Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease.
Botulinum neurotoxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X-ray crystal structures suggest that the active sites of the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate-induced conformational changes for enzyme activation, we synthesized a series of transition-state analog inhibitors in which the dipeptide cleavage site is replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. In this paper, we report our efforts to design inhibitors of BoNT/A metalloprotease. We confirm that an effective substrate sequence for BoNT/A metalloprotease is a 17-mer peptide corresponding to residues 187-203 of SNAP-25. A more stable substrate, Nle202SNAP-25 [187-203] was synthesized in order to develop an assay for proteolytic activity of BoNT/A metalloprotease that can be used to monitor time-dependent inhibition. Alpha-thiol amide analogs of Gln-197 were incorporated via solid-phase peptide synthesis into both 17-mer minimal peptide substrate sequences. The synthesis, characterization and inhibition kinetics for the alpha-thiol amide analogs of holotoxin A substrate are described. These substrate-derived inhibitors were shown to be submicromolar inhibitors of BoNT/A catalytic activity.
School of Pharmacy, University of Wisconsin-Madison, 777 Highland Ave, Madison, WI 53705, USA.
C
C
Sukonpan
T
T
Oost
M
M
Goodnough
W
W
Tepp
E A
EA
Johnson
D H
DH
Rich
D000595:Amino Acid Sequence; D000603:Amino Acids, Sulfur / Q000737:chemistry; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020134:Catalytic Domain; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011480:Protease Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D050683:R-SNARE Proteins; D013379:Substrate Specificity
D000603:Amino Acids, Sulfur; D008565:Membrane Proteins; D010455:Peptides; D011480:Protease Inhibitors; D050683:R-SNARE Proteins; D045726:Metalloproteases; D019274:Botulinum Toxins, Type A