Buforin I, a natural peptide, inhibits botulinum neurotoxin B activity in vitro

Abstract

Botulinum neurotoxin B (BoNT/B) serotype specifically cleaves between the amino acids glutamine and phenylalanine (Q and F bond) in position 76–77 of synaptobrevin (VAMP2). We evaluated peptides that contain the QF cleavage site but are not identical in primary structure to the VAMP2 sequence surrounding the QF site for both inhibition of BoNT/B proteolytic activity and as substrates for BoNT/B. A reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was used to measure digested peptides. A dose as high as 600 μM of substance P, and 11‐amino acid peptide containing the QF bond, was neither a substrate nor inhibitor of BoNT/B in our assay, suggesting that more than the QF bond is required to be recognized by BoNT/B. Buforin I (B‐I, QF site 24–25) is 39 amino acids in length, and sequence comparison of B‐I and VAMP2 indicated a similarity of 18% for conserved amino acids around the QF site. Furthermore, computer‐aided secondary structure computations predict α‐helical structures flanking the QF site for VAMP2 and for the upstream sequence of B‐I. Although predictions for the downstream sequence give nearly equal tendencies for α‐helical and β‐sheet structures, Yi et al. showed that the downstream sequence is likely to be the α‐helix based on their examination of buforin II (B‐II, a 21‐amino acid subset of B‐I (16–36)), which includes the QF site and the downstream sequence of B‐I. Buforin I was found not to be a substrate for BoNT/B; however, B‐I dose dependently and competitively inhibited BoNT/B activity, yielding IC50 = 1 × 10−6 M. In contrast, B‐II was not a substrate for BoNT/B and exhibited only 25% of the B‐I inhibition of BoNT/B. Two additional B‐I deletion peptides were tested for inhibition of BoNT/B proteolysis: peptide 36 (36 mer; containing B‐I amino acids 1–36) and peptide 24 (24 mer; B‐I amino acids 16–39). Peptide 24 had a similar inhibitory effect to B‐II (ca. 25% of B‐I) but peptide 36 was almost 50% as potent as B‐I. These findings suggest that the buforin tertiary structure is important for the inhibitory activity of these peptides for BoNT/B.

The evolving field of biodefence: therapeutic developments and diagnostics

“…However, two non-peptidic inhibitors have been described 46,47 (TABLE 6). With regard to pseudopeptides, phosphoramidon and three of its synthetic derivatives were found to be weak inhibitors 48 , whereas buforin I has also shown activity against the BoNT/B LC 49 . Recently, a Cys-containing peptide inhibitor was also reported 50 .…”

Inhibition of the Protease Activity of the Light Chain of Type A Botulinum Neurotoxin by Aqueous Extract from Stinging Nettle (Urtica dioica) Leaf

“…Because of availability of reagents, research has mostly been focused on finding inhibitors of the protease activity. As a result, peptide inhibitors (Schmidt et al 1998;Garcia et al 1999;Schmidt & Stafford 2002) and small molecule inhibitors (Burnett et al 2003) of type A and B light chain proteolytic activities have been reported.…”

Antimicrobial Peptides: New Recognition Molecules for Detecting Botulinum Toxins

“…On the other hand, cecropin A had the highest affinity for labeled toxoid E; its K d (10 -10 M) was at least ten-fold lower than all others measured and was due to both significantly higher k on and lower k off rates when compared to other AMPs and the other toxins (P < 0.01). These K d s were significantly lower than the IC 50 s measured for buforin-II and botulinum toxin B in inhibition assays [ 24 , 25 ], as well as the inhibition or Michaelis constants of other peptide substrate analogs and inhibitors of botulinum neurotoxins [ 36 - 40 ]. However, a number of peptide inhibitors with nanomolar dissociation constants have recently been developed [ 41 ].…”

“…It is widely held that the natural mechanistic targets for the microbiocidal activity of many AMPs are cellular membranous structures; a large body of work describes the theory and practical aspects of AMP-membrane interactions [ 19 - 23 ]. It was therefore surprising when Garcia and colleagues found that the AMP buforin-I ( Table 1 ) inhibited the protease activity of botulinum neurotoxin B in solution [ 24 ]. In large part because of Garcia's original observations and subsequent work [ 25 , 26 ], we attempted to assess the potential of a number of other AMPs unrelated to buforins to detect inactivated botulinum toxins A, B, and E in rapid assays.…”