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High Throughput Drug Discovery in S. Cerevisiae: the Characterisation of FC-592 and FC-888

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thesis
posted on 12.11.2021, 20:25 by Sampson, Liam D P

The discovery and characterisation of novel small molecule drug candidates is a medical priority. Recent advances in synthetic organic chemistry allow the de novo production of diversity oriented synthetic compound libraries and synthetic modification of natural products to provide candidate compounds for screening as potential therapeutics, bioactive agents or genetic probes. Small drugs function through interaction with complex genetic networks and pathways. However, it is difficult to characterise these interactions on a genome wide level to achieve understanding of drug mechanism. Here, discovery based approaches are utilised to achieve system wide parsing of biological mechanism, in an attempt to characterise the action of novel synthetic compounds and natural product derivatives. Chemical genomic analysis allows for such understanding by examining growth profiles of a genomic deletion library of Saccharomyces cerevisiae mutants in the presence of sub-inhibitory concentrations of drug. The gene targets of small molecule compounds can be identified by noting deletion strains which display increased sensitivity, indicating chemical interaction with the associated gene network. In addition, the development and characterisation of resistant mutants can be used to identify putative drug targets. In this strategy, characterisation of the mechanism of resistance gives insight into drug mode-of-action. This study develops a high throughput yeast inhibition assay to identify bioactive compounds from a synthetic organic compound library, and attempts to characterise mechanism of action by establishing a profile of each compound’s interaction with these gene networks; and mapping a resistance mutation to provide evidence of inhibitory mechanism. Two candidate compounds are identified, FC-592 and FC-888. FC-592 displayed cytostatic inhibition. Further, yeast tag microarray homozygous profiling (HOP), chemical structure analysis, and cell-cycle analysis via flow cytometry for this compound provided evidence for a mechanism of poor specificity that targets glycoprotein biosynthesis and the secretory (Sec) pathway, as well as the cell-division cycle (CDC) pathway. Attempts to characterise a mutant resistant to this compound via synthetic genetic array mapping were unsuccessful when the resistance mutation proved to mediate a slow growth phenotype, abrogating the Synthetic Genetic Array Mapping approach utilised. Pending further analysis, it is suggested that this compound could have a role as a genetic probe in future exploration of the Sec and CDC pathways. Chemical structure analysis and a non-specific HOP screen chemigenomic profile suggested that FC-888 is an alkylating agent with a broad affinity for cellular nucleophiles. The compound demonstrates cytotoxic activity, and its efflux is not mediated by the pleiotropic drug resistance (PDR) network. It is suggested that the compound could find utility as a probe dissecting processes related to cellular defence against non-DNA specific alkylation.

History

Copyright Date

01/01/2012

Date of Award

01/01/2012

Publisher

Te Herenga Waka—Victoria University of Wellington

Rights License

Author Retains Copyright

Degree Discipline

Biotechnology

Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level

Masters

Degree Name

Master of Science

Victoria University of Wellington Item Type

Awarded Research Masters Thesis

Language

en_NZ

Victoria University of Wellington School

School of Biological Sciences

Advisors

Atkinson, Paul; Bellows, David