Therapeutic Role of Pharmacological Chaperones in Lysosomal Storage Disorders: A Review of the Evidence and Informed Approach to Reclassification.

The treatment landscape for lysosomal storage disorders (LSDs) is rapidly evolving. An increase in the number of preclinical and clinical studies in the last decade has demonstrated that pharmacological chaperones are a feasible alternative to enzyme replacement therapy (ERT) for individuals with LSDs. A systematic search was performed to retrieve and critically assess the evidence from preclinical and clinical applications of pharmacological chaperones in the treatment of LSDs and to elucidate the mechanisms by which they could be effective in clinical practice. Publications were screened according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) reporting guidelines. Fifty-two articles evaluating 12 small molecules for the treatment of seven LSDs are included in this review. Overall, a substantial amount of preclinical and clinical data support the potential of pharmacological chaperones as treatments for Fabry disease, Gaucher disease, and Pompe disease. Most of the available clinical evidence evaluated migalastat for the treatment of Fabry disease. There was a lack of consistency in the terminology used to describe pharmacological chaperones in the literature. Therefore, the new small molecule chaperone (SMC) classification system is proposed to inform a standardized approach for new, emerging small molecule therapies in LSDs.

The critical role of hinge-region expulsion in the induced-fit heparin binding mechanism of antithrombin.

Antithrombin (AT) is the most important inhibitor of coagulation proteases. Its activity is stimulated by glycosaminoglycans, such as heparin, through allosteric and template mechanisms. AT utilises an induced-fit mechanism to bind with high affinity to a pentasaccharide sequence found in about one-third of heparin chains. The conformational changes behind this mechanism have been characterised by several crystal structures of AT in the absence and in the presence of pentasaccharide. Pentasaccharide binding ultimately results in a conformational change that improves affinity by about 1000-fold. Crystal structures show several differences, including the expulsion of the hinge region of the reactive centre loop from beta-sheet A, which is known to be critical for the allosteric activation of AT. Here, we present data that reveal an energetically distinct intermediate on the path to full activation where the majority of conformational changes have already occurred. A crystal structure of this intermediate shows that the hinge region is in a native-like state in spite of having the pentasaccharide bound in the normal fashion. We engineered a disulfide bond to lock the hinge in its native position to determine the energetic contributions of the initial and final conformational events. Approximately 60% of the free-energy contribution of conformational change is provided by the final step of hinge-region expulsion and subsequent closure of the main beta-sheet A. A new analysis of the individual structural changes provides a plausible mechanism for propagation of conformational change from the heparin binding site to the remote hinge region in beta-sheet A.

Helix D elongation and allosteric activation of antithrombin.

Antithrombin requires allosteric activation by heparin for efficient inhibition of its target protease, factor Xa. A pentasaccharide sequence found in heparin activates antithrombin by inducing conformational changes that affect the reactive center of the inhibitor resulting in optimal recognition by factor Xa. The mechanism of transmission of the activating conformational change from the heparin-binding region to the reactive center loop remains unresolved. To investigate the role of helix D elongation in the allosteric activation of antithrombin, we substituted a proline residue for Lys(133). Heparin binding affinity was reduced by 25-fold for the proline variant compared with the control, and a significant decrease in the associated intrinsic fluorescence enhancement was also observed. Rapid kinetic studies revealed that the main reason for the reduced affinity for heparin was an increase in the rate of the reverse conformational change step. The pentasaccharide-accelerated rate of factor Xa inhibition for the proline variant was 10-fold lower than control, demonstrating that the proline variant cannot be fully activated toward factor Xa. We conclude that helix D elongation is critical for the full conversion of antithrombin to its high affinity, activated state, and we propose a mechanism to explain how helix D elongation is coupled to allosteric activation.