This project resulted in the development high throughput screens for the targets Cbl-b and tyrosyl-DNA phosphodiesterase-2 in collaboration with Drs. Stanley Lipkowitz and Yves Pommier (CCR). The PCMBS, in collaboration with the laboratory of Dr. Lipkowitz (WMB) is developing a high throughput screen (HTS) of both synthetic compounds and natural products extracts for the discovery and characterization of Cbl-b specific biochemical inhibitors. The WMB has been at the forefront of characterizing and understanding the biochemistry of Cbl-b ubiquitin ligase activity and has established the framework for a cell-free screening assay that the PCMBS has been able to adapt for use in an HTS. As a dimeric RING finger E3 ubiquitin ligase, Cbl-b coordinates the interaction between an ubiquitin-containing E2 enzyme and the final target protein to be ubquitinated (the substrate). While Cbl-b itself does not transfer ubiquitin, it is the single component of the ubiquitination cascade that confers substrate specificity. Therefore, Cbl-b is an attractive target for pharmacologic intervention The structure of Cbl-b contains seven different motifs, including an ubiquitin-associated (UBA) domain which has been shown to have a high affinity and specificity for polyubiquitin chains. The PCMBS has previously had success executing an ubiquitin ligase assay which visualized the catalytic activity of the E3 ligase MDM2 using electrochemiluminescence. In the Cbl-b assay, the Cbl-b UBA domain is used to capture ubiquitinated products of Cbl-b. Enzymatically active E1, E2, and Cbl-b are incubated under ubiquitin ligase competent conditions (with ATP and biotinylated ubiquitin) in the presence of test samples. Enzyme activity is determined by application of streptavidin-labeled horseradish peroxidase and a fluorogenic substrate, coupling ubiquitination to fluorescence intensity. The ubiqiuitin ligase reaction is performed in assay plates pre-coated with the Cbl-b UBA, to allow for binding and retention of the polyubiquitinated (and hence biotinylated) reaction products. In addition to the increased stringency of designing the HTS to include washout steps, we have also optimized the assay for the inclusion of both a nonionic detergent (0.01% Triton X-100) and excipient protein (0.5 mg/mL gelatin). We anticipate that the use of these two agents will reduce the detection of false positive results from aggregating or non-specific protein binding compounds. In collaboration with Dr. Yves Pommier (DTB) a chromogenic assay for discovery of natural products based inhibitors of Tdp2 activity was redesigned from an assay developed by Ogilvie et al.). The assay was designed to measure the inhibition of Tdp2 activity by measuring the decrease in the generation of p-nitrophenol from a substrate, 4-NPPP. The absorbance based assay would employ clear, untreated 384-well plates for detection at 405nm, the absorption maxima of p-nitrophenol. The decrease in absorbance signal would correlate directly with the potency of putative inhibitors. Secondary assays with a larger tyrosinylated DNA construct are taking place in the DTB. DTB provided the TTRAP/Tdp2 enzyme construct (produced by Protein Expression Laboratory, NCI at FNL, Frederick, MD) and all initial enzyme quality assurance was evaluated at the MTL. The Tdp2 activity against the 4-NPPP substrate was mapped to similar values of Vmax, Km, and Kcat as that of the the Manchester group. Our assay concentration of 4-NPPP was set below the observed Km value (Km= 115 mM) primarily due to detector saturation at concentrations 100mM or above and to lengthen the time of the assay conditions beyond 20 minutes. Currently, we have screened all available MTL pure compound and synthetic libraries totaling 69,793 compounds with an initial hit rate of 0.22% (hit requirement of 50% inhibition activity) (Fig.4). A representative assay plate result (Fig.4) show the excellent both signal to background and signal to noise (S/B8 and S/N 50 that were consistently observed during the course of the screening process. HTS for CBL-b and Tdp2 inhibitory compounds is ongoing, and will include testing of MTL synthetic compound and natural product extract libraries Additional collaborations on DNAJ-PKA, RNA binding molecules and Uch37 are also underway with Drs. Ping Zhang, Stuart Legrice and Kylie Walters.
