Materials and Methods for Measuring the Amyloid Cascade By Water Proton Relaxation And

Their Uses in the Diagnosis and Treatment of Amyloid Diseases

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application for Patent, Serial No. 62/358,135, filed July 4, 2016, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention received no federally sponsored research or development funds. FIELD OF THE INVENTION

[0002] The invention provides for materials and methods for measuring the effects of test compounds on the amyloid cascade using nuclear magnetic resonance water proton relaxation measurements.

BACKGROUND OF THE INVENTION

[0003] As used in this description and claims, "amyloid" is a polypeptide that forms aggregates with dimensions that make it visible upon light microscopy, and that has a betasheet structure indicated by the binding of betasheet-binding dyes like Congo Red or the Thioflavins. Amyloid is an abnormal (disease associated), intracellular or extracellular polypeptide aggregate that can be seen as either fibrils or aggregates upon light microscopy.

[0004] The "amyloid cascade", shown in Figure 1, refers to the in vitro conversion of native monomeric proteins or peptides into improperly folded monomers, small aggregates or dimers, then to tetramers, oligomers, and finally into the amyloids that are a common hallmark of different diseases. Amyloid cascades are only examined in vitro using pure monomers. [0005] With Alzheimer's disease, peptides of 40 or 42 amino acids (so-called "ABeta peptides") form amyloid aggregates. With Parkinson's disease, intracellular amyloid structures called Lewy bodies consist of largely aggregates of alpha synuclein, a 140 amino acid protein.

[0006] A protein called "tau" forms amyloid in Alzheimer's disease and in neurodegenerative diseases (NDD's), termed "tauopathies." Tau is often present in amyloid structures termed "neurofibrillary tangles" (NFTs). In Huntington's disease, amyloid is comprised of abnormal forms of the Huntingtin protein.

[0007] Systemic amyloidoses also result from amyloid forming biomolecules, such as β2- microglobulin (dialysis-related amyloidosis), transthyretin (TTR, familial amyloidosis), and immunoglobulin light chain (AL, light chain amyloidosis). Systemic amyloidoses have amyloid deposits in many organs, while in NDDs amyloid is restricted to the CNS. Neurodegenerative diseases and their associated amyloids are discussed in Stefani, 2003 ¹ ; systemic amyloidosis and their amyloids are discussed in Lavatelli, 2015 ¹ . A unique case is type II diabetes, where the amyloid (called amylin or IAPP peptide) is produced in and restricted to the pancreas.

[0008] A major approach in the pharmacotherapy of amyloid related disease is the development of approaches for inhibiting or reversing the amyloid cascade. For example, the low molecular weight drug Tafamidis inhibits the cascade of the transthyretin (TTR) amyloidoses.

[0009] Amyloid cascades and betasheet-binding compounds

[0010] Figure 1 shows a general outline of amyloid cascades, where native proteins unfold, dimerize, oligomerize and finally form mature fibrils containing the characteristic betasheet formation (not shown in Figure).

[0011] As shown in Figure 2A (for the ABeta peptide amyloid cascade) and in Figure 2B (for the tau protein amyloid cascade), the initial phases of the cascades involve small or soluble aggregates that lack a betasheet structure. As the cascades progress, aggregates and fibrils form with the characteristic betasheet structure. [0012] Amyloid cascades are often assayed by the binding of small molecule reporters (SMRs), to the common the betasheets characteristic of amyloid aggregates or amyloid fibrils. As shown in Figure 3A, ABeta peptides form "hairpin loops" and then betasheets fibrils. (Figure 3 is modified from reference .) As shown in Figure 3B, the fluorochrome thioflavin T (ThT), a small molecule reporter, binds in crevices formed by amino acid side chains of the ABeta betasheets formed by multiple ABeta peptides.

[0013] Table 1 provides a listing of amyloid diseases, the amyloid-forming molecule, and Small Molecule Reporters that have been shown to bind to the amyloid-forming molecules. These Small Molecule Reporters include Congo Red, thioflavin T and ¹²⁵ I-IMPY.

[0014] Upon binding to betasheets, some small molecule reporters activate the fluorescence of fluorochromes, like Thioflavin T, which are used assays for inhibitors of betasheet formation, and which are used to visualize fibrils by microscopy. For radiolabeled small molecule reporters, binding to betasheets must be measured after separation of unbound molecule (free/bound separation) using separation techniques like filtration.

[0015] The time course of a typical small molecule reporter fluorescent assay for the amyloid cascade used to screen for inhibitors of the amyloid cascade is shown in Figure 3B. During the lag phase, small aggregates are formed, as shown in Figures 2A and 2B, which do not bind Thioflavin T, and no fluorescence is observed. During the elongation phase, fluorescence increases, reflecting the presence of increasing amounts of betasheet. Figure 3B is a representation of an SMR, such as Thioflavin T, binding to betasheet structures.

[0016] However, small molecule reporter assays have limitations. Small molecule reporter assay methods using betasheet binding reporter molecules detect the presence of the betasheet structure only, and not the biomolecule aggregation that occurs before formation of the betasheet. Thus, the initial, small aggregates of the cascade before betasheet formation cannot be assessed with small molecule reporter type assays.

[0017] These SMR assays have additional limitations: small molecules reporters can interact with test amyloid inhibitors or promote amyloid formation themselves (e.g. thioflavin with alpha- synuclein, ¹⁴ ).

[0018] The fluorescence of possible amyloid cascade inhibitors (e.g. curcumin and quercetin) can interfere with SMR fluorescence ¹⁵ . Some SMRs like Congo Red may bind multiple aggregate forms along the amyloid cascade ¹⁶ .

[0019] There is a need for a methodology that can be used to measure all steps of amyloid cascades in a betasheet independent fashion, and the effect of test molecules on those cascades.

SUMMARY OF THE INVENTION

[0020] A first aspect of the invention is a method for determining the effects of a test molecule on the amyloid cascade by providing a test molecule, adding the test molecule to a solution containing amyloid forming biomolecules, permitting the test molecule to interact with the amyloid forming biomolecules, and measuring the change in water proton relaxation as a result of the interaction of the test molecule on the amyloid cascade.

[0021] Another aspect of the invention is a method for determining the effects of a test molecule on an amyloid cascade by providing a test molecule, adding the test molecule to a solution containing amyloid forming biomolecules, adding a detection agent capable of binding to the amyloid forming biomolecules to the test solution, permitting the test molecule to interact with the amyloid forming biomolecules, and measuring the change in water proton relaxation as a result of the interaction of the test molecule on the amyloid cascade. In additional aspects of the invention, a biotinylated amyloid forming biomolecule is used and the detection agent is streptavidin-magnetic particles.

[0022] In another aspect of the invention, the amyloid forming biomolecule is a fluorescein labeled amyloid forming biomolecule, and the detection agent is anti-fluorescein magnetic particles.

[0023] In another aspect of the invention, the amyloid forming biomolecule is a hapten- modified amyloid forming biomolecule and the detection agent bears antibodies to the hapten. The detection agent can be magnetic particles bearing antibodies to the hapten.

[0024] In another aspect of the invention, the amyloid forming biomolecule is an epitope- bearing amyloid forming biomolecule and the detection agent bears antibodies to the epitope.

[0025] In other aspects of the invention, the amyloid forming biomolecule is ABeta peptide 1-40, ABeta 1-42, Alpha synuclein, Huntingtin, Superoxide Dismutase (SOD) and Transerythrin. In other aspects of the invention the detection agent is selected from the group consisting of Gadolinium chelates and magnetic particles.

[0026] Another aspect of the invention includes methods where an amyloid forming biomolecule to which is attached a paramagnetic Gadolinium chelate or a magnetic particle, see Figure 6. The formation of aggregates changes the way the Gadolinium or magnetic particles alter water proton relaxation rates. Test molecules can therefore affect the way the detection agent-biomolecule participates in the amyloid cascade. ^17"18

[0027] Another aspect of the invention includes methods where the aggregation of biomolecules in the amyloid cascade results in pools of water whose proton relaxation properties are different from bulk water. In this aspect of the invention, the proton relaxation rates are measured without the addition of magnetic particles and paramagnetic chelates. Again, measurements plus and minus a test substance are employed.

[0028] Another aspect of the invention includes methods of measuring the effects of a plurality of test molecules, such as a library of test molecules on the amyloid cascade. The method includes providing a plurality of test molecules, adding the test molecules to a solutions containing amyloid forming biomolecules, permitting the test molecules to interact with the amyloid forming biomolecules, and measuring the changes in water proton relaxation as a result of the interaction of the test molecules on the amyloid cascade.

[0029] Another aspect of the invention is a method for determining the effects of plurality of test molecules on an amyloid cascade by providing a plurality of test molecules, adding the test molecules to solutions containing amyloid forming biomolecules, adding a detection agents capable of binding to the amyloid forming biomolecules to the test solutions, permitting the test molecules to interact with the amyloid forming biomolecules, and measuring the changes in water proton relaxation as a result of the interaction of the test molecule on the amyloid cascade.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows a general outline of amyloid cascades. Native proteins unfold, dimerize, oligomerize into multiple forms of soluble oligomers, and finally form mature fibrils (with betasheet structures not shown). ¹⁹

[0031] Figure 2 shows the betasheet structures that appear late in the amyloid cascade. Figure 2A shows the ABeta peptide cascade with the late cascade stages involving the formation of betasheets. Figure 2B shows the tau protein, with late cascade stages involving the formation of betasheets. ²⁰

[0032] Figure 3 shows the betasheet dependence of a fluorescent assay for an ABeta peptide amyloid cascade using Thioflavin T (ThT) (Figure is from Noel, 2013 ³ .) Figure 3A shows a representation of the binding mode of ThT to the ABeta peptide. Single ABeta peptides (top) form hairpin loops, which are organized into extended betasheet fibrils along the x-axis. ThT molecules bind in crevasses formed by amino acid side chains (dark and light grey ovals extending in the z direction) of the betasheet formed by many ABeta peptides. Figure 3B shows a representative time course of fluorescence in a ThT assay for betasheet formation. During the lag phase, small aggregates are formed, which, because they lack the betasheet structure, do not bind ThT, and so no fluorescence is seen. As the cascade continues, florescence is observed, which corresponds to fibril elongations.

[0033] Figure 4 shows the ability of the water proton relaxation assay methodology to distinguish biotin monomers from biotin dimers in an assay for protease activity. In Step 1, protease (if present) cleaves a dibiotinylated peptide substrate ("biotin dimers") in solution. The dipeptide, shown on the left side of Figure 4, is represented as a grey oval. The biotins on dipeptide are shown as two smaller lighter grey ovals. The cleaved peptide on the right is shown as two half ovals, each bearing the smaller ovals. In Step 2, a detection agent is added to the solution. Here, the detection agent is a magnetic particle ("MP") (dark circle) with streptavidin molecules attached (crescents) (left panel). The monobiotinylated peptide products of protease activity ("biotin monomers") bind strep-MP but do not cause strep-MP aggregation (right panel, "dispersed MPs"). Dibiotin peptides that are not cleaved by protease bind to the strep-MP and aggregate; those strep-MPs that bind peptides that have been cleaved by the protease, i.e, that are now monomers or monobiotinylated peptides, remain dispersed. In Step 3, water proton relaxation measurements compare aggregated and dispersed magnetic particles (strep-MPs). [0034] Figure 5 shows water proton relaxation assays for test molecule inhibition of the cascade using a detection agent, a protein-MP detection agent, which is added to the assay after the amyloid cascade has occurred. In Step 1, an amyloid forming biomolecule (dark oval) bearing a single epitope or hapten (smaller circle) forms aggregates of an amyloid cascade. When a test molecule (not shown) blocks the amyloid cascade, monomers remain. In Step 2, the protein- MP detection agent is added. Protein-MPs here consist of magnetic particles (black circle) with attached proteins (crescents). Monomers bind to protein-MPs, however, no protein-MP aggregation occurs (left panel). When the test molecule fails to alter the amyloid cascade, amyloid aggregates with many haptens or epitopes per aggregate are formed. Now, protein-MP aggregation results. In Step 3, aggregation of the protein-MPs detection agent (or lack of detection agent aggregation) is determined by water proton relaxation measurements.

[0035] Figure 6 shows two water proton relaxation assays for test molecules where detection agents are present during the amyloid cascade. In Figure 6A, the detection agent consists of a Gd-chelate (circle) attached to an amyloid forming biomolecule (oval). In Step 1, the Gd-biomolecule detection agent participates in an amyloid cascade in a solution, with and without test molecule in the solution. Test molecule may block the aggregation of Gd-chelate forming biomolecules (block the amyloid cascade), In Step 2, water proton relaxation measurements of separate solutions with and without test molecule determine the effect of a test molecule on the cascade.

[0036] In Figure 6B, the detection agent is a magnetic particle (black circle) with multiple amyloid forming biomolecules attached (gray ovals). As in Figure 6A in Step 2, water proton relaxation measurements of separate solutions with and without test molecule determine the effect of a test molecule on the cascader

[0037] Figure 7 shows two water proton relaxation assays for the effects of test molecules on amyloid cascades using the exchange of water pools in the solution. Figure 7A shows an assay taking advantage of rapid water exchange in a single pool. In a solution of monomers that participate in the amyloid cascade, a change in water proton relaxation occurs due to the progression of the amyloid cascade. Measurements of the effect of test molecule on the amyloid cascade using solutions with and without the test molecule are made by water proton relaxation. Here, with rapid exchange of only a single pool of water, with a single example, T2 spin-spin relaxation time is obtained.

[0038] In Figure 7B, water proton relaxation measurements take advantage of the slow exchange of water entrapped in monomers and bulk pools of water. A change of water proton relaxation occurs due to the amyloid cascade, and in the presence (or lack of) a test molecule. Here with slow exchange, multiple pools of water exist and multiple relaxation processes are obtained, e.g. multiple T2 measurements over time.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The following Terms have the definition as provided. All other terms not defined in this specification are given their usual and customary definitions as used by one of ordinary skill in the field.

[0040] Amyloid: a disease-associated aggregate seen with light microscopy in tissue sections (histology), consisting of more than about 10 polypeptides with a betasheet structure that binds betasheet-binding dyes like Congo Red or the Thioflavins.

[0041] Amyloid cascade: the aggregation of proteins or peptides into small aggregates, oligomers and finally amyloid that occurs in vitro and is believed to represent with amyloid formation in vivo.

[0042] Amyloid-forming biomolecule: A peptide or protein component of an amyloid seen on histology (in vivo) and which undergoes an amyloid cascade in vitro.

[0043] Bulk water: the majority of water in a sample and which is the water not bound to components, like proteins, in the sample. [0044] Detection agent: a paramagnetic, ferromagnetic or superparamagnetic agent that binds to various intermediates of an amyloid cascade or which participates in an amyloid cascade. Paramagnetic detection agents can employ metal chelates like those of Gadolinium while superparamagnetic agents can employ magnetic particles having a high magnetic moment. Other metal ions include Manganese and Iron.

[0045] Excitatory RF pulse: a radiofrequency pulse applied to a sample in a magnetic field, which excites or is absorbed by water protons.

[0046] Emitted RF signal: a radiofrequency signal from water protons emitted after an excitatory RF pulse.

[0047] Hapten: a small molecule attached to a larger molecule that can bind to an active site on an antibody or protein (e.g. streptavidin).

[0048] High throughput screening ("HTS"): a process of determining the effects on a large number of biomolecules on a biological process. HTS systems can use robotics and other automation techniques to quickly measure interactions between large numbers of test molecules and substrates.

[0049] Library: a collection of different test molecules.

[0050] Magnetic particle (MP): Materials with diameters between 10 nm and 5000 nm that have a strong, positive magnetic susceptibility. Addition of magnetic particles to a sample increases proton relaxation rates (1/T's) and shortens corresponding proton relaxation times (T's). MPs with diameters of 5-200 nm are sometimes termed "nanoparticles" and those with diameters of 200 to 5000 nm termed "microparticles" or micron-sized particles.

[0051] Magnetic particle size: the diameter of a particle, which includes all components of the particle (e.g. magnetic metal or metal oxides, polymers). Magnetic particle size can be determined by light scattering.

[0052] Non-exchanging water: A small pool of water associated with a biomolecule that exchanges slowly or not at all with water in other pools, including the bulk pool. [0053] Paramagnetic chelate: a complex consisting of paramagnetic metal ion (e.g. Gadolinium ⁺ , and small organic molecule to which the metal ion binds (e.g. DOTA, DTP A).

[0054] Polypeptide: a peptide chain for any length or molecular weight, made either synthetically or in a biological system.

[0055] Small Molecule Reporter (SMR): a molecule (<1000 daltons) that binds to a betasheet structure. Binding can be accompanied by fluorescence. When a radiolabeled SMR is used, SMR binding to betasheet is determined by separating bound and free and determining the bound SMR radioactivity.

[0056] Test molecule: a molecule whose effect on an amyloid cascade is to be assessed using water protein relaxometry. Test molecules can be less than about 500 Da or biological materials, such as antibodies and can bind to any portion of an amyloid forming biomolecule

[0057] Water proton relaxation (WPR): a magnetic resonance measurement whereby a radiofrequency pulse excites water protons and after the excitatory pulse, a radiofrequency signal is emitted and its decay (relaxation) is recorded. Water proton relaxation is also referred to as time domain NMR.

[0058]

[0059] The aspects of the invention provide for materials and methods for measuring the amyloid cascade, the various steps along the cascade, and the effects of test molecules on the cascade, using water proton relaxation measurements.

[0060] In one embodiment, a reaction where biomolecule undergoing an amyloid cascade proceeds in two solutions, one solution having a test molecule and one solution without a test molecule. An identical detection agent is then added to both tubes. The detection agent can bind monomers and aggregates of the amyloid forming biomolecule, but different components of the cascade induce different aggregation states of the detection agent, and these, in turn, are sensed by water proton relaxation. Examples of this embodiment are the use of a strep-MP detection agent (Figures 4 & 5, example 1), the use of a monoclonal Ab-MP detection agent (Figure 5, example 4), and the use of Gd-ABeta detection agent (Figure 5, example 3).

[0061] In another embodiment, an amyloid cascade with a detection agent present is run plus and minus a test molecule. The water proton relaxation properties of both solutions are determined and effects of the test compound analyzed by water proton relaxation. Since the detection agent participates in the formation of cascade aggregates and since water proton relaxation measurements do not affect the cascade, many time points per tube can be obtained. An example of a detection agent for this embodiment is the Gd:Chelate-ABeta peptide, see Figure 6A and example 3.

[0062] In another embodiment, the cascade is run plus and minus a test compound (with no detection agent). Water proton relaxation measurements are made and reflect interactions between water and the biomolecules of the cascade. See Figure 7 and examples 5 and 6.

[0063] Advantages of the invention include the ability to interrogate the amyloid without reliance on betasheet formation or betasheet binding molecules.

[0064] The assays and methods of the invention can measure early stages of an amyloid cascade where dimers, small aggregates, or oligomers have been formed. Detection is not limited to betaheet structures:

[0065] The materials and methods described and claimed herein are bidirectional, that is, they are able to measure aggregation or disaggregation of amyloid cascades.

[0066] The materials and methods described and claimed herein use penetrating radiofrequency radiation and are indifferent to light based interferences ²¹ .

[0067] The methods herein described and claimed herein are homogeneous mix and read type methods, lacking a separation of free and bound materials. They lack the separation step required with radioactive assays.

[0068] The materials and methods described and claimed can use low protein concentrations and small volumes. [0069] The materials and methods described and claimed herein can measure aggregation of multiple component systems. For example, biotin-ABeta aggregation can be measured with added ABeta peptide or added tau protein.

[0070] Embodiments of the invention can identify test molecules that alter the amyloid cascade which can be useful for as in vitro or in vivo diagnostic agents, or therapeutic drugs, or as leads for further drug development.

[0071] Water proton relaxometry: Water proton relaxometry (WPR) water proton relaxation methods have measured the presence of analytes ranging from dimers to large viruses ^{22 24} but have not been applied to the subject of the current invention, measuring amyloid cascades and the effect of test substances on those cascades. These studies indicate the water proton relaxation assays have attributes of these assays make them unique for measuring amyloid cascades and the effects of test substances on those cascades

[0072] Water proton relaxation methods apply a brief excitatory radiofrequency pulse to an aqueous sample in a homogeneous magnetic field. With a permanent magnet of 0.47 Tesla, the pulse used to excite water protons is at 20 MHz. Water proton relaxation methods then record the relaxation or decay of the post excitatory pulse signal emitted by solvent water protons. Water proton relaxation employs the RF signal emitted by water protons rather than a signal from any of the many nuclei that are part of a biomolecules participating in amyloid cascades. Water proton relaxation methods use the large NMR signal from water protons (55 Molar H ₂ 0 is 110 Molar protons). Water proton relaxation methods have been termed "time domain NMR," and do not yield the spectra of nuclei in slightly different chemical environments that are obtained with many NMR methods.

[0073] Instrumentation and measurement of water proton relaxation: Satisfactory instrumentation for water proton relaxation is the Bruker minispec mq series made by Bruker, such as the mq20 (20 MHz, 0.47 T), although similar instruments can be used. [0074] Water proton relaxation measurements: The effect of test substances on the amyloid cascade can be evaluated using various water proton relaxation processes including T2 (CPMG pulse sequence), T2* (free induction decay), or Tl (inversion recovery). These pulse sequence feature different TE's (echo times), TR (repetition times), which Bruker minispec can be programmed to use. In general T2 or T2* sequences are employed when MP detection agents are employed and Tl sequences used with Gd detection agents. Tl or T2 sequences can be with bulk water measurements. The change in a water proton relaxation process produced by increasing concentrations of a test molecule can be analyzed using a four-parameter fit (maximum value, minimum value, concentration midpoint, n (slope)).

[0075] Water proton relaxation (WPR) assays can distinguish biotin monomers from biotin dimers and this indicates their ability to analyze early steps of the amyloid cascades (shown in Figures 2 and 3A), which comprise the lag phase shown in Figure 3B. The ability of water proton relaxation methods to distinguish species with one or two biotins per mole has been shown in studies of protease activity ²⁵ . As shown in Figure 4, in step 1 a protease is allowed to cleave a peptide substrate with two biotins per mole. A streptavidin-MP is then added. In the absence of protease, dibiotinylated peptides induce clustering of streptavidin-MP s added in step 2. A protease (if present) generates monobiotinylated peptides ("monomers") that bind to the strepavidin-MP but do induce MP clustering.

[0076] Water proton relaxation methods can be adapted to high throughput screening, see example 7.

[0077] Magnetic Particle (MP) Detection Agents: Magnetic Particle detection agents are Magnetic Particles that can bind to components of an amyloid cascade (Figure 5) or participate in an amyloid cascade (Figure 6). Magnetic Particle detection agents consist of a Magnetic Particle and a polypeptide or protein (e.g. streptavidin, antibody, ABeta peptide). Magnetic Particles (nanoparticles and micron-sized particles) are available from many commercial sources. The theoretical basis for how Magnetic Particles of different sizes alter water proton relaxation upon aggregation (or dispersion) is well understood by those of skill in the art. ^26"27

[0078] Conjugation chemistry for attaching peptide and proteins to Magnetic Particles can be accomplished by diverse reactions ^28"29 . Separation of small molecules from nanoparticles (diameters of 5-200 nm) is best accomplished with gel chromatography, while separation of small molecules from micron-sized particles (200-5000 nm) is best accomplished with permanent magnets. These methods are well known in the art.

[0079] A kit for conjugating antibodies to Magnetic Particles is available (Innova Biosciences). Strep-MPs are available commercially from Thermofisher, Milteny or NE Biolabs. Fluorescein-MPs are available from Polysciences, Bangs Lab or Miltenyi Biotech.

[0080] There are two design considerations for protein-MP detection agents that are added after the amyloid cascade has proceeded. First, the protein-MP must bind to single site on the amyloid forming monomer, so that detection agent aggregation occurs only with the formation of aggregates of an amyloid cascade. Second, the protein-MP detection agent must bind to a site on the aggregate that is exposed after aggregate formation occurs. An ABeta peptide with a single biotin attached to the N-terminus meets these conditions (example 1), because the N-terminal amino acids (1- 16) do not participate in betasheet formation. See Figure 2A.

[0081] With amyloid forming proteins from biological sources, the design of protein-MP detection agents meeting these requirements requires selecting monoclonal antibodies that bind to specific epitopes (sites on the amyloid forming protein) that meet these conditions. The amino acid sequences of amyloid forming biomolecules can be divided into betasheet forming sequences and nonbetasheet forming sequences (Table 2). Protein-MP detection agents can employ monoclonal antibodies that bind to non-betasheet epitopes exposed on amyloid aggregates and which are present as one site per monomer.

Amyloid Forming Biomolecules: Beta-Sheet Forming Sequences Some Non Beta-Sheet Epitopes Recognized By Antibodies Amyloid Forming Betasheet Non betasheet Antibody Reference Protein/Peptide & forming Epitope, amino acid designation

Length sequence numbers

(amino acids, "aa")

ABeta, 17-40 or 17-42, 1-5 3D6 30

40 or 42 aa's See Figures 2A

6-10 6E10

& 3

ASyn, 36-95, see ³¹ 115-122 LB 509 ³² /Biolegend 140 aa's

103-108 4B 12 Biolegend

Tau, 244-372 2-23 Tau 12 Biolegend 441 aa's 20-35 Tau 13 Biolegend

Amylin (IAPP), 12-37 ³³ 1-12 None available

37 aa's

[0082] Paramagnetic Chelate Detection Agents: These detection agents consist of Gd ⁺ binding chelates attached to an amyloid forming biomolecule. The site of attachment must not interfere with the amyloid cascade. For example, with the ABeta peptide, the chelate can be attached to the N-terminus. Paramagnetic detection agents are added during the amyloid cascade. Paramagnetic detection agents utilize the fact that the Tl potency of paramagnetic chelates, referred to as the T 1 relaxivity, reflects the motion of the chelate in solution, which is different for monomers and aggregates. Attachment of biomolecules to paramagnetic chelate detection agents is well known in the art.

[0083] Amyloid-forming biomolecules: A variety of amyloid forming biomolecules can be used in practicing the invention. These can be chemically synthesized peptides such as the ABeta peptides (1-40 or 1-42), a peptide with amino acids numbering 71-82 from ASyn ³⁴ , or polyglutamates of 8-24 amino acids in length (for the Huntingtin protein) ³⁵ . An advantage of using synthetic peptides for amyloid cascades is that haptens (e.g. biotin, fluorescein) or chelates binding Gd ⁺ (e.g. DTPA, DOTA) can be placed at specific positions of peptide, and these can sites that are known not to affect the aggregation of the cascade [0084] Proteins can be used as amyloid forming biomolecules and are typically produced in and purified from microorganisms like E.Coli. Alphasynuclein is available from the Michael J. Fox foundation, Proteos or rPeptides. Various isoforms of the Tau protein can be obtained from Enzolife sciences, Anaspec and Sigma Aldrich.

[0085] Some amyloid forming biomolecules will aggregate upon storage and care must be taken to use monomers for the amyloid cascades of the invention. For ABeta peptides, methods of preparing monomers have been given ³⁶ . Methods of preparing and using monomeric ASyn in the amyloid cascade are available from the Michael J. Fox Foundation. Amyloid forming biomolecules can be characterized to determine its state of aggregation by size exclusion chromatography, gel electrophoresis or light scattering.

[0086] Techniques like the inverse Laplace transforms of CPMG spectra are used to resolve the multi-exponentials that result from multiple, slowly exchanging water compartments, a situation occurs with blood coagulation ⁷ and with brain tissue from animals induced to have experimental allergic encephalitis ⁸ Intrinsic water proton amyloid cascade assays can also use changes in bulk water relaxation characteristics, that is, from the analysis of a single T2 ⁹ .

Examples

Example 1: Use of Biotin-ABeta Peptides and Streptavidin-Magnetic Particles to Determine the Effects of Test Molecules on the Amyloid

Cascade

[0087] General: Biotin-ABeta peptides are allowed to participate in the amyloid cascade plus and minus test molecules and then streptavidin-MPs are added, see Figure 4. A detection agent, Streptavidin-MP, is then added, which aggregates upon binding cascade aggregates bearing two or more biotins per mole of aggregate.

[0088] Materials: N-terminal biotinylated ABeta peptide (1-40) is from Anaspec, while streptavidin-MPs are from Miltenyi Biotech. T2 relaxation times are measured with a Bruker minispec 20 MHz relaxometer (0.47T) with a CPMG pulse sequence. [0089] Procedure, Step 1, run the amyloid cascade in the presence of a test molecule: The test molecule (20 uL, variable concentrations in DMSO) is added to a 12 by 75 mm tube. To this is added 400 uL of Biotin- ABeta in PBS at 1- 100 ug/mL of peptide, preferably at about 10 ug/mL. The mixture is incubated at 37 °C and the amyloid cascade proceeds. Lower Biotin- ABeta peptide concentrations, lower temperatures, and a lack of divalent cations (addition of EDTA) will slow the cascade.

[0090] Step 2, add the streptavidin-MP detection agent: Some 100 uL of Stept-MP is added. The Strep-MP concentration adjusted so that its addition brings the T2 of solution to 100 to 200 msec. The mixture is incubated for 20-30 minutes at 37 °C, to allow strep-MP aggregation by reaction with the Biotin- ABeta peptide aggregates of the amyloid cascade.

[0091] Step 3, determine water proton relaxation properties: Place about 500 uL of solution in an NMR tube and determine T2 relaxation times using the Bruker minispec with a CPMG pulse sequence.

[0092] Step 4, data interpretation: A delta T2 is plotted versions test molecule concentration using a 4 parameter fit with a program like Priszm.

Example 2: Use of ABeta peptide-Magnetic Particles to Measure the

Effects of Test Molecules on the Amyloid Cascade

[0093] General: ABeta peptide-MPs, a detection agent, are synthesized and allowed to participate in the amyloid cascade plus and minus test molecules as shown in Figure 5A.

[0094] ABeta peptide-MPs are synthesized by (i) synthesizing an azide bearing ABeta peptide (N ₃ -Abeta peptide), (ii) synthesizing an alkyne functionalized MP (alkyne-MP) and (iii) attaching the N3-Abeta peptide to the alkyne-MP using a copper mediated click reaction.

[0095] The conjugation of ABeta peptides to MPs is based on ⁴⁰ and uses the Feraheme (ferumoxytol) magnetic particles from AMAG Pharmaceuticals. Ferumoxytol has diameter of less than about 100 nm and can be termed a nanoparticle. Separation of small molecules from ferumoxytol magnetic particles is by gel filtration, ultrafiltration or dialysis rather than by magnetic separation. (Alternatively, alkyne-MPs can be purchased from Click Chemistry Tools.)

[0096] To prevent ABeta peptide-Magnetic Particles from aggregating on storage and before exposure to a test substances two strategies can be employed. Purification and concentration or ABeta peptide-Magnetic Particles can be done in the solvent, DMSO. Alternatively, purification and concentration or ABeta peptide-MPs can be done strongly basic aqueous media, see ⁴¹ . Ferumoxytol is stable in strong base.

[0097] Procedure, synthesis of N ₃ -ABeta peptide: ABeta peptide (40 amino acids) is synthesized with an additional N-terminal amino acid, a click reactive Lys(N3) with Fmoc chemistry using (Fmoc)Lys(N ₃ )-OH (Anaspec). The peptide, denoted N ₃ -ABeta peptide, has 41 amino acids. The N-terminal amino acids do not participate in betasheet formation, while those towards the C-terminus do, see Figure 3A.

[0098] Procedure, synthesis of alkyne-MPs: To 2 mL of ferumoxytol (AMAG Pharmaceuticals) at 6 mg Fe/mL in 0.1 M pH 6.0 MES buffer is added HOBT (10.55 mg, 0.078 mmol), and EDC (55.27 mg, 0.288 mmol) with incubation (room temperature, 20 min). A solution of alkyne-PEG4-Amine (Clickchem Tools) in DMSO (0.1 M, 800 μΐ) is added (50 °C, 2 h). Purification of alkyne-MP is by gel filtration and/or ultrafiltration.

[0099] Procedure, synthesis of ABeta-peptide-MP s: A Cu catalyst stock solution of CuS0 ₄ (8.85 mg, 0.055 mmol) is dissolved in deoxygenated PBS (5 ml). After (BimC4A) ₃ (Tripotassium 5,5',5"-[2,2',2"-nitrilotris(methylene) tris(lH-benzimidazole-2, l-diyl)]tripentanoate hydrate) (44.5 mg, 0.054 mmol) is added, a gel-like light blue colloid is obtained, clarified by an addition of sodium L-ascorbate (397.6 mg, 2 mmol).

[00100] Alkyne-MP (0.2 mg Fe), N ₃ -ABeta (4.6 μΐ, 10 mM in DMSO), Cu stock solution (5 μΐ) and 200 μΐ DPBS are incubated (room temperature, 15 min.) The ABeta-MP is purified by gel filtration in DMSO, concentrated if necessary by ultrafiltation, and stored at about 500 ug Fe/mL or greater at -80 °C [00101] The number of ABeta-peptides per MP can altered by adjusting the ratio of N ₃ -ABeta peptides to alkyne-MPs in the synthesis of ABeta-peptide-MPs. With ferumoxytol magnetic particles, a satisfactory of number of peptides per magnetic particle is between 2 and 8 per particle. To determine the number of ABeta-peptides per magnetic particle, reaction with Azide- Cy5.5 can be used and quantified spectrophotometrically. See the Before and After Fluorochrome Reaction Method of ⁴⁰ .

[00102] Procedure, Step 1, run the amyloid cascade in the presence of a test substance: The test molecule (variable concentrations in DMSO, 20 uL) is added to a 12 by 75 mm tube. Some 400 uL of PBS are added. Some 10 uL of ABeta-peptide-MPs (at about 500 ug Fe/mL in DMSO) are then added to 400 uL PBS to achieve final concentration of about 10 Fe ug/mL. Tubes are incubated at 37C°C.

[00103] Procedure, Step 2, determine water proton relaxation: An amyloid cascade/aggregation NP concentration results and is monitored by from the T2 relaxation time of the solution using a CPMG pulse sequence.

[00104] The concentration of ABeta-MPs employed can be adjusted to optimize conditions. Lower concentrations will produce slower aggregation and higher T2 values.

[00105] Data reduction is as in example 1.

Example 3: Use of Gd-ABeta Peptides to measure the Effects of Test

Molecules on the Amyloid Cascade

[00106] General: Figure 6A shows a representation of the process for using Gd-ABeta to measure the effect of test substances on the amyloid cascade. With Gd-ABeta, Tl measurements are used, since these are sensitive to tumbling of the Gd chelates like the Gd-ABeta peptide. Therefore Tl reflects the participation of Gd-ABeta in the amyloid cascade. An inversion recovery pulse is used.

[00107] Procedure, synthesis of Gd-ABeta peptide: A DOTA-Beta peptide is synthesized with an additional N-terminal amino acid, Lys(DOTA). Fmoc chemistry and with a commercially available (Fmoc)Lys(DOTA)-OH (PE-Biosciences) is used. The peptide has 41 amino acids. Gd addition and chelation is at pH 6.5, with EDTA neutralization and RP-HPLC purification as in section 3.1.10 from ⁴² . Gd-ABeta peptide monomers can be generated using base, if necessary ⁴¹ .

[00108] Procedure, Step 1, run the amyloid cascade in the presence of a test substance: The test molecule (variable concentrations in DMSO, 20 uL) is added to a 12 by 75 mm tube. To this is added 500 uL of Gd-ABeta in PBS at 50 ug/mL and the mixture is incubated at 37°C.

[00109] Procedure, Step 2, determine water proton relaxation.

Example 4: Use of Antibody-Magnetic Particles To Measure the Effects

of Test Molecules on the Amyloid Cascade

[00110] General: Figure 5 shows a representative process for using antibody-magnetic particles to measure the amyloid cascade. The selection of an antibody (Ab) is guided by Table 2. A wide range of MPs and conjugation chemistries can be used.

[00111] Procedure, synthesis of the Ab-MP detection reagent. The anti-alphasynuclein monoclonal antibody LB509 (Table 2) is coupled to the MyOne carboxy terminated magnetic microparticles using the manufacturer's protocol. Briefly, 3 mg of beads in pH 6.5 MES buffer are reacted with 150 ug of antibody using EDC (l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide). Magnetic separators for 1.5 mL tubes (Sigma-Aldrich) are used to separate magnetic particles from small molecules.

[00112] Procedure, Step 1, run the amyloid cascade with alphasynuclein (ASyn) in the presence of a test substance: The test molecule (variable concentrations in DMSO, 20 uL) is added to a 12 by 75 mm tube. To this is added 400 uL at concentrations of 0.1, 1, 10 ng/mL and the mixture is incubated at 37°C. Higher concentrations will give faster amyloid cascades.

[00113] Step 2, add the Ab-MP detection agent: Some 100 uL of Ab-MP is added. The Ab- MP concentration adjusted so that its addition brings the T2 of solution to 100 to 200 msec. The mixture is incubated for 20-30 minutes at 37 °C, to allow Ab-MP aggregation by reaction alphasynuclein aggregates of the amyloid cascade.

[00114] Steps 3 and 4 are from example 1.

Example 5: Determining the Effect of Test Substances on the

Amyloid Cascade From a Single Relaxation Process

[00115] General: With the rapid exchange condition (Figure 7A), the effects of test molecules on the amyloid cascade can be obtained from the effects of cascade biomolecules on water proton relaxation, as is done with studies of proton aggregation ⁹ ' ⁴ and Tl or T2 measurements. Here a magnetic detection agent is not employed.

[00116] Procedure, Step 1, run the amyloid cascade with test molecule: To test substance (variable concentrations in 20 uL in DMSO) is added ABeta peptide monomer (see Ryan) at 50 uM peptide and the mixture is incubated for two to 48 hours at 37 °C.

[00117] Step 2, determine WPR: Measure both T2 and Tl relaxation times and select the relaxation process with largest change with incubation time in the absence of test molecule.

[00118] Data analysis for relaxation processes uses the four parameter fit, above.

Example 6: Determining the Effect of Test Substances on the Amyloid Cascade From Multiple Water Proton Relaxation Processes

[00119] General: A slow exchange condition is needed for determination of multiple WPR processes related to an amyloid cascade as shown in Figure 7B.

[00120] Procedure, Step 1 : See example 5 for concentration of ABeta peptide and variable concentrations of test molecules

[00121] Procedure, Step 2: A CPMG pulse sequence with an interecho spacing of 500 microseconds and, a repetition time of 2-10 seconds is employed. Time dependent decay of the MR signal for up to 5 seconds is recorded. This generates a signal versus time curve from which multiple T2 values are extracted. A CONTIN program can be used to analyze relaxation data via the inverse Laplace transformation processing using the multi-exponential equation below. The CONTIN program has been used to analyze multiple relaxation spectra ⁴⁴ .

[00122] With the above equation, S(t) is the relaxation signal acquired over time, Ai is the signal amplitude, T2i is the relaxation time constant, and O is the offset term. Kinetic spectra will consist of a series of amplitudes (Ai) and corresponding spin-spin relaxation times T2i for each amplitude. Using these methods generated relaxation spectra of the type shown in Figure lb of ³⁷ .

[00123] Data analysis: A relaxation time for bound or occluded water, that is water specifically associated with aggregation and different from the relaxation time of bulk water, is obtained. This is plotted versus test substance concentration.

Example 7: High Throughput Screening Of Effects Of Many Test

Molecules On The Amyloid Cascade

[00124] Examples 1 or 2 can be run in a 96 well microtiter plate format where each well contains a test molecule. The test molecules can be purchased as a focused library (Chemdiv, Chembridge, Selleckchem) and the T2s of the wells of the plate determined simultaneously by the positional encoding techniques of MRI. See figure 4 of ⁴⁵ . T2 weighted pulse sequences are those that have long repetition times (TRs) and long echo times (TEs) and allow the signal intensity (image brightness) of wells to be determined largely by T2. The effects of molecules on the amyloid cascade can then be determined directly from signal intensity data rather than from values of T2. Obtaining values of the T2 relaxation time from an NMR instrument requires imaging with many different pulse sequences. References

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