M4K2234

M4K2234 is a reasonable cellular probe to interrogate ALK1/2 biology in cells. The data reported for this compound in the JMC 2020 publication confirms good on-target dual selectivity, however, there are off-targets that need to be noted. In the publication and probe package TNIK is listed as an important off-target, however, also the selectivity over ALK6 is poor. While the authors argue > 25% remaining activity at 1 uM is an appropriate 'filter', the determined IC50 on ALK6 with a 6x selectivity over ALK2 calls for additional kinases to be considered as off-targets, depending on the dose used. A concentration above 1 uM was consequently not recommended. The recently reported much more selective tool molecule (ALK2 over 1) from Incyte provides an additional compound in this family that could be valuable to explore side-by-side with M4K2234. While in vivo use @ 10 mpk is suggested for this probe, the data presented is inconclusive and thus does not support in vivo use. With a short t1/2 of the compound and no information on protein binding, it cannot be assessed whether (and how long) meaningful free drug concentrations are reached to engage the target in vivo. Without additional information there is insufficient data supporting the utility of M4K2234 in vivo.

Cellular off-target data against ALK3, ALK6 and TNIK is a desirable addition to the data package, since these kinases have in vitro IC50s in the double digit nM range. The cut-off range for off-target activity (< 75% inhibition at 1µM in vitro) is quite generous, and thus additional off-target activity may be an issue. Supplementing experiments with this probe with e.g. an inhibitor-resistant mutant rescue is recommended. The authors show that the probe has oral bioavailability and characterize the P.K. properties at the recommended dose. The brain exposure of these molecules is modest, and K,p,uu is not reported. Biomarker effects or in tissue target engagement were not measured in vivo. Therefore, it is recommended to characterize these parameters to aid interpretation and dosing in biological studies using this compound in vivo.

M4K2234 is a relatively selective ALK1, TINK, and ALK2 inhibitor. The compound's utility is improved when paired with M4K2234NC, a non-active control compound. M4K2234 can be used to selectively modulate ALK1, TINK, and ALK2, however, care must be exercised in the dosing (both in cells and in vivo models). I would suggest a concentration < 1 µM, ideally 100-500 nM for cells and a dose that achieves < 1 µM Cmax for animal studies.