[Screening of potential non-azole inhibitors of lanosterol14-alpha demethylase (CYP51) of Candida fungi]. / Skrining potentsial'nykh neazol'nykh ingibitorov 14-al'fademetilazy (CYP51) gribov roda Candida.

Currently, opportunistic fungi of the genus Candida are the main causative agents of mycoses, which are especially severe upon condition of acquired immunodeficiency. The main target for the development of new antimycotics is the cytochrome P450 51 (CYP51) of the pathogenic fungus. Due to the widespread distribution of Candida strains resistancy to inhibitors of the azole class, the screening for CYP51 inhibitors both among non-azole compounds and among clinically used drugs repurposing as antimycotics is becoming urgent. To identify potential inhibitors from the non-azole group, an integrated approach was applied, including bioinformatics analysis, computer molecular modeling, and a surface plasmon resonance (SPR) technology. Using in silico modeling, the binding sites for acetylsalicylic acid, ibuprofen, chlorpromazine and haloperidol (this compounds, according to the literature, showed antimycotic activity) were predicted in the active site of CYP51 of Candida albicans and Candida glabrata. The Kd values of molecular complexes of acetylsalicylic acid, ibuprofen and haloperidol with CYP51, determined by SPR analysis, ranged from 18 µM to 126 µM. It was also shown that structural derivatives of haloperidol, containing various substituents, could be positioned in the active site of CYP51 of Candida albicans with the possible formation of coordination bonds between the hydroxyl groups of the derivatives and the iron atom in the heme of CYP51. Thus, the potential basic structures of non-azole compounds have been proposed, which can be used for the design of new CYP51 inhibitors of Candida fungi.

[SPR analysis of protein-protein interactions with P450 cytochromes and cytochrome b5 integrated into lipid membrane]. / SPR analiz belok-belkovykh vzaimodeistvii s uchastiem tsitokhromov R450 i tsitokhroma b5, vstroennykh v bisloinuiu lipidnuiu membranu.

Identification of new protein-protein interactions (PPI) and characterization of quantitative parameters of complex formation represent one of central tasks of protein interactomics. This work is a logical continuation of the cycle of our previous works devoted to the study of PPIs among the components of cytochrome P450-dependent monooxygenase system. Using an optical biosensor of Surface Plasmon Resonance (SPR biosensor), a comparative analysis on the determination of kinetic and equilibrium parameters of complex formation between the membrane-bound hemoprotein cytochrome b5 with cytochrome P450s was performed using two different protocols for protein immobilization: 1) covalent non-oriented one on to the carboxymethyl dextran chip type CM and 2) non-covalent oriented immobilization in the lipid environment on the chip type L1 with internal control of liposomes surface distribution. In the second protocol it was shown that the complex formation was characterized by 2.5 times higher affinity due to an decrease in rate dissociation constants. The appropriateness of using both experimental models is discussed.

[Interaction of prostacyclin synthase with cytochromes P450]. / Vzaimodeistvie prostatsiklinsintazy s tsitokhromami P450.

Biosensor experiments on investigation of interaction between prostacyclin synthase (PGIS) and different proteins of the cytochrome P450 monooxygenase systems were perfomed. Interaction of PGIS with microsomal (CYP21A2, CYP2E1) and mitochondrial (CYP27A1, CYP11B1, CYP11B2, CYP11A1) cytochrome P450s was detected. Kinetic and equilibrium parameters of protein complexes formation were determined. Data obtained suggest an essential role of these hemoproteins interaction in regulation of prostacyclin and thromboxane A2 biosynthesis.

[Proteomic analysis of contaminants in recombinant membrane hemeproteins expressed in E. coli and isolated by metal affinity chromatography].

Contaminating proteins have been identified by "shotgun" proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.

[The screening of the inhibitors of the human cytochrome P450(51) (CYP51A1): the plant and animal structural lanosterol's analogs].

The cholesterol biosynthesis regulation is the important part of the hypercholesterolemia diseases therapy. The inhibition of the post-squalene cholesterol biosynthesis steps provide the alternative to classic statin therapy. Sterol-14a-demethylase (CYP51) is one of the hypothetical targets for it. In this work the screening of the ability to interact with human CYP51 (CYP51A1) for the nature low-weight compounds with steroid-like scaffold were performed by integration of the surface plasmon resonance biosensor and spectral titration methods. The results of the selection were 4 compounds (betulafolientriol, holothurin A, teasaponin, capsicoside A) witch had high affinity to the CYP51A1 active site. These data extend the range of compounds which may be used as specific inhibitors of CYP51 and give the permission to suggest the dynamic of the enzyme.