The unfolded protein response modulators GSK2606414 and KIRA6 are potent KIT inhibitors

Abstract

IRE1, PERK, and ATF6 are the three transducers of the mammalian canonical unfolded protein response (UPR). GSK2606414 is a potent inhibitor of PERK, while KIRA6 inhibits the kinase activity of IRE1. Both molecules are frequently used to probe the biological roles of the UPR in mammalian cells. In a direct binding assay, GSK2606414 bound to the cytoplasmic domain of KIT with dissociation constants ( K d ) value of 664 ± 294 nM whereas KIRA6 showed a K d value of 10.8 ± 2.9 µM. In silico docking studies confirmed a compact interaction of GSK2606414 and KIRA6 with KIT ATP binding pocket. In cultured cells, GSK2606414 inhibited KIT tyrosine kinase activity at nanomolar concentrations and in a PERK-independent manner. Moreover, in contrast to other KIT inhibitors, GSK2606414 enhanced KIT endocytosis and its lysosomal degradation. Although KIRA6 also inhibited KIT at nanomolar concentrations, it did not prompt KIT degradation, and rescued KIT from GSK2606414-mediated degradation. Consistent with KIT inhibition, nanomolar concentrations of GSK2606414 and KIRA6 were sufficient to induce cell death in a KIT signaling-dependent mast cell leukemia cell line. Our data show for the first time that KIT is a shared target for two seemingly unrelated UPR inhibitors at concentrations that overlap with PERK and IRE1 inhibition. Furthermore, these data underscore discrepancies between in vitro binding measurements of kinase inhibitors and inhibition of the tyrosine kinase receptors in living cells.

Astrocyte Unfolded Protein Response Induces a Specific Reactivity State that Causes Non-Cell-Autonomous Neuronal Degeneration

“…Trazodone, a drug that acts downstream of eIF2a-P to reverse PERK-eIF2a-P signaling (Halliday et al, 2017), also reduced the elevated expression of Cxcl10, Lcn2, and Vim, as well as C3, on Tg treatment ( Figure S1I). To exclude any off-target effects of GSK2606414 (Mahameed et al, 2019;Rojas-Rivera et al, 2017), we genetically modulated PERK-eIF2a signaling using small interfering RNA (siRNA) targeting PERK expression. A 73% knockdown of PERK expression significantly reduced the levels of eIF2a-P on Tg treatment and, crucially, lowered C3, Cxcl10, Lcn2, and Vim, with 61%, 51%, 82%, and 60% reductions in transcript levels, respectively ( Figure S2).…”

Molecular modeling provides a structural basis for PERK inhibitor selectivity towards RIPK1

“…12 Independently, GSK414 was shown to also inhibit tyrosine kinase receptor c-KIT. 13 Interestingly, the structurally different PERK inhibitor AMG44 does not appear to inhibit RIPK1 or c-KIT. 11,13 Hence, AMG44 is proposed to be a better tool compound to probe for PERK signaling despite the lack of tissue distribution data of the compound.…”

“…13 Interestingly, the structurally different PERK inhibitor AMG44 does not appear to inhibit RIPK1 or c-KIT. 11,13 Hence, AMG44 is proposed to be a better tool compound to probe for PERK signaling despite the lack of tissue distribution data of the compound. On the other hand, GSK414 crosses the blood-brain barrier (BBB) when orally delivered, making it a better choice of inhibitor for central nervous system (CNS) applications.…”

Broad kinase inhibition mitigates early neuronal dysfunction and cognitive deficits in tauopathy

“…GSK2606414 ameliorates molecular underpinnings of neurodegeneration including neuronal loss, cognitive dysfunction, and pathological protein accumulation in a variety of disease models (11)(12)(13)(14)(15)(16)18). These studies established that the benefits of GSK2606414 were conferred by inhibition of its primary target, PERK, which was upregulated in disease.…”

“…This compound potently inhibits multiple tyrosine and serine/threonine kinases at low micromolar concentrations (10) and ameliorates phenotypes in a variety of neurological disorder and neurodegenerative models (11)(12)(13)(14)(15)(16). The targets of 414 include kinases involved in tauopathy pathogenesis, including protein kinase R-like endoplasmic reticulum kinase, or PERK (10)(11)(12), the MAPK cascade (10), receptor-interacting serine/threonine-protein kinase 1, or RIPK1 (17), and KIT (18). We show that brain atrophy and phenotypes of cognitive dysfunction evident in this mouse model of tauopathy are substantially reduced by treatment with 414.…”