YY1 Is a Structural Regulator of Enhancer-Promoter Loops

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 FKBP12F36V with expression of FKBP12F36V 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 KRASG12V 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.

The dTAG system for immediate and target-specific protein degradation

Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

Targeted protein degradation: elements of PROTAC design.

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.

Transcription control by the ENL YEATS domain in acute leukaemia

Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anticancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected polypharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here, we report that benzo[e]pyrimido-[5,4- b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases, including several previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity, as well as how to direct selectivity toward inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 μM BRD4 IC50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.

Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains Structure–activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure–activity data An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement Given these findings, novel and easily synth

Nana K. Offei-Addo

Papers

Transcription control by the ENL YEATS domain in acute leukaemia

Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen