Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics.

The conjugation of proteins with ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous malignancies. For this reason, there is great interest in targeting the ubiquitin-proteasome system in cancer. Several classes of proteasome inhibitors, which block degradation of ubiquitylated proteins, are widely used in research, and one, Bortezomib, is now in clinical use. Despite the well-defined and central role of the ubiquitin-activating enzyme (E1), no cell permeable inhibitors of E1 have been identified. Such inhibitors should, in principle, block all functions of ubiquitylation. We now report 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41) as the first such inhibitor. Unexpectedly, in addition to blocking ubiquitylation, PYR-41 increased total sumoylation in cells. The molecular basis for this is unknown; however, increased sumoylation was also observed in cells harboring temperature-sensitive E1. Functionally, PYR-41 attenuates cytokine-mediated nuclear factor-kappaB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of TRAF6, which is essential to IkappaB kinase activation. PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of IkappaBalpha. Furthermore, PYR-41 inhibits degradation of p53 and activates the transcriptional activity of this tumor suppressor. Consistent with this, it differentially kills transformed p53-expressing cells. Thus, PYR-41 and related pyrazones provide proof of principle for the capacity to differentially kill transformed cells, suggesting the potential for E1 inhibitors as therapeutics in cancer. These inhibitors can also be valuable tools for studying ubiquitylation.

In vitro screening for substrates of the N-end rule-dependent ubiquitylation.

We describe a systematic, high-throughput approach to the discovery of protein substrates of ubiquitylation. This method uses a library of cDNAs in combination with a reticulocyte lysate-based, transcription-translation system that acts as both an excellent means for high-throughput protein expression and a source of ubiquitylation enzymes. Ubiquitylation of newly expressed proteins occurs in this milieu from the action of any one of a number of E3 ligases that are present in the lysate. Specific detection of ubiquitylated proteins is carried out using electrochemiluminescence-based assays in conjunction with a multiplexing scheme that provides replicate measurements of the ubiquitylated products and two controls in each well of a microtiter plate. We used this approach to identify putative substrates of the N-end rule-dependent ubiquitylation (mediated by the UBR family of ubiquitin ligases), a system already well known to have high endogenous activity in reticulocyte lysates. We screened a library of approximately 18,000 cDNA clones, one clone per well, by expressing them in reticulocyte lysate and measuring the extent of modification. We selected approximately 500 proteins that showed significant ubiquitylation. This set of modified proteins was redacted to approximately 60 potential substrates of the N-end rule pathway in a secondary screen that involved looking for inhibition of ubiquitylation in reticulocyte lysates supplemented with specific inhibitors of the N-end rule ubiquitylation. We think our system provides a general approach that can be extended to the identification of substrates of other E3 ligases.

Assays for high-throughput screening of E2 AND E3 ubiquitin ligases.

We developed a series of assays for biochemical activities involving ubiquitin. These assays use electrochemiluminescence detection to measure the ubiquitylation of target proteins. To enable electrochemiluminescence detection, the target proteins were prepared as bacterially expressed fusion proteins and captured on the surface of specially designed microtiter plates having integrated electrodes. Ubiquitylation was quantitated directly, through the use of ubiquitin labeled with an electrochemiluminescent label, or indirectly, through the use of labeled antiubiquitin antibodies. Assays were carried out in both 96-well and 384-well plates. The success of the assay with this variety of formats allowed the selection of optimal work flows for specific applications on the basis of ease of use and overall reagent consumption and availability. We used our ubiquitylation assays to measure the activities of E2 ubiquitin-conjugating enzymes and E3 class ubiquitin ligases. Signal/background ratios for many of our assays were greater than 50, significantly facilitating their conversion to high-throughput practice in a convenient manner. The speed, sensitivity, and convenience of the assay formats makes them well suited for comprehensive interrogations of libraries of compounds or genes in applications like drug and substrate discovery for ubiquitin ligases.

Small molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate p53 in cells.

The p53 tumor suppressor protein is regulated by its interaction with HDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. We have identified a family of small molecules (HLI98) that inhibits HDM2's E3 activity. These compounds show some specificity for HDM2 in vitro, although at higher concentrations effects on unrelated RING and HECT domain E3s are detectable, which could be due, at least in part, to effects on E2-ubiquitin thiol-ester levels. In cells, the compounds allow the stabilization of p53 and HDM2 and activation of p53-dependent transcription and apoptosis, although other p53-independent toxicity was also observed.