2 research outputs found
Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains
This is the author accepted manuscript. The final version is available from American Chemical Society via the DOI in this recordBromodomains have been pursued intensively over the past several years as emerging targets for the devel-opment of anti-cancer 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 poly-pharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selec-tive target profile is desired. Here we report that benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones, versatile ATP-site di-rected kinase pharmacophores utilized in the development of inhibitors of multiple kinases including a number of previ-ously 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 di-rect selectivity towards inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first report-ed 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 rec-ognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid dock-ing studies.This work was supported by NIH (Grant No. U54HL127365, to N.S.G. and J.W.; No. NIH P50 GM107618, to X.X. and S.C.B.; Nos. NIH U54 HD093540 and P01 CA066996, to J.Q.), the Medical Research Council (No. MC_UU_12016/2, to D.R.A.), the Spanish Ministerio de Economia y Competitividad (MINECO) (Grant No. SAF2015-60268R, to J.M.L.), and Fondo Europeo de Desarrollo Regional (FEDER) funds (to J.M.L.). D.L.B. was supported as a Merck Fellow of Damon Runyon Cancer Research Foundation (No. DRG-2196-14)
An Initial Characterization of Terminal Uridylyl Transferase 2, a Non-Canonical Poly(A) Polymerase
Located at the 3’ end of mRNA transcripts, the poly-adenosine (poly(A)) tail determines if a mRNA will be translated. With a shortened poly(A) tail, mRNAs in the cytoplasm are dormant and prone to degradation. However, mRNAs can be reactivated by non-canonical poly(A) polymerases, which adenylate mRNAs to lengthen their poly(A) tails. The enzyme Tut2 polyadenylates mRNAs yet monoadenylates their antagonist miRNAs, to stabilize both against degradation. Since Tut2’s activation and regulation are not fully known, we endeavored to characterize Tut2 further through solving its apo-crystal structure, to visualize Tut2 by itself. While the structure was not obtained in this study, in developing a soluble construct, we have shown human Tut2 does not require an accessory protein to bind it for activation, as GLD3 was initially thought to do. This implies human Tut2, unlike C. elegans GLD2, is catalytically active on its own. This conclusion was demonstrated in Tut2’s high affinity for RNA and how having functional active site rendered Tut2 insoluble for purification. With the soluble construct developed in this study, future efforts will be directed towards completing the characterization of Tut2 by solving Tut2’s apo-crystal structure