Many human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes. Collectively, lysosomal storage disorders (LSDs) result from genetic mutations in the genes that encode specific lysosomal enzymes, leading to a deficiency in essential enzymatic activity and cellular accumulation of the respective substrate. To date, over 50 different LSDs have been identified, several of which are treated clinically with enzyme replacement therapy or substrate reduction therapy, although insufficiently in some cases. Importantly, a wide range of in vitro assays are now available to measure mutant lysosomal enzyme interaction with and stabilization by PCs, as well as subsequent increases in cellular enzyme levels and function. The application of these assays to the identification and characterization of candidate PCs for mutant lysosomal enzymes will be discussed in this review. In addition, considerations for the successful in vivo use and development of PCs to treat LSDs will be discussed.

Parkinson’s disease (PD) may share an etiological basis with Gaucher disease, as GBA1 mutations lead to Gaucher disease when homozygous, and are associated with increased risk for PD when heteroor homozygous. Neuronopathic forms of Gaucher disease and PD also share the therapeutic challenge of targeting the central nervous system (CNS). Pharmacological chaperones (PC) are orally-available, small molecules that represent an innovative approach to specifically increase the activity of target enzymes. AT2101 is a PC developed to enhance the enzyme deficient in Gaucher disease, glucocerebrosidase (GCase). The accumulation of α-synuclein in the CNS is a hallmark of PD. We have successfully utilized AT2101 for proof-of-concept studies in which administration of AT2101 prevented the accumulation of α-synuclein in the brain of two murine PD models that overexpress human α-synuclein. We reasoned that the CNS exposure and other properties of AT2101 could be further improved while maintaining a good safety profile. For instance, AT2101 inhibits targets other than GCase and has a relatively long lysosomal half-life. An assessment of nearly 200 analogs of AT2101 led to the identification of several new PCs with superior characteristics, including greater CNS penetration, increased potency for enhancement of enzyme activity, accelerated efflux from both tissues and lysosomes, and improved target specificity. These new PCs are currently under investigation in models of Parkinson’s and Neuronopathic Gaucher disease.