The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis.

TRIM9 and TRIM67 are neuronally enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified putative neuronal TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high-priority candidates was validated, including Myo16, Coro1A, MAP1B, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized total internal reflection fluorescence microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated that the RNA interference-based knockdown of the unconventional myosin Myo16 in cortical neurons altered growth cone filopodia density and axonal branching patterns in a TRIM9- and netrin-1-dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function. [Media: see text].

PKIS deep dive yields a chemical starting point for dark kinases and a cell active BRSK2 inhibitor.

The Published Kinase Inhibitor Set (PKIS) is a publicly-available chemogenomic library distributed to more than 300 laboratories by GlaxoSmithKline (GSK) between 2011 and 2015 and by SGC-UNC from 2015 to 2017. Screening this library of well-annotated, published kinase inhibitors has yielded a plethora of data in diverse therapeutic and scientific areas, funded applications, publications, and provided impactful pre-clinical results. GW296115 is a compound that was included in PKIS based on its promising selectivity following profiling against 260 human kinases. Herein we present more comprehensive profiling data for 403 wild type human kinases and follow-up enzymatic screening results for GW296115. This more thorough investigation of GW296115 has confirmed it as a potent inhibitor of kinases including BRSK1 and BRSK2 that were identified in the original panel of 260 kinases as well as surfaced other kinases that it potently inhibits. Based on these new kinome-wide screening results, we report that GW296115 is an inhibitor of several members of the Illuminating the Druggable Genome (IDG) list of understudied dark kinases. Specifically, our results establish GW296115 as a potent lead chemical tool that inhibits six IDG kinases with IC50 values less than 100 nM. Focused studies establish that GW296115 is cell active, and directly engages BRSK2. Further evaluation showed that GW296115 downregulates BRSK2-driven phosphorylation and downstream signaling. Therefore, we present GW296115 as a cell-active chemical tool that can be used to interrogate the poorly characterized function(s) of BRSK2.