Microradioactive binding assay for ligand screening

Abstract

The present disclosure relates to binding assays that can measure the binding of ligands to specific protein targets in a microradiometric binding assay. In particular, the present disclosure relates to microradiometric binding assays that are useful for low abundance proteins, such as recombinant or tissue-derived proteins isolated from samples of healthy or diseased human donors.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 909,101, filed October 1, 2019, and U.S. Provisional Application No. 62 / 970,977, filed February 6, 2020, each of which is incorporated herein by reference in its entirety for all purposes.

[0002] This application relates to compositions and methods for microradiometric binding assays for characterizing and screening ligands for proteins immobilized on coated surfaces. [Background technology]

[0003] Radioactive binding assays are intended to determine the binding parameters that govern the interaction between a ligand and a target. Such assays can be used in a variety of experimental paradigms, including saturation, competitive, and kinetic binding experiments, to define distinct parameters of the ligand-target interaction.

[0004] There is a need for highly sensitive assays for low abundance biological targets. Summary of the Invention

[0005] Saturation assays are d The purpose is to measure the affinity of a ligand for a target called K d is the dissociation constant at equilibrium, defined as the concentration of ligand required to occupy 50% of the binding sites of a given target. K dTo determine the K of a ligand, the target protein is incubated with a radiolabeled test ligand, where the protein is at a constant (fixed) concentration while the concentration of the radiolabeled test ligand is varied. At equilibrium, the amount of bound ligand is quantified for each concentration of radiolabeled test ligand until saturation occurs. The resulting data can be expressed as the molar amount of ligand bound to the target protein, thus determining the K of the ligand. d The second type of radioactive binding experiment is the competitive radioactive binding assay. In the competitive format, the radioactive binding assay measures the ability of a non-radiolabeled (cold) test ligand to displace a radiolabeled test ligand. The radiolabeled test ligand is then mixed with its K d By using a fixed concentration near K and using non-radiolabeled test ligands at various concentrations, the inhibition (or displacement) constant (K) can be determined. This competitive format can also be used as a screening assay, in which one or more non-radiolabeled test ligands / test compounds are tested at a single concentration or multiple concentrations for their ability to displace a common radiolabeled tool ligand. Following calculation of the percentage of competition (if the test ligand is used at a single concentration) or K (if the test ligand is used at multiple concentrations), the test ligands / test compounds can be ranked according to their strength in displacing the radiolabeled tool ligand. Ranking can be used to identify potent ligands for a defined target protein, thus driving discovery programs.

[0006] In radioactive binding assays, radiolabeled ligands are labeled with radioisotopes, allowing for quantification of their fraction bound to the target. This is achieved by measuring the ligand's specific ionizing radioactivity with a detector containing a photomultiplier tube device. Estimating the amount of ligand bound to the target at equilibrium requires separation of the target-ligand complex (the bound fraction of ligand) from the unbound ligand (the free fraction of ligand). In classical radioactive binding assays, physical separation is usually achieved by filtration, where a filter, typically made of nitrocellulose or glass fiber, retains only the bound ligand-target complex while the free ligand is removed by passing through the filter. The bound fraction of ligand can then be quantified. Classical filter-based radioactive binding assays require large amounts of target protein to achieve the required protein concentration in the large volumes required for the filtration process. The substantial protein required limits the use of the assay, especially when the target protein needs to be isolated from human tissue samples where it may be present at low levels.

[0007] The microradiolind binding assays described herein allow for the characterization of ligand binding to low-abundance proteins, such as proteins derived from brain or other patient tissues or fluids, including, but not limited to, proteins associated with neurodegenerative diseases. Indeed, these proteins are known to undergo conformational changes that lead to protein deposits, the accumulation of which is directly linked to the onset and progression of the disease. Examples of these proteins are amyloid beta (Aβ) and tau (the deposits of which are characteristic of Alzheimer's disease (AD), Down's syndrome, and other tauopathies), α-synuclein (a-syn) (the deposits of which are characteristic of Parkinson's disease (PD) and dementia with Lewy bodies), and TAR DNA-binding protein 43 (TDP-43) (the deposits of which are characteristic of amyotrophic lateral sclerosis (ALS) and TDP-frontotemporal lobar degeneration (TDP-FTLD)) (Serrano-Pozo et al., 2011, Spillantini et al., 1997, Neumann et al., 2006, and Nelson et al., 2019). Deposits of these pathological proteins can be artificially generated in vitro from recombinant proteins, but it is widely recognized that deposits (e.g., aggregates) generated in vitro differ in conformation from proteins isolated from patient tissues. Therefore, discovery programs aimed at targeting these protein deposits (e.g., aggregates) with therapeutic or diagnostic agents would ideally use brain-derived protein samples as targets for pharmacological assays to ensure the generation of preclinical data with higher exploratory value.

[0008] The need to minimize the amount of biological target required in traditional filter-based radioactive binding assays led to the development of microarray technology to investigate ligand binding to protein G protein-coupled receptors (GPCRs) isolated from cell lines (Posner et al., 2007). However, there remains a need for accurate and sensitive assays adapted to low-abundance pathological proteins, such as those derived from human brain samples.

[0009] This application describes a miniaturized radioactive binding assay specifically designed for low-abundance protein targets, making it particularly suitable for pathological protein deposits derived from patient brain samples. The ability to screen compounds against pathological protein deposits of human origin while minimizing the amount of patient-derived tissue required represents a major limitation of commonly used filter-based radioactive binding assays and a major advantage of the microradioactive binding assay described herein. The microradioactive binding assay allows for the use of very small amounts of protein target, using up to 500 times less protein target material than classical filter-based radioactive binding assays. This assay is characterized by the K d and Ki values, as well as being used in a high-throughput assay for screening ligand libraries. This assay was successfully validated by direct comparison with classical filter-based radioactive binding assays.

[0010] The methods described herein use microarrays with localized microsamples of pathological proteins on a coated surface. In some embodiments, biochemically enriched samples of pathological protein targets are spotted onto a coated surface (e.g., a coated glass surface) to form a pathological protein array with spots at well-defined locations. In some embodiments, the brain-derived protein sample is subjected to an enrichment step to concentrate protein deposits (to ensure an appropriate signal from the assay) and generate an enriched sample with a viscosity suitable for appropriate dispensing or spotting onto the coated surface. Signal detection is achieved by fluorescent imaging, exposing the dried coated surface to a fluorescent imaging film or screen at the end of various incubation steps. After exposing the surface to the screen or film for an appropriate time, the screen is scanned with a fluorescent imaging scanner, and the signal is quantified using image analysis software such as ImageJ-win 64 software.

[0011] In some aspects, the present disclosure provides a method for determining the binding affinity (K) of a test ligand to a pathological protein in an enriched biological sample. d ), comprising the steps of: contacting a plurality of aliquots of the enriched biological sample on the microarray with a saturating fixed concentration of cold test ligand; contacting the aliquots with multiple concentrations of a radiolabeled test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from the aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; From the detected signal in each aliquot, K d and The present invention relates to a method, comprising:

[0012] In some aspects, the present disclosure provides a method for determining the binding affinity (K) of a test ligand to a pathological protein in an enriched biological sample. d ), comprising the steps of: contacting a plurality of aliquots of the enriched biological sample on the microarray with a plurality of concentrations of a radiolabeled test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; contacting the aliquot with a saturating fixed concentration of cold test ligand; removing unbound radiolabeled test ligand from the aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; From the detected signal in each aliquot, K d and The present invention relates to a method, comprising:

[0013] In some aspects, the present disclosure provides a method for determining the binding affinity (K) of a test ligand to a pathological protein in an enriched biological sample. d ), comprising the steps of: contacting multiple aliquots of the enriched biological sample on the microarray with multiple concentrations of radiolabeled test ligand and a saturating fixed concentration of cold test ligand to form radiolabeled complexes between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from the aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; From the detected signal in each aliquot, K d and The present invention relates to a method, comprising:

[0014] In some aspects, the disclosure provides a method for determining the inhibition constant (K) of a test ligand for a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray were analyzed by K d contacting the aliquots with a fixed concentration of radiolabeled test ligand approximately equal to the concentration of the pathological protein in each aliquot to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; contacting the aliquots with multiple concentrations of cold test ligand; removing unbound radiolabeled test ligand from the aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; calculating K from the detected signal in each aliquot; The present invention relates to a method, comprising:

[0015] In some aspects, the disclosure provides a method for determining the inhibition constant (K) of a test ligand for a pathological protein in an enriched biological sample, comprising: contacting a plurality of aliquots of the enriched biological sample on the microarray with a plurality of concentrations of cold test ligand; The aliquots were analyzed by the K d contacting the aliquots with a fixed concentration of radiolabeled test ligand approximately equal to the concentration of the pathological protein in each aliquot to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from the aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; calculating K from the detected signal in each aliquot; The present invention relates to a method, comprising:

[0016] In some aspects, the disclosure provides a method for determining the inhibition constant (K) of a test ligand for a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray were analyzed by K dand a plurality of concentrations of cold test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from the aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; calculating K from the detected signal in each aliquot; The present invention relates to a method, comprising:

[0017] In some aspects, the disclosure provides a method for evaluating a test compound for its ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, comprising: Multiple aliquots of enriched biological samples on the microarray were analyzed by K-reagents of the tool ligands. d contacting the pathological protein in each aliquot with a fixed concentration of radiolabeled tool ligand approximately equal to the concentration of the radiolabeled tool ligand to form a radiolabeled complex between the radiolabeled tool ligand and the pathological protein in each aliquot; contacting the aliquot with a single concentration or multiple concentrations of cold test compound; removing unbound radiolabeled tool ligand from the aliquot; detecting a signal from the radiolabeled tool ligand in the radiolabeled complex in each aliquot; (a) calculating the percent competition for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at a single concentration, or (b) calculating the K for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at multiple concentrations; The present invention relates to a method, comprising:

[0018] In some aspects, the disclosure provides a method for evaluating a test compound for its ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, comprising: contacting multiple aliquots of the enriched biological sample on the microarray with a single concentration or multiple concentrations of cold test compound; Aliquot the K of the tool ligand d contacting the pathological protein in each aliquot with a fixed concentration of radiolabeled tool ligand approximately equal to the concentration of the radiolabeled tool ligand to form a radiolabeled complex between the radiolabeled tool ligand and the pathological protein in each aliquot; removing unbound radiolabeled tool ligand from the aliquot; detecting a signal from the radiolabeled tool ligand in the radiolabeled complex in each aliquot; (a) calculating the percent competition for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at a single concentration, or (b) calculating the K for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at multiple concentrations; The present invention relates to a method, comprising:

[0019] In some aspects, the disclosure provides a method for evaluating a test compound for its ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, comprising: Multiple aliquots of enriched biological samples on the microarray are incubated with a single or multiple concentrations of cold test compounds and tool ligands. d contacting the pathological protein in each aliquot with a fixed concentration of radiolabeled tool ligand approximately equal to the concentration of the radiolabeled tool ligand to form a radiolabeled complex between the radiolabeled tool ligand and the pathological protein in each aliquot; removing unbound radiolabeled tool ligand from the aliquot; detecting a signal from the radiolabeled tool ligand in the radiolabeled complex in each aliquot; (a) calculating the percent competition for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at a single concentration, or (b) calculating the K for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at multiple concentrations; The present invention relates to a method, comprising: [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows the configuration of a microradiometric binding assay. A solid surface (e.g., a glass slide coated with a hydrophobic adhesive surface such as aminopropylsilane (APS)) (A) is used as a support for spotting protein targets. After spotting proteins onto the surface using an automated spotting device, 64 pads of 9 spots each are obtained (B). In some embodiments, protein targets are manually spotted onto a surface (C) with a watertight chamber above it (D). [Figure 2] Figure 2 shows the results of determining the binding affinity (Kd) of a tool ligand for tau deposits from AD human brain using a classical filter-based radioactive binding assay (A) and a microradiometric binding assay (B). The Y-axis in Figure 2A represents a measurement of the amount of specific radiolabeled ligand bound to the target, expressed in counts per minute (cpm). The Y-axis in Figure 2B represents a quantitative value of the intensity of the signal present on the film, which is proportional to the signal obtained by the amount of specific radiolabeled ligand bound to the target. The tool ligand compound exhibited a Kd of 11.8 nM by the filter-based assay (A) and a Kd of 7.9 nM by the microradiometric binding assay (B). Both Kds had good fits (R2 = 0.97 for (A) and R2 = 0.85 for (B)). [Figure 3]Figures 1A and 1B show the results of determining the binding constants (Kd) for test compounds against a-syn and TDP-43 deposits from human brains with PD and frontotemporal dementia (FTD), respectively, using a microradiometric binding assay (Compound 3, a-syn, A; Compound 2, a-syn, B; Compound 3, TDP-43, C). The Y-axis in each figure represents the quantitative value of the signal intensity present on the film, which is proportional to the signal obtained by the amount of specific radiolabeled ligand bound to the target. Compound 3 exhibited a Kd of 10.8 nM for a-syn derived from PD brains, with a good fit (R2 = 0.87; A), and a Kd of 138 nM for TDP-43 derived from FTD brains, with a good fit (R2 = 0.79; C). Compound 2 exhibited a Kd of 7.8 nM for a-syn derived from PD brains, with a good fit (R2 = 0.80; B). [Figure 4] Figure 1 shows the results of determining the displacement potency, as measured by K, of a tritiated tool ligand for tau deposits from AD human brain (A) and for compound 3 for a-syn deposits from PD human brain (B) using a microradiometric binding assay. The Y-axis in each figure represents the displacement of the labeled compound expressed as a percentage, where 100% corresponds to complete displacement. The tool ligand exhibited a K of 1 nM with good fit (R2 = 0.97). Compound 3 exhibited a K of 41 nM with good fit (R2 = 0.84). [Figure 5] Figures 1A, 1B, and 1C show the results of screening compounds 4, 5, and 6 (A, B, and C, respectively) in a microradiometric binding assay using radiolabeled compound 3 as the tool ligand. The Y-axis in each figure represents the displacement of the labeled compound expressed as a percentage, where 100% corresponds to complete displacement. The K values ​​for compounds 4, 5, and 6 were measured at 13 nM, 37 nM, and 147 nM, and all had good fits (R = 0.97, 0.80, and 0.64, respectively). DETAILED DESCRIPTION OF THE INVENTION

[0021]

[0013] Additional aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein illustrative aspects of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various aspects, all without departing from the present disclosure. Accordingly, this detailed description is to be regarded as illustrative in nature and not as limiting.

[0022] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0023] Pathological proteins are proteins that produce pathological effects upon abnormal accumulation in human tissues or bodily fluids. In some embodiments, pathological proteins are proteins that upon such accumulation form deposits, such as filaments, tangles, or other aggregates, which cause dysfunction and disease progression. In some embodiments, the pathological proteins used in the assays described herein are generated by methods known to those of skill in the art. In some embodiments, the pathological proteins used herein are derived from a human biological sample. In some embodiments, the pathological proteins are present in a human biological sample, which is enriched by methods known to those of skill in the art, resulting in a more concentrated biological sample for use in the assays described herein. In some embodiments, the enriched biological sample contains about 1 mg / mL to about 6.5 mg / mL of total protein (pathological protein target plus other sample proteins). In some embodiments, the enriched biological sample contains about 1 mg / mL to about 2 mg / mL of total protein (pathological protein target plus other sample proteins). In some embodiments, the enriched biological sample contains about 3.5 mg / mL to about 6.5 mg / mL of total protein (pathological protein target plus other sample proteins). In some embodiments, the enriched biological sample also contains lipids, RNA, DNA, or other cellular components.

[0024] In some embodiments, the human biological sample is a human body fluid (e.g., nasal secretions, urine sample, blood sample, plasma sample, serum sample, interstitial fluid (ISF) sample, or cerebrospinal fluid (CSF) sample) or a human tissue sample (e.g., tissue from the heart, muscle, brain, etc.). In other embodiments, the human biological sample is a blood sample or a cerebrospinal fluid sample. In some embodiments, the human biological sample is a brain sample, such as a cerebral cortex sample or a hippocampus sample. In some embodiments, the pathological protein is associated with a neurodegenerative disease. In some embodiments, the enriched biological sample is derived from a human biological sample from a patient suffering from a neurodegenerative disease or from a deceased patient who suffered from a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease, Down's syndrome, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, dementia with Lewy bodies, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), or traumatic brain injury, limbic-predominant age-related TDP-43 encephalopathy (LATE), or chronic traumatic encephalopathy (CTE). In some embodiments, the pathological protein is tau, Aβ, α-synuclein, an inflammasome component (including, but not limited to, ASC), C9orf72, or a dipeptide repeat (DPR) derived from TDP-43. In preferred embodiments, the pathological protein is tau, Aβ, α-synuclein, or TDP-43.

[0025] In some embodiments, the microarray is fabricated by repeatedly dispensing aliquots of the enriched biological sample onto a solid support. In some embodiments, the aliquots are dispensed onto a solid support. In some embodiments, the aliquots are spotted onto a solid support. Thus, in some embodiments, the methods described herein further include fabricating a microarray by dispensing aliquots of the enriched biological sample onto a glass slide. In some embodiments, the aliquots of the enriched biological sample are substantially dried on the microarray. In some embodiments, the microarray comprises at least 25 spots, or at least 50 spots, or at least 100 spots, or at least 200 spots, or at least 300 spots, or at least 400 spots, or at least 500 spots, or 250 to 600 spots, or 500 to 600 spots. In some embodiments, the spots are grouped in pad profiles comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 spots, or 4-10 spots, or 9 spots. In some embodiments, the solid support of the microarray is divided into multiple chambers, where each chamber comprises a pad profile defined as the number of spots in the chamber. In some embodiments, the separate chambers are configured such that different fluids or reagents can be added to each individual chamber but do not mix between chambers. In some embodiments, the microarray comprises at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 40, or at least 45, or at least 50, or at least 55, or at least 60, or about 64 chambers. In some embodiments, different known concentrations of test ligands are used in different aliquots, spots, pad profiles, or chambers in the microarray.In some embodiments, contacting the aliquots with multiple concentrations comprises contacting each chamber in the microarray with a different concentration, and when each chamber contains multiple aliquots or spots, the aliquots or spots serve as replicates for the test condition (e.g., test compound or test concentration) for that chamber.

[0026] In some embodiments, dispensing or spotting of aliquots of pathological proteins from the enriched biological sample is performed using a spotting device, such as an automated spotting device (e.g., Nano-Plotter), or by manual pipetting. In some embodiments, spotting is performed using Nano-Plotter 2.1™ (GESIM; Germany). In some embodiments of the present invention, the volume of the enriched biological sample containing pathological proteins to be arrayed is at least 300 picoliters, or at least 1 nanoliter, or at least 10 nanoliters, or at least 36 nanoliters, or in the range of about 200 picoliters to about 36 nanoliters, or about 200 picoliters to about 10 nanoliters, or about 200 picoliters to about 1 nanoliter.

[0027] The microarray comprises a coated solid support, which can be any suitable solid material, such as glass or a polymer. In some embodiments, the solid support is a glass slide. In some embodiments, the microarray solid support is coated with an adherent. In some embodiments, the adherent is a silane, a thiol, a disulfide, an epoxide, and / or a polymer. In some embodiments, the adherent is a silane. In some embodiments, the adherent is an aminopropylsilane. In some embodiments, the microarray solid support is a glass slide coated with aminopropylsilane.

[0028] The ligand, tool ligand, test compound, or test ligand is an organic compound, an antigen, an antibody, a peptide, a protein, or a protein captured by an antibody. In some embodiments, the ligand, tool ligand, test compound, or test ligand is an organic compound, such as a chemical compound or a small molecule compound. In some embodiments, the tool ligand and the test ligand are both small molecule compounds. In some embodiments, the tool ligand and the test compound are both small molecule compounds.

[0029] A labeled ligand, radiolabeled ligand, labeled tool ligand, radiolabeled tool ligand, labeled test ligand, labeled test compound, radiolabeled test compound, or radiolabeled test ligand is an organic compound, antigen, antibody, peptide, protein, or protein captured by an antibody that contains a label that allows for quantification of the ligand, tool ligand, test compound, or test ligand. In some embodiments, the label allows for quantification of the amount of ligand, tool ligand, test compound, or test ligand bound to the pathological protein. The type of label is not particularly limited and will depend on the detection method selected. The position at which the detectable label is attached to the ligand of the present invention is particularly limited.

[0030] In some embodiments, the radiolabeled test ligand is a radiolabeled test ligand. In some embodiments, the radiolabeled tool ligand is a radiolabeled known ligand. In some embodiments, the tool ligand or radiolabeled tool ligand is a known ligand for a pathological protein of interest. Exemplary radiolabeled tool ligands include Aβ ([ 11 C]PiB (Pittsburgh Compound B), [ 18 F] florbetapir, 18 F] florbetaben, or [ 18 F]Flutemetamol), Tau ([ 18 F]T-807 (also known as AV1451), flortaucipir[ 18 F]MK-6240,[18 F]RO6958948,[ 18 F]PI-2620,[ 18 F]-GTP-1, 18 F]JNJ-067,[ 18 F]PM-PBB3, or [ 11 C]PBB3), THK-5351, THK-5562, or α-synuclein ([ 3 3H]SIL26). Exemplary tool ligands include unlabeled versions of these exemplary radiolabeled tool ligands.

[0031] Exemplary labels include isotopes such as radionuclides, positron emitters, or gamma-ray emitters, as well as fluorescent labels, luminescent labels, and / or chromogenic labels. As used herein, radioisotope labels are present in an abundance ratio that is not identical to the natural abundance ratio of the radioisotope. Furthermore, the amount used should allow its detection by the selected detection method. In some embodiments, the label is a radionuclide label. Examples of suitable radioisotopes as radionuclides include: 2 H, 3 H, 18 F, 123 I, 124 I, 125 I, 131 I, 11 C. 13 N, 15 O, and 77 In some embodiments, the radionuclide label is 2 H, 3 H, 11 C. 13 N, 15 O, or 18 F. In some embodiments, the radionuclide label is 2 H, 3 H, and 18 F. In some embodiments, the radionuclide label is 3H. The radiolabeled compounds described herein are generally prepared by conventional procedures known to those skilled in the art using appropriate isotopic variations of suitable reagents that are commercially available or prepared by known synthetic techniques.

[0032] The tool ligand, radiolabeled tool ligand, test ligand, test compound, and radiolabeled test ligand may also be provided in the form of a composition comprising one or more blocking agents, diagnostically acceptable carriers, diluents, additives, or buffers. In some embodiments, the composition comprises a blocking agent. In some embodiments, the blocking agent is bovine serum albumin (BSA), casein, or albumin from chicken egg white. In some embodiments, the blocking agent is BSA. The blocking agent blocks nonspecific binding sites on the pathological protein and reduces background signal. In some embodiments, the method comprises treating an aliquot of the enriched biological sample with a blocking agent prior to or simultaneously with initial contact of the aliquot. In some embodiments, treating the aliquot with a blocking agent comprises treating the aliquot with an assay buffer containing the blocking agent, where, optionally, the assay buffer comprises Tris-HCl or phosphate-buffered saline (PBS).

[0033] As used herein, "saturating fixed concentration" means a concentration that saturates specific binding to a particular protein.

[0034] As used herein, contacting aliquots on a microarray with "multiple concentrations" of a ligand or compound means contacting different aliquots or sets of aliquots with different concentrations of the ligand or compound. When a set of aliquots on a microarray is contacted with a given concentration, the aliquots in that set serve as replicates for the test concentration. An aliquot or set of aliquots can be isolated from other aliquots or sets of aliquots on the microarray, for example, within individual chambers. For methods involving determining binding affinity, in some embodiments, a suitable range of test concentrations is at least 50-fold lower than the saturating fixed concentration.

[0035] In a method for determining the inhibition constant of a test ligand, an aliquot is added to the K d The K for the test ligand refers to within approximately two times the d The sample is contacted with a radiolabeled test ligand at a fixed concentration close to .

[0036] In some embodiments, removing unbound ligand (e.g., test ligand, test compound, or radiolabeled ligand) comprises washing the microarray to remove ligand that is not bound to the protein target (unbound ligand). In some embodiments, washing comprises washing with a buffer. In some embodiments, the buffer is PBS.

[0037] In some embodiments, detecting involves exposing a microarray containing a complex comprising a radiolabeled tool ligand or a radiolabeled test ligand to film and then detecting a signal on the film. In some embodiments, the film is a phosphoscreen film. Quantification of the signal in some embodiments is achieved by scanning or by using photoimager software such as Phosphoimager Typhoon IP. Images can be quantified using image analysis software such as ImageJ-win 64 software. In some embodiments, detecting involves exposing a microarray containing a radiolabeled test ligand or a radiolabeled tool ligand to a film such as a phosphoscreen film, thereby generating a signal on the film, and quantifying the signal on the film. In some embodiments, detecting involves measuring the radioactive signal (decay number) by exposing a microarray containing a complex comprising a radiolabeled tool ligand or a radiolabeled test ligand to a real-time autoradiography system based on a new generation gas detector (e.g., a BeaQuant instrument [ai4R], BetaIMAGER [Biospace Lab]). In some embodiments, signal quantification is performed by digital imaging. In some embodiments, images can be quantified using image analysis software (Beamage [ai4R], M3 Vision [Biospace Lab]). In some embodiments, images can be exported to an image processing tool and quantified using image analysis software such as ImageJ-win 64 software.

[0038] In some embodiments, spotting the pathological protein onto a glass support, such as an aminopropylsilane (APS) coated glass slide, in a pad profile; contacting the spotted protein with an unlabeled (cold) ligand to form a complex between the ligand and the protein; contacting the complex with a labeled ligand to form a labeled complex between the labeled ligand and the protein; Washing the labeled complex with a buffer, for example, PBS buffer; drying the glass support, for example at room temperature or under a stream of argon; exposing the glass support to a film, such as a phosphor screen film; quantitating the signal on the film after exposure of the labeled ligand bound to the protein; In some embodiments, the spotted proteins are contacted with a blocking agent. In some such embodiments, the blocking agent is present in an assay buffer containing the cold ligand and / or an assay buffer containing the labeled ligand.

[0039] In some embodiments, the method involves quantifying the signal on a film after exposure of the labeled ligand bound to the protein and determining the binding affinity (K), e.g., by plotting the quantified values ​​on a graph, e.g., by using image software analysis to plot the values ​​on a graph. d and determining a value of

[0040] In some embodiments, the method includes spotting a pathological protein onto a glass support, particularly an aminopropylsilane (APS)-coated glass slide, organized in a pad profile; contacting a composition comprising a labeled ligand with the spotted protein; allowing the labeled ligand to complex with the protein; contacting a composition comprising an unlabeled (cold) ligand with the complex comprising the protein and the labeled ligand; washing with a buffer, such as PBS buffer; optionally drying the APS-coated glass support, such as a glass slide, at room temperature or under a stream of argon; exposing the APS-coated glass support, such as a glass slide, to a film, such as a phosphoscreen film; quantifying the signal on the film after exposure of the labeled ligand bound to the protein; and determining the inhibition constant (Ki), preferably by plotting the quantified signal on a graph, more preferably by plotting the quantified value on a graph using image software analysis.

[0041] In some embodiments, the method includes spotting a pathological protein onto a glass support organized in a pad profile, such as an aminopropylsilane (APS)-coated glass slide; contacting a composition containing a labeled ligand with the spotted pathological protein to complex the labeled ligand with the protein; contacting a composition containing an unlabeled ligand with the complex containing the protein and the labeled ligand; washing with a buffer, such as PBS; optionally drying the glass support at room temperature or under an argon stream; exposing the glass support to a film, such as a phosphorscreen film; quantifying the signal on the film after exposure of the labeled ligand bound to the protein; and determining the inhibitory capacity (inhibition constant, K) by, for example, plotting the quantified signal on a graph or plotting the quantified value on a graph using image software analysis. In some embodiments, the steps included before drying are repeated at least 6, at least 8, or at least 12 times. In some embodiments, the amount of ligand is increased / decreased each time these steps are repeated. In some embodiments, the K value is used to assess whether a compound has the ability to compete with the binding of a labeled ligand to a protein, hi some embodiments, the K value is used to rank the tested compounds according to their K value.

[0042] Also disclosed herein are kits for use in screening or evaluating test ligands / test compounds for their ability to bind to a target or to compete with the binding of a labeled ligand to a target. Such kits include components for carrying out the methods described herein, such as, for example, buffers, detectable dyes, lab equipment, reaction vessels, instructions for use, etc.

[0043] In some embodiments, the present disclosure provides methods for measuring the binding affinity (K dIn other embodiments, the disclosure provides assays for determining the inhibition constant (Ki) for a test ligand / test compound against a pathological protein target. In some aspects, the disclosure provides assays for evaluating, selecting, and / or screening a test ligand / test compound or a series of test ligands / test compounds, where the test ligand / test compound is selected or the test ligands / test compounds are ranked according to the assay results.

[0044] In some methods of evaluating or screening test compounds for their ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, the method comprises: (a) contacting the aliquots with a plurality of cold test compounds, each at a single concentration; or (b) contacting the aliquot with multiple concentrations of a cold test compound; In some embodiments, the method includes ranking the plurality of test compounds according to the calculated percent competition or K for each test compound. In some embodiments, the plurality of cold test compounds is at least 2, at least 5, at least 10, at least 25, at least 50, or at least 100 cold test compounds, or between 2 and 100, or between 5 and 100, or between 10 and 100, or between 25 and 100, or between 50 and 100 cold test compounds.

[0045] In any of the methods described herein, contacting the coated surface onto which multiple aliquots of enriched biological sample are spotted with the non-radioactively labeled ligand can be performed before, simultaneously with, or after contacting the coated surface with the radioactively labeled ligand. [Section 1] The binding affinity (K) of the test ligand to the pathological protein in the enriched biological sample d ), comprising the steps of: contacting a plurality of aliquots of the enriched biological sample on the microarray with a saturating fixed concentration of cold test ligand; contacting the aliquots with multiple concentrations of a radiolabeled test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; From the detected signal in each aliquot, K d and A method comprising: [Section 2] The binding affinity (K) of the test ligand to the pathological protein in the enriched biological sample d ), comprising the steps of: contacting multiple aliquots of the enriched biological sample on the microarray with multiple concentrations of a radiolabeled test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; contacting said aliquot with a saturating fixed concentration of cold test ligand; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; From the detected signal in each aliquot, K d and A method comprising: [Section 3] The binding affinity (K) of the test ligand to the pathological protein in the enriched biological sample d ), comprising the steps of: contacting multiple aliquots of the enriched biological sample on the microarray with multiple concentrations of radiolabeled test ligand and a saturating fixed concentration of cold test ligand to form radiolabeled complexes between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; From the detected signal in each aliquot, K d and A method comprising: [Section 4] 1. A method for determining the inhibition constant (Ki) of a test ligand for a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray were analyzed by K d contacting the aliquots with a fixed concentration of radiolabeled test ligand approximating a fixed concentration of 0.1 to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; contacting the aliquots with multiple concentrations of cold test ligand; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; calculating K from the detected signal in each aliquot; A method comprising: [Section 5] 1. A method for determining the inhibition constant (Ki) of a test ligand for a pathological protein in an enriched biological sample, comprising: contacting a plurality of aliquots of the enriched biological sample on the microarray with a plurality of concentrations of cold test ligand; The aliquot was analyzed by K d contacting the aliquots with a fixed concentration of radiolabeled test ligand approximating a fixed concentration of 0.1 to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; calculating K from the detected signal in each aliquot; A method comprising: [Section 6] 1. A method for determining the inhibition constant (Ki) of a test ligand for a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray were analyzed by K d and a plurality of concentrations of cold test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; calculating K from the detected signal in each aliquot; A method comprising: [Section 7] 1. A method for evaluating a test compound for its ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray are subjected to K d contacting the aliquots with a fixed concentration of radiolabeled tool ligand approximating a fixed concentration of the radiolabeled tool ligand to form a radiolabeled complex between the radiolabeled tool ligand and the pathological protein in each aliquot; contacting the aliquot with a single concentration or multiple concentrations of cold test compound; removing unbound radiolabeled tool ligand from said aliquot; detecting a signal from the radiolabeled tool ligand in the radiolabeled complex in each aliquot; A method comprising: (a) calculating the percentage of competition for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at a single concentration; or (b) calculating the Ki for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at multiple concentrations. [Section 8] 1. A method for evaluating a test compound for its ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, comprising: contacting multiple aliquots of the enriched biological sample on the microarray with a single concentration or multiple concentrations of cold test compound; The aliquot was analyzed by the K d contacting the aliquots with a fixed concentration of radiolabeled tool ligand approximating a fixed concentration of the radiolabeled tool ligand to form a radiolabeled complex between the radiolabeled tool ligand and the pathological protein in each aliquot; removing unbound radiolabeled tool ligand from said aliquot; detecting a signal from the radiolabeled tool ligand in the radiolabeled complex in each aliquot; A method comprising: (a) calculating the percentage of competition for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at a single concentration; or (b) calculating the Ki for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at multiple concentrations. [Section 9] 1. A method for evaluating a test compound for its ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray are incubated with a single concentration or multiple concentrations of cold test compound and the K of the tool ligand. d contacting the aliquots with a fixed concentration of radiolabeled tool ligand approximating a fixed concentration of the radiolabeled tool ligand to form a radiolabeled complex between the radiolabeled tool ligand and the pathological protein in each aliquot; removing unbound radiolabeled tool ligand from said aliquot; detecting a signal from the radiolabeled tool ligand in the radiolabeled complex in each aliquot; A method comprising: (a) calculating the percentage of competition for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at a single concentration; or (b) calculating the Ki for the cold test compound from the detected signal in each aliquot when the cold test compound is contacted at multiple concentrations. [Section 10] (a) contacting the aliquots with a plurality of cold test compounds, each at a single concentration; or (b) contacting the aliquot with a plurality of concentrations of a cold test compound; [Section 11] 11. The method of claim 10, comprising ranking the plurality of test compounds according to the percent competition or Ki calculated for each test compound. [Section 12] Item 12. The method according to any one of items 1 to 11, comprising contacting an aliquot of the enriched biological sample with a blocking agent. [Section 13] 13. The method of claim 12, wherein the aliquot is contacted with the blocking agent prior to or simultaneously with the radiolabeled tool ligand, cold tool ligand, and / or cold test compound. [Section 14] 14. The method of claim 13, wherein the blocking agent is present in one or more assay buffers that also contain the radiolabeled tool ligand, the cold tool ligand, or the cold test compound. [Section 15] 15. The method of claim 14, wherein the assay buffer comprises Tris-HCl or phosphate buffered saline (PBS). [Section 16] Item 16. The method according to any one of Items 12 to 15, wherein the blocking agent is BSA, casein, or albumin derived from chicken egg white. [Section 17] Item 17. The method according to any one of Items 1 to 16, wherein the pathological protein is selected from Aβ, tau, α-synuclein, and TDP-43. [Section 18] Item 18. The method according to any one of Items 1 to 17, wherein each aliquot of the enriched biological sample contains about 3.5 mg / mL to about 6.5 mg / mL of total protein. [Section 19] Item 19. The method according to any one of items 1 to 18, wherein each aliquot is a spot. [Section 20] 20. The method of any one of paragraphs 1 to 19, wherein the microarray comprises at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 40, or at least 45, or at least 50, or at least 55, or at least 60, or about 64 chambers. [Section 21] 21. The method of claim 20, wherein each chamber contains at least one aliquot, or at least six aliquots, or at least nine aliquots, or nine aliquots of the enriched biological sample. [Section 22] 22. The method of claim 20 or 21, wherein contacting the aliquot with a plurality of concentrations comprises contacting each chamber in the microarray with a different concentration. [Section 23] Item 23. The method according to any one of Items 1 to 22, wherein the detection comprises exposing the microarray to a film and then detecting the signal on the film. [Section 24] 24. The method of claim 23, wherein the film is a phosphorscreen film. [Section 25] Item 25. The method according to any one of Items 1 to 24, wherein the microarray is a glass slide. [Section 26] Item 26. The method according to Item 25, wherein the slide glass is an aminopropylsilane-coated slide glass. [Section 27] 27. The method of claim 25 or 26, comprising dispensing an aliquot of the enriched biological sample onto the glass slide to create the microarray. [Section 28] 28. The method of any one of paragraphs 1 to 27, comprising drying the plurality of aliquots on the microarray prior to contacting with a radiolabeled test ligand or a cold test ligand or cold test compound. [Section 29] Item 29. The method according to any one of items 1 to 28, comprising drying before detecting a signal. [Example]

[0046] The following examples are included to further illustrate some embodiments of the present disclosure and should not be used to limit the scope of the present disclosure. These examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0047] While aspects of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous modifications, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the aspects of the disclosure described herein may be used in practicing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0048] Example 1: Preparation of proteins for microarray fabrication a) Pathological tau protein from AD brain Tau paired helical filaments (PHFs) from AD brains were enriched from postmortem brain samples of a single patient with Alzheimer's disease (AD) obtained from an external source (Tissue Solutions, UK). The enrichment procedure was adapted from Jicha et al., 1997 and Rostagno and Ghiso, 2009, and adapted from Spillantini et al., 1998, which described the extraction of scattered a-syn filaments from PD brains by applying a procedure originally developed to extract scattered paired helical filaments and non-necked filaments from AD brains (Greenberg, SG et al., 1990; Goedert, et al., 1992). Briefly, tissues were homogenized in a glass Dounce homogenizer at a weight-to-volume ratio of 1:4 tissue to homogenization buffer volume [0.75 M NaCl in RAB buffer (100 mM 2-(N-morpholino)ethanesulfonic acid (MES), 1 mM EGTA, 0.5 mM MgSO4, 2 mM DTT, pH 6.8) supplemented with protease inhibitors (Complete; Roche 11697498001)]. The homogenate was then incubated at 4°C for 20 minutes to depolymerize any remaining microtubules before being transferred to polycarbonate centrifuge bottles (16 mm × 76 mm; Beckman 355603) and centrifuged at 11,000 g (12,700 RPM) for 20 minutes at 4°C in a pre-chilled 70.1 rotor (Beckman, 342184) in an ultracentrifuge (Beckman, XL100K). The pellet was kept on ice. The supernatant was pooled in polycarbonate bottles and centrifuged again at 100,000 g (38,000 RPM) for 1 hour at 4°C in a 70.1 Ti rotor to separate the PHF-enriched pellet, while soluble tau remained in the supernatant. The pellets from the first and second centrifugations were resuspended in 120 mL of extraction buffer [10 mM Tris-HCl (pH 7.4), 10% sucrose, 0.85 M NaCl, 1% protease inhibitor (Calbiochem 539131), 1 mM EGTA, 1% phosphatase inhibitor (Sigma P5726 and P0044)].The solution was then transferred into polycarbonate centrifuge bottles (16 mm × 76 mm; Beckman 355603) and centrifuged at 15,000 g (14,800 RPM) for 20 minutes at 4°C in an ultracentrifuge (Beckman, XL100K) using a 70.1 Ti rotor. Low-speed centrifugation in the presence of 10% sucrose resulted in the majority of PHFs remaining in the supernatant, while intact or fragmented NFTs and larger PHF deposits / aggregates were pelleted. The pellet was discarded. 20% Sarkosyl (Sigma L7414-10ML) was added to the supernatant to a final concentration of 1% and stirred at room temperature for 1 hour. The solution was then centrifuged in polycarbonate bottles at 100,000 g (38,000 RPM) in a 70.1 Ti rotor for 1 hour at 4° C., and the pellet containing PHF-rich material was resuspended in PBS at a 1:0.1 tissue-to-PBS weight-to-volume ratio, aliquoted, and stored at −80° C. Samples were analyzed for tau by Western blot.

[0049] b) a-syn protein from PD brain The procedure was adapted from the protocol described in Spillantini et al., 1998. Frozen tissue blocks from either the temporal cortex or amygdala brain region were thawed on ice, and white matter was removed using a scalpel. The tissue was homogenized using a glass Dounce homogenizer at a weight-to-volume ratio of 1:4 tissue to homogenization buffer volume. For homogenization, RAB buffer (100 mM 2-(N-morpholino)ethanesulfonic acid (MES), 1 mM EGTA, 0.5 mM MgSO4, 2 mM DTT, pH 6.8) containing 0.75 mM NaCl and 1× protease inhibitor (Complete; Roche 11697498001) was used. The homogenate was then incubated at 4°C for 20 minutes to depolymerize any remaining microtubules before being transferred to polycarbonate centrifuge bottles (16 mm × 76 mm; Beckman 355603) and centrifuged at 11,000 g (12,700 RPM) in a pre-chilled 70.1 rotor (Beckman 342184) in an ultracentrifuge (Beckman XL100K) for 20 minutes at 4°C. The pellet was kept on ice, while the supernatant was pooled in polycarbonate bottles and centrifuged again at 100,000 g (38,000 RPM) in a 70.1 Ti rotor for 1 hour at 4°C to separate a-syn deposits / aggregates from soluble a-syn. The pellets from the first and second centrifugations were resuspended in extraction buffer (10 mM Tris-HCl (pH 7.4), 10% sucrose, 0.85 mM NaCl, 1% protease inhibitor (Calbiochem 539131), 1 mM EGTA, and 1% phosphatase inhibitors (Sigma P5726 and P0044) at a 1:10 (weight-to-volume, wt / v) ratio. The solution was then transferred to polycarbonate centrifuge bottles (16 mm x 76 mm; Beckman 355603) and centrifuged at 15,000 x g (14,800 RPM, 70.1 Ti rotor) for 20 minutes at 4°C. The pellet was discarded, and sarkosyl (20% stock solution, Sigma L7414) was added to the supernatant to a final concentration of 1% and stirred at room temperature for 1 hour.The solution was then transferred to a polycarbonate bottle and centrifuged at 100,000 g (38,000 RPM, 70.1 Ti rotor) for 1 hour at 4°C. The pellet containing enriched a-syn deposits / aggregates was resuspended in PBS at a tissue-to-PBS weight-to-volume ratio of 1:0.1, aliquoted, and stored at -80°C until use. The final fractions obtained by this procedure were analyzed biochemically (e.g., AlphaLISA, Western blot, and dot blot) using antibodies against a-syn to confirm enrichment of a-syn deposits / aggregates.

[0050] c) TDP-43 protein from frontotemporal dementia (FTD) brain Brain tissue (cortex) sections from human brains with TDP-43 pathology were dissected with a scalpel in a P2 laboratory, and the tissue was weighed on a Petri dish. The tissue was transferred with tweezers to a 2 ml homogenization tube (CKmix). Homogenization buffer containing protease inhibitors was added to the dissected tissue at a 1:4 (wt / vol) ratio to obtain a 20% brain homogenate. The suspension was homogenized at 4°C using a precellys at 5000 rpm for three 30-second cycles with a 15-second pause between each cycle. The homogenized tissue was pooled and resuspended in a 5 ml Eppendorf tube. 600 μl aliquots of the homogenized brain were prepared, frozen on dry ice, and stored at -80°C. Solubilization was performed in 1.5 ml protein low-binding tubes (Eppendorf).

[0051] Brain homogenates were thawed on ice and resuspended in HS buffer to a final concentration of 2% sarkosyl, 1 unit / μL benzonase, and 1 mM MgCl2, and incubated at 37°C for 45 minutes under constant shaking at 600 rpm in a thermomixer. The supernatant was collected in a new tube. The pellet was resuspended in 1000 μl of myelin suspension buffer and centrifuged at 20,000 g for 60 minutes at 4°C in a benchtop centrifuge. The supernatant was carefully removed using a 1000 μl tip to remove all floating lipids. If the lipids could not be removed in a single centrifugation step, the resuspension, centrifugation, and removal of the supernatant were repeated. The resulting pellet was washed with PBS and centrifuged at 4°C for 30 minutes in a benchtop centrifuge. The pellet was then resuspended in 200 μl of PBS. All enriched material was pooled and frozen at -80°C.

[0052] Samples were analyzed by Western blot (phosphorylated TDP-43, TDP-43, histone H3, Aβ).

[0053] Example 2: Fabrication of microarrays for pathological proteins a) Method 1 - Automated Spotting Protein samples were diluted 1:3 (volume / volume) in PBS or assay buffer (50 mM Tris-HCl (pH 7.5) in 0.9% NaCl, 0.1% BSA) and homogenized in 1.5 ml Eppendorf tubes by pipetting with a P200 (Eppendorf). The samples were then ready for automated spotting onto aminopropylsilane (APS)-coated 64-pad microarray glass slides (Lucerna-Chem, no. 63475) using the automated spotting device, a non-contact piezoelectric printer called Nano-Plotter 2.1 (GeSiM; Germany). The automated spotting device is a versatile non-contact array printer capable of dispensing small amounts of liquid (picoliters to nanoliters) using electrical pulses.

[0054] APS slides (Figure 1A) were manually placed on the automatic trail and properly secured to ensure high spotting reproducibility between slides and proper droplet positioning when each slide contained a chamber. A piezoelectric tip was used to pipette the appropriate volume from the loading plate. The system was optimized to dispense 12 × 3 nL droplets per spot, with nine spots per pad, for a total of 64 pads on each slide (Figure 1B). Sample spotting was performed in a humidity-controlled atmosphere at 65% relative humidity. Prior to spotting, the homogenization quality of the dispensed droplets was assessed to ensure that the volume and density of the sample were consistent throughout the entire dispense. To this end, each droplet was measured as it was dispensed from the tip, and the dispersion of the droplets was measured under specific voltages. Once spotted onto the slides, the chambers were assembled in a Proplate multi-well chamber 64 well (25 mm x 75 mm glass microscope slides), and two stainless steel ProPlate™ clips on either side of the slide were used to ensure watertightness of the compartments. This system contained 64 individual wells. Samples were allowed to dry in a humidified chamber for 15 minutes before being stored at 4°C until use.

[0055] b) Method 2 - Manual Spotting Protein samples were manually spotted onto chambered glass slides by pipetting 1 μL using a micropipette p2 (Eppendorf) (Figures 1C and 1D). Only one drop was pipetted per location, one drop corresponding to a spot on the slide. The resulting slides were allowed to dry for at least 2 h at room temperature in a conventional lab hood.

[0056] Example 3: Preparation of cold samples Cold compounds (test ligands or test compounds) were resuspended as 2.5 mM or 10 mM stock solutions in 100% DMSO. Dilutions of cold compounds were obtained by performing a 12-point serial dilution series with 2- to 3-fold dilutions. Dilutions were performed in 100% DMSO to ensure a constant final DMSO concentration of 1% to 2.5% in the binding assay reaction volume. The maximum concentration of cold compound used was 2 μM or 3 μM, depending on the target, and these conditions were also used to determine maximal signal displacement.

[0057] Example 4: Preparation of labeled samples Labeled compounds (radiolabeled test ligand or radiolabeled tool ligand; 1 mCi / mL) were synthesized and dissolved in 100% ethanol. d Labeled compounds were diluted in assay buffer at a range of concentrations appropriate for experiments determining the potency of the antibody, or at a fixed concentration for experiments used to assess the strength of displacement.

[0058] Example 5: Tau binding affinity (K) by microradiometric binding assay d ) determination The chambers containing the spotted pathological tau protein samples were mounted and filled with assay buffer (50 mM Tris (pH 7.5), 138 mM NaCl, 0.1% BSA) containing 2 μM cold test ligand. The chambers were incubated at room temperature for 120 min. Sealing film was used to prevent evaporation. Equal volumes of tritiated test ligand in assay buffer at various concentrations were added to each chamber, mixed well, and incubated at room temperature. The final reaction volume was 40 μL. After 60 min of incubation, the reaction solution containing the radioactive material was collected in an appropriate container. The chambers were washed five times with ice-cold wash buffer. The ProPlate™ chambers were removed from the slides, and the slides were washed with double-distilled H2O. The slides were dried under a stream of argon in a chemical hood. Films were exposed for at least 3 days on a Fujifilm BAS-IP TR 2025 in a hypercassette (Amersham, RPN 11643). Films were scanned using a Phosphoimager Typhoon IP at a resolution of 50 μm and a sensitivity of 4000. Images were then analyzed and quantified using ImageJ-win 64 software. Graphs were generated using GraphPad Prism 7.03. A K of 7.9 nM was obtained with a good fit for the tool ligand and tau deposits / aggregates. d was determined (Figure 2B).

[0059] Example 6: Comparison of microradioactive binding assay with filter-based radioactive binding assay Using this classical filter-based assay and the microradioactive binding assay described above, dA direct comparison of the determinations was performed to assess the differences between these methods. To perform the filter-based assay, tau from AD brains was diluted 1 / 80 and incubated with tritiated test ligands (known tau binders) at concentrations ranging from 1 nM to 50 nM, with or without a fixed concentration of cold test ligand at 2 μM, for 120 min at 25°C. A volume of 35 μL of each sample was filtered under vacuum onto a GF / C filter plate (PerkinElmer 6005174) to capture the test ligand-bound AD brain tau. The GF / C filters were then vacuum-dried, 50 μL of scintillation fluid (Ultimate Gold MB, PerkinElmer) was added to each well, and the filters were analyzed in a Microbeta2 device. Nonspecific signal was determined using samples containing an excess of cold test ligand (2 μM), and specific binding was calculated by subtracting nonspecific signal from total signal. All measurements were performed in at least two technical replicates. K was calculated by nonlinear regression using Prism V7 (GraphPad) with one-site specific binding. d The value was calculated to be 11.8 nM. d was obtained (FIG. 2A). Using the same test ligand, we found that very similar binding affinity values ​​for independent AD brain-derived tau deposits / aggregates were obtained using the two methods (11.8 nM (FIG. 2A) vs. 7.9 nM (FIG. 2B, described in Example 5)). This result demonstrates that the microradiometric binding assay method is a robust alternative to classical filter-based radioactive binding assays.

[0060] Example 7: K for a-syn and TDP-43 by microradiometric binding assay d Decision The method described in Example 5 was also used to measure the binding constants (K) of test ligands (Compound 2 (see PCT Application WO 2019 / 234243) and Compound 3) to the protein targets a-syn (for Compound 2 and Compound 3) and TDP-43 (for Compound 3).d ) were determined. TDP-43-enriched fractions isolated from FTD brains or a-syn-enriched fractions isolated from PD brains were treated with increasing concentrations (1 nM to 300 nM or 1 nM to 30 nM, respectively) of radiolabeled [ 3 H] compound 3 with or without a 2 μM fixed amount of cold compound 3. Similarly, a-syn-enriched fractions isolated from PD brains were incubated with increasing concentrations (1 nM to 30 nM) of radiolabeled [ 3 H] Compound 2 was incubated with or without a fixed amount of cold Compound 2 at 2 μM. Nonspecific binding was determined using a fixed excess concentration of cold Compound 2 (2 μM) or cold Compound 3 (2 μM). Compound 3 had K values ​​of 10.8 nM (Figure 3A) and 138 nM (Figure 3C) for a-syn and TDP-43 proteins, respectively. d The K value was determined to be 7.8 nM for compound 2 against a-syn. d These results demonstrate that the K values ​​obtained by the described microradiometric binding assay are significantly higher for several target proteins known to be present at low or relatively low abundance in biological tissues (Figure 3B). d For example, pathological a-syn and pathological TDP-43 appear to be present in lower abundance than pathological tau in the brains of diseased humans.

[0061] Example 8: Use of a microradioactive binding assay to determine the inhibition constant (Ki) of a test ligand A microradiometric binding assay was used to determine the inhibition constants (Ki) of test ligands against tau deposits / aggregates from AD brains and a-syn deposits / aggregates from PD brains. Proteins were prepared and spotted onto coated glass slides as described in Example 1 (steps a and b) and Example 2 (step a).

[0062] 3 nM tritiated test ligand (known tau binder) was incubated with spotted tau deposits / aggregates and cold test ligand at concentrations ranging from 10 pM to 3 μM (Figure 4A). Maximum signal (100% binding) was obtained in the absence of cold tool ligand, whereas maximum displacement was obtained in the presence of 3 μM cold tool ligand. Ki values ​​were calculated by one-site-fit Ki using Prism V7 (GraphPad). Ki values ​​for test ligands were measured at 1 nM and R 2 =0.97 for good fit.

[0063] Cold compound 3 was injected into a-syn deposits / aggregates spotted at concentrations ranging from 50 pM to 2 μM (or 10 nM to 3 μM) with 40 nM [ 3 H] was incubated with compound 3. The maximum signal (100% binding) was obtained in the absence of cold compound 3, whereas maximum displacement was obtained in the presence of 2 μM cold compound 3. The K value for compound 3 was measured at 41 nM with a good fit (Figure 4B).

[0064] These results demonstrate that the described microradiometric binding assay can determine the self-displacement capacity of compounds (as determined by the calculated K) for several protein targets known to be present in low or relatively low abundance in biological tissues.

[0065] Example 9: Microradiometric binding assay to rank the activity of library compounds Test compounds were administered to a-syn deposits / aggregates derived from the brains of PD patients. 3H] Compound 3 (radiolabeled tool ligand) binding was screened for their ability to compete with the binding. Displacement of test compounds can be assessed in a screening format to rank test compounds based on their ability to displace the radiolabeled tool ligand (ranking based on calculated K values). Protein sample preparation and spotting were performed as described in Example 1(b) and Example 2(a) above.

[0066] Test compounds were tested in duplicate in two independent experiments, with mean values ​​± SEM shown in Figures 5A-5C. 3 Test compounds were screened using [H] compound 3 at concentrations ranging from 50 pM to 2 μM. Representative competition curves are shown for the following compounds: compound 4 (Figure 5A, Ki 13 nM, strong binder), compound 5 (Figure 5B, Ki 37 nM, medium binder), and compound 6 (Figure 5C, Ki 147 nM, weak binder). Collectively, these results demonstrate that the described microradiometric binding assay can be used to measure the displacement ability (Ki) of test compounds in a screening format, allowing for the ranking of screened test compounds according to their calculated Ki values, e.g., from weakest to strongest binders. Test compounds exhibiting lower Ki values ​​are considered stronger binders and represent potential hit compounds for the tested protein target. Furthermore, the ability of a test compound to displace a radiolabeled tool ligand indicates that the test compound binds to the protein target at a site that overlaps with the protein binding site of the radiolabeled tool ligand.

[0067] While aspects of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous modifications, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the aspects of the disclosure described herein may be employed in practicing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0068] References Greenberg, SG and P. Davies, "A preparation of Alzheimer paired helical filaments that displays distinct tau proteins by polyacrylamide gel electrophoresis," Proc. Natl. Acad. Sci. USA 1990, 87(15), 5827-31. Goedert, M. et al., "Cloning of a big tau microtubule-associated protein characteristic of the peripheral nervous system," Proc. Natl. Acad. Sci. USA 1992, 89, 1983-1987. Jicha, GA et al., "A Conformation- and Phosphorylation-Dependent Antibody Recognizing the Paired Helical Filaments of Alzheimer's Disease," J. Neurochem. 1997, 69, 2087-2095. Mandelkow, E. and E. Mandelkow, "Tau in Alzheimer's disease," Trends Cell Biol, 8(11), 425-427. Neumann, M. et al., "Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis," Science 2006, 314 (5796), 130-133. Nelson, P.T. et al., "Limbic-predominant age-related TDP-43 encephalopathy (LATE): consensus working group report," Brain 2019, 142(6), 1503-27. Posner, B. et al., "Multiplexing G protein-coupled receptors in microarrays: A radioligand-binding assay," Anal. Biochem. 2007, 365, 266-73. Rostagno, A. and J. Ghiso, "Isolation and biochemical characterization of amyloid plaques and paired helical filaments," Curr. Protoc. Cell Biol. 2009, 44(1), 3.33.1-3.33.33. Spillantini, M.G. et al., "α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies," Proc. Natl. Acad. Sci. USA 1998, 95, pp. 6469-6473. Serrano-Pozo et al., "Neuropathological alternations in Alzheimer disease," Cold Spring Harb. Perspect. Med. 2011, 1, a006189.

Claims

1. The binding affinity (K) of the test ligand to the pathological protein in the enriched biological sample ｄ ) a method for determining contacting a plurality of aliquots of the enriched biological sample on the microarray with a saturating fixed concentration of a non-radiolabeled test ligand; contacting the aliquots with multiple concentrations of a radiolabeled test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; From the detected signal in each aliquot, K ｄ and A method comprising:

2. The binding affinity (K) of the test ligand to the pathological protein in the enriched biological sample ｄ ) a method for determining contacting multiple aliquots of the enriched biological sample on the microarray with multiple concentrations of a radiolabeled test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; contacting the aliquot with a saturating fixed concentration of a non-radiolabeled test ligand; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; From the detected signal in each aliquot, K ｄ and A method comprising:

3. The binding affinity (K) of the test ligand to the pathological protein in the enriched biological sample ｄ ) a method for determining contacting multiple aliquots of the enriched biological sample on the microarray with multiple concentrations of a radiolabeled test ligand and a saturating fixed concentration of a non-radiolabeled test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; From the detected signal in each aliquot, K ｄ and A method comprising:

4. 1. A method for determining the inhibition constant (Ki) of a test ligand for a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray were analyzed by K ｄ contacting the aliquots with a fixed concentration of radiolabeled test ligand approximating a fixed concentration of 0.1 to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; contacting the aliquots with multiple concentrations of a non-radiolabeled test ligand; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; calculating K from the detected signal in each aliquot; A method comprising:

5. 1. A method for determining the inhibition constant (Ki) of a test ligand for a pathological protein in an enriched biological sample, comprising: contacting multiple aliquots of the enriched biological sample on the microarray with multiple concentrations of a non-radiolabeled test ligand; The aliquot was analyzed by K ｄ contacting the aliquots with a fixed concentration of radiolabeled test ligand approximating a fixed concentration of 0.1 to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; calculating K from the detected signal in each aliquot; A method comprising:

6. 1. A method for determining the inhibition constant (Ki) of a test ligand for a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray were analyzed by K ｄ and a plurality of concentrations of a non-radiolabeled test ligand to form a radiolabeled complex between the radiolabeled test ligand and the pathological protein in each aliquot; removing unbound radiolabeled test ligand from said aliquot; detecting a signal from the radiolabeled test ligand in the radiolabeled complex in each aliquot; calculating K from the detected signal in each aliquot; A method comprising:

7. 1. A method for evaluating a test compound for its ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray are then subjected to the K ｄ contacting the aliquots with a fixed concentration of radiolabeled tool ligand approximating a fixed concentration of the radiolabeled tool ligand to form a radiolabeled complex between the radiolabeled tool ligand and the pathological protein in each aliquot; contacting the aliquot with a single concentration or multiple concentrations of a non-radiolabeled test compound; removing unbound radiolabeled tool ligand from said aliquot; detecting a signal from the radiolabeled tool ligand in the radiolabeled complex in each aliquot; (a) calculating the percent competition for the non-radiolabeled test compound from the detected signal in each aliquot when the non-radiolabeled test compound is contacted at a single concentration, or (b) calculating the Ki for the non-radiolabeled test compound from the detected signal in each aliquot when the non-radiolabeled test compound is contacted at multiple concentrations.

8. 1. A method for evaluating a test compound for its ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, comprising: contacting multiple aliquots of the enriched biological sample on the microarray with a single concentration or multiple concentrations of a non-radiolabeled test compound; The aliquot was then subjected to a K ｄ contacting the aliquots with a fixed concentration of radiolabeled tool ligand approximating a fixed concentration of the radiolabeled tool ligand to form a radiolabeled complex between the radiolabeled tool ligand and the pathological protein in each aliquot; removing unbound radiolabeled tool ligand from said aliquot; detecting a signal from the radiolabeled tool ligand in the radiolabeled complex in each aliquot; (a) calculating the percent competition for the non-radiolabeled test compound from the detected signal in each aliquot when the non-radiolabeled test compound is contacted at a single concentration, or (b) calculating the Ki for the non-radiolabeled test compound from the detected signal in each aliquot when the non-radiolabeled test compound is contacted at multiple concentrations.

9. 1. A method for evaluating a test compound for its ability to displace a radiolabeled tool ligand in a radiolabeled complex with a pathological protein in an enriched biological sample, comprising: Multiple aliquots of the enriched biological sample on the microarray are then subjected to a single or multiple concentrations of non-radiolabeled test compound and the K of the radiolabeled tool ligand. ｄ contacting the aliquots with a fixed concentration of radiolabeled tool ligand approximating a fixed concentration of the radiolabeled tool ligand to form a radiolabeled complex between the radiolabeled tool ligand and the pathological protein in each aliquot; removing unbound radiolabeled tool ligand from said aliquot; detecting a signal from the radiolabeled tool ligand in the radiolabeled complex in each aliquot; (a) calculating the percent competition for the non-radiolabeled test compound from the detected signal in each aliquot when the non-radiolabeled test compound is contacted at a single concentration, or (b) calculating the Ki for the non-radiolabeled test compound from the detected signal in each aliquot when the non-radiolabeled test compound is contacted at multiple concentrations.

10. (a) contacting the aliquots with a plurality of non-radiolabeled test compounds, each at a single concentration; or 10. The method of any one of claims 7 to 9, comprising: (b) contacting the aliquot with a plurality of concentrations of a non-radiolabeled test compound.

11. 11. The method of claim 10, comprising ranking the plurality of test compounds according to the percent competition or Ki calculated for each test compound.

12. The method of any one of claims 1 to 11, comprising contacting an aliquot of the enriched biological sample with a blocking agent.

13. 13. The method of claim 12, wherein the aliquots are contacted with the blocking agent prior to or simultaneously with contacting the aliquots with a radioactively labeled tool ligand, a radioactively labeled test ligand, a non-radioactively labeled test ligand, and / or a non-radioactively labeled test compound.

14. 14. The method of claim 13, wherein the blocking agent is present in one or more assay buffers that also contain the radiolabeled tool ligand, the radiolabeled test ligand, the non-radiolabeled test ligand, or the non-radiolabeled test compound.

15. 15. The method of claim 14, wherein the assay buffer comprises Tris-HCl or phosphate buffered saline (PBS).

16. The method according to any one of claims 12 to 15, wherein the blocking agent is BSA, casein, or albumin from chicken egg white.

17. The method of any one of claims 1 to 16, wherein the pathological protein is selected from Aβ, tau, α-synuclein, and TDP-43.

18. 18. The method of any one of claims 1 to 17, wherein each aliquot of the enriched biological sample contains from about 3.5 mg / mL to about 6.5 mg / mL of total protein.

19. The method of any one of claims 1 to 18, wherein each aliquot is a spot.

20. 20. The method of any one of claims 1 to 19, wherein the microarray comprises at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 40, or at least 45, or at least 50, or at least 55, or at least 60, or about 64 chambers.

21. 21. The method of claim 20, wherein each chamber contains at least one aliquot, or at least six aliquots, or at least nine aliquots, or nine aliquots of the enriched biological sample.

22. 22. The method of claim 20 or 21, wherein contacting the aliquot with a plurality of concentrations comprises contacting each chamber in the microarray with a different concentration.

23. The method of any one of claims 1 to 22, wherein the detecting comprises exposing the microarray to a film and then detecting the signal on the film.

24. 24. The method of claim 23, wherein the film is a phosphorscreen film.

25. The method according to any one of claims 1 to 24, wherein the microarray is a glass slide.

26. 26. The method of claim 25, wherein the slide is an aminopropylsilane-coated slide.

27. 27. The method of claim 25 or 26, comprising creating the microarray by dispensing an aliquot of the enriched biological sample onto the glass slide.

28. 28. The method of any one of claims 1 to 27, comprising drying the plurality of aliquots on the microarray prior to contacting with a radiolabeled test ligand or a non-radiolabeled test ligand or a non-radiolabeled test compound.

29. The method of any one of claims 1 to 28, comprising drying before detecting the signal.

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