Tuberculosis persists as a major global health threat, significantly exacerbated by the rise of drug-resistant strains. Cytochrome P450 of 124 family CYP124 from Mycobacterium tuberculosis (CYP124), implicated in host sterol metabolism and bacterial virulence, represents an emerging and promising therapeutic target. While its precise physiological role was previously debated, CYP124's confirmed ability to metabolise immunomodulatory host sterols underscores its pharmacological relevance. Utilizing surface plasmon resonance binding assays and UV-Vis spectral titration screening, we identified nine novel non-azole ligands for CYP124 from a library of 32 plant-derived and marine natural compounds. Among these hits, (25S)-5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol (termed 15β-octaol) and henricioside H2 (HD-4) induced characteristic difference spectra and formed long-lived inhibitory complexes with CYP124, exhibiting dissociation half-lives of 181 min and 65 min, respectively. However, their inhibitory potency was moderate, with IC50 values of approximately 86 μM for 15β-octaol and exceeding 100 μM for HD-4. Complementary in silico molecular docking and analysis identified key conserved hydrophobic residues within the CYP124 active site crucial for ligand binding, suggesting a shared pharmacophore. Furthermore, structural similarity analysis revealed that 37 human endogenous metabolites, including known immunoregulatory sterols, bear resemblance to the identified CYP124 ligands. This finding points towards a potential sterol-mediated interplay at the host-pathogen interface. Collectively, these results provide a foundation for the future development of mechanism-based CYP124 inhibitors as therapeutics against multidrug-resistant tuberculosis.

Limited availability of Western scientific equipment, explains a growing need to switch to using scientific instruments made in China. This is especially true in the case of optical biosensors operating on the surface plasmon resonance (SPR) effect. However, comparability of experimental data obtained using Western and Chinese biosensors has not been investigated yet. In this work we have comparedresults of SPR analysis of the kinetics and affinity of interaction of IgG2a and IgG1 antibodies with protein A. Two biosensos Biacore X-100 (Cytiva, USA) and MI-S200D (Inter-Bio, China) have been used. It was shown that the values of the association rate constants obtained on both devices for two antibodies were close within one order of magnitude. For IgG1 antibodies, the dissociation rate constants were almost identical. Both devices provide high-quality data that are well described by a simple 1:1 model (Langmuir binding).

Dehydroepiandrosterone (DHEA), a precursor of steroid sex hormones, is synthesized by steroid 17-alpha-hydroxylase/17,20-lyase (CYP17A1) with the participation of microsomal cytochrome b5 (CYB5A) and cytochrome P450 reductase (CPR), followed by sulfation by two cytosolic sulfotransferases, SULT1E1 and SULT2A1, for storage and transport to tissues in which its synthesis is not available. The involvement of CYP17A1 and SULTs in these successive reactions led us to consider the possible interaction of SULTs with DHEA-producing CYP17A1 and its redox partners. Text mining analysis, protein–protein network analysis, and gene co-expression analysis were performed to determine the relationships between SULTs and microsomal CYP isoforms. For the first time, using surface plasmon resonance, we detected interactions between CYP17A1 and SULT2A1 or SULT1E1. SULTs also interacted with CYB5A and CPR. The interaction parameters of SULT2A1/CYP17A1 and SULT2A1/CYB5A complexes seemed to be modulated by 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Affinity purification, combined with mass spectrometry (AP-MS), allowed us to identify a spectrum of SULT1E1 potential protein partners, including CYB5A. We showed that the enzymatic activity of SULTs increased in the presence of only CYP17A1 or CYP17A1 and CYB5A mixture. The structures of CYP17A1/SULT1E1 and CYB5A/SULT1E1 complexes were predicted. Our data provide novel fundamental information about the organization of microsomal CYP-dependent macromolecular complexes.

It is known that intermolecular interactions of proteins and peptides play a critical role in life processes. Such interactions can be either directly related to the implementation of various functions or play the role of a regulator. Currently, there is no doubt that the majority of proteins function as part of various molecular complexes, the formation of which occurs due to protein-protein interactions (PPIs), the totality of which can be defined as the “protein interactome”. Protein subinteractome studies are critical for studying the functions and regulatory mechanisms of unknown or poorly annotated proteins, understanding the architecture of intracellular molecular machines, and the design of PPI modulators. Previously, we used combinations of experimental approaches, as well as analytical and preparative methods, to study the subinteractomes of functionally different cellular proteins, which allowed us to identify the protein subinteractomes of several clinically significant human proteins. The purpose of this work was to conceptualize the principles of the experimental platform we developed for studying protein subinteractomes and to describe its features in detail.

Cytochromes P450 (CYP) are a family of membrane proteins involved in the production of endogenous molecules and the metabolism of xenobiotics. It is well-known that the composition of the membrane can influence the activity and orientation of CYP proteins. However, little is known about how membrane composition affects the ligand binding properties of CYP. In this study, we utilized surface plasmon resonance and fluorescence lifetime analysis to examine the impact of membrane micro-environment composition on the interaction between human microsomal CYP51 (CYP51A1) and its inhibitor, luteolin 7,3 '-disulphate (LDS). We observed that membranes containing cholesterol or sphingomyelin exhibited the lowest apparent equilibrium dissociation constant for the CYP51A1-LDS complex. Additionally, the tendency for relation between kinetic parameters of the CYP51A1-LDS complex and membrane viscosity and overall charge was observed. These findings suggest that the specific composition of the membrane, particularly the presence of cholesterol and sphingomyelin, plays a vital role in regulating the interaction between CYP enzymes and their ligands.

Currently, users of Biacore SPR biosensors (Cytiva, USA) are faced with sanctions restrictions on the purchase of consumables (primarily optical chips) for this type of equipments. In this regard, the use of commercially available analogues of the optical chips has become relevant. In this work, a comparative study of molecular interactions was performed on a Biacore X100 SPR biosensor using an original Biacore CM5 optical chip (Cytiva, USA) and its analogue CMD500M (XanTec bioanalytics GmbH, Germany). Protein A was immobilized on both chips as a molecular ligand, often used in scientific research and biotechnological works to immobilize antibodies on various carriers (biosensor chips, sorbents, nano- and microparticles). An IgG antibody was used as a protein analyte. A comparative study of the interaction of various concentrations of antibodies with protein A immobilized on two versions of the chips was carried out. The values of the kinetic rate constants for the association (kon) and dissociation (koff) of complexes, as well as the equilibrium dissociation constant (Kd), were calculated from the obtained sensorgrams using the interaction model 1:1 (Langmuir) binding. The results of comparative measurements showed similar values of the rate constants and interaction affinities. The differences between the values of kon, koff and Kd were 18%, 10% and 9%, respectively. Thus, this study confirmed the interchangeability of the original SPR chips CM5 and their analogues CMD500M.

The opportunistic fungus Candida krusei is the causative agent of nosocomial infections characterized by high mortality and development of resistance to drugs of the azole class. Therefore, develjoment of non-azole antifungal agents against resistant fungal strains is extremly important. Lanosterol 14-alpha demethylase (CYP51) is a well-known antifungal target. The optical SPR biosensor is a universal tool for screening studies in search of new drug prototypes. This paper presents the methodological aspects of high-hroughput SPR based screening of a library of low molecular weight compounds of natural origin for their interaction with C. krusei CYP51. It has been shown that when performing high-throughput screening, a researcher should pay special attention to the degree of a sensorgram curvature in the association phase. The described approaches to the analysis of high throughput screening data can be useful for researchers working with SPR biosensors from various manufacturers.

Traditional antiviral vaccines are currently created by inactivating the virus chemically, most often using formaldehyde or β-propiolactone. These approaches are not optimal since they negatively affect the safety of the antigenic determinants of the inactivated particles and require additional purification stages. The most promising platforms for creating vaccines are based on pseudoviruses, i.e., viruses that have completely preserved the outer shell (capsid), while losing the ability to reproduce owing to the destruction of the genome. The irradiation of viruses with electron beam is the optimal way to create pseudoviral particles. In this review, with the example of the poliovirus, the main algorithms that can be applied to characterize pseudoviral particles functionally and structurally in the process of creating a vaccine preparation are presented. These algorithms are, namely, the analysis of the degree of genome destruction and coimmunogenicity. The structure of the poliovirus and methods of its inactivation are considered. Methods for assessing residual infectivity and immunogenicity are proposed for the functional characterization of pseudoviruses. Genome integrity analysis approaches, atomic force and electron microscopy, surface plasmon resonance, and bioelectrochemical methods are crucial to structural characterization of the pseudovirus particles.

Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein–protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321.

Ferredoxins are small iron-sulfur proteins and key players in essential metabolic pathways. Among all types, 3Fe-4S ferredoxins are less studied mostly due to anaerobic requirements. Their complexes with cy tochrome P 450 redox partners have not been structurally characterized. In the present work, we solved the structures of both 3Fe-4S ferredoxins from M. tuberculosis - Fdx alone and the fusion FdxE–CYP143. Our SPR analysis demonstrated a high affinity binding of FdxE to CYP143. According to SAXS data, the same complex is present in solution. The structure reveals extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE–CYP143 and modeled Fdx–CYP51 complexes further revealed the specificity of ferredoxins. Our results illuminate the diversity of electron transfer complexes for the production of different secondary metabolites.

The interaction of the neurotrophin BDNF dipeptide mimetic, compound GSB-106, with the tyrosine kinase TrkB receptor specific for the fullsized neurotrophin was studied using surface plasmon resonance. The significant decrease in the binding of BDNF to TrkB, which was preincubated with GSB-106, was shown. The obtained data indicate the interaction of GSB-106 with the TrkB receptor.

The fractions of 26S and 20S proteasomes have been isolated from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to the proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes involved in various metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.

The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻⁶ M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻⁸ M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.

Most proteins function as part of various complexes, forming via stable and dynamic protein-protein interactions (PPIs). The profiling of PPIs expands the fundamental knowledge about the structures, functions, and regulation patterns of protein complexes and intracellular molecular machineries. Protein interactomics aims at solving three main tasks: (1) identification of protein partners and parts of complex intracellular structures; (2) analysis of PPIs parameters (affinity, molecular-recognition specificity, kinetic rate constants, and thermodynamic-parameters determination); (3) the study of the functional role of novel PPIs. The purpose of this work is to update the current state and prospects of multi-omics approaches to profiling of proteins involved in the formation of stable complexes. Methodological paradigm includes a development of protein-extraction and -separation techniques from tissues or cellular lysates and subsequent identification of proteins using mass-spectrometry analysis. In addition, some aspects of authors' experimental platforms, based on high-performance size-exclusion chromatography, procedures of molecular fishing, and protein identification, as well as the possibilities of interactomic taxonomy of each protein, are discussed.

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.

Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson’s disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle plays an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al., Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, (2020), vol. 14, pp. 297−305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and cell division and differentiation. Selected biosensor validation confirmed the interaction of the proteasome Rpn13 subunit with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) recognized during proteomic profiling. The results obtained suggest that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.

Widespread pathologies such as atherosclerosis, metabolic syndrome and cancer are associated with dysregulation of sterol biosynthesis and metabolism. Cholesterol modulates the signaling pathways of neoplastic transformation and tumor progression. Lanosterol 14-alpha demethylase (cytochrome P450(51), CYP51A1) catalyzes one of the key steps in cholesterol biosynthesis. The fairly low somatic mutation frequency of CYP51A1, its druggability, as well as the possibility of interfering with cholesterol metabolism in cancer cells collectively suggest the clinical importance of CYP51A1. Here, we show that the natural flavonoid, luteolin 7,3′-disulfate, inhibits CYP51A1 activity. We also screened baicalein and luteolin, known to have antitumor activities and low toxicity, for their ability to interact with CYP51A1. The Kd values were estimated using both a surface plasmon resonance optical biosensor and spectral titration assays. Unexpectedly, in the enzymatic activity assays, only the water-soluble form of luteolin—luteolin 7,3′-disulfate—showed the ability to potently inhibit CYP51A1. Based on molecular docking, luteolin 7,3′-disulfate binding suggests blocking of the substrate access channel. However, an alternative site on the proximal surface where the redox partner binds cannot be excluded. Overall, flavonoids have the potential to inhibit the activity of human CYP51A1 and should be further explored for their cholesterol-lowering and anti-cancer activity.

Opportunistic fungi of the genus Candida are currently considered as the major causative agents of mycoses, which are characterized by an especially severe course under conditions of acquired immunodeficiency. The main target for the development of new antimycotics is the cytochrome P450 51 (CYP51) of the pathogenic fungus. The widespread distribution of Candida strains resistant to the azole class of CYP51 inhibitors point to the clear need for the screening for CYP51 inhibitors both among non-azole compounds and among clinically used drugs, which would be repositioned as antimycotics. In this study an integrated approach including bioinformatics analysis, computer molecular modeling, and a surface plasmon resonance (SPR) technology was employed to identify potential inhibitors from the non-azole group. Using in silico modeling, the binding sites for acetylsalicylic acid, ibuprofen, chlorpromazine and haloperidol (these compounds, according to the literature, showed antimycotic activity) were predicted in the active site of CYP51 from 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 Candida albicans CYP51 with possible formation of coordination bonds between the hydroxyl groups of the derivatives and the heme iron atom of CYP51. Thus, the potential lead structures of non-azole compounds have been proposed; they can be used for the design of new CYP51 inhibitors of Candida fungi.

Microsomal systems of human cytochrome P450 consist of three components, which are membrane proteins: cytochrome P450 hemoprotein (CYP), NADPH-dependent cytochrome P450 reductase (CPR), and a small regulatory heme-containing protein cytochrome b5 (CYB5A). In the study of the cytochrome P450 system functioning the study of intermolecular interactions both with partner proteins and with possible drug prototypes is of great importance. Surface plasmon resonance (SPR) is a powerful and reliable tool for studying intermolecular interactions. However, there is a problem of immobilization of membrane proteins on the optical chip of the SPR biosensor. It is important to immobilize such proteins in native conditions with respect to the correct orientation of the protein globule to the surface of sensor. Previously, we have developed and described a method involving direct native immobilization of membrane proteins into a planar bilayer lipid membrane on the surface of a biosensor chip. At the same time, one of the commonly used approaches to working with membrane proteins using various methods is the construction of proteoliposomes containing membrane proteins. In this work, using CYP3A4 and CYB5A as protein partners, we evaluated two approaches to the creation of proteoliposomes: incorporation of a membrane protein into liposomes saturated with detergents and incorporation of a membrane protein into the forming proteoliposomes by the mechanism of micellar coalescence. The interaction of CYP3A4 with proteoliposomes obtained by incorporating CYB5A into detergent-saturated liposomes was shown. On the contrary, interaction between CYP3A4 and proteoliposomes containing CYB5A, obtained by the method of micellar coalescence, was not detected. Thus, it was shown that the incorporation of the membrane protein into liposomes saturated with a detergent was a more preferable method for working with an SPR biosensor as compared to the method of proteoliposomes formation by micellar coalescence. Detailed protocols for the creation of proteoliposomes and SPR-analysis can be useful to a wide range of researchers.

Protein–protein interactions (PPIs) are crucial for the successful realization of many metabolic and signaling pathways. Of particular interest are PPIs of membrane proteins, which form stable and/or transient complexes for the signal transduction, ion transport, and electron transfer within electron transfer chains. The Surface Plasmon Resonance (SPR) allows analyzing the thermodynamic and kinetic parameters ( k on and k off ) and equilibrium constants ( K d ) of PPI, as well as the assessment of the effects of low molecular weight compounds. The aim of the present study was the adaptation of the SPR protocols for PPIs involving mitochondrial cytochrome P450 CYP11A1, mitochondrial cytochrome b5 (CYB5B), and adrenodoxin (Adx). We found that the Adx–CYP11A1 and CYB5B–CYP11A interactions depend on the method of protein immobilization and on the microenvironment. For example, the k off values for complexes Adx–CYP11A1 and CYB5B–CYP11A1 in the aqueous environment were similar (1.5 ± 0.2) × 10 –3 s –1 , while the values of k on for these complexes differed from each other by almost one order of magnitude ((6.5 ± 0.5) × 10 4 and (0.30 ± 0.03) × 10 4 M –1 s –1 , respectively). This is in good agreement with the known high affinity of CYP11A1 to its cognate redox partner Adx. In the lipid environment, the rate of complex dissociation was higher than that in the aqueous environment with k off value equal to (9.1 ± 0.3) × 10 –3 s –1 . For the CYP11A1–CYB5B complex, the parameters of interaction in the lipid phase were not determined due to unspecific binding of the proteins used as analytes to the lipids immobilized on the L1 chip. We show for the first time that SPR method could be used for the detection and quantitative analysis of PPI involving mitochondrial cytochromes (CYP) with their redox partners in both aqueous and lipid environment. Experimental protocols developed and validated in our previous work and in this one can serve as valuable tools for studies of interactions of CYP proteins and could also be applied for other membrane proteins.