Endogenous Metabolite
The primary targets of 5'-GTP trisodium salt hydrate are signal-transducing G proteins, which it activates by binding to the G protein alpha subunit and promoting the exchange of GDP for GTP. This activation is a key step in GPCR signaling pathways. Additionally, 5'-GTP serves as a substrate for various enzymes including guanylate kinases, GTPases (such as Ras and Rho family proteins), and DNA/RNA polymerases. As a precursor for RNA synthesis, it is incorporated into RNA molecules during transcription. The compound also serves as an energy source for various cellular processes, similar to ATP.

The extracellular guanosine 5'-triphosphate, GTP, has been demonstrated to be an enhancer of myogenic cell differentiation in a murine cell line, not yet in human muscle cells. Our hypothesis was that GTP could influence also human skeletal muscle regeneration, specifically in the first phases. We tested GTP stimulus on human muscle precursor cells established in culture by human satellite cells derived from Vastus Lateralis of three young male. Our data show that extracellular GTP (a) up-regulated miRNA (specifically miR133a and miR133b) and myogenic regulator factor and (b) induces human myogenic precursor cells to release exosomes stuffed with guanosine based molecules (mainly guanosine) in the extracellular milieu. We think that probably these exosomes could be addressed to influence by means of their content (mainly guanosine) in paracrine or autocrine manner the surrounding cells and/or at distance other muscles or tissues[1].

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In vitro, 5'-GTP trisodium salt hydrate is used as a substrate in numerous biochemical assays. It is essential for in vitro transcription reactions, where it is incorporated into RNA by RNA polymerases. The compound is also used in GTPase activity assays to measure the hydrolysis of GTP to GDP by GTPases. In GPCR signaling studies, 5'-GTP is used to assess G protein activation and receptor-mediated nucleotide exchange. It serves as a cofactor for various enzymes and is used in kinase assays. The compound's activity in these assays depends on its purity and stability in solution. It is typically used at concentrations ranging from micromolar to millimolar.

In vivo, 5'-GTP trisodium salt hydrate functions as a critical nucleotide for RNA synthesis and G protein signaling. However, as an exogenous compound, it is not typically administered as a therapeutic agent. Instead, it is used as a research tool to study nucleotide metabolism and signaling in cells and tissues. When used in cell culture or in vivo experiments, 5'-GTP can be taken up by cells and incorporated into metabolic pathways. Its role in G protein activation makes it useful for studying GPCR signaling in various physiological contexts. The compound's effects in vivo would depend on its stability and cellular uptake.

For in vitro enzyme/receptor binding assays, 5'-GTP trisodium salt hydrate is used as a substrate in GTPase activity assays, which measure the hydrolysis of GTP to GDP by GTPases. These assays typically use recombinant GTPases or cell lysates and quantify the release of inorganic phosphate using colorimetric or fluorometric methods. The compound is also used in nucleotide exchange assays to measure the exchange of GDP for GTP on G protein alpha subunits. In in vitro transcription assays, 5'-GTP is incorporated into RNA by RNA polymerases, and the reaction can be monitored by radioactive labeling or fluorescence. Standard assay conditions include physiological buffer systems with appropriate concentrations of magnesium ions, which are essential for nucleotide binding and catalysis.

The MPCs were plated at a confluence of 15,000 cells/cm2 and maintained for 2 days in Growth Medium. After 2 days in GM, the cells were cultivated with fresh GM for additional 24 h (CTR-undiff) or stimulated by addition of 500 μM GTP for the following 24 h (GTP-undiff). Differentiation was induced by replacing the GM with the Differentiation Medium (DM) on cells plated on growth condition 3 days before. The differentiating cells were regularly cultivated for 24 h (CTR-diff) or stimulated by addition of 500 μM GTP for 24 h (GTP-diff)[1].

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For in vitro cellular experiments, 5'-GTP trisodium salt hydrate can be added to cell culture media to study its effects on cellular metabolism, signaling, and nucleic acid synthesis. Cells are cultured in appropriate media and treated with various concentrations of the compound (typically ranging from micromolar to millimolar). Cellular uptake of GTP can be assessed using labeled or fluorescent nucleotide analogs. Effects on cell viability, proliferation, and metabolic activity can be measured using standard assays. GTP-dependent signaling pathways, such as those mediated by GPCRs and small GTPases, can be studied by measuring downstream effectors. The compound's stability in cell culture medium should be monitored, as phosphatases may degrade GTP over time.

For in vivo animal experiments, 5'-GTP trisodium salt hydrate is not typically administered as a therapeutic agent but may be used as a research tool to study nucleotide metabolism and signaling. When administered to animals, it would likely be rapidly metabolized or cleared. The compound could be used in studies of G protein signaling by administering it along with GPCR agonists or other modulators. However, due to its charged nature and rapid metabolism, alternative approaches such as using cell-permeable GTP analogs are often preferred for in vivo studies. Animal studies should follow appropriate ethical guidelines and use suitable control groups for comparison.

Pharmacokinetic properties of 5'-GTP trisodium salt hydrate are not well characterized, as it is primarily used as a research reagent rather than a drug candidate. As a highly charged nucleotide, it has very low cell permeability and would be expected to have poor oral bioavailability. When administered intravenously, it would likely be rapidly cleared from circulation by phosphatases and other nucleotide-metabolizing enzymes. The compound would be metabolized to guanosine and inorganic phosphate. Its half-life in plasma would be very short. For these reasons, 5'-GTP is typically used in in vitro assays or in cell culture rather than in vivo. Cell-permeable GTP analogs are available for intracellular studies.

Toxicological data for 5'-GTP trisodium salt hydrate are limited, as it is a naturally occurring nucleotide and a normal cellular metabolite. At physiological concentrations, it is well-tolerated. However, high concentrations could potentially disrupt cellular nucleotide balance and signaling. Standard toxicological assessments would include cytotoxicity screening in relevant cell lines. The compound is generally considered to have low toxicity for research use. As with all research chemicals, appropriate safety precautions should be taken when handling 5'-GTP trisodium salt hydrate, including the use of personal protective equipment and adherence to institutional safety guidelines. The compound should be stored properly to maintain stability.

[1]. Extracellular Guanosine 5'-Triphosphate Induces Human Muscle Satellite Cells to Release Exosomes Stuffed With Guanosine. Front Pharmacol. 2018 Mar 16;9:152.
[2]. Functionally nonequivalent interactions of guanosine 5'-triphosphate, inosine 5'-triphosphate, and xanthosine 5'-triphosphate with the retinal G-protein, transducin, and with Gi-proteins in HL-60 leukemia cell membranes. Biochem Pharmacol. 1997 Sep 1;54(5):551-62.

protein-mediated signal transduction from receptors to effector systems. The α subunit of a G protein activates the effector system via guanosine 5'-triphosphate (GTP) binding. Termination of G protein activation is accomplished by a high-affinity GTPase [EC 3.6.1.-] of its α subunit. Similar to GTP, inosine 5'-triphosphate (ITP) and xanthine 5'-triphosphate (XTP) also promote effector system activation. We investigated the interactions of GTP, ITP, and XTP with retinal G protein transducer (TD) and G proteins on the HL-60 leukemia cell membrane. The order of TD hydrolysis of nucleoside 5'-triphosphate (NTP) was GTP > ITP > XTP. The order of NTP elution of TD from the extracellular disc membrane of rod cells was also the same. ITP and XTP competitively inhibited TD-catalyzed GTP hydrolysis. In the HL-60 cell membrane, the chemokines N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and leukotriene B4 (LTB4) effectively activate Gi protein-mediated GTP and ITP hydrolysis. The activation potency of fMLP and LTB4 for ITPase is at least 10 times that for GTPase. Complement C5a effectively activates the GTPase activity of Gi protein, but its activation effect on ITPase is weaker. C5a has similar activating potency for GTP and ITP hydrolysis. The Km value of fMLP-activated GTPase is lower than that of fMLP-activated ITPase, while the Vmax value is the opposite. None of fMLP, C5a, or LTB4 activates XTP hydrolysis. Our data collectively indicate that GTP, ITP, and XTP have different binding affinities to G proteins, different efficiencies in G protein hydrolysis of NTPs, and that chemokines stimulate Gi proteins to hydrolyze GTP and ITP in a receptor-specific manner. Based on our results and literature data, we propose the following hypothesis: GTP, ITP, and XTP function as different signal amplifiers and signal sorters at the G protein level. [2]

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5'-GTP trisodium salt hydrate is a biochemical research reagent used in studies of G protein signaling, nucleic acid synthesis, and enzyme assays. No clinical trials or regulatory approvals have been reported for this compound as a therapeutic agent. It is available from various chemical suppliers for research purposes only. The compound serves as an activator of signal-transducing G proteins and as a precursor for RNA synthesis. 5'-GTP is widely used in molecular biology and biochemistry laboratories. Its stability in solution should be monitored, as GTP can be hydrolyzed over time. The compound is typically stored as a dry powder at -20degC to maintain stability.

C10H19N5NAO15P3

565.193415880203

563.99

207300-85-4

Guanosine 5'-triphosphate trisodium salt;36051-31-7;Guanosine-5'-triphosphate disodium salt;56001-37-7

168011740

White to off-white solid powder

9

17

8

34

927

4

O[C@@H]1[C@@H]([C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C=NC2C(NC(N)=NC1=2)=O)O.[NaH].O

DTXOVEQJIGBCCS-LGVAUZIVSA-N

InChI=1S/C10H16N5O14P3.Na.H2O/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21;;/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18);;1H2/q;+1;/t3-,5-,6-,9-;;/m1../s1

sodium;[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate;hydrate

207300-85-4; ((2R,3S,4R,5R)-5-(2-Amino-6-oxo-3,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate, sodium salt xhydrate; 36051-31-7; sodium triphosphate, guanosine hydrate

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)

H2O : ≥ 150 mg/mL (~254.61 mM)

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.)