Chimeric enkephalin-cystatins: opioid binding and structure-activity relationships of inhibitory domains.
A chimeric inhibitor of cysteine proteinases, YGGFLQVVAGK.amide, was synthesized for use in examining SAR of its cystatin and opioid domains. Analogs were prepared by solid-phase syntheses and evaluated for inhibition of rat brain cathepsin L (E.C. 3.4.22.15) and papain (E.C.3.4.22.2), or binding to rat brain opioid receptors using tritiated DSLET (delta), DAGO (mu) and U69593 (kappa) in competitive binding assays. Cystatin consensus analogs QVVAGK- or N-Ac.QVVK.amide were weakly active as inhibitors, but were enhanced 10-fold to 20-fold with N-terminal L-, FL-, GFL-, GGFL-, or YGGFL, yielding Ki 3 microM and 26 microM for cathepsin L and papain, respectively. YGGFL itself was inactive, but expressed inhibition with N-terminal Q-, QV-, QVV-, etc. Highest activity was found with YGGFLQVK.amide (Ki, 2 microM). N-Dns analogs differentially increased inhibition for papain, whereas an N-Ac-cyclic peptide, N-Ac-CKYGGFLQVVKC.amide, was 2-fold to 10-fold less potent than the two-domain inhibitor. Chimeric peptides containing YGGFL were equipotent as delta-ligands (Ki, 1.7-3.7 nM), but were 2-fold to 10-fold more potent as mu ligands (3.7-11 nM) versus YGGFL itself (37 nM). Two analogs with -QVVAK- and -QVK.amide expressed kappa-binding properties. These data demonstrate the feasibility of using chimeric peptides as probes for exploring enzyme catalysis, and the potential for targeting inhibitors to endosomal or lysosomal compartments via receptor-mediated uptake in cells.
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Nathan S. Kline Institute, Center for Neurochemistry, Orangeburg, NY 10962.
Berg
M J
MJ
Makofske
R C
RC
Swistok
J
J
Simon
E J
EJ
Ofri
D
D
Del Compare
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K
Danho
W
W
D000595:Amino Acid Sequence; D000818:Animals; D015891:Cystatins / Q000737:chemistry / Q000378:metabolism*; D004723:Endorphins / Q000378:metabolism*; D004745:Enkephalins / Q000737:chemistry / Q000378:metabolism*; D004798:Enzymes / Q000032:analysis; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D015891:Cystatins; D004723:Endorphins; D004745:Enkephalins; D004798:Enzymes; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins