Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation.
The deuterohemin-peptide conjugate, DhHP-6 (Dh-β-AHTVEK-NH(2)), is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species (ROS). In this study, specific multi-site N-methylated derivatives of DhHP-6 were designed and synthesized to improve metabolic stability and intestinal absorption, which are important factors for oral delivery of therapeutic peptides and proteins. The DhHP-6 derivatives were tested for (1) scavenging potential of hydrogen peroxide (H(2)O(2)); (2) permeability across Caco-2 cell monolayers and everted gut sacs; and (3) enzymatic stability in serum and intestinal homogenate. The results indicated that the activities of the DhHP-6 derivatives were not influenced by N-methylation, and that tri-N-methylation of DhHP-6 could significantly increase intestinal flux, resulting in a two- to threefold higher apparent permeability coefficient. In addition, molecules with N-methylation at selected sites (e.g., Glu residue) showed high resistance against proteolytic degradation in both diluted serum and intestinal preparation, with 50- to 140-fold higher half-life values. These findings suggest that the DhHP-6 derivatives with appropriate N-methylation could retain activity levels equivalent to that of the parent peptide, while showing enhanced intestinal permeability and stability against enzymatic degradation. The tri-N-methylated peptide Dh-β-AH(Me)T(Me)V(Me)EK-NH(2) derived from this study may be developed as a promising candidate for oral administration.
National Engineering Laboratory for AIDS Vaccine, College of Life Science, Jilin University, Qianjin Street 2699, Changchun, 130012, Jilin, People's Republic of China.
Qing-Guang
QG
Dong
Yong
Y
Zhang
Meng-Shu
MS
Wang
Jiao
J
Feng
Hai-Hong
HH
Zhang
Yong-Ge
YG
Wu
Tie-Jun
TJ
Gu
Xiang-Hui
XH
Yu
Chun-Lai
CL
Jiang
Yan
Y
Chen
Wei
W
Li
Wei
W
Kong
D000818:Animals; D018938:Caco-2 Cells; D004795:Enzyme Stability; D006427:Hemin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000737:chemistry / Q000378:metabolism*; D008745:Methylation; D051379:Mice; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010539:Permeability; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D009842:Oligopeptides; C552681:deuterohemin-alanyl-histidyl-threonyl-valyl-glutamyl-lysine; D006427:Hemin; D009195:Peroxidase