New Irreversible α-l-Iduronidase Inhibitors and Activity-Based Probes

Abstract

Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity-based glycosidase probes (ABPs). Direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination of l-ido-configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α-l-iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity-based manner with human recombinant α-l-iduronidase (rIDUA, Aldurazyme^® ). The structures of IDUA when complexed with the inhibitors in a non-covalent transition state mimicking form and a covalent enzyme-bound form provide insights into its conformational itinerary. Inhibitors 1-3 adopt a half-chair conformation in solution (⁴ H₃ and ³ H₄ ), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1-3 may need to overcome an energy barrier in order to switch from the ⁴ H₃ conformation to the transition state (^{2, 5} B) binding conformation before reacting and adopting a covalent ⁵ S₁ conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.

Keywords: activity-based protein profiling; conformational analysis; cyclophellitol aziridines; glycosidase; irreversible inhibitors.

Figure 1

A) Koshland double‐displacement mechanism employed by retaining α‐l‐iduronidase, showing the ²S₀→^2, 5B→⁵S₁→^2, 5B→²S₀ conformational reaction itinerary from the Michaelis complex, transition state 1, covalent substrate–enzyme intermediate, and transition state 2, to the hydrolyzed product. B) Proposed inhibition mechanism of aziridine‐based inhibitor 1 and ABPs 2 and 3. C) Chemical structures of α‐l‐iduronic‐configured mechanism‐based irreversible inhibitor 1 and ABPs 2 and 3 described in this work.

Figure 2

Synthesis of α‐l‐iduronic‐configured inhibitors and ABPs 1–3. Reagents and conditions: a) DBBT, Et₃N, CH₂Cl₂, −78 °C to −20 °C, 5 h, 60 %; b) LiBH₄, THF, rt, 2 h, 99 %; c) Grubbs II catalyst, CH₂Cl₂, 40 °C, 18 h, 98 %; d) BnBr, TBAI, NaH, DMF, rt, 18 h, 79 %; e) PhI(OAc)₂, CH₂Cl₂, rt, 48 h, 43 %; f) Li, NH₃, THF, −60 °C, 1 h, 93 %; g) 8‐azido‐1‐iodooctane, K₂CO₃, DMF, 55 °C, 24 h, 12: 22 %; h) TEMPO, NaBr, NaOCl, H₂O, 0 °C, 3 h, 14 %; i) CuSO₄, NaAsc, rt, 18–48 h, 2: 22 %, 3: 34 %.

Figure 3

Labeling of rIDUA with ABPs 2 and 3. A) Cy5 ABP 2 labels rIDUA (10 ng) in a concentration‐dependent (left, at 4 h incubation time) and time‐dependent (right, at 50 μm) manner. Labeling signals were quantified (below each gel image) and fitted with a one‐phase association equation. Dotted lines represent 95 % CI. B) ABP 2 labels rIDUA in a pH‐dependent manner. The quantified labeling signals were compared to data obtained with 4‐MU substrate assay (below; error range=SD from technical triplicates). C) ABP 2 labels rIDUA and negative controls: without rIDUA, without ABP 2 or with SDS (upper panel), competition with 4‐MU‐α‐l‐iduronide (middle panel) or 1 (lower panel). D) MS/MS pattern of a sample containing rIDUA Asn297–Leu303 active site peptide labeled with biotin ABP 3 at Glu299, showing peaks corresponding to the detected fragments. Actx=active site peptide. E) Percentages of rIDUA labeling at different time points and at different concentrations of ABP 2. Data were quantified from three sets of fluorescent gels containing rIDUA labeled with ABP 2 under the depicted conditions to derive a rate constant k for each ABP 2 concentration. F) Left, k vs. [inhibitor] plot. Data were curve‐fitted with the Michaelis–Menten equation to obtain kinetic parameters. Right, calculated kinetic parameters for ABP 2 labeling of rIDUA. Error range=SD from the three sets.

Figure 4

Structural insights into raIDUA complexed with ABPs. A) Structure of raIDUA complexed with a fragment of ABP 1, which is covalently linked to the nucleophile Glu299. The maximum likelihood/σ _A weighted 2F _obs−F _calc electron density map (gray) is contoured at 1.2 sigma. B) Structure of raIDUA covalently complexed with a fragment of ABP 1, illustrating the active site residues that interact with the pseudo‐glycoside. C) Structure of raIDUA complexed with a fragment of ABP 3. The nucleophile Glu299 is shown. The maximum likelihood/σ _A weighted 2F _obs−F _calc electron density map (gray) is contoured at 1.0 sigma. D) Structure of raIDUA complexed with a fragment of ABP 3, illustrating the active site residues that interact with the pseudo‐glycoside. E) Superposition of raIDUA covalently complexed with fragments of ABP 1 (green) and 2F‐IdoA (pink; PDB code 4KH28). F) Superposition (based on alignment of protein main‐chain atoms) of raIDUA complexed with a fragment of ABP 1 (covalent, green) and a fragment of ABP 3 (transition state, cyan). G) Superposition (based on alignment of C3 and C4 atoms of each molecule) of raIDUA complexed with a fragment of ABP 1 (covalent, green), a fragment of ABP 3 (transition state, cyan), and IdoA‐DNJ (Michaelis complex, yellow; PDB code 4KGL).8

Figure 5

rIDUA visualization in human fibroblasts by confocal fluorescence microscopy. From top to bottom: NHDF, human normal dermal fibroblasts; MPS I, patient fibroblasts with mucopolysaccharidosis type I, and ML II, patient fibroblasts with mucolipidosis type II. From left to right: cells were incubated without (Ctrl) or with ABP 2‐prelabeled rIDUA (+rIDUA, with successive zoomed‐in images from areas within the indicated white squares), or pre‐treated with mannose‐6‐phosphate prior to rIDUA incubation (+M6P+rIDUA). Color legend: nuclei were stained with DAPI (blue), lysosomes with immunostaining of lysosomal‐associated membrane protein 1 (LAMP1) (green), and rIDUA was labeled with ABP 2 (red). Scale bar=25 μm.