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.

Background/

Objectives

The human cathelicidin LL-37 (37 residues; net charge +6) couples broad-spectrum antimicrobial activity with immunomodulatory function, but rapid proteolysis and peptidylarginine deiminase (PAD)-mediated citrullination limit its therapeutic use. We aimed to enhance enzymatic stability and to test whether substitution with 2,3-diaminopropionic acid (Dap) building blocks bearing an oxa-acid spacer (DAPEG) shifts the functional profile of the LL-37 scaffold.

Methods

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. Identity, purity and secondary structure were assessed via mass spectrometry, ultra-performance liquid chromatography and circular dichroism. Proteolytic stability against human proteinase 3 (PR3), resistance to PAD2/PAD4 citrullination, DNA binding (dynamic light scattering), antibacterial activity, MTT cytotoxicity, and lipopolysaccharide-induced degranulation (PR3 and myeloperoxidase) in differentiated HL-60 cells were evaluated.

Results

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. DNA binding was qualitatively retained, although dynamic light scattering showed weaker complex compaction for lysine analogs. All six analogs lost direct antibacterial activity and were non-cytotoxic at 1-10 µM. 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.

Conclusions

The DAPEG framework shifts LL-37 from microbicidal toward degranulation-suppressive activity, providing a modular route to tune cathelicidin function. The intracellular mechanism (TLR4/FPR2 engagement, cytokine signaling) was not assayed.