scholarly journals Abstract P-11: Microscale Thermophoresis of Mycobacterial Cytochrome P450 with Azole Drugs

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S15-S16
Author(s):  
Ivan Kapranov ◽  
S Bukhdruker ◽  
M Karpova ◽  
Yulia Zagryadskaya ◽  
Ivan Okhrimenko ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Unsaturated Fatty Acids ◽  
Dissociation Constants ◽  
Cytochromes P450 ◽  
Microscale Thermophoresis ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Cytochrome P450 Family ◽  
The Hill

Background: Cytochrome P450 family members are found in most organisms where they are involved in the metabolism and synthesis of steroids, bile acids, unsaturated fatty acids, phenolic metabolites as well as exogenic chemicals. Drugs targeting cytochrome P450 have been shown to inhibit the growth of Mycobacterium tuberculosis, the causative agent of one of the deadliest diseases – tuberculosis. Recently, we showed that CYP124, CYP125, and CYP142 can bind and metabolize a panel of human immunoactive oxysterols in vitro (Varaksa et al., 2021) and one of them (CYP124) can metabolize antituberculosis drugs (Bukhdruker et al., 2020). Thus, inhibition of cytochrome P450 is a promising strategy for the development of new anti-tubercular drugs. The existing methods used to assess protein-ligand interactions for cytochromes P450 (spectral titration and Surface Plasmon Resonance) have a number of limitations. In this regard, we used an alternative approach for this purposes – microscale thermophoresis (MST) which was not previously used for proteins of the cytochrome P450 superfamily Methods: Here we show that MST can be used to determine the micromolar-range dissociation constants (Kd) of membrane-associated mycobacterial cytochrome CYP124 with small-molecule azole drugs. CYP124 was fluorescently labeled with Cy3-NHS and MST curves were collected at Monolith NT.115 instrument (blue/green channel, NanoTemper Technologies) in presence of various concentrations of azole compounds: econazole, ketoconazole, itraconazole, and miconazole. The experimental results were approximated by the second-order bimolecular binding equation as well as by the Hill-Langmuir equation. Results: Therefore, MST is a valuable method for the assessment of cytochrome P450 binding to their ligands for cases when traditional approaches are not applicable. The binding regime of CYP124 with azole derivatives was characterized by the structure of the CYP124 complex with carbethoxyhexyl imidazole solved with ~1Å resolution.

Anti-Müllerian hormone reduces growth rate without altering follicular survival in isolated caprine preantral follicles cultured in vitro

2017 ◽  
Vol 29 (6) ◽  
pp. 1144 ◽  
Author(s):  
R. M. P. Rocha ◽  
L. F. Lima ◽  
I. R. Brito ◽  
G. M. Silva ◽  
H. H. V. Correia ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cytochrome P450 ◽  
Hormone Receptors ◽  
Mrna Levels ◽  
Follicular Growth ◽  
Steroid Secretion ◽  
Preantral Follicles ◽  
Treatment Groups ◽  
Cytochrome P450 Family

The aim of the present study was to evaluate the effect of anti-Müllerian hormone (AMH), with and without FSH, on the in vitro development of isolated caprine preantral follicles, as well as follicular steroid production and mRNA levels of AMH, hormone receptors (AMH and FSH), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP17 (cytochrome P450, family 17, subfamily A, polypeptide 1), HSD3B (3-beta-hydroxysteroid dehydrogenase) and Myc (myelocytomatosis oncogene). Isolated secondary follicles were cultured in minimum essential medium alpha (α-MEM+) alone or supplemented with 50 ng mL–1 AMH and/or 100 ng mL–1 FSH added sequentially on different days of culture. Follicles were cultured for a total of 18 days, with different media during the first (Days 0–9) and second (Days 10–18) halves of the culture period, resulting in six treatment groups, as follows: α-MEM+/α-MEM+, FSH/FSH, AMH/AMH, AMH+FSH/AMH+FSH, AMH/FSH, and FSH/AMH. Follicle development was evaluated on the basis of follicular growth, oocyte maturation and steroid secretion. There was a decrease in follicular growth rate in the AMH, AMH + FSH and AMH/FSH treatment groups compared with α-MEM+ and FSH treatment groups (P < 0.05). However, the different culture conditions had no effect on rates of meiotic resumption and steroid secretion (P > 0.05). Moreover, follicles cultured in the presence of FSH had lower levels of AMH receptor type II (AMHRII) mRNA compared with non-cultured control (freshly isolated follicles), and the AMH and AMH/FSH treatment groups. In conclusion, AMH reduces the follicular growth rate of isolated goat preantral follicles in vitro without affecting follicular survival.

scholarly journals Phytoconstituents from Markhamia Tomentosa Bind To HPV Oncoprotein with Apoptogenic Potential: A Molecular Modeling Approach

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mutiat B. Ibrahim ◽  
Adeola T. Kola-Mustapha ◽  
Niyi S. Adelakun ◽  
Neil A. Koorbanally
Keyword(s):  
Drug Targets ◽  
Active Sites ◽  
Caspase 3 ◽  
Cancer Cell Line ◽  
Hpv 16 ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Potent Growth ◽  
The Stability

Abstract Markhamia tomentosa crude extract and fractions exhibited potent growth inhibitory effects capable to induce apoptosis in cervical (HeLa) cancer cell line via in vitro model. Presently, interaction of M. tomentosa phytoconstituents with molecular drug targets to exert its anticancer property is evaluated via in silico study. Identified phytoconstituents from M. tomentosa were retrieved from PubChem database and docked in active sites of HPV 16 E6, caspase -3 and caspase -8 targets using AutoDockVina from PyRx software. Screening for druglikeness; and absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions was carried out with the use of SwissADME and pkCSM web servers. Standard melphalan and co-crystallized ligands of caspases -3 and -8 enzymes were used to validate protein-ligand interactions. Molecular dynamic simulation was used to validate the stability of the hit molecules complexed with caspases -3 and -8. All identified phytoconstituents from M. tomentosa showed binding affinity for HPV with docking scores range of - 5.4 to -2.6 kcal/mol. Ajugol, carnosol, luteolin and phytol showed good docking energy range of -6.8 to -3.6 kcal/mol; and -4.8 to -1.9 kcal/mol for the active sites of caspases -3 and -8 targets respectively. Based on docking scores; drug-likeliness; and ADMET predictions; luteolin and carnosol were selected as hit compounds. These molecules were found to be stable within the binding site of caspase -3 target throughout the 40ns simulation time. These findings identified hit ligands from M. tomentosa phytoconstituents that inhibit HPV 16 E6 oncogene expression with stimulation of caspases -3 and -8 targets.

Modern Biophysical Approaches to Study Protein–Ligand Interactions

2018 ◽  
Vol 13 (04) ◽  
pp. 133-155
Author(s):  
Priyanka Biswas
Keyword(s):  
Single Molecule ◽  
Analytical Ultracentrifugation ◽  
Correlation Spectroscopy ◽  
Titration Calorimetry ◽  
Resonance Fluorescence ◽  
Biological Interactions ◽  
Protein Ligand Interactions ◽  
Dual Polarization Interferometry ◽  
Ligand Interactions

Protein–ligand interactions act as a pivot to the understanding of most of the biological interactions. The study of interactions between proteins and cellular molecules has led to the establishment and identification of various important pathways that control biological systems. Investigators working in different fields of biological sciences have an intrinsic interest in this field and complement their findings by the application of different biophysical approaches and tools to quantify protein–ligand interactions that include protein–small molecules, protein–DNA, protein–RNA, protein–protein both in vitro and in vivo. In this paper, the various biophysical techniques that can be employed to study such interactions will be discussed. In addition to native gel electrophoresis and fluorescence-based methods, more details will be discussed, on the broad range of modern day biophysical tools such as Circular Dichroism, Fourier Transform Infrared (FTIR) Spectroscopy, Isothermal Titration Calorimetry, Analytical Ultracentrifugation, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy, Differential Scanning Fluorimetry, Nuclear Magnetic Resonance, Mass Spectroscopy, Single Molecule Spectroscopy, Dual Polarization Interferometry, Micro Scale Thermophoresis and Electro–switchable Biosensors that can be used to study the different aspects of protein–ligand interactions.

scholarly journals Footprinting of Inhibitor Interactions ofIn SilicoIdentified Inhibitors of Trypanothione Reductase ofLeishmaniaParasite

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Santhosh K. Venkatesan ◽  
Vikash Kumar Dubey
Keyword(s):  
Virtual Screening ◽  
Leishmania Infantum ◽  
Trypanothione Reductase ◽  
Inhibition Kinetics ◽  
Screening Process ◽  
Protein Ligand Interactions ◽  
Enzymatic Reduction ◽  
Ligand Interactions ◽  
First Time

Structure-based virtual screening of NCI Diversity set II compounds was performed to indentify novel inhibitor scaffolds of trypanothione reductase (TR) fromLeishmania infantum. The top 50 ranked hits were clustered using the AuPoSOM tool. Majority of the top-ranked compounds were Tricyclic. Clustering of hits yielded four major clusters each comprising varying number of subclusters differing in their mode of binding and orientation in the active site. Moreover, for the first time, we report selected alkaloids and dibenzothiazepines as inhibitors ofLeishmania infantumTR. The mode of binding observed among the clusters also potentiates the probablein vitroinhibition kinetics and aids in defining key interaction which might contribute to the inhibition of enzymatic reduction of T[S] 2. The method provides scope for automation and integration into the virtual screening process employing docking softwares, for clustering the small molecule inhibitors based upon protein-ligand interactions.

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