition, a chiral preference of the CAL-B active site for the R-enantiomer was rationalized by in sillico docking studies.

Evaluation of a new protocol for enzymatic dynamic kinetic resolution of 3-hydroxy-3-(aryl)propanoic acids.

The application of tandem metal-enzyme dynamic kinetic resolution (DKR) is a powerful tool for the manufacture of high-value chemical commodities. A new protocol of kinetic resolution based on irreversible enzymatic esterification of carboxylic acids with orthoesters was introduced to obtain optically active ß-hydroxy esters. This procedure was combined with metal catalyzed racemization of the target substrate providing both (R) and (S) enantiomers of ethyl 3-hydroxy-3-(4-nitrophenyl)propanoate with a high yield of 89% at 40 °C. A substantial influence of the enzyme type, organic co-solvent, and metal catalyst on the conversion and enantioselectivity of the enzymatic dynamic kinetic resolution was noted.

First chemoenzymatic stereodivergent synthesis of both enantiomers of promethazine and ethopropazine.

Enantioenriched promethazine and ethopropazine were synthesized through a simple and straightforward four-step chemoenzymatic route. The central chiral building block, 1-(10H-phenothiazin-10-yl)propan-2-ol, was obtained via a lipase-mediated kinetic resolution protocol, which furnished both enantiomeric forms, with superb enantioselectivity (up to E = 844), from the racemate. Novozym 435 and Lipozyme TL IM have been found as ideal biocatalysts for preparation of highly enantioenriched phenothiazolic alcohols (up to >99% ee), which absolute configurations were assigned by Mosher's methodology and unambiguously confirmed by XRD analysis. Thus obtained key-intermediates were further transformed into bromide derivatives by means of PBr3, and subsequently reacted with appropriate amine providing desired pharmacologically valuable (R)- and (S)-stereoisomers of title drugs in an ee range of 84-98%, respectively. The modular amination procedure is based on a solvent-dependent stereodivergent transformation of the bromo derivative, which conducted in toluene gives mainly the product of single inversion, whereas carried out in methanol it provides exclusively the product of net retention. Enantiomeric excess of optically active promethazine and ethopropazine were established by HPLC measurements with chiral columns.