VSPPLTLGQLLS is a small peptide FGFR3 inhibitor, a peptide P3, which can inhibit the phosphorylation of FGFR3. VSPPLTLGQLLS inhibits 9-cisRA-induced tracheal lymphangiogenesis and blocks lymphatic endothelial cell (LEC) proliferation, migration, and tube formation.

Physicochemical Properties

Molecular Formula	C56H97N13O17
Molecular Weight	1224.44629454613
Exact Mass	1223.712
CAS #	1206896-24-3
Related CAS #	VSPPLTLGQLLS TFA
PubChem CID	165437304
Appearance	White to off-white solid powder
LogP	-3.2
Hydrogen Bond Donor Count	15
Hydrogen Bond Acceptor Count	18
Rotatable Bond Count	36
Heavy Atom Count	86
Complexity	2370
Defined Atom Stereocenter Count	12
SMILES	C(N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)O)([C@@H]1CCCN1C(=O)[C@H](CO)NC(=O)[C@@H](N)C(C)C)=O
InChi Key	TUBMLSKMKPKVBN-KYMYZPJVSA-N
InChi Code	InChI=1S/C56H97N13O17/c1-27(2)20-34(46(75)59-24-43(74)60-33(16-17-42(57)73)47(76)61-35(21-28(3)4)48(77)62-36(22-29(5)6)49(78)66-39(26-71)56(85)86)64-53(82)45(32(11)72)67-50(79)37(23-30(7)8)63-51(80)40-14-12-18-68(40)55(84)41-15-13-19-69(41)54(83)38(25-70)65-52(81)44(58)31(9)10/h27-41,44-45,70-72H,12-26,58H2,1-11H3,(H2,57,73)(H,59,75)(H,60,74)(H,61,76)(H,62,77)(H,63,80)(H,64,82)(H,65,81)(H,66,78)(H,67,79)(H,85,86)/t32-,33+,34+,35+,36+,37+,38+,39+,40+,41+,44+,45+/m1/s1
Chemical Name	(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-1-[(2S)-1-[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-3-hydroxypropanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-5-oxopentanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxypropanoic acid
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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
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	FGFR3 K650M-FGFR3 K644E FGFR3
ln Vitro	Human primary lymphatic endothelial cell (LEC) proliferation, migration, and tubule formation are inhibited by VSPPLTLGQLLS (5 μM and 10 μM; 24 h and 48 h)[1]. In addition to being successfully shown to inhibit FGFR3 phosphorylation in LECs, VSPPLTLGQLLS has also been shown to be effective in explanted metatarsal bone cultures, 293T cells, ATDC5 chondrogenic cells, and an in vivo mouse model of thanatophoric dysplasia II[1][2]. Tyrosine kinase activity of FGFR3 and its typical downstream molecules, extracellular signal-regulated kinase/mitogen-activated protein kinase, are inhibited by VSPPLTLGQLLS (10 μM; 6 h)[2]. Additionally, VSPPLTLGQLLS (0, 1, 10, and 50 μM; 24 hours and 3 or 7 days, respectively) stimulates the growth and chondrogenic differentiation of ATDC5 chondrogenic cells that have been cultured[2]. The ERK/MAPK pathway is inhibited in the FGFR3-expressing chondrocytic cell line ATDC5 by VSPPLTLGQLLS (10 μM; 0-60 min)[2].
ln Vivo	VSPPLTLGQLLS (1 mM; intranasal dropping; onced daily for 7 d) blocks 9-cisRA-induced lymphangiogenesis in vivo, while 9-cisRA is an isoform of vitamin A involving in AIDS-related Kaposi Sarcoma[1]. VSPPLTLGQLLS alleviates the bone growth retardation in bone rudiments from mice mimicking human thanatophoric dysplasia type II (TDII), reversed the neonatal lethality of TDII mice[2].
Cell Assay	Western Blot Analysis[1][2] Cell Types: Human primary lymphatic endothelial cell (LEC) s; ATDC5 chondrogenic cells Tested Concentrations: 10 μM Incubation Duration: 0, 5, 10, 30, 45, 60 min Experimental Results: Inhibited FGFR3 phosphorylation at Tyr 724 in human primary lymphatic endothelial cells (LEC)s. Inhibited the FGF2-mediated ERK/ MAPK phosphorylation in FGFR3-expressing chondrocytic cell line ATDC5. Cell Proliferation Assay[1] Cell Types: Human primary lymphatic endothelial cell (LEC)s Tested Concentrations: 2.5 μM, 5 μM, and 10 μM Incubation Duration: 15 min for pre-incubation and co-incubation with 1 μM 9-cisRA for 48 hr Experimental Results: Inhibited LEC proliferation. Cell Migration Assay [1] Cell Types: Human primary lymphatic endothelial cells (LEC)s Tested Concentrations: 5 μM Incubation Duration: 15 min for pre-incubation and co-incubation with 1 μM 9-cisRA for 24 hr; observed at 0, 24, 48 hr Experimental Results: Inhibited LEC migration tubule formation.
Animal Protocol	Animal/Disease Models: Lymphatic reporter mice, Prox1-GFP model[1] Doses: 1 mM Route of Administration: Intranasal dropping; one time/day for 7 days; accompanied with 1 mM 9-cisRA or not Experimental Results: Dramatically inhibited total lymphatic vessel length and number of sprouts compared increase induced by 9-cisRA. Animal/Disease Models: Fgfr3Neo-K644E/+EIIa-Cre mice (TDII mice ) from Fgfr3Neo-K644E/+ mice crossed with heterozygous EIIa-Cre mice[2] Doses: 10 μM Route of Administration: Treated for 7 days Experimental Results: Suppressed FGFR3-mediated growth inhibition in cultured murine metatarsal bones. Rescued the lethal phenotype in thanatophoric dysplasia type II (TDII) mice. Rescued the abnormal growth plate and the lung phenotypes in the TDII mice.
References	[1]. Small Peptide Modulation of Fibroblast Growth Factor Receptor 3-Dependent Postnatal Lymphangiogenesis. Lymphat Res Biol. 2019 Feb;17(1):19-29. [2]. A novel FGFR3-binding peptide inhibits FGFR3 signaling and reverses the lethal phenotype of mice mimicking human thanatophoric dysplasia. Hum Mol Genet. 2012 Dec 15;21(26):5443-55.

Solubility Data

Solubility (In Vitro)

H2O: 50 mg/mL (40.83 mM)

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	0.8167 mL	4.0835 mL	8.1669 mL
	5 mM	0.1633 mL	0.8167 mL	1.6334 mL
	10 mM	0.0817 mL	0.4083 mL	0.8167 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.