β‑Glucocerebrosidase Modulators Promote Dimerization of β‑Glucocerebrosidase and Reveal an Allosteric Binding Site

β-Glucocerebrosidase (GCase) mutations cause Gaucher’s disease and are a high risk factor in Parkinson’s disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, <b>JZ-4109</b>, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (<b>JZ-5029</b>) for <i>in vitro</i> mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher’s disease and Parkinson’s disease

ITI treatment regimen which includes rituximab, methotrexate and intravenous immunoglobulin (IVIG).

<p>This short course of ITI regimen (5 weeks) needs to be started together with the first dose of ERT. IVIG is administered on a monthly basis for a period of 5–6 months.</p

Representative Western gel blot showing CRIM negative status of four patients (lanes 3–6).

<p>Lane 1- protein magic marker; lane 8 -CRIM negative control cell line; lane 10 - normal human fibroblast (NHF) control; Lanes 2, 7 and 9 - left empty. 20 ug of skin fibroblast cell protein extract was loaded for each patient lane and 2.5 ug protein was loaded for NHF. Western blot was probed with anti-GAA antibody and ß-Actin was used as a protein loading control.</p

An algorithm for the management of cross-reactive immunologic material (CRIM)-negative (CN) infantile Pompe disease patients.

<p>^*Institutional review board (IRB) approved study (NCT01665326; <a href="http://www.clinicaltrials.gov" target="_blank">www.clinicaltrials.gov</a>) for rapid determination of CRIM status and long-term follow-up of response to treatment and ITI in Pompe disease. ^†CN status determination from an established CRIM negative mutation database, which allows prediction of CN status in more than 90% cases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Bali1" target="_blank">[15]</a>. ^‡ITI regimen is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone-0067052-g002" target="_blank">Figure 2</a>. ^§Based on the literature antibody titers sustained at ≥6,400 results in a suboptimal therapeutic response to ERT. For that reason, 6,400 was used a cutoff for further intervention <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Banugaria1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Abbott1" target="_blank">[19]</a>. **Based on the half-life of rituximab, CD19% recovery is typically noted around 5 months. ^††The decision to repeat the same ITI regimen (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone-0067052-g003" target="_blank">figure 3</a>) or to administer ITI with a plasma-cell-targeting agent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Banugaria4" target="_blank">[20]</a> should be based on multiple factors including, but not limited to, patients clinical status, CD19% and Fc_γ receptor polymorphism. ^‡‡ITI regimen with plasma cell targeting agent such as bortezomib has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Banugaria4" target="_blank">[20]</a>.</p

Clinical parameters.

<p>Clinical parameters.</p

Details of patient demographics, genotype and immune tolerance induction (ITI) regimen.

*<p>Patient 7 died at the age of 15 months (48 weeks into ERT); mo-months; IVIG-intravenous immunoglobulin.</p

Comparison of median left ventricular mass index (LVMI) values seen over time in CRIM-negative (CN) ERT monotherapy (n = 11) versus CN ERT+ITI (n = 7) treated patients.

<p>The upper limit of normal LVMI is 64 g/m² (represented by a horizontal dashed line).</p

Kaplan-Meier survival curve showing comparison of ventilator-free survival CRIM-negative (CN) ERT monotherapy (n = 11) versus CN ERT+ITI (n = 7) treated patients.

<p>*Three patients in the CN ERT+ITI group began the study invasively ventilated, became ventilator-free with treatment, and are currently still alive and ventilator-free. In contrast, all CN patients in ERT monotherapy treated group were invasive ventilator-free at baseline. This observation suggests that in some cases ERT+ITI can even reverse ventilator dependence in CN Pompe patients.</p

Additional file 1: of Increased Wnt and Notch signaling: a clue to the renal disease in Schimke immuno-osseous dysplasia?

Supplementary Methods, Tables, and Figures. (PDF 45904 kb