Data collection and refinement statistics of the dCK/UDP/EdC crystal structure.

Mechanism of action of compounds identified in PRISM screen as top hits with similar efficacy and sensitivity profile as EdC.

ADME and PK properties of 1µM EdC in vitro.

Individual blood concentrations (ng/mL) and pharmacokinetic parameters for EdC after IP injection.

Additional EdC metabolism data.

Supplementary Figure Legends

Studies have demonstrated the impact of alterations of glutamate homeostasis, involving particularly, its excitotoxicity effects in neurological diseases. As an essential neurotransmitter, glutamate is crucial in several physiological functions, and is regulated by two essential transporters, cystine-glutamate antiporter (xCT) and astrocytic glutamate transporter 1 (GLT-1). Dysfunction of these transporters is associated with a variety of neurological disorders as well as neurotoxic outcomes. This in-silico study evaluated selected novel GLT-1 modulators, and a chemical library for their neuroprotective potential through dual targeting of GLT-1 and xCT. Three novel GLT-1 enhancers and a total of 483 chemical compounds from Selleckchem were screened; of the latter, fifty-three compounds were selected based on favorable blood-brain barrier permeability, Lipinski’s rule of five, and lipophilicity. We docked these 53 compounds and ultimately selected two candidates (MC-350013 and corylin) and MC-100093 as reference for molecular simulation studies based on their pharmacokinetic assessment, toxicity profile, and docking scores. MC-350013 showed the strongest binding affinities for both GLT-1 and xCT, with values of -9.5 and − 9.6 kcal/mol; corylin came second with − 9.0 and − 8.8 kcal/mol, respectively. Molecular simulations confirmed the stability of ligand-protein complexes, especially with MC-350013. Further, MM/PBSA analyses support the potential of MC-350013, with values of -17.95 kcal/mol for GLT-1 and − 28.11 kcal/mol for xCT; the corresponding values for corylin were − 26.59 and − 20.81 kcal/mol, respectively. These findings suggest that MC-350013 and corylin could be potential neuroprotective agents for modulating glutamate neurotoxicity, highlighting the value of in-silico drug discovery in identifying molecules targeting glutamate transporters.

Schematic of semi-unbiased screening approach.

Sequential steps and intermediates for synthesis of EdC (compound 13).

RNA-seq analysis of T-ALL Jurkat and DLBCL SUDHL-10 after 16-hour treatment with 1µM EdC.

Evaluation of in vitro ADME properties and in vivo pharmacokinetics of EdC.

Mutation-based sensitivity patterns and correlation analysis of CRISPR knock-out with EdC sensitivity.

EdC sensitivity correlates with sensitivity to replication stress-inducing compounds.

Additional methods for PRISM screen and EdC synthesis.

Dual leucine-zipper kinase (DLK) is implicated in at least two distinct processes that drive neurodegeneration: retrograde (axon-to-soma) signaling to activate pro-degenerative transcription programs, and axon-intrinsic action to drive Wallerian and Wallerian-like axon degeneration. Inhibiting DLK-dependent signaling is thus an attractive neuroprotective strategy, but compounds that inhibit all cellular pools of DLK cause unintended side effects. We recently successfully deployed a complementary approach to identify compounds that selectively block DLK retrograde (axon-to-soma) signaling and subsequent neurodegeneration by inhibiting acute, axonal palmitoylation of DLK. Here, we explored chemical space for one of our two most effective compounds and identified multiple analogs that are equally neuroprotective in a model of transcription-dependent neurodegeneration that requires DLK-dependent retrograde signaling. In contrast, our original hits and these additional analogs had minimal effect in two models of DLK-dependent, but transcription-independent, distal axon degeneration. Moreover, our original hits did not phenocopy the stabilization of axon survival factor proteins that is a well-described effect of conventional DLK kinase domain inhibitors. These findings reveal additional potential neuroprotective compounds and further support the notion that the pool of DLK that conveys axonal retrograde signals can be selectively targeted therapeutically.

Antibodies and experimental conditions used in these studies.

Inhibiting dual leucine-zipper kinase (DLK) could potentially ameliorate diverse neuropathological conditions, but a direct inhibitor of DLK's kinase domain caused unintended side effects in human patients, indicative of neuronal cytoskeletal disruption. We sought a more precise intervention and show here that axon-to-soma pro-degenerative signaling requires acute, axonal palmitoylation of DLK. To identify potential modulators of this modification, we screened >28,000 compounds using a high-content imaging readout of DLK's palmitoylation-dependent subcellular localization. Several hits alter DLK localization in non-neuronal cells, reduce DLK retrograde signaling and protect cultured dorsal root ganglion neurons from neurodegeneration. Mechanistically, the two most neuroprotective compounds selectively prevent DLK's stimulus-dependent palmitoylation and subsequent recruitment to axonal vesicles, but do not affect palmitoylation of other axonal proteins assessed and avoid the cytoskeletal disruption associated with direct DLK inhibition. Our hit compounds also reduce pro-degenerative retrograde signaling in vivo, revealing a previously unrecognized neuroprotective strategy.

PRKN-HMGB1 stimulation of IFN signaling.

PRKN ubiquitination of HMGB proteins.

Immuno-gold labeling of HMGB1-associated membranous structures.