Seetharaman, J.

Abstract Background: Kisspeptins (Kp) are a group of hypothalamic peptides that aid in regulating pubertal development and sex hormone signaling. In our recent studies, we observed the important role of Kp-10 (Kp peptide) and its receptor KISS1R signaling in regulating the airway smooth muscle (ASM) structure and function. In these studies, we observed significantly reduced expression of KISS1R during asthma pathophysiology suggesting a loss of protective mechanism and exogeneous Kp-10 may be protective in the context of asthma. Studies have shown that specific modifications to the amino acids of Kp peptides can result in increased binding efficiency and receptor activation. Accordingly, we developed three novel Kp peptides, named KpSATs (13A, 13B, and 13C), and tested the potential effects/efficiency on ASM cell remodeling. Methods: The Molecular docking of the KpSAT peptides (13A, 13B and 13C) with the receptor KISS1R were performed using HPEPDOCK. KpSATs were synthesized by GenScript (New Jersey, US) with purity of ≥97%. Primary human ASM cells isolated from surgical lung resections (Mayo Clinic IRB-approved) were cultured in DMEM-F12. Cells were treated with PDGF (2ng/mL) and/or TGF-β (2ng/mL) with/without treatments of KpSATs (13A, 13B, and 13C: 250nM-1µM) and Kp-10 (1µM). ASM cell proliferation was measured using MTT. Extracellular matrix (ECM) deposition (fibronectin and collagen-I/III) studies were performed using In-Cell Western (LiCor-odyssey). The mechanistic signaling pathway (phospho-PKC-δ) were measured using Western blot analysis. Results: In the novel KpSATs, we substituted three amino acids in the endogenous Kp-13 sequence, Y10W and P12T for KpSAT-13A; S6A and P12T for KpSAT-13B; and S6A and Y10W for KpSAT-13C. The peptides dock in the orthosteric ligand binding pocket attained with docking scores ranging from -250 to -280. The MTT assay indicated a reduction in PDGF-induced ASM cell proliferation upon exposure to KpSATs. Notably, we found that KpSAT-13A and KpSAT-13B had significant effects in reducing proliferation at lower concentrations of 500nM compared to 1µM of Kp-10. Furthermore, ECM deposition studies showed a significant reduction of TGF-β1-induced collagen-I/III and fibronectin in ASM cells exposed to KpSAT-13A and 13B. In addition, we observed significant inhibition of PDGF-induced phospho-PKC-δ by KpSAT-13A and 13B. Conclusion: Overall, these findings highlight that modification in amino acid sequence in endogenous Kp-13 sequence significantly increases the potency of synthesized novel KpSATs on ASM cells. In addition, KpSATs showed better inhibitory effects with a lower concentration compared to Kp-10 and could be considered as a promising therapeutic strategy to mitigate asthma.