Microbial transformation of steroids and evaluation of their biotransformed products as antiproliferative agents and acetylcholinesterase inhibitors (in-vitro and insilico studies) / Sharifah Nurfazilah Wan Yusop

Natural products account for 60% of the total market, making them a major source of
drug discovery. Some of these are sourced from the cultivation of microorganisms.
Microbial transformation is an example of the application of cultivation of
microorganisms. It is a method of modifying the chemical structure of compounds such
as steroids by microorganisms. The diversity of the possible reactions types in microbial
transformation includes the process of oxidation, hydroxylation, esterification,
isomerization, reduction, acetylation, hydrogenation and glycosylation. Therefore, the
present study aims to screen new microbial strains which can carry out steroid
bioconversions, and isolate prospective biologically active biotransformed products. No
report on biotransformation of medroxyprogesterone has been published to date
although biotransformations of medroxyprogesterone acetate have been studied by
several groups. To date, only one study has published information on the microbial
transformation of ethynodiol diacetate (ED), which is achieved by Cunninghamella
elegans. In this study, the biotransformations of steroids medroxyprogesterone, and
ethynodiol diacetate by a series of fungi (ATCC, endophytes and Antarctic) have been
investigated. The literature on manipulations of the psychrotolerant fungi, specifically
the Antarctic fungi for biotransformations is almost non-existent. Screening
experiments were performed in 100 ml conical flasks containing 40 ml media and were
autoclaved at 121°C for 15 minutes. After 3 days of inoculation, substrates were
introduced aseptically into fermented liquid media and further fermentations were
allowed for 4-12 days. Metabolic changes were observed by comparing the HPLC
chromatograms of starting compounds, control cultures and fermented extracts. For
medroxyprogesterone, Trichothecium roseum ATCC 13411, R3-2 SP17, Mucor
plumbeus ATCC 47400, and Cunninghamella elegans ATCC 36112 were found to be
suitable for large scale productions. Botrytis cinerea ATCC 36112, Trichothecium
roseum ATCC 13411, and R3-2 SP17 were selected to proceed with large scale
fermentation of ethynodiol diacetate. After separation and purification of the mixtures,
the complete spectrometric analysis was performed to verify the structures of
transformed products. The products with satisfactory quantity along with their
precursors were subjected to anti-proliferative assay and acetylcholinesterase inhibition
assay. From the analysis of the above data, one of the IC50 of the biotransformed
products (the new biotransformed product, MP 2) is close to the limits of the active
cytotoxic limit of a pure compound defined by the American National Cancer Institute,
which is 4 μg/mL or less. Modification of steroid has also revealed to improve its antiacetylcholinesterase
activity to some extent than its precursor specifically in the MP
series. This is observed in MP 7, the new isolated biotransformed metabolite. The
activities reported here deserve attention, and they are good examples in supporting the
application of microbial transformation as a viable method of future development of
anti-proliferative drug candidates, and acetylcholinesterase inhibitors.