Evaluation of Luminogenic Substrates as Probe Substrates for Bacterial Cytochrome P450 Enzymes: Application to Mycobacterium tuberculosis
| Autor: | James J. Cali, Dongping Ma, Sandra Ortega Ugalde, Jan N. M. Commandeur |
|---|---|
| Přispěvatelé: | AIMMS, Molecular and Computational Toxicology |
| Rok vydání: | 2019 |
| Předmět: |
cytochrome P450
High-throughput screening high-throughput screening 01 natural sciences Biochemistry Analytical Chemistry Hydroxylation 03 medical and health sciences chemistry.chemical_compound SDG 3 - Good Health and Well-being Luciferase 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology Lanosterol Cytochrome P450 Mycobacterium tuberculosis bioluminescence Luciferin 3. Good health 0104 chemical sciences 010404 medicinal & biomolecular chemistry Enzyme tuberculosis chemistry biology.protein Molecular Medicine Adenosine triphosphate Biotechnology |
| Zdroj: | Ortega Ugalde, S, Ma, D, Cali, J J & Commandeur, J N M 2019, ' Evaluation of Luminogenic Substrates as Probe Substrates for Bacterial Cytochrome P450 Enzymes : Application to Mycobacterium tuberculosis ', SLAS Discovery, vol. 24, no. 7, pp. 745-754 . https://doi.org/10.1177/2472555219853220, https://doi.org/10.1177/2472555219853220 SLAS Discovery, 24(7), 745-754. SAGE Publications Inc. |
| ISSN: | 2472-5552 |
| DOI: | 10.1177/2472555219853220 |
| Popis: | Several cytochrome P450 enzymes (CYPs) encoded in the genome of Mycobacterium tuberculosis (Mtb) are considered potential new drug targets due to the essential roles they play in bacterial viability and in the establishment of chronic intracellular infection. Identification of inhibitors of Mtb CYPs at present is conducted by ultraviolet-visible (UV-vis) optical titration experiments or by metabolism studies using endogenous substrates, such as cholesterol and lanosterol. The first technique requires high enzyme concentrations and volumes, while analysis of steroid hydroxylation is dependent on low-throughput analytical methods. Luciferin-based luminogenic substrates have proven to be very sensitive substrates for the high-throughput profiling of inhibitors of human CYPs. In the present study, 17 pro-luciferins were evaluated as substrates for Mtb CYP121A1, CYP124A1, CYP125A1, CYP130A1, and CYP142A1. Luciferin-BE was identified as an excellent probe substrate for CYP130A1, resulting in a high luminescence yield after addition of luciferase and adenosine triphosphate (ATP). Its applicability for high-throughput screening was supported by a high Z’-factor and high signal-to-background ratio. Using this substrate, the inhibitory properties of a selection of known inhibitors could be characterized using significantly less protein concentration when compared to UV-vis optical titration experiments. Although several luminogenic substrates were also identified for CYP121A1, CYP124A1, CYP125A1, and CYP142A1, their relatively low yield of luminescence and low signal-to-background ratios make them less suitable for high-throughput screening since high enzyme concentrations will be needed. Further structural optimization of luminogenic substrates will be necessary to obtain more sensitive probe substrates for these Mtb CYPs. |
| Databáze: | OpenAIRE |
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