Fluorescent peptomer substrates for differential degradation by metalloproteases

Abstract

Proteases, especially MMPs, are attractive biomarkers given their central role in both physiological and pathological processes. Distinguishing MMP activity with degradable substrates, however, is a difficult task due to overlapping substrate specificity profiles. Here, we developed a system of peptomers (peptide-peptoid hybrids) to probe the impact of non-natural residues on MMP specificity for a MMP peptide consensus sequence. Peptoids are non-natural, N-substituted glycines with a large side chain diversity. Given the presence of a hallmark proline residue in the P3 position of MMP consensus sequences, we hypothesized that peptoids may offer N-substituted alternatives to generate differential interactions with MMPs. To investigate this hypothesis, peptomer substrates were exposed to five different MMPs, as well as bacterial collagenase, and monitored by fluorescence resonance energy transfer and liquid chromatography-mass spectrometry to determine the rate of cleavage and the composition of degraded fragments, respectively. We found that peptoid residues are well-tolerated in the P3 and P3' substrate sites and that the identity of the peptoid in these sites displays moderate influence on the rate of cleavage. However, peptoid residues were even better tolerated in the P1 substrate site where activity was more strongly correlated with sidechain identity than sidechain position. All MMPs explored demonstrated similar trends in specificity for the peptomers but exhibited different degrees of variability in proteolytic rate. These kinetic profiles served as "fingerprints" for the proteases and yielded separation by multivariate data analysis. To further demonstrate practical application of this tunability in degradation kinetics, peptomer substrates were tethered into hydrogels and released over distinct timescales. Overall, this work represents a significant step toward the design of probes that maximize differential MMP behavior and presents design rules to tune degradation kinetics with peptoid substitutions, which has promising implications for diagnostic and prognostic applications using array-based sensors.

Microbial proteases and their applications

“…More sensitive methods are needed when the quantity or activity of protease enzymes are low, and sensitive fluorescent peptide substrates are available, through which the limit of detection reaches the ng level (Austin et al, 2022).…”

“…However, when detection is performed using a single fluorescently labeled protease substrate, the product and substrate must be separated, and the pH needs to be adjusted to enhance the detection signal. The detection steps remain relatively complex (Twining, 1984;Austin et al, 2022).…”

Oxidative degradation of sequence-defined peptoid oligomers

“…The fact that the N-substitution in LPro did not have this effect suggests that the NAla residue specifically alters trypsin degradation behavior. Studies have explored oligomers with non-natural residues to leverage alternative backbone interactions and side-chains that change recognition by proteases, [46][47][48] which could be a reason for this altered cleavage behavior.…”