High-Throughput Screen for the Oncoprotein MYC - ABSTRACT MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. More importantly, MYC has been directly implicated in over 50% of human cancers and is recognized as a general hallmark of cancer initiation and maintenance. Despite this prevalence, there are few functional chemical probes for MYC and no therapeutics that target it. We have discovered a compound, KJ-Pyr-9, that binds to MYC with high potency and specificity, downregulates the transcriptional activities of MYC and, most importantly, is the first MYC ligand that shows efficacy in vivo. However, while SAR efforts were able to improve the pharmacokinetic and pharmacodynamic properties of the scaffold, it remains insufficient for therapeutic use. This endeavor did yield several valuable probes, one of which, RSH470, exhibits an increase in fluorescence in the presence of MYC. HDX-MS experiment revealed that RSH470 binds a novel site in the critical bHLH-LZ motif of MYC. Excitingly, modeling and single amino acids mutations of the site have validated this finding and provided a structural explanation of the inhibitor mechanism. Utilizing RSH470, we have developed the first fluorescence-based HTS screening competition assay that specifically identifies MYC inhibitors and does not require protein modification, DNA binding, or the complimentary dimer partner MAX. Furthermore, it is simple, inexpensive, and free of proprietary restrictions that limit available HTS assays for MYC. The effectiveness of this assay has been validated by established orthogonal methods (BLI, Bio-FET, SPR) and cellular oncogenic transformation experiments. Furthermore, structurally distinct compounds, with specific cellular activity, have been discovered by pilot screens performed on both Scripps Research campuses. In this proposal, we present a strategy to screen of the entire >665,000 Scripps Drug Discovery Library (SDDL) to identify novel MYC inhibitor scaffolds. A secondary HTS with and without MYC will determine whether hit activity is MYC dependent. Hits selected by a medicinal chemist will then be validated by bio-layer interferometry (BLI), surface plasmon resonance (SPR), and field-effect transistor analysis (Bio- FET). Cellular potency and MYC specificity will be established through oncogenic transformation assays with orthogonal oncogenes as well as with inhibitor resistant MYC mutants cell lines. The pharmacokinetics properties of leads compounds will be assessed in vitro before their final evaluation in an established xenograft model. This research program will produce a set of precisely characterized chemical leads with a strong correlation between in vitro and in vivo efficacy. Not only will these compounds be beneficial in the study MYC functions, but they will may lead to a therapeutic strategy for MYC driven cancers.
