Small-Molecule Disruptors of Mutant Huntingtin-Calmodulin Protein-Protein Interaction Attenuate Deleterious Effects of Mutant Huntingtin.

Huntington's disease is a progressive and lethal neurodegenerative disease caused by an increased CAG repeat mutation in exon 1 of the huntingtin gene (mutant huntingtin). Current drug treatments provide only limited symptomatic relief without impacting disease progression. Previous studies in our lab and others identified the abnormal binding of mutant huntingtin protein with calmodulin, a key regulator of calcium signaling. Disrupting the abnormal binding of mutant huntingtin to calmodulin reduces perturbations caused by mutant huntingtin in cell and mouse models of Huntington's disease and importantly normalizes receptor-stimulated calcium release. Using a series of high-throughput in vitro and cell-based screening assays, we identified numerous small-molecule hits that disrupt the binding of mutant huntingtin to calmodulin and demonstrate protective effects. Iterative optimization of one hit resulted in nontoxic, selective compounds that are protective against mutant huntingtin cytotoxicity and normalized receptor-stimulated intracellular calcium release in PC12 cell models of Huntington's disease. Importantly, the compounds do not work by reducing the levels of mutant huntingtin, allowing this strategy to complement future molecular approaches to reduce mutant huntingtin expression. Our novel scaffold will serve as a prototype for further drug development in Huntington's disease. These studies indicate that the development of small-molecule compounds that disrupt the binding of mutant huntingtin to calmodulin is a promising approach for the advancement of therapeutics to treat Huntington's disease.

Discovery of sultam-containing small-molecule disruptors of the huntingtin-calmodulin protein-protein interaction.

The aberrant protein-protein interaction between calmodulin and mutant huntingtin protein in Huntington's disease patients has been found to contribute to Huntington's disease progression. A high-throughput screen for small molecules capable of disrupting this interaction revealed a sultam series as potent small-molecule disruptors. Diversification of the sultam scaffold afforded a set of 24 analogs or further evaluation. Several structure-activity trends within the analog set were found, most notably a negligible effect of absolute stereochemistry and a strong beneficial correlation with electron-withdrawing aromatic substituents. The most promising analogs were profiled for off-target effects at relevant kinases and, ultimately, one candidate molecule was evaluated for neuroprotection in a neuronal cell model of Huntington's disease.

Receptor-stimulated transamidation induces activation of Rac1 and Cdc42 and the regulation of dendritic spines.

Regulation of dendritic spines is an important component of synaptic function and plasticity whereas dendritic spine dysregulation is related to several psychiatric and neurological diseases. In the present study, we tested the hypothesis that serotonin (5-HT)2A/2C receptor-induced Rho family transamidation and activation regulates dendritic spine morphology and that activation of multiple types of receptors can induce transglutaminase (TGase)-catalyzed transamidation of small G proteins. We previously reported a novel 5-HT2A receptor downstream effector, TGase-catalyzed serotonylation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. We now extend these findings to rat primary cortical cultures which develop dendritic spines; stimulation of 5-HT2A/2C receptors increased transamidation of Rac1 and Cdc42, but not RhoA. Inhibition of TGases significantly decreased transamidation and activation of Rac1 and Cdc42, suggesting that transamidation led to their activation. In primary cortical cultures, stimulation of 5-HT2A/2C receptors by 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) caused a transient dendritic spine enlargement, which was blocked by TGase inhibition. Stimulation of both 5-HT2A and 5-HT2C receptors contributed to DOI-induced Rac1 transamidation in primary cortical cultures as demonstrated by selective antagonists. Furthermore, stimulation of muscarinic acetylcholine receptors and NMDA receptors also increased TGase-catalyzed Rac1 activation in SH-SY5Y cells and N2a cells, respectively. Receptor-stimulated TGase-catalyzed transamidation of Rac1 occurs at Q61, a site previously reported to be important in the inactivation of Rac1. These studies demonstrate that TGase-catalyzed transamidation and activation of small G proteins results from stimulation of multiple types of receptors and this novel signaling pathway can regulate dendritic spine morphology and plasticity.