Convergent activation of Ca2+ permeability in two-pore channel 2 through distinct molecular routes.

TPC2 is a pathophysiologically relevant lysosomal ion channel that is activated directly by the phosphoinositide PI(3,5)P2 and indirectly by the calcium ion (Ca2+)-mobilizing molecule NAADP through accessory proteins that associate with the channel. TPC2 toggles between PI(3,5)P2-induced, sodium ion (Na+)-selective and NAADP-induced, Ca2+-permeable states in response to these cues. To address the molecular basis of polymodal gating and ion-selectivity switching, we investigated the mechanism by which NAADP and its synthetic functional agonist, TPC2-A1-N, induced Ca2+ release through TPC2 in human cells. Whereas NAADP required the NAADP-binding proteins JPT2 and LSM12 to evoke endogenous calcium ion signals, TPC2-A1-N did not. Residues in TPC2 that bind to PI(3,5)P2 were required for channel activation by NAADP but not for activation by TPC2-A1-N. The cryptic voltage-sensing region of TPC2 was required for the actions of TPC2-A1-N and PI(3,5)P2 but not for those of NAADP. These data mechanistically distinguish natural and synthetic agonist action at TPC2 despite convergent effects on Ca2+ permeability and delineate a route for pharmacologically correcting impaired NAADP-evoked Ca2+ signals.

Developing HDAC4-Selective Protein Degraders To Investigate the Role of HDAC4 in Huntington's Disease Pathology.

Huntington's disease (HD) is a lethal autosomal dominant neurodegenerative disorder resulting from a CAG repeat expansion in the huntingtin (HTT) gene. The product of translation of this gene is a highly aggregation-prone protein containing a polyglutamine tract >35 repeats (mHTT) that has been shown to colocalize with histone deacetylase 4 (HDAC4) in cytoplasmic inclusions in HD mouse models. Genetic reduction of HDAC4 in an HD mouse model resulted in delayed aggregation of mHTT, along with amelioration of neurological phenotypes and extended lifespan. To further investigate the role of HDAC4 in cellular models of HD, we have developed bifunctional degraders of the protein and report the first potent and selective degraders of HDAC4 that show an effect in multiple cell lines, including HD mouse model-derived cortical neurons. These degraders act via the ubiquitin-proteasomal pathway and selectively degrade HDAC4 over other class IIa HDAC isoforms (HDAC5, HDAC7, and HDAC9).

Structure-Based Exploration of Selectivity for ATM Inhibitors in Huntington's Disease.

Our group has recently shown that brain-penetrant ataxia telangiectasia-mutated (ATM) kinase inhibitors may have potential as novel therapeutics for the treatment of Huntington's disease (HD). However, the previously described pyranone-thioxanthenes (e.g., 4) failed to afford selectivity over a vacuolar protein sorting 34 (Vps34) kinase, an important kinase involved with autophagy. Given that impaired autophagy has been proposed as a pathogenic mechanism of neurodegenerative diseases such as HD, achieving selectivity over Vps34 became an important objective for our program. Here, we report the successful selectivity optimization of ATM over Vps34 by using X-ray crystal structures of a Vps34-ATM protein chimera where the Vps34 ATP-binding site was mutated to approximate that of an ATM kinase. The morpholino-pyridone and morpholino-pyrimidinone series that resulted as a consequence of this selectivity optimization process have high ATM potency and good oral bioavailability and have lower molecular weight, reduced lipophilicity, higher aqueous solubility, and greater synthetic tractability compared to the pyranone-thioxanthenes.

Optimization of Potent and Selective Ataxia Telangiectasia-Mutated Inhibitors Suitable for a Proof-of-Concept Study in Huntington's Disease Models.

Genetic and pharmacological evidence indicates that the reduction of ataxia telangiectasia-mutated (ATM) kinase activity can ameliorate mutant huntingtin (mHTT) toxicity in cellular and animal models of Huntington's disease (HD), suggesting that selective inhibition of ATM could provide a novel clinical intervention to treat HD. Here, we describe the development and characterization of ATM inhibitor molecules to enable in vivo proof-of-concept studies in HD animal models. Starting from previously reported ATM inhibitors, we aimed with few modifications to increase brain exposure by decreasing P-glycoprotein liability while maintaining potency and selectivity. Here, we report brain-penetrant ATM inhibitors that have robust pharmacodynamic (PD) effects consistent with ATM kinase inhibition in the mouse brain and an understandable pharmacokinetic/PD (PK/PD) relationship. Compound 17 engages ATM kinase and shows robust dose-dependent inhibition of X-ray irradiation-induced KAP1 phosphorylation in the mouse brain. Furthermore, compound 17 protects against mHTT (Q73)-induced cytotoxicity in a cortical-striatal cell model of HD.