The bromodomain‐containing protein BRD9, a subunit of the human BAF (SWI/SNF) nucleosome remodeling complex, has emerged as an attractive therapeutic target in cancer. Despite the development of ...chemical probes targeting the BRD9 bromodomain, there is a limited understanding of BRD9 function beyond acetyl‐lysine recognition. We have therefore created the first BRD9‐directed chemical degraders, through iterative design and testing of heterobifunctional ligands that bridge the BRD9 bromodomain and the cereblon E3 ubiquitin ligase complex. Degraders of BRD9 exhibit markedly enhanced potency compared to parental ligands (10‐ to 100‐fold). Parallel study of degraders with divergent BRD9‐binding chemotypes in models of acute myeloid leukemia resolves bromodomain polypharmacology in this emerging drug class. Together, these findings reveal the tractability of non‐BET bromodomain containing proteins to chemical degradation, and highlight lead compound dBRD9 as a tool for the study of BRD9.
With structural guidance alongside comparative biochemical and biological assays, an iterative design strategy resulted in the development of small‐molecule protein degraders that rapidly, potently, and selectively eliminate bromodomain‐containing protein BRD9 from the BAF complex. These first in class non‐BET bromodomain degraders show significant potency improvements over existing BRD9 probes in models of acute myeloid leukemia.
Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription ...elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.
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•Competitive BET bromodomain inhibition differs from BET protein degradation•BET proteins are master regulators of productive transcription elongation•BET protein degradation is inconsequential for CDK9 recruitment•BET degradation provokes an assembly defect of a transcription elongation complex
Winter et al. delineate fundamental differences in the molecular pharmacology of BET bromodomain inhibition and BET protein degradation. Comparative studies led to the identification of BET proteins as master regulators of transcription elongation. Acute BET protein degradation prompts a global collapse of productive elongation that is independent of CDK9 recruitment.
Thorough preclinical target validation is essential for the success of drug discovery efforts. In this study, we combined chemical and genetic perturbants, including the development of a novel ...selective maternal embryonic leucine zipper kinase (MELK) inhibitor HTH-01-091, CRISPR/Cas9-mediated MELK knockout, a novel chemical-induced protein degradation strategy, RNA interference and CRISPR interference to validate MELK as a therapeutic target in basal-like breast cancers (BBC). In common culture conditions, we found that small molecule inhibition, genetic deletion, or acute depletion of MELK did not significantly affect cellular growth. This discrepancy to previous findings illuminated selectivity issues of the widely used MELK inhibitor OTSSP167, and potential off-target effects of MELK-targeting short hairpins. The different genetic and chemical tools developed here allow for the identification and validation of any causal roles MELK may play in cancer biology, which will be required to guide future MELK drug discovery efforts. Furthermore, our study provides a general framework for preclinical target validation.
Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, ...and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12
with expression of FKBP12
in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRAS
loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.
I. Targeted protein degradation of TIP60 to regulate Treg cell activity in lung cancerRegulatory T (Treg) cells are significant contributors to immune system suppression. Foxp3 is the key ...transcriptional regulator of Treg cells, responsible for their development and function. Foxp3 is a target of acetylation by lysine acetyltransferases (KATs), such as TIP60 and P300. The acetylation of Foxp3 regulates Treg cells activity, leading to a suppression of immune responses. TIP60 is the major protein that has been shown to regulate the Treg cell level by interacting with Foxp3. Therefore, targeting TIP60 in a selective manner is significant for the regulation of immune system activity. We designed a library of TIP60 degraders to recruit the Cereblon (CRBN) E3 ubiquitin ligase and promote ligand-induced dimerization of TIP60 and CRBN in cells to degrade TIP60 by the proteasome. By varying linker composition and length, we were able to assess conditions that allow for effective degradation of TIP60 in vitro and in vivo.II. Synthesis of small molecule inhibitors for Gαi-GIV protein-protein interaction to target cell migration in breast cancer.G-protein coupled receptors (GPCRs) are the largest, most diverse group of membrane receptors in eukaryotes, with a crucial role in cell function and activity. Heterotrimeric G proteins are highly utilized signaling nodes and achieving and controlling their activation have been of interest for decades. GIV, a metastasis-related protein expressed in highly invasive cancer cells, binds, and activates Gα, resulting in the dissociation of Gβγ which leads to cell migration in cancer. Therefore, we designed small molecule modulators to target the Gαi-GIV interaction. NMR binding studies, along with computational modeling enabled us to synthesize a library of fluorene-sulfonamide analogues. In vivo studies after treating the MDA-MB-231 bearing mice with the most potent analogue from the library showed a decrease in tumor cell migration.
Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of ...chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.
The bromodomain‐containing protein BRD9, a subunit of the human BAF (SWI/SNF) nucleosome remodeling complex, has emerged as an attractive therapeutic target in cancer. Despite the development of ...chemical probes targeting the BRD9 bromodomain, there is a limited understanding of BRD9 function beyond acetyl‐lysine recognition. We have therefore created the first BRD9‐directed chemical degraders, through iterative design and testing of heterobifunctional ligands that bridge the BRD9 bromodomain and the cereblon E3 ubiquitin ligase complex. Degraders of BRD9 exhibit markedly enhanced potency compared to parental ligands (10‐ to 100‐fold). Parallel study of degraders with divergent BRD9‐binding chemotypes in models of acute myeloid leukemia resolves bromodomain polypharmacology in this emerging drug class. Together, these findings reveal the tractability of non‐BET bromodomain containing proteins to chemical degradation, and highlight lead compound dBRD9 as a tool for the study of BRD9.
Niedermolekulare Abbaureagentien, die schnell, wirksam und selektiv das Bromodomänenprotein BRD9 aus dem BAF‐Komplex eliminieren, wurden mithilfe einer iterativen, strukturbasierten Designstrategie entwickelt. In Modellen akuter myeloischer Leukämie zeigen diese ersten Abbaureagentien für Bromodomänen außerhalb der BET‐Familie eine deutlich höhere Wirkung als existierende BRD9‐Sonden.
Activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of cell signaling widely targeted by clinically approved drugs. However, it has ...become evident that heterotrimeric G-proteins can also be activated via GPCR-independent mechanisms that remain untapped as pharmacological targets. GIV/Girdin has emerged as a prototypical non-GPCR activator of G proteins that promotes cancer metastasis. Here, we introduce IGGi-11, a first-in-class small-molecule inhibitor of noncanonical activation of heterotrimeric G-protein signaling. IGGi-11 binding to G-protein α-subunits (Gαi) specifically disrupted their engagement with GIV/Girdin, thereby blocking noncanonical G-protein signaling in tumor cells and inhibiting proinvasive traits of metastatic cancer cells. In contrast, IGGi-11 did not interfere with canonical G-protein signaling mechanisms triggered by GPCRs. By revealing that small molecules can selectively disable noncanonical mechanisms of G-protein activation dysregulated in disease, these findings warrant the exploration of therapeutic modalities in G-protein signaling that go beyond targeting GPCRs.
I. Targeted protein degradation of TIP60 to regulate Treg cell activity in lung cancer;
;
Regulatory T (Treg) cells are significant contributors to immune system suppression. Foxp3 is the key ...transcriptional regulator of Treg cells, responsible for their development and function. Foxp3 is a target of acetylation by lysine acetyltransferases (KATs), such as TIP60 and P300. The acetylation of Foxp3 regulates Treg cells activity, leading to a suppression of immune responses. TIP60 is the major protein that has been shown to regulate the Treg cell level by interacting with Foxp3. Therefore, targeting TIP60 in a selective manner is significant for the regulation of immune system activity. We designed a library of TIP60 degraders to recruit the Cereblon (CRBN) E3 ubiquitin ligase and promote ligand-induced dimerization of TIP60 and CRBN in cells to degrade TIP60 by the proteasome. By varying linker composition and length, we were able to assess conditions that allow for effective degradation of TIP60 in vitro and in vivo.;
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II. Synthesis of small molecule inhibitors for Gαi-GIV protein-protein interaction to target cell migration in breast cancer.;
;
G-protein coupled receptors (GPCRs) are the largest, most diverse group of membrane receptors in eukaryotes, with a crucial role in cell function and activity. Heterotrimeric G proteins are highly utilized signaling nodes and achieving and controlling their activation have been of interest for decades. GIV, a metastasis-related protein expressed in highly invasive cancer cells, binds, and activates Gα, resulting in the dissociation of Gβγ which leads to cell migration in cancer. Therefore, we designed small molecule modulators to target the Gαi-GIV interaction. NMR binding studies, along with computational modeling enabled us to synthesize a library of fluorene-sulfonamide analogues. In vivo studies after treating the MDA-MB-231 bearing mice with the most potent analogue from the library showed a decrease in tumor cell migration.
2023-03-30T00:00:00Z
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