Abstract
We developed and implemented a reconstituted system to screen for modulators of the ubiquitination of proliferating cell nuclear antigen, a process that activates pathways of DNA damage tolerance and drug resistance. We identified the primary putatively health-beneficial green tea polyphenol epigallocatechin gallate (EGCG) and certain related small molecules as potent inhibitors of ubiquitination. EGCG directly and reversibly targets the ubiquitin-activating enzyme Uba1, blocking formation of the Uba1~ubiquitin thioester conjugate and thus ubiquitination and in the cell. Structure–activity relationship profiles across multiple biochemical and cellular assays for a battery of EGCG analogues revealed distinct chemical and mechanism-of-action clusters of molecules, with catechin gallates, alkyl gallates, and myricetin potently inhibiting ubiquitination. This study defines a number of related though distinct first-in-class inhibitors of ubiquitination, each series with its own unique activity pattern and mechanistic signature.
Highlights
Substantial compelling evidence suggests inhibition of the DNA repair and damage tolerance machinery is a viable strategy for the development of therapeutics against cancers and other diseases
It consists of a reconstituted system containing biotin-conjugated ubiquitin, FLAG-tagged proliferating cell nuclear antigen (PCNA), nicked circular pUC19 plasmid, replication factor C (RFC), Uba[1], Rad[6], and Rad[18], adapted from a previously published assay[34], with ubiquitination of PCNA by Rad6–Rad[18] being specific for the K164 residue, as K164R mutant PCNA protein is not ubiquitinated[34,35]
Singlet oxygen diffuses away and reacts with a thioxene derivative in the amplified luminescent proximity homogeneous assay (Alpha) acceptor beads, producing a chemiluminescent emission that excites fluors present in the acceptor beads, yielding fluorescence detected by a photomultiplier tube
Summary
Substantial compelling evidence suggests inhibition of the DNA repair and damage tolerance machinery is a viable strategy for the development of therapeutics against cancers and other diseases. We are interested in discovering and leveraging small molecules that modulate the function of proteins involved in the DNA damage response as potential drug leads and research probes. Such agents would ideally limit the proliferation of and induce apoptotic death in diseased cells—either as standalone drugs or in synergistic combination with other treatments, such as chemotherapy, radiotherapy, and targeted therapies—and reverse loss of efficacy in cases of therapeutic resistance. Our results delineate pharmacophores that may serve in the potential development of more selective agents as tools for research and chemotherapeutics against cancers and other diseases
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