Optimisation of Cellular thermal shift assay (CETSA) for the examination of novel substrate engagement to integral protein transport UapA

Abstract

Chronic invasive pulmonary infections caused by A. fumigatus airborne spores are an increasing with its high mortality rate and difficulty of treatment. Treating fungal infections requires a new way to not only manage infections but eliminate them. The solved structure of UapA, uric acid-xanthine/H+ symporter, found in model organism in Aspergillus nidulans provides a target for novel antifungal compound synthesis. 3-benzylxanthine bulky synthetic compound is generated and requires testing the engagement of the compound to UapA. Cellular thermal shift assay (CETSA) is required. This paper looks to optimise experimental conditions of CETSA including detergent for membrane solubilisation, heat shock temperature for heat shock. results also explored using fluorescence size-exclusion chromatography (FSEC). Promising results show a preference for 1% DDM solubilisation with heat shock at 45°C. however, this was difficult to decipher with FSEC, with an issue of reproducibility coming up. Preliminary data on 3-benzylxanthine effect on thermostability UapA wildtype, as well as more stable mutant constructs G411V and Q408E, is also presented.