PKIS deep dive yields a chemical starting point for dark kinases and a cell active BRSK2 inhibitor

Abstract

The Published Kinase Inhibitor Set (PKIS) is a publicly-available chemogenomic library distributed to more than 300 laboratories by GlaxoSmithKline (GSK) between 2011 and 2015 and by SGC-UNC from 2015 to 2017. Screening this library of well-annotated, published kinase inhibitors has yielded a plethora of data in diverse therapeutic and scientific areas, funded applications, publications, and provided impactful pre-clinical results. GW296115 is a compound that was included in PKIS based on its promising selectivity following profiling against 260 human kinases. Herein we present more comprehensive profiling data for 403 wild type human kinases and follow-up enzymatic screening results for GW296115. This more thorough investigation of GW296115 has confirmed it as a potent inhibitor of kinases including BRSK1 and BRSK2 that were identified in the original panel of 260 kinases as well as surfaced other kinases that it potently inhibits. Based on these new kinome-wide screening results, we report that GW296115 is an inhibitor of several members of the Illuminating the Druggable Genome (IDG) list of understudied dark kinases. Specifically, our results establish GW296115 as a potent lead chemical tool that inhibits six IDG kinases with IC50 values less than 100 nM. Focused studies establish that GW296115 is cell active, and directly engages BRSK2. Further evaluation showed that GW296115 downregulates BRSK2-driven phosphorylation and downstream signaling. Therefore, we present GW296115 as a cell-active chemical tool that can be used to interrogate the poorly characterized function(s) of BRSK2.

Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms

“…We next attempted to validate these in vitro observations in a human cell line. As there are currently no suitable endogenous substrates known to be specifically or exclusively phosphorylated by BRSK1 or 2, or that are not regulated by redox themselves (Tamir et al 2020), we employed a GFP-Tau overexpression system in HEK-293T cells to monitor intracellular BRSK activity. BRSKs have previously been shown to increase the phosphorylation of Tau at Ser 262 (Yoshida and Goedert 2012).…”

“…Full length BRSK kinases share similar domain architecture to other ARK family members, including a ubiquitin associate domain (UBA) and kinase associated domain (KA1) following their kinase domain (Fig 1a). Due to the absence of known endogenous substrates selectively phosphorylated by BRSK1 or 2 (Tamir et al 2020), we utilized a EGFP-Tau overexpression system in HEK-293T cells to assess BRSK activity (Yoshida and Goedert 2012). BRSK1 and 2, when co-expressed with EGFP-Tau, induced substantial phosphorylation of Tau at Ser 262, a modification lost in kinase-dead (KD) mutants with the catalytic aspartate in the ‘HRD’ motif mutated to alanine (D146 BRSK1 or D141 BRSK2 ), as shown in Figure 1b.…”

“…This insight provides a new understanding of BRSK regulation at the structural level and has potential implications in cellular signaling and diseases that warrant further investigation. Validation of these reversibly oxidized Cys species is also of interest relevance as this may implicate a mechanistic role for ROS sensing in the largely obscure BRSK signaling pathways that operate in different cell types, including those that impact on canonical redox pathways that lead to NRF2 inactivation in cells (Tamir et al 2020).…”

“…BRSK proteins expressed from bacteria are unphosphorylated and catalytically inactive, and were activated by incubation with 10 ng of purified LKB1/STRADα/MO25α holoenzyme complex in the presence of 1 mM ATP and 10 mM MgCl 2 for 18 h at 4°C. Phosphorylation of BRSK proteins was verified by mass spectrometry and/or Western blotting analysis using a pThr 172 AMPKα antibody, which demonstrates cross-reactivity for BRSK1/2 T-Loop phosphorylation (Tamir et al 2020).…”

“…The human AMPK-related kinase (ARK) family, consisting of 14 members (termed BRSK1-2, NUAK1-2, SIK1-3, MARK1-4, MELK, and AMPKα1 and AMPKα2) are fundamental regulators of cellular metabolism, growth, differentiation, and polarity (Shao et al 2014; Byrne et al 2020; Shirwany and Zou 2014; Zmijewski et al 2010), and BRSK1/2 function upstream of redox-based signaling to the pleiotropic transcription factor Nrf2 (Tamir, Drewry, et al 2020; Tamir, Bowman, et al 2020). Like other ARK members, BRSK1/2 possess similar structural organization, consisting of an N-terminal serine/threonine catalytic (kinase) domain, which is followed by a ubiquitin-associated (UBA) domain, a C-terminal spacer, and in some members, a kinase-associated (KA1) domain (Bright, Thornton, and Carling 2009) (Fig 1a).…”