Partial Restoration of Mutant Enzyme Homeostasis in Three Distinct Lysosomal Storage Disease Cell Lines by Altering Calcium Homeostasis

Abstract

A lysosomal storage disease (LSD) results from deficient lysosomal enzyme activity, thus the substrate of the mutant enzyme accumulates in the lysosome, leading to pathology. In many but not all LSDs, the clinically most important mutations compromise the cellular folding of the enzyme, subjecting it to endoplasmic reticulum–associated degradation instead of proper folding and lysosomal trafficking. A small molecule that restores partial mutant enzyme folding, trafficking, and activity would be highly desirable, particularly if one molecule could ameliorate multiple distinct LSDs by virtue of its mechanism of action. Inhibition of L-type Ca²⁺ channels, using either diltiazem or verapamil—both US Food and Drug Administration–approved hypertension drugs—partially restores N370S and L444P glucocerebrosidase homeostasis in Gaucher patient–derived fibroblasts; the latter mutation is associated with refractory neuropathic disease. Diltiazem structure-activity studies suggest that it is its Ca²⁺ channel blocker activity that enhances the capacity of the endoplasmic reticulum to fold misfolding-prone proteins, likely by modest up-regulation of a subset of molecular chaperones, including BiP and Hsp40. Importantly, diltiazem and verapamil also partially restore mutant enzyme homeostasis in two other distinct LSDs involving enzymes essential for glycoprotein and heparan sulfate degradation, namely α-mannosidosis and type IIIA mucopolysaccharidosis, respectively. Manipulation of calcium homeostasis may represent a general strategy to restore protein homeostasis in multiple LSDs. However, further efforts are required to demonstrate clinical utility and safety. Lysosomes are organelles that contain more than 50 hydrolytic enzymes that break down macromolecules in a cell. A lysosomal storage disease results from deficient activity of one or more of these enzymes, leading to the accumulation of corresponding substrate(s). Currently, lysosomal storage diseases are treated by enzyme replacement therapy, which can be challenging because the enzyme has to enter the cell and the lysosome to function; in neuropathic diseases, enzyme replacement is not useful because recombinant enzymes do not enter the brain. We have shown that diltiazem and verapamil, potent US Food and Drug Administration–approved L-type Ca²⁺ channel blocker drugs, increased the endoplasmic reticulum (ER) folding capacity, trafficking, and activity of mutant lysosomal enzymes associated with three distinct lysosomal storage diseases. These compounds appear to function through a Ca²⁺ ion–mediated up-regulation of a subset of cytoplasmic and ER lumenal chaperones, possibly by activating signaling pathways that mitigate cellular stress. We have shown that increasing ER calcium levels appears to be a relatively selective strategy to partially restore mutant lysosomal enzyme homeostasis in diseases caused by the misfolding and degradation of nonhomologous mutant enzymes. Because diltiazem crosses the blood–brain barrier, it may be useful for the treatment of neuropathic lysosomal storage diseases, and possibly other loss-of-function diseases, although efficacy needs to be demonstrated.