Studies of Thioamide Effects on Serine Protease Activity Enable Two-Site Stabilization of Cancer Imaging Peptides

Abstract

Thioamide substitutions in peptides can be used as fluorescence quenchers in protease sensors and as stabilizing modifications of hormone analogs. To guide these applications in the context of serine proteases, we here examine the cleavage of several model substrates, scanning a thioamide between the P3 and P3′ positions, and identify perturbing positions for thioamide substitution. While all serine proteases tested were affected by P1 thioamidation, certain proteases were also significantly affected by other thioamide positions. We demonstrate how these findings can be applied by harnessing the combined P3/P1 effect of a single thioamide on kallikrein proteolysis to protect two key positions in a neuropeptide Y-based imaging probe, increasing its serum half-life to >24 h while maintaining potency for binding to Y1 receptor expressing cells. Such stabilized peptide probes could find application in imaging cell populations in animal models or even in clinical applications such as fluorescence-guided surgery.

Light-Emitting Probes for Labeling Peptides

“…Fluorescent quenchers, such as thioamides, can be used to track and reduce proteolytic degradation, thus stabilizing and increasing the in vivo serum half-life of peptides. 37 , 38 Cysteine protease can be tracked through the synthesis of thioamide-labeled peptides. 39 When the peptide is cleaved, the thioamide group stops quenching and fluorescence can be observed.…”

Rational design of thioamide peptides as selective inhibitors of cysteine protease cathepsin L

“…We have previously observed similar multiple-site stabilization with thioamide substrates of serine proteases, which we were able to exploit to stabilize cancer cell imaging peptides at two positions with a single thioamide modication. 22 Overall, having a thioamide at the P1 position here rendered the K S 3A and R S 3A peptides completely resistant to proteolysis by Cts V, Cts K, and Cts S, while signicantly slowing the rate of proteolysis by Cts L (Table 1). The only exception to this P1 thioamide effect was with Cts B, where the K S 3A and R S 3A peptides were cleaved at the penultimate C-terminal alanine residue as indicated by the slashes -mHLFK S AA/Am or mHLFR S AA/Am (Tables S7, S9 and Fig.…”

“…21 Motivated by these results showing thioamide stabilization effects at P2 and P1 positions (positions numbered from the scissile bond by convention), our laboratory developed a uorescence sensor design to systematically study the positional effects of thioamide substitution against different cysteine proteases (papain, cathepsins L, V, K, B, and S) and serine proteases (trypsin, chymotrypsin, and kallikrein). [22][23][24] Intriguingly, we found that thioamide positional effects differ not only between serine proteases and cysteine proteases, but also between members of the same protease family despite their high homology (31-59% sequence identity) and mechanistic similarity. 22,24 We also successfully utilized data from these systematic studies to design a two-site stabilized thioamide peptide specically targeting neuropeptide Y 1receptor expressing MCF-7 breast cancer cells.…”

“…[22][23][24] Intriguingly, we found that thioamide positional effects differ not only between serine proteases and cysteine proteases, but also between members of the same protease family despite their high homology (31-59% sequence identity) and mechanistic similarity. 22,24 We also successfully utilized data from these systematic studies to design a two-site stabilized thioamide peptide specically targeting neuropeptide Y 1receptor expressing MCF-7 breast cancer cells. 22 With the experimental data from these systematic studies, we recently developed a Rosetta machine learning model that accurately classies positional effects of thioamides on proteolysis by these cysteine and serine proteases which can be used to rationally design stabilized peptides for therapeutic and imaging applications.…”

Side‐chain thioamides as fluorescence quenching probes

“…Recently, thioamides have been used in a variety of biochemical contexts, including conformational stabilization of peptide macrocycles, [6] conferring protease resistance to peptides, [7] and as fluorescence turn-on probes for proteases. [8][9][10] However, it has also been shown that thioamide substitution in the backbone can have negative effects on protein stability. [11] Additionally, positioning of thioamides near the scissile bond of protease substrates must be evaluated carefully.…”