Patent Description:
For example, treatment of Alzheimer's disease (AD) is an emerging field, which affects millions of human beings worldwide. The disease progression is characterized by the formation of plaques and tangles in the brain, which are based on aggregation processes of the Aβ peptide and Tau protein. The protein aggregation is driven by structural transition into β-sheet enriched peptides or protein species. The drug development against the Alzheimer's disease is challenging. Many promising drug candidates failed in the clinical trials (<NPL>);<NPL>); <NPL>); <NPL>);<NPL>)) and until now no drug is available on the market to cure early/mid stages of Alzheimer's disease nor the late stages. In these stages at which clinical symptoms appear the brain is already irreversible damaged. Therefore, an early diagnosis before clinical symptoms appear is a prerequisite. Such diagnostic tool is patented (previous patent application <CIT>). The patent application here shows that this tool can be used to monitor also the intervention of a drug on the secondary structure distribution and its ability to refold the pathological species into harmless forms and monitor thereby its efficacy. Techniques like surface plasmon resonance (SPR), surface acoustic waves (SAW) or quartz crystal microbalance (QCM) are used to analyze protein-ligand or protein drug interactions. Since, these techniques only provide kinetical information, but no spectral or structural resolution; they are not able to monitor secondary structure distributions of proteins. A related approach is the use of high-throughput chemical microarray surface plasmon resonance (HT-CM-SPR), which is in principle a reversed SPR system, because the potential drugs are immobilized and the target protein is flushed over the surface (<NPL>)). This approach was successfully employed to identify small molecules that bind to monomeric Tau (<NPL>)), but an effect on the secondary structure cannot be detected due to the lack of spectral and structural resolution. Further techniques like surface enhanced Infrared absorption (SEIRA) spectroscopy provide spectral resolution, but the reproducibility of the measurements is very challenging due to the preparation of the rough gold surfaces and thus does not provide a robust platform for a protein-drug analysis. Moreover, secondary structure analysis of proteins by Fourier-transform infrared (FTIR-) spectroscopy has been described for the analysis of recombinant and purified proteins and peptides, i.e. for assessment of the anti-amyloidogenic activity of G. acerosa against the Aβ peptide <NUM>-<NUM> (<NPL>). The state of the art in the clinical diagnostics are Positron emission tomography (PET) and Magnetic resonance tomography (MRT) to detect aggregates (accumulated from β-sheet enriched proteins) such as plaques in the human brain. The drug induced dissociation of such aggregates appearing at late stages can be monitored. Nevertheless, PET and MRT are very expensive and time-consuming techniques, which cannot be employed as mass screening method nor for preselection of drug candidates. A further disadvantage is in the case of PET the usage of contrast agents, which also stress the patients. However, these techniques provide the analysis of a drug effect in vivo at later stages of the disease. The presented invention is able to reveal the drug effect by analysing body fluids as shown here for cerebrospinal fluid (CSF). Besides the already mentioned techniques, fluorescence based immuno assays are also an emerging field, especially Enzyme Linked Immunosorbent Assay (ELISA) and surface-based fluorescence intensity distribution analysis (sFIDA). However, in contrast these assays are not label-free as the invention, because the need of fluorescent labelled antibodies which may influence the secondary structure distribution of the target protein (<NPL>); <NPL>)). Most importantly, ELISA and sFIDA do not provide direct information about the secondary structure distribution or changes of the secondary structure distribution by the drug. But this information is crucial for the analysis of a drug effect as for example in neurodegenerative diseases. Common assays like Western blots also require labelled secondary antibodies and provide indirect information on the aggregation state of the protein based on the molecular weight only. Due to the preparation process of the Western blot the native secondary structure of the protein is lost. Thus ELISA, sFIDA, or Western blots are not able to measure directly the intervention of a potential drug on the secondary structure distribution of a target protein. The presented invention overcomes all the mentioned limitations and provides evidence on the drug efficacy in vitro. Detailed information about the surface chemistry and the general set-up is described in the literature by our self (<NPL>); <NPL>); <NPL>);<NPL>); <NPL>); <NPL>)) and in the patent application <CIT>.

As an example for a drug candidate methylene blue is used. It is a compound that is applied in many different scientific fields (<NPL>); <NPL>); <NPL>)). The aggregation of the Tau protein is associated with several diseases such as Alzheimer's disease (AD), Huntigton disease (HD), or Pick disease (PiD) (<NPL>); <NPL>); <NPL>)). Claude Wischik showed in <NUM> the selective inhibition of the Tau protein aggregation by methylene blue (<NPL>)). In the last decades methylene blue was then investigated in several studies and is nowadays analyzed in a clinical phase III trial (<NPL>); <NPL>); <NPL>); Šimić et al. , Biomolecules <NUM>(<NUM>):<NUM> doi:<NUM>/biom6010006 (<NUM>)). In <NUM> the oxidation of the Cys residue was found to be the mechanistic reason for the inhibition of Tau aggregation (<NPL>)). We here describe a screening method utilizing an ATR-FTIR sensor that measures directly and label-free the effect of potential drugs on the secondary structure distribution of disease related target proteins as schematically shown in <FIG>. A requirement for the assay is the secondary structural change within the protein during the disease progression. Many neurodegenerative diseases ideally fulfil this requirement, because the disease progression is often characterized by aggregation of a specific protein or peptide. Since, the most common cause of dementia is Alzheimer's disease, we investigated the drug interaction with the two major biomarkers Tau and Aβ<NUM>-<NUM>. In case of the Tau protein, which is involved in the formation of neurofibrillary tangles in the brain of AD patients, methylene blue is a promising drug that is currently evaluated in clinical phase III trial (<NPL>); <NPL>); Šimić et al. , Biomolecules <NUM>(<NUM>):<NUM> doi:<NUM>/biom6010006. We demonstrated the potential of our screening method using two different drug candidates methylene blue and berberine. The intervention of methylene blue on the secondary structure distribution of the human Tau protein extracted from CSF from AD patients was measured (<FIG>). The potential drug berberine is a multiple target drug that originally comes from traditional Chinese medicine (<NPL>)). The broad usage of berberine in medical applications is nicely summarized in the review by Ahmed et al. Especially, in the treatment of AD berberine shows promising effects (<NPL>); <NPL>); <NPL>); <NPL>)). Berberine shows a positive effect on the memory function in rat models (<NPL>); <NPL>); <NPL>)). Due to the broad spectrum of treatment by berberine, there are many pathways and receptors that might play a crucial role (<NPL>)). The molecular mechanism on the memory effect that could be important in AD treatment is not completely understood so far.

<NPL>) relate to screening strategies to identify small molecular inhibitors of protein aggregation for possible therapy against deposition diseases. On the other hand, methods for analyzing the secondary structure distribution of a specific protein in bodily fluids are known in the art. In said methods, the protein of interest is selectively bound within the surface layer, which is achieved with an antibody-functionalized internal reflection element (IRE) (<NPL>)). This method was applied for the extraction and determination of the secondary structure distribution of the soluble Aβ fraction from CSF and blood plasma for moderate AD and disease control differentiation (<NPL>); <NPL>)).

<CIT> provides a biosensor for conformation and secondary structure analysis, notably for the direct non-invasive qualitative secondary structure analysis of a single selected protein within a complex mixture, as e.g. a body fluid, by vibrational spectroscopic methods. For the analysis it is not required that the selected substance is isolated, concentrated, or pretreated by a special preparative procedure. The biosensor is suitable for the determination of a disease, in which a conformational transition of a candidate biomarker protein is associated with disease pathology, wherein a shift of the amide I band maximum of the biomarker protein is indicative for the disease. It is moreover emphasized that such biosensor could potentially also be used to monitor the therapeutic efficacy of drug candidates which support the refolding of Aβ back to the less neurotoxic α-helical form (<NPL>)).

It was now found that - with such sensor and with an appropriate assay setting - an intervention of berberine on the Aβ<NUM>-<NUM> secondary structure distribution in a complex body fluid, which closely resembles the Aβ situation in vivo, can in fact be observed. In particular, we found that berberine decelerates the auto-induced fibrilization of Aβ<NUM>-<NUM> at high concentrations and may therefore be an interesting drug candidate for further investigations. The two examples show that the ATR-FTIR sensor can be used as an universal in-vitro screening assay in complex bodily fluids to preselect potential drugs for the treatment of neurodegenerative diseases, especially Alzheimer's disease.

The subject matter of the invention is as defined in the appended claims.

The present invention provides a screening assay utilizing an infrared sensor element for the direct analysis of potential drugs inducing a secondary structural change in the target protein (hereinafter also referred to as "biomarker", "candidate biomarker" and "candidate biomarker protein") that correlates with the efficacy of the drug. It is based on a chemically modified (for example silanes or thiols) germanium surface, which is terminated with covalently attached antibodies. The principle is universal, thus any capture antibody against a desired target protein can be applied and furthermore also any potential drug (small molecule, therapeutic antibody) can be in principle screened with the developed assay. Target proteins can be either immobilized from purified samples or be extracted out of a complex fluid like human CSF. The analysis of the potential drug is done in real-time, label-free and gives evidence of the efficacy in vitro. The invention thus provides.

The invention describes a method for the preselection of potential drugs against pathological misfolded protein targets, such as in many neurodegenerative diseases. The method comprises the steps:.

wherein the target protein undergoing conformational transitions is alpha-Synuclein.

According to the invention the infrared transparent material of the IR cell is selected from gallium arsenide, silicon, germanium, zinc selenide and diamond, and preferably is germanium. Further, the candidate biomarker protein alpha-Synuclein undergoes conformational transitions and is an amyloidogenic peptide or a (poly-) peptide of health-status dependent, characteristic secondary structure composition. Further comparative examples of candidate biomarker proteins include Amyloid-beta (Aβ) peptides and Tau protein associated with Alzheimer's disease, Prion protein associated with Creutzfeldt-Jakob disease, or Huntingtin protein associated with Huntington's disease. Moreover, the sample with candidate biomarker protein may be a purified sample of the biomarker or may be a complex body fluid comprising the biomarker including human CSF. Other suitable complex bodily fluids are human serum, blood plasma, lacrimal fluid and nipple aspirate fluid.

It is preferred that said infrared sensor element comprises a germanium internal reflection element being of trapezoid or parallelogram shape and being transparent in the infrared with sufficient signal to noise ratio to detect the amide I band beyond large background absorbance, and at least one receptor for the biomarker protein being antibodies capable of specific and conformational independent binding to the biomarker protein, and being directly grafted to at least one surface, preferably to at least two or three surfaces of said internal germanium reflection element, by silanization with short silane linkers or by thiolation with short thiol linkers, reacting freely accessible amine groups of said at least one receptor with amine-reactive groups on the short silane/thiol linkers, and blocking remaining amine-reactive groups on the short silane/thiol linkers with a blocking substance not cross-reacting with the biomarker protein.

According to the invention it is particularly preferred that the internal reflection element is a germanium monocrystal, preferably is a trapezoid cut germanium monocrystal. It is further preferred that the germanium crystal allows for or provides for one, more than one, or more than three reflections of the infrared light through the reflection element, particularly preferred are more than five reflections or even more than twenty reflections (preferred are <NUM> reflections with <NUM> actively sensed reflections). Even more, it is particularly preferred that the internal reflection element is suitable for the parallel analysis by another optical method including detection of fluorescence at different wavelengths. Finally, it is crucial that the blocking substance is not cross-reacting with the biomarker protein, which is selected from casein, ethanolamine, L-lysine, polyethylene glycols, albumins and derivatives thereof.

The silane and thiol linkers for the grafting include homogenous silane and thiol linkers, mixtures of silane linkers and mixtures of thiol linkers, and have an effective linker chain length (combined number carbon and heteroatoms) of not more than <NUM> atoms or not more than <NUM> atoms, preferably.

wherein W is H or R<NUM>S-, X at each occurrence is independently selected from halogen and C<NUM>-<NUM> alkoxy, n is an integer of <NUM> to <NUM>, n' is an integer of <NUM> to <NUM>; R<NUM> at each occurrence is independently selected from C<NUM>-<NUM> alkyl, Y is selected from a chemical bond, -O-, -CO-, -SO<NUM>-, -NR<NUM>-, -S-, -SS-, -NR<NUM>CO-, -CONR<NUM>-, -NR<NUM>SO<NUM>- and -SO<NUM>NR<NUM>-(wherein R<NUM> is H or C<NUM>-<NUM> alkyl), and Z is an amine-reactive group including -CO<NUM>H, - SO<NUM>H and ester derivatives thereof. The halogen within the present invention includes a fluorine, chlorine, bromine and iodine atom. C<NUM>-<NUM> alkyl and C<NUM>-<NUM> alkoxy includes straight, branched or cyclic alkyl or alkoxy groups having <NUM> to <NUM> carbon atoms that may be saturated or unsaturated. In case of cyclic alkyl and alkoxy groups, this refers to those having <NUM> to <NUM> carbon atoms. Suitable C<NUM>-<NUM> alkyl and C<NUM>-<NUM> alkoxy groups include, among others, methyl and methoxy, ethyl and ethoxy, n-propyl and n-propoxy, isopropyl and iso-propoxy, cyclopropyl and cyclopropoxy, n-butyl and n-butoxy, tert-butyl and tert-butoxy, cyclobutyl and cyclobutoxy, n-pentyl and n-pentoxy, cyclopentyl and cycloppentoxy, n-hexyl and n-hexoxy, cyclohexyl and cyclohexoxy, and so on. The amine-reactive group Z includes all types of functional groups that are reactive with a free amino group. Among those, -CO<NUM>H, -SO<NUM>H and ester derivatives thereof (including active esters) are particularly preferred.

The -(CH<NUM>)n- and -(CH<NUM>)n'- structural elements in the above formulas may also contain one or more double and/or triple bonds and may be substituted with one or more halogen atoms such as fluorine or with deuterium.

When the infrared sensor element is obtainable by silanization, it is then preferred that in the linkers of formulas (i) to (iii) above X is independently selected from C<NUM>-<NUM> alkoxy-groups, preferably from methoxy and ethoxy groups, Y is -NHCO-, Z is -CO<NUM>H or an ester derivative thereof, and n is an integer of <NUM> to <NUM> and n' is an integer of <NUM> to <NUM>, preferably n is <NUM> and n' is <NUM>.

When the infrared sensor element is obtainable by thiolation, it is then preferred that in the linker of formula (iv) above W is H, Y is a chemical bond, Z is -CO<NUM>H or an ester derivative thereof, and n is an integer of <NUM> to <NUM> and n' is an integer of <NUM> to <NUM>, preferably n is <NUM> and n' is <NUM>.

In one disclosure, the biomarker protein is an Aβ peptide and the receptor binding to the Aβ peptide is an antibody, preferably is an antibody specifically binding to the central epitope of the Aβ peptide, including antibody A8978.

In a further disclosure, the biomarker protein is a Tau protein and the receptor binding to the Tau protein is an antibody, preferably is an antibody specifically binding to a epitope accessible for all Tau variants (phosphorylated, truncated, <NUM> to <NUM> repeat regions etc., isoforms), including antibody Tau-<NUM>.

In the method of the invention, the concentration of the potential drug in the solution is either below the detection limit of the IR determination or can be easily subtracted by reference spectra of the potential drug.

In the method of the invention, when the potential drug possesses amide bands, such as antibodies, the method further comprises subtracting a reference spectrum of the potential drug for detecting the shift of the amide I band of the target protein.

In one particular disclosure, when the target protein is an Aβ peptide, a shift of the amide I band, preferably a shift of the amide I band maximum, to any value indicative for the Aβ peptide secondary structure is indicative for the efficacy of the potential drug. Notably, for a fibrillary fraction of the Aβ peptide a shift from of <NUM>-<NUM> to <NUM>-<NUM> and for the total fraction of the Aβ peptide a shift from <NUM>-<NUM> to <NUM>-<NUM> is indicative for the efficacy of the potential drug.

In a further disclosure, when the target protein is a Tau protein, a shift of the amide I band, preferably a shift of the amide I band maximum, to any value indicative for the Tau protein secondary structure is indicative for the efficacy of the potential drug. Notably, for a fibrillary fraction of the Tau protein a shift from of <NUM>-<NUM> to <NUM>-<NUM> and for the total fraction of the Tau protein a shift from <NUM>-<NUM> to <NUM>-<NUM> is indicative for the efficacy of the potential drug.

The present invention is based on the detection of secondary structural changes induced by the potential drug by means of vibrational spectroscopy. The invention uses in principle the same experimental set-up as our previous patent application <CIT>. Instead of a 70V (Bruker) we employed an 80V FTIR spectrometer (Bruker) to improve the signal to noise ratio of the measurements. As internal reflection element germanium crystal were chemically modified with NHS-silanes, which function as anchors for the covalent attachment of the desired antibodies. After blocking the surface with casein the surface is ready for capturing of the target protein alpha-Synuclein protein. Further disclosed is the capturing of Tau or Aβ<NUM>-<NUM>. The Tau protein was directly extracted out of human CSF. This is a great advantage since no purified protein samples are required and no pretreatment of CSF is needed, which makes the assay easier accessible for the application in clinics or clinical labs. The target protein was analyzed in the presence of the potential drug and the effect was monitored by the change in the amide I band. As shown for the Tau protein the effect of the potential drug methylene blue was directly monitored (<FIG>). The change of the secondary structural distribution is characterized by the drug induced shift of the amide I band from <NUM>-<NUM> (untreated) to <NUM>-<NUM> (treated). The effect becomes even more evident in the double difference spectrum (<FIG>), which clearly shows a negative band at <NUM>-<NUM> indicative for the disappearance of β-sheet and a positive band at <NUM>-<NUM> typical for α-helix. In a control without drug the amide I band absorbance maximum of the Tau protein remains stable (<FIG>). Thus, for the pre-selection of drug candidates in the treatment of neurodegenerative diseases the invention provides an ideal platform. Another disclosed example is the study of the second important biomarker Aβ<NUM>-<NUM>. Since the developed sensor is universal, the antibody A8978 against the epitope <NUM>-<NUM> of Aβ<NUM>-<NUM> could be applied for the analysis of the potential drug berberine. To monitor a potential effect of berberine the surface was loaded with synthetic Aβ<NUM>-<NUM>. Without any incubation the auto-induced fibrilization process at high concentrations leads to a broad secondary structure distribution of Aβ<NUM>-<NUM> dominated by β-sheet (<FIG>, dashed light grey spectrum). Addition of berberine shifts the amide I maximum of the broad secondary structure distribution from <NUM> to <NUM>-<NUM> indicating mainly α-helical or monomeric species (<FIG>, dashed black spectrum). Thus, berberine seems to decelerate the auto-induced fibrilization process. It may be an interesting target for further investigations. In conclusion, the method provides label-free direct information about the intervention of the secondary structure distribution of the target protein by a drug candidate as demonstrated for Tau and Aβ<NUM>-<NUM>. It resolves the different intervention of the drug on the same target protein. This universal approach can in principle be transferred to any protein and small molecule (potential drug) and has therefore a very high potential for pharmaceutical applications.

The invention is further disclosed in the following Examples, which are however not to be construed so as to limit the application.

Materials and Methods: The same experimental set-up is used as in applicant's previous patent application <CIT>.

Sampling and pretreatment: CSF was drawn by lumbal puncture and aliquoted at the university hospital Essen, snap-frozen in liquid nitrogen, shipped and stored at -<NUM>. Samples were not pretreated before the measurement, only thawed at <NUM> for <NUM> and kept on ice until used.

Phosphate buffered saline (PBS-buffer): <NUM> sodium chloride (NaCl), <NUM> potassium chloride (KCI), <NUM> total-phosphate (in the form of Na<NUM>HPO<NUM> and NaH<NUM>PO<NUM>), pH <NUM>.

Casein blocking-solution: <NUM> sodium hydroxide (NaOH), <NUM> % (w/v) casein from bovine milk (powder), pH adjusted with H<NUM>PO<NUM> to <NUM>.

Silanization-solution: The used NHS-silane (N-(<NUM>,<NUM>,<NUM>-triethoxysilanebutyl)succinamic acid <NUM>,<NUM>-dioxopyrrolidin-<NUM>-yl ester) was synthesized and characterized as described (<NPL>).

Antibody: For the analysis of Aβ<NUM>-<NUM> the antibody A8978 (lot no: 061M4773, Sigma Aldrich) was employed. In case of the Tau protein the antibody Tau-<NUM> (AHB0042, Thermo Fisher Scientific) was used.

AB<NUM>-<NUM>: The human Aβ-peptide was purchased from Sigma-Aldrich (A9810, Amyloid-beta-Protein fragment <NUM>-<NUM>).

Potential drugs: Methylene blue (methylthionine hydrochloride, lot no: <NUM>) and berberine chloride (lot no: B3251) were purchased by Sigma Aldrich.

Performing the measurement: The general procedure is identical to the patent application <CIT>. IR-measurements were performed on a Vertex 80V spectrometer (Bruker Optics GmbH, Ettlingen, Germany) with liquid nitrogen cooled mercury-cadmium-telluride (MCT) detector. Double-sided interferograms were recorded in forward-backward interferometer movement at a <NUM> data rate with a spectral resolution of <NUM>-<NUM>, Blackman-Harris-<NUM>-Term-apodisation, Mertz-phase correction and <NUM> times zero filling. Reference spectra were recorded as an average of <NUM>, sample spectra of <NUM> interferograms. Recording reference single channel spectra of the blank sensor, sensor with <NUM>-propanol, the silanized surface, the buffers, antibody or casein coated surface in equilibrium states enabled high sensitivity difference spectroscopy based on Lambert-Beer law (E=-log(I/I<NUM>). The absorbance of the state change is the negative decadic logarithm of the intensity relation before and after the change.

Tau-protein treated with methylene blue: The Tau antibody (Tau-<NUM>) from Thermo Fisher Scientific was covalently attached to the germanium surface as described for other antibodies by Nabers et al. After blocking the Tau-antibody terminated surface was incubated with <NUM>µl of human CSF till the Tau protein was successfully immobilized (about <NUM>). In the next step, <NUM> of a <NUM> methylene blue solution (PBS, pH <NUM>) was flushed over the surface till the system was equilibrated (<NUM>) and then circulated for <NUM>. The effect on the secondary structure of tau was directly monitored by the band position and shape of the amide I.

Aβ-peptide treated with berberine: The antibody A8979 (Sigma-aldrich) was employed for capturing the Aβ-peptide (<NPL>)). The Aβ-peptide (Aβ<NUM>-<NUM>, synthetic, Sigma-aldrich, Taufkrichen, Germany) was monomerized by incubation with hexafluoro-<NUM>-propanol as described elsewhere (<NPL>)). For the analysis <NUM>µg of Aβ<NUM>-<NUM> were circulated over the antibody terminated sensor for <NUM> to ensure that the drug is not interfering with the immobilization process. In the control experiments the immobilization of Aβ<NUM>-<NUM> was monitored for further <NUM> (total <NUM>) in the presence of the potential drug, to follow the auto-induced fibrilization process (<FIG>, dashed light grey spectrum). To analyze the effect of the potential drug berberine the same protocol as in the control experiment was used. The only difference was the addition of <NUM> berberine after the <NUM> immobilization of Aβ<NUM>-<NUM> (PBS, pH <NUM>) by equilibrating with <NUM> of <NUM> berberine solution and subsequently circulating the system in the presence of <NUM> berberine (<FIG>, dashed black spectrum). The effect on the secondary structure of Aβ<NUM>-<NUM> was directly monitored by the band position and shape of the amide I. The corresponding dashed spectra show intermediates of the folding processes for each experiment after a total time of <NUM>.

Pretreatment of the spectra: By scaled subtraction of a reference spectrum water vapor was removed. Spectra were baseline corrected, a sliding average was performed as described (<NPL>)) and normalized to the same amide I signal intensity in the region <NUM> till <NUM>-<NUM> depending on the observed secondary structure.

To monitor the drug effect of methylene blue the invented method was employed. We previously invented an immuno-ATR sensor, which differentiates AD with an accuracy of <NUM> % based on CSF and <NUM> % based on blood plasma analyzes (<NPL>)). First, we employed silane chemistry to modify the germanium surface (<NPL>)). Second, the monoclonal IgG1 antibody Tau-<NUM> was covalently immobilized on the germanium surface. The immobilization is completed after <NUM> hours as presented in <FIG> by reaching an absorbance of <NUM> mOD. After washing the surface with binding buffer <NUM> the antibody remains stable (<FIG>). To obtain a highly specific surface the saturation with casein is crucial (<NPL>)). Finally, a complex sample such as cerebrospinal fluid (CSF) is flushed over the sensor. The resulting monoexponential binding kinetics of the Tau protein is presented in <FIG>. With the immobilized Tau fraction it is now possible to analyze the effect of the potential drug methylene blue. A <NUM> solution of methylene blue was flushed over the surface and after equilibration the system was circulated. The above mentioned immuno-ATR-FTIR sensor (<CIT>) for the diagnosis of Alzheimer's disease uses for the diagnosis a simple threshold classifier with a value at <NUM>-<NUM> for AD and disease control differentiation, which can also be transferred to the Tau protein (unpublished data, patent application in preparation). The black spectrum in <FIG> shows an amide I maximum of <NUM>-<NUM> indicating a higher amount of disease related β-sheet enriched isoforms, which would be diagnosed as diseased by our immuno-IR-sensor (<NPL>). Upon methylene blue incubation a significant shift to higher wavenumbers was observed within <NUM> (<FIG>, dashed spectrum), thus a secondary structure change to an disordered or α-helical conformation was induced by the potential drug methylene blue. This is in consistence with the in vivo studies of the group of Claude Wischik, which demonstrated the reduction of the Tau associated tangles in the human brain (Šimić et al. , Biomolecules <NUM>(<NUM>):<NUM> doi:<NUM>/biom6010006 (<NUM>); <NPL>)). The patient would now be diagnosed as healthy by our developed immuno-ATR-FTIR sensor (<FIG>, dashed spectrum) (<NPL>)). Thus, the presented approach has a very high potential as prescreening tool for the selection of candidate drugs against the AD and also against other neurodegenerative diseases. By subtraction of the drug treated state minus the untreated state the secondary structural change becomes even more obvious as indicated by the negative band at <NUM>-<NUM> and the positive band at <NUM>-<NUM> (<FIG>). To prove that the changes are really caused by methylene blue a control without methylene was performed (<FIG>). The amide I maximum is stable and only differs about ±<NUM> wavenumbers without the drug incubation (<FIG>, dashed light grey line), whereas with the presence of the drug a clear shift to higher wavenumbers is observed (<FIG>, solid black line).

The second important marker protein for the Alzheimer's disease is Aβ<NUM>-<NUM>. We analyzed the fibrilization process with the described method. Synthetic Aβ<NUM>-<NUM> was monomerized with hexafluoro-<NUM>-propanol. A solution of monomerized Aβ<NUM>-<NUM> was flushed over the sensor and specifically immobilized with antibody A8979 (<NPL>)). The spontaneous fibrilization was monitored over <NUM> resulting in an amide I maximum of <NUM>-<NUM> (<FIG>, dashed light grey spectrum). The same experiment was done in the presence of <NUM> berberine showing a <NUM>-<NUM> shift of the amide I maximum to <NUM>-<NUM> (<FIG>, dashed black spectrum). This indicates that berberine directly decelerates the aggregation process of Aβ<NUM>-<NUM>. A small amount of β-sheet enriched isoforms is observed as a shoulder at <NUM>-<NUM>, but the dominating conformation is monomeric Aβ<NUM>-<NUM>. This suggests a direct interaction of berberine and AB<NUM>-<NUM> that could be applied as drug to prevent the initial processes of AD and thus might be useful to slow down disease progression.

In addition, the effect of methylene blue on Aβ<NUM>-<NUM> was investigated under the same conditions as for berberine. The obtained spectrum clearly shows a fibril (<FIG>, dashed light grey spectrum), which is in consistence with the literature (<NPL>)). The effect is discussed to prevent the formation of toxic oligomers and therefore might have a potential in treating Alzheimer's disease (<NPL>)). This shows that the method works very efficient and gives direct information regarding the molecular mechanism of the drug.

Claim 1:
A drug-screening assay method for determining the efficacy of a potential drug on a target protein undergoing secondary structural changes associated with a disease with misfolded target proteins in a complex body fluid, comprising the steps:
(a) conducting, in an IR cell comprising an infrared sensor element having an internal reflection element with a core of an infrared transparent material and at least one receptor for the target protein directly grafted to at least one surface of said core, said at least one receptor for the target protein being antibodies capable of specific and conformationally independent binding to the target protein, a flux of a sample of the complex body fluid with soluble target protein; submitting an IR beam through said IR cell; and obtaining an infrared spectrum therefrom;
(b) conducting, in the same IR cell of step (a), wherein the receptors for the target protein grafted to the surface of the core are loaded with the target protein, a flux of a solution with potential drug; submitting an IR beam through said IR cell; and obtaining an infrared spectrum therefrom;
(c) subtracting a reference spectrum of the potential drug for detecting the shift of the amide I band of the target protein; and
(d) analyzing the obtained infrared spectra to evaluate the effect of the potential drug by determining the secondary structure distribution of the soluble target protein in the sample and after application of the potential drug, wherein an upshift or disappearance of the amide I band characteristic for β-sheets in the spectrum of (b) relative to the corresponding amide I band in (a) is indicative for the efficacy of the potential drug;
wherein the target protein undergoing conformational transitions is alpha-Synuclein.