B-isoxazole derivatives, diagnostic compositions; and methods related thereto

Abstract

The present application relates to novel b-isox analogs for use in detecting misfolded proteins associated with neurodegenerative diseases, such as ALS, PD, AD, FTLD, and LATE. The present inventors identified and modified a specific site on the b-isox molecule, which significantly enhances detection capabilities. This modification also allows for the selection of analogs that either exhibit low background interference or alter cellular targets, based on the alteration of a functional group at critical site.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority benefit of U.S. Provisional Application no. 63 / 608,665, filed December 11, 2023, the entire content of which is hereby incorporated herein by reference. INTRODUCTION

[0002] Conformational diseases, also known as protein misfolding diseases, occur when proteins fold incorrectly, leading to various biological dysfunctions. Protein misfolding can result from genetic mutations, environmental factors, or age-related changes. Misfolded proteins tend to aggregate, forming insoluble fibrils or plaques, which disrupt cellular functions. This can lead to toxic gain of function, loss of normal function, cellular stress, and in some cases, neurodegeneration. These diseases include Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease, ALS, Prion Diseases, Systemic Amyloidosis, Cystic Fibrosis, Type 2 Diabetes, and others.

[0003] Misfolded proteins play a critical role in various diseases. In Alzheimer’s Disease, the proteins Amyloid-beta and Tau are known to misfold. Parkinson’s Disease is associated with the misfolding of Alpha-synuclein. Huntington’s Disease involves the protein Huntingtin, which misfolds due to expanded polyglutamine repeats. Other proteins like SOD1, TDP-43, and C9orf72 are implicated in diseases such as ALS. Prion diseases are characterized by the misfolding of the Prion protein (PrP). Systemic Amyloidosis involve misfolded Immunoglobulin light chains and transthyretin. In Cystic Fibrosis, the CFTR protein misfolds. Type 2 Diabetes is associated with the misfolding of lAPP. Additionally, diseases like Light Chain Amyloidosis, Hereditary ATTR Amyloidosis, Lewy Body Dementia, and Frontotemporal Dementia are also linked to protein misfolding.

[0004] Biotinylated isoxazole (b-isox), or 6-(5-(Thiophen-2-yl) isoxazole-3-carboxamido) hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d] imidazol-4-yl) pentanoate, is a specialized small molecule renowned for its ability to precipitate RNA-binding proteins predominantly found in stress granules and RNA granules. The majority of proteins that bind to b-isox possess low-complexity (LC) domains, which are critical for their protein interaction. These LC domains, characterized by an intrinsically disordered structure, facilitate selfinteractions. A notable feature of this type of self-interaction domain is its capacity to transiently form a cross-P polymeric condensed phase, playing a vital role in various crucial biological processes. These processes include DNA transcription and replication, chromatin remodeling, nuclear pore transit, signal transduction, synaptic transmission, and cytoskeleton regulation, primarily through homotypic or heterotypic cross-P multimeric interactions. Significantly, in contrast to pathological cross-P aggregates that are rigid and stacked inregister, the physiological cross-P multimers formed by LC domains are loose and reversible, distinguishing them from their pathological counterparts.

[0005] Current diagnostics for neurodegenerative diseases like Alzheimer's, Parkinson's, and ALS primarily use clinical assessments, imaging, and sometimes cerebrospinal fluid analysis to confirm diagnoses. Early and accurate detection remains difficult, leading to research into more definitive methods, including blood-based biomarkers. SUMMARY

[0006] The newly developed derivatives retain the detection capabilities of the original compounds for Amyotrophic Lateral Sclerosis (ALS) and Parkinson's Disease (PD), while surpassing them by showing lower readings in healthy individuals and higher readings in patients. This dual characteristic highlights their enhanced accuracy and efficiency in diagnosing neurodegenerative diseases. Additionally, the required volume of plasma was also reduced to 50 pl from the previous 100 pl.

[0007] The present application provides OG-01, -02, -03, -08 novel derivatives of b-isox. OG-01, -02, -03, -08 exhibits improved size of isoxazole precipitates from the plasma of patients, and demonstrates enhanced diagnostic efficacy in neurodegenerative disease, such as ALS and PD. Significantly, the absence of pellets in the plasma of healthy individuals indicates an all-or-none detection, providing a clear and accurate assessment of the data.

[0008] The present application provides OG-04, -05 novel derivatives of b-isox. Compound OG-04, -05 exhibits improved size of isoxazole generated precipitates in healthy people, and could be used as healthy controls for b-isox precipitation diagnostics.

[0009] The present inventors determined that modifications to the thiazole in the b-isox have led to a significant increase in both the binding to and precipitation of cross-P rich complexes.

[0010] The OG-series compounds, designed for neurodegenerative disease detection through chemical ELISA, not only retain but greatly surpass the detection capabilities of their predecessors b-isox for ALS, PD, and AD. Compounds OG-01, OG-07, OG-08 and OG-10 demonstrate a marked enhancement in sensitivity, evidenced by substantially higher readings in disease states and notably lower readings in healthy individuals, indicating a major advancement in the detection of disease-specific proteins.

[0011] The present application provides OG-04, a novel derivative of b-isox. Compound OG-04 exhibits improved detection sensitivity of normal conformation of SMN, but decrease of p-TDP-43 by using b-isoxELISA. OG-04 could be used as healthy controls of b-isoxELISA diagnostic.

[0012] In one aspect, the application provides a method for detecting a human disease in a human subject, comprising: obtaining a biofluid sample from the subject; adding an isoxazole to the obtained biofluid sample to form a biofluid isoxazole composition in the biofluid sample; and detecting a presence of the biofluid isoxazole composition.

[0013] In one embodiment, isoxazole is biotin-isoxazole, (6-(5-(Thiophen-2-yl)isoxazole-3-carboxamidojhexyl           5-((3alS,,4S,6aA)-2-oxohexahydro-l / / -thieno[3,4-<f|imidazol-4- yl)pentanoate), or its salt or an analog thereof, especially biotin-isoxazole and very especially biotin-isoxazole.

[0014] In other embodiments, the biofluid sample is urine, whole blood, plasma, or serum, cerebrospinal fluid (CSF), saliva, or mucosa, such as, urine, saliva, CSF or plasma, and especially CSF or plasma.

[0015] In other embodiments, the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), frontotemporal dementia (FTLD), diabetes, cancer, infectious disease, huntington disease, schizophrenia, aging- associated disease, or protienopathies, especially wherein the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

[0016] In other embodiments, the method further comprising thereafter treating the human subject for the human disease or thereafter changing an existing treatment of the human subject for the human disease based on the detecting the presents of the biofluid isoxazole composition, such as a treatment including pharmaceutical therapy for the human disease, and optionally further comprising diagnosing the human disease in the human subject.

[0017] In other embodiments, the concentration of isoxazole ranges from 0.0 ImM to 0.3 mM, preferably from 0.100 mM to 0.200mM, in the biofluid sample. 10018] In other embodiments, the biofluid isoxazole composition is in a precipitate.

[0019] In other embodiments, the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

[0020] In other embodiments, the human disease is ALS, AD, DLB, MSA or PDD or PDnD.

[0021] In other embodiments, the human disease is ALS, such as sporadic ALS.

[0022] In other embodiments, the method further comprises monitoring the size of the precipitate to monitor the progression of ALS in the subject.In other embodiments, the method further comprises adding a polypeptide to biofluid isoxazole composition in the biofluid sample to facilitate detection by an immune assay, such as a blot assay, a chemiluminescence immunoassay, an enzyme-linked immunosorbent assay (ELISA), a light scattering immunoassay, a radiolabeled immunoassay, in particular, ELISA or a Western blot.In other embodiments, the human conformational disease is Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD)In other embodiments, the polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof which binds to the biofluid isoxazole composition.

[0023] In other embodiments, the human diseases is ALS, and wherein the polypeptide for detecting ALS is an antibody against SOD1, C9orf72 dipeptide repeats, PFN1, PRDX2, phospho-TDP-43, CAI, MYL12B, CD14, ANXA5, STOM, SMN, ACTB, or GLUT1, such as SOD1, MYL12B, CD14 and p-TDP-43, and especially SOD1 and p-TDP-43.

[0024] In other embodiments, the human diseases is AD, and wherein polypeptide for detecting AD is an antibody against APP, phospho-TDP-43, TDP-43, Tau, STOM, or ANK1.

[0025] In other embodiments, the human diseases is PD, and wherein polypeptide for detecting PD is an antibody against synuclein, CHL1, NELL2, p-TDP-43, NrCAM, ANK1, STOM, PRDX2, CAI, CD 14, and RUVBL1.

[0026] In other embodiments, the biofluid is plasma or CSF.

[0027] In other embodiments, the method is ELISA, such as direct, indirect, sandwich, or competitive ELISA.

[0028] In other embodiments, use of an isoxazole to detect a human disease in any of the methodology.

[0029] In another aspect, provided is a pharmaceutical composition comprising a compound selected from

[0030] OCL01:6-(5-(thiophen-3-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno [3,4-d]imidazol-4-y l)pentanoate

[0031] OCL02:6-(5-(furan-2-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate

[0032] QG-03: 6-(5-(5-chlorothiophen-2-yl)isoxazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate

[0033] OG204:6-(5-(benzo[b]thiophen-2-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate

[0034] OG4)5:6-(5-(thiazol-2-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate

[0035] OG206:6-(5-(thiazol-5-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno [3,4-d]imidazol-4-y l)pentanoate

[0036] QG-07: 6-(5-(thiophen-2-yl)-l,2,4-oxadiazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate

[0037] QG-08: 6-(5-(thiophen-2-yl)-l,2,4-oxadiazole-3-carboxamido)hexyl 5- ((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate

[0038] OG-09:6-(2-methvl-5-(thiophen-2-vl)furan-3-carboxamido)hexvl 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate

[0039] OG40:6-(5-(thiophen-2-yl)isoxazole-4-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate

[0040] OGG l:4-(5-(thiophen-2-yl)isoxazole-3-carboxamido)butyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno [3,4-d]imidazol-4-y l)pentanoate and

[0041] OG-12:2-(5-(thior>hen-2-vl)isoxazole-3-carboxamido)ethyl 5-((3aS,4S,6aR)-2-oxohexahydro-1 H-thieno [3,4-d]imidazol-4-yl)pentanoate.

[0042] In other embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0043] In another aspect, provided is a series of novel b-isox analogs for use in diagnostic assays for neurodegenerative diseases.

[0044] In other embodiments, the assay compounds are OG-01, OG-02, OG-03 and OG-08.

[0045] In other embodiments, the size of the diagnostic precipitates in patients with ALS, PD and AD is increased than that of b-isox, providing enhanced diagnostic accuracy.

[0046] In other embodiments, the assay compounds are OG-04 and OG-05.

[0047] In other embodiments, the size of the diagnostic precipitates in health individuals is increased but absent in patients, providing negative controls to improve diagnostic accuracy.

[0048] In other embodiments, the assay compounds are OG-01, OG-07, OG-08 and OG-10.

[0049] In other embodiments, the plasma levels of disease proteins are increased in ALS, PD and AD patients, providing enhanced diagnostic accuracy.

[0050] In other embodiments, the b-isox analogs are used for detecting and monitoring ALS, PD and AD.

[0051] In other embodiments, the assay compounds have a similar detection capability. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Chemical composition of b-isox analogs: Detailed chemical structuresof OG series, ranging from OG-01 to OG-12 and comparison of the structure with b-isox, were listed in Figure 1.

[0053] Comparative data analysis of chemical precipitation: Charts illustrating the advantages of OG-01, OG-02, OG-03, and OG-08 over b-isox, showcasing significantly increase precipitates in ALS patients (Fig. 2c). Notably, the absence of pellets in the plasma from healthy individuals in OG series suggests an all-or-none detection method, potentially leading to a precise and reliable evaluation of the data.

[0054] Comparative Data Analysis of Chemical ELISA: Charts illustrating the advantages of OG-01, OG-07, OG-08, and OG-10 over b-isox, showcasing reduced background interference and improved detection accuracy for poly(GR) and / or p-TDP-43 (Fig. 3c, 3d, 8b and 10b).

[0055] Illustrative embodiments of the present invention are described in detail below with reference to the following Figures:

[0056] Fig. 1 comprises a collection of images showcasing the detailed chemical structures of the newly developed b-isox analogs in the OG series, ranging from OG-01 to OG-12.

[0057] Fig. 2 is an assembly of images illustrating an analysis of OG series (OG-01 to OG-12) precipitates from the plasma of healthy individuals and patients with ALS. Panel (a) displays a flowchart detailing the chemical precipitation process. Panel (b) provides a statistical analysis of the size of chemical precipitates extracted from ALS patients' plasma using both the OG series and b-isox, with a dashed line indicating the size of b-isox precipitates. Panel (c) shows a statistical comparison of the sizes of chemical precipitates isolated from 50 pl plasma samples of healthy individuals and ALS patients, using compounds OG-01 through OG-04 and b-isox. Panel (d) presents a statistical analysis of the size of precipitates isolated from 100 pl plasma samples of both healthy individuals and ALS patients, processed with the OG series and b-isox. Panel (e) presents a statistical analysis comparing the size of chemical precipitates extracted from 50 pl and 100 pl plasma samples of healthy individuals and ALS patients, using OG-04 over reaction times of 20, 40, and 60 minutes. Panel (f) shows a statistical comparison of the sizes of chemical precipitates isolated from 100 pl plasma samples of healthy individuals and PD patients with or without family history, using compounds OG-01.

[0058] Fig. 3 displays a collection of images that detail the comparative effectiveness of the OG-01 assay in healthy individuals and patients with ALS. Panel (a) shows a flowchart outlining the chemical ELISA protocol utilizing the OG series compounds. Panel (b) presents comparative data on the assay efficiency of OG-01 in healthy subjects versus ALS patients. Panel (c) offers a comparison of the assay efficiencies of OG-01 and b-isox in the same ALS patients. Panel (d) offers a comparison of the assay efficiencies of OG-01 and b-isox in health individuals. Panel (e) presents comparative data on the assay efficiency of OG-01 in healthy subjects versus PD patients. Panel (f) presents the variance in levels of amyloid oligomer and amyloid between healthy subjects and patients diagnosed with AD, and a comparison of the amyloid assay efficiencies of OG-01 and b-isox in the same AD patient.

[0059] Fig. 4 displays a collection of images that detail the comparative effectiveness of the OG-02 assay in healthy individuals and patients with ALS.

[0060] Fig. 5 displays a collection of images that detail the comparative effectiveness of the OG-03 assay in healthy individuals and patients with ALS.

[0061] Fig. 6 displays a collection of images that detail the comparative effectiveness of the OG-04 assay in healthy individuals and patients with ALS.

[0062] Fig. 7 displays a collection of images that detail the comparative effectiveness of the OG-05 and OG-06 assay in healthy individuals and patients with ALS.

[0063] Fig. 8 displays a collection of images that detail the comparative effectiveness of the OG-07 and OG-08 assay in healthy individuals and patients with ALS. Panel (a) presents comparative data on the assay efficiency of OG-07 and OG-08 in healthy subjects versus ALS patients. Panel (b) offers a comparison of the assay efficiencies of OG-07, OG-08 and b-isox in the same ALS patient.

[0064] Fig. 9 displays a collection of images that detail the comparative effectiveness of the OG-09 and OG10 assay in healthy individuals and patients with ALS. Panel (a) presents comparative data on the assay efficiency of OG-09 and OG-10 in healthy subjects versus ALS patients. Panel (b) offers a comparison of the assay efficiencies of OG-10 and b-isox in the same ALS patient.

[0065] Fig. 10 displays a collection of images that detail the comparative effectiveness of the OG-11 and OG-12 assay in healthy individuals and patients with ALS. DETAILED DESCRIPTION

[0066] b-isox is recognized for its effectiveness in plasma diagnostics for conformational diseases, yet its application faces challenges due to limited sensitivity. To address these limitations, the OG-series, derived from b-isox, has been developed. These derivatives are tailored to not only overcome the challenges associated with b-isox, but also are engineered to yield minimal baseline interference in healthy subjects.

[0067] Crucially, the present inventors developed OG-04 and OG-05, compounds specifically designed to prefer physiological conformers and act as negative controls. This strategy is intended to significantly improve the accuracy and dependability of plasma diagnostic techniques in the field of conformational disease research.

[0068] The disclosed method encompasses detection, diagnosis, or treatment of a human disease in a subject. This method involves introducing isoxazole analogs into a collected biofluid sample, thereby creating a biofluid isoxazole composition. Subsequent detection of this composition in the biofluid sample is indicative of the presence or progression of the disease. While the disclosed methods are applicable to various purposes, their primary utility lies in differential diagnostics, real-time pathophysiological monitoring, presymptomatic diagnostics, and measurement of pharmacoresponse. These applications are particularly relevant in the context of conformational diseases and proteinopathies, including but not limited to neurodegenerative diseases, diabetes, cancer, psychiatric disorders, and processes related to aging.

[0069] Reference will now be made in detail to the present embodiment(s) (exemplary embodiments) of the invention, an example(s) of which is (are) illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0070] Methods for the detection of conformational diseases and proteinopathies, such as neurodegenerative diseases, are described herein by using a small-molecule to generate visual precipitates.

[0071] In addition, applicants identified novel and specific biofluid biomarkers for differential diagnosis and monitoring pathophysiology in patients with neurodegenerative diseases, including ALS, AD and PD.

[0072] Also described are novel methods for the plasma diagnosis of neurodegenerative diseases. The novel method named b-isox-ELISA, is combination of b-isox chemicalprecipitation and immunoassay with specific biomarkers of ALS, AD or PD. b-isox ELISA can be used to screen the risks of ALS, AD and PD. This invention can be used to distinguish between AD, TDP-43 proteinopathies, PD, and dementia with Lewy bodies, ALS subtyping, real-time readout of pharmacoresponse, and monitoring the relief of pathological burden of misfolded disease proteins in clinical trial, preclinical diagnosis and clinical practice.

[0073] As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

[0074] As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. A. Comparative Analysis Reveals Enhanced Detection Accuracy of b-isox Precipitation by OG-01, -02, -03, -04 and -08 Compounds

[0075] Applicants developed a chemical ELISA method, termed b-isox ELISA, for detecting the levels of specific proteins in b-isox precipitates in previous works. Applicants found the chemical ELISA detected the levels of specific b-isox captured proteins tested in plasma, and significantly differed between the neurodegenerative disease groups and HCs.

[0076] Applicants have further modified the b-isox compound to create new analogs. These innovations are aimed at enhancing the size of precipitates and minimizing the volume of plasma required for effective use (Fig. 1).

[0077] The compounds from the thiazole variations, specifically OG-01 to OG-04, are noted to increase the size of these precipitates compared to the b-isox, as depicted in Figures 2b.

[0078] Applicants identified b-isox analogs OG-01, -02, -03 and -08 were successful in producing visible precipitates exclusively in the 50 ul plasma of ALS patients, signifying a specific reaction under pathological conditions, unlike the b-isox compound which failed to produce precipitates in similar circumstances. The results suggest that OG-01, -02, -03 and -08 could be more effective for clinical applications, particularly in the context of neurodegenerative diseases like ALS (Fig. 2b-d and f).

[0079] Uniquely, the applicants observed that the OG-04 and -05 compounds formed pellets predominantly in healthy individuals, rather than in ALS patients (Fig. 2b and e). This observation suggests that these modifications of b-isox compound alter its target selection, favoring physiological conformations characterized by cross-P rich structures.

[0080] Finally, we investigated the precipitation effect of OG-1 in patients with PD and individuals with or without a family history of PD. Our study revealed that OG-1 formed larger pellets in the plasma samples from PD patients and individuals with a family history of PD, in contrast to individuals without PD family history. These observations are depicted in Figure 2f.

[0081] The analogs showed different behaviors in patients with ALS and PD compared to healthy individuals, highlighting the potential for these compounds to be used in distinguishing or treating neurodegenerative diseases. B. Comparative Analysis Reveals Enhanced Detection Accuracy of Plasma Disease Proteins by OG-01, -07, -08, and -10 Compounds

[0082] A schematic diagram of the OG-series ELISA process is detailed in Figure 3a. Plasma is mixed with OG series (OGx: OG-01 to OG-12) compounds to create OGx-bound complexes. These complexes are then captured using streptavidin, followed by a traditional ELISA (Enzyme-Linked Immunosorbent Assay) that uses a specific antibody to detect target proteins. Plasma is mixed with OGx compounds to create OGx-bound complexes. These complexes are then captured using streptavidin, followed by a traditional ELISA (Enzyme-Linked Immunosorbent Assay) that uses a specific antibody to detect target proteins.

[0083] The proteins analyzed included poly(GR), SOD1, p-TDP-43, CD14, and STOM. There was an observed increase in the levels of these proteins in the plasma of ALS patients.

[0084] Most new analogs demonstrate a capability comparable to the original compound bisox in detecting ALS (Fig. 3-10).

[0085] A comparative study was conducted to assess the efficiency of the assay using b-isox and OGs in the same cohort of ALS patients. OG-01 demonstrated greater efficiency than bisox, evidenced by lower readings in healthy individuals and higher readings in patients (Fig. 3c). OG-07 and OG-08 exhibits increased efficiency in detecting poly(GR), while OG-10 demonstrates higher efficiency in identifying p-TDP-43, compared to b-isox (Fig. 8b and 9b repectively). C. Materials and Methods Used in the Examples

[0086] The following materials and methods were used in the Examples described below.

[0087] Reagents and Antibodies: b-isox was purchased from Sigma and Dalton dissolved in dimethyl sulfoxide (DMSO). Primary antibodies against SMN were purchased from BD Bioscience. The primary antibodies against CD14 (#17000-I-AP), GR repeat proteins (#23978-l-AP), phospho-TDP-43 (Ser 409 / 410) (#22309-1-AP), were purchased from Proteintech. The primary antibodies against P-actin (#A1978) was purchased from Sigma. The primary antibodies against SOD1 (#A2770) was purchased from Cell signaling.

[0088] Chemical Precipitation: 10 mM biotinylated isoxazole and OG series compounds were added to the human blood plasma or CSF to a final concentration of 100 to 200 pM. The mixtures were then incubated at 4°C for 60 min, centrifuged at 15000 rpm for 15 min at 4°C, and the supernatant was discarded. The diameters of b-isox precipitates were measured.

[0089] Chemical Based Enzyme-linked immunosorbent assay (Chemical ELISA) Blood samples from patients and healthy control were firstly collected through Blood Collection Tubes. Centrifugation of the tubes for 15 min at 2,200 'g. The resulting supernatant (upper layer) as the plasma sample. Before immunoassay, gently mixed 50-100mL plasma (or CSF) with 0.5-lmL b-isox or OG series compoundst hrough pipetting and rotated for Ih at 4°C. Wash each streptavidin-coated microwell 3 times by 200mL wash buffer (WB / (25mM Tris, 150mM NaCl; pH 7.2), 0.1% BSA, 0.05% Tween®-20) (do not allow wells to dry). Add lOOpL of the reaction mixtures to each well and incubate for 2h with shaking (~60 rpm) at room temperature (RT). Wash 3 times with 200pL WB when reaction finished. Add 100mL primary antibody diluent (appropriate primary antibody dilution in WB), incubating for Ih at RT with shaking. At this step, a no primary antibody control should be included (Add antibody diluent alone in a sample well). Wash 3 times with 200u L WB. Add 1 OOpL antibody diluent with appropriately diluted HRP-conjugated secondary antibody, incubating for Ih at RT with shaking. Equilibrate the TMB substrate solution to RT at this step. Wash 3 times with 200pL WB. Add lOOpL of the TMB Substrate Solution to each microplate well, incubating for 15-30 min until the color develops. Stop the reaction by adding lOOpL 2M sulfuric acid (or 2N HC1). Measure the optical density of at 450 nm by a microplate reader. EXAMPLES The following Examples are illustrative and do not limit the disclosure. Example 1: Chemical structure of OG series OG-01 to OG-12

[0090] Figure 1 presents the chemical structures of the OG series compounds, ranging from OG-01 to OG-12. These analogs are categorized into three groups based on their modification sites: thiazole variations, isoxazole variations, and alkyl chain variations. Each compound is distinctly identified, showcasing the variations in their molecular structures, particularly at the modification sites which serve as the basis for their classification into the respective groups. Example 2: Analysis of chemical precipitation efficiency for the OG series, from OG-01 to OG-12

[0091] Blood plasma samples, 50 or 100 pL, from patients with ALS were incubated with bisox and OG series compounds, followed by centrifugation to precipitate cross-P rich complexes, as illustrated in Figure 2a. The precipitates formed by the b-isox and OG series compounds were visually observable in samples from ALS patients. Significantly, thiazole variations, specifically OG-01 to OG-04, resulted in larger precipitates compared to those formed by b-isox, as depicted in Figures 2b.

[0092] To optimize the conditions for chemical precipitation, we reduced plasma volume to 50 pL and incubated plasma samples with thiazole variations or OG-08 compounds. The statistical analysis of the size of the chemical precipitates formed in the plasma of ALS patients using compounds OG-01, -02, -03, -04, -08, and b-isox is presented in Figure 2d. Notably, OG-01, -02, -03, and -08 successfully produced visible precipitates specifically in the plasma of ALS patients, but not in that of healthy individuals. In contrast, b-isox did not generate any pellets. OG-04 produced pellets of a size that did not allow differentiation between ALS patients and healthy individuals. These findings suggest that OG-01, -02, -03, and -08 are more effective compounds for clinical use in the treatment of neurodegenerative diseases.

[0093] In Fig 2c, OG-05 uniquely induced pellet formation in healthy individuals, rather than in ALS patients. 10094] We then sought to determine the optimal conditions for using OG-04. Through testing various plasma volumes and reaction times, it was found that OG-04, similarly to OG-05, could generate pellets in healthy individuals but not in ALS patients. This was observed at a concentration range of 50 to 100 pM, with reaction times between 20 and 40 minutes, and using 50 to 100 pL of plasma, as illustrated in Figure 2e. The optimal condition for OG-04 in clinical applications is likely to be at a concentration of 50 pM, with 50 pL of plasma and a reaction time of 20 minutes.

[0095] We assessed the precipitation response of OG-1 in three distinct groups: patients diagnosed with Parkinson's Disease (PD), individuals who have a family history of PD, and those without any known PD family history. The experiment revealed a notable difference in response to OG-1: significantly larger pellets were formed in the plasma of PD patients with or without a PD family history, and individuals with a PD family history. No pellets were detected in healthy controls without a family history of PD. These findings are comprehensively illustrated in Figure 2f. Example 3: Evaluating the efficiency of the b-isoxELISA assay with OG-01 in healthy individuals and ALS patients, and comparing its effectiveness to b-isox in ALS, PD and AD patients.

[0096] Figure 3a presents a schematic diagram of the OG-series compounds’ ELISA. In this assay, plasma is mixed with OG compounds to form OG-bound complexes. These complexes are then captured using streptavidin, followed by a traditional ELISA procedure using a specific antibody targeting proteins of interest.

[0097] Initially, we conducted an analysis of the plasma levels of poly(GR), SOD 1, p-TDP-43, CD14, and STOM in both healthy controls and ALS patients using OG-01, as depicted in Figure 3b. Our findings reveal that OG-01, similar to b-isox, significantly detects the increased levels of these proteins in the plasma of ALS patients.

[0098] Additionally, we conducted a comparative study to evaluate the assay efficiency between b-isox and OG-01. The results, illustrated in Figure 3c, demonstrate that OG-01 exhibits higher efficiency compared to b-isox. This was exemplified by two specific cases presented in figure 3c.

[0099] Figure 3d. shows a comparison of the assay efficiencies of OG-01 and b-isox in health individuals. OG-01 has a lower nonspecific reactivity in healthy individuals, thus improving diagnostic specificity. fOOlOO] Next, we conducted an analysis of the plasma levels of synuclein, NrCAM, CHL1, STOM and RUVBL1 in both healthy controls and PD patients using OG-01, as depicted in Figure 3e. Our findings reveal that OG-01, similar to b-isox, significantly detects the levels of these proteins in the plasma of PD patients.

[00101] Next, we conducted an analy sis of the plasma levels of amyloid oligomers and amyloid in both healthy controls and AD patients using OG-01, as depicted in Figure 3f. Our findings reveal that OG-01, similar to b-isox, significantly detects the levels of these proteins in the plasma of AD patient. Additionally, we conducted a comparative study to evaluate the assay efficiency of amyloid between b-isox and OG-01. The results, illustrated in Figure 3f, demonstrate that OG-01 exhibits higher efficiency of detecting amyloid compared to b-isox. Example 4: b-isoxELISA assay efficiency of OG-02 in healthy and ALS patients.

[00102] We analyzed the plasma levels of poly(GR), SOD1, p-TDP-43, CD14, and STOM in both healthy controls and ALS patients using OG-02, as illustrated in Figure 3b. Our findings demonstrate that OG-02 detects a noticeable increase in the levels of these proteins in the plasma of ALS patients, a result similar to that observed with b-isox. Example 5: b-isoxELISA assay efficiency of OG-03 in healthy and ALS patients.

[00103] We conducted an analysis of the plasma levels of poly(GR), SOD1, p-TDP-43, CD 14, and STOM in both healthy controls and ALS patients using OG-03, as depicted in Figure 5. Our findings reveal that similar to using b-isox, significantly elevates the levels of these proteins in the plasma of ALS patients by b-isox OG-03. Example 6: b-isoxELISA assay efficiency of OG-04 in healthy and ALS patients.

[00104] We conducted an analysis of the plasma levels of poly(GR), p-TDP-43, SMN, and Actin in both healthy controls and ALS patients using OG-04, as depicted in Figure 6. Our findings reveal that the reduced levels of pTDP-43 proteins, but increased levels of SMN and Actin in the plasma of ALS patients by OG-04. Example 7: b-isoxELISA assay efficiency of OG-05 and OG-06 in healthy and ALS patients.

[00105] We conducted an analysis of the plasma levels of poly(GR) and p-TDP-43 in both healthy controls and ALS patients using OG-05 and OG-06, as depicted in Figure 7. Our findings reveal that OG-05 and G-06, similar to b-isox, significantly detects the levels of these proteins in the plasma of ALS patients. Example 8: Evaluating the efficiency of the b-isoxELISA assay with OG-07 and OG-08 in healthy individuals and ALS patients, and comparing its effectiveness to b-isox in ALS patients.

[00106] Initially, we conducted an analysis of the plasma levels of poly(GR) and p-TDP-43 in both healthy controls and ALS patients using OG-07 and OG-08, as depicted in Figure 8a. Our findings reveal that OG-7 and OG-08, similar to b-isox, significantly detects the levels of these proteins in the plasma of ALS patients.

[00107] Additionally, we conducted a comparative study to evaluate the assay efficiency between b-isox and OG-07 and OG-08 in the same ALS patient. The results, illustrated in Figure 8b, demonstrate that OG-07 and OG-08 exhibits higher efficiency of poly(GR) compared to b-isox. Example 9: b-isoxELISA assay efficiency of OG-09 and OG-10 in healthy and ALS patients, and its comparative analysis with b-isox in ALS patients.

[00108] We conducted an analysis of the plasma levels of poly(GR) and p-TDP-43, in both healthy controls and ALS patients using OG-09 and OG-10, as depicted in Figure 9a. Our findings reveal that OG-09 and OG-10, similar to b-isox, significantly detects the increased levels of p-TDP-43 proteins in the plasma of ALS patients.

[00109] Additionally, we conducted a comparative study to evaluate the assay efficiency between b-isox and OG-10 in the same ALS patient. The results, illustrated in Figure 9b, demonstrate that OG-10 exhibits higher efficiency of p-TDP-43 detection compared to b-isox. Example 10: b-isoxELISA assay efficiency of OG-11 and OG-12 in healthy and ALS patients, and its comparative analysis with b-isox in ALS patients.

[00110] We conducted an analysis of the plasma levels of poly(GR) and p-TDP-43 in both healthy controls and ALS patients using OG-11 and OG-12, as depicted in Figure 10. Our findings reveal that OG-11 and OG-12, similar to b-isox, significantly detects the increased levels of p-TDP-43 proteins in the plasma of ALS patients.

[00111] References 1.     Prof et al., Conformational disease, 1997, The Lancet, vol. 350, pp.134-138. 2.     Kiernan et al., Amyotrophic lateral sclerosis, 2017, The Lancet. Vol. 390, pp. 2084 2098. 3. Rosen et al., Mutations in Cu / Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, 1993, Nature. Vol. 362(6415), pp. 59-62. 4. Neumann et al., Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis, 2006, Science, vol. 314, pp. 130-133. 5. Wang et al., TDP-43: an emerging new player in neurodegenerative diseases, 2008, Trends Mol. Med., vol. 14, pp.479-485. 6. DeJesus-Hemandez et al., Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS, 2011, Neuron, vol.72, pp.245-56. 7. Achmitz et al., Emerging Perspectives on Dipeptide Repeat Proteins in C9ORF72 ALS / FTD, 2021, The Lancet, vol. 397, pp. 1577-1590. 8.     Scheltens et al., Alzheimer's disease, 2021, The Lancet, vol. 397, pp.1577-1590. 9.     Braak et al., Staging of brain pathology related to sporadic Parkinson's disease, 2003, Neurobiol. Aging, vol. 24(2), pp. 197-211. 10. Wang et al. The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies, 2012, Nature Commun., Vol. 3, pp. 766. 11. Kato et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels, 2012, Cell, vol. 149, pp. 753-767. 12. Han et al. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies, 2012, Cell, vol. 149, pp. 768-779. 13. Brangwynne et al. Germline P granules are liquid droplets that localize by controlled dissolution / condensation, 2009, Science vol. 324, pp. 1729-1732.

[00112] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only.

Claims

1. A method for detecting a human disease in a human subject, comprising,(a) obtaining a biofluid sample from the subject;(b) adding an isoxazole to the obtained biofluid sample to form a biofluid isoxazole composition in the biofluid sample; and(c) detecting a presence of the biofluid isoxazole composition.

2. The method of claim 1, wherein the isoxazole is biotin-isoxazole, (6-(5-(Thiophen-2-yl)isoxazole-3-carboxamido)hexyl 5-((3a.S',4.S'.6aA,)-2-oxohexahydro-l / f-ihieno|3,4-ri]imidazol-4-yl)pentanoate), or its salt or an analog thereof, especially biotin-isoxazole and very especially biotin-isoxazole.

3. The method of Claims 1-2, wherein the biofluid sample is urine, whole blood, plasma, or serum, cerebrospinal fluid (CSF), saliva, or mucosa, such as, urine, saliva, CSF or plasma, and especially CSF or plasma.

4. The method of Claims 1-3, wherein the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), frontotemporal dementia (FTLD), diabetes, cancer, infectious disease, huntington disease, schizophrenia, aging- associated disease, or protienopathies, especially wherein the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

5. The method of claims 1-4, further comprising thereafter treating the human subject for the human disease or thereafter changing an existing treatment of the human subject for the human disease based on the detecting the presents of the biofluid isoxazole composition, such as a treatment including pharmaceutical therapy for the human disease, and optionally further comprising diagnosing the human disease in the human subject.

6. The method of claims 1-5, wherein the concentration of isoxazole ranges from O.OlmM to 0.3 mM, preferably from 0.100 mM to 0.200mM, in the biofluid sample.

7. The method of claims 1-6, wherein the biofluid isoxazole composition is in a precipitate.

8. The method of claim 7, wherein the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

9. The method of claim 8, wherein the human disease is ALS, AD, DLB, MSA or PDD or PDnD.

10. The method of claim 9, wherein the human disease is ALS, such as sporadic ALS.

11. The method of claims 7-10, further comprising monitoring the size of the precipitate to monitor the progression of ALS in the subject.12.The method of claims 1-6, further comprising adding a polypeptide to biofluid isoxazole composition in the biofluid sample to facilitate detection by an immune assay, such as a blot assay, a chemiluminescence immunoassay, an enzyme-linked immunosorbent assay (ELISA),a light scattering immunoassay, a radiolabeled immunoassay, in particular, ELISA or a Western blot.

13. The method of claim 12, wherein the human conformational disease is Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

14. The method of claims 12-13, wherein the polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof which binds to the biofluid isoxazole composition.

15. The method of Claim 14, wherein the human diseases is ALS, and wherein the polypeptide for detecting ALS is an antibody against SOD1, C9orf72 dipeptide repeats, PFN1, PRDX2, phospho-TDP-43, CAI, MYL12B, CD14, ANXA5, STOM, SMN, ACTB, or GLUT1, such as SOD1, MYL12B, CD14 and p-TDP-43, and especially SOD1 and p-TDP-43.

16. The method of Claim 14, wherein the human diseases is AD, and wherein polypeptide for detecting AD is an antibody against APP, phospho-TDP-43, TDP-43, Tau, STOM, or ANK1.

17. The method of Claim 14, wherein the human diseases is PD, and wherein polypeptide for detecting PD is an antibody against synuclein, CHL1, NELL2, p-TDP-43, NrCAM, ANK1, STOM, PRDX2, CAI, CD14, and RUVBL1.

18. The method of claims 15-17, wherein biofluid is plasma or CSF.

19. The method of claims 12-18, wherein the method is ELISA, such as direct, indirect, sandwich, or competitive ELISA.

20. Use of an isoxazole to detect a human disease in any method of claims 1-19.

21. A pharmaceutical composition comprising a compound selected fromOCL01:6-(5-(thiophen-3-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoateOCL02:6-(5-(furan-2-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoateOG-03: 6-(5-(5-chlorothiophen-2-yl)isoxazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1 H-thieno [3,4-d]imidazoL4-yl)pentanoateOCL04:6-(5-(benzo[b]thiophen-2-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoateOG205:6-(5-(thiazol-2-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoateOCL06:6-(5-(thiazol-5-yl)isoxazole-3-carboxamido)hexyl5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoateOG-07: 6-(5-(thiophen-2-yl)-l,2,4-oxadiazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoateQG-08: 6-(5-(thiophen-2-yl)-l,2,4-oxadiazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahy dro-1 H-thieno [3,4-d]imidazol-4-yl)pentanoateOG-09:6-(2-methyl-5-(thiophen-2-vl)furan-3-carboxamido)hexvl 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoateQG-10:6-(5-(thiophen-2-vl)isoxazole-4-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1 H-thieno [3,4-d]imidazoL4-yl)pentanoateOG-ll:4-(5-(thiophen-2-yl)isoxazole-3-carboxamido)butyl 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate andOG-l2:2-L5-(thiophen-2-vl')isoxazole-3-carboxamido')ethv1 5-((3aS,4S,6aR)-2-oxohexahydro-1 H-thieno [3,4-d]imidazoL4-yl)pentanoate.

22. The pharmaceutical composition of claim 21, comprising a pharmaceutically acceptable carrier.

23. A series of novel b-isox analogs for use in diagnostic assays for neurodegenerative diseases.

24. The b-isox analogs of claim 23, wherein the assay compounds are OG-01, OG-02, OG-03 and OG-08.

25. The b-isox analogs of claim 24, wherein the size of the diagnostic precipitates in patients with ALS, PD and AD is increased than that of b-isox, providing enhanced diagnostic accuracy.

26. The b-isox analogs of claim 23, wherein the assay compounds are OG-04 and OG-05.

27. The b-isox analogs of claim 26, wherein the size of the diagnostic precipitates in health individuals is increased but absent in patients, providing negative controls to improve diagnostic accuracy.

28. The b-isox analogs of claim 23, wherein the assay compounds are OG-01, OG-07, OG-08 and OG-10.

29. The b-isox analogs of claim 28, wherein the plasma levels of disease proteins are increased in ALS, PD and AD patients, providing enhanced diagnostic accuracy.

30. The use of the b-isox analogs of claim 29 in the detection and monitoring of ALS, PD and AD.

31. The b-isox analogs of claim 23, wherein the assay compounds have a similar detection capability.

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