A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase

Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source

Fluorescent amino acids as versatile building blocks for chemical biology

Fluorophores have transformed the way we study biological systems, enabling non-invasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein–protein interactions in situ or imaging nanoscopic events in real time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of fluorescent amino acids and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using site-specific genetic encoding and bioorthogonal approaches, and their application to design novel artificial proteins, as well as to investigate biological processes in cells by means of optical imaging. [Figure not available: see fulltext.]

Synthesis and Evaluation of Hybrid Structures Composed of Two Glucosylceramide Synthase Inhibitors

Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, type2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism - glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2) - is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39-compound library does not contain new GCS inhibitors; however, a potent (200nm) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator. Taking the best of both: Two established glucosylceramide synthase (GCS) inhibitors were merged via convergent synthesis to obtain hybrid compounds. Members of this 39-compound library have characteristics of both parent GCS inhibitors. No new GCS inhibitors were established, but a potent (200nm) acid glucosylceramidase (GBA1) inhibitor was identified. This adamantanemethyloxypenanoic acid pyrrolidene-substituted derivative of eliglustat can serve as a lead for further biomedical development of selective GBA1 modulators

Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutic

Identification and Development of Biphenyl Substituted Iminosugars as Improved Dual Glucosylceramide Synthase/Neutral Glucosylceramidase Inhibitors

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of d-<i>gluco</i> and l-<i>ido</i>-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring <i>N</i>-pentyloxy­methylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (d-<i>gluco</i> and l-<i>ido</i>) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted l-<i>ido</i>-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their d-<i>gluco</i>-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics