Research Projects

Introduction: HPV induced cancers, particularly cervical cancers are a major problem in the Indian population and all of the developing world. In spite of the known addiction of such cancers on the oncogenic HPV-E6 and HPV-E7 proteins no specific drugs exist against such cancers. Rationale: (1) The dependence of HPV induced cancers on oncogenic E6 and E7 can be exploited to treat such cancers by inhibiting their oncogenic partners in the host cells. Recently we have discovered that HPV induced cancers are dependent on the deubiquitinase USP46, that is a druggable target owing to presence of a distinct catalytic pocket. (2) Depletion of USP46 leads to loss of tumor growth and accordingly small molecule inhibitors of USP46 will be potent drugs against such cancers (3) HPV-E6 forms an E6-USP46 complex which targets substrates for deubiquitination. This binding of HPV-E6 to USP46 is dependent on a modification on USP46. Characterization of this modification and identification of its substrates of this complex will substantiate the oncogenic mechanisms and drug candidature of the E6-USP46 complex in HPV induced cancers. Objectives: (1) Identify inhibitors of USP46 by screening small molecule libraries against a deubiquitinase assay that we have established. (2) Identify modifications on USP46 that facilitate the formation of E6-USP46 complex and its physiological targets in HPV induced cancers. Earlier work: (1) We have established a deubiquitinase assay with a Z’ of 0.84 that will be used for small molecule libraries. (2) For objective-2 we have established HPV-positive and negative cell lines where USP46 gene is tagged to Strep tag-II by CRISPR-HDR. These cells express the endogenous USP46 protein as USP46-Strep-tag-II fusion protein. This approach maintains the stoichiometry of the target protein and allows affinity purification of the interacting complex using the Strep-Tactin beads. Methodology: (1) The deubiquitinase assay will be applied to a library of small molecule inhibitors to identify potent inhibitors of the USP46. Identified molecules will be further shortlisted on the basis of specificity to USP46 by testing them against other deubiquitinases and their efficacy against HPV induced cancers. (2) For identifying the modification on USP46 and cellular targets of the E6-USP46 complex, we will affinity purify the USP46-Strep-tag-II from HPV positive and negative cell lines and identify associated proteins by LC/MS. Protein targets specifically present in HPV positive cells will be validated. Significance: USP46 inhibitors identified will be useful in treatment of HPV induced cancers either alone or in combination with existing therapies. Identifying additional targets of the E6-USP46 complex and modification on USP46 that facilitates this complex, will potentiate USP46 candidature as a drug target for HPV induced cancers.