Thiazovivin (TZV) is a novel, selective, and cell-permeable small molecule ROCK (Rho-associated kinase) inhibitor with an IC50 of 0.5 μM in a cell-free assay. It can protect human embryonic stem cells (hESC). Thiazovivin promotes hESC survival after single-cell dissociation and improves stemness maintenance of embryo-derived stem-like cells under chemically defined culture conditions in cattle. Thiazovivin directly targets ROCK and increases expression of pluripotency factors. The process using thiazovivin could be easier, faster and more cost effective than transgene integration into somatic cells.

Physicochemical Properties

Molecular Formula	C15H13N5OS
Molecular Weight	311.36
Exact Mass	311.084
CAS #	1226056-71-8
Related CAS #	1226056-71-8
PubChem CID	46209426
Appearance	Light yellow to brown solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.691
LogP	2
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	5
Heavy Atom Count	22
Complexity	364
Defined Atom Stereocenter Count	0
InChi Key	DOBKQCZBPPCLEG-UHFFFAOYSA-N
InChi Code	InChI=1S/C15H13N5OS/c21-14(17-8-11-4-2-1-3-5-11)12-9-22-15(19-12)20-13-6-7-16-10-18-13/h1-7,9-10H,8H2,(H,17,21)(H,16,18,19,20)
Chemical Name	N-(phenylmethyl)-2-(4-pyrimidinylamino)-4-thiazolecarboxamide
Synonyms	Thiazovivin; TZV
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Thiazovivin specifically targets Rho-associated coiled kinase (ROCK) isoforms ROCK1 and ROCK2 (ROCK1 IC50 = 0.5 nM; ROCK2 IC50 = 0.3 nM) [1] Thiazovivin shows no significant inhibition of other kinases (PKA, PKC, MLCK: IC50 > 10 μM) [1]
ln Vitro	One ROCK inhibitor is thiazovivin. Thiazovivin (2 μM) preserves human embryonic stem cells (hESC) via blocking ROCK activity. After separation, thiazovivin dramatically prolongs hESCs' lifespan while preserving their pluripotency. Thiazovivin improves integrin signaling mediated by cell-ECM adhesion. Additionally, thiazovivin stabilizes E-cadherin upon cell dissociation to prevent hESCs from dying in the absence of extracellular matrix [1]. Thiazovivin promotes primary colony development and cell attachment of bovine embryonic-derived stem cell-like cells (eSLC) on feeder layers. Thiazovivin promotes the growth of eSLC cultures during passage and augments putative colony growth. Moreover, in bovine eSLC, thiazovivin increases the expression of genes unique to the ectodermal lineage [2]. In human pluripotent stem cells (PSCs), Thiazovivin (1 μM) promotes cell survival and self-renewal. It reduces apoptosis by 68% compared to control, with Annexin V-positive cells decreasing from 22% to 7% after 72 hours. It also upregulates stemness markers Oct4 (2.3-fold), Sox2 (2.1-fold), and Nanog (1.9-fold) at mRNA level, and maintains colony morphology of PSCs [1] - In cattle embryo-derived stem-like cells (EDSCs), Thiazovivin (0.5 μM) maintains stemness under chemically defined culture conditions. After 14 days of culture, the proportion of Oct4-positive cells remains at 85% (vs. 42% in vehicle group), and clone formation rate is increased by 55%. It inhibits spontaneous differentiation, with reduced expression of differentiation markers (α-SMA, Nestin) [2] - In PSCs, Thiazovivin (1 μM) inhibits ROCK-mediated phosphorylation of myosin light chain (MLC) by 75% at Ser19, blocking actin cytoskeleton rearrangement associated with apoptosis [1]
ln Vivo
Enzyme Assay	ROCK1/ROCK2 kinase activity assay: Purified recombinant human ROCK1 or ROCK2 was incubated with MLC-derived substrate peptide and Thiazovivin (0.01 nM-10 nM) in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM ATP) at 30°C for 60 minutes. Phosphorylated substrate was detected by radiolabeled ATP counting, and IC50 values were calculated from dose-response curves [1] - ATP competition assay: ROCK2 was incubated with increasing concentrations of ATP (0.05-1 mM) and fixed Thiazovivin (0.3 nM). Kinase activity was measured to confirm competitive binding to the ATP-binding pocket of ROCK [1] - Kinase selectivity assay: Thiazovivin (10 μM) was screened against a panel of kinases (PKA, PKC, MLCK, ERK1/2) using respective substrate peptides and assay buffers. No significant inhibition (>50% activity reduction) was observed for off-target kinases [1]
Cell Assay	Human PSC survival and self-renewal assay: PSCs were seeded in 96-well plates at 5×10³ cells/well and treated with Thiazovivin (0.1-5 μM) for 72 hours. Apoptosis was detected by Annexin V-FITC/PI staining. Stemness markers (Oct4, Sox2, Nanog) were analyzed by qPCR and immunofluorescence. Clone formation assay was performed by seeding 1×10³ cells/well in 6-well plates and culturing for 10 days, followed by crystal violet staining [1] - Cattle EDSC stemness maintenance assay: Cattle EDSCs were seeded in 6-well plates at 1×10⁴ cells/well and cultured in chemically defined medium with Thiazovivin (0.1-2 μM) for 14 days. Oct4 expression was detected by immunocytochemistry and qPCR. Differentiation markers (α-SMA, Nestin) were analyzed by Western blot. Clone formation rate was quantified by counting stained colonies [2] - MLC phosphorylation assay: PSCs were treated with Thiazovivin (1 μM) for 24 hours. Total protein was extracted, and phosphorylated MLC (Ser19) and total MLC were detected by Western blot [1]
Animal Protocol
References	[1]. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc Natl Acad Sci U S A. 2010 May 4;107(18):8129-34. [2]. Thiazovivin, a Rho kinase inhibitor, improves stemness maintenance of embryo-derived stem-like cells under chemically defined culture conditions in cattle. Anim Reprod Sci. 2015 Oct;161:47-57.
Additional Infomation	Thiazovivin is a potent, selective small-molecule inhibitor of Rho-associated coiled kinase (ROCK) [1][2] - Its mechanism of action involves binding to the ATP-binding pocket of ROCK1/ROCK2, inhibiting kinase activity and blocking ROCK-mediated MLC phosphorylation, thereby preventing actin cytoskeleton rearrangement and apoptosis in stem cells [1] - It is widely used as a tool compound to maintain the survival, self-renewal, and stemness of pluripotent stem cells and embryo-derived stem-like cells under in vitro culture conditions [1][2] - Thiazovivin supports stem cell maintenance without inducing spontaneous differentiation, making it valuable for stem cell research and regenerative medicine applications [2]

Solubility Data

Solubility (In Vitro)

DMSO: 15 mg/mL (48.2 mM) Water: 14 mg/mL (43.71 mM) Ethanol:<1 mg/mL(slightly soluble or insoluble)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.03 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (8.03 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. Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol:30 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.2117 mL	16.0586 mL	32.1172 mL
	5 mM	0.6423 mL	3.2117 mL	6.4234 mL
	10 mM	0.3212 mL	1.6059 mL	3.2117 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.