Parallel Optimization of Potency and Pharmacokinetics Leading to the Discovery of a Pyrrole Carboxamide ERK5 Kinase Domain Inhibitor

Abstract

The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.

Modulation of ERK5 Activity as a Therapeutic Anti-Cancer Strategy

“…A study in HeLa cells also linked an ERK5-dependent proliferative response to an upstream growth factor stimulus with the activation of ERK5 being induced by epidermal growth factor (EGF) treatment . Despite these observations, the proliferation of many tumor cells is not impacted by the genetic perturbation of ERK5, including cells where ERK5 is overexpressed or MAPK7 is amplified . There are also reports of ERK5 perturbation with shRNA stimulating the growth of breast cancer xenografts. , Clearly, while there is experimental evidence linking ERK5 to tumor cell proliferation in some cell models, it is not a universal phenomenon.…”

“…Further elaboration of the amide substituent by appending a basic aliphatic heterocycle onto the aryl amide enabled identification of highly potent inhibitors such as 10 (K d < 10 nM). 28 However, the pharmacokinetic parameters were compromised for these basic compounds with low oral bioavailability due to poor passive permeability and high efflux. Activity against these kinases should be taken into consideration when interpreting results from biological studies using this compound, as off-target activities could influence observed results (e.g., inflammation or angiogenesis).…”

ERK5 Signalling and Resistance to ERK1/2 Pathway Therapeutics: The Path Less Travelled?

“…A range of ERK5 kinase domain inhibitors have recently been shown to bind to ERK5 and cause a conformational change that leads to the exposure of the NLS, nuclear localisation, and paradoxical activation of the C-terminal TAD ( Lochhead et al, 2020 ). All ERK5i tested to date, including XMD8-92, XMD17-109 (compound 26, ERK5-IN-1), XMD17-26 (compound 25), AX15836, compound 46, BAY-885, and compound 34b, cause paradoxical activation of the ERK5 TAD, albeit to varying degrees ( Cook et al, 2020 ; Lochhead et al, 2020 ; Miller et al, 2022 ) ( Figure 5A ). These inhibitors are type I (that is, they bind the active kinase conformation) ATP-competitive inhibitors, and it remains possible that other classes of ERK5i may not have the same effect.…”

“…Since then more than 70 protein kinase inhibitors have been approved for clinical use ( Cohen et al, 2021 ). Therefore, it is not surprising that when seeking to target ERK5 in cancer (or other indications) there has been a lot of interest in developing small molecule inhibitors of the kinase domain, including our own collaborations with excellent drug discovery teams ( Myers et al, 2019 ; Miller et al, 2022 ). However, kinase inhibitor programmes have experienced common setbacks including 1) innate or acquired resistance to the kinase inhibitor due to mutation of the intended target, mutation of other pathway components or pathway remodelling (as exemplified above in melanoma and PDAC) and 2) unintended activation of the target pathway, either by inhibition of negative feedback pathways or through inhibitor binding to the kinase resulting in paradoxical activation (the latter termed “inhibitor hijacking of kinase activation”).…”

“…Unfortunately a range ERK5i are capable of inducing a paradoxical effect on the ERK5 TAD function ( Cook et al, 2020 ; Lochhead et al, 2020 ). At present AX15836, compound 46, compound 34b, and BAY-885 are the most potent and selective inhibitors of ERK5 kinase activity but they are all able to drive paradoxical activation of the C-terminal TAD ( Lochhead et al, 2020 ; Miller et al, 2022 ). Clearly a priority is to develop selective (non-BRD4 binding) ERK5i that act as “paradox breakers” by inhibiting kinase activity without causing paradoxical activation of the TAD.…”