(647e) Development of Proteolytically Selective Peptoid-Based Biomaterials

Sequence-controlled synthetic biomaterials offer a platform to mimic the bioactivity of natural matrices while engineering properties by design (e.g., enhanced biostability, reduced cross-reactivity, etc.). Peptoids, or N-substituted glycines, are peptidomimetics with sidechains affixed to the amide backbone nitrogen, rather than the α-carbon. Peptoids can be synthesized with full sequence definition using the submonomer technique, which affords incorporation of any sidechain available as a primary amine. Thus, peptoids provide a versatile non-natural platform for advancing biomaterials, but their functional diversity elicits investigation of their biological response, especially in conjunction with peptides. N-substitution enacts proteolytic resistance, increasing biostability versus their peptide counterparts and providing an approach to engineer the proteolytic susceptibility of a substrate. By generating peptide-peptoid hybrid (termed “peptomer”) sequences, the role of each residue can be individually explored and leveraged to uniquely direct biochemical recognition. In this work, we therefore used substitutions with non-natural peptoid residues to enhance specificity over classic amino acid sequences and develop substrates with programmed biodegradation.

Here, we targeted the matrix metalloproteinase (MMP) class of proteases. MMPs are enzymes predominantly responsible for remodeling of the extracellular matrix. The misregulation of distinct MMP types has been linked to the pathology of various diseases, but their efficacy as biomarkers is hindered by overlapping substrate specificity profiles. Beginning with a consensus sequence for collagenases (PAN↑LVA, where ↑ indicates the scissile site), we incorporated systematic peptoid substitutions to modulate substrate recognition and degradation rate. Our first-generation substrate library consisted of the native peptide sequence, six peptomers with a single peptoid substitution in each position, and one full peptoid sequence wherein each residue was converted to a peptoid analogue. The substrates were functionalized with a 7-methoxycoumarin-4-acetyl fluorophore and a 2,4-dinitrophenyl quencher, which enabled cleavage tracking in real time. In addition, the absorbance maximum of the dinitrophenyl group at 363 nm afforded identification and quantification of cleavage products by liquid chromatography and mass spectrometry. As a preliminary screening, substrates were incubated with collagenase type I from Clostridium histolyticum. Each substrate demonstrated a distinct response to the collagenase with variable cleavage rates and scissile sites, prompting experimentation with isolated human MMPs in the collagenase subfamily. Notably, we identified that N-substitution within the P1 site generates a substrate that is selectively cleaved by MMP-13 over other collagenases. We then generated a second library with expanded side chain functionalities in the P1 site to further optimize this sequence. Selectivity was further investigated using multiple types of isolated MMPs, as well as with cell-secreted MMP-13 in vitro. We believe this systematic approach is broadly applicable and will assist in understanding overall proteolysis specificity trends, identifying selective substrates beyond the collagenase MMPs, and functionalizing biomaterials with programmed degradation behavior.