OST/oligosaccharyltransferase
Oligosaccharyltransferase (OST) complex, including catalytic subunits STT3A and STT3B [1]
Oligosaccharyltransferase (OST) complex [2]
Oligosaccharyltransferase (OST) complex [3]

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.

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Effect on Lassa virus (LASV) and arenaviruses: Effectively reduced the infectivity of recombinant arenavirus rLCMV/LASV GPC without affecting cell viability. Caused hypoglycosylation of LASV glycoprotein (GP) and inhibited virus propagation. The STT3B-dependent N-glycosylation inhibition of GP was conserved among both Old World and New World arenaviruses. Tested in HEK293T, A549, HeLa, Huh7, and HEK293 cells with consistent antiviral effects [1]
- Inhibition of receptor tyrosine kinase (RTK) signaling in NSCLC cells: Blocked cell-surface localization and signaling of epidermal growth factor receptor (EGFR) glycoprotein. Selectively arrested proliferation in EGFR- or FGFR-dependent non-small-cell lung cancer (NSCLC) cell lines (PC9, H1581, H2444), inducing G1 cell-cycle arrest, p21 induction, autofluorescence, and senescence-related morphology changes. Had minimal effect on AR-negative Hela cells or non-RTK-dependent NSCLC cells (A549) [2]
- Modulation of RTK activity and radiosensitivity in glioma cells: Reduced glycosylation, protein levels, and activation of multiple RTKs (EGFR, ErbB2, ErbB3, MET, PDGFR, FGFR1) in glioma cell lines (D54, SKMG3, U251, T98G, 42-MG) with elevated ErbB family activation. Enhanced radiosensitivity (increased γH2AX foci and G1 arrest) and cytotoxic effects of chemotherapy (temozolomide, etoposide) in ErbB-high glioma cells, but not in cells with low RTK activation or PTEN loss. Expression of glycosylation-independent CD8-EGFR reversed these effects, confirming RTK inhibition as the core mechanism [3]

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]

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Inhibition of glioma xenograft growth: Nude mice bearing subcutaneous D54 or SKMG3 glioma xenografts were treated with NGI-1 nanoparticle formulation (20 mg/kg i.v. every other day for 3 doses) combined with fractionated radiation (10 Gy total, 2 Gy daily). Tumor volume was measured twice weekly, and combined treatment significantly reduced tumor growth compared to radiation alone or control nanoparticles. No significant weight loss or other toxicity was observed in treated animals [3]
- Validation of glycosylation inhibition in vivo: D54-ERLucT xenograft-bearing mice treated with NGI-1 nanoparticles (20 mg/kg i.v.) showed significant induction of bioluminescence at 24 and 48 hours (1.7-fold increase), confirming in vivo inhibition of N-linked glycosylation [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.

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Cell-free OST activity assay: Rabbit reticulocyte lysates supplemented with canine pancreas rough microsomes were used to translate Saposin-DDK-His6 mRNA for 60 minutes in the presence of ML414 (NGI-1) or tunicamycin. OST activity was evaluated by ^{35}S metabolic labeling to detect glycosylation of the translated protein [2]
- Cellular thermal shift assay (CETSA): 293T cells were treated with 100 μM ML414 (NGI-1) for 30 minutes, followed by thermal treatment. The binding of ML414 (NGI-1) to OST subunits was assessed by detecting changes in protein thermal stability via Western blot [2]

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.

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Arenavirus infection and glycosylation assay: HEK293T, A549, HeLa, Huh7, and HEK293 cells were infected with rLCMV/LASV GPC virus at an MOI of 0.01. After virus entry, ML414 (NGI-1) at different concentrations was added, with DMSO as control. Cells and supernatants were harvested at 36 hpi. Glycosylation patterns of viral GP were analyzed by Western blot, and viral titers (immunological plaque assay) and RNA copy numbers (qRT-PCR) were determined [1]
- NSCLC cell RTK signaling and proliferation assay: CHO-Lec15, CHO-Lec35, HeLa, HEK293, and NSCLC cells (PC9, A549, H1581, H2444) were treated with ML414 (NGI-1) (1–10 μM) for 24–72 hours. Western blot was used to detect EGFR glycosylation, phosphorylation, and downstream signaling; surface biotinylation and confocal microscopy analyzed EGFR localization; MTT assay measured cell proliferation; flow cytometry determined cell cycle distribution; qRT-PCR quantified cyclin D1 mRNA levels [2]
- Glioma cell RTK activation and radiosensitivity assay: Glioma cells (D54, SKMG3, U251, T98G, 42-MG) were pre-treated with 10 μM ML414 (NGI-1) for 48 hours, then exposed to radiation (0–6 Gy) or chemotherapy (temozolomide 10 μM, etoposide 0.1 μM). Clonogenic survival assay evaluated radiosensitivity; CellTiter 96 assay measured proliferation; Western blot detected RTK glycosylation, phosphorylation, and γH2AX (DNA damage marker); immunofluorescence quantified γH2AX foci; flow cytometry analyzed cell cycle distribution [3]

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]

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Glioma xenograft therapeutic assay: Nude mice were subcutaneously injected with 1×10^6 D54 or SKMG3 glioma cells into the hind limb. Four days later, mice were randomized into four groups: control nanoparticles, ML414 (NGI-1) nanoparticles (20 mg/kg i.v. every other day for 3 doses), sham irradiation, or radiation (10 Gy total, 2 Gy daily). Tumor size was measured twice weekly, and tumor volume was calculated using the formula π/6 × (large diameter) × (small diameter)² [3]
- In vivo glycosylation inhibition validation assay: Mice bearing D54-ERLucT xenografts (1×10^7 cells subcutaneously) were treated with control nanoparticles, ML414 (NGI-1) nanoparticles (20 mg/kg i.v.), or tunicamycin (1 mg/kg i.v.). At 5–30 minutes after intraperitoneal injection of luciferin (150 mg/kg), bioluminescent signal intensity was quantified at 24 and 48 hours to confirm glycosylation inhibition [3]

In vitro toxicity: At concentrations that inhibit viral infection, ML414 (NGI-1) had no effect on the viability of HEK293T, A549, HeLa, Huh7, and HEK293 cells [1]
- In vivo toxicity: Mice treated with ML414 (NGI-1) nanoparticles (20 mg/kg intravenously) did not show significant weight loss or other significant toxic side effects [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 pathogen of a deadly hemorrhagic fever in humans. LASV glycoproteins (GPs) mediate viral entry into host cells, and proper processing and modification of GPs by host factors are prerequisites for viral replication. This study used affinity purification-mass spectrometry (AP-MS) to identify 591 host proteins that interact with LASV GPs. Functional annotation of these proteins by gene ontology analysis revealed a high enrichment of the oligosaccharide transferase (OST) complex. Functional studies using CRISPR-Cas9-mediated gene knockout technology showed that two catalytically active isoforms of the OST complex, STT3A and STT3B, are crucial for the proliferation of the recombinant arenavirus rLCMV/LASV glycoprotein precursor, primarily through influencing viral infectivity. Knockout of STT3B (but not STT3A) resulted in insufficient LASV GP glycosylation, indicating that LASV preferentially depends on the STT3B-OST isoform. Furthermore, double knockout of two specific subunits of STT3B-OST—magnesium transporter 1 (MAGT1) and tumor suppressor candidate gene 3 (TUSC3)—also leads to insufficient LASV GP glycosylation and affects viral replication. Site-directed mutagenesis analysis showed that the CXXC oxidoreductase active site motif of MAGT1 or TUSC3 is crucial for LASV GP glycosylation. The small molecule OST inhibitor ML414 (NGI-1) effectively reduced viral infectivity without affecting cell viability. STT3B-dependent GP N-glycosylation is also conserved in other arenavirus genera, including Old World and New World arenaviruses. Our study systematically analyzed the interaction between Lassa virus (LASV) glycoprotein (GP) and the host and revealed the preferential dependence of STT3B on LASV GP N-glycosylation. Importance: Glycoproteins play a crucial role in the life cycle of arenaviruses, facilitating viral entry into host cells and participating in viral budding. N-glycosylation of glycoproteins is key to their normal function; however, little is known about virus-dependent host factors. This study comprehensively characterized the interaction genome of LASV GP and further found that arenavirus GP preferentially depends on STT3B-dependent N-glycosylation, and that this process is essential for viral infectivity. Two specific thioredoxin subunits, MAGT1 and TUSC3, of the STT3B-OST complex were found to be essential for viral GP N-glycosylation. In addition, the small molecule thioredoxin inhibitor ML414 (NGI-1) also showed significant inhibitory effects against arenavirus. Our study provides new insights into the interaction between Lassa virus glycoprotein (LASV GP) and the host and expands the potential targets for the development of novel therapies for Lassa fever. [1]

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Asparagine (N)-linked glycosylation is a protein modification that is essential for glycoprotein folding, stability and cellular localization. To identify novel small molecules capable of inhibiting this biosynthetic pathway, we initiated a cell-based high-throughput screening and lead compound optimization program, ultimately yielding a cell permeability inhibitor, ML414 (NGI-1). ML414 (NGI-1) targets oligosaccharide transferases (OSTs), heterooligomeric enzymes with multiple isoforms capable of transferring oligosaccharides to receptor proteins. In non-small cell lung cancer cells, ML414 (NGI-1) blocks the cell surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively inhibits the proliferation of EGFR-dependent (or fibroblast growth factor, FGFR) cell lines. In these cell lines, OST inhibition leads to cell cycle arrest, accompanied by p21 induction, autofluorescence, and altered cell morphology—all hallmarks of aging. These results suggest that OST inhibition is a potential therapeutic approach for receptor tyrosine kinase-dependent tumors and provides a chemical probe for reversibly modulating N-linked glycosylation in mammalian cells. [2] Parallel signal transduction reduces the efficacy of receptor tyrosine kinase (RTK) therapy in gliomas. We hypothesized that inhibition of protein N-linked glycosylation (an endoplasmic reticulum co-translational and post-translational modification crucial for RTK maturation and activation) could provide a novel approach for radiosensitization in gliomas. Experimental Design: We investigated the effects of the oligosaccharide transferase small molecule inhibitor ML414 (NGI-1) on EGFR family receptors MET, PDGFR, and FGFR1. We determined the effects of glycosylation status on tumor cell radiosensitivity, chemotherapy-induced cytotoxicity, DNA damage, and cell cycle arrest, and correlated these effects with glioma cell receptor expression profiles. We tested the effect of ML414 (NGI-1) on xenograft tumor growth using a nanoparticle formulation validated by in vivo molecular imaging. The mechanistic role of the RTK signaling pathway was assessed by expressing a glycosylation-independent CD8-EGFR chimera. Results: ML414 (NGI-1) reduced the glycosylation, protein levels, and activation of most RTKs. ML414 (NGI-1) also enhanced the radiosensitivity and cytotoxicity of glioma cells with increased ErbB family activation, but had no effect on cells with low RTK activation. The radiosensitizing effect of ML414 (NGI-1) was associated with increased DNA damage and G1 phase cell cycle arrest. Compared with the control group, fractionated radiotherapy combined with ML414 (NGI-1) significantly inhibited tumor growth in glioma xenografts. The expression of CD8-EGFR eliminated the effect of ML414 (NGI-1) on G1 phase arrest, DNA damage and cell radiosensitivity, thus identifying RTK inhibition as the main mechanism of action of ML414 (NGI-1). [3] ML414 (NGI-1) is a cell-permeable small molecule oligosaccharide transferase (OST) inhibitor, which catalyzes N-linked glycosylation of proteins. It reversibly modulates N-linked glycosylation in mammalian cells without completely inhibiting OST activity [2]
ML414 (NGI-1) targets two catalytic subunits (STT3A and STT3B) of the OST complex, disrupting the maturation and function of glycoproteins (e.g., RTKs, viral glycoproteins) that rely on N-linked glycosylation for stability and localization [1][2][3]
ML414 (NGI-1) is a potential therapeutic for RTK-driven tumors (e.g., non-small cell lung cancer, glioma) and arenavirus infections (e.g., Lassa fever) by targeting common biosynthetic pathways essential for pathogen/virus survival and tumor progression [1][2][3]
In glioma cells,ML414 (NGI-1) primarily enhances radiosensitivity by inhibiting ErbB family RTKs. Signal transduction, such as the rescue effect of glycosylation-independent CD8-EGFR, has been demonstrated [3]. ML414 (NGI-1) exhibits selectivity for RTK-dependent cells and minimal impact on the viability of non-RTK-dependent cells or normal cells, indicating a favorable therapeutic window [2][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.)