Biochemical characterisation of human KifC1 and Eg5, two potential targets for drug development in cancer chemotherapy

• Main Author: Tham, Chuin Lean
• Format: Dissertation
• Language: English
• Published: UCL (University College London) 2019

Kinesins are ATP-dependent molecular motors that mostly travel unidirectionally along microtubules (MTs) to fulfil their roles in intracellular transport or cell division. Those involved in cell division, known as mitotic kinesins have been implicated in a variety of cancers. As they are crucial for cell division, certain mitotic kinesins such as Eg5 and KifC1 have been considered for their potential as novel drug targets. This is with the hope that these drugs will have fewer toxic side effects than conventional cancer therapies using taxanes and vinca alkaloids. Today, several inhibitors targeting Eg5 have entered Phase I, II and III clinical trials either as monotherapies or in combination with other drugs. As new members are continually being validated, KifC1 is also becoming increasingly important as a potential new target. My PhD thesis focuses on the characterisation of human mitotic kinesins KifC1 and Eg5, two potential targets for drug development in cancer chemotherapy. The first part of this thesis (chapter 3) covers the investigation of KifC1. Firstly, the development and optimisation of the protocols for the expression and purification of several KifC1 constructs covering full-length KifC1 is presented. Then, secondary structure and biophysical characterisation of each KifC1 domain is examined. Furthermore, biochemical characterisation of KifC1 is described and the kinetic properties of each construct in relation to the biophysical data such as oligomeric state is discussed. After this, attempts to crystallise the KifC1 motor domain are described. Finally, inhibitor screening of 15,000 small molecules for KifC1-specific hits are reported. The results provide a basis for future characterisation of KifC1. The second part of this thesis (chapter 4) involves the investigation of K858, a novel inhibitor of Eg5 bearing the 1,3,4-thiadiazole scaffold, an important core moiety in many clinical drug candidates targeting a variety of diseases. Biochemical and biophysical characterisation of K858 binding to Eg5 is reported. 1.8 Å resolution crystal structure of the Eg5-K858 complex (PDB ID: 6G6Y) is presented, providing the first structural evidence of how the thiadiazole containing scaffolds ARRY-520 is so successful in advancing into phase III clinical trials. Finally, structure-activity relationship (SAR) study of 22 K858 analogues is reported.