Building Blocks of 2,3-Diaminopropionic Acid Functionalized with an Oxa-Acid Spacer Shift the Functional Profile of Human Cathelicidin LL-37: Enhanced Enzymatic Stability and Selective Suppression of Neutrophil Degranulation.

In vitro synthesis and biological characterization of six DAPEG-modified LL-37 analogs

“Here we report the synthesis and biological characterization of six DAPEG-modified analogs of LL-37 in which (i) all five Arg residues (positions 7, 19, 23, 29, 34), (ii) all six Lys residues (positions 8, 10, 12, 15, 18, 25) or (iii) all three Ile residues (positions 13, 20, 24) were substituted with Dap derivatives bearing either a one-unit or a two-unit oxa-spacer.”

Six DAPEG-modified LL-37 analogs, native LL-37, reference bacterial strains, and differentiated HL-60 cells

“Six LL-37 analogs were synthesized using Fmoc/tBu solid-phase peptide synthesis, replacing all five arginine, all six lysine, or three isoleucine residues with Dap bearing a one- or two-unit oxa-acid spacer.”

Six analogs tested with three biological replicates per condition

“n = 3 biological replicates with technical duplicates per condition.”

Assays conducted over time points ranging from 1 hour to 24 hours

“Aliquots were taken at 0, 1, 2, 4, 6 and 24 h and analyzed using liquid chromatography–mass spectrometry (LC-MS; Bruker HCT Ultra ion trap; Phenomenex Gemini-NX 5µ C18, 4.6 × 150 mm; 10–90% acetonitrile/0.1% formic acid over 50 min).”

Arginine-substituted Dap(GO1) analog showed 5.7-fold increased proteolytic stability against PR3

“Proteolytic stability against PR3 increased 5.7-fold for the arginine-substituted Dap(GO1) analog, and both arginine analogs resisted PAD2- and PAD4-mediated citrullination, with Dap(GO2) being the most resistant.”

All six analogs lost direct antibacterial activity

“All six analogs lost direct antibacterial activity and were non-cytotoxic at 1–10 µM.”

Lysine-substituted analogs suppressed LPS-induced degranulation of PR3 and MPO

“In differentiated HL-60 cells, the lysine-substituted Dap(O1) and Dap(O2) analogs suppressed lipopolysaccharide-induced secretion of both PR3 and myeloperoxidase from azurophilic granules, whereas isoleucine analogs were suppressed partially and arginine analogs behaved like native LL-37.”

Proteolytic stability tested only with isolated PR3; intracellular mechanisms not assayed; no validation in primary neutrophils

“The intracellular mechanism (TLR4/FPR2 engagement, cytokine signaling) was not assayed.”