Yaroslav V. Faletrov

22-NBD-cholesterol as a novel fluorescent substrate for cholesterol-converting oxidoreductases.

Docking simulations and experimental data indicate that 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD-cholesterol), a common fluorescent sterol analog, binds into active sites of bovine cytochrome P450scc and microbial cholesterol dehydrogenases (CHDHs) and then undergoes regiospecific oxidations by these enzymes. The P450scc-dependent system was established to realize N-dealkylation activity toward 22-NBD-cholesterol, resulting in 7-nitrobenz[c][1,2,5]oxadiazole-4-amine (NBD-NH(2)) formation as a dominant fluorescent product. Basing on LC-MS data of the probes derivatized with hydroxylamine or cholesterol oxidase, both pregnenolone and 20-formyl-pregn-5-en-3β-ol were deduced to be steroidal co-products of NBD-NH(2), indicating intricate character of the reaction. Products of CHDH-mediated conversions of 22-NBD-cholesterol were defined as 3-oxo-4-en and 3-oxo-5-en derivatives of the steroid. Moreover, the 3-oxo-4-en derivative was also found to be formed after 22-NBD-cholesterol incubation with pathogenic bacterium Pseudomonas aeruginosa, indicating a possible application of the reaction for a selective and sensitive detection of some microbes. The 3-keto-4-en derivative of 22-NBD-cholesterol may be also suitable as a new fluorescent probe for steroid hormone-binding enzymes or receptors.

Evaluation of the fluorescent probes Nile Red and 25‐NBD‐cholesterol as substrates for steroid‐converting oxidoreductases using pure enzymes and microorganisms

The fluorescent probes Nile Red (nonsteroidal dye) and 25‐{N‐[(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐methyl]amino}‐27‐norcholesterol (25‐NBD‐cholesterol) (a cholesterol analog) were evaluated as novel substrates for steroid‐converting oxidoreductases. Docking simulations with autodock showed that Nile Red fits well into the substrate‐binding site of cytochrome P450 17α‐hydroxylase/17,20‐lyase (CYP17A1) (binding energy value of −8.3 kcal·mol−1). Recombinant Saccharomyces cerevisiae and Yarrowia lipolytica, both expressing CYP17A1, were found to catalyze the conversion of Nile Red into two N‐dealkylated derivatives. The conversion by the yeasts was shown to increase in the cases of coexpression of electron‐donating partners of CYP17A1. The highest specific activity value (1.30 ± 0.02 min−1) was achieved for the strain Y. lipolytica DC5, expressing CYP17A1 and the yeasts NADPH‐cytochrome P450 reductase. The dye was also metabolized by pure CYP17A1 into the N‐dealkylated derivatives, and gave a type I difference spectrum when titrated into low‐spin CYP17A1. Analogously, docking simulations demonstrated that 25‐NBD‐cholesterol binds into the active site of the microbial cholesterol oxidase (CHOX) from Brevibacterium sterolicum (binding energy value of −5.6 kcal·mol−1). The steroid was found to be converted into its 4‐en‐3‐one derivative by CHOX (Km and kcat values were estimated to be 58.1 ± 5.9 μm and 0.66 ± 0.14 s−1, respectively). The 4‐en‐3‐one derivative was also detected as the product of 25‐NBD‐cholesterol oxidation with both pure microbial cholesterol dehydrogenase (CHDH) and a pathogenic bacterium, Pseudomonas aeruginosa, possessing CHOXs and CHDHs. These results provide novel opportunities for investigation of the structure–function relationships of the aforementioned oxidoreductases, which catalyze essential steps of steroid bioconversion in mammals (CYP17A1) and bacteria (CHOX and CHDH), with fluorescence‐based techniques.

Uptake and metabolism of fluorescent steroids by mycobacterial cells.

HighlightsMycobacteria can convert BODIPY‐cholesterol and similar NBD‐labeled steroids.4‐En‐3‐one derivatives are major fluorescent products of the bioconversion.NBD fluorophore of the compounds undergoes degradation in the mycobacteria.The steroids cause staining of envelope lipids and intracellular lipid droplets.3‐NBD‐cholestane don’t converted by M. tuberculosis H37Rv and M. smegmatis mc2 155. Abstract Fluorescent steroids BODIPY‐cholesterol (BPCh) and 7‐nitrobenzoxadiazole‐4‐amino‐(NBD)‐labeled 22‐NBD‐chelesterol (22NC) as well as synthesized 20‐(NBD)‐pregn‐5‐en‐3&bgr;‐ol (20NP) were found to undergo bioconversions by Mycobacterium tuberculosis H37Rv and M. smegmatis mc2 155. The major fluorescent products were determined to be 4‐en‐3‐one derivatives of the compounds. Degradation of NBD fluorophore was also detected in the cases of 22NC and 20NP, but neither NBD degradation nor steroidal part modification were observed for the synthesized 3‐(NBD)‐cholestane. Mycobacterial 3&bgr;‐hydroxysteroid dehydrogenases were concluded to be responsible for the formation of the 4‐en‐3‐one derivatives. All the compounds tested were found to cause staining both membrane lipids and cytosolic lipid droplets when incubated with mycobacteria in different manner, demonstrating ability of the steroids to reside in the compartments. The findings reveal a potential of the compounds for monitoring of steroid interactions with mycobacteria and provide information for design of new probes for this purpose.

Effect of the NBD-group position on interaction of fluorescently-labeled cholesterol analogues with human steroidogenic acute regulatory protein STARD1

Steroidogenic acute regulatory protein (StAR, STARD1) is a key factor of intracellular cholesterol transfer to mitochondria, necessary for adrenal and gonadal steroidogenesis, and is an archetypal member of the START protein family. Despite the common overall structural fold, START members differ in their binding selectivity toward various lipid ligands, but the lack of direct structural information hinders complete understanding of the binding process and cholesterol orientation in the STARD1 complex in particular. Cholesterol binding has been widely studied by commercially available fluorescent steroids, but the effect of the fluorescent group position on binding remained underexplored. Here, we dissect STARD1 interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different positions, namely, with 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence response. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and substantially increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP slightly outperforms commonly used 22NC and can thus be used for screening of various potential ligands by a competition mechanism in the future.

Solution Structure Of Human STARD1 Protein And Its Interaction With Fluorescently-Labeled Cholesterol Analogues With Different Position Of The NBD-Group

Intracellular cholesterol transfer to mitochondria, a bottleneck of adrenal and gonadal steroidogenesis, relies on the functioning of the steroidogenic acute regulatory protein (StAR, STARD1), for which many disease-associated mutations have been described. Despite significant progress in the field, the exact mechanism of cholesterol binding and transfer by STARD1 remains debatable, and the solution conformation of STARD1 is insufficiently characterized, partially due to its poor solubility. Although cholesterol binding to STARD1 was widely studied by commercially available fluorescent NBD-analogues, the effect of the NBD group position on binding remained unexplored. Here, we analyzed in detail the hydrodynamic properties and solution conformation of STARD1 and its interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different position, namely 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). The small-angle X-ray scattering (SAXS)-based modeling and docking simulations show that, apart from movements of the flexible Ω1-loop, STARD1 unlikely undergoes significant structural rearrangements proposed earlier as a gating mechanism for cholesterol binding. While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence yield. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP outperforms commonly used 22NC and is recommended for future studies. Our study benefits from state-of-the-art techniques and revisits the results of the STARD1 research over the last 20 years, revealing important novel information.