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I began my PhD as a mature student studying the antimalarial target, lactate dehydrogenase from Plasmodium falciparum with Professors Holbrooke and Clarke in 1997. During this time, I designed, cloned, expressed and purified a stable enzyme construct which provided the basis for a full kinetic and mechanistic characterization using a range of biophysical techniques. Crucially, I learned the importance of experimental rigor in all things from my supervisors. In addition to methods such as absorption and fluorescence spectroscopy under transient and steady state conditions, I used FACSIMILE (Atomic Energy Authority) to simulate data for different enzyme mechanisms to compare with my experimentally derived data. I also produced and characterized slow turnover mutants for use in combined crystallography/microspectroscopy experiments.
I then worked on a variety of crystallographic projects, some aimed at novel antimalarials (Plasmodial LDH) or anticancer (human LDH) therapeutics. I used CCP4 programs to solve the crystal structures of proteins including human and plasmodial LDHs (mutants and wild-type) in apo, binary and ternary forms and in complex with inhibitory compounds. I solved the novel maleylpyruvate isomerase enzyme from the proteobacterium Ralstonia using molecular replacement methods and in silico molecular modelling techniques to provide insights into the enzyme mechanism.
I then moved on to a drug discovery project targeting the cholinergic supporting TrkA receptor towards an Alzheimer’s disease (AD) therapeutic, which I later returned to as a nociceptor target for analgesia. I selected compounds for testing in vitro, first by in silico means and then by visual inspection and predicted ADMET criteria. This resulted in an excellent 5 – 10% in vitro hit-rate. I used SAR and molecular modelling methods to predict the binding site of the first family of compounds, subsequently confirmed by NMR (HSQC) once amide peak assignments had been made. I designed a biologically active, TrkAIg2 construct for NMR and crystallography that overcame the problem of strand-swapping which occludes native ligand binding at high protein concentrations (95% amide peaks, 80% side chains assigned). I performed a range of in silico and in vitro techniques, spanning tissue culture, automated immunofluorescence, Western blot, radioligand competition assays, surface plasmon resonance, preparing samples for NMR and assisting in collecting/interpreting spectra. I used molecular dynamics simulations (GROMACS) to provide insights into compound/TrkAIg2 interactions to inform design for improved affinity. I identified compounds for expansion into families and selected eight compounds for efficacy testing, five of which were non-sedating, dose-dependent, analgesics at 1 - 4mg/kg. During this project I gained valuable experience in managing data from diverse sources. The efficient collection, collation, interpretation and feedback of data to the rest of the team contributed to the successful completion of this milestone-driven project.
I was then invited to consult for a project with the MRC Prion Unit to find compounds to stabilize the native fold of the prion protein, towards a therapeutic for Creutzfeldt-Jakob disease. I used our Bristol University Docking Engine (BUDE) docking software on the EMERALD supercomputer (South West GPGPU consortium) to search through 8 million virtual compounds in the ZINC database, to predict binding energies for all compounds docked into a hotspot on the prion protein (identified by BUDE). I wrote code to handle and analyse the ~5 terabytes of data output from this project and selected compounds based on a combination of predicted binding energies and visual inspection of docked poses. Compounds tested in vitro had an impressive ~30% hit rate.
Whilst researching for a grant proposal for Alzheimer’s I stumbled upon tantalizing links between oral bacteria and risk for (AD). In 2012 I designed a study to explore these links experimentally which was funded by the BRACE charity. Having attained ethics approval and confirmed the presence of more, including oral, bacterial DNA in AD brain samples than control subjects (SouthWest Brain bank), I developed a biomarker to monitor oral bacterial levels in the mouth and set up a collaboration with the Bristol Dental Hospital for a feasibility study with people in the early stages of AD. This was the first in a series of trials to ascertain whether reducing oral bacterial load slows cognitive decline. The clinical trials continue.
I now provide molecular modeling support for synthetic biology projects by assisting in the design and understanding of the behaviour of proteins designed de novo, using modelling and atomistic dynamic simulation techniques and docking software. I have been directly involved in many of the diverse projects within BrisSynBio as well as forging and supporting collaborations within the University, across the UK and beyond.
Research Interests
Papers共 70 篇Author StatisticsCo-AuthorSimilar Experts
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Diabetesno. 4 (2024): 565-571
Journal of molecular cell biologyno. 3 (2023)
Journal of Cystic Fibrosis (2023): S44-S45
RSC Medicinal Chemistryno. 8 (2022): 929-943
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