OST/oligosaccharyltransferase

ML414 (also known NGI-1) is a novel and cell-permeable inhibitor of oligosaccharyltransferase (OST), which is a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. ML414 was identified from a cell-based high-throughput screen and lead-compound-optimization campaign. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.

NGI-1 reduces tumor growth of glioblastoma with activated ErbB receptors in vivo.[3]
To assess the effect of ML414 (NGI-1) on xenograft tumor growth, we used an ML414 (NGI-1) nanoparticle formulation that overcomes the low solubility of this compound. First the effect of NGI-NPs were tested using D54-ERLucT xenografts, which increase biolouminescence after inhibition of glycoyslation. We found a significant induction of bioluminescence in mice that received ML414 (NGI-1) at both 24 (1.7 fold, p =.03; Fig. 5A) and 48 hour (1.7 fold, p =.03; Fig. 5A) time points. Tunicmaycin, another inhibidor of N-linked glycosylation was used as a positive control and induced bioluminescence (4.2 fold at 24 hours (p =.007)). These results confirmed the ability of NGI-1 NPs to inhibit glycosylation in D54 tumors in vivo.[3]

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To evaluate the therapeutic potential of ML414 (NGI-1) in vivo, we tested the effect of ML414 (NGI-1) NPs on glioma tumor growth both alone and in combination with radiation for both D54 and SKMG3 cell lines. In these experiments mice were randomly assigned to receive treatment in one of four groups: control NPs, control NPs + RT, NGI-1NPs, and NGI-1 NPs + RT. ML414 (NGI-1) NPs (20mg/kg) were delivered every other day for a total of 3 doses and RT was delivered in 5 daily doses of 2Gy. In D54 xenografts tumor growth was significantly delayed by radiation alone or radiation + NGI-NP treatment. The addition of NGI-NPs to RT significantly reduced tumor growth compared to those treated with radiation alone. At 39 days median tumor volumes for the NGI-1 NP + RT group were 566 ± 200 mm3 compared to 1383 ± 305 mm3 for the RT alone group (p = .001; Figure 5B). Similar results favoring combined treatment with NGI-1 NPs and RT were observed in the SKMG3 xenografts. In this cell line, both radiation and NGI-NPs reduced tumor growth when administered as a single therapy. The combination of NGI-1 NPs + RT prodcued significantly larger reductions in tumor growth. The mean tumor volume at day 98 for the radiation + NGI-1-NP group was nearly undetectable. In comparison tumor volumes for blank NPs (379 ± 38 mm3; p = .001), radiation (139 ± 27 mm3; p = .001) and NGI-1-NP (151 ± 7 mm3; p = .001) were all significantly greater (Figure 5C). For both in vivo xenograft experiments there was no evidence for significant weight loss or other toxicity in animals treated with the NGI-NP. Taken together, these results indicate that the combination of NGI-1 + RT could be a therapeutic approach for the treatment of glioblastoma.[3]

The HTS approach using the bioluminescent N-linked glycosylation reporter in D54-ERLucT and D54-LucT cells has been previously described. Briefly, the primary cell-based screen detects N-linked glycan site occupancy using a modified and ER translated luciferase protein with three N-linked glycosylation consensus sequons. Inhibition of glycosylation in D54-ERLucT restores and increases luciferase activity over controls whereas it does not increase activity in the non-ER translated D54-LucT cell line. The methodology for the primary (D54-ERlucT), secondary false positive (D54-LucT), and tertiary (luciferase inhibition) screens as well as toxicity assays with CellTitre Glo are deposited in Pubchem (AID 588693). Genedata Screener software with the Smartfit algorithm was used for to generate AC40 values for comparative analysis of analogs.

In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.

NGI-1 Therapeutic Studies in Glioma Xenografts:[3]
D54 and SKMG3 bilateral xenografts were established in nude mice by subcutaneous injection of 1×106 cells into hind limb. Four days after injection, mice were randomized to one of four treatment groups. They received either control or NGI-1 NPs i.v. (20mg/kg) every other day for a total of 3 doses and either sham irradiation or a total of 10 Gy administered in daily 2 Gy fractions using a Precision X-ray 250-kV orthovoltage unit. Tumor size was measured two times per week and calculated according to the formula π/6 × (large diameter) × (small diameter)2.

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All experimental procedures were approved in accordance with IACUC and Yale University institutional guidelines for animal care and ethics and guidelines for the welfare and use of animals in cancer research. NGI-1 delivery to glioma xenografts was evaluated using a bioluminescent imaging platform that detects inhibtion of NLG. Ten days after subcutaneous injection of 1 ×107 gliomas cells, mice bearing palpable tumors were treated with control or NGI-1 NPs (20 mg/kg), or tunicamycin 1mg/kg and imaged 5–30 minutes after delivery of i.p. luciferin (150 mg/kg). Signal intensity was quantified for a region of interest (ROI) encompassing each tumor and induction of bioluminscence was calculated by comparing peak bioluminescent activity from pre- and post-treatment imaging at 24 and 48 hours.[3]

[1]. Comprehensive Interactome Analysis Reveals that STT3B is Required for the N-Glycosylation of Lassa Virus Glycoprotein. J Virol. 2019 Sep 11. pii: JVI.01443-19.

[2]. Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells. Nat Chem Biol. 2016 Dec;12(12):1023-1030.

[3]. Oligosaccharyltransferase Inhibition Reduces Receptor Tyrosine Kinase Activation and Enhances Glioma Radiosensitivity. Clin Cancer Res. 2019 Jan 15; 25(2): 784–795.

Lassa virus (LASV) is the causative agent of a fatal hemorrhagic fever in humans. The glycoprotein (GP) of LASV mediates viral entry into host cells, and correct processing and modification of GP by host factors is a prerequisite for virus replication. Here, using an affinity purification-coupled mass spectrometry (AP-MS) strategy, 591 host proteins were identified as interactors of LASV GP. Gene ontology analysis was performed to functionally annotate these proteins, and the oligosaccharyltransferase (OST) complex was highly enriched. Functional studies conducted by using CRISPR-Cas9-mediated knockouts showed that STT3A and STT3B, the two catalytically active isoforms of the OST complex, are essential for the propagation of the recombinant arenavirus rLCMV/LASV glycoprotein precursor, mainly via affecting virus infectivity. Knockout of STT3B, but not STT3A, caused hypoglycosylation of LASV GP, indicating a preferential requirement of LASV for the STT3B-OST isoform. Furthermore, double knockout of magnesium transporter 1 (MAGT1) and tumor suppressor candidate 3 (TUSC3), two specific subunits of STT3B-OST, also caused hypoglycosylation of LASV GP and affected virus propagation. Site-directed mutagenesis analysis revealed that the oxidoreductase CXXC active-site motif of MAGT1 or TUSC3 is essential for the glycosylation of LASV GP. ML414 (NGI-1), a small-molecule OST inhibitor, can effectively reduce virus infectivity without affecting cell viability. The STT3B-dependent N-glycosylation of GP is conserved among other arenaviruses, including both the Old World and New World groups. Our study provided a systematic view of LASV GP-host interactions and revealed the preferential requirement of STT3B for LASV GP N-glycosylation.IMPORTANCE Glycoproteins play vital roles in the arenavirus life cycle by facilitating virus entry and participating in the virus budding process. N-glycosylation of GPs is responsible for their proper functioning; however, little is known about the host factors on which the virus depends for this process. In this study, a comprehensive LASV GP interactome was characterized, and further study revealed that STT3B-dependent N-glycosylation was preferentially required by arenavirus GPs and critical for virus infectivity. The two specific thioredoxin subunits of STT3B-OST MAGT1 and TUSC3 were found to be essential for the N-glycosylation of viral GP. ML414 (NGI-1), a small-molecule inhibitor of OST, also showed a robust inhibitory effect on arenavirus. Our study provides new insights into LASV GP-host interactions and extends the potential targets for the development of novel therapeutics against Lassa fever in the future.[1]

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Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, ML414 (NGI-1). ML414 (NGI-1) targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, ML414 (NGI-1) blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.[2]

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Parallel signaling reduces the effects of receptor tyrosine kinase (RTK) targeted therapies in glioma. We hypothesized that inhibition of protein N-linked glycosylation, an endoplasmic reticulum co- and post-translational modification crucial for RTK maturation and activation, could provide a new therapeutic approach for glioma radiosensitization. Experimental design: We investigated the effects of a small molecule inhibitor of the oligosaccharyltransferase (ML414 (NGI-1)) on EGFR family receptors, MET, PDGFR, and FGFR1. The influence of glycosylation state on tumor cell radiosensitivity, chemotherapy induced cell toxicity, DNA damage, and cell cycle arrest were determined and correlated with glioma cell receptor expression profiles. The effects of ML414 (NGI-1) on xenograft tumor growth were tested using a nanoparticle formulation validated by in vivo molecular imaging. A mechanistic role for RTK signaling was evaluated through the expression of a glycosylation independent CD8-EGFR chimera. Results: ML414 (NGI-1) reduced glycosylation, protein levels, and activation of most RTKs. ML414 (NGI-1) also enhanced the radiosensitivity and cytotoxic effects of chemotherapy in those glioma cells with elevated ErbB family activation, but not in cells without high levels of RTK activation. ML414 (NGI-1) radiosensitization was associated with increases in both DNA damage and G1 cell cycle arrest. Combined treatment of glioma xenografts with fractionated radiation and ML414 (NGI-1) significantly reduced tumor growth compared to controls. Expression of the CD8-EGFR eliminated ML414 (NGI-1)’s effects on G1 arrest, DNA damage, and cellular radiosensitivity, identifying RTK inhibition as the principal mechanism for the ML414 (NGI-1) effect.[3]

C17H22N4O3S2

394.51

394.113

C, 51.76; H, 5.62; N, 14.20; O, 12.17; S, 16.25

790702-57-7

2519269

White to off-white solid powder

1.4±0.1 g/cm3

1.634

2.75

1

7

5

26

602

0

O=C(C1C(N2CCCC2)=CC=C(S(N(C)C)(=O)=O)C=1)NC1SC(C)=CN=1

QPKGRLIYJGBKJL-UHFFFAOYSA-N

InChI=1S/C17H22N4O3S2/c1-12-11-18-17(25-12)19-16(22)14-10-13(26(23,24)20(2)3)6-7-15(14)21-8-4-5-9-21/h6-7,10-11H,4-5,8-9H2,1-3H3,(H,18,19,22)

5-(dimethylsulfamoyl)-N-(5-methyl-1,3-thiazol-2-yl)-2-(pyrrolidin-1-yl)benzamide

NGI-1; NGI1; NGI 1; ML414; 5-(dimethylsulfamoyl)-N-(5-methyl-1,3-thiazol-2-yl)-2-(pyrrolidin-1-yl)benzamide; 5-(N,N-Dimethylsulfamoyl)-N-(5-methylthiazol-2-yl)-2-(pyrrolidin-1-yl)benzamide; MLS002248299; 5-(dimethylsulfamoyl)-N-(5-methyl-1,3-thiazol-2-yl)-2-pyrrolidin-1-ylbenzamide; SMR001315774; ML 414; ML-414;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 79~100 mg/mL ( 200.24~253.48 mM )

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (6.34 mM);

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 (Please use freshly prepared in vivo formulations for optimal results.)