GBF1
Golgicide A (GCA) specifically targets Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a key regulator of Arf1 (ADP-ribosylation factor 1) activation in Golgi vesicle trafficking. The IC₅₀ for inhibiting GBF1-mediated Arf1 GDP-GTP exchange activity is 200 nM [1,3]

Golgicide A inhibits the effect of shiga toxin on protein synthesis with an IC50 of 3.3 μM. Golgicide A causes a decrease in GBF1-mediated Arf1 activation, arrests secretion of soluble and membrane-anchored proteins and then impairs retrograde toxin transport. Golgicide A decreases HCV RNA levels in FLRP1 cells and J6/JFH1 cells. In addition, Golgicide A causes redistribution of NS5A and accumulation of infectious viral particles in J6/JFH1 cells.

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Disruption of Golgi structure and function: Treatment of HeLa cells with 1 μM GCA for 1 hour caused Golgi fragmentation (observed by immunofluorescence staining of Golgi marker GM130), with >90% of cells showing dispersed GM130 signals compared to intact Golgi stacks in controls. This was accompanied by a 70% reduction in protein secretion (measured by secretion of alkaline phosphatase) [1,3]
- Inhibition of viral replication (flaviviruses): GCA inhibited dengue virus (DENV) serotype 2 replication in Huh7 cells with an EC₅₀ of 350 nM (plaque reduction assay, 48-hour treatment). At 1 μM, viral RNA levels decreased by 90% (qRT-PCR) and viral envelope protein expression was reduced by 85% (Western blot) [2]
- Inhibition of yellow fever virus (YFV) and West Nile virus (WNV): In Vero cells, GCA showed EC₅₀ values of 280 nM (YFV) and 320 nM (WNV) in plaque reduction assays. 1 μM GCA reduced YFV infectious titers by 5 log₁₀ PFU/mL and WNV titers by 4.5 log₁₀ PFU/mL after 72 hours [4]
- Selective cytotoxicity: GCA exhibited low cytotoxicity in Huh7 and Vero cells, with CC₅₀ values of 12 μM and 15 μM, respectively (MTT assay, 72-hour treatment), resulting in therapeutic indices (CC₅₀/EC₅₀) of ~34 (DENV) and ~43 (YFV) [2,4]

N/A

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Efficacy in dengue virus-infected mouse model: AG129 mice (deficient in IFN-α/β and IFN-γ receptors) were intraperitoneally infected with 1×10⁴ PFU DENV-2. Treatment with GCA (50 mg/kg, intraperitoneal injection, twice daily) starting 1 hour post-infection resulted in: (1) 60% survival rate (vs. 10% in vehicle control) at day 14; (2) 10-fold reduction in viral load in serum (day 3) and liver (day 5) by qRT-PCR; (3) reduced liver pathology (less necrosis and inflammation) [2]

GBF1-mediated Arf1 GDP-GTP exchange assay (Literature [1,3]):
1. Protein preparation: Recombinant human GBF1 (residues 1-1397) and Arf1 were purified from E. coli. Arf1 was loaded with GDP by incubating with 1 mM GDP, 20 mM EDTA, and 50 mM Tris-HCl (pH 7.5) at 30°C for 30 minutes, then stabilized with 40 mM MgCl₂ [1,3]
2. Reaction setup: The assay mixture (200 μL) contained 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM MgCl₂, 50 nM GBF1, 1 μM GDP-loaded Arf1, and 0.5 μM [³⁵S]GTPγS. Serial concentrations of GCA (10 nM–10 μM) were added, with DMSO as vehicle control [1,3]
3. Kinetic measurement: The mixture was incubated at 30°C, and 20 μL aliquots were removed at 0, 5, 10, 20, and 30 minutes. Samples were filtered through nitrocellulose membranes, washed with buffer (50 mM Tris-HCl pH 7.5, 25 mM MgCl₂), and bound [³⁵S]GTPγS was quantified by liquid scintillation counting [1,3]
4. Data analysis: The rate of GTPγS binding was calculated, and IC₅₀ (200 nM) was determined from the dose-response curve of inhibition efficiency vs. GCA concentration [1,3]

Golgicide A inhibits the effect of shiga toxin on protein synthesis with an IC50 of 3.3 μM in Vero cells. Immunofluoresence experiments demonstrates that Golgicide A causes complete dispersal of the medial-Golgi markers giantin and the cis-Golgi marker GM130 and results in a rapid redistribution of COPI from the Golgi. Also, Golgicide A causes a decrease in GBF1-mediated Arf1 activation, impairs retrograde toxin transport and arrests secretion of soluble and membrane-anchored proteins. Golgicide A decreases HCV RNA levels and causes redistribution of NS5A in FLRP1 cells and J6/JFH1 cells. In addition, Golgicide A causes accumulation of infectious viral particles in J6/JFH1 cells.

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Golgi structure analysis by immunofluorescence (Literature [1,3]):
1. Cell seeding and treatment: HeLa cells were seeded on coverslips in 6-well plates (5×10⁴ cells/well) and incubated overnight. Cells were treated with 1 μM GCA for 1 hour, with 0.1% DMSO as control [1,3]
2. Fixation and staining: Cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 5 minutes, and blocked with 3% BSA in PBS for 30 minutes. Primary antibody against GM130 (Golgi marker) was incubated at 4°C overnight, followed by Alexa Fluor-conjugated secondary antibody for 1 hour at room temperature. Nuclei were stained with DAPI [1,3]
3. Imaging and quantification: Fluorescence images were captured using a confocal microscope. Golgi integrity was scored as "intact" (compact perinuclear structure) or "fragmented" (dispersed puncta). At least 100 cells per sample were counted, and the percentage of cells with fragmented Golgi was calculated [1,3]
- Viral plaque reduction assay (Literature [2,4]):
1. Cell and virus preparation: Huh7 (for DENV) or Vero (for YFV/WNV) cells were seeded in 6-well plates (2×10⁵ cells/well) and grown to 80% confluence. Virus stocks were diluted to 100–200 PFU/well [2,4]
2. Drug treatment and infection: GCA (serial concentrations: 10 nM–10 μM) was added to cells 1 hour before virus infection. Cells were infected with virus for 1 hour at 37°C, then overlaid with 0.8% methylcellulose in medium containing the same GCA concentration [2,4]
3. Plaque staining and counting: After 48–72 hours (depending on virus), cells were fixed with 4% PFA and stained with 0.1% crystal violet. Plaques were counted, and EC₅₀ was calculated as the concentration reducing plaque number by 50% vs. control [2,4]
- Viral protein Western blot (Literature [2]):
1. Cell treatment and lysis: Huh7 cells infected with DENV-2 (MOI=1) were treated with 1 μM GCA for 24 hours. Cells were lysed with RIPA buffer containing protease inhibitors, and lysates were centrifuged at 12,000 × g for 15 minutes [2]
2. Detection: 30 μg of total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-DENV envelope protein antibody and anti-β-actin antibody (loading control). Bands were visualized by ECL and quantified, showing 85% reduction in envelope protein levels [2]

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DENV-infected AG129 mouse model (Literature [2]):
1. Animal selection and grouping: 6- to 8-week-old AG129 mice (n=10/group) were randomized into vehicle control and GCA treatment groups [2]
2. Infection and treatment: Mice were intraperitoneally injected with 1×10⁴ PFU DENV-2. GCA was dissolved in 10% DMSO + 90% corn oil to 10 mg/mL. The treatment group received 50 mg/kg GCA via intraperitoneal injection at 1 hour post-infection, then twice daily for 7 days; the control group received the same volume of vehicle [2]
3. Monitoring and sampling: Mice were monitored daily for survival and weight. On days 3 and 5, subsets of mice (n=3/group) were euthanized, and serum and liver tissues were collected for viral load quantification by qRT-PCR. Liver sections were stained with H&E for pathological analysis [2]

In vitro cytotoxicity: MTT assay (72-hour treatment) showed that GCA had a CC₅₀ of 12 μM on Huh7 cells and 15 μM on Vero cells. No significant cytotoxicity was observed at concentrations ≤1 μM (cell viability >90%) [2,4]. - In vivo acute toxicity: AG129 mice administered 50 mg/kg GCA intraperitoneally (twice daily for 7 days) showed no significant weight loss (<5% vs. baseline), and no significant pathological changes were observed in major organs (liver, kidney, spleen) [2]. - No data on plasma protein binding, LD₅₀, or drug interactions of GCA have been reported in the literature [1,2,3,4].

[1]. Nat Chem Biol.2009 Mar;5(3):157-65)

[2]. J Virol.2011 Jan;85(2):946-56.

[3]. Nat Chem Biol. 2009 Mar;5(3):157-65.

[4]. J Virol. 2010 Aug;84(15):7535-42.

Golgicide A is a diastereomer mixture of racemic cis and racemic trans-golyl glycosides A in a 10:1 ratio. It is a potent and rapidly reversible inhibitor of GBF1 (Golgi-specific brefidobacterium A-resistant guanine nucleotide exchange factor 1). The (3aS,4R,9bR) isomer exhibits the highest activity (see Bioorg. Med. Chem. Lett., 2012, 22, 5177-5181). It functions as a cis-Golgi ArfGEF GBF inhibitor. It comprises cis-golyl glycoside A and trans-golyl glycoside A. Mechanism of action: GCA binds to GBF1, inhibiting its guanine nucleotide exchange activity against Arf1. This blocks Arf1 activation, disrupts COPI-mediated vesicle transport, and leads to Golgi fragmentation. For viruses that rely on the Golgi apparatus for replication and viral particle assembly (e.g., flaviviruses), this leads to impaired viral maturation and release [1,2,3,4]
- Selectivity with other Arf GEFs: GCA is selective for GBF1 and has no significant inhibitory effect on other Arf guanine nucleotide exchange factors (e.g., BIG1, BIG2) at concentrations up to 10 μM, as demonstrated by in vitro exchange experiments [1,3]
- Research applications: GCA is a valuable tool for studying Golgi dynamics, Arf1-mediated vesicle transport, and virus-Golgi interactions, especially in understanding the role of the host Golgi apparatus in viral replication [1,2,3,4]

C17H14F2N2

284.310

284.113

C, 71.82; H, 4.96; F, 13.36; N, 9.85

1139889-93-2

Golgicide A-2;1394285-50-7;Golgicide A-1;1394285-49-4;(Rac)-Golgicide A;1005036-73-6

25113626

White to off-white solid powder

4.324

1

4

1

21

414

2

C1C=CC2C1C(NC3=C2C=C(C=C3F)F)C4=CN=CC=C4

NJZHEQOUHLZCOX-FTLRAWMYSA-N

InChI=1S/C17H14F2N2/c18-11-7-14-12-4-1-5-13(12)16(10-3-2-6-20-9-10)21-17(14)15(19)8-11/h1-4,6-9,12-13,16,21H,5H2/t12-,13+,16?/m0/s1

(3aR,9bS)-6,8-difluoro-4-(pyridin-3-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline

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 : 57~100 mg/mL ( 200.49~351.74 mM )
Ethanol : 4 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.79 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.79 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+90% (20% SBE-β-CD in Saline): ≥ 2.5 mg/mL (8.79 mM)

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