Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II.
MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity.
Avrutina
Olga
O
Institute for Organic and Biomolecular Chemistry, Georg-August-University, D-37077 Göttingen, Germany.
Schmoldt
Hans-Ulrich
HU
Gabrijelcic-Geiger
Dusica
D
Le Nguyen
Dung
D
Sommerhoff
Christian P
CP
Diederichsen
Ulf
U
Kolmar
Harald
H
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D002864:Chromogranins / Q000737:chemistry; D019662:Cucurbitaceae / Q000201:enzymology*; D003500:Cyclization; D047169:Cyclotides / Q000138:chemical synthesis* / Q000494:pharmacology; D008239:Lysine / Q000737:chemistry; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism*
D002864:Chromogranins; D047169:Cyclotides; C046560:beta-granins; C409923:trypsin inhibitor MCoTI-II; D001120:Arginine; D014357:Trypsin; D008239:Lysine; D000409:Alanine