Functional and Structural Characterization of human Rvb1 and Rvb2 and Screening for Inhibitors of their ATPase Activity

Rvb1 and Rvb2 are highly conserved and essential eukaryotic AAA+ proteins linked to a wide range of cellular processes. AAA+ proteins are ATPases associated with diverse cellular activities. Both proteins have been found to be part of critical cellular complexes and processes in many different species: the chromatin remodeling complexes Ino80 and SWR-C, the telomerase complex, mitotic spindle assembly, as well as phosphatidylinsitol 3-kinase-related protein kinase (PIKK) signaling. Rvb1 and Rvb2 are linked to DNA damage sensing and repair, transcriptional regulation and cell growth, therefore, implicating them in carcinogenesis. Due to wide range of Rvb1 and Rvb2 involvement, their pivotal roles and overexpression in several cancers, investigating their molecular structure and activity will aid in understanding their physiological function and explain their multifaceted role. This project sheds light on the structural and functional characteristics of human Rvb1 and Rvb2 that have been highly controversial as per several independent studies. Rvb1 and Rvb2 were shown to form a hetero-hexameric ring complex that exhibits enhanced ATPase activity in comparison to the individual proteins, with WB motif of both subunits being essential for activity. Our investigation of human Rvbs’ oligomerization using multiple techniques has shown that an N-terminal tag induced dodecamerization of human Rvb1/Rvb2 complex and affected the oligomerization of human Rvb2 alone. A mutation in the DLLDR motif was found to cause a significant enhancement in the ATPase activity of human Rvb2. In addition, our high-throughput screen for inhibitors of the ATPase activity of human Rvbs led to the discovery of sorafenib as a weak inhibitor of human Rvb2 and human Rvb1/2 complex. Sorafenib was characterized to be a mixed-non competitive inhibitor of human Rvb2 with an IC50 of 3.1 µM and Kd of ~ 22 µM. Our work suggests that sorafenib binds to human Rvb2 through the insertion domain (DII) as shown by SPR and ATPase assays, and that it possibly has a physiological role in the SHQ1-DKC1 disassembly. This work advances our understanding of human Rvb1 and Rvb2 and lays out a strong foundation for targeting their ATPase activity as a strategy for the identification of novel cancer therapeutics.Ph.D.2020-11-19 00:00:0

Sorafenib as an Inhibitor of RUVBL2

RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various complexes and cellular processes, several of which are closely linked to oncogenesis. The proteins were implicated in DNA damage signaling and repair, chromatin remodeling, telomerase activity, and in modulating the transcriptional activities of proto-oncogenes such as c-Myc and β-catenin. Moreover, both proteins were found to be overexpressed in several different types of cancers such as breast, lung, kidney, bladder, and leukemia. Given their various roles and strong involvement in carcinogenesis, the RUVBL proteins are considered to be novel targets for the discovery and development of therapeutic cancer drugs. Here, we describe the identification of sorafenib as a novel inhibitor of the ATPase activity of human RUVBL2. Enzyme kinetics and surface plasmon resonance experiments revealed that sorafenib is a weak, mixed non-competitive inhibitor of the protein’s ATPase activity. Size exclusion chromatography and small angle X-ray scattering data indicated that the interaction of sorafenib with RUVBL2 does not cause a significant effect on the solution conformation of the protein; however, the data suggested that the effect of sorafenib on RUVBL2 activity is mediated by the insertion domain in the protein. Sorafenib also inhibited the ATPase activity of the RUVBL1/2 complex. Hence, we propose that sorafenib could be further optimized to be a potent inhibitor of the RUVBL proteins

Sorafenib as an Inhibitor of RUVBL2

RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various complexes and cellular processes, several of which are closely linked to oncogenesis. The proteins were implicated in DNA damage signaling and repair, chromatin remodeling, telomerase activity, and in modulating the transcriptional activities of proto-oncogenes such as c-Myc and β-catenin. Moreover, both proteins were found to be overexpressed in several different types of cancers such as breast, lung, kidney, bladder, and leukemia. Given their various roles and strong involvement in carcinogenesis, the RUVBL proteins are considered to be novel targets for the discovery and development of therapeutic cancer drugs. Here, we describe the identification of sorafenib as a novel inhibitor of the ATPase activity of human RUVBL2. Enzyme kinetics and surface plasmon resonance experiments revealed that sorafenib is a weak, mixed non-competitive inhibitor of the protein’s ATPase activity. Size exclusion chromatography and small angle X-ray scattering data indicated that the interaction of sorafenib with RUVBL2 does not cause a significant effect on the solution conformation of the protein; however, the data suggested that the effect of sorafenib on RUVBL2 activity is mediated by the insertion domain in the protein. Sorafenib also inhibited the ATPase activity of the RUVBL1/2 complex. Hence, we propose that sorafenib could be further optimized to be a potent inhibitor of the RUVBL proteins