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

Abstract

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 IC (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.

Discovery of a selective inhibitor of doublecortin like kinase 1

“…ERK5 32 P-ATP kinase assay was performed as previously described, 12 with minor modifications to the reported protocol, with 50 μM [γ− 32 P]-ATP (500 cpm/pmol) and 200 μM PIMtide (ARKKRRHPSGPPTA) used as substrates. Briefly, compounds were tested in 12-point dose response, at a maximum concentration of 10 μM.…”

“…These molecules were initially developed to target the unrelated kinases LRRK2 and ERK5, respectively, and later discovered to also inhibit BET bromodomains. 11 12 LRRK2-IN-1 and XMD8–92 have been shown to reduce proliferation of human pancreatic cancer cell lines and murine Kras G12D pancreatic spheroids, and to exert cytostatic effects in murine xenograft models. 13 5 However, the pleiotropic effects of BRD4 bromodomain inhibition 14 and the abstruse role of ERK5 in oncogenesis 15 confound interpretation of these results.…”

Small molecule ERK5 kinase inhibitors paradoxically activate ERK5 signalling: be careful what you wish for…

“…This study also suggested that the ERK5 C-terminal domain is important for the biological function of ERK5 [64]. Heedful researchers have since used BRD4 inhibitors, such as JQ1, to delineate the role of BRD4 in their systems [25,45,55,56] and testing of ERK5 inhibitors against BRD4 is now an essential step in ERK5 drug discovery. Adding to the complexity, we have found that cpd 26 (XMD17-109) and AX15836, cause a conformational change in the kinase domain which leads to exposure of the C-terminal NLS and paradoxical activation of the ERK5 TAD [55] (see below and Figure 2).…”

“…MEK5-catalysed phosphorylation of the activation-loop TEY motif activates the ERK5 kinase domain, which subsequently drives autophosphorylation at multiple sites within the Continued C-terminus (Figures 1 and 2). This multi-site phosphorylation causes a pronounced reduction in mobility of ERK5 on SDS-PAGE gels [44,45]. Since this ERK5 'band shift' reflects kinase domain-catalysed autophosphorylation, its loss in cells treated with an ERK5i reports inhibition of ERK5 ( Figure 1A).…”

“…The kinase selectivity data for JWG-071 have been published, showing that it only inhibits three other kinases (LRRK2, PLK2, DCAMKL1 and 2) at low micro molar concentrations. In cell-based assays, an inhibitor that blocks LRRK2 activity (such as JWG-048 [45]), and not ERK5 activity, could be used to confirm ERK5 kinase involvement. A multi-site collaboration reported cpd 46; this inhibitor is distinct from the XMD8-92 series, has no activity towards BRD4 or LRRK2, is highly selective for ERK5 and is also suitable for use in animal studies [56].…”