Discovery and development of human origin recognition complex (ORC) inhibitors as a new class of anti-cancer agents

Johns Hopkins University, Baltimore MD

Investigators

Abstract

Project Summary/Abstract The origin recognition complex (ORC) plays a critical role in DNA replication initiation by facilitating the ATP- dependent loading of MCM2-7 helicases onto DNA to form pre-replicative complexes. This process, known as origin licensing, ensures that DNA replication is tightly controlled and occurs only once per cell cycle. However, in cancer cells, this replication control system is often disrupted, contributing to uncontrolled proliferation. Cancer cells that are highly proliferative or harbor mitotic checkpoint defects often exhibit a strong dependency on replication initiation factors such as ORC, making them particularly vulnerable to inhibitors targeting this process. Consequently, ORC represents an attractive therapeutic target for cancer treatment. Despite its importance, there are currently no approved small molecules that specifically target ORC. The development of such inhibitors could selectively induce replication stress and cell death in cancer cells with minimal impact on normal cells. This paradigm is exemplified by a previously identified replication licensing inhibitor, called RL5a, which I have biochemically established to inhibit ORC-DNA binding. A promising avenue for targeting ORC involves the use of rapafucins, a novel class of macrocycles inspired by the natural product rapamycin, designed to target difficult- to-drug protein-protein interactions. Rapafucins have shown potential in cancer-related targets, making them an excellent candidate for developing selective ORC inhibitors that exploit cancer-specific vulnerabilities. In the present proposal, I aim to identify and characterize novel inhibitors of ORC to disrupt origin licensing and selectively target cancer cells. My approach integrates high-throughput biochemical screening with structural and functional studies to uncover the mechanisms underlying ORC inhibition. I have established the utility of the rapafucin library using a pilot microarray screen of a subset (~20%) of the full library, identifying several compounds that selectively bind to ORC and inhibit its binding to DNA. In Aim 1, I plan to conduct high- throughput biochemical screens of the rapafucin library to identify additional compounds that inhibit ORC-DNA binding as well as ORC ATPase activity. Promising hits will be evaluated for their ability to selectively impair the growth of cancer cells, particularly in those with known initiation factor dependencies to identify rapafucin leads with a favorable therapeutic index. In Aim 2, I will use cryo-EM to determine high resolution structures of ORC in complex with RL5a, as well as with rapafucins identified from the microarray that inhibited ORC-DNA binding or impaired cancer cell growth. These structural studies will provide detailed insights into the binding modes of these inhibitors and help identify key residues involved in ORC-inhibitor interactions. My planned work will further establish a pipeline for determining how any new hits obtained as part of Aim 1 also act on ORC. Ultimately, these combined effects will lay the foundation for the development of effective therapies that target the replication initiation process in cancer cells.