A practical synthesis of nitrone-derived C5a-functionalized isofagomines as protein stabilizers to treat Gaucher disease.

Isofagomine (IFG) and its analogues possess promising glycosidase inhibitory activities. However, a flexible synthetic strategy toward both C5a-functionalized IFGs remains to be explored. Here we show a practical synthesis of C5a-S and R aminomethyl IFG-based derivatives via the diastereoselective addition of cyanide to cyclic nitrone 1. Nitrone 1 was conveniently prepared on a gram scale and in high yield from inexpensive (-)-diethyl D-tartrate via a straightforward method, with a stereoselective Michael addition of a nitroolefin and a Nef reaction as key steps. A 268-membered library (134 × 2) of the C5a-functionalized derivatives was submitted to enzyme- or cell-based bio-evaluations, which resulted in the identification of a promising ß-glucocerebrosidase (GCase) stabilizer demonstrating a 2.7-fold enhancement at 25 nM in p.Asn370Ser GCase activity and a 13-fold increase at 1 µM in recombinant human GCase activity in Gaucher cell lines.

Mechanistic Insights into Dibasic Iminosugars as pH-Selective Pharmacological Chaperones to Stabilize Human α-Galactosidase.

The use of pharmacological chaperones (PCs) to stabilize specific enzymes and impart a therapeutic benefit is an emerging strategy in drug discovery. However, designing molecules that can bind optimally to their targets at physiological pH remains a major challenge. Our previous study found that dibasic polyhydroxylated pyrrolidine 5 exhibited superior pH-selective inhibitory activity and chaperoning activity for human α-galactosidase A (α-Gal A) compared with its monobasic parent molecule, 4. To further investigate the role of different C-2 moieties on the pH-selectivity and protecting effects of these compounds, we designed and synthesized a library of monobasic and dibasic iminosugars, screened them for α-Gal A-stabilizing activity using thermal shift and heat-induced denaturation assays, and characterized the mechanistic basis for this stabilization using X-ray crystallography and binding assays. We noted that the dibasic iminosugars 5 and 20 protect α-Gal A from denaturation and inactivation at lower concentrations than monobasic or other N-substituted derivatives; a finding attributed to the nitrogen on the C-2 methylene of 5 and 20, which forms the bifurcated salt bridges (BSBs) with two carboxyl residues, E203 and D231. Additionally, the formation of BSBs at pH 7.0 and the electrostatic repulsion between the vicinal ammonium cations of dibasic iminosugars at pH 4.5 are responsible for their pH-selective binding to α-Gal A. Moreover, compounds 5 and 20 demonstrated promising results in improving enzyme replacement therapy and exhibited significant chaperoning effects in Fabry cells. These findings suggest amino-iminosugars 5 and 20 as useful models to demonstrate how an additional exocyclic amino group can improve their pH-selectivity and protecting effects, providing new insights for the design of pH-selective PCs.

Harnessing polyhydroxylated pyrrolidines as a stabilizer of acid alpha-glucosidase (GAA) to enhance the efficacy of enzyme replacement therapy in Pompe disease.

To discover small molecules as acid alpha-glucosidase (GAA) stabilizers for potential benefits of the exogenous enzyme treatment toward Pompe disease cells, we started from the initial screening of the unique chemical space, consisting of sixteen stereoisomers of 2-aminomethyl polyhydroxylated pyrrolidines (ADMDPs) to find out two primary stabilizers 17 and 18. Further external or internal structural modifications of 17 and 18 were performed to increase structural diversity, followed by the protein thermal shift study to evaluate the GAA stabilizing ability. Fortunately, pyrrolidine 21, possessing an l-arabino-typed configuration pattern, was identified as a specific potent rh-GAA stabilizer, enabling the suppression of rh-GAA protein denaturation. In a cell-based Pompe model, co-administration of 21 with rh-GAA protein significantly improved enzymatic activity (up to 5-fold) compared to administration of enzyme alone. Potentially, pyrrolidine 21 enables the direct increase of ERT (enzyme replacement therapy) efficacy in cellulo and in vivo.

Discovery of small-molecule protein stabilizers toward exogenous alpha-l-iduronidase to reduce the accumulated heparan sulfate in mucopolysaccharidosis type I cells.

Synthesis of a series of l-iduronic acid (IdoA)- and imino-IdoA-typed C-glycosides for modulating α-l-iduronidase (IDUA) activity is described. In an enzyme inhibition study, IdoA-typed C-glycosides were more potent than imino-IdoA analogs, with the most potent IdoA-typed C-glycoside 27c showing an IC50 value of 1 µM. On the other hand, co-treatment of 12 with rh-α-IDUA in mucopolysaccharidosis type I (MPS I) fibroblasts exhibited a nearly 3-fold increase of the IDUA activity, resulting in a clear reduction of the accumulated heparan sulfate (HS) compared to the exogenous enzyme treatment alone. This is the first report of small molecules facilitating IDUA stabilization, enhancing enzyme activity, and reducing accumulated HS in MPS I cell-based assays, which reveals that small molecules as rh-α-IDUA stabilizers to improve enzyme replacement therapy (ERT) efficacy toward MPS I is feasible and promising.

Synthesis of Nitrone-derived Pyrrolidine Scaffolds and Their Combinatorial Libraries to Develop Selective α-l-Rhamnosidase Inhibitors.

A general and flexible approach toward the development of α-l-rhamnosidase (α-l-Rha-ase) inhibitors is described. Five enantiopure poly-substituted pyrrolidine-based scaffolds bearing the C1-aminomethyl moiety were designed and synthesized from five-membered cyclic nitrones. Each structurally diversified amide library of these scaffolds was rapidly generated via combinatorial parallel synthesis and applied for in-situ inhibition study against α-l-Rha-ase, allowing us to efficiently identify new inhibition hits. Surprisingly, all promising inhibitors are derived from the same scaffold 3. Among them, the most potent and selective inhibitor is pyrrolidine 19 with Ki =0.24 µM, approximately 24-fold more potent than the reference compound DAA (Ki =5.7 µM). It is the first study to comprehensively prepare pyrrolidine-based scaffolds and libraries for inhibition study against α-l-Rha-ase.

A combinatorial approach towards the synthesis of non-hydrolysable triazole-iduronic acid hybrid inhibitors of human α-l-iduronidase: discovery of enzyme stabilizers for the potential treatment of MPSI.

Preparation of substituent-diverse, triazole-iduronic acid hybrid molecules by click reaction of an azido iduronic acid derivative with randomly chosen alkynes is described. Library members were screened for their ability to inhibit α-l-iduronidase, and hit molecules and analogues were then investigated for their ability to stabilize rh-α-IDUA in a thermal denaturation study. This work resulted in the discovery of the first small molecules that can be used to stabilize exogenous rh-α-IDUA protein in vitro.

Synthesis of (3S,4S,5S)-trihydroxylpiperidine derivatives as enzyme stabilizers to improve therapeutic enzyme activity in Fabry patient cell lines.

A series of 3S,4S,5S-trihydroxylated piperidines bearing structural diversity at C-2 or C-6 positions has been synthesized and tested to determine their ability to stabilize the activity of recombinant human α-Galactosidase A (rh-α-Gal A). Hit molecules were identified by rapid inhibitory activity screening, and then further investigated for their ability to protect this enzyme from thermo-induced denaturation and enhance its activity in Fabry patient cell lines. Our study resulted in the identification of a new class of small molecules as enzyme stabilizers for the potential treatment of Fabry disease. Of these, stabilizer 21 was the most effective, showing a 12-fold increase in rh-α-Gal A activity in Fabry disease cell lines.

Rapid preparation of (3R,4S,5R) polyhydroxylated pyrrolidine-based libraries to discover a pharmacological chaperone for treatment of Fabry disease.

The rapid discovery of a pharmacological chaperone toward human α-Gal A for the treatment of Fabry disease is described. Two polyhydroxylated pyrrolidines with the (3R,4S,5R) configuration pattern underwent rapid substituent diversity by conjugating the primary aminomethyl moiety of each with a variety of carboxylic acids to generate two libraries (2 × 60 members). Our bioevaluation results showed one member with the (2R,3R,4S,5R) configuration pattern and bearing a 5-cyclohexylpentanoyl group as a substituent moiety possessed sufficient chaperoning capability to rescue α-Gal A activity in the lymphocyte of the N215S Fabry patient-derived cell line and other α-Gal A mutants in COS7 cells.

Bioevaluation of sixteen ADMDP stereoisomers toward alpha-galactosidase A: Development of a new pharmacological chaperone for the treatment of Fabry disease and potential enhancement of enzyme replacement therapy efficiency.

A unique molecular library consisting of all sixteen synthetic ADMDP (1-aminodeoxy-DMDP) stereoisomers has been prepared and evaluated for inhibitory activity against α-Gal A, and ability to impart thermal stabilization of this enzyme. The results of this testing led us to develop a novel pharmacological chaperone for the treatment of Fabry disease. 3-Epimer ADMDP was found to be an effective pharmacological chaperone, able to rescue α-Gal A activity in the lymphoblast of the N215S Fabry patient-derived cell line, without impairment of cellular ß-galactosidase activity. When 3-epimer ADMDP was administered with rh-α-Gal A (enzyme replacement therapy) for the treatment of Fabry patient-derived cell lines, improvements in the efficacy of rh-α-Gal A was observed, which suggests this small molecule can also provide clinical benefit of enzyme replacement therapy in Fabry disease.

Synthesis of novel polyhydroxylated pyrrolidine-triazole/-isoxazole hybrid molecules.

A straightforward synthesis of novel, 2-heterocyclyl polyhydroxylated pyrrolidines is described. Stereocontrolled additions of nucleophiles to cyclic nitrones generated the corresponding 2,3-trans adducts, allowing the synthesis of the corresponding pyrrolidines via key intermediates bearing an alkyne and a nitrile oxide. Three hybrid systems, including a pyrrolidine with two isoxazoles and one triazole, are efficiently prepared via 1,3-dipolar cycloaddition. Biological testing of the product alkaloids showed that subtle structural variations have drastic effects on their inhibitory activities against glucosidases.

Evolution of the complete photonic bandgap of two-dimensional photonic crystal.

In this paper, the complete photonic bandgap (CPBG) of two-dimensional photonic crystals (PCs), which are formed by a square array of solid or hollow dielectric rods connected with dielectric veins, are numerically investigated using the plane wave expansion method. It is clearly demonstrated how the CPBG evolves as the pattern of veins or the type of rods changes. An optimal structure with an ultralarge CPBG is found, whose CPBG reaches Δω=0.22374 (2πc/a), which is larger than those reported in literatures. The proposed structure seems to have promising applications due to its ultralarge CPBG and large fabrication tolerance.