Multilevel structure–activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism

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. Structureactivity 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.

Robust high-throughput assays to assess discrete steps in ubiquitination and related cascades

“…We found the high-throughput Alpha assays for the different steps in the PCNA ubiquitination cascade to be robust, reliable, and sensitive. We found half-maximal inhibitory concentration (IC 50 ) values and structure-activity relationship trends for Uba1 inhibitors we previously reported [19] as determined by the Alpha assay generally corresponding to other more traditional methods in terms of both inhibition of the overall PCNA ubiquitination cascade and Uba1 specifically. Thus, the system performs well as a screening tool for this reaction sequence and as a legitimate quantitative method for measuring ubiquitination and formation of thioester conjugates.…”

“…Starting from conditions we optimized previously for Western blot analysis and quantitation of PCNA ubiquitination, as well as gel-based analyses of the formation of Uba1~ubiquitin thioester and Rad6~ubiquitin thioester conjugates [19], we further refined the reconstituted systems for the more sensitive Alpha technology. We sought to reduce the concentrations of the components in the reactions and also optimize the subsequent detection step of the assays.…”

“…As a departure point, we began with the conditions we had found provided virtually complete ubiquitination of PCNA readily detectable by Western blot analysis [19]. We individually varied concentrations of FLAG-PCNA (Fig.…”

“…We developed three additional Alpha assays to probe other steps in the PCNA ubiquitination cascade. First, we adapted the Alpha system for quantitative evaluation of Uba1~ubiquitin thioester formation, again starting with the conditions we previously worked out for gel-based detection [19] and then refined for Alpha detection. We used a FLAG-Uba1 construct and individually varied concentrations of the analytes, FLAG-Uba1 (Fig.…”

“…Second, we developed an Alpha assay for detection of the Rad6~ubiquitin thioester conjugate, again starting from conditions previously developed for gel-based detection [19]. We made a FLAG-Rad6 construct and performed reactions with non-FLAG-tagged Uba1 and biotin-ubiquitin.…”