Method for the preselection of drugs for protein misfolding diseases
Inventors
GERWERT, Klaus • Nabers, Andreas • SCHARTNER, Jonas
Assignees
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Publication Number
US-11592451-B2
Publication Date
2023-02-28
Expiration Date
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.
Core Innovation
The invention relates to a label-free infrared (IR) drug-preselection or drug-screening assay for diseases associated with protein misfolding and neurodegenerative diseases. A sample of complex body fluid comprising a target protein is contacted with a potential drug, and an IR spectrum is obtained from the target protein in a sensor cell before and after drug application.
The sensor cell includes an IR sensor element with an internal reflection element comprising a core of an IR transparent material and antibodies capable of specific and conformationally independent binding to the target protein. Antibodies are covalently attached to a surface of the internal reflection element and are loaded by contacting with the target protein from the complex body fluid.
After submitting an IR beam through the IR cell, a first infrared spectrum is obtained, and after contacting the IR cell with a solution comprising a potential drug, a second infrared spectrum is obtained and compared with a reference spectrum of the potential drug via subtraction. The target protein undergoes secondary structure changes associated with disease, and the assay determines a secondary structure distribution from IR spectral features.
Efficacy is evaluated by analyzing secondary-structure-dependent IR changes, in particular via an upshift or disappearance of an amide I band characteristic for β-sheets in the second infrared spectrum after subtraction relative to the corresponding amide I band in the first spectrum.
Claims Coverage
The independent claim covers a drug-screening assay using an IR cell with an internal reflection element whose surface has covalently attached, conformationally independent antibodies, with spectra acquired before and after potential drug exposure and analyzed by subtracting a reference drug spectrum to determine secondary-structure distribution. The inventive features are concentrated in the antibody-functionalized IR sensor element, the before/after spectral workflow with reference subtraction, and the efficacy criterion based on β-sheet amide I band behavior.
Conformationally independent covalently attached antibodies on an IR internal reflection element
An IR cell comprising an infrared sensor element with an internal reflection element including 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 the internal reflection element.
Before/after IR spectra with reference drug subtraction
Contacting a sample of complex body fluid comprising a target protein with the IR cell and obtaining a first infrared spectrum, then contacting the IR cell with a solution comprising a potential drug, obtaining a second infrared spectrum, subtracting a reference spectrum of the potential drug from the second infrared spectrum, and analyzing the first spectrum and the second-after-subtraction spectrum.
Efficacy evaluation by secondary-structure distribution via β-sheet amide I band upshift or disappearance
Analyzing 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 relative to the corresponding amide I band in the first spectrum is indicative of efficacy.
Potential drug constrained to antibodies or amide bonds
Wherein the potential drug is an antibody or comprises amide bonds.
Across the covered independent claim, efficacy is determined from secondary-structure-dependent IR changes of a disease-associated target protein measured before and after potential drug exposure in an IR cell having conformationally independent covalently attached antibodies, with a reference spectrum subtraction step, and an amide I β-sheet upshift or disappearance criterion.
Stated Advantages
Provides a label-free infrared (IR) drug-screening assay for evaluating the effect of a potential drug on secondary structure changes associated with a disease.
Enables evaluation of drug efficacy by determining a secondary structure distribution from IR spectra, using an amide I band characteristic for β-sheets as an efficacy indicator.
Uses subtraction of a reference spectrum of the potential drug from the second infrared spectrum to support analysis of drug effects on the target protein.
Documented Applications
Drug preselection or drug-screening for protein misfolding and neurodegenerative diseases using complex body fluid samples comprising disease-associated target proteins such as Tau and Aβ1-42.
Measurement of drug effects on Tau and Aβ1-42 secondary structure distribution using IR (ATR-FTIR) amide I band behavior, including examples with methylene blue and berberine.
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