The addressable pocket of a protein is often not functionally relevant in disease. This is true for the multidomain, bromodomain-containing transcriptional regulator TRIM24. TRIM24 has been posited as a dependency in numerous cancers, yet potent and selective ligands for the TRIM24 bromodomain do not exert effective anti-proliferative responses. We therefore repositioned these probes as targeting features for heterobifunctional protein degraders. Recruitment of the VHL E3 ubiquitin ligase by dTRIM24 elicits potent and selective degradation of TRIM24. Using dTRIM24 to probe TRIM24 function, we characterize the dynamic genome-wide consequences of TRIM24 loss on chromatin localization and gene control. Further, we identify TRIM24 as a novel dependency in acute leukemia. Pairwise study of TRIM24 degradation versus bromodomain inhibition reveals enhanced anti-proliferative response from degradation. We offer dTRIM24 as a chemical probe of an emerging cancer dependency, and establish a path forward for numerous selective yet ineffectual ligands for proteins of therapeutic interest.
High cancer death rates indicate the need for new anti-cancer therapeutic agents. Approaches to discover new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds by phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the phosphodiesterase 3A gene, PDE3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells while others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggesting a neomorphic activity. Co-expression of SLFN12 with PDE3A correlates with DNMDP sensitivity, while depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.
Neglected tropical disease drug discovery requires application of pragmatic and efficient methods for development of new therapeutic agents. In this report we describe our target repurposing efforts for the essential phosphodiesterase (PDE) enzymes TbrPDEB1 and TbrPDEB2 of Trypanosoma brucei, the causative agent for human African trypanosomiasis (HAT). We describe protein expression and purification, assay development, and benchmark screening of a collection of 20 established human PDE inhibitors. We disclose that the human PDE4 inhibitor piclamilast, and some of its analogs, show modest inhibition of TbrPDEB1 and B2, and quickly kill the bloodstream form of the subspecies T. brucei brucei. We also report the development of a homology model of TbrPDEB1 that is useful for understanding the compound-enzyme interactions and for comparing the parasitic and human enzymes. Our profiling and early medicinal chemistry results strongly suggest that human PDE4 chemotypes represent a better starting point for optimization of TbrPDEB inhibitors than those that target any other human PDEs.
A fundamental biological understanding of the individual contributions of functional domains within multidomain proteins is critical to inform therapeutic approaches to targeting mechanisms driving human disease. TRIM24 is a multi-domain protein that has been broadly characterized as a co-regulator of transcription. It is therapeutically relevant as it is implicated as a dependency in many human cancers, however, the potent and selective inhibitors of the TRIM24 bromodomain do not have well-characterized phenotypic consequences. Where inhibition of one activity of a protein does not appear to be efficacious, chemical knockdown tools allow for the acute depletion of the entire protein, therefore eliminating all protein activities. In this study, we have used chemical degradation as one strategy to target the entire TRIM24 protein, where we have shown that a bifunctional degrader molecule hijacks the ubiquitin ligase machinery for targeted TRIM24 degradation. We have shown that TRIM24 degradation is required to perturb the oncogenic state in leukemia. In this context, TRIM24 degradation rather than bromodomain inhibition alone is required to displace TRIM24 from chromatin. Using this probe, a further understanding of the contribution of TRIM24 domains to its transcriptional activation function will provide mechanistic insight as to how TRIM24 promotes a gene expression program permissive of the oncogenic state, as well as inform a therapeutic approach to target multidomain proteins, such as TRIM24, that are tightly linked to disease. Citation Format: Lara N. Gechijian, Dennis Buckley, Matthew Lawlor, Thomas Scott, Joshiawa Paulk, Jaime Reyes, Georg Winter, Michael Erb, Chris Ott, Sirano Dhe-Paganon, James Bradner. The design and characterization of a selective TRIM24 degrader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 981. doi:10.1158/1538-7445.AM2017-981
Recent large sequencing and cancer dependency studies have accelerated the identification of candidate targets for precision medicine. However, the current drug development paradigm starting with target identification and validation can be slow and has thus far yielded a limited variety of successful targets. We sought to return to an empirical approach to drug discovery and performed a high throughput screen to identify small molecules that were both potent and selective. In a primary screen of 2000 compounds in two cell-lines: A549 and H1734, three compounds only affected H1734 viability. One of which validated in a dose-response experiment with great potency and specificity, we called this small molecule ‘Compound 1B’. In an effort to identify the target of Compound 1B, we profiled 766 genomically-characterized cancer cell lines and found that approximately 4% were sensitive to our compound. Sensitivity was not restricted to a particular tissue of origin. Interestingly, expression of Phosphodiesterase 3A (PDE3A) correlated with cytotoxicity. We further showed that Compound 1B specifically inhibited the enzymatic activity of PDE3A and PDE3B in a panel of 11 different phosphodiesterase family members. However, only a subset of other PDE3 inhibitors shared the same cytotoxic phenotype of Compound 1B. In a rescue screen of 1600 bioactive compounds, we identified the non-lethal PDE3 inhibitors as compounds that were able to rescue cell death induced by Compound 1B. Biochemical assays showed that both Compound 1B, cytotoxic and non-cytotoxic PDE3 inhibitors compete for binding to PDE3A. Knockdown of PDE3A did not affect cell viability and inhibited response of sensitive cell lines to Compound 1B. Thus we have identified a potent and selective small molecule that likely acts through PDE3A to induce cancer cell-line cytotoxicity. Our data suggest a hyper- or neomorphic function of PDE3A induced upon binding of Compound 1B. By cross-referencing integrative datasets with compound-sensitivity data, we show that reversal of the current drug-development paradigm can elucidate novel cancer targets, which are not yet identifiable by analysis of large next-generation sequencing datasets. Citation Format: Luc M. de Waal, Tim A. Lewis, Lara Gechijian, Aviad Tsherniak, Willmen Youngsaye, Matthew Rees, Oliver Mikse, Mark Hickey, Patrick Faloon, Nicola Tolliday, Angela Koehler, Monica Schenone, Kwok Wong, Alykhan Shamji, Benito Munoz, Stuart L. Schreiber, Heidi Greulich, Matthew L. Meyerson. An integrated genomic characterization of the target of a small molecule identifies a novel cancer dependency. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4596. doi:10.1158/1538-7445.AM2014-4596
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