The Botulinum J., Vol. 1, No. 3, 2009
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Structure-based design of peptide inhibitors of botulinum neurotoxin serotype A proteolytic activity Matthew L. Ludivico Department of Cell Biology and Biochemistry, Integrated Toxicology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, USA E-mail:
[email protected]
Leonard A. Smith Medical Countermeasures Technology, US Army Medical Research and Materiel Command, Fort Detrick, MD 21702-5011, USA Fax No. 301-619-2348 E-mail:
[email protected]
S. Ashraf Ahmed* Department of Cell Biology and Biochemistry, Integrated Toxicology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, USA Fax: (301)-619-2348 E-mail:
[email protected] *Corresponding author Abstract: P1’arginine of the substrate SNAP-25 is an absolute requirement for catalysis, and the active site of botulinum neurotoxin serotype A (BoNT/A) is populated by acidic residues. We reasoned that arginine derivatives, and basic peptides might bind to the proteolytic domain, and act as inhibitors. A systematic investigation of amino acids, derivatives and basic peptides provided proofs of our concept. D-arginine, arginine hydroxamate and basic peptides effectively inhibited BoNT/A activity. Optimisation of the peptide inhibitors resulted in a RRGC tetrapeptide displaying 90% inhibition at 20 microM. Our studies provide an inhibitor scaffold for potential development as a BoNT/A drug candidate. Keywords: botulinum neurotoxin catalytic domain; basic tetrapeptides; peptide inhibitors; protease inhibitors; tetrapeptide inhibitors; botulinum neurotoxin inhibitors; arginine hydroxamate. Reference to this paper should be made as follows: Ludivico, M.L., Smith, L.A. and Ahmed, S.A. (2009) ‘Structure-based design of peptide inhibitors of botulinum neurotoxin serotype A proteolytic activity’, The Botulinum J., Vol. 1, No. 3, pp.297–308.
Copyright © 2009 Inderscience Enterprises Ltd.
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M.L. Ludivico et al. Biographical notes: Matthew L. Ludivico received a BSc in Molecular Biology from Lehigh University, PA. He later joined the US Army, received training as a Medical Laboratory Technician, and was stationed at the US Army Medical Research Institute of Infectious Diseases (USAMRIID). For six years, he worked on structural and functional aspects of botulinum neurotoxin catalytic domain, and on development of inhibitors against it. Leonard A. Smith received his PhD in Biochemistry from Georgetown University School of Medicine. He is the Senior Research Scientist (ST) for Medical Countermeasures Technology at the US Army Research Institute of Infectious Diseases. His research efforts focus on the development of toxin therapeutics and toxin vaccine products. He currently is the Chairman of the NATO HFM-ET91 working group. S. Ashraf Ahmed is a Research Chemist at USAMRIID. He had his MSc from Dhaka University, Bangladesh, and a PhD in microbial biochemistry from Kyoto University, Japan. He extensively worked on structure and mechanism of enzymes of amino acid and oxygen metabolism and of botulinum neurotoxin. A Research Chemist, his current research is focused on therapeutic development against botulism. He has published over 70 peer reviewed scientific papers.
1
Introduction
Of all the natural and synthetic toxins, botulinum neurotoxins are the most lethal. There are seven immunologically distinct botulinum neurotoxins (BoNT), designated BoNT/A-G, produced by strains of Clostridium botulinum (see Montecucco and Schiavo, 1995; Schiavo et al., 1995 for a review). While they are potential military threats and bioterrorism agents (Arnon et al., 2001), these neurotoxins are also widely used as laboratory research tools (Steinhardt et al., 1994), as clinical drugs in a variety of neuromuscular disorders and in cosmetic applications (Verheyden et al., 2001; Brin et al., 1999). BoNTs are expressed by bacteria as 150-kDa single polypeptides. Post-translational cleavage by a trypsin-like protease generates a 50-kDa N-terminal Light Chain (LC) and a 100-kDa C-terminal Heavy Chain (HC) that are still connected by a disulfide bond. The 100-kDa HC can further be proteolysed into a 50-kDa N-terminal membrane spanning domain (Hn) and a 50-kDa C-terminal receptor-binding domain (Hc). The LC possesses the toxic, zinc endopeptidase catalytic domain, but in the absence of HC, it is non-toxic. Because of its potential bio-weapon and bio-terrorism use, these neurotoxins are targets of inhibitor and drug design. Since the full-length BoNT is highly toxic but its separated catalytic LC moiety is not, we expressed BoNT/A LC in Eschericia coli and purified it to homogeneity from inclusion bodies (Ahmed and Smith, 2000) or soluble extracts (Ahmed et al., 2001; Jensen et al., 2003) as stable and highly active in vitro and in vivo (Alderton et al., 2000), and then characterised them further (Ahmed et al., 2003). In characterising a recombinant BoNT/A LC, we demonstrated earlier that organic and inorganic mercury compounds such as p-chloro-mercuribenzoate and mercuric chloride completely inhibited (Ki 90% by (Quality Controlled Biochemicals, Hampton, MS).
3
Methods
3.1 Protein concentration To determine protein concentration and to assess purity, UV-visible absorption spectra were recorded at 22°C with a Hewlett-Packard 8452 diode array spectrophotometer. LC concentration was determined using A0.1% (1 cm light path) value of 1.0 at 278 nm or by BCA assay (Pierce) with Bovine Serum Albumin (BSA) as standard. Both methods give the same result (Ahmed et al., 2001).
3.2 Enzymatic activity assays The enzymatic assay was based on HPLC separation and measurement of the cleaved products from a 17-residue C-terminal peptide corresponding to residue #187-203 of SNAP-25 (Ahmed and Smith, 2000; Schmidt and Bostian, 1995). A master reaction mixture lacking the LC was made and aliquots were stored at –20°C. At the time of assay, an aliquot of the master mix was thawed and 25 µl was added to 5 µl of the LC (see above) to initiate the enzymatic reaction. Components and final concentration in this 30 µl reaction mixture were 0.9 mM substrate peptide, 0.25 mM ZnCl2, 5 mM dithiothreitol, and 50 mM Na-HEPES, pH 7.4. Amino acids, their derivatives and peptides were added to the reaction mixtures at the indicated concentrations before starting the reaction by adding the substrate.
4
Results
4.1 Amino acids We measured the BoNT/A LC activity using a 17-residue synthetic peptide substrate (Schmidt and Bostian, 1997) encompassing the glutamine-arginine cleavage site of SNAP-25 in the presence of all natural L-amino acids and their D-enantiomers. Neither L-glutamine nor L-arginine, the two residues N- and C-terminal, respectively, to the BoNT/A cleavage site, was an inhibitor (Table 1). Although L-arginine was not an inhibitor, D-arginine inhibited the reaction by 50%. We have no explanation why D-arginine is an inhibitor and not L-arginine, but find an analogy with a peptide having D-cysteine as a better inhibitor than the one having L-cysteine in the sequence CRATKML (Schmidt and Stafford, 2002). Except for D- and L-cysteine, L-serine, and D-glutamate, inhibition by other L-amino acids was insignificant. Combining the results with L- and D-amino acids, the following generalisation of amino acids as inhibitors of BoNT/A protease activity was made: D-arginine > D-glutamate = D-cysteine > D-tryptophan > L-serine = D-aspartate = L-cysteine. Inhibitory action of cysteine is most likely due to its interaction with the active-site zinc (Ahmed and Smith, 2000; Schmidt and Stafford, 2002).
Structure-based design of peptide inhibitors Table 1
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Effects of L- and D-amino acids on the catalytic activity of BoNT/A LC Percent activity
Amino acid
L-
Control
100
100
Arginine
90
50
D-
Lysine
105
110
Histidine
115
120
Proline
105
100
Hydroxyproline
125
n.d.
Alanine
120
115
Valine
118
n.d.
Leucine
120
110
Isoleucine
120
135
Norleucine
115
n.d.
Methionine1
105
115
Glycine
120
Cysteine
80
70
Cystine2
n.d.
90
Serine
80
115
100
100
1003
1304
90
145
Tryptophan
125
75
Aspartate
120
80
Glutamate
120
70
Asparagine
95
105
Glutamine
95
130
Threonine Tyrosine Phenylalanine
1
Each amino acid (1 mM) was incubated at 37°C for 5 min in a 30 µl reaction mixture containing 1 mM substrate peptide, 0.375 µg of LC and 50 mM HEPES pH 7.4. The control 100% activity represents a specific activity of 3.8 µmol/min/mg. Results represent average of three assays that was rounded to the nearest 5. 1 0.5 mM; 20.2 mM; 30.1 mM; 40.3 mM.
4.2 Arginine derivatives and hydroxamates Because arginine was shown to be an essential residue in the BoNT/A substrate (Schmidt and Bostian, 1997), and D-arginine displayed significant inhibition, we investigated several commercially available arginine and guanido derivatives in order to identify more potent inhibitors (Table 2). Among all the compounds tested, hydroxamates of both enantiomers of arginine were equally effective and most potent inhibitors. Although a derivative of dipeptide hydroxamate was reported to be ineffective as a BoNT inhibitor (Deshpande et al., 1995), hydroxamates are generally known as
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metalloprotease inhibitors (Butler et al., 2004). We measured the activity of BoNT/A LC as a function of substrate concentration at several fixed concentrations of L-arginine hydroxamate (Figure 1(a)). With the limitation of sub-Km concentration of substrate employed in this experiment, the reciprocal data points in the Linewever-Burke plot appeared to intersect on the y-axis suggesting a competitive inhibition. Replot of the apparent Km calculated from Figure 1(a) as a function of L-hydroxamate concentration (Figure 1(b)) was linear yielding an average a Ki of 0.16 mM. Lack of significant effects of hydroxamates of lysine, aspartate, glutamate or nicotinic acid (Table 2) suggest that inhibition by arginine hydroxamate is specific for BoNT/A LC. Figure 1
(a) Double reciprocal plots of reaction rate vs. substrate concentration at fixed L-arginine hydroxamate concentrations of 0. mM (open circle), 0.083 mM (closed circle), 0.167 mM (open triangle), and 0.33 mM (closed triangle) and (b) the apparent Km values calculated from (a) are plotted against L-arginine hydroxamate concentration. An average Ki of 0.16 mM was calculated from the relation Ki = (Km*[inhibitor]/(apparent Km – Km)
(a) Table 2
(b)
Effects of arginine and its derivatives on BoNT/A LC activity
Arginine derivative
% Activity
Control
100
L-Arginine
105
D-Arginine
50
L-Argininamide
110
L-Arginine ethyl ester
80
L-arginine p-nitroanilide
95
L-arginino succinate
80
L-arginine 7-amido-4 methyl
100
L-Arginine N-phosphate
100
L-arginine hydroxamate N-α-benzoyl DL-arginine
20 1
40 100
N-α-acetyl DL-arginine 1
80 Nω−Nitro D-arginine Me-ester Final concentration of each compound was 1 mM in the assay mixture. The control 100% activity represents a specific activity of 3.8 µmol/min/mg. Results represent average of three assays that was rounded to nearest 5. 1 0.1 mM.
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Because inhibition by D-arginine and hydroxamates of D- or L-arginine but not by D-lysine or other hydroxamates (Tables 1 and 3) may suggest a specific inhibition of BoNT/A LC activity, we measured the activity of BoNT/B LC in the presence of these compounds. Our results showed that neither enantiomer of arginine and lysine nor arginine hydroxamate was an inhibitor of BoNT/B activity. This result also suggested that although the overall structures are very similar (Swaminathan and Eswaramoorthy, 2000), the active sites of BoNT/A and BoNT/B LCs are different enough to show this discrepancy. Arphamenine A and arphamenine B are extremely potent inhibitors of arginine aminopeptidase having very low (2.5–0.84 nM) Ki (Harbeson and Rich, 1988). Because both arginine aminopeptidase and BoNT/A LC share arginine (in the P1 and P1′ positions, respectively) in the cleavable bond, we tested the effects of arphamenine A & B on the activity of BoNT/A LC: at 4.5 mM concentration, they decreased the activity by 25–35% only. This result may serve as an indirect proof that inhibition of BoNT/A LC activity by D-arginine and hydroxamates of L- and D-arginine is specific. Moreover, arginine aminopeptidase is not known to be inhibited by L- or D-arginine. Table 3
Effects of hydroxamates on BoNT/A LC activity
Hydroxamate Control L-arginine hydroxamate* D-arginine hydroxamate* L-lysine hydroxamate
% Activity 100 20 20 110
D-lysine hydroxamate
140
DL-aspartate hydroxamate
100
D-aspartate β-hydroxamate
90
L-glutamate γ-hydroxamate
100
L-Tryptophan hydroxamate
90
DL-phenylalanine hydroxamate DL-tryptophan hydroxamate L-tyrosine hydroxamate
105 90 65
DL-methionine hydroxamate
110
Nicotinic acid hydroxamate
75
Final concentration of each compound was 1.0 mM in the assay mixture. The control 100% activity represents a specific activity of 3.8 µmol/min/mg. Results represent an average of three assays that was rounded to nearest 5. *0.1 mM.
We also investigated many commercially available guanidine derivatives and sulfur containing compounds (Table 4) as probable inhibitors of BoNT/A LC activity, and compared their effects with that of L-arginine hydroxamate and cysteine, respectively. None of the compounds tested were better than L-arginine hydroxamate as inhibitors. Cysteine is an inhibitor (Table 1) that acts as a chelator of zinc at the active site of BoNT/A LC (Ahmed and Smith, 2000; Schmidt and Stafford, 2002). The small protein (∼60 amino acids), metallothionein, containing 20 cysteine residues is well characterised physiological metal chelators including zinc (Chen and Song, 2009).
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Metal free form of this protein was also ineffective as an inhibitor (Table 4). All these results thus proved our prediction that arginine or its derivative(s) should act as inhibitor(s) of BoNT/A LC activity. Indeed, recent X-ray crystallographic studies provided structural proof of the inhibition showing that arginine hydroxamate binds at the active site of BoNT/A LC with its hydroxamate chelating the active site zinc (Fu et al., 2006; Silvaggi et al., 2007). Table 4
Guanidino derivatives and sulphur containing compounds
Compound
% Activity
Control
100
L-Arginine hydroxamate1 Nγ-aminoflavinate L-arginine
20 2
100
α-Guanidino glutarate
105
α-Guanidino butyrate
45
4-Guanidino benzoate
2
Guanidino succinate
60 75
L-Citruline
185
D-Citruline
185
Guanidine thiocyanate2
80
D-Cysteine
70
Thioglycollate
70
Thiazolidine COOH2
140
Metallothionein1
75
δ-Amino levulinic acid
50
Final concentration of each compound was 1 mM in the assay mixture. The control 100% activity represents a specific activity of 3.4 µmol/min/mg. Results represent average of three assays that was rounded to nearest 5. 1 0.2 mM; 20.1 mM.
4.3 Protamine sulphate and basic peptides Protamine sulphate is a small protein of 5100 Da that is often used in precipitating nucleic acids from cell extracts. When used at 20 µM concentration with standard BoNT/A LC assay mixtures, it inhibited 85% of the activity. We next tried two basic peptides, one GRKKRRQRRRPPQC, is derived from HIV-tat (Albini et al., 1998), and the other a polymer of arginine. As shown in Table 5, both inhibited the activity remarkably. These basic peptides might bind at the acidic access route to the active site restricting substrate access. Because these peptides contain arginine residues, they might also inhibit by interacting with the active site residues normally occupied by P1’ arginine of the substrate. Figure 2 shows that inhibition of BoNT/A LC activity by the GRKKRRQRRRPPQC peptide is biphasic probably suggesting a high affinity and a low affinity binding of the inhibitor to the protein.
Structure-based design of peptide inhibitors Table 5
305
Basic protein and peptides
Peptide
% Activity
Control
100
Protamine sulfate
15
GRKKRRQRRRPPQC-amide
10
RRRRRRRRGC-amide
35
RKRKRKRKGC-amide
60
dRKdRKdRKdRKGC-amide
45
dRdKdRdKdRdKdRdKGC-amide
35
RRGC-amide
10
Final concentration of each peptide was 20 µM in the assay mixture. Results represent an average of five assays that was rounded to nearest 5. Figure 2
Inhibition of BoNT/A LC activity by the peptide GRKKRRQRRRPPQC at a substrate concentration of 0.9 mM
Because the GRKKRRQRRRPPQC peptide containing both arginine and lysine residues was a better inhibitor than the peptide with eight arginine residues, we modified the latter peptide with alternating arginine and lysine residues both as D- and L-enatiomers. However, none of these modifications yielded any inhibitor better than the HIV-tat peptide. We also varied the length of the eight arginine containing peptide (not shown), and found that a basic peptide containing two arginine residues was as effective as the GRKKRRQRRRPPQC peptide. All of the peptides listed in Table 5 are highly soluble in aqueous buffers. The RRGC tetrapeptide competitively inhibits BoNT/A LC with a Ki of 157 nM (Kumaran et al., 2008b). The sequence of this peptide suggested that it could mimic the corresponding sequence of the substrate, QRAT, and might inhibit the BoNT/A LC reaction by competing with the substrate. Indeed, our recent high resolution 3-dimensional structure showed that RRGC was bound at the active site of BoNT/A LC that would normally be expected to be occupied by the substrate sequence (Kumaran et al., 2008). Moreover, the peptide was bound with extensive polar interactions with the protein residues.
5
Discussions
The enzymatic assay in this study used a very dependable quantitative method on a 17-mer SNAP-25 derived substrate (Schmidt and Bostian, 1995) instead of the
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full length SNAP-25. Therefore, some of the inhibitors may or may not be behave the same way on these two substrates. As potential drugs, peptides often exhibit highly desirable physical and pharmacological characteristics, such as good aqueous solubility, low toxicity, and high specificity for the targeted disease agent (Ayoub and Scheidegger, 2006; Marx, 2005). They are easily synthesised by conventional techniques, readily soluble in aqueous buffers, and are target-specific. Our results provide a proof of the concept that arginine derivatives and basic peptides should act as inhibitors of BoNT/A LC activity. In addition, low Ki of the tetrapeptide RRGC make it one of the highest affinity inhibitor thus far demonstrated for BoNT/A. Thus, this tetrapeptide or any of its variants (Kumaran et al., 2008b) will be a good candidate for drug development. Poor oral bioavailability of peptides may be a concern in their use as drugs (Hamman et al., 2005). In such a situation, the structural coordinates of the optimised tetrapeptide inhibitor should provide a starting point to design high affinity small molecule inhibitors.
Acknowledgement and disclaimer The research described herein was supported by JSTO-CBD Project number 3.10012_06_RD_B. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army.
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