Patent Application
20230048462
The present disclosure generally relates to screening methods for identifying compounds useful as anti-clostridial neurotoxin agents. Specifically disclosed are polypeptide and nucleic acid constructs and methods for detecting agents that inhibit the binding of clostridial neurotoxins to their cellular receptor.
References considered to be relevant as background to the presently disclosed subject matter are listed below:
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
Clostridial neurotoxins (for example Botulinum neurotoxins, BoNTs and tetanus toxin) are among the most poisonous substances in nature. Botulinum neurotoxins are produced by the gram-positive spore-forming bacterium Clostridium botulinum. There are eight serotypes of BoNTs designated A-H, and antibodies raised against one serotype can neutralize only the toxic effects of the BoNT serotype against which it was raised and not that of the other serotypes.
All BoNT serotypes share a common architecture that consists of the following three domains responsible for the different steps in the intoxication mechanism: 1. The receptor binding domain (also known as the Hc-fragment); 2. The translocation domain; and 3. The catalytic domain. Following exposure of a subject to BoNTs, the BoNTs bind specific receptors on motor neurons and disable the ability of the cells to transmit the neurotransmitter acetyl choline to muscle cells. This results in flaccid muscle paralysis and can lead to respiratory failure and eventually to death.
Currently, the only approved therapy for botulinum intoxication is antitoxin, an antibody preparation originating from vaccinated horses, which has the capacity to neutralize the botulinum toxin at the bloodstream. Although effective, antitoxin therapy suffers from several drawbacks. First, the administration of a large dose of foreign protein, namely, an equine (horse-derived) antibody, can cause severe side effects, including an anaphylactic shock. In addition, due to secondary immunologic reactions against the equine antibodies, the antitoxin can be administered only for a single intoxication event per patient. Second, antitoxin therapy is very expensive due to the requirement for horses and for restricted safety facilities dictated by an activity involving the hazardous neurotoxin. Third, antibodies are thermo-labile and require a so called “cold chain delivery”, which limits antitoxin distribution.
Due to at least the above reasons, there is a motivation for developing next generation therapy for botulinum intoxication (botulism) based on small molecules that inhibit the neurotoxin. The production costs of small molecules are relatively low. In addition, the immune system does not react against small molecules, and therefore such therapy is safer than an antitoxin therapy and can be administered repeatedly and even in a prophylactic manner. Furthermore, small molecules are generally stable and do not require a cold chain delivery.
Small molecules can target each one of the neurotoxin domains that are responsible for the different intoxication steps i.e.: receptor binding, toxin translocation, and enzymatic cleavage of the cytoplasmic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Many efforts are being presently made for finding small molecule inhibitors that target the catalytic domain of the toxin that will inactivate intracellular toxin (1, 2). The effect of small molecules on the catalytic domain can be evaluated by measuring the enzymatic activity of the catalytic domain on synthetic substrates, in the presence of potential inhibitors. Indeed, many small molecules were found to inhibit the catalytic domain of BoNTs, with dissociation constants in the nanomolar range (1, 2). However, only a limited therapeutic effect was reported thus far in vivo for catalytic domain inhibitors, due to their limited cell entry.
Targeting the receptor binding domain of botulinum neurotoxins is an additional approach for generating therapy for botulism, as the receptor binding domain may be encountered by inhibitors while in the blood stream and thus the inhibitors are not required to enter the cells. The publications WO 2007/050390 and U.S. Pat. No. 8,476,024 relate inter alia to methods for identifying inhibitors of Botulinum neurotoxin A (BoNT/A) binding to the synaptic vesicle glycoprotein 2C (SV2C), the receptor for botulinum neurotoxin A. Methods for identifying inhibitors of Botulinum neurotoxin serotypes other than BoNT/A have also been reported (5).
In a first aspect the present disclosure provides a method for identifying an agent that reduces binding of a clostridial neurotoxin to synaptic vesicle glycoprotein 2 (SV2), wherein said method comprises the following steps:
(a) bringing into contact a first fusion protein, said first fusion protein comprising a receptor binding domain of Botulinum neurotoxin (BoNT Hc fragment) fused to a reporter protein, with a second protein, said second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or a fragment thereof optionally fused to an expression facilitating domain;
(b) measuring the binding of said first fusion protein to said second protein in the presence of a sample; and
(c) comparing the measured binding to the binding performed in a control measurement in the absence of said sample, wherein a lower binding than that of the control measurement indicates that said agent reduces binding of said clostridial neurotoxin to SV2.
In some embodiments the method according to the present disclosure is wherein the agent is a small molecule.
In other embodiments the method according to the present disclosure is wherein the agent is a polypeptide.
In further embodiments the method according to the present disclosure is wherein the agent is an antibody.
In specific embodiments the method according to the present disclosure is wherein the first fusion protein further comprises a linker situated between said reporter protein and said BoNT Hc fragment.
In other specific embodiments the method according to the present disclosure is wherein said first fusion protein further comprises at least one heterologous amino acid sequence at its N- and/or C-terminus. In still further embodiments the method according to the present disclosure is wherein said first fusion protein comprises a His tag sequence at its C-terminus.
In some embodiments the method according to the present disclosure is wherein the reporter protein is situated N-terminal to said BoNT Hc fragment. In specific embodiments the method according to the present disclosure is wherein said reporter protein is a beta-galactosidase domain.
In other specific embodiments the method according to the present disclosure is wherein said first fusion protein comprises a receptor binding domain of at least one of Botulinum neurotoxin A (BoNT/A Hc fragment), or Botulinum neurotoxin E (BoNT/E Hc fragment), or Botulinum neurotoxin D (BoNT/D Hc fragment) or Botulinum neurotoxin F (BoNT/F Hc fragment) or tetanus neurotoxin.
In other embodiments the method according to the present disclosure is wherein the first fusion protein comprises a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment).
In further embodiments the method according to the present disclosure is wherein said clostridial neurotoxin is a botulinum neurotoxin or a tetanus neurotoxin.
In further embodiments the method according to the present disclosure is wherein the synaptic vesicle glycoprotein 2 is synaptic vesicle glycoprotein 2C (SV2C).
In some embodiments the method according to the present disclosure is wherein said expression facilitating domain is a glutathione-s-transferase (GST).
In other embodiments the method according to the present disclosure is wherein the method is for identifying an agent that reduces binding of BoNT/A to the BoNT receptor SV2C.
In further embodiments the method according to the present disclosure is wherein the first fusion protein comprises a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) having an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 2 and a reporter protein which is a beta-galactosidase domain having an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 1.
In still further embodiments the method according to the present disclosure is wherein the first fusion protein comprises a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) having the amino acid sequence denoted by SEQ ID NO. 2 and a reporter protein which is a beta-galactosidase domain having the amino acid sequence denoted by SEQ ID NO. 1.
In specific embodiments thereof the present disclosure encompasses a method wherein the first fusion protein comprises from an amino-terminal position to a carboxyl-terminal position a reporter protein which is a beta-galactosidase domain having the amino acid sequence denoted by SEQ ID NO. 1, a linker having the amino acid sequence denoted by SEQ ID NO. 3, a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) having the amino acid sequence denoted by SEQ ID NO. 2 and a His tag having the amino acid sequence denoted by SEQ ID NO. 4.
In some embodiments the method according to the present disclosure is wherein the first fusion protein comprises an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 5. In other embodiments the method according to the present disclosure is wherein the first fusion protein comprises or consists of the amino acid sequence denoted by SEQ ID NO. 5.
In other embodiments the method according to the present disclosure is wherein the second protein comprises the fourth luminal loop of the BoNT receptor SV2C having the amino acid sequence denoted by SEQ ID NO. 13. In further embodiments the method according to the present disclosure is wherein the second protein is a fusion protein comprising an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 15. In still further embodiments the method according to the present disclosure is wherein said second protein is a fusion protein comprising or consisting of the amino acid sequence denoted by SEQ ID NO. 15.
In some embodiments the method according to the present disclosure is wherein at least one of the first fusion protein or the second protein is immobilized onto a solid surface.
In specific embodiments the method according to the present disclosure is wherein the second protein is immobilized onto a solid surface.
In other specific embodiments the method according to the present disclosure is wherein the step of measuring the binding comprises adding at least one substrate of the reporter protein of said first fusion protein.
In further specific embodiments the method according to the present disclosure is wherein the reporter protein is beta-galactosidase domain and said at least one substrate is 4-methylumbellyferyl-galactopyranoside (4-MUG), ortho-nitrophenyl-galactopyranoside (oNPG), fluorescein Di-β-D-Galactopyranoside or naphthofluorescein di-(beta-D-galactopyranoside).
By another one of its aspects the present disclosure provides a method for identifying at least one agent that reduces binding of Botulinum neurotoxin A (BoNT/A) to the synaptic vesicle glycoprotein 2C (SV2C), wherein said method comprises the following steps:
(a) contacting a first fusion protein, said first fusion protein comprising BoNT/A Hc fragment and a beta-galactosidase domain with at least one sample, thereby obtaining a mixture;
(b) contacting said mixture obtained in step (a) with a second protein optionally attached to a surface, said second protein being a fusion protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2C (SV2C) and a glutathione-s-transferase domain (GST);
(c) adding at least one substrate of the beta-galactosidase domain of said first fusion protein; and
(d) determining the signal;
wherein a signal lower than a threshold signal indicates that said agent reduces binding of BoNT/A to SV2C.
In some embodiments the method according to the present disclosure further comprising at least one washing step with a wash solution between steps (a) and (b) and/or steps (b) and (c). In other embodiments the method according to the present disclosure further comprising adding a stop solution after step (c). In further embodiments the method according to the present disclosure is wherein the first fusion protein is contacted with the at least one sample for one hour at room temperature.
In specific embodiments the method according to the present disclosure is wherein the second protein is contacted with the mixture obtained in step (a) for one hour at 37° C.
In further specific embodiments the method according to the present disclosure is wherein the method is used for high-throughput screening (HTS).
It is to be understood that said sample referred to above is a sample that potentially comprises said agent.
In specific embodiments the method according to the present disclosure is wherein the sample is a biological sample obtained from a subject and wherein the agent is an antibody.
By another one of its aspects the present disclosure provides a kit comprising:
(a) a first fusion protein, said first fusion protein comprising a receptor binding domain of Botulinum neurotoxin (BoNT Hc fragment) fused to a reporter protein or a composition comprising said first fusion protein and a suitable carrier or excipient;
(b) a second protein, said second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof optionally fused to an expression facilitating domain or a composition comprising said second protein and a suitable carrier or excipient; optionally, said second protein is attached to a solid surface; and optionally
(c) instructions for use.
In some embodiments the kit according to the present disclosure is wherein the reporter protein is a beta-galactosidase domain. In other embodiments the kit according to the present disclosure further comprises at least one substrate for said beta-galactosidase domain.
In other embodiments the kit according to the present disclosure is wherein the first fusion protein comprises a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment).
In further embodiments the kit according to the present disclosure is wherein the second protein comprises a BoNT receptor which is synaptic vesicle glycoprotein 2C (SV2C).
In specific embodiments the kit according to the present disclosure is wherein the expression facilitating domain is a glutathione-s-transferase (GST).
In further specific embodiments the kit according to the present disclosure is wherein the first fusion protein comprises an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 5. In still further specific embodiments, the kit according to the present disclosure is wherein the first fusion protein comprises or consists of the amino acid sequence as denoted by SEQ ID NO. 5.
In some embodiments the kit according to the present disclosure is wherein the second protein comprises the fourth luminal loop of SV2C having the amino acid sequence denoted by SEQ ID NO. 13.
In other embodiments the kit according to the present disclosure is wherein the second protein is a fusion protein comprising an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 15. In further embodiments the kit according to the present disclosure is wherein the second protein is a fusion protein comprising or consisting of the amino acid sequence denoted by SEQ ID NO. 15.
In specific embodiments the kit according to the present disclosure is wherein the at least one substrate is 4-methylumbellyferyl-galactopyranoside (4-MUG), ortho-nitrophenyl-galactopyranoside (oNPG), fluorescein Di-β-D-Galactopyranoside or naphthofluorescein di-(beta-D-galactopyranoside).
In another one of its aspects the present disclosure provides an isolated fusion protein comprising the receptor binding domain of Botulinum neurotoxin (BoNT Hc fragment) and a reporter protein, or tetanus neurotoxin.
In certain embodiments the isolated fusion protein according to the present disclosure comprises the receptor binding domain of at least one of Botulinum neurotoxin A (BoNT/A Hc fragment), or Botulinum neurotoxin E (BoNT/E Hc fragment), or Botulinum neurotoxin D (BoNT/D Hc fragment) or Botulinum neurotoxin F (BoNT/F Hc fragment).
In some embodiments the isolated fusion protein according to the present disclosure comprises the receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment).
In other embodiments the isolated fusion protein according to the present disclosure is wherein the reporter protein is a beta-galactosidase domain. In specific embodiments the isolated fusion protein according to the present disclosure is wherein said beta-galactosidase domain is situated N-terminal to said BoNT/A Hc fragment.
In further specific embodiments the isolated fusion protein according to the present disclosure further comprises a linker situated between said beta-galactosidase domain and said BoNT/A Hc fragment.
In some embodiments the isolated fusion protein according to the present disclosure further comprises at least one heterologous amino acid sequence at its N- and/or C-terminus.
In other embodiments the isolated fusion protein according to the present disclosure comprises a His tag sequence at its C-terminus.
In further embodiments the isolated fusion protein according to the present disclosure comprises the BoNT/A Hc fragment having an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 2 and wherein said reporter protein is a beta-galactosidase domain having the amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 1.
In still further embodiments the isolated fusion protein according to the present disclosure comprises the BoNT/A Hc fragment having the amino acid sequence denoted by SEQ ID NO. 2 and wherein said reporter protein is a beta-galactosidase domain has the amino acid sequence denoted by SEQ ID NO. 1.
In specific embodiments the isolated fusion protein according to the present disclosure comprises from an amino-terminal position to a carboxyl-terminal position a beta-galactosidase domain having the amino acid sequence denoted by SEQ ID NO. 1, a linker having the amino acid denoted by SEQ ID NO. 3, a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) having the amino acid sequence denoted by SEQ ID NO. 2 and a His tag having the amino acid sequence denoted by SEQ ID NO. 4.
In specific embodiments the isolated fusion protein according to the present disclosure comprises an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 5. In still further specific embodiments, the isolated fusion protein according to the present disclosure comprises or consists of the amino acid sequence denoted by SEQ ID NO. 5.
By another one of its aspects the present disclosure provides an isolated nucleic acid sequence encoding the fusion protein as defined herein. The present disclosure further provides a vector comprising the nucleic acid sequence as herein defined, and a host cell comprising the vector as herein defined.
The present disclosure further provides a composition comprising the isolated fusion protein as defined in the present disclosure and a suitable carrier or excipient.
For better understanding the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
The present disclosure is based on the development of a screening method for identifying agents that inhibit the binding of Botulinum neurotoxin A (BoNT/A) to its receptor, the synaptic vesicle protein SV2C. The method is generally based on the design of a chimeric protein construct comprising the receptor binding domain of BoNT/A (also referred herein as the Hc-fragment) fused to the reporter enzyme beta-galactosidase from Escherichia coli and on an additional protein construct comprising SV2C, thereby allowing high throughput screening (HTS) of a compound library.
Generally, high throughput screening (HTS) of a compound library is an important tool for discovery of new drugs for a desired activity and can be accomplished only if an efficient, sensitive, and specific assay is available. Currently, there are no HTS assays to assist in screening for inhibitors of Botulinum neurotoxin (BoNT) binding to their receptors. Assays based on the enzymatic activity of BoNT could have been potentially used to detect inhibitors of the receptor-bound toxin. However, such assays are either inadequate for HTS (as they require detection of the specific endoproteinase activity of BoNT using mass spectrometry (Endopep-MS)), or suffer from a limited sensitivity, and most importantly, such assays obligate safety restrictions and are associated with high costs due to the involvement of the hazardous toxin.
Alternatively, assays designed for monitoring receptor binding can be based on the receptor-binding domain of the toxin only. Since the receptor-binding domain of the toxin (the Hc fragment) is non-toxic, special safety restrictions are not required and such an assay can potentially be conducted at any laboratory. However, the absence of the catalytic domain prevents facile detection of receptor-bound Hc fragment.
For overcoming this obstacle, the assay can be based on an enzyme-linked immunosorbent assay (ELISA) format, in which receptor-bound Hc-fragment is identified with specific antibodies, which are in turn recognized by secondary antibodies that are conjugated to peroxidase or alkaline phosphates. Addition of chromogenic substrate results in a signal whose intensity is proportional to the amount of receptor-bound Hc-fragment. Nevertheless, such an assay requires multiple steps and is time consuming. In addition, this type of an assay will not allow examination of antitoxin antibodies preparations since the Hc-fragment will already be blocked by the tested antibodies and therefore will not be available for the identifying antibody. Furthermore, it may suffer from low sensitivity due to non-specific interaction of the antibodies.
For overcoming all of the above-mentioned obstacles and limitations the inventors have designed, as detailed below, a chimeric protein construct comprising the receptor binding domain of BoNT/A (also termed herein the Hc-fragment) fused to the reporter enzyme beta-galactosidase from Escherichia coli. The codon usage of both proteins was optimized for expression and a flexible linker with the sequence (GGGGS)3 was positioned as a connector between the Hc-fragment and beta-galactosidase proteins. In addition, the inventors have used a receptor construct based on the synaptic vesicle protein (SV2C) conjugated to a Glutathione S-Transferase (GST) protein to facilitate expression of the SV2C. Detection of receptor-bound Hc fragment is accomplished with the addition of the chromogenic substrate ortho-nitrophenyl-ß-galactoside (oNPG) or the fluorogenic substrate 4-methylumbelliferyl-ß-galactoside (4-MUG) that provides even a higher sensitivity. This assay includes a small number of steps and presents a high sensitivity (˜2fM of the chimeric protein), making it suitable for HTS. Since the chimeric protein comprising the Hc fragment is non-toxic the assay can be conducted in fact in any laboratory, without requiring special equipment or taking any special safety measures and restrictions.
In addition to screening of small molecule inhibitors, the assay can be used to evaluate the capacity of an anti-BoNT/A antibody preparation to prevent binding of the Hc fragment to the receptor.
As shown in the Examples below, by using the chimeric protein assay the inventors were able to screen a compound library in an HTS approach and discover several compounds that inhibited BoNT/A binding to its receptor SV2C. Three of the compounds also demonstrated a therapeutic effect in-vivo.
Furthermore, the assay was used to evaluate the potency of botulinum antitoxin preparations to neutralize botulinum toxin.
Therefore, in one of its aspects, the present invention provides a method for identifying an agent that reduces binding of a clostridial neurotoxin to synaptic vesicle glycoprotein 2 (SV2), wherein said method comprises the following steps:
(a) bringing into contact a first fusion protein, said first fusion protein comprising a receptor binding domain of Botulinum neurotoxin (BoNT Hc fragment) fused to a reporter protein, with a second protein, said second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or a fragment thereof optionally fused to an expression facilitating domain;
(b) measuring the binding of said first fusion protein to said second protein in the presence of a sample potentially comprising said agent; and
(c) comparing the measured binding to the binding performed in a control measurement in the absence of said sample, wherein a lower binding than that of the control measurement indicates that said agent reduces binding of said clostridial neurotoxin to SV2.
In other words, the present invention provides a method for identifying an agent that reduces the binding of any clostridial neurotoxin to its receptor, namely to synaptic vesicle glycoprotein 2 (SV2).
Clostridial neurotoxins are bacterial proteins produced by clostridia, a genus of Gram-positive bacteria, which includes several human pathogens, among these the causative agents of botulism and tetanus. Clostridium species inhabit soils and the intestinal tract of animals, including humans. Clostridium include around 100 species, inter alia, Clostridium botulinum which can produce botulinum toxin in food or wounds and can cause botulism; Clostridium perfringens which causes a wide range of symptoms, from food poisoning to cellulitis, fasciitis, and gas gangrene; Clostridium tetani which can produce tetanus neurotoxin and causes tetanus or the exotoxin tetanolysin, a hemolysin that causes destruction of tissues; and Clostridium sordellii that can cause a fatal infection in exceptionally rare cases after medical abortions.
In certain embodiments the present invention relates to a clostridial neurotoxin which is a Botulinum neurotoxin, a tetanus neurotoxin, or a combination thereof.
Botulinum neurotoxins (BoNTs) as known in the art and as used herein are bacterial proteins that cause the life-threatening disease botulism and are designated by the Centers for Disease Control and Prevention (CDC) “category A” agents. Eight antigenically distinct serotypes (designated A to H) are produced by several anaerobic species: Clostridium botulinum, Clostridium butyricum, Clostridium baratii and Clostridium argentinense. BoNTs A, B, E, and rarely, F serotypes are primarily related to human illness. The neurotoxin is a di-chain polypeptide consisting of a 100-kDa heavy chain (HC) joined by a disulfide bond to a 50-kDa light chain (LC). All BoNT serotypes exert similar mechanisms of action on their target nerve cells, namely: initial binding of the C-terminal portion of the HC through ganglioside and protein receptors on the presynaptic cell surface, followed by internalization and translocation within the nerve ending, mediated by the N-terminal portion of the HC. Inside the nerve terminus, the neurotoxin LC, cleaves the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), which involved in the fusion and release of acetylcholine. As a result, acetylcholine transmission across neuromuscular junctions is blocked and symmetric descending flaccid paralysis occurs.
In some specific embodiments the present invention relates to Botulinum neurotoxin produced by Clostridium botulinum. In other embodiments the present invention relates to Botulinum neurotoxin A, B, C, D, E, F, G and H and to any combination thereof.
In specific embodiments the present invention relates to each one of Botulinum neurotoxin A, E, D and F.
In still further embodiments the present invention relates to Botulinum neurotoxin A.
As detailed above BoNTs are composed of a 150-kDa protein consisting of a 100-kDa heavy chain (HC) joined by a disulfide bond to a 50-kDa light chain (LC). The C-terminal half of the heavy chain (referred to herein as the “Hc fragment”) functions in binding of the toxin to specific receptors and the N-terminal half of the heavy chain mediates the translocation of the light chain into the cytoplasm of the neuron.
Therefore, as used herein the term “receptor binding domain of Botulinum neurotoxin” (also referred to herein as “BoNT Hc fragment”) refers to the C-terminal region of BoNT heavy chain which functions in binding to the receptor synaptic vesicle glycoprotein 2 (SV2). The present invention further encompasses any fragment of the receptor binding domain of Botulinum neurotoxin which maintains its binding to the receptor thereof. The preparation of a receptor binding domain of Botulinum neurotoxin or any fragment thereof is known to a person of skill in the art.
In some embodiments the present invention relates to each one of the receptor binding domains of Botulinum neurotoxin A, E, D and F.
As non-limiting examples, the amino acid sequence of the receptor binding domain of C. botulinum Botulinum neurotoxin A (C. botulinum BoNT/A He fragment) is denoted herein by SEQ ID NO. 2 and the nucleic acid sequence encoding it is denoted herein by SEQ ID NO. 8.
Following are further non-limiting examples of amino acid and nucleic acid sequences of other botulinum neurotoxin receptor binding domains. The amino acid sequence of C. botulinum B (gene bank code M81186, amino acids 860-1291) is denoted herein by SEQ ID NO. 16 and the nucleic acid sequence encoding thereof is denoted herein by SEQ ID NO. 17. The amino acid sequence of C. botulinum C (gene bank code AB745658.1, amino acids 843-1291) is denoted herein by SEQ ID NO. 18 and the nucleic acid sequence encoding thereof is denoted herein by SEQ ID NO. 19. The amino acid sequence of C. botulinum D (gene bank code X54254.1, amino acids 861-1276) is denoted herein by SEQ ID NO. 20 and the nucleic acid sequence encoding thereof is denoted herein by SEQ ID NO. 21. The amino acid sequence of C. botulinum E (gene bank code X62683.1, amino acids 845-1252) is denoted herein by SEQ ID NO. 22 and the nucleic acid sequence encoding thereof is denoted herein by SEQ ID NO. 23. Furthermore, the amino acid sequence of C. botulinum F (gene bank code GU213203.1, amino acids 860-1278) is denoted herein by SEQ ID NO. 24 and the nucleic acid sequence encoding thereof is denoted herein by SEQ ID NO. 25. In addition, the amino acid sequence of C. botulinum G (gene bank code X74162.1, amino acids 860-1297) is denoted herein by SEQ ID NO. 26 and the nucleic acid sequence encoding thereof is denoted herein by SEQ ID NO. 27. The above sequences denoted herein by SEQ ID NO. 16 to SEQ ID NO. 27 are listed in Table 7 below.
In specific embodiments the present invention relates to the receptor binding domain of Botulinum neurotoxin A.
In further specific embodiments the present invention relates to the receptor binding domain of Botulinum neurotoxin A BoNT/A having the amino acid sequence which is at least 70% identical to the amino acid sequence denoted by SEQ ID NO. 2.
By the term “sequence which is at least 70% identical to a sequence” it is meant that the sequence referred to is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence indicated as a reference sequence.
Thus, in still further specific embodiments the present invention relates to the receptor binding domain of Botulinum neurotoxin A BoNT/A having the amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence denoted by SEQ ID NO. 2.
As detailed above, the present invention provides a method for identifying an agent that reduces binding of a clostridial neurotoxin to synaptic vesicle glycoprotein 2 (SV2). The “receptor synaptic vesicle glycoprotein 2”, also referred to herein as “SV2” as known in the art is a family of transporter-like proteins located in synaptic neurotransmitter-containing vesicles. The three SV2 genes in mammals encode three isoforms, namely SV2A, SV2B and SV2C.
SV2s are membrane proteins, found in synaptic and endocrine secretory vesicles of vertebrates. The proteins have 12 putative transmembrane domains, and five loops are directed towards the vesicle lumen. Dong et. al. (6) demonstrated that BoNT/A binds the fourth luminal loop of the synaptic vesicle glycoprotein 2C with the highest affinity. Specifically, the binding site of BoNT/A was shown to be located on amino acids 529-566 of SV2C.
BoNT/E uses the fourth luminal loop of SV2 as the protein receptor as well. The isoforms SV2A and SV2B are the preferred target for BoNT/E, which was shown to be dependent on the glycosylation of N573 in SV2A for its biological activity (9). Additionally, it is believed that SV2s are the protein receptor for BoNT/D, BoNT/F, and tetanus toxin.
The present invention provides a method for identifying an agent that reduces binding of any clostridial neurotoxin to any type of the synaptic vesicle glycoprotein 2 (SV2) receptor. In specific embodiments, the present invention relates inter alia to the synaptic vesicle glycoprotein 2 (SV2) types SV2A, SV2B and SV2C.
By way of example, the amino acid sequence of the synaptic vesicle glycoprotein 2 type A (SV2A, Mus musculus, NCBI Reference Sequence: NP_071313.1) is denoted herein by SEQ ID NO. 28, the amino acid sequence of the synaptic vesicle glycoprotein 2 type B (SV2B, Mus musculus, NCBI Reference Sequence: NP_001347503.1) is denoted herein by SEQ ID NO. 29 and the amino acid sequence of the synaptic vesicle glycoprotein 2 type C (SV2C, Mus musculus, GenBank: AAI37862.1) is denoted herein by SEQ ID NO. 30. The above amino acid sequences are listed in Table 8 below.
In specific embodiments, the present invention relates to SV2C. In other specific embodiments, the present invention relates to SV2A. In specific embodiments, the present invention relates to SV2B. In other words, in some embodiments the method as herein defined is wherein said receptor is synaptic vesicle glycoprotein 2C (SV2C).
The present invention further provides a method as herein defined for identifying an agent that reduces binding of a tetanus neurotoxin to synaptic vesicle glycoprotein 2 (SV2).
Furthermore, in the above and other embodiments the method according to the present invention is wherein said method is for identifying an agent that reduces binding of BoNT/A to the BoNT receptor SV2C.
In other words, the present invention further provides a method for identifying an agent that reduces binding of Botulinum neurotoxin A (BoNT/A) to synaptic vesicle glycoprotein 2C (SV2C), wherein said method comprises the following steps:
(a) bringing into contact a first fusion protein, said first fusion protein comprising a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) fused to a reporter protein, with a second protein, said second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2C (SV2C) or fragment thereof optionally fused to an expression facilitating domain;
(b) measuring the binding of said first fusion protein to said second protein in the presence of a sample potentially comprising said agent; and
(c) comparing the measured binding to the binding performed in a control measurement in the absence of said sample, wherein a lower binding than that of the control measurement indicates that said agent reduces binding of BoNT/A to SV2C.
By the term “agent” in the context of the present invention (i.e., an agent that reduces binding of a clostridial neurotoxin to SV2), it is referred to any molecule. In specific embodiments the term “agent” as used herein relates to a small molecule, a polypeptide (e.g., an antibody) or to a nucleic acid or to any combination thereof. In further specific embodiments the agent as herein defined is a small molecule. In still further embodiments the agent as herein defined is a polypeptide. In other specific embodiments the agent as herein defined is an antibody.
By the term “identifying” as used herein it is meant to determine, find, select, or establish an agent that reduces binding of a clostridial neurotoxin to synaptic vesicle glycoprotein 2 (SV2).
The binding (or engagement, or association) of a clostridial neurotoxin to its SV2 receptor may be monitored by any method known to a person of skill in the art, inter alia as exemplified in the present invention. By the term “reduce” binding it is referred to any reduction, diminishing, inhibition, lessening in the binding of a clostridial neurotoxin to its SV2 receptor, by at least about 1%-100%, about 5%-95%, about 10%-90%, about 15%-85%, about 20%-80%, about 25%-75%, about 30%-70%, about 35%-65%, about 40%-60% or about 45%-55%. Said reduction, diminishing, inhibition, lessening in the binding of a clostridial neurotoxin to its SV2 receptor may also be by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100%.
As detailed above, the present invention provides a method for identifying an agent that reduces binding of a clostridial neurotoxin to SV2, wherein said method comprises, among others, the step of bringing into contact (contacting) a first fusion protein, said first fusion protein comprising a receptor binding domain of Botulinum neurotoxin (BoNT Hc fragment) fused to a reporter protein, with a second protein, said second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof fused to an expression facilitating domain.
By the term “fusion protein” it is meant that at least two protein (polypeptide) fragments of the same or different origin are connected, combined, or integrated by a peptide bond. The terms “polypeptide” and “protein” are used interchangeably.
The first fusion protein according to the present invention comprises a receptor binding domain of Botulinum neurotoxin (BoNT Hc fragment) fused to a reporter protein.
The present invention encompasses a first fusion protein that comprises an Hc fragment of Botulinum neurotoxin of any serotype, for example the receptor binding domain of Botulinum neurotoxin A, B, C, D, E, F, G and H. In specific embodiments the present invention relates to a first fusion protein comprising a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment). In further specific embodiments the present invention relates to a first fusion protein comprising a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) having the amino acid sequence which is at least 70%, 75%, 80%, 85%, 905, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence as denoted by SEQ ID NO. 2.
By the term “reporter protein” it is referred to any protein or polypeptide that allows determining, analyzing, or monitoring the binding of a clostridial neurotoxin to its SV2 receptor. Examples of reporter proteins include but are not limited to the beta-galactosidase domain, green fluorescent protein (GFP), luciferase, or a protein tag that can be detected with antibodies, such as FLAG-tag, HA-tag.
In some embodiments the method according to the present invention is wherein said reporter protein is situated N-terminal to said BoNT Hc fragment. In certain specific embodiments the method according to the present invention is wherein said reporter protein is a beta-galactosidase domain.
As known in the art, by the term “beta-galactosidase domain” as used herein it is referred to β-galactosidase, also called lactase, beta-gal, or β-gal, which is a glycoside hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides through the breaking of a glycosidic bond. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins.
In specific embodiments the beta-galactosidase domain is derived from E. coli.
In further specific embodiments the reporter protein according to the present invention is a beta-galactosidase domain which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence having an amino acid sequence denoted by SEQ ID NO. 1.
In some embodiments, the first fusion protein according to the present invention comprises, from an amino-terminal position to a carboxyl-terminal position, a reporter protein (e.g., a beta-galactosidase domain) and a receptor binding domain of Botulinum neurotoxin (e.g., BoNT/A Hc fragment).
In further specific embodiments, the first fusion protein according to the present invention comprises, from an amino-terminal position to a carboxyl-terminal position, a beta-galactosidase domain which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence having the amino acid sequence as denoted by SEQ ID NO. 1 and a receptor binding domain of Botulinum neurotoxin A having the amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence as denoted by SEQ ID NO. 2.
The first fusion protein according to the present invention may further comprise additional amino acid sequences, domains, or fragments. In some embodiments the method according to the present invention is wherein said first fusion protein further comprises a linker situated between said reporter protein and said BoNT Hc fragment.
By the term “linker” it is meant to refer to a chain of at least one, two, three, four, five, six, seven, eight, nine, ten or more amino acid residues, such as 2-15, 3-12, 4-10, 5-10 amino acid residue-long fragment, comprising for example flexible amino acids, such as Glycine (Gly, G), Alanine (Ala, A), Glutamic acid (Glu, E), Lysine (Lys, K) to name but few. The linker can be flexible, having, for example, a sequence such as (GGGGS)4 denoted by SEQ ID NO. 31 or Gn (n=5-10). The linker can also be rigid, with a defined structure, having, for example, a sequence of (EAAAK)n (n=1-3) wherein the sequence of the monomeric unit EAAAK is denoted herein by SEQ ID NO. 32, or for example A(EAAAK)4ALEA(EAAAK)4A denoted by SEQ ID NO. 33.
In specific embodiments the present invention relates to a linker which has the amino acid sequence of GGGGSGGGGSGGGGS as denoted herein by SEQ ID NO. 3.
In additional embodiments the first fusion protein according to the present invention further comprises at least one heterologous amino acid sequence at its N- and/or C-terminus. By the term “heterologous amino acid sequence” it is meant to refer to an amino acid sequence that is non-native to either one of the protein components of the first fusion protein of the present invention.
In some embodiments the method according to the present invention is wherein said first fusion protein further comprises at least one heterologous amino acid sequence at its N- and/or C-terminus. In other specific embodiments the method according to the present invention is wherein said first fusion protein comprises a His tag sequence at its C-terminus. In the above and other embodiments, the present invention relates to a heterologous amino acid sequence which has the amino acid sequence of HHHHHH as denoted herein by SEQ ID NO. 4.
By the term “at least one” it is referred to one, two, three, four, five, six, seven, eight, nine, ten or more amino acid residues.
In further specific embodiments the first fusion protein according to the present invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence denoted by 5.
In certain specific embodiments the method according to the present invention is wherein said first fusion protein comprises a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) having an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence denoted by SEQ ID NO. 2 and a reporter protein which is a beta-galactosidase domain having an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence denoted by SEQ ID NO. 1.
In some specific embodiments the method according to the present invention is wherein said first fusion protein comprises a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) having the amino acid sequence denoted by SEQ ID NO. 2 and a reporter protein which is a beta-galactosidase domain having the amino acid sequence denoted by SEQ ID NO. 1.
In other specific embodiments the method according to the present invention is wherein said first fusion protein comprises from an amino-terminal position to a carboxyl-terminal position a reporter protein which is a beta-galactosidase domain having the amino acid sequence denoted by SEQ ID NO. 1, a linker having the amino acid sequence denoted by SEQ ID NO. 3, a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) having the amino acid sequence denoted by SEQ ID NO. 2 and a His tag having the amino acid sequence denoted by SEQ ID NO. 4.
In further embodiments the method according to the present invention is wherein said first fusion protein comprises an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence denoted by SEQ ID NO. 5.
In the above and other embodiments, the method according to the present invention is wherein said first fusion protein comprises or consists of the amino acid sequence denoted by SEQ ID NO. 5.
As detailed above, the method for identifying an agent that reduces binding of a clostridial neurotoxin to SV2 provided by the present invention, comprises bringing into contact a first fusion protein as herein described with a second protein. The second protein comprises the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof optionally fused to an expression facilitating domain.
As detailed above, the term receptor synaptic vesicle glycoprotein 2 (SV2) refers to a family of transporter-like proteins located in synaptic neurotransmitter-containing vesicles, of the three isoforms SV2A, SV2B, and SV2C. In specific embodiments the second protein according to the present invention comprises SV2C, SV2A or SV2B or a fragment thereof. In other specific embodiments the second protein according to the present invention comprises the receptor SV2C or a fragment thereof.
As detailed below, a fusion protein comprising the SV2C fragment referred to as the “fourth luminal loop of the synaptic vesicle glycoprotein 2C ((SV2C)” was prepared by the inventors.
By the term, the “fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2)” it is referred to the binding site of BoNT/A and/or BoNT/E on SV2, which was shown to be located on amino acids 529-566 of SV2C. Therefore, the term “fourth luminal loop of SV2” is defined as comprising a contiguous sequence of amino acids in SV2 encompassing the binding site of BoNT/A and BoNT/E, or any fragments of said fourth luminal loop of SV2 that maintain the binding ability to the toxin. Preparation of a fusion protein comprising the fourth luminal loop of SV2C is known to a person of skill in the art, for example based on the examples below.
In still further specific embodiments, the present invention relates to a second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2. In other specific embodiments the present invention relates to a second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2C. In still further specific embodiments, the second protein as herein defined comprises the fourth luminal loop of the synaptic vesicle glycoprotein 2C (SV2C) having the amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence denoted by SEQ ID NO. 13.
In specific embodiments the method according to the present invention is wherein said second protein comprises the fourth luminal loop of the BoNT receptor SV2C having the amino acid sequence denoted by SEQ ID NO. 13.
As indicated above the second protein comprises the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof optionally fused to an expression facilitating protein. In certain embodiments, said second protein is a fusion protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof fused to an expression facilitating protein. The term “expression facilitating protein” or “expression facilitating domain” refers to any protein that facilitates the recombinant expression and/or purification of the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof. Any expression facilitating protein known in the art is encompassed by the present invention. Commonly used expression facilitating proteins include but are not limited to glutathione-s-transferase domain (GST), Thioredoxin (Trx), maltose binding protein (MBP) and poly (NANP). The preparation of such fusion proteins is within the skills of a person of ordinary skill in the art.
As exemplified herein the second protein as herein defined comprises the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof fused to an expression facilitating protein which is a glutathione-s-transferase domain (GST).
Therefore, in specific embodiments the expression facilitating protein according to the present invention is a glutathione-s-transferase (GST).
By the term glutathione-s-transferase domain (GST) as known in the art and as herein defined it is referred to a family of eukaryotic and prokaryotic phase II metabolic isozymes best known for their ability to catalyze the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification.
In some embodiments the second protein as herein defined is a fusion protein which comprises GST having the amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence denoted herein by SEQ ID NO. 12.
In further specific embodiments the method according to the present invention is wherein said second protein is a fusion protein that comprises an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence denoted by SEQ ID NO. 15.
In still further embodiments the method as herein defined is wherein said second protein is a fusion protein that comprises or consists of the amino acid sequence denoted by SEQ ID NO. 15.
The term “protein” or “polypeptide” as used interchangeably herein and as known in the art refers to a molecular chain of amino acid residues, which can be optionally modified at one or more of its amino acid residues, for example by manosylation, glycosylation, amidation (for example C-terminal amides), carboxylation or phosphorylation. The polypeptide of the present disclosure may be obtained synthetically, through genetic engineering methods, expression in a host cell, or through any other suitable means as known to those of skill in the art. Methods for producing peptides or polypeptides are well known in the art, for example as exemplified below.
Amino acids as used herein, encompass both naturally occurring and synthetic amino acid residues, as well as amino acid analogs and amino acid mimetics. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate and O-phosphoserine. The term amino acid encompasses L-amino acids and D-amino acids, which are mirror images of L-amino acids, where the chirality at carbon alpha has been inverted.
The terms “amino acid sequence” or “polypeptide sequence” also relate to the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins. The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group.
As indicated above the present invention encompasses sequences which is at least 70% identical to a reference indicated sequence by at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence indicated as a reference sequence.
Thus, the present invention encompasses derivatives of the amino acid and nucleic acid disclosed herein namely fusion proteins, polypeptides or nucleic acid sequences which comprise any one of the amino acid sequences denoted herein but differ in one or more amino acid or nucleic acid residues in their overall sequence, namely, which have deletions, substitutions (e.g., replacement of at least one amino acid by another amino acid), inversions or additions within the overall sequence. This also encompasses the replacement of at least one amino acid residue in the overall sequence by its respective D amino acid residue.
By way of example, amino acid “substitutions” are the result of replacing one amino acid with another amino acid, for example with another amino acid that has similar structural and/or chemical properties (conservative amino acid replacements). Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, each of the following eight groups contains amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M).
Measuring the binding between the first fusion protein to the second protein as herein defined may be performed by any method known in the art, for example as exemplified below. Particularly, measuring the binding between the first fusion protein to the second protein as herein defined may be performed by exposure to an appropriate substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
For example, according to the method provided by the present disclosure, the resulting binding measured between the first fusion protein to the second protein as herein defined is compared to the binding performed in a control measurement in the absence of the sample as herein defined, wherein a lower binding than that of the control measurement indicates that said agent reduces binding of a clostridial neurotoxin to SV2.
By the term “control measurement” it is meant that the method is performed in the absence of the sample. In other words, the control measurement relates to measuring the binding between the first fusion protein and the second protein as herein defined under such conditions that allow binding to occur between the two fusion proteins.
In some embodiments the method according to the present invention is wherein at least one of said first fusion protein and said second protein is immobilized onto a solid surface.
By the term “solid surface” it is meant to refer to a solid phase support, solid phase carrier, solid support, solid carrier, support, or carrier namely any support or carrier capable of binding at least one of said first fusion protein and said second protein. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support or carrier configuration may be spherical, as in a bead, cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding at least one of said first fusion protein and said second protein or will be able to ascertain the same by use of routine experimentation.
In further embodiments the method according to the present invention is wherein said second protein is immobilized onto a solid surface.
As detailed above, measuring the binding between the first fusion protein to the second protein as herein defined may be performed by any method known in the art. In certain embodiments the method according to the present invention is wherein said step of measuring the binding comprises adding at least one substrate of the reporter protein of said first fusion protein.
By the term “substrate” in the context of the present invention it is meant to include any molecule which will react with the reporter protein in such a manner to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric, luminometric, or by visual means.
As detailed above, when the reporter protein is a beta-galactosidase domain, detection of receptor-bound Hc fragment may be accomplished by the addition of beta-galactosidase substrates, namely the chromogenic substrate ortho-nitrophenyl-ß-galactoside (oNPG) or the fluorogenic substrate 4-methylumbelliferyl-ß-galactoside (4-MUG).
Therefore, if the reporter protein used is a beta-galactosidase domain, the substrate may be any molecule cleaved thereby, for example but not limited to ortho-nitrophenyl-ß-galactoside (oNPG), 4-methylumbelliferyl-ß-galactoside (4-MUG), fluorescein Di-ß-D-Galactopyranoside or naphthofluorescein di-(beta-D-galactopyranoside).
In other exemplary embodiments, the reporter protein is Beta-galactosidase and the substrate(s) thereof is oNPG/4-MUG, in further exemplary embodiments, the reporter protein is Luciferase and the substrate thereof is luciferin, in still further embodiments the reporter protein is FLAG-tag/HA-tag and the substrate is detected using as anti-FLAG/anti HA antibodies and in further exemplary embodiments, the reporter protein is GFP without requiring any substrate.
In specific embodiments the method as herein defined is wherein the reporter protein is beta-galactosidase domain and the at least one substrate is 4-methylumbellyferyl-galactopyranoside (4-MUG) or ortho-nitrophenyl-galactopyranoside (oNPG).
In an additional aspect, the present invention provides a method for identifying at least one agent that reduces binding of Botulinum neurotoxin A (BoNT/A) to the synaptic vesicle glycoprotein 2C (SV2C), wherein said method comprises the following steps:
(a) contacting a first fusion protein, said first fusion protein comprising BoNT/A Hc fragment and a beta-galactosidase domain with at least one sample potentially comprising said agent, thereby obtaining a mixture;
(b) contacting said mixture obtained in step (a) with a second protein optionally attached to a surface, said second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2C (SV2C) and optionally a glutathione-s-transferase domain (GST);
(c) adding at least one substrate of the beta-galactosidase domain of said first fusion protein; and
(d) determining the signal;
wherein a signal lower than a threshold signal indicates that said agent reduces binding of BoNT/A to SV2C.
By the term “signal” as used herein it is meant to encompass any detectable indication or sign for the activity of the reporter protein (or residual activity as termed in the examples below), resulting from the metabolites formed during activity of the reporter protein on the substrate thereof. The signal may be, for example, a fluorescent signal or colorimetric signal depending on the nature of the substrate and resulting metabolite.
The signal obtained upon adding at least one substrate of the reporter protein, for example of the beta-galactosidase domain, may be determined or measured by any method known in the art (e.g., using a spectrophotometer) based on the type of substrate (and resulting metabolite) used. For example, when the product of the substrate is fluorescent (e.g., as in the case of the substrate 4-MUG) the signal is fluorescence and when the metabolite of the substrate is colorimetric (e.g., as in the case of the substrate oNPG), the signal is colorimetric.
The term “threshold signal” as herein defined refers to a positive control signal or residual activity of the reporter protein, namely to a signal obtained in the absence of an agent, under conditions that allow binding of the first fusion protein as herein defined, which comprises a BoNT Hc fragment to the second protein as herein defined, which comprises SV2 or any fragment thereof. Threshold signal (or positive control) may be determined under the conditions described herein, based on the signal obtained upon adding at least one substrate of the reporter protein (e.g., a beta-galactosidase domain).
As detailed above, by using the method defined herein the inventors were able to screen a compound library and discover several compounds that inhibited BoNT/A binding to its receptor SV2C. As shown below, for these compounds a signal lower than the threshold signal was obtained. Briefly, as shown in Table 4 below, wells in column 12 (E-H) were all positive control (“no-compound” wells or threshold signal). While in most of the wells a similar signal (also termed “residual activity value” in the examples below) was obtained, a substantially lower signal (residual activity) was present in well F5, and therefore it contained a potential inhibitor of the binding between the ß-gal-Hc/A fusion protein and the SV2C fusion receptor protein, namely a potential inhibitor of botulinum toxin A binding to the SV2C receptor.
The term “lower” or “lower signal” in the context of the present invention means a signal decreased, reduced, lessened, or diminished by at least about 1%-100%, about 5%-95%, about 10%-90%, about 15%-85%, about 20%-80%, about 25%-75%, about 30%-70%, about 35%-65%, about 40%-60% or about 45%-55%. Said decreased, reduced, lessened or diminished signal may also be by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100%.
When performing the method as herein described, a signal lower than a threshold signal indicates that said agent reduces binding of a clostridial neurotoxin (e.g., BoNT/A) to SV2 (e.g., SV2C). In other words, when a signal (e.g., fluorescence or colorimetric) lower than a threshold signal (e.g., fluorescence or colorimetric, respectively) indicates that said agent reduces binding of a clostridial neurotoxin to SV2 (e.g., BoNT/A to SV2C).
In some embodiments the method according to the present invention further comprises at least one washing step with a wash solution between steps (b) and (c). The preparation of a wash solution may be determined by a person of skill in the art, for example based on the examples below.
In other embodiments the method according to the present invention further comprises adding a stop solution after step (c). The preparation of a stop solution may be determined by a person of skill in the art, for example based on the examples below.
In certain embodiments the method according to the present invention is wherein said first fusion protein is contacted with at least one sample for one hour at room temperature. In other embodiments the method according to the present invention is wherein said second protein is contacted with said mixture obtained in step (a) for one hour at 37° C.
As detailed herein, by using the method described herein the inventors were able to screen a compound library in a high-throughput screening (HTS) approach and discover several compounds that inhibited BoNT/A binding to its receptor SV2C. Therefore, in some embodiments the method according to the present invention is wherein said method is used for high-throughput screening (HTS).
By the term “high-throughput screening (HTS)” it is referred to rapid, robust, reliable, and miniaturized methods to test thousands of compounds on a target in a short period, in an attempt to find active drugs. HTS relates to the identification of active compounds within large compound libraries.
As detailed above measuring the binding between the first fusion protein to the second protein as herein defined is performed according to the method provide by the present invention in the presence of a sample potentially comprising an agent.
The term “sample” is used in its broadest sense. In one sense it can refer to a culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. These examples are not to be construed as limiting the sample types applicable to the present invention. In specific embodiment the sample as used herein is a solution comprising an agent or a biological sample obtained from a subject.
In certain embodiments, the method of the invention may be particularly suitable for examination of a biological sample obtained from a subject, such as blood, lymph, serum, milk, plasma, plural effusion, urine, faeces, semen, saliva, brain extracts, spinal cord fluid (SCF), appendix, spleen, and tonsillar tissue extracts.
In some non-limiting examples, the sample encompassed by the present invention may be a blood, fractionated blood or plasma sample obtained from a subject that has been previously administered with a Botulinum neurotoxin A.
As known in the art, injections of Botulinum neurotoxin A (also referred to as “BOTOX”) are available for cosmetic and medical purposes (such as uncontrolled muscle spasms). Botox is used for three main purposes: muscle spasm control, severe underarm sweating, and cosmetic improvement. Injection of BOTOX (BoNT/A) during a cosmetic or medical procedure may result in generating neutralizing antibodies in the subject's body, such that will eventually neutralize the action of further injections of BoNT/A and render the cosmetic or medical procedure comprising thereof non-useful to the subject being treated.
By specifically applying the method as herein defined on a sample obtained from a subject that has been previously administered with BOTOX (Botulinum neurotoxin A), the method herein defined may be used for diagnosis of neutralizing antibodies in samples obtained from a subject that has been previously administered with a botulinum neurotoxin A.
Therefore, in some embodiments the method as herein defined is for identifying a neutralizing antibody directed against BoNT/A in a biological sample.
In certain embodiments the method according to the present invention is wherein said sample is a biological sample obtained from a subject and wherein said agent is an antibody.
By the term “subject” as used herein it is referred to warm-blooded animals, such as for example rats, mice, dogs, cats, guinea pigs, primates, and humans. In certain embodiments, the subject as referred by the present invention is a subject that has been previously administered with BOTOX (Botulinum neurotoxin A) for at least one of muscle spasm control, severe underarm sweating, and cosmetic improvement.
The present invention therefore encompasses a method for selecting a subject suitable for repeated BoNT/A injection. By the term “selecting” it is meant applying the methods as herein defined to identify a neutralizing antibody directed against BoNT/A in a biological sample obtained from the subject, wherein such neutralizing antibody is detected, the subject is considered not suitable for repeated BoNT/A injection.
The term “antibody” refers to a polypeptide encoded by an immunoglobulin gene or functional fragments thereof that specifically binds and recognizes an antigen, namely botulinum toxin. Methods for identifying antibodies are known to those of skill in the art.
By another one of its aspects the present invention provides a kit comprising:
(a) a first fusion protein, said first fusion protein comprising a receptor binding domain of Botulinum neurotoxin (BoNT Hc fragment) fused to a reporter protein or a composition comprising said first fusion protein and a suitable carrier or excipient;
(b) a second protein, said second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof optionally fused to an expression facilitating domain or a composition comprising said second protein and a suitable carrier or excipient; optionally, said second protein is attached to a solid surface; and optionally
(c) instructions for use.
In certain embodiments the kit according to the invention is for use in identifying an agent that reduces binding of a clostridial neurotoxin (for example Botulinum neurotoxin A, E, D, F, or tetanus neurotoxin) to synaptic vesicle glycoprotein 2 (SV2).
In some embodiments the kit according to the present invention is wherein said reporter protein is a beta-galactosidase domain.
In other embodiments the kit according to the present invention further comprises at least one substrate as herein defined for said beta-galactosidase domain.
In further embodiments the kit according to the present invention is wherein said first fusion protein comprises a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment).
In still further embodiments the kit according to the present invention is wherein said second protein comprises the BoNT receptor synaptic vesicle glycoprotein 2C (SV2C).
In specific embodiments the kit according to the present invention is wherein said expression facilitating domain is a glutathione-s-transferase (GST).
In certain embodiments the kit according to the present invention is wherein said first fusion protein comprises an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 5.
In some embodiments the kit according to the present invention is wherein said first fusion protein comprises or consists of the amino acid sequence as denoted by SEQ ID NO. 5.
In other embodiments the kit according to the present invention is wherein said second protein comprises the fourth luminal loop of SV2C having the amino acid sequence denoted by SEQ ID NO. 13.
In further embodiments the kit according to the present invention is wherein said second protein is a fusion protein comprising an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 15.
In still further embodiments the kit according to the present invention is wherein said second protein is a fusion protein comprising or consisting of the amino acid sequence denoted by SEQ ID NO. 15.
In certain embodiments the kit according to the present invention is wherein said at least one substrate is 4-methylumbellyferyl-galactopyranoside (4-MUG) or ortho-nitrophenyl-galactopyranoside (oNPG).
By another one of its aspects the present invention provides an isolated fusion protein comprising the receptor binding domain of Botulinum neurotoxin and a reporter protein as herein defined.
The term “isolated” refers to molecules, such as the amino acid sequences described herein, peptides or polypeptides that are removed from their natural environment, isolated, or separated.
In certain embodiments the isolated fusion protein according to the present invention comprises the receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment), Botulinum neurotoxin E (BoNT/E Hc fragment), Botulinum neurotoxin D (BoNT/D Hc fragment) or of Botulinum neurotoxin F (BoNT/F Hc fragment).
In specific embodiments the isolated fusion protein according to the present invention is wherein said isolated fusion protein comprises the receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment).
In other embodiments the isolated fusion protein according to the present invention is wherein said reporter protein is a beta-galactosidase domain as herein defined.
In further embodiments the isolated fusion protein according to the present invention is wherein said beta-galactosidase domain is situated N-terminal to said BoNT/A Hc fragment.
In still further embodiments the isolated fusion protein according to the present invention is wherein said isolated fusion protein further comprises a linker as herein defined situated between said beta-galactosidase domain and said BoNT Hc fragment.
In certain embodiments the isolated fusion protein according to the present invention is wherein said isolated fusion protein further comprises at least one heterologous amino acid sequence as herein defined at its N- and/or C-terminus.
In specific embodiments the isolated fusion protein according to the present invention is wherein said isolated fusion protein comprises a His tag sequence as herein defined at its C-terminus.
In certain embodiments the isolated fusion protein according to the present invention is wherein said isolated fusion protein comprises the BoNT/A Hc fragment having an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 2 and wherein said reporter protein is a beta-galactosidase domain having the amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 1.
In certain embodiments the isolated fusion protein according to the present invention is wherein said isolated fusion protein comprises the BoNT/A Hc fragment having the amino acid sequence denoted by SEQ ID NO. 2 and wherein said reporter protein is a beta-galactosidase domain has the amino acid sequence denoted by SEQ ID NO. 1.
In specific embodiments the isolated fusion protein according to the present invention is wherein said isolated fusion protein comprises from an amino-terminal position to a carboxyl-terminal position a beta-galactosidase domain having the amino acid sequence denoted by SEQ ID NO. 1, a linker having the amino acid denoted by SEQ ID NO. 3, a receptor binding domain of Botulinum neurotoxin A (BoNT/A Hc fragment) having the amino acid sequence denoted by SEQ ID NO. 2 and a His tag having the amino acid sequence denoted by SEQ ID NO. 4.
In some embodiments the isolated fusion protein according to the present invention is wherein said isolated fusion protein comprises an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 5.
In other embodiments the isolated fusion protein according to the present invention is wherein said isolated polypeptide comprises or consists of the amino acid sequence denoted by SEQ ID NO. 5.
The present invention further provides an isolated nucleic acid sequence encoding the fusion protein as herein defined.
The term “nucleic acid” or “nucleic acid molecule” as herein defined refers to polymer of nucleotides, which may be either single- or double-stranded, which is a polynucleotide such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. The term DNA used herein also encompasses cDNA, i.e. complementary or copy DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase).
Still further the present invention provides a vector comprising the nucleic acid sequence as herein defined.
The term “vector” sometimes referred to as “expression vehicle”, “expression construct”, or “expression vector” as used herein, encompasses vectors such as plasmids, viruses, bacteriophage, DNA fragments capable of being integrated, and other vehicles, which enable the integration of DNA fragments into the genome of the host. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. The expression vector in accordance with the invention may be competent with expression in bacterial, yeast, or mammalian host cells, to name but few.
The present invention further provides a host cell comprising the vector as herein defined.
The term “host cell” or the term “host cells” as used herein refers to cells which are susceptible to the introduction of the isolated nucleic acid molecule according to the present invention or with the expression vector according to the invention. Preferably, said cells are cells used in the present disclosure. Transfection of the isolated nucleic acid molecule or the expression vector according to the invention to the host cell may be performed by any method known in the art.
By still another one of its aspects the present invention provides a composition comprising the isolated fusion protein as herein defined and a suitable carrier or excipient.
The composition of the present invention generally comprises the first fusion protein and/or or the second protein as herein defined and a buffering agent, an agent which adjusts the osmolarity of the composition and optionally, one or more pharmaceutically acceptable carriers, excipients and/or diluents as known in the art. Supplementary active ingredients can also be incorporated into the compositions, e.g., antibiotics.
As used herein the term “suitable carrier or excipient” includes any solvents, dispersion media, coatings, antibacterial and antifungal agents and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
The term “about” as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range.
It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.
Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present disclosure to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way.
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.
A chimeric (fusion) protein construct was prepared as described below, consisting of two proteins: (1) beta-galactosidase from Escherichia coli; and (2) the receptor binding domain of BoNT/A, also designated the “Hc fragment” (or the C-terminal fragment of BoNT/A heavy chain). The fusion protein construct is referred to herein inter alia as the “I3-gal-Hc/A”. The sequence of the beta-galactosidase protein originating from E. coli BL21 is publicly available, for example as detailed in gene bank code CAQ30819 and is denoted herein by SEQ ID NO. 1. The sequence of the Hc fragment protein of BoNT/A originating from Clostridium botulinum strain 62A is publicly available, for example as detailed in gene bank code M30196, amino acids 872-1296, and is denoted herein by SEQ ID NO. 2. Beta-galactosidase (also referred herein as beta-gal or (3-gal) was set (i.e. positioned) on the N-terminus of the chimeric protein construct and the Hc-fragment on its C-terminus. The two proteins were connected by a flexible linker with the sequence (GGGGS)3, denoted herein by SEQ ID NO. 3, and a Hisx6 tag (denoted herein by SEQ ID NO. 4) was added to the C-terminus of the chimeric protein construct (namely C-terminal to the Hc-fragment) in order to facilitate its purification thereof using a nickel (Ni) column. The amino acid sequence of the chimeric protein construct prepared as described above is denoted by SEQ ID NO. 5 and a schematic presentation of the chimeric protein construct is shown in
Genes of clostridial origin are AT rich and their codon usage is different from that of E. coli. Therefore, a synthetic gene with optimized codon usage for protein expression in E. coli was prepared (GenScript). The gene was cloned into the expression vector pET-9a to obtain the plasmid pET-9a-ß-gal-Hc/A. The nucleic acid sequence encoding the chimeric protein construct prepared as described above, which was inserted into the plasmid pET-9a-ß-gal-Hc/A, is denoted herein by SEQ ID NO. 9.
An E. coli nucleic acid sequence encoding the beta-galactosidase protein is denoted herein by SEQ ID NO. 6. A nucleic acid sequence encoding the beta-galactosidase protein with optimized codon usage for protein expression in E. coli is denoted herein by SEQ ID NO. 7 and a nucleic acid sequence encoding the Hc fragment of BoNT/A is denoted herein by SEQ ID NO. 8.
The sequences detailed herein in connection with the preparation of the ß-gal-Hc/A fusion protein construct are detailed in Table 2 below.
E. coli β-gal
C.
botulinum
E. coli β-gal
E. coli β-gal
The plasmid pET-9a-ß-gal-Hc/A was transformed into E. coli BL21(DE3). A starter culture was prepared by inoculating a colony into 40 ml of TB media (comprising 24 g/l yeast extract; 12 g/l tryptone; 4 g/l glycerol; 89 mM potassium phosphate) with kanamycin (30 μg/ml) in an Ultra Yield 250 ml (Thomson Instrument Company) shake flask, and the flasks were incubated at 37° C., 250 rounds per minute (rpm). After 8 hours the starter culture was used to inoculate 1 liter of TB media with kanamycin, in two Ultra Yield 2.5 liter shake flasks, and the flasks were incubated at 18° C., 250 rpm for 40 hours. The culture was harvested by centrifugation.
The cell pellet was re-suspended in 100 ml binding buffer (20 mM imidazole; 20 mM sodium phosphate; 0.5 M NaCl; pH 7.4) and the cells were disrupted by sonication (Cole Parmer ultrasonic processor; 60% amplitude, 15 minutes). The cell extract was clarified by centrifugation (14,000 g, 30 min) and loaded onto a HisTrap FF 1 ml column (GE Healthcare) mounted on an AKTA Explorer FPLC system (GE Healthcare). The column was washed with 10 column volumes (CV) of binding buffer and 10 CV of binding buffer containing 40 mM imidazole. The protein was eluted from the column with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM Imidazole, pH 7.4). The pure protein was dialyzed against 50 mM sodium citrate buffer, pH 5.5, and stored at −70° C.).
The protein receptor for BoNT/A is Synaptic vesicle glycoprotein 2C (SV2C). Specifically, the toxin binds the fourth luminal loop of SV2C (6). A fusion receptor protein comprising SV2C and glutathione-s-transferase (GST) was prepared as follows. The gene encoding the fourth luminal loop of sv2c (having a nucleic acid sequence denoted herein as SEQ ID NO. 10) was fused to glutathione-s-transferase (gst) gene (having a nucleic acid sequence denoted herein as SEQ ID NO. 11) to facilitate purification and enhance its solubility. The amino acid (protein) sequence of GST was taken from pET-41a vector and has a sequence denoted herein as SEQ ID NO. 12. The amino acid (protein) sequence for the fourth luminal loop of SV2C was taken from Mus musculus and has a sequence denoted herein as SEQ ID NO. 13. The resulting fusion receptor polypeptide consists of GST on its N-terminus and the fourth luminal loop of SV2C on its C-terminus (GST-SV2C). Next, a synthetic gene with optimized codon usage for expression in E. coli was prepared for optimal expression. The gene encoding for the fusion receptor protein GST-SV2C, having a sequence denoted herein as SEQ ID NO. 14, was cloned into the expression vector pET-9a.
The plasmid pET-9a-gst-sv2c was transformed into E. coli BL21(DE3). A starter culture was prepared by inoculating a colony into 40 ml of TB media with kanamycin in an Ultra Yield 250 ml shake flask, and the flasks were incubated at 37° C., 250 rpm. After 8 hours the starter culture was used to inoculate 1 liter of TB media with kanamycin, in two Ultra Yield 2.5 liter shake flasks, and the flasks were incubated at 18° C., 250 rpm for 40 hours. The culture was harvested by centrifugation.
The cell pellet was re-suspended in 100 ml PBS and the cells were disrupted by sonication. The cell extract was clarified by centrifugation (14,000 g, 30 min) and loaded onto a GSTrap FF 5 ml column (GE Healthcare) mounted on an AKTA Explorer FPLC system (GE Healthcare). The column was washed with 15 CV of binding buffer to remove unbound proteins. The protein was eluted from the column with elution buffer (50 mM tris-HCl; 10 mM reduced glutathione, pH 8.0). The amino acid sequence of the GST-SV2C fusion receptor protein obtained is denoted herein as SEQ ID NO. 15.
The sequences detailed herein in connection with the preparation of the fusion receptor protein GST-SV2C are detailed in Table 3 below.
M.
musculus
Binding Analysis of β-gal-Hc/A to GST-SV2C
Analysis of binding of the fusion proteins ß-gal-Hc/A to GST-SV2C was performed as follows: a 96-well plate was coated with 50 μl per well of the fusion receptor protein GST-SV2C diluted 1:200 in coating buffer (50 mM Na2CO3, pH 9.6) and incubated overnight at 4° C. GST-SV2C was immobilized to the plate (using Na2CO3). The plate was then washed with wash solution (NaCl 0.9%, Tween 20 0.05%) and blocked for one hour at 37° C. with TSTA buffer (50 mM Tris, 0.9% NaCl, 0.05% Tween, 2% BSA, 200 μl per well). After washing, the plates were incubated for one hour at 37° C. with serial dilutions (from 200 to 204800) of the fusion protein ß-gal-Hc/A in TSTA, 50 μl per well. The plate was then washed and incubated with ortho-nitrophenyl-galactopyranoside (oNPG, sigma) solution (1 mg/ml) in Z-buffer (100 mM sodium phosphate, 10 mM KCl, 1 mM MgSO4, 50 mM ß-mercaptoethanol, pH 7.0) for one hour at 37° C. The reaction was stopped with stop solution (1 M sodium carbonate), and absorbance was measured at 420 nm. Alternatively, the fluorogenic substrate 4-methylumbellyferyl-galactopyranoside (4-MUG, sigma) (0.5 mg/ml; excitation 355 nm, emission 455 nm) was used.
A 96-well plate was coated with the GST-SV2C fusion receptor protein as detailed above and incubated overnight at 4° C. The plate was then washed with wash solution and blocked for one hour at 37° C. with TSTA buffer. In a different (separate) 96-wells polypropylene plate, the ß-gal-Hc/A fusion protein diluted 1:6000 with TSTA was incubated for one hour at room temperature with one type of the following horse antibodies (prepared by the inventors): anti BoNT/A, BoNT/B, BoNT/E or naive serum, diluted 1:1650 with TSTA. The ß-gal-Hc/A fusion protein—antitoxins mixtures were then transferred into the GST-SV2C coated plate, 50 μl per well, and the plate was incubated for one hour at 37° C. The plate was then washed and incubated with oNPG solution in Z-buffer (prepared as detailed above) for one hour at 37° C. The reaction was stopped with stop solution, and absorbance was measured at 420 nm. The residual activity of ß-gal was calculated by dividing the absorbance obtained in each one of the wells by the absorbance in a control well that did not contain antitoxin.
Binding assay in the presence of neutralizing antibodies
The assay described above was also performed in the presence of several monoclonal antibodies (A-1-A-8) directed against the receptor binding domain of BoNT/A, as well as in the presence of monoclonal antibodies directed against PA (anthrax protective antigen), BoNT/B and antibodies directed against BoNT/E. The monoclonal antibodies were produced in the Israel Institute for Biological Research (IIBR). The preparation of the antibodies is described in Diamant et al. (10).
Determination of neutralizing antibody concentration (NAC) was performed by incubating serial dilutions of a neutralizing antibody (directed against BoNT/A) sample for one hour with a test dose of botulinum toxin (type A), and then injecting the mixture to groups of four mice (CD-1, Charles River). The survival order of the injected groups was compared to that of groups injected with serial dilutions of a World Health Organization (WHO) standard with known NAC. One international unit (IU) of antitoxin neutralizes at least 104 LD50 toxin.
Plates (96-well) were coated with the Hc fragment of BoNTA (Hc/A, having the amino acid sequence denoted herein by SEQ ID NO. 2) solution (10 μg/ml in coating buffer, 50 μl per well) and incubated overnight at 4° C. Following washing, the plates were blocked with TSTA (200 μl per well). The plates were then washed and loaded with serial dilution of plasma samples containing horse anti BoNT/A antibodies (50 μl per well). Following incubation (37° C., 1 hour) the plates were washed and incubated with alkaline phosphatase conjugated goat anti horse IgG (Jackson ImmunoResearch). Finally, the plates were washed with wash solution, and the colorimetric reaction was developed using the substrate p-nitrophenyl phosphate (1 mg/ml in 0.2 M Tris buffer). Following 15 minutes of incubation (37° C.) absorbance was measured at 405 nm. The titers were defined as the highest dilution of the samples for which the signal was above 0.4.
High throughput screening (HTS) was conducted for the Library Of Pharmacologically Active Compounds (LOPAC1280, Sigma), which contains 1280 active compounds in DMSO (10 mM solutions). The screening procedure was performed as follows: first, a dilution plate of the tested compounds was prepared by 10-fold dilution to 1 mM with a solution of 50% DMSO and 50% PBS. Next, a polypropylene 96-wells plate was filled (90 μl per well) with the fusion protein ß-gal-Hc/A (diluted 1:90,000 in TSTA). The diluted compounds (10 μl per well) were then mixed with the ß-gal-Hc/A fusion protein solution and the plate was incubated for 1 hour at 25° C. with shaking (200 rpm). Control wells were prepared as follows: 1. No compound—100% activity; 2. No ß-gal-Hc/A fusion protein—zero activity. The mixtures of ß-gal-Hc/A fusion protein with the tested compounds were then transferred (50 μl per well) into 96-wells polystyrene plates coated with the fusion receptor protein GST-SV2C, and the plate was incubated one hour at 37° C. The wells were washed with a wash solution and a substrate solution (50 μl per well of 4-MUG 0.5 mg/ml in buffer Z) was next added, and the plate was further incubated one hour at 37° C. The reaction was stopped with stop solution (50 μl per well) and the fluorescence was measured. For each one of the wells the residual ß-gal activity was calculated using the following equation:
Residual activity=100×(WF−BF)/PCF
Where WF is the fluorescence of the well, BF is the fluorescence of the blank, and PCF is the fluorescence of the positive control 100% activity.
A ß-gal-Hc/A chimeric (fusion) protein construct was designed, composed of the receptor binding domain of BoNT/A (also termed herein the “Hc-fragment”) fused to the reporter enzyme beta-galactosidase derived from Escherichia coli, as detailed above. The amino acid sequence of the chimeric ß-gal-Hc/A protein construct comprising the receptor binding domain of BoNT/A and Escherichia coli beta-galactosidase is denoted herein by SEQ ID NO. 5 and is detailed in Table 2 above.
In addition, the inventors have used a GST-SV2C fusion receptor protein construct, namely a receptor construct based on the fourth luminal loop of the synaptic vesicle protein (SV2C) conjugated to a GST protein. The amino acid sequence of the GST-SV2C receptor construct is denoted herein by SEQ ID NO. 15 and is detailed in Table 3 above.
Detection of receptor-bound Hc fragment was accomplished with the addition of the chromogenic substrate ortho-nitrophenyl-ß-galactoside (oNPG) or the fluorogenic substrate 4-methylumbelliferyl-ß-galactoside (4-MUG) as detailed above. When the colorimetric substrate oNPG was used for measuring the presence and activity of the ß-gal-Hc/A construct, the recorded absorbance was proportional to the ß-gal-Hc/A fusion protein dilution from a dilution of 1:200 to 1:6400. Above a dilution of 1:6400 the absorbance was similar to blank wells with TSTA buffer alone, as shown in
Specificity of Anti-BoNT Antibodies in Prevention of ß-Gal-Hc/A Binding to GST-SV2C
The neutralizing mechanism of BoNTs by antitoxins (namely by antibodies) is mainly based on prevention of binding of the toxins to their receptors (2, 7). According to the definition of BoNT serotypes, antibodies generated towards a single serotype are able to neutralize only this specific serotype, and not different serotypes. Therefore, as a means for evaluating the binding assay described herein, as well as the specificity thereof, the ability of the following horse-derived antibodies, anti BoNT/A, anti BoNT/B and anti BoNT/E antibodies to prevent binding of ß-gal-Hc/A to GST-SV2C was evaluated.
The binding assay in the presence of horse anti BoNT/A, anti BoNT/B and anti BoNT/E antibodies was conducted as described above. As demonstrated in
As an additional means for evaluating the binding assay described herein, the inventors determined the neutralizing antibody concentration (NAC) of antibodies specific to BoNT/A using the binding assay, as described below, and compared the results obtained thereby to results obtained by other acceptable evaluation methods.
First, the potency of botulinum antitoxin (antibodies) preparation was determined using a pharmacopoeial mouse neutralization assay (MNA, (8)), as detailed above. The MNA assay suffers from several drawbacks, as follows: use of high toxin doses dictates stringent safety restriction, the assay raises ethical issues as it uses numerous laboratory animals, assays conducted in animals are generally characterized by high variability and finally, the duration of the mouse neutralization assay is very lengthy (i.e. four days).
In the currently available in vitro antibody evaluation method, the ELISA titers of antibodies which bind BoNTs do not correlate with NAC and therefore no simple in vitro assay is available in the prior art to determine NAC. Without wishing to be bound by theory, the reason for the poor correlation is that binding of antibodies alone is not enough for toxin neutralization. ELISA measures binding of any antibody to any epitope of BoNT. It is currently known that not any antibody-epitope binding event eventually results in neutralization of BoNT. Most of the neutralizing antibodies directed against BoNTs bind the Hc fragment and thereby prevent binding of the toxin to its receptor. Since the ß-gal-Hc/A assay specifically measures the binding of the receptor binding domain of BoNT/A to the receptor SV2C, it may be used to identify neutralizing antibodies and determine neutralizing antibody concentration.
Thus in order to evaluate the in vitro method described herein, plasma samples of horse vaccinated against BoNT/A and antitoxin standard (namely antibodies) of known potency (330 IU/ml) were serially diluted (dilution factor of 1.5) in TSTA and mixed in 1:1 ratio with the fusion protein ß-gal-Hc/A solution (diluted 1:1000 with TSTA). The mixtures were incubated for one hour at room temperature, then transferred to 96-wells plates coated with the fusion receptor protein GST-SV2C and the plate was incubated one hour at 37° C. After incubation, the plate was washed with wash solution and a substrate solution (oNPG 1 mg/ml in buffer Z) was added. Following incubation for one hour at 37° C. the reaction was stopped by addition of stop solution and absorbance at 420 nm was measured. The NAC in the unknown samples (horse vaccinated samples) were determined based on interpolation from the standard curve prepared for the antitoxin (antibodies) of known potency.
The chimeric (fusion) protein ß-gal-Hc/A prepared as described above allows fast and simple detection and quantitation of the binding between the receptor binding domain of BoNT/A and its receptor SV2C, while using standard equipment (e.g. a plate reader) and without use of toxic substances. The above characteristics enabled the development of a high-throughput screening (HTS) assay for identifying binding inhibitors from a compound library. In other words, the HTS assay described herein may be used inter alia for screening of agents that interfere with binding of botulinum toxin A to the SV2C receptor. Briefly, the ß-gal-Hc/A fusion protein was mixed with the examined compounds and then incubated with the GST-SV2C fusion receptor protein. Following removal of unbound ß-gal-Hc/A the activity of ß-galactosidase was determined. Inhibitors of toxin-receptor binding will decrease ß-gal activity in comparison to a positive control.
Since in most compound libraries the compounds are dissolved in DMSO, the susceptibility of the ß-gal-Hc/A fusion protein to DMSO was first examined. The ß-gal-Hc/A fusion protein was diluted 1:100,000 with TSTA and then mixed 1:1 with PBS and DMSO solutions to obtain final DMSO concentrations in the range of 0-50%. The mixtures were incubated one hour at room temperature, afterwards the content was transferred into 96-wells plate coated with the GST-SV2C fusion receptor protein and the plate was incubated one hour at 37° C. Following washing of the plate with wash solution, a substrate solution (4-MUG, 0.5 mg/ml) was added to the wells. The plate was incubated for one hour at 37° C., afterwards the stop solution was added and fluorescence was measured. Reduction in fluorescence was obtained for DMSO concentration higher than 8%, and for 5% DMSO the fluorescence intensity was similar to a control sample without DMSO.
Next, the HTS was conducted for the Library Of Pharmacologically Active Compounds (LOPAC1280, Sigma), as detailed above.
Table 4 below shows the results of exemplary residual activity obtained for plate 1 of LOPAC1280. Wells in column 1 (A-H) were all blank wells. Wells in column 12 (E-H) were all positive control (“no-compound” wells). In most of the wells similar residual activity values were obtained. Interestingly, a substantially lower residual activity was present in well F5, and therefore it contained a potential inhibitor of the binding between the ß-gal-Hc/A fusion protein and the SV2C fusion receptor protein, namely a potential inhibitor of botulinum toxin A binding to the SV2C receptor.
Additional results of exemplary residual activities obtained for compounds of the LOPAC1280 library are shown below in tables 5 and 6. As shown both in Table 4 and in table 5, substantially lower residual activity was present in well H4 of both plates, therefore these wells measured the activity of potential inhibitors of the binding between the ß-gal-Hc/A fusion protein and the SV2C fusion receptor protein, namely further potential inhibitors of botulinum toxin A binding to the SV2C receptor.
Most neutralizing antibodies of BoNT are directed towards the receptor binding domain of the toxin. Since the assay presented in this invention measures the binding of BoNT/A to its receptor SV2C, it can be applied to evaluate neutralizing antibodies.
The assay presented above was next used to determine the inhibition potential of BoNT/A binding to its receptor SV2C by several monoclonal antibodies, as shown in
As demonstrated in
In contrast, only a minor interference to ß-gal-Hc/A-GST-SV2C interaction was observed for antibodies directed against PA (anthrax protective antigen), BoNT/B or BoNT/E, demonstrating the assay specificity.
To evaluate neutralizing antibodies in patients, samples (such as human serum or plasma) is serially diluted with TSTA buffer in 96-well plate. Afterwards, ß-gal-Hc/A is added to the wells and the plate is incubated for one hour at 25° C. This step allows the neutralizing antibodies to bind their epitopes on the Hc/A fragment. Then, the mixtures of the serum (or plasma) with ß-gal-Hc/A are transferred to another 96-well plate previously coated with GST-SV2C, and the plate is incubated for one hour at 37° C. This step allows non-neutralized Hc/A fragment to bind SV2C. Following removal of unbound ß-gal-Hc/A, by washing the plate with wash solution as described above, a synthetic substrate for β-galactosidase, such as 4-MUG or oNPG, is added to the plate and the plate is incubated for one hour at 37° C. The reactions are stopped by addition of stop solution and absorbance or fluorescence are measured, using a plate reader, as described above. To translate absorbance (or fluorescence) values to neutralizing antibody concentration the assay includes a standard curve. The standard sample consists of a well-characterized serum, having a known neutralizing antibody concentration, and it is serially diluted in a similar manner to the samples.
botulinum B
botulinum B
botulinum C
botulinum C
botulinum D
botulinum D
botulinum E
botulinum E
botulinum F
botulinum F
botulinum G
botulinum G
musculus)
musculus)
musculus)
| Number | Date | Country | Kind |
|---|---|---|---|
| 272002 | Jan 2020 | IL | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2021/050039 | 1/13/2021 | WO |
