First-in-human single-dose study of nizubaglustat, a dual inhibitor of ceramide glucosyltransferase and non-lysosomal glucosylceramidase: Safety, tolerability, pharmacokinetics, and pharmacodynamics of single ascending and multiple doses in healthy adults.

Nizubaglustat is a novel, orally available, brain penetrant, potent, and selective dual inhibitor of ceramide glucosyltranferase and non-lysosomal neutral glucosylceramidase (NLGase), which is currently under development for the treatment of subjects with neurological manifestations in primary and secondary gangliosidoses. The objectives of this first-in-human study were to evaluate the safety and tolerability, pharmacokinetics, and pharmacodynamics (PD) of single oral doses of nizubaglustat after single (1, 3, and 9 mg) and multiple oral doses (9 mg once per day (QD) over 14 days) in healthy adults. Nizubaglustat was rapidly absorbed and systemic exposure was dose-proportional. Steady-state was achieved after three days of QD multiple dosing with minimal accumulation. Renal clearance accounted for around 15% of nizubaglustat elimination. Following multiple dosing, plasma concentrations of glucosylceramide (GlcCer), lactosylceramide (LacCer), and monosialodihexosylganglioside (GM3) decreased to a nadir at Day 10. PD target engagement of GCS inhibition was shown by a median decrease from baseline of plasma concentrations of GlcCer, LacCer, and GM3 ganglioside by 70%, 50%, and 48%, respectively. NLGase inhibition was also manifested by increased concentrations of GlcCer in cerebrospinal fluid from Day 1 to Day 14. Nizubaglustat was safe and well-tolerated at all doses tested. Consistent with the high selectivity, and the absence of intestinal disaccharidases inhibition, no cases of diarrhea were reported. No decreased appetite or weight loss was noted. Only treatment-emergent adverse events with preferred terms belonging to the system organ class skin and subcutaneous disorders of mild intensity were reported as drug-related in the nizubaglustat arm, in line with the pharmacological mechanism targeting glucosylceramide metabolism. Taken together, these data support QD dosing of nizubaglustat and its ongoing development in patients with primary and secondary forms of gangliosidoses.

Potent and Selective Cell-Active Iminosugar Inhibitors of Human α-N-Acetylgalactosaminidase (α-NAGAL).

Glycoside hydrolases (GHs) are a class of enzymes with emerging roles in a range of disease. Selective GH inhibitors are sought to better understand their functions and assess the therapeutic potential of modulating their activities. Iminosugars are a promising class of GH inhibitors but typically lack the selectivity required to accurately perturb biological systems. Here, we describe a concise synthesis of iminosugar inhibitors of N-acetyl-α-galactosaminidase (α-NAGAL), the GH responsible for cleaving terminal α-N-acetylgalactosamine residues from glycoproteins and other glycoconjugates. Starting from non-carbohydrate precursors, this modular synthesis supported the identification of a potent (490 nM) and α-NAGAL selective (∼200-fold) guanidino-containing derivative DGJNGuan. To illustrate the cellular activity of this new inhibitor, we developed a quantitative fluorescence image-based method to measure levels of the Tn-antigen, a cellular glycoprotein substrate of α-NAGAL. Using this assay, we show that DGJNGuan exhibits excellent inhibition of α-NAGAL within cells using patient derived fibroblasts (EC50 =150 nM). Moreover, in vitro and in cell assays to assess levels of lysosomal ß-hexosaminidase substrate ganglioside GM2 show that DGJNGuan is selective whereas DGJNAc exhibits off-target inhibition both in vitro and within cells. DGJNGuan is a readily produced and selective tool compound that should prove useful for investigating the physiological roles of α-NAGAL.

Synthesis of Uronic Acid 1-Azasugars as Putative Inhibitors of α-Iduronidase, ß-Glucuronidase and Heparanase.

1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited ß-glucuronidase (ß-GLU). The d-GlcA-configured 9 was a potent inhibitor of ß-GLU and a moderate inhibitor of the endo-ß-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile.

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.

Comparative genomic analysis of azasugar biosynthesis.

Azasugars are monosaccharide analogs in which the ring oxygen is replaced with a nitrogen atom. These well-known glycosidase inhibitors are of interest as therapeutics, yet several aspects of azasugars remain unknown including their distribution, structural diversity, and chemical ecology. The hallmark signature of bacterial azasugar biosynthesis is a three gene cluster (3GC) coding for aminotransferase, phosphatase, and dehydrogenase enzymes. Using the bioinformatics platform Enzyme Similarity Tool (EST), we identified hundreds of putative three gene clusters coding for azasugar production in microbial species. In the course of this work, we also report a consensus sequence for the aminotransferase involved in azasugar biosynthesis as being: SGNXFRXXXFPNXXXXXXXLXVPXPYCXRC. Most clusters are found in Bacillus and Streptomyces species which typically inhabit soil and the rhizosphere, but some clusters are found with diverse species representation such as Photorhabdus and Xenorhabdus which are symbiotic with entomopathogenic nematodes; the human skin commensal Cutibacterium acnes, and the marine Bacillus rugosus SPB7, a symbiont to the sea sponge Spongia officinalis. This pan-taxonomic survey of the azasugar 3GC signature may lead to the identification of new azasugar producers, facilitate studies of their natural functions, and lead to new potential therapeutics.

Synthesis of "All-Cis" Trihydroxypiperidines from a Carbohydrate-Derived Ketone: Hints for the Design of New ß-Gal and GCase Inhibitors.

Pharmacological chaperones (PCs) are small compounds able to rescue the activity of mutated lysosomal enzymes when used at subinhibitory concentrations. Nitrogen-containing glycomimetics such as aza- or iminosugars are known to behave as PCs for lysosomal storage disorders (LSDs). As part of our research into lysosomal sphingolipidoses inhibitors and looking in particular for new ß-galactosidase inhibitors, we report the synthesis of a series of alkylated azasugars with a relative "all-cis" configuration at the hydroxy/amine-substituted stereocenters. The novel compounds were synthesized from a common carbohydrate-derived piperidinone intermediate 8, through reductive amination or alkylation of the derived alcohol. In addition, the reaction of ketone 8 with several lithium acetylides allowed the stereoselective synthesis of new azasugars alkylated at C-3. The activity of the new compounds towards lysosomal ß-galactosidase was negligible, showing that the presence of an alkyl chain in this position is detrimental to inhibitory activity. Interestingly, 9, 10, and 12 behave as good inhibitors of lysosomal ß-glucosidase (GCase) (IC50 = 12, 6.4, and 60 µM, respectively). When tested on cell lines bearing the Gaucher mutation, they did not impart any enzyme rescue. However, altogether, the data included in this work give interesting hints for the design of novel inhibitors.

Intestinal Absorption and Tissue Distribution of Aza-Sugars from Mulberry Leaves and Evaluation of Their Transport by Sugar Transporters.

Mulberry leaves are rich in aza-sugars, particularly 1-deoxynojirimycin (DNJ), fagomine, and 2-O-α-d-galactopyranosyl-1-deoxynojirimycin (GAL-DNJ), which have antidiabetes and antiobesity properties. To help us understand the mechanisms of action of aza-sugars, pharmacokinetic studies are necessary. Therefore, in this study, we evaluated and compared the absorption and organ distribution of these aza-sugars in rats. Following oral intake, DNJ exhibited the highest plasma concentration followed by fagomine and GAL-DNJ. Meanwhile, similar amounts of DNJ and fagomine were present in organs, while GAL-DNJ was hardly detected, suggesting the diversity in absorption and distribution characteristics of these aza-sugars. We then investigated the role of the sodium-glucose cotransporter and the glucose transporter (GLUT) in the transport of aza-sugars and found that both are involved in DNJ transport, while transport of fagomine is solely facilitated by the GLUT. These findings provide insight into the bioavailability and bioactive mechanisms of these aza-sugars.

Synthesis of 1,4-imino-L-lyxitols modified at C-5 and their evaluation as inhibitors of GH38 α-mannosidases.

A synthetic approach to 1,4-imino-L-lyxitols with various modifications at the C-5 position is reported. These imino-L-lyxitol cores were used for the preparation of a series of N-(4-halobenzyl)polyhydroxypyrrolidines. An impact of the C-5 modification on the inhibition and selectivity against GH38 α-mannosidases from Drosophila melanogaster, the Golgi (GMIIb) and lysosomal (LManII) mannosidases and commercial jack bean α-mannosidase from Canavalia ensiformis was evaluated. The modification at C-5 affected their inhibitory activity against the target GMIIb enzyme. In contrast, no inhibition effect of the pyrrolidines against LManII was observed. The modification of the imino-L-lyxitol core is therefore a suitable motif for the design of inhibitors with desired selectivity against the target GMIIb enzyme.

Improved production of 1-deoxynojirymicin in Escherichia coli through metabolic engineering.

Azasugars, such as 1-deoxynojirymicin (1-DNJ), are associated with diverse pharmaceutical applications, such as antidiabetic, anti-obesity, anti-HIV, and antitumor properties. Different azasugars have been isolated from diverse microbial and plant sources though complicated purification steps, or generated by costly chemical synthesis processes. But the biosynthesis of such potent molecules using Escherichia coli as a heterologous host provides a broader opportunity to access these molecules, particularly by utilizing synthetic biological, metabolic engineering, and process optimization approaches. This work used an integrated approach of synthetic biology, enzyme engineering, and pathway optimization for rational metabolic engineering, leading to the improved production of 1-DNJ. The production of 1-DNJ in recombinant E. coli culture broth was confirmed by enzymatic assays and mass spectrometric analysis. Specifically, the pathway engineering for its key precursor, fructose-6-phosphate, along with optimized media condition, results in the highest production levels. When combined, 1-DNJ production was extended to ~ 273 mg/L, which is the highest titer of production of 1-DNJ reported using E. coli.

Conformational Behaviour of Azasugars Based on Mannuronic Acid.

A set of mannuronic-acid-based iminosugars, consisting of the C-5-carboxylic acid, methyl ester and amide analogues of 1deoxymannorjirimicin (DMJ), was synthesised and their pH-dependent conformational behaviour was studied. Under acidic conditions the methyl ester and the carboxylic acid adopted an "inverted" 1 C4 chair conformation as opposed to the "normal" 4 C1 chair at basic pH. This conformational change is explained in terms of the stereoelectronic effects of the ring substituents and it parallels the behaviour of the mannuronic acid ester oxocarbenium ion. Because of this solution-phase behaviour, the mannuronic acid ester azasugar was examined as an inhibitor for a Caulobacter GH47 mannosidase that hydrolyses its substrates by way of a reaction itinerary that proceeds through a 3 H4 transition state. No binding was observed for the mannuronic acid ester azasugar, but sub-atomic resolution data were obtained for the DMJ⋅CkGH47 complex, showing two conformations-3 S1 and 1 C4 -for the DMJ inhibitor.

Exploiting the hydrophobic terrain in fucosidases with aryl-substituted pyrrolidine iminosugars.

Fucosidase inhibition shows potential in numerous therapeutic contexts. Substitution of fucose-like iminosugars with hydrophobic "aglycons" yields significant improvements in potency of fucosidase inhibition. Here we have prepared three new 2-aryl-3,4-dihydroxy-5-methylpyrrolidines featuring phenyl substituents in variable orientations with respect to the iminocyclitol core and at various distances from it to explore the key binding interactions that stabilise the enzyme-inhibitor complex. The presence of a triazole linker in one structure resulted in nanomolar inhibition of the fucosidase from bovine kidney (Ki =4.8 nM), thus giving rise to one of the most potent pyrrolidine-type inhibitors of this enzyme known to date.

A concise synthesis of N-substituted fagomine derivatives and the systematic exploration of their α-glycosidase inhibition.

A novel and concise scheme has been developed successfully for the syntheses of N-substituted fagomine derivatives. The transformation of lactone (2) to 1,5-diol (3) was carried on with high yield (93-95%). The cyclization of 4 to 5 is a high stereoselective reaction (de value > 98%). It is disclosed that bulky substituent at N atom of the piperidine decreases the inhibition activity except those substituents having the ability of solvation or forming disulfide bond with M444 at the active site of α-glycosidase, which enhance the interaction with enzyme. Compounds with S-configuration at C-3 show greater activity than those with R-configuration. The structure-activity relationship study is also supported by molecular docking analysis.

Matrix metalloproteinases and their role in psoriasis.

This review summarizes the contribution of matrix metalloproteinases to the pathogenesis of psoriasis. In psoriasis, matrix metalloproteinases are involved in the structural changes of the epidermis via the modification of intracellular contacts and the composition of the extracellular matrix, promoting angiogenesis in the dermal blood vessels and the infiltration of immune cells. Moreover, some matrix metalloproteinases become differentially expressed during the disease eruption and their expression correlates with the clinical score. A separate section of the review is dedicated to the pharmacological approaches that are used to control matrix metalloproteinases, such as oral metalloproteinase inhibitors, such as azasugars and phosphonamides. The aim of this manuscript is to assess the role of matrix metalloproteinases in the physiological processes that accompany the disease. Moreover, it is especially important to evaluate progress in this field and characterize recently appeared medicines. Because any experimental drugs that target matrix metalloproteinases are involved in active clinical trials, this manuscript also reviews the latest experimental data regarding distribution and expression of matrix metalloproteinases in healthy skin and lesional skin. Therefore, the performed analysis highlights potential problems associated with the use of metalloproteinase inhibitors in clinical studies and suggests simple and easy understandable criteria that future innovative metalloproteinase inhibitors shall satisfy.

N-Heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid a novel scaffold for the design of uncompetitive α-glucosidase inhibitors.

The enzyme α-glucosidase has attracted interest owing to its involvement in the digestive process of carbohydrate, its role in intracellular glycoprotein trafficking, tumorigenesis and viral infection. In this study, several members of a new family of N-heteroarylmethyl substituted azasugars were synthesized and evaluated as α-glucosidase inhibitors. We systematically investigated the effect of different N-substituents as well as the role of hydroxyl and carboxylate moieties on the piperidine ring. The compounds N-heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid emerged as potent α-glucosidase inhibitors. Unlike Acarbose and other clinically relevant α-glucosidase inhibitors, these compounds act through a reversible uncompetitive mechanism of inhibition which make them attractive candidates for drug development.

Metathesis access to monocyclic iminocyclitol-based therapeutic agents.

By focusing on recent developments on natural and non-natural azasugars (iminocyclitols), this review bolsters the case for the role of olefin metathesis reactions (RCM, CM) as key transformations in the multistep syntheses of pyrrolidine-, piperidine- and azepane-based iminocyclitols, as important therapeutic agents against a range of common diseases and as tools for studying metabolic disorders. Considerable improvements brought about by introduction of one or more metathesis steps are outlined, with emphasis on the exquisite steric control and atom-economical outcome of the overall process. The comparative performance of several established metathesis catalysts is also highlighted.