The human vaccinia-related kinase 1 (VRK1), is a mitotic kinase, involved in cell division regulation and carcinogenesis. VRK1 phosphorylates the histone H3 at Thr3 and Ser10, which is a critical event for initiating chromosome condensation during mitosis. The deregulation of VRK1 can affect chromatin architecture and remodeling during mitosis, which could result in aneuploidy and genome instability, leading to cancer. Recent studies have shown that high levels of VRK1 is directly correlated to poor clinical outcomes in breast cancer patients, apart from colorectal, prostate and lung cancers among others. Hence, inhibiting VRK1 is considered a viable approach toward developing cancer drugs. VRK1 can be targeted... More
The human vaccinia-related kinase 1 (VRK1), is a mitotic kinase, involved in cell division regulation and carcinogenesis. VRK1 phosphorylates the histone H3 at Thr3 and Ser10, which is a critical event for initiating chromosome condensation during mitosis. The deregulation of VRK1 can affect chromatin architecture and remodeling during mitosis, which could result in aneuploidy and genome instability, leading to cancer. Recent studies have shown that high levels of VRK1 is directly correlated to poor clinical outcomes in breast cancer patients, apart from colorectal, prostate and lung cancers among others. Hence, inhibiting VRK1 is considered a viable approach toward developing cancer drugs. VRK1 can be targeted by ATP-competitive inhibitors, commonly known as Type I inhibitors, as it possesses a conserved ATP-binding domain. Currently, there are ~37 FDA-approved kinase inhibitors available, and most of them belong to this category, implying the importance of kinase inhibitor discovery and at the same time posing a challenge toward designing specific kinase inhibitors. Therefore, a three-dimensional portrait of VRK1 with its substrate will provide structural insights into developing specific inhibitors for VRK1 which lacks cross-reactivity with other kinases. To-date, the structural basis of VRK1-ATP interaction is not available, hindering the progress toward designing VRK1 specific Type I inhibitors. Moreover, the molecular basis of histone H3 substrate recognition on VRK1 remain poorly understood, which is also impeding our efforts in developing alternative inhibitors against VRK1. Thus, in this thesis, I have focused on the structural characterization of (a) ATP-binding to VRK1 and (b) VRK1’s interaction with the nucleosome, for the understanding of VRK1-mediated histone H3 regulation. In this direction, first the crystallographic structure of VRK1 in complex with AMP-PNP was determined at a resolution of 2.07Å, revealing several key residues such as Asp132, Phe134, Met131, Ser181 and Val196 of VRK1 important for ATP interaction, supported by NMR studies. A structural comparison with the inhibitor-bound VRK1, its paralogs and other mitotic kinases enabled us to identify hotspots to design more specific ATP-competitive VRK1 inhibitor. In the second part of thesis, the molecular interaction of VRK1 with histone H3 was characterized. To this end, attempts were made to obtain nucleosome core particle (NCP) in complex with VRK1. While our preliminary studies using mobility shift electrophoretic assay indicated complex formation, nonetheless, obtaining the homogenous NCP-VRK1 complex remained a challenge. To achieve this purpose, further optimization is required to unravel the VRK1 and histone H3 interaction mechanism, which together with the ATP binding studies, can offer more strategies for the development of novel VRK1 inhibitors with better specificity and potency in the future.
