Lending a helping hand, screening chemical libraries for compounds that enhance β‐hexosaminidase A activity in GM2 gangliosidosis cells

Abstract

Enzyme enhancement therapy is an emerging therapeutic approach that has the potential to treat many genetic diseases. Candidate diseases are those associated with a mutant protein that has difficulty folding and/or assembling into active oligomers in the endoplasmic reticulum. Many lysosomal storage diseases are candidates for enzyme enhancement therapy and have the additional advantage of requiring only 5–10% of normal enzyme levels to reduce and/or prevent substrate accumulation. Our long experience in working with the β‐hexosaminidase (EC 3.2.1.52) isozymes system and its associated deficiencies (Tay‐Sachs and Sandhoff disease) lead us to search for possible enzyme enhancement therapy‐agents that could treat the chronic forms of these diseases which express 2–5% residual activity. Pharmacological chaperones are enzyme enhancement therapy‐agents that are competitive inhibitors of the target enzyme. Each of the known β‐hexosaminidase inhibitors (low µm IC50) increased mutant enzyme levels to ≥ 10% in chronic Tay‐Sachs fibroblasts and also attenuated the thermo‐denaturation of β‐hexosaminidase. To expand the repertoire of pharmacological chaperones to more ‘drug‐like’ compounds, we screened the Maybridge library of 50 000 compounds using a real‐time assay for noncarbohydrate‐based β‐hexosaminidase inhibitors and identified several that functioned as pharmacological chaperones in patient cells. Two of these inhibitors had derivatives that had been tested in humans for other purposes. These observations lead us to screen the NINDS library of 1040 Food and Drug Administration approved compounds for pharmacological chaperones. Pyrimethamine, an antimalarial drug with well documented pharmacokinetics, was confirmed as a β‐hexosaminidase pharmacological chaperone and compared favorably with our best carbohydrate‐based pharmacological chaperone in patient cells with various mutant genotypes.

Diltiazem, a L-type Ca2+ channel blocker, also acts as a pharmacological chaperone in Gaucher patient cells

“…5). We then applied a more general test for potential PC-activity, the ability to attenuate the heat inactivation of the target enzyme [29], at pH 4.5 as compared to pH 7.0. Interestingly at 50 °C, Gcc has a half-life (T 1/2 ) of 3.3 min at pH 4.5, but at pH 7.0 it drops to only 1.7 min.…”

“…Both approaches have been reported to target the ERQC in different ways, and if their proposed mechanisms of action are correct, they could act additively or even synergistically. The aim of EET is to increase the stability of the native conformation of the mutant enzyme in the ER, allowing more of it to be engaged by the ER transport machinery for transport to the lysosome [27,29]. To date successful chaperones have also been competitive inhibitors of their target enzymes, i.e., pharmacological chaperones (PCs).…”

Crystal Structure of β-Hexosaminidase B in Complex with Pyrimethamine, a Potential Pharmacological Chaperone

“…These screens have led to the discovery that pyrimethamine (PYR, 5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine) is also a good candidate for a PC. 9 PYR is more widely recognized as an antimalarial drug that targets dihydrofolate reductase (DHFR). The structure of the DHFR:PYR complex revealed that the drug binds to the active site of DHFR and acts as a competitive inhibitor.…”

Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy

“…The second small molecule approach is enzyme enhancement therapy (EET) [7,8], which is still under investigation, but has shown some promising preclinical results in at least four enzyme deficiencies [3,9] with several Phase I and Phase II clinical trials being completed (e.g. [10]).…”

“…The attractiveness of this strategy resides in its applicability to a wide range of both inherited and acquired pathologic conditions associated with protein misfolding; e.g. Adult Tay-Sachs [7] and ischemic diseases [16], respectively. On the negative side, each target protein requires a different molecule and not all mutations, not even all missense mutations, will be responsive.…”