HIF-1α/hypoxia inducible factor 1 α

Strong broad-spectrum antibiotic activity is exhibited by Gramicidin A against Gram-positive strains, including strains that are resistant to multiple drugs[1]. One drawback of gramicidin A is its high hemolytic activity[1].
Similar to monensin (HY-N4302), gramicidin A (0.1 nM–10 μM, 72 h) decreases the viability of RCC cell lines[2]. Neither VHL nor HIF-1α expression significantly affects the cellular sensitivity of gramicidin A[2]. In RCC cells, gramicidin A (1 and 10 μM, 48 or 72 h) causes nonapoptotic cell death[2]. In RCC cells, gramicidin A (0–10 μM, 24 h) causes metabolic dysfunction and depletes cellular energy[2].
HIF-1α and HIF-2α protein expression, HIF transcriptional activity, and target gene expression are all decreased by gramicidin A (0–1 μM, 24-72 h)[3].

RCC tumor xenografts are inhibited in growth by gramicidin A (0.11 mg/kg; intratumoral injection; twice weekly for 14 days)[2].
For 26 days, gramicidin A (0.22 mg/kg; intraperitoneal injection; three times weekly) prevents VHL-expressing RCC tumor xenografts from growing and angiogenizing[3].

Cell Line: A498, 786-O, Caki-1, SN12C, ACHN, UMRC6, UMRC6+VHL, HEK293T+pcDNA3, HEK293T+HA-HIF-1α, HEK293T+HA-HIF-1α-mut
Concentration: 0.1 nM-10 μM
Incubation Time: 72 h
Result: decreased the viability against A498, 786-O, Caki-1, SN12C, ACHN, UMRC6, UMRC6+VHL, HEK293T+pcDNA3, HEK293T+HA-HIF-1α, and HEK293T+HA-HIF-1α-mut cells with IC50s of 0.420, 0.430, 0.228, 0.104, 0.783, 0.253, 0.425, 0.057, 0.058, and 0.067 μM, respectively.

Animal Model: Six to eight weeks old, female Nu/J mice without hair were given a subcutaneous injection of a 1.0 × 10^6 SN12C cell suspension in a 50% reduced growth factor Matrigel solution[2].
Dosage: 0.11 mg/kg body weight
Administration: Intratumoral injection, twice weekly for 14 days
Result: The average tumor mass was reduced by approximately 40% without significant toxicity.

16132269 Mice oral LD50 1 gm/kg CRC Handbook of Antibiotic Compounds, Volume 1-, Berdy, J., Boca Raton, FL, CRC Press, 1980, 4(1)(240), 1980
16132269 Mice intraperitoneal LD50 60 mg/kg CRC Handbook of Antibiotic Compounds, Volume 1-, Berdy, J., Boca Raton, FL, CRC Press, 1980, 4(1)(240), 1980
16132269 Mice intravenous LD50 5 mg/kg CRC Handbook of Antibiotic Compounds, Volume 1-, Berdy, J., Boca Raton, FL, CRC Press, 1980, 4(1)(240), 1980

[1]. Discovery of gramicidin A analogues with altered activities by multidimensional screening of a one-bead-one-compound library. Nat Commun. 2020 Oct 1;11(1):4935.

[2]. Gramicidin A induces metabolic dysfunction and energy depletion leading to cell death in renal cell carcinoma cells. Mol Cancer Ther. 2013 Nov;12(11):2296-307.

[3]. Gramicidin A blocks tumor growth and angiogenesis through inhibition of hypoxia-inducible factor in renal cell carcinoma. Mol Cancer Ther. 2014 Apr;13(4):788-99.

group of peptide antibiotics produced by Bacillus brevis. Gram-C or S is a cyclic ten-amino acid polypeptide, while Gram-A, B, and D are linear. Gram-C is one of the two major components of tyrosinins. Gram-A (1) is a peptide antibiotic that disrupts transmembrane ion concentration gradients by forming ion channels in the lipid bilayer. Although it has been used clinically for many years, its application has been limited by its strong hemolytic activity and cytotoxicity to mammals (possibly due to their shared ion transport mechanism). This paper reports an integrated high-throughput strategy for discovering analogs of Compound 1 with different bioactivity profiles. We designed 4096 analog structures to maintain the charge neutrality, hydrophobicity, and channel-forming properties of Compound 1. Through analog synthesis, tandem mass spectrometry sequencing, and three micro-screenings, we finally identified 10 representative analogs. Resynthesis and detailed functional evaluation revealed that all 10 analogs had similar ion channel functions, but differed in cytotoxicity, hemolytic activity, and antibacterial activity. Our large-scale structure-activity relationship study suggests that it is feasible to develop compound 1 analogs that can selectively induce toxicity in target organisms. [1]

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Ion carriers are lipid-soluble organic molecules that disrupt the transmembrane potential of cells by making biological membranes permeable to specific ions. They include mobile carriers that complex with metal cations and channel-forming agents that insert into the membrane to form hydrophilic pores. Although mobile carriers have anticancer properties, research on channel-forming agents is limited. Here, we used the channel-forming ion carrier gramin A to investigate its effects on the growth and survival of renal cell carcinoma (RCC) cells. RCC is a highly histologically heterogeneous malignant tumor that is highly resistant to conventional therapies. We found that gramin A reduced the in vitro viability of various renal cell carcinoma (RCC) cell lines at sub-micromolar concentrations (all IC50 < 1.0 μmol/L). Gram-A toxicity to RCC cells is independent of histological subtype, expression of von Hippel-Lindau tumor suppressor gene and its downstream target gene hypoxia-inducible factor-1α. Gram-A’s effect on reducing cell viability is comparable to or stronger than that of the carrier monensin, depending on the cell line. Mechanistic studies have shown that Gram-A blocks ATP production by inhibiting oxidative phosphorylation and glycolysis, leading to cell energy depletion and non-apoptotic cell death. In addition, Gram-A can also effectively inhibit the growth of in vivo RCC xenograft tumors. These results reveal the new application prospects of Gram-A as a potential therapeutic agent for RCC. [2]

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Ion carriers are hydrophobic organic molecules that disrupt the transmembrane potential of cells by increasing the permeability of the cell membrane to specific ions. Gram-A is a channel-forming ion carrier that can form hydrophilic membrane pores that allow monovalent cations to pass through rapidly. Previously, we found that Gram-A can induce cell energy stress and cell death in renal cell carcinoma (RCC) cell lines. Renal cell carcinoma (RCC) is a refractory cancer characterized by constitutive activation of the transcription factor hypoxia-inducible factor (HIF). In this study, we demonstrated that grammidine A can inhibit HIF in RCC cells. We found that under both normoxic and hypoxic conditions, grammidine A destabilized HIF-1α and HIF-2α proteins, thereby reducing HIF transcriptional activity and the expression of various hypoxia-responsive genes. Mechanistic studies revealed that grammidine A accelerates O₂-dependent HIF downregulation by upregulating the expression of von Hippel-Lindau (VHL) tumor suppressor protein, and VHL targets hydroxylated HIF for proteasomal degradation. Furthermore, grammidine A inhibited the growth of human renal cell carcinoma xenografts without significant toxicity in mice. Tumors treated with grammidine A also exhibited physiological and molecular characteristics consistent with HIF-dependent angiogenesis inhibition. In summary, these results indicate that Gram-A, as a potent HIF inhibitor, plays a novel role in inhibiting tumor growth and angiogenesis in renal cell carcinoma expressing VHL. [3]

C99H140N20O17

1882.29000

1881.07

C, 63.17; H, 7.50; N, 14.88; O, 14.45

11029-61-1

Gramicidin A TFA

16132269

White to off-white solid powder

11.262

21

17

52

136

3980

14

OCCNC([C@@H](NC(CNC([C@@H](NC(CNC([C@@H](NC(CNC([C@@H](NC([C@H](NC([C@@H](NC)C(C)C)=O)C(C)C)=O)CC1C2=CC=CC=C2NC=1)=O)=O)CC1C2=CC=CC=C2NC=1)=O)=O)CC1C2=CC=CC=C2NC=1)=O)=O)CC1C2=CC=CC=C2NC=1)=O

ZWCXYZRRTRDGQE-LUPIJMBPSA-N

InChI=1S/C99H140N20O17/c1-51(2)37-73(109-86(123)59(17)107-81(122)49-105-96(133)82(55(9)10)106-50-121)89(126)108-60(18)87(124)117-84(57(13)14)98(135)119-85(58(15)16)99(136)118-83(56(11)12)97(134)116-80(44-64-48-104-72-34-26-22-30-68(64)72)95(132)112-76(40-54(7)8)92(129)115-79(43-63-47-103-71-33-25-21-29-67(63)71)94(131)111-75(39-53(5)6)91(128)114-78(42-62-46-102-70-32-24-20-28-66(62)70)93(130)110-74(38-52(3)4)90(127)113-77(88(125)100-35-36-120)41-61-45-101-69-31-23-19-27-65(61)69/h19-34,45-48,50-60,73-80,82-85,101-104,120H,35-44,49H2,1-18H3,(H,100,125)(H,105,133)(H,106,121)(H,107,122)(H,108,126)(H,109,123)(H,110,130)(H,111,131)(H,112,132)(H,113,127)(H,114,128)(H,115,129)(H,116,134)(H,117,124)(H,118,136)(H,119,135)/t59-,60-,73+,74+,75+,76+,77-,78-,79-,80-,82-,83+,84+,85-/m0/s1

(2R)-2-[[(2S)-2-[[2-[[(2S)-2-formamido-3-methylbutanoyl]amino]acetyl]amino]propanoyl]amino]-N-[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-(2-hydroxyethylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxopropan-2-yl]-4-methylpentanamide

Valinegramicidin A; Valyl gramicidin A; Gramicidin A; 11029-61-1; 1-L-Valinegramicidin A; 4419-81-2; Gramicidin A, 1-L-valine-; GNF-Pf-2578; 1-L-Valinegramicidin A;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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