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Title: Inhibition of enzymes from mycobacteria using fragment-based approaches
Author: Whitehouse, Andrew John
ISNI:       0000 0004 7972 991X
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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The work described in this thesis is focused on the application of fragment-based approaches for two essential mycobacterial target proteins, fumarate hydratase (fumarase) from Mycobacterium tuberculosis (Mtb) and tRNA (m1G37) methyltransferase (TrmD) from Mycobacterium abscessus (Mab). With Mtb fumarase a high-throughput screening (HTS) hit was used to design a small library of fragments in a deconstruction-reconstruction approach. These fragments were screened using a range of both biochemical and biophysical methods. The resultant fragments showed evidence of weak protein binding. As an alternative strategy, derivatives of the HTS hit were synthesised and screened by a biochemical assay, which identified nanomolar inhibitors of this enzyme. In addition, X-ray crystallography was also carried out with a range of these compounds. Selected compounds were subsequently screened by collaborators at the NIH against Mtb. With the enzyme TrmD from Mab, the fragment hits identified were used as the basis of a fragment-merging approach to develop potent inhibitors, guided by structural biology. In the implementation of this approach, synthesis and biophysical techniques were extensively utilised, including both differential scanning fluorimetry and isothermal titration calorimetry. This approach led to the development of novel inhibitors with low nanomolar affinity. Select compounds were screened by collaborators against both Mab and Mtb in vitro. In light of encouraging activity against Mtb, the TrmD homolog in Mtb was expressed and screened against select compounds to demonstrate the broader applicability of the lead series.
Supervisor: Abell, Chris ; Coyne, Anthony Sponsor: EPSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Mycobacterium abscessus ; Mycobacterium tuberculosis ; TrmD ; fragment-based ; fumarate hydratase ; fumarase ; tRNA (m1G37) methyltransferase ; differential scanning fluorimetry ; isothermal titration calorimetry ; nontuberculous mycobacteria ; allosteric ; deconstruction-reconstruction ; X-ray crystallography ; fragment-merging ; tuberculosis ; cystic fibrosis