Research data supporting '2,3-dihydropyrrolo[1,2-a]quinazolin-5(1H)-one inhibitors targeting bromodomains within the Switch/Sucrose Non-Fermenting complex'.

Data in support of the characterisation of compounds and their biological binding to proteins in the above manuscript. For synthesised compounds infra-red characterisation traces, HPLC traces where performed, and processed NMR files are provided. Raw files for the interaction of key compounds with PB1(5) and SMARCA2 and SMARCA4 proteins as measured by isothermal calorimetry are included. X-ray crystallography files are available through the Protein Data Bank.This work was supported by the EPSRC [grant numbers EP/K099494/1 EP/K039520/1], Wellcome Trust [grant number 092809/Z/10/Z] and the Cambridge PhD Training Programme in Molecular Medicine

An Activity-based Probe Targeting Non-catalytic, Highly Conserved Amino Acid Residues Within Bromodomains

Bromodomain-containing proteins are epigenetic modulators involved in a wide range of cellular processes, from physiological recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl-lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule based approach to covalently modify recombinant and endogenous bromodomain-containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag, via copper-catalyzed alkyne azide coupling, to an alkyne handle on the probe allowed in-gel visualization and selective pull-down of the desired bromodomains using both recombinant and endogenous proteins.</b

Identification and Development of 2,3-Dihydropyrrolo[1,2‑<i>a</i>]quinazolin-5(1<i>H</i>)‑one Inhibitors Targeting Bromodomains within the Switch/Sucrose Nonfermenting Complex

Bromodomain containing proteins PB1, SMARCA4, and SMARCA2 are important components of SWI/SNF chromatin remodeling complexes. We identified bromodomain inhibitors that target these proteins and display unusual binding modes involving water displacement from the KAc binding site. The best compound binds the fifth bromodomain of PB1 with a <i>K</i>_D of 124 nM, SMARCA2B and SMARCA4 with <i>K</i>_D values of 262 and 417 nM, respectively, and displays excellent selectivity over bromodomains other than PB1, SMARCA2, and SMARCA4

Discovery of an MLLT1/3 YEATS Domain Chemical Probe

YEATS domain (YD) containing proteins are an emerging class of epigenetic targets in drug discovery. Dysregulation of these modified lysine binding proteins has been linked to the onset and progression of cancers. We herein report the discovery and characterisation of the first small molecule chemical probe, SGC-iMLLT, for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). SGC-iMLLT is a potent and selective inhibitor of MLLT1/3 -histone interactions. Excellent selectivity over other human YD proteins (YEATS2/4) and bromodomains was observed. Furthermore, our probe displays cellular target engagement of MLLT1 and MLLT3. The first small molecule X-ray co-crystal structures with the MLLT1 YD are also reported. This first in class probe molecule can be used to understand MLLT1/3 associated biology and the therapeutic potential of small molecule YD inhibitors.</p

[1,2,4]Triazolo[4,3‑<i>a</i>]phthalazines: Inhibitors of Diverse Bromodomains

Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]­triazolo­[4,3-<i>a</i>]­phthalazines are potent inhibitors of both the BET bromodomains such as BRD4 as well as bromodomains outside the BET family such as BRD9, CECR2, and CREBBP. This new series of compounds is the first example of submicromolar inhibitors of bromodomains outside the BET subfamily. Representative compounds are active in cells exhibiting potent cellular inhibition activity in a FRAP model of CREBBP and chromatin association. The compounds described are valuable starting points for discovery of selective bromodomain inhibitors and inhibitors with mixed bromodomain pharmacology

Discovery and Optimization of Small-Molecule Ligands for the CBP/p300 Bromodomains

Small-molecule inhibitors that target bromo­domains outside of the bromo­domain and extra-terminal (BET) sub-family are lacking. Here, we describe highly potent and selective ligands for the bromo­domain module of the human lysine acetyl transferase CBP/p300, developed from a series of 5-isoxazolyl-benz­imid­azoles. Our starting point was a fragment hit, which was optimized into a more potent and selective lead using parallel synthesis employing Suzuki couplings, benz­imid­azole-forming reactions, and reductive aminations. The selectivity of the lead compound against other bromo­domain family members was investigated using a thermal stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromo­domain of BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues, selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (<i>K</i>_d = 21 nM) and selective, displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching (FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nano­molar affinity for CBP/p300; therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300 bromo­domains as therapeutic targets

A Chemical Probe For Tudor Domain Protein Spindlin1 to Investigate Chromatin Functions

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a \u27chromatin or histone code\u27. Proteins that contain \u27reader\u27 domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyllysine/arginine reader domains and was identified as a putative oncogene and transcriptional co-activator. Here we report a SPIN1 chemical probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, identified genes which are transcriptionally regulated by SPIN1 in squamous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis.<br /