# Patent

## Title
Methods for identifying inhibitors of botulinum neurotoxins

## Abstract

A system and method for identifying a botulinum neurotoxin inhibitor employing a botulinum neurotoxin substrate complex having a peptide substrate, preferably SNAP-25, a reporter domain on one side of said peptide substrate and an immobilization domain on the opposite side of said peptide substrate. The botulinum neurotoxin inhibitor is identified by its ability to decrease the relative amount of cleaved complex, detected through measuring a decrease in complex bound to a solid support. The method of the present invention also utilizes novel cells that express a botulinum neurotoxin substrate complex. The methods of the present invention are adapted for cell based screening to monitor the catalytic activity of a BoNT in living cells and to identify molecules that inhibit the catalytic activity of a BoNT in living cells. Also provided are novel stable cell lines that express the botulinum toxin substrate complex and viral vectors capable of efficiently expressing an active light chain of the BoNT within mammalian cells.

## Background
PRIORITY
This application is continuation of PCT Application No. PCT/US2003/030899 filed Oct. 1, 2003, which claims the benefit of U.S. Provisional Application No. 60/415,177 filed Oct. 1, 2002, both incorporated herein by reference in their entirety.


BACKGROUND
1. Field of Invention
This invention relates to a method for identifying inhibitors of botulinum neurotoxins.
2. Background of the Invention
Botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise two polypeptide chains connected via a disulfide linkage. The light chain ( ̃50 kDa) is disulfide linked to a heavy chain ( ̃100 kDa). The anaerobic bacterium Clostridium botulinum produces seven immunologically distinct but structurally similar neurotoxins designated BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F and BoNT/G (collectively, “BoNTs”). After synthesis, highly active neurotoxin is generated by proteolytic cleavage of the clostridial neurotoxins.
These neurotoxins inhibit neurotransmitter release at distinct synapses, which causes two severe neuroparalytic diseases, tetanus and botulism. Many aspects of the cellular and molecular modes of action of these toxins have been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Internalization of toxin is a rapid event and the toxin shows persistent catalytic activity within neurons. Subsequently, the light chain of the neurotoxin is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: (1) synaptosomal associated protein of 25 kDa (SNAP-25); (2) synaptobrevin, also called vesicle associated membrane protein (VAMP); and (3) syntaxin. Specifically, BoNT/A, BoNT/E and BoNT/C cleave SNAP-25; BoNT/C also cleaves syntaxin. BoNT/B, BoNT/D, BoNT/F, BoNT/G cleave synaptobrevin/VAMP. Tetanus neurotoxin cleaves synaptobrevin/VAMP, at the same cleavage site as BoNT/B. See, Schmidt J J, et al., supra; Anne C, et al., Anal Biochem (2001 291:253-61).
The location of the enzymatic subunit of the clostridial neurotoxins has been mapped to the light chain, which has Zn endopeptidase activity. The binding and translocation motifs in a BoNT are located within the heavy (H) chain. All of the BoNT serotypes bind to receptors/acceptors on the presynaptic terminals of motor neurons at the neuromuscular junction. Schiavo G, et al., (1993) FEBS Lett 335:99-103. The binding of the BoNT to the presynaptic terminal is mediated by the C-terminal domain of the heavy chain (HC) of the toxin. Schiavo G, et al., J Biol Chem (1993) 268: 23784-7 and Schiavo G, et al., Nature (1992) 359: 832-5. Binding is followed by endocytosis of the toxin into vesicles at the presynaptic terminal. As the endocytotic vesicle is acidified, the N-terminus of the HC forms a pore in the vesicle membrane. The light chain (LC) disassociates from HC to act as a zinc-dependent protease that cleaves and inactivates SNARE proteins essential for exocytosis of neurotransmitter. Arnon S S, et al., JAMA 2001, 285:1059-70. In the case of BoNT/A (the most potent and persistent of the BoNTs) the substrate is SNAP-25, a SNARE protein which resides on the cytoplasmic surface of the presynaptic membrane. See, Foran P, et al., Biochemistry (1996) 35:2630-6; Lewis J, et al., Nat Med (1999) 5:832-5; and Schmidt J J, et al., Anal Biochem (2001) 296:130-7.
The botulinum neurotoxin cleaves the substrate proteins at highly specific sites. BoNT/A cleaves SNAP-25 at residues 197/198 (amino acids QR). See, Foran P, et al., Biochemistry (1996) 35:2630-6; and Lewis J, et al., (1999) supra. BoNT/E cleaves SNAP-25 at residues 180/181 (amino acids RI).
The unique specificities of BoNT/A and BoNT/E for SNAP-25 was suggested to be directed through the recognition of a nine residue sequence, termed the SNARE motif. The SNARE motif is about 50 amino acids in length and assumes a coiled confirmation. The SNARE motif in SNAP-25 is common to the other two SNARE proteins: VAMP and syntaxin. SNAP-25, VAMP and syntaxin are the only known substrates of the seven clostridial neurotoxins. There are four copies of the SNARE motif present in SNAP-25. Studies on the interaction of SNAP-25 with BoNT/A and BoNT/E showed that a single copy of the motif is sufficient for BoNT/A and BoNT/E to recognize SNAP-25. Washbourne P et al., FEBS Lett. (1997) 418:1-5. The full kinetic activity of BoNT/A and BoNT/E for SNAP-25 requires at least one SNARE motif. Although the copy of the SNARE motif that is proximal to the SNAP-25 cleavage site is clearly involved in recognition with BoNT/A and BoNT/E, in its absence, other more distant copies of the motif are able to support proteolysis. Id.
The proteolytic attack at specific sites in the protein targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics. These alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release caused by the neurotoxins.
The botulinum neurotoxins (BoNTs) are some of the most potent and persistent toxins known and can be delivered by an oral or inhalation route. These properties have contributed to attempts by others to use BoNT as a bioweapon. No effective antidote for BoNT intoxication is available. Current therapy consists primarily of long term ventilator support, although early administration of hyperimmune antiserum within the first 12 hours can shorten the duration of paralysis. This therapy currently involves administration of horse serum derived antibodies with the risks of anaphylactic reaction. Human hyperimmune antiserum is used to treat infantile botulism. Human hyperimmune antiserum is too limited a source for use in a bioterrorism attack involving BoNT. Monoclonal IgG antitoxins are being pursued for BoNT therapy, but at least three different monoclonal antibodies are required to inhibit each of the serotypes of botulinum neurotoxin. The cost of producing an oligoclonal treatment consisting of 15-18 monoclonal antibodies is not commercially feasible.
Immunization is currently the major biodefense strategy against BoNT attacks. Although vaccination can clearly protect against the paralytic effects of the toxin, there are clear limitations to this strategy which include: 1) the need to vaccinate a large at risk population to prevent disease in even a small number of exposed individuals; 2) active vaccination must be accomplished well before exposure to the toxin; 3) strains of BoNT can be engineered for bioterrorism, that can evade immune defense or delivered by viral vector overcoming host immunity (See Fishman P S, et al., Nat Toxins 1999, 7:151-6), and; 4) vaccination will interfere with the potential future use of BoNT for medical conditions and deny the current standard of care to immunized patients. Oyler G A, et al., IBRCC (2001).
An alternative strategy to vaccination against BoNT is the development of a clinically useful antidote. Oyler G A, et al., Interagency Botulinum Research Coordinating Committee, 2001. This strategy opens a wide array of possibilities based on the understanding of the molecular pathogenesis of intoxication.
Methods to detect botulinum neurotoxin's catalytic activity have been based on detecting SNARE protein cleavage products in vitro. See, for example, Schmidt U.S. Pat. No. 5,965,699, the contents of which are hereby incorporated by reference in their entirety.
The blocking proteolytic activity of the catalytic light chain is a candidate for treatments to inhibit and terminate the action of the toxin. SNARE protein cleavage is a late event in intoxication.
Rapid replenishment of SNARE proteins normally occurs and could result in early restoration of neuromuscular synaptic function. Inhibitors that are able to reach the site of action in the cytosolic compartment of the pre-synaptic terminal of the neuromuscular junction (unprotected by the blood-brain/nerve-barrier) could decrease the neurotoxin's effect in infected individuals. There is a need for a method to identify a clinically relevant botulinum catalytic inhibitor that penetrates to the intracellular site of action of the toxin and is non-toxic to living cells. Therefore, a need exists for a method for screening inhibitors of botulinum neurotoxin type A (BoNT/A), to identify neurotoxin inhibitors that function both in vitro and in living cells. There is also a need for a method of screening inhibitors of botulinum neurotoxin type E (BoNT/E), type C (BoNT/C), type B (BoNT/B), type D (BoNT/D), type F (BoNT/F) and type G (BoNT/G) that can be used to identify neurotoxin inhibitors that function both in vitro and in living cells. In order to facilitate the identification and development of such botulinum toxin inhibitors, there is a need for a system to rapidly assess botulinum toxin catalytic activity.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a design of an in-vitro assay for BoNT catalytic activity. The in-vitro assay for BoNT activity based on separation of a reporter domain and immobilization domain upon cleavage of SNAP-25 by BoNT. Cleavage of YFP-SNAP-25-His×6 immobilized on metal ion resin by BoNT releases yellow fluorescent protein (YFP) into the supernatant, which can be monitored by YFP fluorescence.
FIG. 2 is comprised of FIGS. 2A, 2B, 2C, 2D, 2E, and 2F. Cleavage of YFP-SNAP-25-His×6 by BoNT/A and E, compared to a Control (C) is shown in FIG. 2E. GST-SNAP-25, GST-SNAP-25-His×6 and YFP-SNAP-25-His×6 were efficiently cleaved by BoNT/A and E in vitro (FIGS. 2A and 2D, respectively). GST-SNAP-25 (1-197) and GST-SNAP-25 (1-180) are recombinant proteins corresponding to the cleaved fragments from BoNT/A and E cleavage, respectively (FIGS. 2B and 2C). GST-SNAP-25 A/NC, which harbors a single point mutation (R198T) that renders it BoNT/A resistant, was cleaved only by BoNT/E in this assay (FIG. 2F).
FIGS. 3A and 3B show the results of the in-vitro assay for BoNT catalytic activity. YFP-SNAP-25-His×6 immobilized on Nickel resin were incubated with BoNT/A at 37° C. for 4 hours without agitation. The amount of YFP fluorescence released into the supernatant was monitored with a fluorescence plate reader. The assay was sensitive enough to detect 0.1 U/mL and 1.0 U/mL (1.0 ng/mL and 5.0 ng/mL) of BoNT/A.
FIG. 4 is comprised of FIGS. 4A and 4B. FIG. 4A shows the results of a assays in which synthetic BoNT/A LC is expressed in mammalian cells and catalytically active. In FIG. 4A, mouse brain extract was incubated with lysates prepared from HEK 293 cells that were transiently transfected with BoNT/A LC. Immunoblots showed that mouse endogenous SNAP-25 was cleaved by the BoNT/A LC expressed in HEK 293 cells. FIG. 4B shows the results of assays employing primary neuronal cultures and HEK 293 cells stably expressing YFP-SNAP-25-His×6. Both types of cell cultures were infected with a Sindbis virus overexpressing BoNT/A LC (moi 5) and analyzed for SNAP-25 cleavage by immunoblot. The synthetic BoNT/A LC was efficiently expressed and cleaved both endogenous neuronal SNAP-25 and YFP-SNAP-25-His×6.
FIG. 5 is a photomicrograph of HEK 293 cells stably expressing YFP-SNAP-25-His×6. Cells were imaged for YFP fluorescence which showed the proper localization of YFP-SNAP-25-His×6 at the cell membranes.
FIG. 6 shows is a schematic illustration of the cell-based assay for BoNT catalytic activity. HEK 293 cells stably expressing YFP-SNAP-25-His×6 are exposed to BoNT and the amount of YFP-SNAP-25-His×6 cleavage monitored by the quantity of YFP fluorescence bound to Nickel resin. A high-throughput cell-based assay uses a similar assay platform to that used in the in-vitro assay, to monitor the neurotoxin proteolytic activity on a substrate located in cells.
FIG. 7 shows the results of a cell-based assay for BoNT catalytic activity in which HEK 293 cell-lines YSH5b and YSH12b were infected with Sindbis virus over-expressing recombinant BoNT/A LC. The expressed BoNT/A LC efficiently cleaves YFP-SNAP-25-His×6 in these cells, resulting in a decrease in YFP fluorescence reporter signal bound a Nickel column.
FIGS. 8A and 8B are the synthetic BoNT/A and BoNT/E sequences, respectively in which the BamHI and AccIII restriction enzymes sites are identified.



DETAILED DESCRIPTION OF THE INVENTION
This present invention is the first system for screening inhibitors of botulinum neurotoxin type A (BoNT/A) for use in both in vitro and in living cells. Such a system can be used to greatly accelerate the search for a clinically useful antidote to botulism.
All references cited herein are hereby incorporated by reference in their entirety.
This is a novel system for monitoring the catalytic activity of a BoNT both in vitro and within living cells. The system is designed to facilitate the identification of clinically useful antidotes for botulinum neurotoxin type A and can be adapted for use as a high throughput screening assay system.
The system of the present invention provides a method for detecting BoNT activity and identifying inhibitors of BoNT activity by monitoring the cleavage of the neurotoxin's endogenous substrate using a novel recombinant protein, referred to as a botulinum neurotoxin substrate complex (or substrate indicator protein). The botulinum neurotoxin substrate complex of the present invention is comprised of: (a) a peptide substrate that is capable of being cleaved at a specific cleavage site by a botulinum neurotoxin; (b) a reporter domain on one side of the peptide substrate; and (c) an immobilization domain on the opposite side of the peptide substrate. The preferred peptide substrates are SNAP-25, a SNAP-25 isoform, syntaxin, a syntaxin isoform, VAMP, a VAMP isoform, and peptides having at least 80% identity to the foregoing as long as the peptide substrate is capable of being cleaved at a specific cleavage site by a botulinum neurotoxin. The more preferred peptide subtrates are SNAP-25, syntaxin and VAMP. The most preferred peptide substrate is SNAP-25 because it is the endogenous substrate of BoNT/A and BoNT/E, which are the two serotypes that account for the majority of botulinum infections. The nucleotide and amino acid sequence encoding murine SNAP-25 is shown in SEQ ID No. 15 and SEQ ID No. 16, respectively.
The system of the present invention for detecting BoNT/A or BoNT/E activity and identifying inhibitors of BoNT/A or BoNT/E activity is based on methods for monitoring the cleavage of their endogenous substrate SNAP-25. The system of the present invention monitors the proteolytic cleavage of SNAP-25 using a novel recombinant protein, referred to as a botulinum neurotoxin substrate complex. In one embodiment of the SNAP-25 botulinum neurotoxin substrate complex (YFP-SNAP-25-His×6), the complex is comprised of the protein substrate SNAP-25, which has a hexahistidine peptide (His×6) immobilization domain at its carboxyl terminus and a yellow fluorescent protein (YFP) reporter domain at its amino-terminus. The YFP-SNAP-25-His×6 example of a botulinum neurotoxin substrate complex is illustrated graphically in FIG. 1. This YFP-SNAP-25-His×6 system can also be used to detect BoNT/C activity and identify inhibitors of BoNT/C.
The YFP-SNAP-25-His×6 complex is capable of binding to nickel resin beads through its C-terminal His×6 immobilization domain of the complex. Nickel resin coated 96 well microtiter plates are suitable for high throughput screening and are commercially available (Pierce). The YFP-SNAP-25-His×6 complex is bound to the nickel resin in the wells of the plate. In the presence of BoNT/A, BoNT/C, or BoNT/E, the complex is cleaved to produce a cleaved complex, liberating the fluorescent indicator YFP reporter domain into the supernatant and leaving the immobilized domain bound to the Nickel. The remaining intact complex (containing the reporter domain) present on the plates and/or the reporter domain released into the supernatant can be monitored; the YFP reporter is monitored by YFP fluorescence. There is an inverse correlation between toxin concentration in the well and YFP fluorescence bound to plate. In other words, the greater the concentration of BoNT, the lower the concentration of YFP-SNAP-25-His×6 complex bound to the plate because the toxin releases the YFP reporter domain from the plate.
This approach confers a substantial advantage over other BoNT assays by capturing the “free” (proteolytically-liberated) portion containing fluorescence, enzyme activity or other detection signature. This strategy improves assay sensitivity and reduces background, thus permitting even very low amounts of the (proteolyzed) product to be measured. The complex is immobilized on a nickel surface through a C-terminal hexahistadine immobilization domain. This approach effectively removes unwanted background materials from the test sample and permits the reduction in bound reporter domain in the immobilized complex to be measured.
The system of the present invention further provides methods adapted for cell based screening to monitor the catalytic activity of a BoNT in living cells and to identify molecules that inhibit the catalytic activity of a BoNT in living cells. The present invention provides novel stable cell lines that express the botulinum toxin substrate complex (e.g., YFP-SNAP25-His×6 or GST-SNAP25-His×6). In one embodiment of present invention, a viral vector capable of efficiently expressing an active light chain of the BoNT within mammalian cells is provided.
Both the botulinum toxin substrate complex component and the BoNT expressing viral vector component of the system are suitable for use in high throughput methods. Commercially available multi-titer plates coated with nickel resin are capable of binding to the substrate indicator protein (i.e., the neurotoxin substrate complex) of the present invention. Stable YFP-SNAP-25-His×6 expressing cell lines will grow consistently within multi-titer plates as well. Plates of such cell lines allow for simultaneous, consistent infection with Sindbis virus expressing the synthetic BoNT LC in all wells. These dually expressing cells create multiple replicates per plate, where each well is available as a test vessel for a putative BoNT inhibitor. Also, lysates from such cells can be incubated and washed in the resin coated wells and the plates can be assessed for bound YFP fluorescence using a multi-well fluourimeter. Libraries of compounds, having established or potential inhibitory properties for metal protease, can be screened for their potency as a BoNT inhibitor. A compound identified as a BoNT inhibitor can be developed for use as a BoNT antidote.
EXAMPLE 1
Construction and Expression of YFP-SNAP-25-His×6
To construct YFP-SNAP-25-His×6, PCR is used to generate His×6 tag (histidine tag) at the carboxyl terminus of mouse SNAP-25 (shown in SEQ ID No. 16) and ligated into EYFP-C1 vector (Clontech). Similar constructs can be made with GST reporter domain encoding vectors. For bacterial expression, PCR is used to generate YFP-SNAP-25-His×6 with appropriate restriction sites and cloned into pGEX4T3 vector (Amersham Pharmacia Biotech). The protein is expressed in BL21(DE3) (Stratagene) bacteria and purified with glutathione sepharose 4B (AP Biotech) and the GST motif removed with thrombin cleavage. YFP-SNAP-25-His×6 is also cloned into pET vector (Novagen), expressed in BL21(DE3) bacteria and purified by nickel affinity chromatography. The in-vitro assay described below uses the protein purified from pGEX vector.
EXAMPLE 2
Generation of YFP-SNAP-25-His×6 Cell-Lines
HEK 293 cells were cultured in Minimum Essential Medium (GIBCO) supplemented with 10% fetal bovine serum, L-glutamine (10%) and pen/strep antibiotics (1%). HEK 293 cells were transfected with YFP-SNAP-25-His×6 plasmid and cultured in media containing G418 until isolated foci emerged. Isolated foci were selected for expansion and screened by immunoblots to obtain clonal cell-lines stably expressing YFP-SNAP-25-His×6.
EXAMPLE 3
Viral RNA Transcription, Transfection and Plague Assays
Purified plasmid DNAs were linearized by digestion with XhoI and transcribed using SP6 polymerase in the presence of cap analog. Transcription reactions were used for transfection of BHK-21 cells using standard methods. BHK-21 cells (ATCC) were cultured in Dubelco's Minimum Essential Medium (GIBCO) supplemented with 10% fetal bovine serum, L-glutamine (10%) and pen/strep antibiotics (1%). Plaque formation was assayed using BHK-21 monolayers.
EXAMPLE 4
In Vitro Assay for BoNT Catalytic Activity
The catalytic activity of BoNT/A was assayed using YFP-SNAP-25-His×6 immobilized on Nickel resin in 96-well plates. Purified YFP-SNAP-25-His×6 protein was immobilized on the resin and washed extensively in PBS. BoNT/A was added in the range of 1-100 U/mL PBS. The plates were incubated at 37° C. without agitation. The reaction was quenched with EDTA and the supernatant monitored for YFP fluorescence in a fluorescence plate reader.
The basis for the detection of BoNT A or E activity is the cleavage of SNAP-25. Cleavage is monitored with a novel recombinant protein where SNAP-25 has a hexahistidine peptide (“His×6” or “histadine tag”) fused to its carboxyl terminus and the yellow fluorescent protein (YFP) fused to its amino-terminus (YFP-SNAP-25-His×6, FIG. 1). The histadine tag molecule binds to nickel resin beads through its C-terminal His×6. Such nickel resin is bound to 96 well microtiter plates, which are commercially available (Pierce) and suitable for high throughput screening. In the presence of BoNT A, C, or E, the bound YFP-SNAP-25-His×6 is cleaved, liberating the fluorescent indicator YFP domain. There is an inverse correlation between toxin concentration in the well and YFP fluorescence bound to plate. In other words, the greater the concentration of toxin, the lower the concentration of YFP-SNAP-25 bound to the plate because the toxin releases the YFP reporter/signal from the plate.
The YFP-SNAP-25-His×6 immobilized on metal ion resin is cleaved by BoNT, which separates the reporter domain from the immobilization domain and releases the YFP reporter domain into the supernatant (leaving the immobilization domain attached to the metal ion resin). The YFP reporter can be monitored by YFP fluorescence.
FIGS. 2A, 2B, 2C, 2D, 2E, and 2F show the results of experiments conducted to determine if the YFP or GST added to the N-terminus of SNAP-25 and the charged hexahistidine group at the C-terminus of SNAP-25 effects the sensitivity of SNAP-25 to BoNT cleavage. BoNT/A cleaves SNAP-25 only 7 amino-acids from its C-terminus. Each of GST-SNAP-25, GST-SNAP-25-His×6 and YFP-SNAP-25-His×6 were purified from bacterial expression.
We first demonstrated that BoNT/A and BoNT/E efficiently cleave each of GST-SNAP-25, GST-SNAP-25-His×6 and YFP-SNAP-25-His×6 in-vitro, as shown in FIGS. 2A, 2D and 2E, respectively. Cleavage of YFP-SNAP-25-His×6 by BoNT/A and E is shown in FIG. 2A, and FIGS. 2D-2F. GST-SNAP-25, GST-SNAP-25-His×6 and YFP-SNAP-25-His×6 were efficiently cleaved by BoNT/A and E in vitro. GST-SNAP-25 (1-197) and GST-SNAP-25 (1-180) are recombinant proteins corresponding to the cleaved fragments from BoNT/A and E cleavage respectively. GST-SNAP-25 A/NC, which harbors a single point mutation (R198T), renders it BoNT/A resistant, was cleaved only by BoNT/E in this assay. GST-SNAP-25 (1-197) is the cleavage fragment from BoNT/A cleavage of the recombinant protein GST-SNAP-25. GST-SNAP-25 (1-180) is the cleavage fragment from BoNT/E cleavage of the recombinant protein GST-SNAP-25.
A single point mutation in SNAP-25 (R198T) renders SNAP-25 resistant to BoNT/A, but sensitive to cleavage by BoNT/E. In this assay, GST-SNAP-25 A/NC is resistant to BoNT/A cleavage, yet sensitive to cleavage by BoNT/E.
A single point mutation in SNAP-25 (D179K) renders SNAP-25 resistant to BoNT/E, but sensitive to cleavage by BoNT/A. An assay employing GST-SNAP-25 (D179K) can be tested to determine if the fusion protein is resistant to BoNT/E cleavage while remaining sensitive to BoNT/A.
A double point mutation in SNAP-25 (D179K and R198T) renders SNAP-25 resistant to both BoNT/A and BoNT/E.
To evaluate the sensitivity of an in-vitro assay based on cleavage of YFP-SNAP-25-His×6, we incubated 96-well plates coated with YFP-SNAP-25-His×6 with 1-100 U/mL BoNT/A and measured the amount of fluorescence released into the supernatant. At 4 hours, YFP fluorescence released into the supernatant increases almost 10 fold over background in the treated wells. Control wells containing no toxin or varying quantities from 0.1 U/mL to 100 U/mL (i.e., 1.0 ng/ml to 100.0 ng/ml) of BoNT/A pre-inactivated by boiling for 5 min show minimal release of YFP fluorescence (FIGS. 3A and 3B). FIGS. 3A and 3B show the in-vitro assay for BoNT catalytic activity. YFP-SNAP-25-His×6 immobilized on Nickel resin were incubated with BoNT/A at 37° C. for 4 hours without agitation. The amount of YFP fluorescence released into the supernatant was monitored with a fluorescence plate reader. The pilot assay format was sensitive enough to detect 0.1 U/mL of BoNT/A. The assay therefore exhibits sensitivity down to 0.1 U/mL of BoNT/A.
High-throughput screening of inhibitors of BoNT can be achieved by incubating YFP-SNAP-25-His×6 coated plates with BoNT and a putative toxin inhibitor. Efficacy of any inhibitor of the catalytic activity of BoNT for SNAP-25 cleavage would be proportional to the increase of bound fluorescence toward that seen in control wells without toxin. This approach confers a substantial advantage over other BoNT assays by capturing the “free” (proteolytically-liberated) portion containing fluorescence, enzyme activity or other detection signature. This strategy improves assay sensitivity and reduces background, thus permitting even very low amounts of the (proteolyzed) product to be measured.
In one embodiment, intact target protein is immobilized on a nickel surface through a C-terminal 6×his tag. This approach effectively removes unwanted background materials from the test sample by measuring the reduction in bound activity in the immobilized complex. The method of the present invention can measure both the loss of fluorescence from the beads as the substrate is cleaved and the increase in free fluorescence in solution. There is also a measurable loss of fluorescence from beads. In one embodiment of the high throughput assay method of the present invention, Nickel beads are incubated with a solution containing excess GST-YFP-SNAP25-6×His before the beads are washed in order to load the beads to maximum capacity. The fluorescence of the loaded beads is measured before they are incubated with Bo/NT and the fluorescence is measured again. The amount of loss of fluorescence is proportional to the amount of Bo/NT added. Also the fluorescence liberated into solution is measure to determine the increase in fluorescence released into solution.
EXAMPLE 5
Cell-Based Assay for BoNT Catalytic Activity
HEK 293 cells stably expressing YFP-SNAP-25-His×6 were infected with Sindbis virus overexpressing catalytic BoNT/A LC at multiplicity of infection of 5. At the termination of such a test run, the cells were lysed in a lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 0.1% NP-40 and protease inhibitors. The lysate was applied to Nickel resin followed by extensive washes in Tris-buffered saline. YFP fluorescence in the flow through or bound to the resin was measured using a fluorescence plate reader. This assay can be performed in multi-well plates and the lysis buffer is added to the wells after the YFP-SNAP-25-His×6 expressing cells are infected with the BoNT/A expressing Sindbis virus. The lysated is withdrawn, and applied to a replicate resin coated plate.
Although Sindbis virus is cytopathic, there is a window of at least 24 hours from the time of Sindbis virus infection where the stably transfected HEK 293 express both the recombinant YFP-SNAP-25-His×6 and BoNT/A can be used to test moieties or compounds for their ability to inhibit the toxin's catalytic activity in the cells. In another embodiment of the present invention, non-cytopathic forms of the Sindbis virus can be used to improve cell viability. In yet another embodiment of the present invention, inducible cell-lines that express YFP-SNAP-25-His×6 and conditionally express the recombinant BoNT LC (in the presence of an inducer) can be developed and used in the cell-based assays of the present invention.
The recombinant BoNT/A light chain is efficiently expressed and catalytically active. To verify that the synthetic BoNT/A light chain LC is catalytically active, HEK 293 cells were transiently transfected using a mammalian expression vector containing BoNT/A LC. Incubation of mouse brain extract with lysates from the transfected cells resulted in cleavage of mouse SNAP-25, as monitored by immunoblots (FIG. 4A). FIGS. 4A and 4 B show that synthetic BoNT/A LC is expressed in mammalian cells and catalytically active. FIG. 4A shows the results from an assay in which mouse brain extract was incubated with lysates prepared from HEK 293 cells transiently transfected with BoNT/A LC. Immunoblots showed that mouse endogenous SNAP-25 was cleaved by BoNT/A LC expressed in HEK 293 cells. (B) Primary neuronal cell cultures and HEK 293 cells stably expressing YFP-SNAP-25-His×6 were infected with a Sindbis virus overexpressing BoNT/A LC (moi 5) and analyzed for SNAP-25 cleavage by immunoblot. The synthetic BoNT/A LC was efficiently expressed and cleaved both endogenous neuronal SNAP-25 and YFP-SNAP-25-His×6.
Infection of primary neuronal cultures with a Sindbis virus overexpressing the synthetic BoNT/A LC also resulted in efficient cleavage of SNAP-25 in these neurons (FIG. 4B).
Cell-Based Assay for BoNT Catalytic Activity
Any inhibitor of the proteolytic activity of BoNT must have both low cytotoxicity and high intracellular penetration to be considered as a potential clinical antidote. Toward the goals of identifying clinically useful agents we have developed a cell based system to monitor the proteolytic activity of BoNT/A and BoNT/E. Two different clonal lines of human embryonic kidney cells (HEK293) were produced by transfecting the HEK293 cells so that they express high levels of YFP-SNAP25-His×6. These two cells lines are identified as YSH5b and YSH12b. HEK 29 cells stably expressing YFP-SNAP-25-His×6 are shown in FIG. 5, imaged for YFP fluorescence showing proper localization of YFP-SNAP-25-His×6 to the cell membranes. SNAP-25 is normally associated with cell membranes in neurons. Although HEK 293 cells do not express SNAP-25 endogenously, YFP-SNAP-25-His×6 expressed in these cells was properly localized to the cell membranes. Since HEK 293 cells do not express receptors for BoNT, a novel route is required to intoxicate these cells. We achieve this using a Sindbis virus vector engineered to express a catalytically active form of the light chain of BoNT (SV-LC).
Both lines of the YFP-SNAP25-His×6 expressing HEK 293 cells (i.e., YSH5b and YSH12b) as well as primary dissociated neurons show cleavage of SNAP-25 when infected with SV-LC (FIG. 4B). Immunoblot analysis of cell lysates reveals that all SNAP-25 associated protein (YFP-SNAP25-6His in HEK293 and native SNAP-25 in neurons) from transfected cells has a molecular weight consistent with BoNT/A cleavage.
The cleavage of YFP-SNAP-25-His×6 in the YSH5b and YSH12b cells by expression of BoNT LC can be monitored quantitatively using the assay system that is similar to the in-vitro methods is illustrated in FIG. 6. FIG. 6 illustrates one embodiment of the cell-based assay for BoNT catalytic activity. HEK 293 cells stably expressing YFP-SNAP-25-His×6 are exposed to BoNT and the amount of YFP-SNAP-25-His×6 cleavage is monitored by the YFP fluorescence bound to Nickel resin. This allow for high-throughput cell-based assay using similar assay platform as the in-vitro assay.
Cleavage of the recombinant YFP-SNAP-25-6His by BoNT LC produces cell lysates containing YFP-SNAP-25 devoid of the His tag, which therefore results in a reduced quantity of fluorescence bound to nickel resin wells (FIG. 7). FIG. 7 shows the results from a cell-based assay for BoNT catalytic activity. HEK 293 cell-lines YSH5b and YSH12b were infected with Sindbis virus over-expressing recombinant BoNT/A LC. The expressed BoNT/A LC efficiently cleaves YFP-SNAP-25-His×6 in these cells resulting in a decrease in YFP fluorescence bound the Nickel column. If a molecule can enter the cells and inhibit activity of the BoNT light chain, resin bound fluorescence will be restored to control (non-SVLC infected) levels.
EXAMPLE 6
Construction of Type A and Type E BoNT Light Chains
Clostridial genes are aberrantly A/T rich and poorly translated in eukaryotic cells. To achieve efficient expression of the BoNT LC, we reconstructed codon-substituted BoNT/A and BoNT/E LC with these criteria: (1) preferred codon usage in E. coli and eukaryotic cells, (2) divide the LC into interchangeable domains to facilitate the design of chimeric BoNT LC, (3) insert restriction sites compatible with several types of expression systems. BoNT/A and /E LC are constructed in PCR reactions by overlap extension of oligonucleotides as building blocks. The synthetic LC are subcloned into appropriate mammalian expression vector and Sindbis virus vector.
Based on these criteria, synthetic BoNT/A and BoNT/E LCs were designed, introducing internal BaM H1 and Acc III sites into the gene to create modules “1”, “2”, and “3” (5′ to 3′). The synthetic genes were engineered to include tandem Xho I, Nhe I, and Sph I sites at their 5′′ ends and Apa I on the 3′ end. The sequence of the synthetic BoNT/A LC gene (SEQ ID No. 1) is shown in FIG. 8A and sequence of the synthetic BoNT/E LC gene (SEQ ID No. 2) is shown in FIG. 8B. FIG. 8A shows the nucleotide sequence (SEQ ID NO: 1) encoding BoNT/A LC optimized for expression in eukaryotic cells in which a BamHI (bold and underlined) and AccIII (italics+double underlined) restriction enzyme sites have been engineered. FIG. 8B shows the nucleotide sequence (SEQ ID NO: 2) encoding BoNT/E LC optimized for expression in eukaryotic cells in which a BamHI (bold and underlined) and AccIII (italics+double underlined) restriction enzyme sites have been engineered.
Oligonucleotides of 50-60 nt were designed in pairs to introduce overlapping regions of 12 nt at their opposing ends. The oligos were optimized for preferred codon usage in E. coli and eukaryotic cells. These pairs were extended and amplified by using PCR to create fragments of  ̃100 nt, which were then utilized as building blocks in successive rounds of PCR with oligos having 12 nt overlaps with the ends of the prior PCR amplification. This type of “overlapping PCR” gene synthesis was utilized to create the entire synthetic gene. To monitor the fidelity of the gene construction process, PCR fragments were cloned into TA TOPO cloning vectors (Invitrogen) at regular intervals and sequenced to obtain template lacking mutations in coding sequence or restrictions sites.
The light chain sequences for BoNT/A and BoNT/E were divided into three sections by creating the internal restriction sites, BamHI (GGATCC (underlined in the sequences above) and AccIII (TCCGGA (double underlined in the sequences above) sites, without changing the amino acid sequence of the light chains (silent mutagenesis). For each serotype, Fragment One is from the ATC codon to the BamHI site (underlined). Fragment Two is from the BamHI site to the AccIII site (double underlined). Fragment Three is from the AccIII site to the final CAT codon.
Synthetic genes for the BoNT/A and BoNT/E LCs were subcloned into appropriate expression systems for biological applications. For those transient recombinant protein expression applications requiring plasmid the completed recombinant genes were transferred to pcDNA 3.1, using unique NheI and ApaI restriction sites in the expression plasmid in the following manner: (a) Fragment One is excised using SpeI/BamHI and ligated into NheI/BamHI sites of pcDNA3.1(+); (b) Fragment Three is cut with SpeI/ApaI and ligated into the XbaI/ApaI sites of pcDNA3.1(+)/Fragment 1; (c) finally, Fragment Two is cloned in using BamHI/AccIII to get the complete light chain sequence. After verification by DNA sequencing of the insert ligation sites, preparative amounts of plasmid were purified.
EXAMPLE 7
Construction of YFP-BoNT/A and YFP-BoNT/E Expressing Vectors
For recombinant protein expression applications requiring introduction of mammalian virus the genes were transferred to pSindREP 5 (Invitrogen), using unique Xba I and Apa I sites in the viral DNA vector (note: Xba I and Nhe I restriction sites have compatible ends for ligation) (NheI/ApaI sites for the insert). pVSindREP5 can be used to make viral replicons (ie. replication-deficient virus). After verification by DNA sequencing of the insert ligation sites, preparative amounts of the DNAs were purified for later expression studies. For studies of expression using fluorescent fusion proteins, the BoNT/A and BoNT/E LC genes were transferred to a plasmid containing the coding sequence for yellow fluorescent protein (YFP) pEYFP (EYFP-C1 from Clontech), using unique Xho I and Apa I sites. After verification by DNA sequencing of the insert ligation sites, preparative amounts of plasmid were purified.
YFP-BoNT/A and YFP-BoNT/E expression vectors are also constructed by ligating the synthetic light chains into the EYFP-C1 vector (Clontech) using XhoI/ApaI sites to facilitate tracking the light chains in separate experiments.
EXAMPLE 8
Construction of Sinbis Viral Vector for Expressing BoNT Light Chains
For recombinant protein expression applications requiring introduction of mammalian virus, the genes are transferred to Sindbis virus vector pVSind 1 using XbaI/NotI sites in the vector (NheI/NotI in the insert). pVSind 1 vector is modified from the TE12Q strain of Sindbis described by Lewis J, et al., Nat Med (1999) 5:832-5, which is hereby incorporated by reference in its entirety. This construct is used to make replication-competent Sindbis virus.
EXAMPLE 9
Construction of pGEX6P2 Vector for Expressing BoNT Light Chains
The BoNT/A is also cloned into pGEX6P2 vector (Amersham Pharmacia Biotech) to express in bacteria as a source of BoNT/A light chain for antibody production and in-vitro assays, so that the risk associated with using the holotoxin can be minimized.
EXAMPLE 10
Construction of Chimeric BoNT/A and BoNT/E LCs
The addition of the unique restriction sites within the BoNT/A and BoNT/E light chains described herein also allows for convenient swapping of domains from BoNT/A and E light chains for creation of chimeric light chains in order to produce light chains having novel properties for use in identifying inhibitors of BoNTs and for use themselves as therapeutic products.
The intermediate constructs are cloned into TA TOPO cloning vectors (Invitrogen) to check for PCR fidelity by sequencing. The completed fragments of the sequence are then ligated using the internal restriction sites in pcDNA3.1(Neo+): (a) Fragment One is excised using SpeI/BamHI and ligated into NheI/BamHI sites of pcDNA3.1(+); (b) Fragment Three is cut with SpeI/ApaI and ligated into the XbaI/ApaI sites of pcDNA3.1(+)/Fragment 1; (c) finally, Fragment Two is cloned in using BamHI/AccIII to product a complete chimeric light chain sequence. The chimeric light chains are constructed by the same procedure with different combinations of Fragments One, Two, Three of BoNT/A and BoNT/E. After each assembly step, verification of correct ligation was carried out by DNA sequencing. The following chimeric LCs were transferred to pCDNA 3.1, using the unique Nhe I and Apa I sites in the expression plasmid: 1) A1-A2-E3, 2) A1-E2-E3, 3) A1-E2-A3, 4) E1-A2-A3, 5) E1-A2-E3, 6) E1-E2-A3. These same chimeric LCs were transferred to the replication-competent Sindbis expression vector pVSind1, using unique Xba 1 and Not 1 sites, the latter derived from the TA vector. In addition to the chimeras, the full-length BoNT/A and BoNT/E LC genes were transferred to the pVSind1 vector to enable comparison with the chimeric forms.
ALTERNATIVE EMBODIMENTS
One can easily use green fluorescent protein (GFP) instead of yellow fluorescent protein (YFP). Furthermore, reporter moieties other than fluorescent markets can be used. For example, calorimetric substrate reactions such as beta-galactosidase, alkaline phosphatase, or glutathione-S-transferase (GST) or other enzymes along with the appropriate substrate or antibody (for an immunoassay) can be used. An absorption assay can be used to detect inhibitors of BoNT activity. Some examples of other enzymes and substrates can be found in U.S. Pat. No. 6,197,534. Any reporter compound which can be detected in an immunoassay, absorption assay, or substrate assay can be used.
A preferred embodiment is described as an indicator for BoNT/A and as an indicator for BoNT/E. The present invention can also be easily adapted by those of skill in the art for monitoring syntaxtin cleaving by BoNT/C or VAMP/synaptobrevin cleaving by BoNT/B, BoNT/D, BoNT/F and BoNT/G.
The nucleic acid and amino acid sequences referenced in the instant specification can be found in the corresponding SEQ ID Numbers, which are identified in Table 1 below, the sequence listing of each of which is hereby incorporated by reference in its entirety.








TABLE 1





SEQ ID No.
Type of Sequence and Protein Encoded
















1
BoNT/A nucleic acid


2
BoNT/E nucleic acid


3
BoNT/A amino acid


4
BoNT/E amino acid


5
BoNT/C nucleic acid


6
BoNT/C amino acid


7
BoNT/B nucleic acid


8
BoNT/B amino acid


9
BoNT/D nucleic acid


10
BoNT/D amino acid


11
BoNT/F nucleic acid


12
BoNT/F amino acid


13
BoNT/G nucleic acid


14
BoNT/G amino acid nucleic acid


15
Murine SNAP-25 nucleic acid


16
Murine SNAP-25 amino acid









In describing representative embodiments of the invention, the specification may have presented the method and/or process of the invention as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. In addition, the claims directed to the method and/or process of the invention should not be limited to the performance of their steps in the order written, to the extent that the method or process does not rely on the particular order of steps, and one skilled in the art can readily appreciate that the sequences may be varied still remain within the spirit and scope of the invention.
The foregoing disclosure of the embodiments of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many variations and modifications of the embodiments described herein will be apparent to one of ordinary skill in the art in light of the above disclosure.
REFERENCES


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    5. Oyler G A, et al., Development of a cell-based high-throughput screening system for inhibitors of Botulinum toxin. Interagency Botulinum Research Coordinating Committee, October 2001.
    6. Simpson L L. Botulinum toxin and tetanus toxin recognize similar membrane determinants. Brain Res 1984, 305: 177-80.
    7. Lalli G, et al., Functional characterization of tetanus and Botulinum neurotoxins binding domains. J Cell Sci 1999, 112: 2715-24.
    8. Oyler G A, et al., The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations. J Cell Biol 1989, 109:3039-52.
    9. Blasi J, et al., Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 1993, 365: 160-3.
    10. Schiavo G, et al., Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBS Lett 1993, 335:99-103.
    11. Schiavo G, et al., Identification of the nerve terminal targets of Botulinum neurotoxin serotypes A, D and E. J Biol Chem 1993: 268: 23784-7.
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## Claims

1. A botulinum neurotoxin substrate complex comprising:
(a) a peptide substrate selected from the group consisting of synaptosomal associated protein of 25 kD (SNAP-25), a SNAP-25 isoform, syntaxin, a syntaxin isoform, vesicle-associated membrane protein (VAMP), a VAMP isoform, and peptides having at least 80% identity to the foregoing, wherein said peptide substrate comprises a motif of soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor (a SNARE motif);
(b) a reporter domain comprising a fluorescent protein covalently attached to one side of said peptide substrate; and
(c) an immobilization domain covalently attached to an opposite side of said peptide substrate.

2. The botulinum neurotoxin substrate complex of claim 1, wherein said reporter domain is on the amino terminal side of said peptide substrate and said immobilization domain is on the carboxy terminal side of said peptide substrate.

3. The botulinum neurotoxin substrate complex of claim 1, wherein the fluorescent protein is selected from the group consisting of yellow fluorescent protein (YFP), blue fluorescent protein (BFP), green fluorescent protein (GFP), red fluorescent protein (RFP) and fluorescent mutants thereof.

4. The botulinum neurotoxin substrate complex of claim 1, wherein said immobilization domain is selected from the group consisting of a polyhistadine, Protein A and a maltose binding protein.

5. The botulinum neurotoxin substrate complex of claim 4, wherein said polyhistadine is hexahistadine.

6. The botulinum neurotoxin substrate complex of claim 1, wherein the SNAP-25 peptide substrate comprises the amino acid sequence of SEQ ID NO: 16.

7. A method for identifying a botulinum neurotoxin inhibitor comprising the steps of:
(i) contacting a botulinum neurotoxin substrate complex with a botulinum neurotoxin, in the presence and absence of a test molecule, wherein said complex comprises:
(a) a peptide substrate selected from the group consisting of synaptosomal associated protein of 25 kD (SNAP-25), a SNAP-25 isoform, syntaxin, a syntaxin isoform, vesicle-associated membrane protein (VAMP), a VAMP isoform, and peptides having at least 80% identity to the foregoing, wherein said peptide substrate comprises a motif of soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor (a SNARE motif);
(b) a reporter domain comprising a fluorescent protein covalently attached to one side of said peptide substrate; and
(c) an immobilization domain covalently attached to an opposite side of said peptide substrate,

wherein said complex is cleaved to produce a cleaved complex in the absence of said test molecule;
(ii) comparing the effect of the presence of said test molecule on the production of said cleaved complex to the production of said cleaved complex in the absence of said test molecule, and
(iii) identifying the test molecule as a botulinum neurotoxin inhibitor if the presence of said test molecule decreases the relative amount of cleaved complex as compared to the amount of cleaved complex in the absence of said molecule.

8. The method of claim 7, wherein said complex is immobilized on a solid support prior to said contacting step (i).

9. The method of claim 7, wherein said peptide substrate is SNAP-25 and said botulinum neurotoxin is BoNT/A.

10. The method of claim 7, wherein said peptide substrate is SNAP-25 and said botulinum neurotoxin is BoNT/E.

11. The method of claim 7, wherein said peptide substrate is SNAP-25 and said botulinum neurotoxin is BoNT/C.

12. The method of claim 7, wherein said peptide substrate is syntaxin and said botulinum neurotoxin is BoNT/C.

13. The method of claim 7, wherein said peptide substrate is VAMP and said botulinum neurotoxin is selected from the group consisting of BoNT/B, BoNT/D, BoNT/F, and BoNT/G.

14. The method of claim 7, wherein said reporter domain is on the amino terminal side of said peptide substrate and said immobilization domain is on the carboxy terminal side of said peptide substrate.

15. The method of claim 7, wherein said fluorescent protein is selected from the group consisting of yellow fluorescent protein (YFP), blue fluorescent protein (BFP), green fluorescent protein (GFP), red fluorescent protein (RFP) and fluorescent mutants thereof.

16. The method of claim 7, wherein the amount of the cleaved complex is determined by measuring the fluorescence of said reporter domain.

17. The method of claim 7, wherein said reporter domain is capable of being detected in a manner to quantitatively measure the quantity of substrate complex bound to or released from a solid support.

18. The method of claim 7, wherein said immobilization domain is selected from the group consisting of a polyhistadine, Protein A and a maltose binding protein.

19. The method of claim 18, wherein said polyhistadine is hexahistadine.

20. The method of claim 7, wherein said test molecule is one of a plurality of compounds having established inhibitory properties for metalloprotease or potential inhibitory properties for metalloprotease.

21. The method of claim 7, wherein said test molecule is one of a plurality of small molecules in a combinatorial library selected from the group consisting of a small molecule combinatorial library and a peptide combinatorial library.

22. The method of claim 7, wherein said peptide substrate is the SNAP-25 peptide having the amino acid sequence represented as SEQ ID NO: 16.

23. The method of claim 7, wherein said substrate complex is a recombinant product wherein said peptide substrate is encoded by the nucleic acid sequence shown in SEQ ID NO: 15.

24. The method of claim 7, wherein said neurotoxin is selected from the group consisting of BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, and BoNT/G.

25. The method of claim 7, wherein said botulinum neurotoxin is an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14.