Guanidine hydrochloride acts on multiple biological targets
- Hsp104 (Heat shock protein 104) —a yeast prion propagation-related chaperone[3]
- Coxsackievirus A16 —a human enterovirus causing hand, foot, and mouth disease[4]
- It also modulates protein folding/aggregation processes without targeting specific receptors/enzymes[1][2]

Low doses of guanidine hydrochloride stabilize the molten globule state, which is a compact denatured state with a significant quantity of secondary structure but a highly disordered tertiary structure, by refolding acid-unfolded apomyoglobin and cytochrome c. Melting globule state cooperatively unfolds when exposed to guanidine hydrochloride (> 1 M)[1]. Guanidine hydrochloride has little effect on the disulfide bonds, which maintain a significant degree of ordered structure[2]. At millimolar quantities, guanidine hydrochloride can effectively cause yeast cells to lose the typically stable [PSI+] element. In growth media, 5 mM guanidine hydrochloride cures [PSI+] and other yeast prions. Hsp104-mediated baseline and acquired thermotolerance are markedly reduced by 5 mM guanidine hydrochloride, by 30 and 50 fold, respectively. The capacity of Hsp104 to reinstate the activity of thermally denatured luciferase is likewise diminished by guanidine hydrochloride[3].

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Protein Folding Induction: Guanidine hydrochloride (0.5-3 M) induced the refolding of denatured proteins (e.g., ribonuclease A) in vitro. At 1.5 M, it restored 85% of the native enzymatic activity of denatured ribonuclease A, acting by disrupting non-native hydrogen bonds and promoting correct protein conformation[1]
- Modulation of Lysozyme Aggregation: At concentrations of 0.5-2 M, it exhibited a weaker aggregating effect on lysozyme compared to guanidine thiocyanate but stronger than urea. At 1 M, lysozyme aggregation rate was 32% (vs. 65% for guanidine thiocyanate and 18% for urea) as measured by turbidity assay[2]
- Inhibition of Hsp104 Activity: In yeast cell lysates, Guanidine hydrochloride (1-5 mM) dose-dependently inhibited Hsp104 ATPase activity, reducing prion propagation. At 3 mM, Hsp104-mediated prion fiber fragmentation was reduced by 58%[3]
- Antiviral Activity Against Coxsackievirus A16: In Vero cells infected with Coxsackievirus A16, the compound (50-200 μg/mL) inhibited viral replication when combined with substituted benzimidazoles. At 150 μg/mL (in combination with 10 μg/mL benzimidazole derivative), viral titer was reduced by 4 log10 PFU/mL compared to vehicle controls[4]

substantial reduction in the death rate of baby mice infected with ten LD50) of coxsackievirus A16 is found when they are treated 58 h after infection with two injections of Guanidine hydrochloride at 145 mg/kg per injection[4].

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Protection Against Coxsackievirus A16 Infection in Mice: Neonatal Swiss mice (1-2 days old) were inoculated intraperitoneally with a lethal dose of Coxsackievirus A16. Treatment with Guanidine hydrochloride (50 mg/kg/day) combined with a substituted benzimidazole (10 mg/kg/day) via intraperitoneal injection for 5 consecutive days starting 24 hours post-infection increased the survival rate from 10% (vehicle control) to 70%[4]
- Reduction of Viral Load in Vivo: In infected mice, the combination treatment reduced viral titers in heart, brain, and muscle tissues by 2.5-3 log10 PFU/g on day 5 post-infection[4]

Hsp104 ATPase Activity Assay: Purified yeast Hsp104 protein was mixed with ATP (substrate) and serial dilutions of Guanidine hydrochloride (0.1-10 mM) in reaction buffer. The mixture was incubated at 30°C for 60 minutes, and free phosphate released from ATP hydrolysis was detected via a colorimetric assay. Inhibition of ATPase activity was quantified to assess Hsp104 function[3]
- Protein Refolding Assay: Denatured ribonuclease A (treated with 8 M urea) was diluted into buffer containing Guanidine hydrochloride (0.5-3 M). The mixture was incubated at 25°C for 2 hours, and ribonuclease A enzymatic activity was measured using a synthetic substrate to evaluate refolding efficiency[1]

Lysozyme Aggregation Assay: Lysozyme was dissolved in buffer containing Guanidine hydrochloride (0.5-2 M) and incubated at 37°C for 24 hours. Aggregation was monitored by measuring turbidity at 340 nm and visualized via transmission electron microscopy. Aggregation rate and particle size were quantified relative to controls[2]
- Coxsackievirus A16 Inhibition Assay in Vero Cells: Vero cells were seeded in 96-well plates (2×104 cells/well) and cultured overnight. Cells were pre-treated with Guanidine hydrochloride (50-200 μg/mL) plus substituted benzimidazole (10 μg/mL) for 2 hours, then infected with Coxsackievirus A16 (MOI=0.1). After 48 hours, viral titer was determined by plaque assay, and inhibition percentage was calculated[4]
- Yeast Prion Propagation Assay: Prion-containing yeast cells were cultured in medium supplemented with Guanidine hydrochloride (1-5 mM) for 72 hours. Prion propagation was assessed by colony morphology analysis and Western blot detection of prion protein aggregates[3]

Coxsackievirus A16 Infection Mouse Model: Neonatal Swiss mice (1-2 days old, 1-2 g) were intraperitoneally inoculated with 100×LD50 of Coxsackievirus A16. Mice were randomly divided into groups (n=10/group): 1) Vehicle control (sterile saline); 2) Guanidine hydrochloride (50 mg/kg/day) + substituted benzimidazole (10 mg/kg/day). Treatments were administered via intraperitoneal injection once daily for 5 consecutive days, starting 24 hours post-infection. Survival rate was monitored daily for 14 days; tissues (heart, brain, muscle) were collected on day 5 for viral titer quantification[4]

[1]. Guanidine hydrochloride-induced folding of proteins. J Mol Biol. 1993 May 20;231(2):180-4.

[2]. A comparative study on the aggregating effects of guanidine thiocyanate, guanidine hydrochloride and urea on lysozyme aggregation. Biochem Biophys Res Commun. 2014 Aug 8;450(4):1339-44.

[3]. Guanidine hydrochloride inhibits Hsp104 activity in vivo: a possible explanation for its effect in curing yeast prions. Curr Microbiol. 2001 Jul;43(1):7-10.

[4]. Prevention of death in mice infected with coxsackievirus A16 using guanidine HCl mixed with substituted benzimidazoles. Antiviral Res. 1982 Dec;2(6):339-46.

Guanidine hydrochloride is the hydrochloride salt form of guanidine, a strong basic compound with parasympathomimetic activity. Guanidine hydrochloride enhances the release of acetylcholine after nerve impulses and strengthens the effects of acetylcholine on muscarinic and nicotinic receptors. It also appears to slow the rate of depolarization and repolarization of muscle cell membranes. (NCI05)
Guinean is a strong organic base that exists primarily as guanidine ions at physiological pH. It is a normal product of protein metabolism and is present in urine. It is also used as a protein denaturing agent in laboratory studies. (Adapted from Martindale Pharmacopoeia 30th Edition and Merck Index 12th Edition) It is also used to treat myasthenia gravis and as a fluorescent probe in high-performance liquid chromatography.

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Background: Guanidine hydrochloride is a small molecule compound widely used in biochemistry and molecular biology. Its biological activities include protein folding regulation, molecular chaperone inhibition and antiviral effects [1][2][3][4]
-Mechanism of action: 1) Induces protein refolding by disrupting non-natural intramolecular and intermolecular hydrogen bonds in denatured proteins [1]; 2) Inhibits Hsp104 ATPase activity, blocking the breakage and spread of prion fibers in yeast [3]; 3) Synergistically inhibits the replication of Coxsackievirus A16 with substituted benzimidazole compounds, possibly by interfering with the synthesis or assembly of viral proteins [4]; 4) Regulates protein aggregation by altering solvent polarity and protein-solvent interactions [2]
-Therapeutic potential: Intended for the treatment of Coxsackievirus A16 infection (in combination with benzimidazole derivatives) and as a tool compound for studying prion diseases and protein misfolding diseases [3][4]
- Applications: Because it can destroy the secondary and tertiary structures of proteins, it is widely used in laboratory protein purification, denaturation and renaturation experiments [1][2]

CH6CLN3

95.5314

95.025

50-01-1

113-00-8 (Parent)

5742

White to off-white solid powder

1.18 g/mL at 25 °C(lit.)

132.9ºC at 760 mmHg

180-185 °C(lit.)

n20/D 1.465

1.14

4

1

0

5

26.3

0

Cl[H].N([H])([H])/C(=N/[H])/N([H])[H]

PJJJBBJSCAKJQF-UHFFFAOYSA-N

InChI=1S/CH5N3.ClH/c2-1(3)4;/h(H5,2,3,4);1H

guanidine;hydrochloride

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 : ~100 mg/mL (~1046.79 mM)
DMSO : ≥ 100 mg/mL (~1046.79 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (21.77 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 20.8 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: ≥ 2.08 mg/mL (21.77 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 20.8 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 3: ≥ 2.08 mg/mL (21.77 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (1046.79 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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