Identification of novel inhibitor molecules for cancer therapeutics : the c-Myc inhibition story (PhD)

Abstract

c-Myc, a member of the basic helix-loop-helix transcription factor family, regulate the expression of nearly 15% of the entire human genome and maintain cellular homeostasis. The c-Myc dimerize with its partner protein, MAX, and bind to the target gene's enhancer box sequence, thereby regulating its expression. The level of c-Myc is tightly regulated in the normal cells; however, a defect in these regulatory checks leads to aberrant expression of c-Myc, leading to oncogenic phenotype. Researchers have employed various transcriptional and post-translational strategies to inhibit the functionality of c-Myc but, no clinically approved c-Myc targeted therapy has been discovered yet. Targeting the c-Myc/MAX heterodimerization is considered a tough job because of the challenges associated with it. The MAX interacting domain of c-Myc is flat, large, and uncategorized to bind small molecules. Additionally, the bHLH domain of c-Myc is disordered in nature and, therefore, displays structure heterogeneity. Due to the highly dynamic structure, discovering inhibitors using the traditional drug discovery approach is not entirely plausible, making the c-Myc an "undruggable" target. In the present study, we have considered all these challenges associated with c-Myc mediated cancer therapeutics and employed various strategies to overcome these challenges to discover novel c-Myc inhibitor molecules. Firstly, we considered the helix-loop-helix structure of c-Myc and identified a potential druggable site. The identified site was further employed to screen molecules that can successfully inhibit the functionality of c-Myc. As an important cancer target, screening of molecules that show efficient binding to the disordered c-Myc will definitely open the scope of c-Myc targeting. Therefore, in the next objective, we study the physical interaction of Salvianolic Acid B with the disordered c-Myc peptide employing several in silico and biophysical experiments. Further, we studied the structural aspects of the c-Myc/MAX interface to identify "hot-spot" residues and considered the disordered nature of c-Myc. The identified hot-spots on the MAX interacting interface of c-Myc were used to discover novel inhibitor molecules by employing the conformation ensemble approach. Using various c-Myc targeting strategies, we have reported four novel molecules as c-Myc inhibitors that showed potential for cancer therapeutics. These molecules showed substantial in vitro potency, evident from their anticancer potential against c-Myc expressing cancer and cancer stem cells with significant IC50 values in the lower micromolar to nanomolar range. The compounds successfully inhibited the regulatory function of c-Myc by abrogating the c-Myc/MAX heterodimerization. Further, using various biophysical and computational studies, the compounds showed an effective binding affinity and stability toward the disordered c-Myc peptide. Our data collectively present four novel c-Myc inhibitors with substantial in vitro potency achieved by functional inhibition of the c-Myc. Further, preclinical studies on these four compounds will establish their in vivo efficacy and pharmacokinetics and render them promising c-Myc targeted antineoplastic drugs.