Xenopus laevis as a chemical genetic screening tool for drug discovery and development.

In this thesis we explore the applicability of the X.laevis chemical genetic screening model towards drug discovery and drug development. The NCI diversity set II compound library was screened to identify abnormal pigmentation generating phenotypes that may have therapeutic application towards the treatment of melanoma cancer. 13 hit compounds identified were shown to have significantly lower IC50’s in the A375 melanoma cell line when compared to two control cell lines. Using the structural data of compounds screened (combined with the phenotypic data generated by the X.laevis screen), a report in which targets were predicted for each phenotypic category is described. Of the 10 targets predicted to generate an abnormal melanophore migration phenotype, six presented abnormal pigmentation phenotypes by compound antagonists. Two of these targets had no known previous link towards melanoma cancer. Many of the identified targets were also predicted to be targeted by nine out of 13 of the identified NCI compounds in the library screen. Thus, through a combination of forward chemical genetic screening, appropriate cell based assays and chemoinformatical analysis we have developed an efficient and effective screening strategy for the rapid identification of hit compounds that are likely to be acting through either well known or novel targets that may have possible implications towards the treatment of melanoma cancer. To assess the applicability of the X.laevis model towards drug development, in collaboration with AstraZeneca we designed a renal function toxicity assay. Renal toxicity is a serious concern in the pharmaceutical industry, being responsible for 7% of preclinical compound dropouts. I developed a biochemical assay in which renal function would be monitored by quantfying the concentration of ammonia excreted by embryos into media. A decrease in ammonia detected in the presence of nephrotoxic compounds was hypothesised to iii represent a decrease in renal function, and therefore indicate toxicity. Despite promising preliminary experiments, the original salicylic acid ammonia assay detection method was inhibited by the presence of the compound solvant DMSO. A second assay (the glutamate dehydrogenase assay (GDH)) was trialled which could not detect a change in renal function in response to nephrotoxic compounds when compared to the vehicle control. In its current form, the X.laevis renal function assay is not capable of identifying nephrotoxic compounds and so further work is required.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of East Anglia

Year dc:date.issued

2014

Author dc:creator

• Hendry, Adam

Language dc:language

en