Enhanced cleavage of diaminopimelate-containing isopeptides by leucine aminopeptidase and matrix metalloproteinases in tumors: application to bioadhesive peptides.
We prepared (2S,6S)-Z-Dpm(Z)(OMe) (4) by protease-mediated hydrolysis of (R,R/S,S)-Z-Dpm(Z)(OMe)-OMe (3), converted it to (2S,6S)-Dpm(Z)(OMe) (6) via PCI5 to an NCA intermediate and hydrolysis, protected the amino group with Boc to give (2S,6S)-Boc-Dpm(Z)(OMe) (7), which upon ammonolysis of the Me ester afforded (2S,6S)-Boc-Dpm(Z)(NH2) (8). Hydrogenolysis of 8 and protection with Fmoc gave (2S,6S)-Boc-Dpm(Fmoc)(NH2)(10). Using 10 and SPPS, we prepared three Dpm-containing peptides and their corresponding Lys peptides. Enzymatic studies with mLAP and cLAP showed that the Leu moiety in Ac-Gly-(2S,6S)-Dpm(Leu)(NH2)-Ala (14) was hydrolyzed 68-fold and >1000-fold more rapidly, respectively, than that in Ac-Gly-Lys(Leu)-Ala (12). The enhanced rate of Leu formation from 14 compared to 12 was also observed with homogenates of mouse C3 sarcomas. This homogenate also hydrolyzed Ac-Gly-(2S,6S)-Dpm(Ac-Gly-Pro-Gln-Gly-Leu)(NH2)-Ala (16) to Ac-Gly-(2S,6S)-Dpm(NH2)-Ala (13), Leu and Ac-Gly-Pro-Gln-Gly (17). This implies the side chain is cleaved first by endopeptidases, such as matrix metalloproteinases (MMPs), and then the remaining Leu is cleaved by LAP-like exopeptidases. The rate of liberation of 17 from 16 and the corresponding Lys isopeptide, Ac-Gly-Lys(Ac-Gly-Pro-Gln-Gly-Leu)-Ala (15), was not significantly different. The rate of formation of 13 was faster from 16 than Ac-Gly-Lys-Ala (11) was from 15. Thus, the entire isopeptide side chain can be removed by the cooperative action of LAP-like and MMP-like peptidases present in tumor tissue, which occurs faster in the Dpm peptide 16 than in the Lys peptide 15. The rate of formation of 13 from 16 by lung, liver, and intestine homogenates (from the same C3 tumor-bearing mice) was comparable to or higher than from the tumor homogenates, but the rate by blood was only 4% the value of the tumor homogenates. Analogs of a bioadhesive fragment from the laminin alpha1 chain were prepared by replacing the essential Lys with Dpm(NH2) (20) and Dpm(Leu)(NH2) (21). Both Dpm-containing peptides were active, although considerably weaker than the corresponding Lys peptides 18 and 19, in a cell attachment assay with human fibrosarcoma HT-1080 cells.
Yamazaki
Y
Y
Department of Pharmaceuticals Sciences, School of Pharmacy, University of Pittsburgh, PA 15261, USA.
Savva
M
M
Kleinman
H K
HK
Oka
S
S
Mokotoff
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D003960:Diaminopimelic Acid / Q000737:chemistry*; D015195:Drug Design; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005354:Fibrosarcoma / Q000188:drug therapy / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects; D007797:Laminin / Q000737:chemistry / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D007797:Laminin; D010455:Peptides; C084098:laminin A; D003960:Diaminopimelic Acid; D007931:Leucyl Aminopeptidase; D008666:Metalloendopeptidases