Bioinformatic identification of novel melanoma drug targets and investigation of potential inhibitors using high throughput screening

Abstract

A vast and growing amount of genomic, molecular and clinical information about melanoma is available to researchers. This information may be especially valuable for the identification of novel drug targets. However, due to the challenge of assembling, integrating and interpreting this dispersed information, it is difficult to leverage to use for drug target prioritisation or to aid rational experimental design. In this project, multiple sources of information about melanoma have been integrated into a comprehensive bioinformatics database. This database allows complex links to be drawn between somatic variants in individual melanomas revealed by DNA sequencing, associations between gene expression in melanoma and patient outcome, data concerning drug targets, biomarkers, protein druggability, the biomedical literature and clinical trials. While recent years have seen the advent of many new therapies for melanoma including the immune checkpoint inhibitors, factors such as tumour heterogeneity and drug resistance mean that treatment remains challenging. Development of new, varied therapeutic options are still urgently required. Therefore, the melanoma database was used to identify a short list of putative melanoma drug targets. From this list, one candidate, the transcription factor YB-1, was selected as a target for high throughput screening. YB-1 expression in melanoma has a strong association with patient outcome and plays distinct roles in multiple aspects of cancer biology. This makes YB-1 a valuable but potentially difficult target, requiring a multi-pronged approach to identify lead compounds. Two novel high throughput assays were developed to detect compounds that might interfere with YB-1 binding to nucleic acid. The first assay is an in vitro cell-based, luciferase reporter gene assay that detects the activation of the E2F1 promoter fragment by YB-1. The second assay is based on the AlphaScreen system, detecting YB-1 binding to a single-stranded DNA oligonucleotide. These orthogonal assays complement one another by measuring the binding of YB-1 to two discrete nucleic acid sequences with completely different read-outs. These assays were used in high throughput screening of over 7,000 small molecule compounds to identify eight compounds for follow-up study and development.