br Conflict of interest br Acknowledgements br Introduction

Conflict of interest
Acknowledgements
Introduction The mitogen-activated protein kinase (MAPK) pathway is an important intracellular signaling system that regulates diverse cellular functions, such as proliferation, differentiation, and apoptosis [1]. Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), is an ubiquitously expressed member of MAPK kinase kinases regulating Jun N-terminal kinase (JNK) and p38 signaling [[2], [3], [4]]. ASK1 is preferentially activated in response to various stimuli including reactive oxygen species (ROS), lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), mitochondrial dysfunction and endoplasmic reticulum (ER) stress [[5], [6], [7], [8]]. The redox regulation of ASK1 occurs via thioredoxin (TRX) which is covalently bound to the N-terminal region of ASK1 through a disulfide bond [9]. Under conditions such as oxidative stress, ROS induced liberation of TRX from ASK1 leads to ASK1 homodimerization [10] and the phosphorylation of the critical threonine residue (Thr838) in the activation loop of ASK1 [11]. A number of studies using ASK1-deficient mice suggest that ASK1 has important functions in oxidative stress-related diseases. ASK1−/− mice display a protective phenotype in multiple disease models including 3CAI [12], acute kidney injury [13], neurodegenerative disorders [8], cardiovascular [14,15], inflammatory [16], and metabolic disorders [17]. Thus, it is thought that small molecule compounds inhibiting ASK1 could be used for the treatment of these pathologies. Recent studies suggest that ASK1 inhibitors may be protective in rodent models of ischemia-reperfusion injury [18], dilated cardiomyopathy [19], diabetic nephropathy [20], and hepatotoxic liver injury [21]. Several ASK1 inhibitors have been disclosed in the public domain (Fig. 1). Imidazopyridine 1 was recently reported to inhibit ASK1 with an IC50 of 14 nM [22]. Subsequently, additional ASK1 inhibitors have been described in the patent literature with compounds 2 [23] and 3 [24] as representative examples from their respective filings, while analog 4 was the sole compound reported in a patent application [25] and was recently reported to be in clinical trials as GS-4997/selonsertib for diabetic kidney disease and nonalcoholic steatohepatitis (NASH) [26].
Structural analysis An analysis of the ATP-binding site revealed that most of the amino acids in each position of the ASK1 active site frequently occur in other kinases as well. For example, 182 of the 442 kinases examined, including ASK1, share a methionine as the “gatekeeper” residue (Fig. 2). In contrast, Gln756 (at the GK+2 position using the nomenclature of Ghose et al. [27]) has a very low abundance in this particular position of the ATP binding site of kinases. Only 3 kinases feature a Gln at the GK+2 position: ASK1 (MEKK5, MAP3K5), TAK1 (MAP3K7), and PIK3R4. Inspection of a crystal structure [22] of imidazopyridine 1 bound in the active site of ASK1 revealed that the side-chain of Gln756 engages the backbone carbonyl of Ile706 in an intra-protein hydrogen bond. It was speculated that this interaction widens the channel at the hinge, allowing the t-Bu phenyl moiety of compound 1 to move past the Gln756 side chain and access space commonly inaccessible. This distinguishing feature of the ASK1 pocket within the kinome was effectively demonstrated from the overlay of crystal structures of ∼4800 kinase inhibitors from the Pfizer database into the ATP active site of ASK1 (Fig. 3; For clarity Fig. 3 was prepared by clustering the 4800 inhibitors to 1000 clusters and the cluster centers were kept). It was noted that inhibitor 1 emerges from this ensemble of kinase inhibitors to occupy this unusual selectivity channel adjacent to Gln756. This observation, in addition to the fact that the GK+2 position is rarely populated by the polar amino acid glutamine led to the design hypothesis that (1) accessing this unique channel, as observed for compound 1, with small molecules while simultaneously (2) interacting with Gln756 at the GK+2 position would lead to potent and highly selective ASK1 inhibitors.