Abstract 1264: Design, synthesis and biological evaluation of new phenothiazine derivatives as potential Tousled-like kinase 1 inhibitors in prostate cancer treatment

Abstract

Abstract Introduction: The standard therapy for advanced Prostate Cancer (PCa) consists of anti-androgens which provide respite from the disease progression, yet ultimately fail and result in the incurable phase of the disease: mCRPC. Targeting PCa cells before their progression to mCRPC would significantly improve the outcome. Untoward toxicity limits the combination therapies targeting the DNA Damage Response (DDR), and hence the goal of clinical trials is to target the DDR more specifically. Androgen deprivation therapy (ADT) in LNCaP cells results in the increased expression of TLK1B, a critical kinase upstream of NEK1 and ATR, thereby mediating a DDR that typically causes a temporary cell cycle arrest of androgen-responsive PCa cells. Following the DNA damage, the addition of a TLK1 specific inhibitor, thioridazine (THD), impairs ATR and Chk1 activation, establishing the existence of an ADT>TLK1>NEK1>ATR>Chk1 DDR pathway, while its abrogation, leads to apoptosis. However, THD is a known anti-psychotic and has undesirable side-effects. Hence, there is a compelling need to design and develop next-generation TLK1 inhibitors to circumvent the adverse effects and advance them in the clinic. Methods: We performed immunoblotting of the tumour tissue phosphoproteins (pATR, pChk1 and pNEK1) and immunohistochemistry analysis of the tissue sections from the LNCaP xenograft models. To identify and develop new potent inhibitors against TLK1, we employed an in-silico homology modelling and molecular docking approach. Based on the protein-ligand binding interactions and the docking score, a handful of compounds were shortlisted, synthesised and screened for the TLK1 inhibition potential in-vitro and using cell-based assays. Results: Our experimental data revealed that the pATR, pChk1, pNEK1, Ki-67 and PCNA were remarkably inhibited when treated with THD in combination with an anti-androgen drug, Bicalutamide (BIC). Moreover, it also induced apoptosis and increased DNA damage as demonstrated by the cleaved PARP, Caspase 3 and γH2AX levels respectively. The new inhibitor screening assay showed J54 compound to be most potent and inhibitory with a logIC50 of 1.1µM. J54 binds to the protein’s allosteric site noncompetitively with ATP and interacts with His504 and Gly630 with a corresponding docking score of -6.736. J54 is found to be non-toxic to normal cells and also suppresses the growth of androgen-dependent colonies of LNCaP cells cultured with BIC. Conclusion: Our preliminary work suggests that targeting the TLK1/NEK1 axis with specific TLK1 inhibitors might be an effective therapy for PCa in combination with standard care, ADT. Citation Format: Siddhant Bhoir, Javeena Hussain, Vibha Singh, Rupesh Chikhale, Richard Bryce, Sivapriya Kirubakaran, Arrigo DeBenedetti. Design, synthesis and biological evaluation of new phenothiazine derivatives as potential Tousled-like kinase 1 inhibitors in prostate cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1264.