G-protein-coupled receptor 54 (GPR54, also known as KISS1R). No IC50, Ki, EC50, or binding affinity values are provided in this manuscript. [1]
The primary target of Kisspeptin-10 is the KISS1 receptor (KISS1R), previously known as GPR54, AXOR12, or hOT7T175. KISS1R is a G protein-coupled receptor belonging to the rhodopsin family, widely distributed in tissues including the hypothalamus, pituitary, placenta, pancreas, testis, liver, and small intestine. Upon KP-10 binding, KISS1R primarily activates the phospholipase C pathway, leading to inositol trisphosphate (IP₃) accumulation and intracellular calcium mobilization, while also activating ERK1/2 and p38 mitogen-activated protein kinase signaling pathways. The receptor is also involved in G protein-coupled receptor signaling, cytoskeleton organization regulation, positive regulation of luteinizing hormone secretion, and MAPK cascade activation.

- Induction of osteogenic marker genes: In C3H10T1/2 cells, treatment with 5 μM or 50 μM KP-10 for 2 days significantly increased mRNA expression of osteogenic markers (Dlx5, Runx2, ALP, OC) in a dose-dependent manner. Treatment with 50 μM KP-10 for 1 or 2 days also increased expression in a time-dependent manner (p < 0.05, p < 0.01, p < 0.005 vs. control). [1]
- ALP activity: KP-10 (50 μM) treatment for 4 days in the presence of ascorbic acid and β-glycerophosphate significantly increased alkaline phosphatase (ALP) staining intensity in C3H10T1/2 cells. [1]
- Mineralization: KP-10 (50 μM) treatment for 20 days increased matrix mineralization as detected by Alizarin Red S staining in C3H10T1/2 cells. [1]
- BMP2 expression: KP-10 (5 or 50 μM) significantly increased BMP2 mRNA levels in a dose-dependent manner. KP-10 (50 μM for 1 or 2 days) increased BMP2 mRNA in a time-dependent manner. KP-10 (50 μM for 4 days) increased BMP2 protein levels as shown by Western blot. [1]
- BMP2 promoter activity: KP-10 (5 or 50 μM) significantly increased BMP2-luciferase reporter activity in a dose-dependent manner (p < 0.01, p < 0.005 vs. control). [1]
- Smad1/5/9 phosphorylation: KP-10 (50 μM) increased phosphorylation of Smad1/5/9 in a time-dependent manner (detected at 0.5, 1, 3, 6, 12, and 24 hours). [1]
- NFATc4 expression: KP-10 (50 μM for 2 days) significantly increased NFATc4 mRNA expression, while expression of NFATc1, NFATc2, and NFATc3 showed slight or no change. KP-10 (50 μM for 1 or 2 days) increased NFATc4 mRNA in a time-dependent manner. [1]
- NFATc4 overexpression: Overexpression of NFATc4 using pCMV-NFATc4 significantly increased BMP2 and osteogenic gene expression (Dlx5, Runx2, ALP, OC). NFATc4 also increased BMP2-luciferase activity with or without KP-10 co-treatment. NFATc4 overexpression increased matrix mineralization in wild-type but not in GPR54−/− cells. [1]
- NFATc4 knockdown: Transfection with siNFATc4 (siNFATc4II or siNFATc4III) suppressed KP-10-induced BMP2 expression (mRNA and promoter activity), confirming that KP-10 induces BMP2 via NFATc4. [1]
- GPR54 dependency: KP-10 treatment increased BMP2 protein expression in wild-type C3H10T1/2 cells but not in GPR54−/− cells. Runx2 expression was also induced by KP-10 in wild-type but not in GPR54−/− cells. [1]
- Autocrine effect of BMP2: Conditioned medium (C.M) from KP-10-treated wild-type cells (50 μM for 12 hours) was collected and added to GPR54−/− cells. This conditioned medium increased Dlx5 and Runx2 expression in GPR54−/− cells, whereas direct KP-10 treatment did not. This demonstrates that KP-10 induces secretion of BMP2, which then acts in an autocrine manner to promote osteogenic gene expression. [1]

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The receptor GPR54 and kisspeptin-10 (KP-10) play a crucial role in controlling the release of GnRH in humans and other mammals. GPR54 binds to the protein kisspeptin-10. Kisspeptin-10, a breast cancer cell metastasis inhibitor, is activated in human lung melanoma [1].

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In vitro studies demonstrate that Kisspeptin-10 significantly inhibits human umbilical vein endothelial cell migration, invasion, and tube formation, processes critical for angiogenesis. In HTR8/SVneo trophoblast cells treated with high glucose (mimicking gestational diabetes conditions), Gpr54 expression is downregulated with impaired glucose uptake and insulin resistance; Kp-10 treatment restores the cAMP/PKA signaling pathway and enhances glucose uptake by upregulating Glut-4, Insr, and Irs1 expression. Kp-10 also inhibits trophoblast invasion and migration (critical regulatory processes during embryo implantation) and suppresses the gelatinolytic activity of isolated trophoblasts. In KISS1R-transfected CHO cells, human Kp-10 exhibits an EC₅₀ value of approximately 1.54-2.6 × 10⁻⁸ M for Ca²⁺ response activation.

Kisspeptin-10 exhibits significant in vivo activity in various animal models. Intravenous injection of 1 μg/kg Kp-10 increases serum LH levels in healthy men from 4.1 ± 0.4 IU/L to 12.4 ± 1.7 IU/L within 30 minutes; continuous infusion at 1.5 μg/kg/h for 22.5 hours increases LH pulse frequency from 0.7 ± 0.1 to 1.0 ± 0.2 pulses/hour and pulse secretory mass from 3.9 ± 0.4 to 12.8 ± 2.6 IU/L. In a rat model of gestational diabetes mellitus, Kp-10 treatment (intravenous, once daily) improves fasting blood glucose levels, insulin sensitivity, and fetal outcomes, including increased fetal weight and decreased fetal blood glucose. In SCID mice xenografted with human prostate cancer cells (PC-3), Kp-10 inhibits tumor growth by suppressing tumor angiogenesis. In both chicken chorioallantoic membrane assays and VEGF-induced mouse corneal micropocket assays, Kp-10 significantly inhibits angiogenesis in vivo.

Cell-free receptor binding studies for Kisspeptin-10 primarily utilize radioligand binding assays. A typical protocol includes: 1) Prepare membrane suspensions expressing KISS1R (using native tissues or KISS1R-transfected cells); 2) Dissolve Kp-10 in binding buffer (containing 50 mM HEPES pH 7.4, 5 mM MgCl₂, 0.1% BSA) to prepare serial concentrations; 3) Add radiolabeled Kp-10 analog (e.g., [¹²⁵I]-Kp-10) and incubate for 60 minutes at room temperature; 4) Terminate the reaction by rapid vacuum filtration and wash filters with ice-cold buffer to remove unbound ligand; 5) Measure membrane-bound radioactivity using a gamma counter; 6) Calculate IC₅₀ and Kᵢ values from competition binding curves. Structure-activity relationship studies show that Kp-10 exhibits a helicoidal structure between residues Asn4 and Tyr10 (mixed α- and 3₁₀-helix characteristics), with alanine substitutions at positions 6 or 10 significantly reducing receptor affinity and functional activity.

- Cell culture: Mouse mesenchymal stem cell line C3H10T1/2 was maintained in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2. Osteoblast differentiation was induced by adding osteogenic medium containing 2% FBS, 50 μg/mL ascorbic acid, and 5 mM β-glycerophosphate, replaced every 2 days. KP-10 was used at 50 μM unless otherwise indicated. [1]
- RT-PCR and real-time PCR: Total RNA was isolated using TRIzol reagent. Reverse transcription was performed using 1 μg total RNA. PCR conditions: initial denaturation at 95°C for 5 min, followed by 30-35 cycles of denaturation at 95°C for 30 s, annealing at optimal temperature for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 5 min. Real-time PCR used a green mix with Hi-ROX. Primer sequences for β-actin, Dlx5, Runx2, ALP, OC, BMP2, NFATc1, NFATc2, NFATc3, and NFATc4 are provided. [1]
- Western blot analysis: Cells were harvested using a lysis kit, centrifuged at 12,000 g for 10 min at 4°C. Proteins were quantified by Bradford assay, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies (1:1000) against BMP2, β-actin, Smad, and phospho-Smad (p-Smad), then with secondary antibodies. Signals were detected using ECL reagent and a FUSION solo analyzer system. [1]
- Luciferase reporter assay: Cells were transfected with BMP2-luc (0.4 μg) for 6 hours, then treated with KP-10 (5 or 50 μM). For NFATc4 studies, cells were co-transfected with BMP2-luc and pCMV-NFATc4 (0.4 μg) or siNFATc4. Luciferase activity was measured. [1]
- ALP staining: Cells were cultured with ascorbic acid (50 μg/mL), β-glycerophosphate (5 mM), and KP-10 (50 μM) for 4 days. Cells were fixed with 10% formaldehyde, rinsed with deionized water, and treated with BCIP/NBT solution for 15 min. [1]
- Alizarin Red S staining: Cells were treated with KP-10 (50 μM) or transfected with pCMV-NFATc4 (0.4 μg) for 20 days, fixed with 4% formaldehyde for 5 min, washed, and stained with 300 μg/mL Alizarin Red S solution for 30 min at room temperature. [1]
- CRISPR/Cas9 generation of GPR54−/− cells: A Cas9-expressing plasmid and sgRNA plasmid targeting exon 1 of GPR54 were co-transfected into C3H10T1/2 cells using a nucleofector system at a 1:2 molecular weight ratio (Cas9:sgRNA). Single cell-derived colonies were selected. Mutant colonies were confirmed by PCR and T7 endonuclease 1 (T7E1) assay. Nested PCR amplified the GPR54 exon 1 region with specific primers. [1]
- siRNA transfection: Three NFATc4 siRNA primers (siNFATc4I, II, III) were designed. Cells were transfected with siNFATc4 for 6 hours, then treated with 50 μM KP-10 for 2 days. The siRNA sequences are provided in the manuscript. [1]
- Conditioned medium (C.M) experiment: Wild-type C3H10T1/2 cells were treated with 50 μM KP-10 for 12 hours, and the conditioned medium was collected. GPR54−/− cells were then treated with either 50 μM KP-10 or the conditioned medium for 2 days, and RT-PCR was performed to assess Dlx5 and Runx2 expression. [1]

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The in vitro cell assay protocol for Kisspeptin-10 is as follows: 1) Seed target cells (such as KISS1R-transfected CHO cells, HTR8/SVneo trophoblast cells, HUVEC endothelial cells, or primary trophoblasts) in culture plates and culture to appropriate density at 37°C with 5% CO₂; 2) Treat cells with various concentrations of Kp-10 (typically 10⁻¹¹ to 10⁻⁶ M); 3) For Ca²⁺ mobilization assays, label cells with fluorescent probes (e.g., Fluo-4 AM) and monitor fluorescence intensity changes in real-time after Kp-10 stimulation using a fluorescence microscope or plate reader; 4) For migration and invasion assays, use Transwell chambers (8 μm pore size), seed cells in the upper chamber with Kp-10-containing medium in the lower chamber, incubate, then stain and count cells that migrated through the membrane; 5) For tube formation assays, seed HUVECs on Matrigel-coated plates, add Kp-10, incubate for 6-18 hours, and observe and count tube-like structures under a microscope; 6) Detect expression and phosphorylation levels of relevant signaling pathway proteins by Western blot.

The in vivo animal assay protocol for Kisspeptin-10 is as follows: 1) Select appropriate animal models: SD rats (gestational diabetes model), SCID mice (tumor xenograft model), Beagle dogs (toxicology studies), or male monkeys (reproductive studies); 2) Group assignment: randomly divide animals into vehicle control and multiple Kp-10 dose groups (e.g., 30, 100, and 1000 μg/kg in dog toxicology studies); 3) Administration routes: intravenous injection, subcutaneous injection, or intracerebroventricular injection; dosing volume typically 2.6 mL/kg, once daily for 14 consecutive days; 4) Sample collection: collect blood samples at multiple time points post-administration (e.g., 5, 15, 30 minutes) and measure LH levels as a pharmacodynamic marker; 5) Behavioral testing: assess anxiety-like behavior and locomotor activity using open field test and novel tank test; 6) Tissue collection: euthanize animals at study termination and collect major organs (such as liver, kidney, spleen, brain, placenta) for histopathological examination; 7) Data acquisition: measure body weight, food consumption, body temperature, electrocardiogram, and respiratory rate.

Kisspeptin-10 exhibits extremely rapid pharmacokinetics in vivo. In in vitro stability studies, the decomposition half-life of Kp-10 in rat plasma is only 1.7 minutes at 37°C, 2.9 minutes at 25°C, and 6.8 minutes at 4°C; the primary decomposition product is the N-terminal tyrosine-deleted des-Tyr1-Kp-10. Following intravenous bolus administration of 1.0 mg/kg Kp-10, low ng/mL levels of Kp-10 are detectable in rat plasma only during the first few minutes, becoming undetectable by 30 minutes post-dose. In humans, the terminal half-life of Kp-10 after intravenous infusion is approximately 3.8-4.1 minutes. Following subcutaneous administration of radiolabeled Kp-10 analogs in dogs, approximately complete recovery of the administered radioactive dose is achieved within 48-72 hours, primarily excreted in urine after extensive metabolism. Due to the rapid clearance of the peptide in vivo, LH levels are commonly used as a surrogate marker for Kp-10 exposure in studies.

Kisspeptin-10 demonstrates a favorable safety profile in preclinical studies. In a 14-day repeat-dose GLP toxicology study in Beagle dogs, once-daily intravenous administration of KP-10 at dose levels of 30, 100, and 1000 μg/kg for 14 days revealed no overt signs of drug-related toxicity. No significant toxicity was observed in clinical signs, body weights, food consumption, clinical pathology, histopathology, urinalysis, electrocardiogram, or respiratory rate. The 1000 μg/kg dose was established as the No Observed Adverse Effect Level (NOAEL). The study also noted a 20-day rat TK study suggesting potential toxicity to skeletal development in juvenile animals, thus the safety of Kp-10 in juvenile animals requires further evaluation. In younger (4-week-old) rats, MCH (monomethylhistamine) levels were slightly increased in female animals. In human studies, no serious adverse events have been reported following Kp-10 administration, with common adverse reactions primarily being mild injection site reactions.

[1]. Kisspeptin-10 (KP-10) stimulates osteoblast differentiation through GPR54-mediated regulation of BMP2 expression and activation. Sci Rep. 2018 Feb 1;8(1):2134.

- Origin and structure: KP-10 is a 10-amino acid peptide (C-terminal region of kisspeptin) derived from the proteolytic cleavage of the 145-amino acid Kiss1 gene product. Other kisspeptins include KP-54, KP-14, and KP-13. [1]
- Biological functions (as background): KP-10 acts as a metastasis suppressor in melanoma and breast cancer. The KP-10/GPR54 system regulates pubertal development, and loss-of-function mutations cause hypogonadotropic hypogonadism. GPR54 also regulates BMP7 expression during embryonic kidney development. [1]
- Proposed mechanism in osteoblast differentiation (Figure 5): KP-10 binds to GPR54, leading to NFATc4-mediated BMP2 expression. Secreted BMP2 acts in an autocrine manner to phosphorylate Smad1/5/9, which then increases expression of osteogenic genes including Dlx5 and Runx2, ultimately promoting osteoblast differentiation and mineralization. [1]
- Novel finding: This is the first study to demonstrate that KP-10/GPR54 signaling induces osteoblast differentiation through NFATc4-mediated BMP2 expression and activation in C3H10T1/2 cells. [1]

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Kisspeptin-10 is being studied in the clinical trial NCT03771326 (KP-10 and male insulin secretion).

C63H83N17O14

1302.44

1301.63

374675-21-5

Kisspeptin-10, human TFA;Kisspeptin-10, rat;478507-53-8

25240297

Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH2
H-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH2
L-tyrosyl-L-asparagyl-L-tryptophyl-L-asparagyl-L-seryl-L-phenylalanyl-glycyl-L-leucyl-L-arginyl-L-phenylalaninamide

YNWNSFGLRF
YNWNSFGLRF-NH2

White to off-white solid powder

3.514

18

16

38

94

2590

9

CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N)NC(=O)CNC(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC3=CNC4=CC=CC=C43)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC5=CC=C(C=C5)O)N

RITKWYDZSSQNJI-INXYWQKQSA-N

InChI=1S/C63H83N17O14/c1-34(2)24-45(58(90)74-43(18-11-23-70-63(68)69)57(89)75-44(54(67)86)26-35-12-5-3-6-13-35)73-53(85)32-72-56(88)46(27-36-14-7-4-8-15-36)77-62(94)50(33-81)80-61(93)49(30-52(66)84)79-59(91)47(28-38-31-71-42-17-10-9-16-40(38)42)78-60(92)48(29-51(65)83)76-55(87)41(64)25-37-19-21-39(82)22-20-37/h3-10,12-17,19-22,31,34,41,43-50,71,81-82H,11,18,23-30,32-33,64H2,1-2H3,(H2,65,83)(H2,66,84)(H2,67,86)(H,72,88)(H,73,85)(H,74,90)(H,75,89)(H,76,87)(H,77,94)(H,78,92)(H,79,91)(H,80,93)(H4,68,69,70)/t41-,43-,44-,45-,46-,47-,48-,49-,50-/m0/s1

(2S)-N-[(2S)-1-[[(2S)-4-amino-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-1-[[(2S)-1-[[(2S)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]-2-[[(2S)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]butanediamide

Kisspeptin-10; 374675-21-5; Kisspeptin-10 (human); FS1N52VS3S; Human metastin 45-54;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O : ≥ 25 mg/mL (~19.19 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.)