Inhibition of enzymes from the non-mevalonate pathway using fragment-based approaches

Abstract

Achieving the world’s development goals is contingent upon realising healthy, sustainable and inclusive food systems and agricultural development is one of the most powerful tools to do this. However, global agriculture is facing many challenges from rising food demands, climate change and the need to improve sustainability. Herbicidal resistance represents one of these major challenges and there has been no herbicide with a novel mode of action commercialised in the last two decades and it is for this reason that there is a crucial need for the design of more potent herbicides with novel mechanisms of action. The work described in this thesis is focussed on the application of fragment-based approaches to identify novel scaffolds as chemical probes against two essential target proteins in the non-mevalonate pathway (MEP), 1-deoxy-D-xylulose 5-phosphate synthase (DXS) from D.radiodurans and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) from E.coli. There have been only a handful of reports of small molecules developed to target the DXS enzyme and this project will be the first to use fragment-based approaches to find small molecules which bind to DXS and DXR. A fragment library screen was employed against both enzymes using a range of both biochemical and biophysical methods. The resultant fragments showed evidence of protein binding with moderate inhibitory activity. X-ray crystallography was attempted in order to ascertain structural binding information for validated fragment hits against both enzymes however, due to difficulties in obtaining crystallographic data alternative strategies were employed. With E.coli DXR validated fragment hits were used to inform an in silico study (in collaboration with industrial sponsors Syngenta AG) which identified a novel class of DXR inhibitors with moderate inhibitory activity. With DXS a proof of concept study evaluating nanobody use as structural chaperones in crystallography experiments opportunistically led to the discovery of a novel class of inhibitors of the enzyme with nanomolar inhibitory activity against A.thaliana DXS.