Talapatra, Sandeep Kumar
Mechanistic investigation of small molecule inhibitors of kinesin-5 and kinesin-6 family members in cancer drug development.
PhD thesis, University of Glasgow.
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The kinesins form a superfamily of molecular motors that use energy from ATP turnover to generate force. They carry out a diverse set of cellular functions by transducing this force to transport cargos along microtubule (MTs) tracks. Kinesins are distributed among 14 families with different structural organisations all sharing a conserved motor domain that is comprised of a catalytic site for ATP hydrolysis and a MT binding site. Kinesins perform different functions with numerous key roles in mitosis.
The kinesin-5 family member, Eg5, has been shown to play a key role in cross-linking and separating anti-parallel MTs. Inhibition of Eg5 leads to the formation of characteristic monoastral spindles, cell cycle arrest and may also lead to apoptotic cell death. This has led to Eg5 being identified as a potential target for drug development in cancer chemotherapy. To understand the mode of interaction between the protein and the potent drug candidate ispinesib, an extensive study using biochemical and biophysical techniques together with crystallography is presented in this thesis.
Sustained chemotherapy imposes selection pressure on cancer cells, and can generate resistant mutants against chemotherapeutic agents. We have employed crystallographic, biochemical and biophysical approaches to understand the underlying molecular mechanism of resistance to Eg5 inhibition. These approaches establish that the point mutations in the inhibitor-binding pocket decrease the potency of SB743921, a potent Eg5 inhibitor binding at a site near loop L5, by more than 1000-fold. By determining crystal structures of the mutant Eg5 motor domains in the presence and absence of SB743921 and combining the results with calorimetric and molecular dynamics studies, we show that the development of resistance is due to changes in the global flexibility of the protein.
In a second approach to address drug resistance towards allosteric inhibitors binding at the loop L5 site, we have also characterised inhibitor binding to a novel and distinct allosteric pocket in Eg5. This is the first experimental characterisation of protein-ligand interactions in this new site. These studies also revealed an unexpected second site. Extensive biophysical characterisation was used to determine the importance of each site to the observed biochemical activity.
Finally, I have investigated kinesin-6 members as new targets for therapeutic intervention. Among kinesins involved in the mitotic phase, the members of kinesin-6 family are of particular interest as potential drug targets because of their important roles during the anaphase to telophase transition until completion of cell division by cytokinesis. This family consists of three members in humans; MKLP-1, MKLP-2 and MPP1 with a unique structural feature of a long insertion of around 100 amino acids in the loop L6 region. This unique feature makes them interesting not only as a drug target but also to understand their role in protein function. The motor domain of the kinesin-6 family members, MKLP-2 and MPP1 were cloned, expressed and purified and crystallisation trials were carried out. Moderately potent inhibitors were identified by biochemical screening of freely available compound collections. Selectivity analysis was also carried out against other kinesin members to provide specificity data about the inhibitor.
In collaboration with Christophe Labiere from Dr. Catherine Guillou's laboratory at the CNRS, Gif-sur Yvette, France, a SAR investigation of the MKLP-2 inhibitor paprotrain was carried out using 135 analogues. This required extensive biochemical screening against MKLP-2 to identify more potent analogues of paprotrain.
The first objective of this research is to investigate the mechanism by which existing Eg5 inhibitors block catalytic activity and the mechanism through which Eg5 develops resistance to these inhibitors. A second objective was to lay foundations for structural and biochemical characterisation of kinesin-6 family members. The results of this thesis provide a detailed understanding of Eg5 inhibition by ispinesib and describe an unexpected resistance mechanism towards allosteric Eg5 inhibitors dependent on unfavourable entropic effects in the mutant. The experimental characterisation of a new allosteric inhibitor binding site presented here, together with the crystal structure of an inhibitor binding at this site, provides a foundation for structure based design approaches. The work also provides an extensive study on MPP1 and MKLP-2 cloning, protein expression and purification together with screening and characterisation of their inhibitors.
||Kinesin, Cancer, Drug development, Crystallography, SPR, ITC, ATPase, Eg5, MKLP-2, MPP1, Structure-Activity Relationship
||R Medicine > RM Therapeutics. Pharmacology
||College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
||Pannifer, Dr. Andrew and Kozielski, Prof. Frank
|Date of Award:
Dr Sandeep K Talapatra
||Copyright of this thesis is held by the author.
||07 Dec 2012
||24 Nov 2015 11:32
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