Pharmacological chaperones (PC) are small molecules that bind and stabilize the folded state of a specific target protein. Stabilization of the folded protein decreases the fraction that is degraded by the cellular quality control system, leading to increased levels of the targeted protein. Early-onset familial Alzheimer's disease (EOFAD) represents ∼1% of the total AD population, and over half of the EOFAD cases are caused by a missense mutation on a single allele of the presenilin 1 (PS1) gene (PSEN1). PS1 contains the catalytic active site of the 106 Da tetrameric protein complex that makes up the aspartyl protease known as gamma secretase. Heterozygous expression of mutant and wild type PS1 results in altered gamma secretase function and increased production of the amyloidogenic peptide Ab42. Our working hypothesis is that PS1/gamma secretase dysfunction in PS1 EOFAD can be normalized by increasing the ratio of wild type to mutant PS1 in the gamma secretase complex. We have synthesized several classes of compounds that selectively target PS1 and tested those compounds for PC properties. We have demonstrated both in vivo and in vitro that treatment with PCs specific for PS1 results in a two-fold increase in protein levels of wild type PS1 and the gamma secretase complex. We have developed a cell-based model for multiple PS1 EOFAD mutations that mimic EOFAD by co-expression of both mutant and wild type PS1. The EOFAD cell models exhibit altered gamma secretase processing, i.e. increased Ab42 production and an unexpected increase in total Ab. PS1 EOFAD cell-based models treated with a PS1 specific PC increased PS1 and gamma secretase levels and reduced Ab42 and total Ab levels in a dose-dependent manner. We also treated an in vivo model of PS1 EOFAD (WTxM146V KI heterozygous mice) with a PS1 specific PC; the treated animals responded in a manner similar to that seen in the in vitro models in that Ab42 and total Ab were reduced. We have shown that pharmacological chaperones specific for PS1 could be a novel approach for the normalization of gamma secretase function and treatment of PS1 EOFAD.

The most frequent cause of Early-onset familial Alzheimer's disease (EOFAD) is the autosomal dominant inheritance of a missense mutation in the presenilin 1 (PS1) gene (PSEN). Since EOFAD results from a mutation in a single allele of PSEN, both wild type and the PSEN variant gene products are co-expressed. The potential for the mutant PS1 protein to affect the function of wild-type PS1 (e.g., a dominant negative effect) and the relative amount of wild type and mutant PS1 incorporated into the gamma secretase complex are important factors that are relevant to the understanding and the treatment of PS1 EOFAD. We have generated five PS1 EOFAD cell lines (M146V, A431E, H163R, delta9, E280A and WT control in H4 neuroglioma cells) in which recombinant mutant PS1 expression, in the presence of endogenous wild type PS1, is under the control of the TETrepressor. We measured Ab42and Ab40 levels as a function of the ratio of wild type to mutant PS1 in the gamma secretase complex and demonstrated that all EOFAD PS1 mutants tested exhibit dominant negative behavior for Ab42 and total Ab production. In contrast, over-expression of wild type PS1 had no effect on Ab42 or total Ab production. Although equal gene dosage of mutant and wild type PS1 is expected in EOFAD, the actual ratio of mutant to wild type PS1 in the gamma secretase complex will be dependent on the relative stability (i.e., multiple factors such as mutant holo-PS1 stability, its ability to interact with other gamma secretase components and the stability of the resulting gamma secretase complex). In PS1 EOFAD, the relative stability of the PS1 variant could impact the degree of a dominant negative effect and thus make a significant contribution to disease severity and age of onset. We determined the relative stability of the PS1 mutants by measuring the amount of mutant PS1 incorporated into the gamma secretase complex as a function of recombinant mRNA levels. Our results suggest that increasing the ratio of wild type to mutant PS1 may promote normalization of gamma secretase function and could be a promising therapeutic approach for PS1 EOFAD.

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.

Heterozygous null mutations in the melanocortin-4 receptor (MC4R) cause early-onset obesity in humans, indicating that metabolic homeostasis is sensitive to quantitative variation in MC4R function. Most of the obesity-causing MC4R mutations functionally characterized so far lead to intracellular retention of receptors by the cell's quality control system. Thus, recovering cell surface expression of mutant MC4Rs could have a beneficial therapeutic value. We tested a pharmacological chaperone approach to restore cell surface expression and function of 10 different mutant forms of human melanocortin-4 receptor found in obese patients. Five cell-permeant MC4R-selective ligands were tested and displayed pharmacological chaperone activities, restoring cell surface targeting and function of the receptors with distinct efficacy profiles for the different mutations. Such mutation-specific efficacies suggested a structure-activity relationship between compounds and mutant receptor conformations that may open a path toward personalized therapy. In addition, one of the five pharmacological chaperones restored function to most of the mutant receptors tested. Combined with its ability to reach the central nervous system and its selectivity for the MC4R, this pharmacological chaperone may represent a candidate for the development of a targeted therapy suitable for a large subset of patients with MC4R-deficient obesity.