# Patent

## Title
Methods and compounds for increasing sensitivity of botulinum assays

## Abstract

Apparatus, systems and methods can provide improved detection of botulinum neurotoxins. In one aspect an isoquinolynyl compound can be used to enhance the sensitivity of both Förster resonance energy transfer (FRET) and non-FRET cell-based assays. In another aspect, non-FRET assays and constructs utilize a reporter that is not coupled with the second fluorophore in a manner that produces significant FRET. In that subject matter an environment cell can include an enzyme that facilitates degradation of the reporter significantly faster after the cleavage than before the cleavage, and presence of the Botulinum toxin correlates with reduction of the signal from a baseline signal. Where the environment is a cell, the cell can advantageously express both the construct that includes the reporter, and an enzyme that facilitates the degradation.

## Background
PRIORITY CLAIM
This application claims priority to U.S. provisional application Ser. No. 61/582,940, filed Jan. 4, 2012, and U.S. utility application Ser. No. 13/485,537, filed May 31, 2012, the disclosures of which is incorporated herein by reference in their entirety. These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.


FIELD OF THE INVENTION
The field of the invention is detection of Botulinum Neurotoxins (BoNTs).
BACKGROUND
Botulinum Neurotoxins are the most lethal substances known, and depending on the serotype, the estimated human lethal dose ranges from 1 to 3 ng toxin per kg body weight. Due to their ease of purification and high potency, BoNTs pose a real and potential threat for use as bioweapons. Further, BoNTs are increasing used for cosmetic and therapeutic purposes. Currently, the majority of BoNT detection and potency measurements completed in government and industrial laboratories are done using animal-based assays that suffer from high costs and poor accuracy.
The primary advantage of using cell-based assays (CBAs) for BoNT potency measurements is that CBA systems closely mimic the individual physiologic steps that occur in neurons during intoxication. BoNTs are zinc-dependent endopeptidases composed of a heavy chain, responsible for neuron-specific receptor binding and cell entry, and a catalytic light chain responsible for synaptic protein cleavage. Upon entry into neurons, the BoNTs specifically disrupt the protein machinery responsible for fusion of synaptic vesicles with the plasma membrane, thereby inhibiting neurotransmitter release into the post-synaptic junction. Because all physiologic steps must be accounted for in CBAs, most assays fail to meet the sensitivity requirements of BoNT activity measurement.
The employment of established, stable model cell lines for the detection of BoNTs have recently been described, however, past CBAs fail to meet the sensitivity requirements for the quantification of pharmaceutical preparations of BoNT or the detection of BoNT clinical samples. Presumably, current established model cell lines lack critical neuronal characteristics required for efficient BoNT uptake. A recent solution to increasing BoNT sensitivity is the use of mouse embryonic stem cells (mESCs). Because these cells can be terminally differentiated into neurons they are more sensitive to BoNT treatment. A major drawback to this method, though, is the multiple weeks required to fully differentiate mESCs.
Thus, there is an urgent need to development cell based assays for assessing and quantifying the potency of BoNT-containing samples for bioweapon defense, food home illness, and therapeutic purposes.
SUMMARY OF THE INVENTION
In one aspect, the inventive subject matter provides apparatus, systems and methods in which isoquinolynyl compounds are used to enhance the sensitivity of cell-based assays for botulinum neurotoxins. In preferred embodiments, the compound comprises 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dichloride (H7). The reporter is preferably an artificial construct expressed in the cell, and having a first fluorophore selected from the group consisting of Yellow Fluorescent Protein (YFP), Citrine, Venus, and a YPet protein, and a he cleavage site comprising at least one of a SNARE protein, motif and mutein.
Also contemplated are assays in which cells are incubated in a culture medium containing B27.
Another aspect of the inventive subject matter involves non-FRET assays and constructs, in which the reporter is not coupled with the second fluorophore in a manner that produces significant Förster resonance energy transfer (FRET). In such embodiments, a cell can advantageously express an enzyme that facilitates degradation of the reporter at least ten times faster after the cleavage than before the cleavage, and presence of the Botulinum toxin correlates with reduction of the signal from a baseline signal. As used herein, the term “non-FRET assays and constructs” refers to assays and constructs either (a) having no detectable FRET signal, or (b) producing a FRET signal with a FRET transfer efficiency of less than 5%, according method E1 (the relative fluorescence of the donor in presence (FDA) and absence (FD) of the acceptor E1=1−FDA/FD) as described in “FRET or No FRET: A Quantitative Comparison”, Claude Berney and Gaudenz Danuser, Biophys J. 2003 June; 84(6): 3992-4010, It is contemplated that any FRET signal FRET having transfer efficiency of less than 5% is at such an insignificant level that it is not practical to use the signal to provide a quantitative measurement on concentration of a FRET-coupled component.
In some embodiments an artificial construct having a reporter-containing portion chemically is coupled to a cleavage site that interacts with an investigational substance in a manner that cleaves the reporter-containing portion from a remainder of the construct, and the reporter-containing portion is degraded in the cytosol.
A particular class of embodiments is directed to methods for the qualitative and quantitative detection of a Botulinum toxin, comprising:

    
    
        (i) providing a composition that includes an artificial construct and an enzyme,
        
            wherein the construct has (a) a reporter-containing portion and (b) a cleavage site that interacts with a portion of the Botulinum toxin in a manner that produces a cleavage of the reporter-containing portion from a remainder of the construct,
            wherein the composition exhibits emissions identifying a baseline signal; and
            wherein the enzyme facilitates degradation of the reporter-containing portion faster by at least two, at least five, at least ten or at least twenty times after the cleavage than before the cleavage;
        
        
        (ii) obtaining first emission measurements from said baseline signal
        (iii) exposing the composition to the Botulinum toxin; and then
        (iv) Obtaining further emission measurements by testing the composition for a reduction of the baseline signal as an indication of
        
            the toxin cleaving the construct at the cleavage site, and
            degradation of the reporter-containing portion;
        
        
        (v) comparing the first emission measurements of step (ii) with the further measurements of step (iv).
    
    


The inventive subject matter for non-FRET assays also relates to an assay for testing presence of an investigative substance, comprising a cell having an artificial construct and an enzyme, wherein the construct has a protected portion and a protecting portion, selected such that the protected portion includes a reporter, and the investigative substance acts to de-protect the protected portion in vitro, wherein the enzyme facilitates degradation of the protected portion by at least two, at least five, at least ten or at least twenty times when de-protected than when protected, thereby reducing a signal obtainable from the reporter which is used for the quantitative determination of the investigative substance.
The cleaved reporter-containing portion is destroyed or otherwise degraded by the local environment, and presence of the investigational substance is then evidenced by reduction in signal from the reporter. In the context of this application, it is contemplated that degradation of the protected portion will typically intracellularly by at least one of two pathways, by the ubiquitin-dependent process that targets proteins to the proteasome, or by the autophagy-lysosomal pathway. In one pathway, the proteasome is the enzyme. In the lysome pathways, it is contemplated that the enzymes of interest are hydrolases, including especially a family of proteases called the cathepsins.
In other aspects of preferred embodiments, the cleavable reporter-containing portion comprises a fluorescent protein, as for example, Yellow Fluorescent Protein (YFP). YFP is a genetic mutant of green fluorescent protein, derived from Aequorea Victoria, and has an excitation peak is 514 nm and emission peak is 527 nm.
Also contemplated for use in the cleavable reporter-containing portion are the closely related Citrine, Venus, and YPet proteins. The modifications have reduced chloride sensitivity, faster maturation, and increased brightness (product of the extinction coefficient and quantum yield). Of course, any of the fluorescent proteins mentioned herein can be modified to include specific characteristics (e.g., spectral) or be truncated to a specific size.
Upon cleavage, the construct is cleaved into two parts, a reporter containing portion that is destroyed or otherwise degraded by the cytosol or other local environment, and a second portion. To normalize the signal detection, that second portion can advantageously include a second fluorescent protein, preferably at an opposite end from the reporter, which can assist in normalizing the assay. The second fluorescent protein can, for example, be Cyan Fluorescent Protein (CFP), mCherry, or mStrawberry. The reporter-containing portion is not coupled with the second fluorophore in a manner that produces Förster resonance energy transfer (FRET).
Thus, prior to exposure of the construct to BoNT or other cleaving substance, the composition containing the construct (whether cell-based or otherwise) exhibits a baseline signal, and then after exposure exhibits a reduced signal. For the measurement of YFP degradation, directly, separately excited YFP emissions (top, Ex500, Em526) and CFP emissions (middle, Ex434, Em470) are collected. Those emissions are then background subtracted and the YFP emission is divided by CFP emission to control for cell density and reporter expression in the individual cells. That emission ratio (YFP/CFP, bottom) is how the assay is reported.
Destruction or other degradation of the reporter-containing portion takes place at a much faster rate post-exposure to BoNT than pre-exposure. In preferred embodiments, it is contemplated that the destruction or other degradation of the reporter containing portion occurs at least 2× (twice) as fast post-exposure as pre-exposure, but more preferably the rate post-exposure rate is at least 5×, at least 10×, at least 100× relative to the pre-exposure rate.
In yet other aspects of preferred embodiments, the local environment is the cytosol of a living cell. For example, YFP can be used as the C-terminal fluorophore, and CFP can be used the N-terminal fluorophore. Experimental work has now verified that in the absence of BoNT/A, YFP can be directly excited and the emission collected. The excitation occurs typically at 505 nm and the corresponding emission at 527 nm. In the presence of BoNT/A, the reporter is cleaved releasing a fragment containing YFP. That fragment is degraded by the cell, destroying the YFP and its emission. Thus, BoNT/A activity is detected by measurements relative to the loss in YFP emission. No FRET is therefore required, what avoids all the limits and the problems indicated above.
Local environments for the non-FRET construct other than living cells are also contemplated, including for example, cytosol of lysed cells, and synthetic media that contains one or more enzymes capable of degrading the cleavable fragment when cleaved from the reporter molecule, but incapable or much less capable of degrading the cleavable fragment prior to cleavage from reporter molecule.
It is further contemplated to provide an isolated polynucleotide molecule encoding a construct described above. The construct is preferably an expression vector comprising the polynucleotide molecule operably linked to a promoter. A preferable promoter is an inducible promoter.
In a further embodiment, a kit comprises a construct as contemplated herein, in a suitable container.
The inventive subject matter also provides a method for screening for an inhibitor of a botulinum neurotoxin, comprising providing a cell genetically engineered to express a construct as described above, exposing said cell to the botulinum neurotoxin in the presence of a candidate inhibitor compound; and detecting a fluorescent or other signal of the cell before and after said exposing to the botulinum toxin, and comparing the signal to that of a cell exposed to the botulinum neurotoxin in the absence of the candidate inhibitor. To the extent that signal reduction was circumvented, the candidate inhibitor would be considered capable of inhibiting the botulinum neurotoxin. In some contemplated embodiments, the candidate compound could be a member of a library of compounds, and the method could be a high throughput method.
Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A, 1B, 1C, 1D, 1E are graphs showing that H7 treatment, but not calphostin, IBMX, db-cAMP, or 8-Br-cAMP treatment, cause an increase in BoCellTM Assay sensitivity.
FIGS. 2A and 2B are graphs showing that the H7 effect is dose dependent, and does not affect cell viability.
FIG. 3 is a graph showing that the effect of H7 pre-treatment (before BoNT/A addition). Here, H7 is included with BoNT/A incubation as well.
FIGS. 4A and 4B are graphs showing that 1) only holotoxin (complete BoNT/A) but not light-chain (BoNT/A fragment) elicits a response in the BoCellTM assay and 2) BoNT/A activity can be blocked by the addition of HcR/A which competes for cellular receptors. Thus, BoNT/A responses in the assay occur through natural mechanisms.
FIG. 5 is a graph showing that multiple H7 lots from different manufacturers all cause an increase in sensitivity. (There is a report in the literature that some H7 lots from certain manufacturers are not actually H7, thus we wanted to confirm the effect with multiple lots.)
FIG. 6 is a graph showing assay response and sensitivity in the presence of H7 following a 24 and 48 hour incubation with BoNT/A. We expect that limits of detection of <3 pM will be possible with assay optimization.
FIG. 7A is a schematic of a BoCellTM construct.
FIG. 7B is a schematic of an exemplary assay in which BoNT/A cell-based reporters are used to detect BoNT/A activity by loss of YFP fluorescence
FIG. 8A is an image of BoNT/A-induced changes in fluorescence responses.
FIG. 8B is a chart showing fluorescence ratios and BoNT/A sensitivities of the cell-based reporters.
FIG. 8C is a blot showing activity of BoNT/A in cells regardless of the reporter.
FIG. 9 comprises multiple charts showing emission data for both YFP degradation and loss of FRET according to the state of the art practice from the exact same plates of cells.



DETAILED DESCRIPTION
Cleavage Sequence(s)
In preferred embodiments, the investigational substance is a Botulinum toxin (BoNT), and the cleavage sequence is appropriately matched with the investigational substance. For example, the BoNT/A, E, and C cleave SNAP-25, and BoNT/B, D, F, G cleave synaptobrevin (Syb), at single but different sites. BoNT/C also cleaves syntaxin in addition to SNAP-25.
Contemplated cleavage site sequences can advantageously comprise (a) a SNARE protein, motif, or mutein, “Muteins” of a protein should be interpreted herein as having at least 30% identity with a corresponding native protein, including for example compositions having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identity with the native protein. Variations from identity can comprise any or more of additions, deletions and substitutions. Contemplated muteins include fragments, truncates and fusion proteins.
Transfected Cells
Hybrid protein(s) that are formed in the transfected cells preferably include a transmembrane domain, which tends to locate to intracellular vesicles for BoNT/B, D, F, and G, and thereby present a vesicle-bound substrate. Heavy chain-mediated endocytosis of the BoNT into the transfected cell is followed by presentation of the light chain on the outer surface of the vesicle, allowing the protease activity of the light chain to cleave the cleavage sequence of the hybrid protein(s), thus cleaving the reporter-containing portion, which then is destroyed or degraded to reduce the signal being tested. Full-length Syb, for example, contains 116 amino acids, and is localized to vesicles through a single transmembrane domain.
Hybrid protein(s) that are formed in the transacted cells preferably include a transmembrane domain, which tends to locate to the plasma membrane for BoNT/A, C, and E. In some contemplated assays, the membrane-anchoring domain comprises a fragment that contains a palmitoylation site. Suitable membrane-anchoring domains are described, for example, in US 20060134722 to Chapman.
While it is especially preferred that the transmembrane domain is the transmembrane domain of synaptobrevin, mutations (e.g., transitions, transversions, insertions, deletions, inversions, etc.) thereof, and even non-synaptobrevin transmembrane domains are also deemed suitable for use herein. Similarly, it should be appreciated that the transmembrane domain may also be replaced by another polypeptide moiety that allows at least temporary anchoring of the hybrid protein to a membrane such that the remainder of the hybrid protein is exposed to the cytosol.
With respect to the transfected cells expressing the hybrid protein it is generally preferred that the cell is stably transfected. Nevertheless, transient transfection is also contemplated. It is still further typically preferred that the transfected cell is a neuronal cell. However, numerous other non-neuronal cells (including mammalian, rodent, and insect cells, and even yeast and bacterial cells) are also contemplated herein. Most typically, the cells will constitutively express the hybrid protein(s) are therefore under appropriate regulatory elements. In alternative aspects, the expression my also be induced.
According to a preferred embodiment, a recombinant nucleic acid molecule, preferably an expression vector, encoding a BoNT substrate polypeptide and a suitable reporter is introduced into a suitable host cell. An ordinarily skilled person can choose a suitable expression vector, preferably a mammalian expression vector, and will recognize that there are enormous numbers of choices. For example, the pcDNA series of vectors, such as pCI and pSi (from Promega, Madison, Wis.), CDM8, pCeo4. Many of these vectors use viral promoters. Preferably, inducible promoters are used, such as the tet-off and tet-on vectors from Clontech (Mountain View, Calif.).
Many choices of cell lines are suitable as the host cell. Preferably, the cell is of a type in which the respective BoNT exhibits its toxic activities. In other words, the cells preferably display suitable cell surface receptors, or otherwise allow the toxin to be translocated into the cell sufficiently efficiently, and allow the toxin to cleave the suitable substrate polypeptide. Specific examples include primary cultured neurons (cortical neuron, hippocampal neuron, spinal cord motor neuron, etc); PC12 cells or derived PC12 cell lines; primary cultured chromaffin cells; several cultured neuroblastoma cell lines, such as murine cholinergic Neuro 2a cell line, human adrenergic SK-N-SH cell line, and NS-26 cell line. See e.g. Foster and Stringer (1999), Genetic Regulatory Elements Introduced Into Neural Stem and Progenitor Cell Populations, Brain Pathology 9: 547-567.
The coding region for the reporter/cleavage site construct is under the control of a suitable promoter. Depending on the types of host cells used, many suitable promoters are known and readily available in the art. Such promoters can be inducible or constitutive. A constitutive promoter may be selected to direct the expression of the desired polypeptide. Such an expression construct may provide additional advantages since it circumvents the need to culture the expression hosts on a medium containing an inducing substrate. Examples of suitable promoters would be LTR, SV40 and CMV mammalian systems; E. coli lac or trp in bacterial systems; baculovirus polyhedron promoter (polh) in insect systems and other promoters that are known to control expression in eukaryotic and prokaryotic cells or their viruses. Examples of strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are those which are Obtainable from the fungal genes for xylanase (xlnA), phytase, ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi), alcohol dehydrogenase (AdhA), .alpha.-amylase (amy), amyloglucosidase (AG—from the glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) promoters. Examples of strong yeast promoters are those obtainable from the genes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase and triosephosphate isomerase. Examples of strong bacterial promoters include SPO2 promoters as well as promoters from extracellular protease genes.
Hybrid promoters may also be used to improve inducible regulation of the expression construct. The promoter can additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a Pribnow Box or a TATA box. The promoter may even contain other sequences to affect (such as to maintain, enhance or decrease) the levels of expression of the nucleotide sequence. For example, suitable other sequences include the Shl-intron or an ADH intron. Other sequences include inducible elements—such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5′ signal sequence (see Sleat, 1987, Gene 217: 217-225; and Dawson, 1993, Plant Mol. Biol. 23: 97).
The expression vector may also contain sequences which act on the promoter to amplify expression. For example, the SV40, CMV, and polyoma cis-acting elements (enhancer) and a selectable marker can provide a phenotypic trait for selection (e.g. dihydrofolate reductase or neomycin resistance for mammalian cells or ampticillin/tetracyclin resistance for E. coli). Selection of the appropriate vector containing the appropriate promoter and selection marker is well within the level of those skilled in the art.
Preferably, the coding region for the construct is under the control of an inducible promoter. In comparison to a constitutive promoter, an inducible promoter is preferable because it allows for suitable control of the concentration of the reporter in the cell, therefore the measurement of changes in signals are greatly facilitated.
For example, expression can be controlled using the Tet-on & Tet-off system Clontech (Mountain View, Calif.). Under the control of this promoter, gene expression can be regulated in a precise, reversible and quantitative manner. Briefly, for Tet-on system, the transcription of downstream gene only happens when doxycycline is present in the culture medium. After the transcription for a certain period of time, one can change culture medium to deplete doxycycline, thus, stop the synthesis of new reporter proteins. Therefore, there is no background from newly synthesized reporter proteins, and one may be able to see a faster change after toxin treatment.
Fluorescent Analysis
Fluorescent analysis can be carried out using, for example, a photon counting epifluorescent microscope system (containing the appropriate dichroic mirror and filters for monitoring fluorescent emission at the particular range), a photon counting photomultiplier system or a fluorometer. Excitation to initiate energy emission can be carried out with an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation in the desired range. It will be apparent to those skilled in the art that excitation/detection means can be augmented by the incorporation of photomultiptier means to enhance detection sensitivity. For example, the two photon cross correlation method may be used to achieve the detection on a single-molecule scale (see e.g. Kohl et al., Proc. Nat.'l. Acad. Sci., 99:12161, 2002).
Sensitizer
In a particular embodiment, inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dichloride (H7) significantly enhances the sensitivity of the BoCellTM model cell line to botulinum neurotoxin type A (BoNT/A).
Pretreatment of the BoCellTM modified neuroblastoma cells with H7 caused rapid increases in both neurite length and neurite number per cells. H7 effect was both time and dose-dependent with maximal effects seen with 1 mM H7 treatment. Further, this phenotypic change could be “rescued” by removal of the H7, Lastly, pretreatment of cells with H7 significantly increased the sensitivity of these cells to BoNT/A treatment as measured by SNAP-25 reporter cleavage.
H7 also inhibits cAMP and cGMP-dependent kinases, though pretreatment of cells with the drug HA1004, a selective inhibitor of cAMP and cGMP kinases failed to induce morphologic changes in neuroblastoma cells, suggesting the effect is specific to PKC inhibition (1990, JBC paper). On the other hand, other molecules that are known neuronal cell differentiators, including some inhibitors of PKA and PKC, do NOT increase the sensitivity of the BoCellTM Assay. The increased sensitivity was not seen with other drugs previously shown to induce neurite formation: Calphostin C, IBMX, dibutyryl-cAMP, and 8-Bromoadenosine-cAMP.
It is thus contemplated that inhibition of specific PKC isoforms with selective isoquinolynyl analogues and other drugs may infer similar increases in BoNT/A sensitivity in established model cell lines by inducing neurite formation.
It is also contemplated that the culture media can include one or both of H7 and B27, and that the osmolarity of the culture media can be modified to optimize effectiveness with respect to specific Botulinum toxins, especially those in Table 1. Osmolarity can be adjusted in according to known principles, including especially modifying the salt and/or sugar content. As used herein, the term “optimization” means taking steps to improve a desired result, which may or may not actually achieve the best possible result.







TABLE 1







Excipient formulations of BoNT/A-based pharmaceuticals. 












Active


Content



ingredient
Supplied
Excipients
concentrations


Therapeutic
per vial
form
per vial
in a 1 ml





Botox ®
100 U
Lyophilized
0.5 mg
100 U BoNT/A,



BoNT/A
powder
HSA, 0.9
 0.05% HSA, 





mg NaCl
15.4 roll NaCl


Dysport ®/
500 U
Lyophilized
125 μg
500 U BoNT/A,


Reloxin ®
BoNT/A
powder
HSA, 2.5
0.0125% HSA,





mg lactose
0.25% lactose


Xeomin ®
100 U
Lyophilized
1 mg HSA,
100 U BoNT/A,



BoNT/A
powder
4.7 mg
  0.1% HSA,





sucrose
0.47% sucrose





Botox ® is manufactured by Allergan Inc. (Irvine, CA), Dysport ® by Ipsen (France), and Xeomin ® by Merz GmbH (Germany).






Sensitizer Experimental Results

Our finding is that treatment of our BoCellTM cells with the compound H7 increases the sensitivity of our BoCellTM assay. From a methods standpoint, the possible technological advantages of H7 treatment include:

    
    
        1. Reduced assay times. With H7 treatment, we get BoCellTM BoNT/A sensitivities after 24 hours of BoNT/A treatment that are equivalent to 72 hour BoNT/A treatments with our current assay conditions. An assay time reduction of 48 hours.
        2. Overall increase in assay sensitivity. Using H7 we can increase the sensitivity of the assay by 0.5 log-1.0 log with a 48 hour BoNT/A treatment compared to our current assay conditions. We expect that further increases in sensitivity with assay optimization.
        3. Increase neuronal cell sensitivity to other BoNT serotypes. We are currently testing this.
    
    


It is thus contemplated that use of the compounds contemplated herein can increase the sensitivity of the assay by at least 0.5 log, at least 0.6 log, at least 0.7 log, at least 0.8 log, at least 0.9 log and at least it 1.0. Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints and open-ended ranges should be interpreted to include only commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.
FIGS. 1-6 show data generated using H7.
In FIGS. 1A-E, BoCells were plated, and pre-treated for 24 hr with A) H7, B) Calphostin C, C) IBMX, D) dibutyryl-cyclic AMP (db-cAMP), or E) 8-brom o-cyclic AMP (8-Br-cAMP) at the indicated concentration. Control cells were treated with vehicle equivalent to the highest drug concentration tested. After pre-treatment, BoCells were treated with BoNT/A containing indicated drug concentration for an additional 24 hr. The assay response was collected by reading the emission at 485 and 535 nm using a TecanTM Infinite 500 series microplate reader. Emission ratios (YFP/CFP) were plotted as a function of BoNT/A concentration. Data represent mean±standard deviation of the mean of samples run in triplicate.
In FIGS. 2A-B, A) BoCell were plated and pre-treated for 24 hr with the indicated concentration of H7. Control cells were treated with vehicle equivalent to the highest drug concentration tested. After pre-treatment. BoCells were treated with BoNT/A containing indicated drug concentration for an additional 24 hr. The assay response was collected by reading the emission at 485 and 535 nm using a TecanTM Infinite 500 series microplate reader. Emission ratios (YFP/CFP) were plotted as a function of BoNT/A concentration. Data represent mean±standard deviation of the mean of samples run in triplicate. B) BoCells were stained with trypan blue then 3× fields of 50 or more cells were counted per treatment and cell viability calculated.
In FIG. 3, BoCells were plated and pre-treated for either 3. 6. 12 or 24 hr with 0.75 mM H7. After pre-treatment. BoCells were treated with BoNT/A containing 0.75 mM H7 for an additional 24 hr. The assay response was collected by reading the emission at 485 and 535 nm using a Tecan Infinite 500 series microplate reader. Emission ratios (YFP/CFP) were plotted as a function of BoNT/A concentration. Data represent mean±standard deviation of the mean of samples run in triplicate.
In FIGS. 4A-B, A) BoCells were plated and pre-treated for 24 hr with 0.75 mM H7. After pre-treatment. BoCells were treated with either BoNT/A holotoxin or BoNT/A light chain containing 0.75 mM 87 for an additional 24 hr. B.) BoCells were pre-treated for 24 hr with 0.75 mM H7.10 mM HcR/A or vehicle was added to wells for 1 hr prior to addition of BoNT/A. The assay response was collected after 24 hrs by reading the emission at 485 and 535 nm using a Tecan Infinite 500 series microplate reader. Emission ratios (YFP/CFP) were plotted as a function of BoNT/A concentration. Data represent mean±standard deviation of the mean of samples run in triplicate.
In FIG. 5, BoCells were pre-incubated with 0.75 mM H7 from three independent manufacturers (listed) for 24 hrs then treated with BoNT/A in the presence of 0.75 mM H7. The assay response was collected after 24 hrs by reading the emission at 485 and 535 nm using a TecanTM Infinite 500 series microplate reader. Emission ratios (YFP/CFP) were plotted as a function of BoNT/A concentration. Data represent mean±standard deviation of the mean of samples run in triplicate.
In FIG. 6, BoCells were pre-incubated with 0.75 mM H7 for 24 hrs then treated with BoNT/A in the presence of 0.75 mM H7. The assay response was collected after 24 hrs and 48 hrs by reading the emission at 485 and 535 nm using a TecanTM Infinite 500 series microplate reader. Emission ratios (YFP/CFP) were plotted as a function of BoNT/A concentration. Data represent mean±standard deviation of the mean of samples run in triplicate.
Non-FRET Based Assays
FIG. 7A depicts BioSentinel's BoCellTM A BoNT/A construct. The reporter fluorophore, YFP, and the normalization fluorophore, CFP, are coupled by a cleavage sequence, SNAP-25 (green). SNAP-25 palmitoylation localizes the reporter to a plasma membrane.
FIG. 7B depicts an exemplary assay in which BoNT/A cell-based reporters are used to detect BoNT/A activity by loss of YFP fluorescence. Here, the YFP moiety is directly excited leading to fluorescence emission in the absence of BoNT/A. Cleavage of the reporter by BoNT/A releases a C-terminal reporter fragment containing the YFP moiety into the cytosol. The fragment is rapidly degraded and, thus, YFP emission is lost. The CFP signal is still used to control for cell-to-cell reporter expression levels and cell density.
Surprisingly, not all fluorescent proteins related to YFP are effective as the reporter fluorophore. For example, FIGS. 8A-8C provide evidence that reporters containing YFP or the closely related derivative Venus can detect BoNT/A activity in cells, but not mCherry or mStrawberry. Here, Neuro2A cells were grown in a 96-well plate to 70% confluency and transiently transfected using Lipofectamine 2000 (InvitrogenTM), with reporters containing the indicated N-terminal and C-terminal (N-term/C-term) fluorophore pairs. After 24 h, cells were incubated in the presence or absence of 10 nM BoNT/A at 37° C. for 72 h in 100 μl of phenol red-free MEM medium.
FIG. 8A shows BoNT/A-induced changes in fluorescence responses. Semi-automated YFP and CFP fluorescence measurements were performed using a NikonTM TE2000-U fluorescent microscope with 20× magnification and Nikon NIS Elements 3.4 software. Shown are randomly selected fields pseudo-colored for the C-terminal/N-terminal fluorescent protein (FP) fluorescence ratio. Ratios were calculated from emissions collected upon direct excitement of each fluorophore.
FIG. 8B represents fluorescence ratios and BoNT/A sensitivities of the cell-based reporters. 30 randomly selected cells per condition were analyzed for fluorescence ratios in the presence or absence of 10 nM BoNT/A. The average signal from the 30 cells from 5 microscopic fields on 3 different wells is shown. Cells exhibiting over-saturated fluorescence were excluded.
FIG. 8C is a blot showing that BoNT/A was active in cells regardless of the reporter. All reporters show some cleavage in the presence of BoNT/A, and all native SNAP25s are cleaved. Cells were transfected and treated with BoNT/A as described above but scaled up into 6-well plates. After 72 h incubation with BoNT/A, cells were washed 3× with serum-free MEM, collected by scraping, and lysed using M-Per Lysis Buffer (PierceTM). 40 μg of cell lysate was subjected to SDS-PAGE before transfer to nitrocellulose paper and immunoblot analysis using an antibody directed against SNAP-25 (clone 71.2, Synaptic Systems). Arrows indicate the position of the full-length (closed) and cleaved (open) forms of the reporters. Full-length (*) and cleaved (**) native SNAP-25 are indicated.
The inventive subject matter can be extended beyond cleavable substrates, to any assay having a construct with a reporter that can be de-protected, and then degraded in some manner by the cytosol or other local environment. For example, a susceptible reporter could be modified to include a ‘bait’ domain that is used to screen against a library of recombinant proteins that could possibly bind with the bait domain. Without the bait domain protected by a binding protein, the susceptible reporter will be degraded. In such an assay, cells expressing binding proteins will form a complex to protect the susceptible reporter from degradation, while cells expressing a binding partner to the bait will light up. The bait domain could advantageously be a small peptide, and the binding partners could be members of a library of proteins (or protein mutants). The system could also be reversed such that there is a library of bait domains tested against a single test protein (or test protein library).
In each of these instances it is considered advantageous to include a second reporter that is not degraded post-exposure by the cytosol or other local environment, or is at least degraded much more slowly post-exposure than the first reporter.
Still further, whereas the reporter can conveniently be selected from suitable fluorophores, it is contemplated that the reporter could be replaced or augmented by any other protein or other component with a defined function that is known to (a) have a relatively fast turnover in the cell without protection, and (b) that can be protected by interaction with a binding partner. Defined functions include transcription activators for reporter gene, repressors for lethal genes, etc (anything that can be easily identified or selected against).
FIG. 9 comprises multiple charts showing emission data for both YFP degradation and loss of FRET according to the state of the art practice from the exact same plates of cells. For YFP degradation, directly and singularly excited YFP emissions (top, Ex500, Em526) and CFP emissions (middle, Ex434, Em470) are collected. Those emission are then background subtracted and the YFP emission is divided by CFP emission to control for cell density and reporter expression in the individual wells. That emission ratio (YFP/CFP, bottom) is then used for the essay report.
For loss of FRET, FRET emissions (top, Ex434, Em526) and CFP emissions (middle, Ex434, Em470) are collected. Those emissions are then background subtracted, and the FRET emission is divided by CFP emission to control for cell density and reporter expression in the individual wells. That emission ratio (FRET/CFP, bottom) is shown here to compare to the normal method.
The key comparison is the loss of directly excited YEP versus the loss of FRET emission. From the comparison between the measurements and the corresponding curves, it becomes immediately apparent that the overall dynamic range for YFP degradation is much larger than the dynamic range of loss of FRET emissions. In some cases, there is no difference, statistically, between cells treated with no BoNT versus sells treated with saturating concentrations of BoNT when looking solely at the raw FRET emissions. For the loss of FRET method, the BoNT dose response only becomes clear after dividing the FRET emission by the CFP (donor) emission. The CFP (donor) emission shows a small increase emission due to de-quenching in response to reporter cleavage.
In summary, the loss of FRET method reports BoNT-induced changes in the reporter very poorly, or not at all, and therefore cannot be therefore used for a correct qualitative and quantitative determination. In contrast, preferred methods contemplated herein have a high degree of specificity and reproducibility, which allow one to rely on the data for both the qualitative and quantitative analysis.
It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

## Claims

1. A method of detecting the presence of a Botulinum toxin, comprising:
providing an assay with a cell having (a) a molecule with a cleavage sequence that is cleaved by a light chain of said Botulinum toxin and (b) a reporter that reports a signal responsive to cleavage of said molecule at said cleavage sequence induced by the light chain of said Botulinum toxin; and
incubating the cell in a culture media that contains a compound effective to increase sensitivity of the assay by at least 0.5 log;
treating the cell with said Botulinum toxin, then detecting the signal, and correlating the signal with the presence or absence of said Botulinum toxin,
wherein said compound is an isoquinolynyl compound that inhibits protein kinase C (PKC), and wherein said cleavage sequence comprises a SNARE protein, SNARE motif, or SNARE mutein.

2. The method of claim 1, wherein said compound comprises 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dichloride (H7).

3. The method of claim 1, wherein osmolarity of the culture media is optimized for characterization of a composition comprising BoNT/A, 0.05% human serum albumin, and 15.4 mM sodium chloride.

4. The method of claim 1, wherein osmolarity of the culture media is optimized for characterization of a composition comprising BoNT/A, 0.0125% human serum albumin, and 0.25% lactose.

5. The method of claim 1, wherein osmolarity of the culture media is optimized for characterization of a composition comprising BoNT/A, 0.1% human serum albumin, and 0.47% sucrose.

6. The method of claim 1, wherein said reporter is an artificial construct expressed in the cell.

7. The method of claim 1, wherein said molecule includes a first fluorophore and the cleavage sequence.

8. The method of claim 7, wherein said first fluorophore is selected from the group consisting of Yellow Fluorescent Protein (YFP), Citrine, Venus, and a YPet protein.

9. The method of claim 7, wherein said first fluorophore comprises Yellow Fluorescent Protein (YFP).

10. The method of claim 7, wherein said molecule comprises the reporter and a second fluorophore.

11. The method of claim 10, wherein said reporter is not coupled with the second fluorophore in a manner that produces Forster resonance energy transfer (FRET).

12. The method of claim 1, wherein said cell expresses an enzyme that facilitates degradation of the reporter at least two times faster after the cleavage than before the cleavage.

13. The method of claim 1, wherein said cell expresses an enzyme that facilitates degradation of the reporter at least five times faster after the cleavage than before the cleavage.

14. The method of claim 1, wherein presence of said Botulinum toxin correlates with reduction of the signal from a baseline signal.