Identification of a retinal neuroprotective kinase inhibitor with preferential activity against DLK compared to LZK

Abstract

Purpose : Dual leucine zipper kinase (DLK, MAP3K12) signaling has been implicated in the death of multiple types of neurons, and inhibitors of DLK promote retinal neuronal survival in a variety of in vitro and in vivo models. Additionally, the homologous protein leucine zipper kinase (LZK, MAP3K13) acts synergistically in inducing retinal ganglion cell (RGC) death. With the goal of developing novel therapeutics for retinal disease, we have been working to develop highly-specific small molecule DLK inhibitors.

Methods : A TR-FRET based high throughput assay was developed and used to assay nearly four million compounds for DLK inhibitory activity. Dissociation constants were determined for selected “hits” using an SPR binding assay. Broader kinase selectivity profiles were assessed (Eurofins). Neuroprotective activity was assessed by measuring the survival of primary mouse RGCs. Medicinal chemistry was used to optimize lead compounds.

Results : The diazepinone BAY-452 inhibits DLK in our biochemical assay, IC₅₀ = 180 nM; similar K_D values were determined at DiscoverX (K_D = 88 nM) and in an SPR assay (K_D = 110 nM). Unlike most other DLK inhibitors, whose LZK inhibitory activity tends to parallel their activity against DLK, BAY-452 is markedly less active against LZK, demonstrating an IC₅₀ of approximately 9 µM. Based upon a co-crystal structure of BAY-452 with DLK’s kinase domain, we developed a hypothesis to explain its preference for DLK over LZK. BAY-452 is only moderately selective – in a test panel of 318 kinases, 50 were inhibited >70% at 1 µM compound concentration. BAY-452 shows mixed PK in vitro, with good permeation in a Caco2 assay, but only intermediate-to-low metabolic stability in rat hepatocytes. Consistent with these results, in an initial PK study in mice, the half-life is 0.76 hr. BAY-452 promotes the survival of RGCs, but with considerably less potency than other molecules with similar DLK and greater LZK inhibitory activity. Genetic disruption of LZK increases BAY-452’s RGC survival promoting activity.

Conclusions : Through a combination of high throughput screening and medicinal chemistry we identified the diazepinone BAY-452 as a potent DLK inhibitor. Compared to other DLK inhibitors, BAY-452 shows selectivity against DLK vs. LZK. Thus BAY-452 may serve as a useful molecular probe to dissect the role of DLZ vs. LZK activity in cellular signaling and neuroprotection.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.