Harnessing natural-product-inspired combinatorial chemistry and computation-guided synthesis to develop N-glycan modulators as anticancer agents

Modulation of N-glycosylation using human Golgi α-mannosidase II (α-hGMII) inhibitors is a potential anticancer approach, but the clinical utility of current α-hGMII inhibitors is limited by their co-inhibition of human lysosomal α-mannosidase (α-hLM), resulting in abnormal storage of oligomannoses. We describe the synthesis and screening of a small library of novel bicyclic iminosugar-based scaffolds, prepared via natural product-inspired combinatorial chemistry (NPICC), which resulted in the identification of a primary α-hGMII inhibitor with 13.5-fold selectivity over α-hLM. Derivatization of this primary inhibitor using computation-guided synthesis (CGS) yielded an advanced α-hGMII inhibitor with nanomolar potency and 106-fold selectivity over α-hLM. In vitro studies demonstrated its N-glycan modulation and inhibitory effect on hepatocellular carcinoma (HCC) cells. In vivo studies confirmed its encouraging anti-HCC activity, without evidence of oligomannose accumulation.


Study of sequence similarity (homology) and 3D molecular structures of -hLM and -hGMII
Up to date, no structure of human lysosomal -mannosidase (-hLM) and human Golgi -mannosidase II (-hGMII) is available. In contrast, the crystal structures of bovine lysosomal -mannosidase (-bLM) (PDB coded 1O7D) and drosophila Golgi -mannosidase II (-dGMII) (PDB coded 3DDF) are accessible. Thus, the accessible crystal structure as the 3D template can be applied to build the target structure of the desired enzyme if two proteins possess more than 30% identical residues over their entire sequence lengths.
In this study. the conventional homology approach (BLAST) was performed, and the results showed that (i) the sequence of -hGMII is approximately 40% homologous to the sequence of -dGMII ( Supplementary   Fig. 6a); (ii) the sequence of -hLM is approximately 80% homologous to the sequence of -bLM ( Supplementary Fig. 6b). In addition, in the 3D molecular structures, both -dGMII and -hGMII are structurally identical in the region having radius of 6 Å around the central Zn 2+ (Supplementary Fig. 7a), so are -bLM and -hLM ( Supplementary Fig. 7b). Therefore, both -dGMII and -bLM, which display high sequence identity with their corresponding human enzymes, are qualified as template models.    Nine molecules (S4-S13) were designed and their -hGMII binding energies were calculated. It was found that (i) cyclohexyl and, to a lesser extent, cyclopentyl rings are favored; (ii) hydrophobic substituents should be located at the para and not meta position; (iii) the n-propyl lipid chain is better than the n-hexyl; and (iv) S15 the thiourea linkage is better than the amide linkage. Thus, 8b-3 (S8) incorporating a 4-propylcyclohexyl moiety was selected for investigations and its molecular models with -hGMII and -hLM were built ( Fig.  3c and 3d). S16     h to quench unreacted isocyanate. The resulting crude products were directly screened in the enzyme-based assay without further purification. Neither quenched iso(thio)cyanate reagents nor DIPEA and DMSO showed any significant inhibition. Across the total set of preliminary (thio)urea-indolizidine library screening (1 M for -hLM and 0.5 M for -hGMII), a potentially -hGMII-selective compound 8b-1 (5G) was found (approximate 60% inhibition against -hGMII at 0.5M) and excitingly display less than 10% inhibition against -hLM at 1 M. The compounds with the best selectivity were selected for re-synthesis and further testing.

Molecular docking studies
Molecular docking studies were performed using the Glide module of the Schrodinger Suite (Schrödinger, LLC, New York). The three-dimensional (3D) structures of the -hGMII and -hLM were created by homology modeling on Swiss-Model, 1 using the crystallized structures of Drosophila -GMII (PDB coded 3DDF) 2 and Bovine -LM (PDB coded 1O7D) 3 as molecular templates, respectively. Both of the 3D structures were refined by Prime with Protein Preparation Wizard (Schrodinger) before molecular docking.
The compound structures were prepared by using LigPrep module (Schrodinger) before being input to Glide.
The OPLS_2005 force field was applied in all procedures. Briefly, a docking grid (size of 46 × 46 × 46 centered at the benzene moiety of (thio)urea and Vander Waal radius scale of 0.90 Å with a partial atomic charge of 0.25) was created in the cleft of the target protein, including the swainsonine (sw) binding site with considering the size of the docking compounds. The extra precision (XP) mode was applied and root mean square deviation (RMSD) from sw was checked to validate the docking pose of each compound. S28

MM-GBSA analysis
The MM-GBSA method in Prime module of Schrödinger was applied to calculate the optimal binding free energies, using the following equation: MMGBSA_ΔG= PrimeEnergy (Optimized Complex) -PrimeEnergy (Optimized Free Ligand) -PrimeEnergy (Optimized Free Receptor). Briefly, the VGSB2 solvent model 4 was applied with OPLS_2005 force field inclusive of an implicit solvent model in addition to physics-based corrections for hydrogen bonding, hydrophobic interactions, π-π interactions and self-contact interactions.

General UPLC condition of glycan-based analysis
The glycan mixture was analyzed by Waters Acquity UPLC equipped with Acquity UPLC Glycan BEH

MALDI-TOF mass analysis of GlcNAcMan 5 GlcNAc 2 -2AB
1 µL of sample was mixed with 1 µl matrix solution consisting of 2, 5-dihydroxybenzoic acid (50 nmol/µL in 50% acetonitrile). Then 1 µl of the resulting mixture was spotted onto the MALDI stainless steel sample plate and allowed to air dry at room temperature. Measurements were performed on an Ultraflex II MALDI-TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Mass spectra were obtained in the range of mass to charge ratio (m/z) from 10,000 to 30,000 with linear mode.

Cell culture
Human normal fibroblasts (08C0015) were seeded in 10 cm culture plates at a density of 4× 10 5 cells/plate.
After 24 h of attachment, cells were treated for 24 h with different concentrations of compound sw and 8b-3 at various concentration.

Isolation of Man 2-9 GlcNAc
After treatment, sw-or 8b-3 treated cells were washed three times with PBS and harvested into centrifuge tubes (one tube per plate) by trypsin treatment. The cell pellets were washed three times with PBS (1 mL) and resuspended in deionized water (200 L). The cells in water suspension were disrupted for 1 min at 460 r/min in BEAD CRUSHER (TAITEC). The supernatant obtained after centrifugation at 15000 g for 10 min was applied to Amicon Ultra-0.5 Centrifugal Filter Unit (3KDa cutoff, Merck, UFC5003) and centrifuge at 15000 g for 15 min to remove molecules with high molecular weight. The filtrate was collected and the released oligomannoses were purified with porous graphitic carbon (PGC, Supelco 57127-U) spin column. Briefly, the PGC spin column was washed with 400 L of 80% acetonitrile (acetonitrile) with 0.1% formic acid (FA) three times and conditioned with 400 L of 0.1% FA three times before use. The released N-glycan mixture was then loaded onto the PGC spin column, and the column was washed with 400 L of 0.1% FA three times.
Finally, N-glycans were eluted with 250 L of 50% acetonitrile with 100 mM of ammonium bicarbonate two times. The eluents were combined, and dried using SpeedVac and analyzed using LC-MS.

N-Glycan profiling assay
Cell culture S32 Cells were seeded in 10 cm culture plates at a density of 10 6 cells/plate for Huh-7 and 7×10 5 cells/ plate for HepG2. After 24 h of attachment, cells were treated for 72 h with different concentrations of compound 8b-3.

Cell Lysis
For cellular (glyco) protein extraction, the 8b-3-treated cells were washed three times with 1 mL of PBS.
After washing, 2 mL of trypsin was added onto a 10 cm tissue culture dish of cells, and the cells were harvested and transferred to a 15 mL centrifuge tube. When necessary, 10 L of the cell suspension was removed for counting, to determine the total number of cells used. The remaining cell suspension was centrifuged at 1000 rpm for 5 min. After centrifugation, the pellet was washed three times with 1 mL of PBS. The cell pellet was transferred to 1.5 mL microcentrifuge tubes for centrifugation at 1000 rpm for 5 min. After removing PBS, for each 60 mg of cell pellet was resuspended with 150 μl of 1x RIPA buffer (10x stock, Merck Millipore, No. 20-188) and incubated on ice for 30 min. The lysate was centrifuged at 16,000 g for 15 min, and the supernatant was collected. The protein concentration of the supernatant was measured by BCA protein assay.

FANGS method for N-glycan release
The N-glycans analysis was performed as described previously. 5 The

Fluorescent imaging
Cells were seeded in 24-well culture plates at a density of 10 5 cells/well for Huh-7 and 1.

MTT cell proliferation assay
Cells were seeded in 96-well plates at a density of 4× 10 3 cells/well for Huh7 and 10 4 cells/well for HepG2. eluents were combined, dried using SpeedVac and the oligomannoses (Man 2-9 GlcNAc) were analyzed by LC-

Statistical analysis
All statistical analysis was conducted using a student's t-test with GraphPad Prism 6 (GraphPad Software, Inc. La Jolla, CA). Data were presented as mean±standard deviations. p values < 0.05 were deemed statistically significant.

Experimental data General
All chemicals were obtained from commercial suppliers and used without further purification. Reactions  to give fully protected iminosugar residue as colorless oil, which was directly used in next step without further purification.
HCOOH (100 mL) was added to an ice-cold solution of fully protected iminosugar residue in dry ethyl ether (120 mL) and the mixture stirred. After 1 h, the ice bath was removed and stirring continued for another 30 min. Then, the reaction mixture was diluted with EtOAc and the reaction was carefully quenched by dilution with NaHCO 3(s) until effervescence ceased. The organic layer was washed with water and brine, dried (MgSO 4 ), concentrated, and purified by column chromatography (n-hexane / EtOAc = 5:1, silica gel) to give S1 (4.5g, 11.0 mmol, 56% over 2 steps) as a white foam. Dess-Martin periodinane (10.7 g, 25.9 mmol) was added to a solution of S1 (7.25 g, 17.3 mmol) in CH 2 Cl 2 (60 mL) and the mixture stirred for 1 h at rt. The reaction solution was filtered through a Celite pad, then diluted with NaHCO 3(aq) and then washed with NaS 2 O 3(aq) and CH 2 Cl 2 . The organic layer was concentrated to give aldehyde residue, which was purified by column chromatography (n-hexane / EtOAc 4:1, silica gel) to give 3 as a white foam (7.16 g, 17.1 mmol, 99%).  82.6, 112.2, 112.3, 153.4, 154.2, 156.1, 156.3, 197.9, 198    Zinc bromide (1.2 g, 5.5 mmol) was added to a stirring solution of 6a (500 mg, 1.1 mmol) in CH 2 Cl 2 (4 mL). The resulting solution was stirred at rt for 8 h. After the reaction was complete, the mixture was filter, concentrated, and purified with column chromatography (MeOH / CH 2 Cl 2 = 1:30, silica gel) to give a diamine crude.

Preparation of compound 4a
The diamine crude in THF (3.6 mL) was treated with benzyl chloroformate (0.6 mL, 4.4 mmol) and conc. NaHCO 3 (aq) (3.6 mL) at rt and stirred for 1h (n-hexane / EtOAc, v/v 1:2, R f = 0.6). After the reaction was complete, the reaction mixture was extracted with EtOAc. The organic layer was separated, and the water layer was extracted with EtOAc three times. The combined organic layer was dried (MgSO 4 ). The solvent was removed, and the product was purified by column chromatography (n-hexane / EtOAc = 5:1, silica gel) to give 7a as a yellow oil (393 mg, 0.76 mmol, 69% over 2 steps).  1, 8 mL). The reaction was stirred at rt for 5 h. After the reaction was complete, H 2 O and CH 2 Cl 2 were added. The organic layer was S42 separated, and the water layer was extracted with CH 2 Cl 2 three times. The combined organic layer was dried (MgSO 4 ). The solvent was removed, and the product was purified by column chromatography (n-hexane /EtOAc = 3:1, silica gel) to give aldehyde (n-hexane / EtOAc, v/v 1:1, R f = 0.1) as a white foam.
The aldehyde in MeOH (5 mL) was added Pd/C (20 mg). The flask was evacuated, filled with H 2 , and stirred for 8 h at rt. The mixture was then filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (MeOH / CH 2 Cl 2 = 1:7, silica gel) to give S14 as a yellow oil (103 mg, 0.43 mmol, 89% over 2 steps). (420 mg, 0.9 mmol) and pyridine (0.37 mL, 4.6 mmol) in CH 2 Cl 2 (9.2 mL) at 0 o C. The reaction was warmed to rt and stirred for 8 h. After the reaction was complete, water and CH 2 Cl 2 were added. The organic layer was separated, and the water layer was extracted with CH 2 Cl 2 three times. The combined organic layer was washed with 1N HCl to remove pyridine. The organic layer was then dried (MgSO 4 ). The solvent was removed, and the product was purified by column chromatography (n-hexane /EtOAc = 2:1, silica gel) to give cyclic carbamate 6b as a yellow foam (247 mg, 0.65 mmol, 73%).  The solution of LiOH•H 2 O (545 mg, 13 mmol) in water (4 mL) was added to a solution of 6b (247 mg, 0.64 mmol) in EtOH (4 mL). The mixture was stirred over 20 h at 90 °C. After the reaction was complete, the reaction mixture was concentrated, and the product was purified by column chromatography (n-hexane / EtOAc = 2:1, silica gel) to give amino alcohol as a white foam (167 mg, 0.47 mmol, 73 %).
The amino alcohol (167.1 mg, 0.47 mmol) in THF (5 mL) and conc. NaHCO 3 (aq) (5 mL) was treated with benzyl chloroformate (0.14 mL, 0.94 mmol) at 0 o C. The reaction was warmed to rt and stirred for 1 h. After S44 the reaction was complete, the reaction mixture was extracted with EtOAc. The organic layer was separated, and the water layer was extracted with EtOAc three times. The combined organic layer was dried (MgSO 4 ).
The solvent was removed, and the product was purified by column chromatography (n-hexane / EtOAc = 7:1, silica gel) to give 7b (218 mg, 95%) as a white solid.