Screening inhibitory peptides against sphingomyelinase D from the livestock tick Rhipicephalus microplus using phage display technology

Abstract

The cattle tick Rhipicephalus microplus, which is distributed worldwide, causes significant economic loss to the livestock industry. A sphingomyelinase (SMase) D-like protein was previously identified in R. microplus saliva (RmSMase) and characterized in terms of its cofactors, optimum pH, and products generated from sphingomyelin hydrolysis. SMases are present in the venom of spiders from Loxosceles spp. and in the digestive system of non-venomous spiders from Uloborus spp. and are secreted by pathogenic bacteria. In this study A peptide library was screened using Phage Display technology and two out of the three peptides synthesized showed inhibitory activity against RmSMase with sphingomyelin as the substrate. Two of the three synthesized peptides showed inhibitory activity against RmSMase. Docking analysis suggested that the peptides block access of the substrate to the catalytic site. The RWLWWLW peptide showed competitive behavior (Ki = 5.35 ± 0.46 μM), whereas WLSWLW showed competitive inhibition behavior (Ki = 29.54 ± 6.11 μM). Both RWLWWLW and WLSWLW also inhibited the activity of SMase C from the bacterial pathogen Bacillus cereus, presenting IC₅₀ values of 4.99 ± 0.07 and 16.94 ± 8.71 μM, respectively. Because SMases have been suggested to be involved in the development of Alzheimer's disease, major depression, and cerebral ischemia, the discovery and characterization of new SMase inhibitors may contribute to the development of alternative treatments. This is the first study to identify peptide inhibitors against tick SMase using phage display technology.