Method for the preselection of drugs for protein misfolding diseases

Abstract

The invention provides a method that gives direct information about the intervention of a potential drug on the secondary structure distribution of a targetbiomolecule, i.e., for a disease with misfolded protein, such as neurodegenerative diseases in a complex body fluid. The secondary structural change is monitored by vibrational spectroscopy. The method can be applied for prescreening of drug candidates for targeting of specific biomolecules. The effect of the drug on the secondary structure distribution is monitored label-free in real time and provides thereby direct information about the efficacy of the potential drug.

Claims

1. A drug-screening assay comprising the steps: contacting a flux of a sample of a complex body fluid comprising a target protein, wherein the target protein undergoes secondary structure changes associated with a disease, with an infrared (IR) cell comprising an infrared sensor element comprising: an internal reflection element comprising a core of an infrared transparent material and at least one antibody capable of specific and conformationally independent binding to the target protein, wherein the antibody is covalently attached to at least one surface of said internal reflection element; wherein said contacting loads said at least one antibody with the target protein; submitting an IR beam through said IR cell; and obtaining a first infrared spectrum therefrom; contacting the IR cell with a solution comprising a potential drug; submitting an IR beam through said IR cell; and obtaining a second infrared spectrum therefrom, and subtracting a reference spectrum of the potential drug from the second infrared spectrum; and analyzing: i) the first infrared spectrum and ii) the second infrared spectrum after subtraction of the reference spectrum of the potential drug, to evaluate the effect of the potential drug by determining the secondary structure distribution of the target protein in the sample before and after application of the potential drug, wherein an upshift or disappearance of an amide I band characteristic for β-sheets in the second infrared spectrum after subtraction of the reference spectrum of the potential drug relative to the corresponding amide I band in the first spectrum is indicative of the efficacy of the potential drug; wherein the potential drug is an antibody or comprises amide bonds.

2. The method of claim 1, wherein (i) the infrared transparent material of the IR cell is independently selected from gallium arsenide, silicon, germanium, zinc, selenide and diamond and/or (ii) the target protein is an amyloidogenic peptide or a (poly-) peptide of health-status dependent, characteristic secondary structure composition; and/or (iii) the sample of the complex body fluid with Tau target protein is human cerebrospinal fluid (CSF), serum or blood plasma.

3. The method of claim 1, wherein 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, and wherein the at least one antibody is and being directly grafted covalently attached to at least one surface of said internal germanium reflection element by a method comprising: silanization with short silane linkers or by thiolation with short thiol linkers, reacting freely accessible amine groups of said at least one antibody 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.

4. The method of claim 1, wherein the internal reflection element (i) is a germanium monocrystal; and/or (ii) allows for or provides for more than one passages of the infrared light through the reflection element; and/or (iii) is further suitable for the parallel analysis by another optical method including detection of fluorescence at different wavelengths; and/or (iv) the blocking substance not cross-reacting with the target protein is selected from casein, ethanolamine, L-lysine, polyethylene glycols, albumins and derivatives thereof.

5. The method of claim 3, wherein the silane and thiol linkers comprise 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 20 atoms or not more than 15 atoms.

6. The method of claim 1, wherein the target protein is a Tau protein and wherein a shift of the amide I band to any value indicative for the Tau protein secondary structure is indicative for the efficacy of the potential drug.

7. The method of claim 1, wherein the potential drug is for the treatment of a neurodegenerative disease.

8. The method of claim 2, wherein the infrared transparent material is germanium.

9. The method of claim 4, wherein the germanium monocrystal is a trapezoid cut germanium monocrystal.

10. The method of claim 5, wherein the silane linkers have one of the following formulas:
X.sub.3Si—(CH.sub.2).sub.n—Y—(CH.sub.2).sub.n′—Z,  (i)
X.sub.2R.sup.1Si—(CH.sub.2).sub.n—Y—(CH.sub.2).sub.n′—Z or  (ii)
X(R.sup.1).sub.2Si—(CH.sub.2).sub.n—Y—(CH.sub.2).sub.n′—Z,  (iii) and the thiol linkers have the following formula:
WS—(CH.sub.2)n-Y—(CH.sub.2)n′-Z,  (iv) wherein W is H or R.sup.1S—, X at each occurrence is independently selected from halogen and C.sub.1-6 alkoxy, n is an integers of 1 to 10, n′ is an integer of 1 to 5; R.sup.1 at each occurrence is independently selected from C.sub.1-6 alkyl, Y is selected from a chemical bond, O—, —CO—, —SO.sub.2—, —NR.sup.2—, —S—, —SS—, —NR.sup.2CO—, —CONR.sup.2—, —NR.sup.2SO.sub.2— and —SO.sub.2NR.sup.2— (wherein R.sup.2 is H or C.sub.1-6 alkyl), and Z is an amine-reactive group including —CO.sub.2H, —SO.sub.3H and ester derivatives thereof.

11. The method of claim 1, wherein the potential drug is an antibody.

12. The method of claim 6, wherein the shift of the amide I band is a shift to the amide 1 band maximum.

13. The method of claim 6, wherein the shift for a fibrillary fraction of the Tau protein from of 1626 cm.sup.−1 to 1655 cm.sup.−1 is indicative of the efficacy of the potential drug.

14. The method of claim 6, wherein the shift for the total fraction of the Tau protein from 1636 cm.sup.−1 to 1655 cm.sup.−1 is indicative of the efficacy of the potential drug.

15. The method of claim 10, wherein X is independently selected from methoxy and ethoxy groups.

16. The method of claim 10, wherein n is 3 and n′ is 2.

17. The method of claim 10, wherein n is 8 and n′ is 4.

18. The method of claim 1, wherein (i) the target protein is an Aβ peptide, or (ii) the target protein is a Tau protein.

19. The method of claim 1, wherein the target protein is an Aβ peptide and a shift of the amide I band to any value indicative for the Aβ peptide secondary structure is indicative of the efficacy of the potential drug.

20. The method of claim 10, wherein the infrared sensor element is obtainable by: (A) silanization and in the linkers of formulas (i) to (iii), wherein X is independently selected from C.sub.1-6 alkoxy-groups, Y is —NHCO—, Z is —CO.sub.2H or an ester derivative thereof, and n is an integer of 1 to 5 and n′ is an integer of 1 to 3; or (B) thiolation and in the linkers of formula (iv), wherein W is H, Y is a chemical bond, Z is —CO.sub.2H or an ester derivative thereof, and n is an integer of 1 to 8 and n′ is an integer of 1 to 5.

21. The method of claim 20, wherein X is independently selected from methoxy and ethoxy groups.

22. The method of claim 20, wherein n is 3 and n′ is 2.

23. The method of claim 20, wherein n is 8 and n′ is 4.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1: Graphical abstract showing the general principle of the invented method.

(2) FIG. 2: (A) Binding of the antibody of Tau-5 via silane-chemistry and final wash with PBS buffer. (B) The antibody specifically captures the Tau protein from human CSF, which remains sufficiently stable after washing the surface with PBS buffer.

(3) FIG. 3: The Tau protein was immobilized on antibody terminated germanium. The amide I of the Tau protein represents a high amount of β-sheets resulting in an absorbance maximum at 1640 cm.sup.−1 (black). Addition of the potential drug methylene blue shifted the amide I band to 1653 cm.sup.−1 indicating a major structural change to α-helix dominated secondary structure distribution (grey).

(4) FIG. 4: The difference spectra between the two spectra shown in FIG. 3 shows mainly a decrease of β-sheet and an increase of α-helix induced by addition of methylene blue (50 μM). This shows that the drug intervention on the secondary structure distribution can be monitored by the invention very precisely.

(5) FIG. 5: Structural shift of the amide I band of the Tau protein from CSF with (black) and without the presence of methylene blue (grey) in dependence of time.

(6) FIG. 6: Aβ.sub.1-42 protein was immobilized on antibody (A8978) terminated germanium surface. The light grey (right) spectrum shows the a broad distribution of different secondary structures with an absorbance maximum at 1634 cm.sup.−1 characteristic for fibrillized Aβ.sub.1-42. Furthermore, the amide I of the intermediate of the folding process (dashed light grey) is shown after 160 min. Addition of the potential drug berberine (100 μM) results in a significant different secondary structure distribution dominated now by an α-helical or mainly monomeric isoform of Aβ.sub.1-42 at 1659 cm.sup.−1 (black spectrum, left), however with a significant shoulder at 1634 cm.sup.−1. The dashed dark grey spectrum shows the amide I band as an intermediate of the folding process after 160 min.

(7) FIG. 7: Comparison of the Aβ.sub.1-42 secondary structure distribution (black) after incubation with methylene blue (light grey), berberine (grey), and without any drug intervention (black). As seen already in FIG. 6 berberine shifts the distribution mainly to α-helix, whereas methylene blue induces β-sheet and fibril formation. This finding is in consistence with the literature (Necula et al., Biochem. 46(30):8850-60 doi:10.1021/bi700411k (2007). This shows that different interventions on the secondary structure distribution by different drugs can be resolved.

(8) FIG. 8: The potential drug berberine (100 μM) did not change the secondary structure distribution of the Tau protein (extracted from CSF) in contrast to methylene blue as shown in FIG. 3. This finding shows that different interventions of different drugs on the same target protein can be resolved.

DETAILED DESCRIPTION OF THE INVENTION

(9) 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:

(10) (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 candidate biomarker directly grafted to at least one surface of said core, a flux of a sample with soluble candidate biomarker protein, submitting an IR beam through said first IR cell, and obtaining an infrared spectrum therefrom;
(b) conducting, in the same IR cell of step (a), wherein the receptors for the candidate biomarker, which are grafted to the surface of the core, are loaded with the candidate biomarker protein, a flux of a solution with potential drug, submitting an IR beam through said IR cell, and obtaining an infrared spectrum therefrom; and
(c) analyzing the obtained infrared spectra to evaluate the effect of the potential drug by determining the secondary structure distribution of the soluble candidate biomarker protein in the sample and after application of the potential drug, wherein an upshift or disappearance of the amide I band in the spectrum of (b) relative to (a) is indicative for the efficacy of the potential drug.

(11) 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 undergoes conformational transitions associated with the disease and is an amyloidogenic peptide or a (poly-) peptide of health-status dependent, characteristic secondary structure composition, including Amyloid-beta (Aβ) peptides and Tau protein associated with Alzheimer's disease, alpha-Synuclein associated with Parkinson's disease, Prion protein associated with Creutzfeldt-Jakob disease, or Huntingtin protein associated with Huntington's disease, and preferably is an Aβ peptide or a Tau protein. 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.

(12) 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.

(13) 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 25 reflections with 13 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 20 atoms or not more than 15 atoms, preferably the silane linkers have one of the following formulas:
X.sub.3Si—(CH.sub.2).sub.n—Y—(CH.sub.2).sub.n′—Z,  (i)
X.sub.2R.sup.1Si—(CH.sub.2).sub.n—Y—(CH.sub.2).sub.n′—Z or  (ii)
X(R.sup.1).sub.2Si—(CH.sub.2).sub.n—Y—(CH.sub.2).sub.n′—Z,  (iii)
and the thiol linkers have the following formula:
WS—(CH.sub.2).sub.n—Y—(CH.sub.2).sub.n′—Z,  (iv)
wherein W is H or R.sup.1S—, X at each occurrence is independently selected from halogen and C.sub.1-6 alkoxy, n is an integers of 1 to 10, n′ is an integer of 1 to 5; R.sup.1 at each occurrence is independently selected from C.sub.1-6 alkyl, Y is selected from a chemical bond, —O—, —CO—, —SO.sub.2—, —NR.sup.2—, —S—, —SS—, —NR.sup.2CO—, —CONR.sup.2—, —NR.sup.2SO.sub.2— and —SO.sub.2NR.sup.2— (wherein R.sup.2 is H or C.sub.1-6 alkyl), and Z is an amine-reactive group including —CO.sub.2H, —SO.sub.3H and ester derivatives thereof. The halogen within the present invention includes a fluorine, chlorine, bromine and iodine atom. C.sub.1-6 alkyl and C.sub.1-6 alkoxy includes straight, branched or cyclic alkyl or alkoxy groups having 1 to 6 carbon atoms that may be saturated or unsaturated. In case of cyclic alkyl and alkoxy groups, this refers to those having 3 to 6 carbon atoms. Suitable C.sub.1-6 alkyl and C.sub.1-6 alkoxy groups include, among others, methyl and methoxy, ethyl and ethoxy, n-propyl and n-propoxy, iso-propyl 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.sub.2H, —SO.sub.3H and ester derivatives thereof (including active esters) are particularly preferred.

(14) The —(CH.sub.2).sub.n— and —(CH.sub.2).sub.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.

(15) 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.sub.1-6 alkoxy-groups, preferably from methoxy and ethoxy groups, Y is —NHCO—, Z is —CO.sub.2H or an ester derivative thereof, and n is an integer of 1 to 5 and n′ is an integer of 1 to 3, preferably n is 3 and n′ is 2.

(16) 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.sub.2H or an ester derivative thereof, and n is an integer of 1 to 8 and n′ is an integer of 1 to 5, preferably n is 8 and n′ is 4.

(17) In a particular preferred embodiment, 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.

(18) In a further particular preferred embodiment, 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, 3 to 4 repeat regions etc., isoforms), including antibody Tau-5.

(19) 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.

(20) 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.

(21) In particular, 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 1626 cm.sup.−1 to 1655 cm.sup.−1 and for the total fraction of the Aβ peptide a shift from 1636 cm.sup.−1 to 1655 cm.sup.−1 is indicative for the efficacy of the potential drug.

(22) Further, 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 1626 cm.sup.−1 to 1655 cm.sup.−1 and for the total fraction of the Tau protein a shift from 1636 cm.sup.−1 to 1655 cm.sup.−1 is indicative for the efficacy of the potential drug.

(23) 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 WO 2015121339. 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 (Tau or Aβ.sub.1-42). 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. 3). The change of the secondary structural distribution is characterized by the drug induced shift of the amide I band from 1640 cm.sup.−1 (untreated) to 1653 cm.sup.−1 (treated). The effect becomes even more evident in the double difference spectrum (FIG. 4), which clearly shows a negative band at 1625 cm.sup.−1 indicative for the disappearance of β-sheet and a positive band at 1655 cm.sup.−1 typical for α-helix. In a control without drug the amide I band absorbance maximum of the Tau protein remains stable (FIG. 5). Thus, for the pre-selection of drug candidates in the treatment of neurodegenerative diseases the invention provides an ideal platform. Another example is the study of the second important biomarker Aβ.sub.1-42. Since the developed sensor is universal, the antibody A8978 against the epitope 13-28 of Aβ.sub.1-42 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.sub.1-42. Without any incubation the auto-induced fibrilization process at high concentrations leads to a broad secondary structure distribution of Aβ.sub.1-42 dominated by β-sheet (FIG. 6, light grey). Addition of berberine shifts the amide I maximum of the broad secondary structure distribution from 1634 to 1659 cm.sup.−1 indicating mainly α-helical or monomeric species (FIG. 6, black). 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.sub.1-42. 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.

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

EXAMPLES

(25) Materials and Methods:

(26) The same experimental set-up is used as in applicant's previous patent application WO 2015121339.

(27) Sampling and Pretreatment:

(28) CSF was drawn by lumbal puncture and aliquoted at the university hospital Essen, snap-frozen in liquid nitrogen, shipped and stored at −80° C. Samples were not pretreated before the measurement, only thawed at 37° C. for 30 s and kept on ice until used.

(29) Phosphate Buffered Saline (PBS-Buffer):

(30) 137 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 12 mM total-phosphate (in the form of Na.sub.2HPO.sub.4 and NaH.sub.2PO.sub.4), pH 7.4.

(31) Casein Blocking-Solution:

(32) 200 mM sodium hydroxide (NaOH), 1% (w/v) casein from bovine milk (powder), pH adjusted with H.sub.3PO.sub.4 to 7.4.

(33) Silanization-Solution:

(34) The used NHS-silane (N-(4,4,4-triethoxysilanebutyl)succinamic acid 2,5-dioxopyrrolidin-1-yl ester) was synthesized and characterized as described (Schartner et al., JACS 135(10):4079-4087 (2013).

(35) Antibody:

(36) For the analysis of Aβ.sub.1-42 the antibody A8978 (lot no: 061M4773, Sigma Aldrich) was employed. In case of the Tau protein the antibody Tau-5 (AHB0042, Thermo Fisher Scientific) was used.

(37) Aβ.sub.1-42:

(38) The human Aβ-peptide was purchased from Sigma-Aldrich (A9810, Amyloid-beta-Protein fragment 1-42).

(39) Potential Drugs:

(40) Methylene blue (methylthionine hydrochloride, lot no: 66720) and berberine chloride (lot no: B3251) were purchased by Sigma Aldrich.

(41) Performing the Measurement:

(42) The general procedure is identical to the patent application WO 2015121339. 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 80 kHz data rate with a spectral resolution of 2 cm.sup.−1, Blackman-Harris-3-Term-apodisation, Mertz-phase correction and 4 times zero filling. Reference spectra were recorded as an average of 1000, sample spectra of 200 interferograms. Recording reference single channel spectra of the blank sensor, sensor with 2-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.sub.0). The absorbance of the state change is the negative decadic logarithm of the intensity relation before and after the change.

(43) Tau-Protein Treated with Methylene Blue:

(44) The Tau antibody (Tau-5) from Thermo Fisher Scientific was covalently attached to the germanium surface as described for other antibodies by Nabers et al. (Nabers et al., J. Biophotonics 9(3):224-34 doi:10.1002/jbio.201400145 (2016)). After blocking the Tau-antibody terminated surface was incubated with 100 μl of human CSF till the Tau protein was successfully immobilized (about 60 min). In the next step, 2 ml of a 50 μM methylene blue solution (PBS, pH 7.4) was flushed over the surface till the system was equilibrated (1 ml) and then circulated for 60 min. The effect on the secondary structure of tau was directly monitored by the band position and shape of the amide I.

(45) Aβ-Peptide Treated with Berberine:

(46) The antibody A8979 (Sigma-aldrich) was employed for capturing the Aβ-peptide (Nabers et al., Anal. Chem. 88(5):2755-62 doi:10.1021/acs.analchem.5b04286 (2016)). The Aβ-peptide (Aβ.sub.1-42, synthetic, Sigma-aldrich, Taufkrichen, Germany) was monomerized by incubation with hexafluoro-2-propanol as described elsewhere (Nabers et al., J. Biophotonics 9(3):224-34 doi:10.1002/jbio.201400145 (2016)). For the analysis 100 μg of Aβ.sub.1-42 were circulated over the antibody terminated sensor for 1 h to ensure that the drug is not interfering with the immobilization process. In the control experiments the immobilization of Aβ.sub.1-42 was monitored for further 17 h (total 18 h) in the presence of the potential drug, to follow the auto-induced fibrilization process (FIG. 6, 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 100 μM berberine after the 1 h immobilization of Aβ.sub.1-42 (PBS, pH 7.4) by equilibrating with 1.5 mL of 100 μM berberine solution and subsequently circulating the system in the presence of 100 μM berberine (FIG. 6, black spectrum). The effect on the secondary structure of Aβ.sub.1-42 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 160 min.

(47) Pretreatment of the Spectra:

(48) By scaled subtraction of a reference spectrum water vapor was removed. Spectra were baseline corrected, a sliding average was performed as described (Schartner et al., Chembiochem 15(17):2529-34 doi:10.1002/cbic.201402478 (2014)) and normalized to the same amide I signal intensity in the region 1730 till 1590 cm.sup.−1 depending on the observed secondary structure.

Example 1: Methylene Blue “Cures” Alzheimer's Disease In Vitro

(49) 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 90% based on CSF and 84% based on blood plasma analyzes (Nabers et al., Anal. Chem. 88(5):2755-62 doi:10.1021/acs.analchem.5b04286 (2016)). First, we employed silane chemistry to modify the germanium surface (Schartner et al., JACS 135(10):4079-87 doi:10.1021/ja400253p (2013)). Second, the monoclonal IgG1 antibody Tau-5 was covalently immobilized on the germanium surface. The immobilization is completed after 2 hours as presented in FIG. 2A by reaching an absorbance of 5 mOD. After washing the surface with binding buffer 1 the antibody remains stable (FIG. 2A). To obtain a highly specific surface the saturation with casein is crucial (Nabers et al., J. Biophotonics 9(3):224-34 doi:10.1002/jbio.201400145 (2016)). 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. 2B. With the immobilized Tau fraction it is now possible to analyze the effect of the potential drug methylene blue. A 50 μM solution of methylene blue was flushed over the surface and after equilibration the system was circulated. The above mentioned immuno-ATR-FTIR sensor (WO 2015121339) for the diagnosis of Alzheimer's disease uses for the diagnosis a simple threshold classifier with a value at 1643 cm.sup.−1 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. 3 shows an amide I maximum of 1640 cm.sup.−1 indicating a higher amount of disease related β-sheet enriched isoforms, which would be diagnosed as diseased by our immuno-IR-sensor (Nabers et al., Anal. Chem. 88(5):2755-62 doi:10.1021/acs.analchem.5b04286 (2016). Upon methylene blue incubation a significant shift to higher wavenumbers was observed within 1 h (FIG. 3, grey 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 6(1):6 doi:10.3390/biom6010006 (2016); Wischik et al., PNAS 93(20):11213-18 (1996)). The patient would now be diagnosed as healthy by our developed immuno-ATR-FTIR sensor (FIG. 3, grey spectrum) (Nabers et al., Anal. Chem. 88(5):2755-62 doi:10.1021/acs.analchem.5b04286 (2016)). 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 1625 cm.sup.−1 and the positive band at 1655 cm.sup.−1 (FIG. 4). To prove that the changes are really caused by methylene blue a control without methylene was performed (FIG. 5). The amide I maximum is stable and only differs about ±1 wavenumbers without the drug incubation (FIG. 5, grey line), whereas with the presence of the drug a clear shift to higher wavenumbers is observed (FIG. 5, black line).

Example 2: Berberine Decelerates the Aggregation of Aβ.SUB.1-42

(50) The second important marker protein for the Alzheimer's disease is Aβ.sub.1-42. We analyzed the fibrilization process with the described method. Synthetic Aβ.sub.1-42 was monomerized with hexafluoro-2-propanol. A solution of monomerized Aβ.sub.1-42 was flushed over the sensor and specifically immobilized with antibody A8979 (Nabers et al., J. Biophotonics 9(3):234-34 doi:10.1002/jbio.201400145 (2016)). The spontaneous fibrilization was monitored over 18 h resulting in an amide I maximum of 1634 cm.sup.−1 (FIG. 6, light grey spectrum). The same experiment was done in the presence of 100 μM berberine showing a 25 cm.sup.−1 shift of the amide I maximum to 1659 cm.sup.−1 (FIG. 6, black spectrum). This indicates that berberine directly decelerates the aggregation process of Aβ.sub.1-42. A small amount of β-sheet enriched isoforms is observed as a shoulder at 1634 cm.sup.−1, but the dominating conformation is monomeric Aβ.sub.1-42. This suggests a direct interaction of berberine and Aβ.sub.1-42 that could be applied as drug to prevent the initial processes of AD and thus might be useful to slow down disease progression.

Example 3: Methylene Blue Affects Also Aβ.SUB.1-42

(51) In addition, the effect of methylene blue on Aβ.sub.1-42 was investigated under the same conditions as for berberine. The obtained spectrum clearly shows a fibril (FIG. 7, light grey spectrum), which is in consistence with the literature (Necula et al., Biochem. 46(30):8850-60 doi:10.1021/bi700411k (2007)). The effect is discussed to prevent the formation of toxic oligomers and therefore might have a potential in treating Alzheimer's disease (Necula et al., Biochem. 46(30):8850-60 doi:10.1021/bi700411k (2007)). This shows that the method works very efficient and gives direct information regarding the molecular mechanism of the drug.

Example 4: Berberine does not Affect Tau

(52) Furthermore, we studied the effect of berberine on the Tau protein (from CSF) under the same conditions as for methylene blue (FIG. 8). The amide band and its maximum are not affected by the berberine treatment, which indicates that berberine has no significant effect on the Tau protein in comparison to methylene blue (FIG. 3 and FIG. 8).