An ALS Therapeutic Assembly Modulator Target in Peripheral Blood Mononuclear Cells: Implications for ALS Pathophysiology, Therapeutics, and Diagnostics

Abstract

Assembly modulators are a new class of allosteric site-targeted therapeutic small molecules, some of which are effective at restoring nuclear localization of TDP-43 in ALS cellular models, and display efficacy in a variety of ALS animal models. One of these compounds has been shown to target a small subfraction of protein disulfide isomerase, a known allosteric modulator implicated in ALS pathophysiology, within a novel, transient, and energy-dependent multi-protein complex that includes other important members of the ALS interactome, such as TDP-43, RanGTPase, and selective autophagy receptor p62/SQSTM1. Building on earlier literature suggesting PBMC dysfunction in ALS, we demonstrate here that a similar multi-protein complex drug target is present in PBMCs with signature alterations in PBMCs from ALS patients compared to PBMCs from healthy individuals. ALS-associated changes in the drug target include increased RanGTPase and MMP9, diminished p62/SQSTM1, and most distinctively, appearance of a 17kDa post-translationally modified form of RanGTPase. Many of these changes are not readily apparent from analysis of whole cell extracts, as a number of the proteins present in the target multi-protein complex, including RanGTPase, comprise a miniscule percent of their total in cell extracts. A small subset of each of these proteins appear to come together in a transient, energy-dependent fashion, to form the drug target. Furthermore, whole blood from ALS patients shows a distinctive degradation of RanGTPase not observed in blood from healthy individuals, which appears to be rescued by treatment with either of two structurally unrelated ALS-active assembly modulators. Our findings are consistent with the hypothesis that ALS is fundamentally a disorder of homeostasis that can be both detected and treated by assembly modulators.