More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.
HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.
Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. The cellular targets of these small molecules have yet to be identified. We have discovered that these molecules target oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L)—proteins not known to be involved in cancer cell survival. OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal transduction, lipid transport and lipid metabolism. The functions of OSBP and the ORPs, however, remain largely enigmatic. Based on our findings, we have named the aforementioned natural products ORPphilins. Here we used ORPphilins to reveal new cellular activities of OSBP. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.