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Malcolm Walkinshaw

Co-workers:

Liz Blackburn,Sandra Bruce, Yiyuan Chen, Jaqueline Dornan, Peter Fernandes, Charis Georgiou, James Kinkead, Divya Malik, Iain McNae, Paul Michels, Matthew Nowicki, Giulia Romanelli, Paul Taylor, Martin Wear, Andromachi Xipnitou, Li-Hsuan Yen, Meng Yuan
Malcolm Walkinshaw

Drug Discovery and Molecular Recognition in Biological Systems

Communication within and between living cells depends on molecular interactions. We are interested in understanding how proteins, small molecule ligands and drug molecules interact and regulate cellular processes. The enzymes in the glycolytic pathway provide an interesting model system, as the ten enzymatic steps that break down glucose to eventually generate pyruvate are all strictly regulated by elaborate feedback mechanisms. The pathway has evolved over a period of 2 billion years and most of the enzymatic steps are well conserved between bacteria, protists and mammals. X-ray structural studies show that the active sites are highly conserved. In contrast, control mechanisms have evolved in a variety of surprisingly different ways. We have studied isoforms of the two main regulatory enzymes in the pathway namely phosphofructokinase (PFK) and pyruvate kinase(PYK). By analysing the differences between the allosteric mechanisms of mammals, bacteria and parasitic protists, we are designing drug-like inhibitors that will specifically inhibit the infectious organism but leave the host unaffected.

The ‘Tri Tryps’ parasitic protists Trypanosoma cruzi, Trypanosoma brucei and Leishmania Mexicana which cause a number of deadly diseases including Chagas disease, sleeping sickness and leishmaniasis are of particular interest; many of the glycolytic steps occur in special organelles called the glycosomes that evolved some 600 million years ago resulting in a very different set of allosteric regulatory mechanisms. Our current focus is on developing a drug against T.brucei which is carried by the tsetse fly and enters the bloodstream of the host when the fly has a blood meal. It relies solely on the glucose in mammalian blood to generate ATP and blocking the glycolytic pathway leads to death within minutes. We have used X-ray structures of the tetrameric TbPFK enzyme to explain its allosteric mode of action. Figure 1 shows the active site of one of the tetramer chains with
the substrate molecules (ATP and glucose-6-phosphate) lined up in the active site. Phospho transfer from ATP to form the product glucose-1,6-phosphate requires a concerted motion of all four subunits and a rearrangement of protein loops. As this mechanism is unique to the parasite enzyme we have been able to design a series of allosteric inhibitors that block this movement and inhibit glycolysis in the parasite without affecting the mammalian enzyme. These compounds are also effective at killing the parasites in infected mice and will hopefully lead to a new class of badly needed trypanocidal drugs.

Selected publications:

Naithani, A., Taylor, P., Erman, B., and Walkinshaw, M. D. (2015) A Molecular Dynamics Study of Allosteric Transitions in Leishmania mexicana Pyruvate Kinase. Biophys J 109, 1149-1156
Blackburn, E. A., Wear, M. A., Landre, V., Narayan, V., Ning, J., Erman, B., Ball, K. L., and Walkinshaw, M. D. (2015) Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90. Biosci Rep 35
Shave,S.,Blackburn,E.A.,Adie,J.,Houston,D.R.,Auer,M.,Webster,S.P.,Taylor, P. and Walkinshaw, M.D., UFSRAT: Ultra-Fast Shape Recognition with Atom Types–The Discovery of Novel Bioactive Small Molecular Scaffolds for FKBP12 and 11βHSD1 (2015),PloS one 10 (2), e0116570


Left: T.brucei phosphofructokinase tetramer showing the substrate molecules ATP and fructose-6-phophate in the active site of one of the chains (highlighted in colour).
Right: close-up of active site showing the drug molecule (purple) in an allosteric pocket locking the enzyme in the inactive conformation