Microbial Natural Product Drug Discovery Through Systematic Sampling of Diverse Texas Soils

Purpose: Microorganisms have long been a valuable source of new pharmaceutically relevant small molecules. Because of their intrinsic need to compete for scarce resources in their microenvironment, they have often adapted to produce secondary metabolites capable of exerting cytotoxic effects against competing microorganisms. Microbial natural products and their derivatives account for nearly half of the currently approved anti-infective and anti-cancer drugs. Because of the State of Texas’ rich soil diversity, systematically collecting and fermenting bacteria and fungi collected around the state should yield a library of interesting and diverse molecules which can then be screened for desirable activities and identified for use as lead compounds in pharmaceutical research. Methods: To validate this project, the chemical crude extracts from 80 fungal species were separated through reverse-phase flash chromatography and screened for cytotoxicity against MIA PaCa-2 (ATCC® CRL-1420), SH-SY5Y (ATCC® CRL-2266), and COLO 829 (ATCC® CRL-1974) cancer cell lines as measured by Promega’s CellTiter-Glo® Luminescent Cell Viability Assay (ATP-Luciferase assay). Subsequently, a short-list of fractions containing promising cytotoxic compounds was identified and those fractions were subjected to further purification through multi-step activity-guided reverse-phase high-performance liquid chromatography (HPLC). Accurate masses and isotope distributions for compounds were assessed through time-of-flight mass spectrometry (TOF MS) and compared to known compound databases for dereplication. The purified compounds then underwent high-resolution proton and carbon NMR studies to determine their structure and novelty. Results: The first 80 fungal species collected from around the state were separated into 800 flash chromatography fractions of which 17 exhibited consistent cytotoxicity against MIA PaCa-2, a human pancreatic carcinoma cell line. Early study of a further narrowed test-group of 8 fungal species has thus far yielded a few interesting compounds. Notably of these species, one produces a compound with a mass of 401.257 which has been observed in multiple HPLC generated fractions exhibiting [greater than] 99% cell clearance after 48 hours. Based upon the accurate mass and isotope distribution, this compound preliminarily does not appear to have been previously described. Sufficient quantities of purified compound have been produced to perform high-resolution proton and carbon NMR to determine compound structure. Conclusions: We have seen from our preliminary study that we can systematically construct and screen a library of natural products derived from Texas microbes as way to identify lead compounds which exert cytotoxic effects on established cancer cell lines. Further, through collaboration, this library can be screened against a wider variety of targets to aid in identifying valuable lead compounds.