| Autor: |
Ilyas H; Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India., van der Plas MJA; Department of Pharmacy, University of Copenhagen, DK-2100 Copenhagen, Denmark.; Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, SE-221 84 Lund, Sweden., Agnoletti M; Department of Pharmacy, University of Copenhagen, DK-2100 Copenhagen, Denmark., Kumar S; Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India., Mandal AK; Division of Molecular Medicine, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India., Atreya HS; NMR Research Center, Indian Institute of Science, Bangalore 560012, India., Bhunia A; Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India., Malmsten M; Department of Pharmacy, University of Copenhagen, DK-2100 Copenhagen, Denmark.; Department of Physical Chemistry 1, University of Lund, SE-22100 Lund, Sweden. |
| Abstrakt: |
Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups ( n = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini. |
