James J. Schmidt

Endoproteinase Activity of Type A Botulinum Neurotoxin: Substrate Requirements and Activation by Serum Albumin

Type A botulinum neurotoxin, a zinc-dependent endoproteinase that selectively cleaves the neuronal protein SNAP-25, can also cleave relatively short peptides. We found that bovine and other serum albumins stimulated the type A-catalyzed hydrolysis of synthetic peptide substrates, through a direct effect on the kinetic constants of the reaction. Furthermore, with bovine serum albumin in the assays, the optimum substrate size was 16 residues (11 on the amino-terminal side of the cleavage site and 5 on the carboxy-terminal side). To further investigate the catalytic requirements of the neurotoxin, peptides were synthesized with various amino acid substitutions at the P5 through P5′ substrate sites. Changes at all of these locations affected values for both kcat and Km. Substitutions at the P2, P1′, and P2′ sites had more pronounced effects on hydrolysis rates than did substitutions at the P1 site. Enzyme–substrate interactions at the P3′ threonine probably involved the side-chain methyl group rather than the hydroxyl group. Replacing the P2′ alanine with leucine eliminated detectable hydrolysis, but not binding, since this peptide was an inhibitor. A negatively charged residue was preferred at P5, but not at P4. The data indicate that type A botulinum neurotoxin has an extended substrate recognition region and a requirement for arginine as the P1′ residue.

Identification of small molecule inhibitors of anthrax lethal factor

The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death. We report here the identification of small molecule (nonpeptidic) inhibitors of LF. Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds. Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository. Three of these compounds have Ki values in the 0.5–5 μM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.

Proteolysis of synthetic peptides by type A botulinum neurotoxin.

Type A botulinum neurotoxin catalyzed the hydrolysis of synthetic peptides based on the sequence of the 25-kD synaptosomal protein SNAP-25. In each peptide, the toxin cleaved at a single glutaminyl-arginine bond corresponding to residues 197 and 198 of SNAP-25, confirming earlier reports on the enzymatic specificity of the toxin in synaptosomal preparations. Metal chelators inhibited catalysis, consistent with a metalloprotease activity. In contrast to tetanus toxin and other botulinum toxin serotypes, type A toxin hydrolyzed relatively short, 17-to 20-residue peptides. In the substrates, SNAP-25 residue 202 and one or more of residues 187–191 were required for efficient hydrolysis, but residues 167–186 and 203–206 were not. The highest rates of hydrolysis were found when the C-terminal residues of the peptides were amidated.

Fluorigenic substrates for the protease activities of botulinum neurotoxins, serotypes A, B, and F

ABSTRACT The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P1 and P3′ residues were substituted with 2,4-dinitrophenyl-lysine and S-(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a Ki value of 4 μM. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.

Inhibition of Metalloprotease Botulinum Serotype A from a Pseudo-peptide Binding Mode to a Small Molecule That Is Active in Primary Neurons

An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (Ki = 330 nm) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institutes Open Repository. Four of the inhibitors possess Ki values ranging from 3.0 to 10.0 μm. Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomal-associated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 μm.

Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1' binding subsite.

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP‐25. Single‐residue changes in a 17‐residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2′ or P3′ cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 cysteine were not cleaved and were inhibitors. Peptides with either d‐ or l‐cysteine as the N‐terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a K i value of 2 μM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active‐site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P1′ inhibitor residue, demonstrating that the S1′ binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.

A high-affinity competitive inhibitor of type A botulinum neurotoxin protease activity

The peptide N‐acetyl‐CRATKML‐amide is an effective inhibitor of type A botulinum neurotoxin (BoNT A) protease activity [Schmidt et al., FEBS Lett. 435 (1998) 61–64]. To improve inhibitor binding, the peptide was modified by replacing cysteine with other sulfhydryl‐containing compounds. Ten peptides were synthesized. One peptide adapted the structure of captopril to the binding requirements of BoNT A, but it was a weak inhibitor, suggesting that angiotensin‐converting enzyme is not a good model for BoNT A inhibitor development. However, replacing cysteine with 2‐mercapto‐3‐phenylpropionyl yielded a peptide with K i of 330 nM, the best inhibitor of BoNT A protease activity reported to date. Additional modifications of the inhibitor revealed structural elements important for binding and supported our earlier findings that, with the exception of P1′ arginine, subsites on BoNT A are not highly specific for particular amino acid side chains.

Computer-Aided Lead Optimization: Improved Small-Molecule Inhibitor of the Zinc Endopeptidase of Botulinum Neurotoxin Serotype A

Optimization of a serotype-selective, small-molecule inhibitor of botulinum neurotoxin serotype A (BoNTA) endopeptidase is a formidable challenge because the enzyme-substrate interface is unusually large and the endopeptidase itself is a large, zinc-binding protein with a complex fold that is difficult to simulate computationally. We conducted multiple molecular dynamics simulations of the endopeptidase in complex with a previously described inhibitor (K i app of 7±2.4 µM) using the cationic dummy atom approach. Based on our computational results, we hypothesized that introducing a hydroxyl group to the inhibitor could improve its potency. Synthesis and testing of the hydroxyl-containing analog as a BoNTA endopeptidase inhibitor showed a twofold improvement in inhibitory potency (K i app of 3.8±0.8 µM) with a relatively small increase in molecular weight (16 Da). The results offer an improved template for further optimization of BoNTA endopeptidase inhibitors and demonstrate the effectiveness of the cationic dummy atom approach in the design and optimization of zinc protease inhibitors.

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins. Toxicon 35, 1193-1203, 1997.-Two sets of proteins have been purified from the venom of the Brazilian coral snake, Micrurus frontalis frontalis. One set has mol. wts, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in the 8000-13,000 range and includes some proteins which are toxic to mice and others which are not. These proteins appear to be isoforms of postsynaptic toxins. The other set shows phospholipase A2 (PLA2) activity and the toxic members of this set promote hemorrhage in mice in a manner closely resembling that produced by PLA2s isolated from the venom of the Australian tiger snake (Notechis scutatus scutatus). These PLA2s migrate on SDS-PAGE with apparent mol. wts in the 18,000-22,000 range which is characteristic of PLA2s that have an alpha-helix D similar to pancreatic PLA2s. Elapid venom PLA2s of the type which typically migrate on SDS-PAGE with mol. wts in the 13,000-16,000 range and do not have alpha-helix D have not been detected in M. f. frontalis venom.

Characterization and amino acid sequences of two lethal peptides isolated from venom of Wagler's pit viper, Trimeresurus wagleri.

Two lethal toxins were isolated from Trimeresurus wagleri venom by fast protein liquid chromatography (molecular sieve) and high performance liquid chromatography (reverse phase). The toxins (termed peptide I and II) had mol. wt of 2504 and 2530, respectively, pIs of 9.6-9.9 and lacked phospholipase A, proteolytic, and hemolytic activity. Lethal peptide I had a murine i.p. LD50 of 0.369 mg/kg, while lethal II had a murine i.p. LD50 of 0.583 mg/kg. Peptide I retained full toxicity after autoclaving at 121 degrees C for 40 min. The lethal activity was found to represent less than 1% of the total venom protein, which was only 62-65% of crude venom. The amino acid sequence of peptide I revealed a proline-rich (over 30% of total sequence) sequence unique among snake venom toxins. Lethal peptide II showed the same sequence except for a second tyrosine in the position of histidine (residue No. 10) in peptide I. The toxin lacked antigenic identity with a number of representative neurotoxins and myotoxins. The crude venom shared at least one antigen with Crotalus scutulatus scutulatus venom. This antigen was not Mojave toxin. The toxin appears symptomatologically suggestive of a vasoactive peptide or neurotoxin.