Richardson, T. I. | Search bioRxiv

bioRxiv2026

Optimization and Characterization of SHIP1 Ligands for Cellular Target Engagement and Activity in Alzheimer's Disease Models

Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1), encoded by the gene INPP5D, is a lipid phosphatase that negatively regulates immune receptor signaling in hematopoietic cells and microglia. Here, we describe a pyridyl-pyrazole-piperidine scaffold and the lead compound 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (32), which demonstrates SHIP1 target engagement, brain exposure, and evidence of a central pharmacodynamic response in vivo. Structure-activity relationship studies, guided by biochemical and cellular assays using multiple human and murine protein constructs and cells, identified SHIP1-active ligands. A thermal shift assay using full-length SHIP1 was used to assess compounds for cellular target engagement, while studies in IL-4 conditioned THP-1 cells was used to demonstrate changes in downstream AKT signaling. Targeted lipidomics revealed changes in the overall phosphoinositide pool consistent with SHIP1 target engagement and reduction of phospho-AKT levels. In a protein-lipid overlay assay, compound 32 induced changes in the relative association of SHIP1 with multiple phosphatidylinositols on a membrane surface. In high-content cellular imaging assays, compound 32 enhanced the uptake of myelin/membrane debris and fibrillar amyloid by primary murine microglia, phenocopying a genetic model with reduced SHIP1 expression. Finally, oral administration of compound 32 resulted in brain exposure sufficient to alter gene expression and reduce IL-1{beta} levels as pharmacodynamic markers of microglial activation and neuroinflammation in an amyloidosis mouse model of Alzheimers disease. Collectively, these results define a scaffold with SHIP1 target engagement, CNS exposure, and in vivo activity, providing a foundation for the optimization of brain-penetrant SHIP1 ligands suitable for further mechanistic studies and therapeutic development for the treatment of Alzheimers disease.

pharmacology and toxicology↗

bioRxiv2024

Target deconvolution of an insulin hypersecretion-inducer acting through VDAC1 with a distinct transcriptomic signature in beta-cells

Obesity, insulin resistance, and a host of environmental and genetic factors can drive hyperglycemia, causing {beta}-cells to compensate by increasing insulin production and secretion. In type 2 diabetes (T2D), {beta}-cells under these conditions eventually fail. Rare {beta}-cell diseases like congenital hyperinsulinism (HI) also cause inappropriate insulin secretion, and some HI patients develop diabetes. However, the mechanisms of insulin hypersecretion and how it causes {beta}-cell dysfunction are not fully understood. We previously discovered small molecules (e.g. SW016789) that cause insulin hypersecretion and lead to a loss in {beta}-cell function without cell death. Here, we uncover the protein target of SW016789 and provide the first time-course transcriptomic analysis of hypersecretory responses versus thapsigargin-mediated ER stress in {beta}-cells. In mouse MIN6 and human EndoC-{beta}H1 {beta}-cells, we identified and validated VDAC1 as a SW016789 target using photoaffinity proteomics, cellular thermal shift assays, siRNA, and small molecule inhibitors. SW016789 raises membrane potential to enhance Ca2+ influx, potentially through VDAC1. Chronically elevated intracellular Ca2+ appears to underpin the negative impacts of hypersecretion, as nifedipine protected against each small molecule hypersecretion inducer we tested. Using time- course RNAseq, we discovered that hypersecretion induced a distinct transcriptional pattern compared to ER stress. Clustering analyses led us to focus on ER-associated degradation (ERAD) as a potential mediator of the adaptive response. SW016789 reduced the abundance of ERAD substrate OS-9 and pharmacological inhibition of ERAD worsened {beta}-cell survival in response to hypersecretory stress. Changes in other ERAD components in MIN6 and EndoC-{beta}H1 at the protein level were minor with either SW016789 or thapsigargin. However, immunostaining for core ERAD components SEL1L, HRD1, and DERL3 in non-diabetic and T2D human pancreas revealed altered distributions of SEL1L/HRD1 and SEL1L/DERL3 rations in {beta}-cells of T2D islets, in alignment with altered ERAD in stressed {beta}-cells. We conclude that hypersecretory stimuli, including SW016789- mediated VDAC1 activation, cause enhanced Ca2+ influx and insulin release. Subsequent differential gene expression represents a {beta}-cell hypersecretory response signature that is reflected at the protein level for some, but not all genes. A better understanding of how {beta}-cells induce hypersecretion and the mechanisms of negative feedback on secretory rate may lead to the discovery of novel therapeutic targets for T2D and HI.

cell biology↗