Screening method for identifying compounds

Abstract

Methods described herein provide procedures that can be used to rapidly screen drugs for high probability of effectiveness as therapy for amyloid associated neurodegenerative diseases, such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, and prion-associated diseases by detecting size and / or aggregation of phage incubated with a target compound. The data generated indicate that bacteriophage or phage capsid subunits can switch conformation to a conformation that mimics the neurodegenerative disease-causing conformation of amyloid proteins. This switch has been observed by incubation of bacteriophage T4 with methylene blue, a compound for which literature data indicates has anti-AD activity.

Description

SCREENING METHOD FOR IDENTIFYING COMPOUNDSRELATED APPLICATION S

[0001] This Application is an International Application claiming priority to US Provisional Patent Application 63 / 572,401 filed April 1, 2024 which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] None.FIELD

[0003] Embodiments of the invention are directed generally to the field of microbiology, neuroscience, and medicine and particularly to methods for identifying treatments for cancerous tumors and amyloid associated neurodegenerative disorders, including tauopathies.BACKGROUND

[0004] Several neurodegenerative diseases, including Alzheimer’s disease (AD), are associated with excess in-brain production of extracellular P-sheet structured protein, usually thought to be causative. This protein is generically called amyloid because of starch-like texture (reviewed in Al Adem and Lee, Protein Sci. 2023, 32, e4639; Miller et al., PNAS USA 2023, 120, e2216234120; Wen et al., Int. J. Mol. Sci. 2023, 24, 8593; Ma et al., Molecules 2022, 27, 1210; Candelise et al., Int. J. Mol. Sci. 2021, 22, 6016). Therapies for AD are usually designed to promote removal or destruction of amyloid protein (Ap in the case of AD) or its cleavage products, based on the hypothesis that the observed amyloid causes disease (amyloid cascade hypothesis (Karran and De Strooper, Nat. Rev. Drug Discov. 2022, 21, 306-18; Selkoe, Nat. Rev. Neurol. 2019, 15, 365-66)). However, these therapies have failed to significantly reverse the course of AD (Selkoe, Nat. Rev. Neurol. 2019, 15, 365-66; Zagorska et al., Pharmaceuticals 2023, 16, 151; Cai et al., Biomolecules 2023, 13, 313; Ostrowitzki et al., JAMA Neurol. 2022 Nov 1;79(11): 1113-21; Mullane and Williams, Biochem. Pharmacol. 2018, 158, 359-75; Mullane and Williams, Biochem. Pharmacol. 2020, 177, 113945).

[0005] The evidence is that P-sheet structured amyloid protein is not in the pathway of disease formation. For example, AD related symptoms sometimes occur without excess p-sheet amyloid protein production (Selkoe, Science 2002, 29S, 789-91; Monsell et al., JAMA Neurol. 2015, 72, 1124-31; Herrup, Nat. Neurosci. 2015, 18, 794-99) and excess P-sheet amyloid (plaque) production sometimes occurs without AD (Davis et al., J. Neuropathol. Exp. Neurol. 1999, 58, 376-88; Jack et al., Lancet Neurol. 2017, 16, 435-44). More likely, P-sheet structured amyloid protein is a dead-end product of protein conformation-based detoxification (Serwer and Wright, Biophysica 2021, 1, 15-21). In other words, AD is a protein conformation-caused and amount-caused disease as would be other amyloid-associated neurodegenerative diseases.

[0006] Given the above scenario, electron microscopic observation of AD-brain might be revealing. The perspective involved has some similarity to the perspective that generated the following quotation attributed to physicist, Richard Feynman. “It is very easy to answer many of these fundamental biological questions; you just look at the thing!” (Cheng, Science 2018, 361, 876-80). One observation made by electron microscopy is that at least some dark lipofuscin of an AD-brain is packed with bent, doubled fibers (Serwer et al., J. Alzheimers Dis. 2022, 88, 429- 38). In contrast, non- AD brain has dark lipofuscin in which bent, doubled fibers appear to be relatively rare (Boellaard and Schlote, Acta Neuropathol. 1986, 71, 285-94).

[0007] There is a need for additional methods and compounds for treating amyloid associated diseases.SUMMARY

[0008] The inventors provide solution(s) to the treatment problems described above by developing an assay system designed to improve identification of compounds with therapeutic potential. This is done by determining the level of interaction of a compound with particles with characteristics of a-sheet structure. The methods described herein provide procedures that can be used to rapidly screen drugs for high probability of effectiveness as therapy for amyloid associated neurodegenerative diseases, such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Huntington’s disease, and prion-associated diseases. The data generated indicate that bacteriophage or phage capsid subunits can switch conformation to a conformation that is the same as or close to the neurodegenerative disease-causing conformation of amyloid proteins. This switch has been observed by incubation of bacteriophage T4 withmethylene blue, a compound for which literature data indicates weak anti-AD activity. Methylene blue is routinely used in human patients for other purposes.

[0009] Embodiments can include (1) incubating test compounds with bacteriophage (e.g., bacteriophage T4 or similar phage) (2) subjecting the incubated sample to size and surface charge density fractionation, e.g., native agarose gel electrophoresis, and (3) identifying compounds that cause aggregation and / or hyper-expansion of the bacteriophage. The assay determines the concentrations of the test compound that are effective in producing conformational changes in the phage. The lower the minimum effective concentration is, the more potent the compound is projected to be in anti-neurodegenerative disease activity.

[0010] Certain embodiments are directed to methods for identifying compounds, these methods comprising: (i) incubating a phage with a test compound forming a test mixture and (ii) subjecting the test mixture to size fractionation and identifying aggregated phage / test compound complexes, the aggregated phage being indicative of a conformational change in the phage. In certain aspects size fractionation is by agarose gel electrophoresis. The agarose gel can be a 0.1% to a 1% gel, in certain aspects a 0.5% agarose gel. Agarose gel electrophoresis can be carried out at a voltage of 0.1-10 volts / cm, in certain aspects 50 volts for 0.25 to 2 hours, in certain aspects 1 hour. The bacteriophage can be T4 bacteriophage. In certain aspects the test compound and the phage are incubated for 30 minutes to an hour. The test compound and the phage can be incubated at a temperature of 10°C to 70°C, in certain aspects at or about 50 °C ± 10°C. The methods can include or further comprise staining a fractionated phage with a DNA stain.

[0011] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0012] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0013] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0014] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0015] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0016] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and / or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps) but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and / or method.

[0017] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim taken as a whole.

[0018] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect thebasic and novel character! stic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0019] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DESCRIPTION OF THE DRAWINGS

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0021] FIG. 1. The disease model deduced from diverse data, as described here (adapted from Serwer and Wright, Biophysica 2021, 1, 15-21). (a) DNA enters the capsid of either a phage (phage T3 is used as the example) or a herpesvirus, (b) Hyper-expansion, with subunit conversion to a-sheet structure, occurs to accelerate DNA packaging when packaging is slowed in (a) (proposed details Serwer and Wright, Biophysica 2021, 1, 15-21). (c) In the case of a phage, packaging finishes and a tail is added to the capsid to form a mature phage particle, (d) In the case of herpesviruses, amyloid proteins block progression of the hyper-expanded capsids by converting to a-sheet structure and then co-assembling with the a-sheet of subunits of the capsid (bottom left), (e) Alzheimer’s disease is initiated by over-production of amyloid protein a-sheet, which is toxic (bottom right). Some a-sheet is subsequently detoxified by being converted to the P-sheet amyloid protein of plaques. However, this conversion is not sufficient to avoid toxicity when Alzheimer’s disease is present.

[0022] FIG. 2a-2b. Native agarose gel electrophoresis (AGE) analysis of the interaction of phage T4 with methylene blue (MB). Phage T4 was prepared and partially purified as previously described for the gated loading of ethidium and bleomycin (URL www.mdpi.com / 2673- 4125 / 2 / 4 / 33). Then, T4 was incubated with MB at 54 °C for 1.0 hr and subjected to horizontalAGE. (a) The gel was photographed after staining with GelStar for 3.0 hr. (b) The same gel was photographed after expulsion of packaged DNA by incubation with 0.002 M EDTA, pH 7.4 at room temperature (22 ±3 °C) (URL www.mdpi.com / 2673-4125 / 2 / 4 / 33). The concentration of MB (mM) is indicated at the top of a lane. The arrowheads indicate the origins of electrophoresis; the arrow indicates the direction of electrophoresis. A phage T3 standard was included (lane labeled T3). Some DNA was spontaneously expelled from both T3 and T4, as indicated by the horizontal lines labeled D at the right.

[0023] FIG. 3. Electron microscopy of MB -reacted, negatively stained phage T4. Phage T4 was incubated with MB as in FIG. 2 and then immediately negatively stained with 1.5% uranyl acetate and observed by conventional transmission electron microscopy.

[0024] FIG. 4. Cryo-electron microscopy of MB-reacted, unstained phage T4. Phage T4 was incubated with MB as for FIG. 2 and observed by cryo-electron microscopy.

[0025] FIG. 5. AGE of T4 vs. [MB] co-incubated with T4 at 58 °C for 1.0 hr. The concentration (pM) of MB is above a lane. Vertical arrow, direction of AGE; (p3, phage T3; cp4, phage T4; H4, T4 head; D4, T4 DNA; D3, T3 DNA; T3, phage T3 used as a standard.

[0026] FIG. 6. AGE of MB-co-incubated T4 vs. dilution of Al l antibody (Invitrogen) subsequently incubated: 1.0 hr. at 50 °C. D4, T4 DNA; cp4, phage T4; H, T4 head.

[0027] FIG. 7. EM of MB -incubated, Al l antibody-stabilized phage T4, negatively stained with 1.5% uranyl acetate.

[0028] FIG. 8. AGE of T4 vs. [acridine orange] coincubated with T4. The concentration [pM] of acridine orange is above a lane. Vertical arrow, direction of AGE; (p3, phage T3; (p4, phage T4; H, T4 head; D4, T4 DNA; D3, T3 DNA, T3, phage T3 used as a standard.DESCRIPTION

[0029] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant onlyto be an example of that embodiment, and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0030] Alzheimer’s disease (AD) is a neurogenerative disorder characterized by a slow decline in cognitive memory and function (De Ture and Dickson, Mol.Neurodegener 2019, 14, 32; Dubois et al., Lancet Neurol. 2021, 20, 484-96; Alzheimers Dement. 2024, 20, 3708-21; Kumar et al., Alzheimer Disease. 2024. In: StatPearls 2024 Jan.). Past research has concentrated on ramifications of the Amyloid-Beta (Cascade) Hypothesis: disease causation via toxicity of 0- sheet structured amyloidbeta (A0) protein (plaque) and tau fibers (Hardy and Higgins, Science 1992, 256, 184-185; Zhang et al., Sig. Transduct. Target Ther. 2023, 8, 248; Ma et al., Molec des 2022, 27, 1210; Thawabteh et al., Molecules 2024, 29, 5131). However, A0 plaquereducing therapies have been, at best, minimally effective (Herrup, Nat. Neurosci. 2015, 18, 794-799; Mullane and Williams, Biochem. Pharmacol. 2018, 158, 359-375; Selkoe, Nat. Rev. Neurol. 2019, 15, 365-366; Kurkinen et al., Biomolecules 2023, 13, 453; Zagorska et al., Pharmaceuticals 2023, 16, 151; Behl, Front. Aging Neurosci. 2024, 16, 1459224). This has led to re-examination of the fundamentals, including the basic conformation-based chemistry of the toxic conformer of A0 protein.

[0031] Most models of Alzheimer’s disease (AD) contradict at least one datum. Adequate therapy does not exist. One concept is that AD is caused by over-activity of a primitive, not-well- studied innate immunity. This concept originates in known descent of amyloid proteins (AB for AD) from virus proteins, with conjectured CRISPR-like, anti-viral amyloid protein function. A central hypothesis is (1) Triggering of AD is sometimes caused by microbial infections, which explains correlation with previous herpesvirus infections. (2) AB innate immune function occurs via conversion of AB to a Pauling-Corey discovered conformation now called alpha-sheet, shown to be toxic. AB then inhibits microbes by co-assembly with microbial alpha-sheet. Packaging of dsDNA in bacteriophage (phage) capsids is associated with capsid hyper-expansion / thinning that is explained by alpha-sheet structured capsid subunits. Because herpesviruses package DNA via a phage-like pathway, the hypothesis includes formation by herpesvirus capsids (and other microbial structures) of alpha-sheet that is targeted by alpha-sheet structured AB. (4) This process causes AD when excessive alpha-sheet structured AB is produced. (5) B-sheet AB of AD plaques is a detoxification product, which explains AD-drug development failures. (6) More general alpha-sheet toxicity does not occur because alpha-sheet longer than 4-6 amino acids isnot normally in human proteins. It was recently found that methylene blue (MB) triggers hyper- expansion / thinning of the phage T4 capsid, seen by cryo-electron microscopy (cryo-EM). MB also has anti-AD activity, possibly because MB selectively binds to alpha-sheet-AB.

[0032] It has been proposed that AD is caused by a toxic protein conformation called an flsheet (Shea et al., PNAS USA 2019, 118, 8895-900; Shea and Daggett, Biophysica 2022, 2, 91- 110; Prosswimmer and Daggett, Open Biol. 2022, 12, 220261), a protein conformation discovered by Pauling and Corey before the discovery of the P-sheet structure (Pauling and Corey, PNAS USA 1951, 37, 251-56; Pauling and Corey, PNAS USA 1951, 37, 256-61). Molecular dynamics simulation has shown that ci-sheet and P-sheet can interconvert (Prosswimmer and Daggett, Open Biol. 2022, 12, 220261; Hayward et al., Proteins 2008, 71, 415-425; Hayward et al., Proteins 2011, 79, 3193-3207), with interconversion stimulated by side chain hydrophilicity (Prosswimmer and Daggett, Open Biol. 2022, 12, 220-61). The proposition of a-sheet as disease causing agent is supported by the observation that the toxic form of in vitro assembled AD amyloid protein (cleaved Ap protein, 40-42 amino acids long (Hayward et al., Proteins 2008, 71, 415-25; Hayward et al., Proteins 2011, 79, 3193-207)) has two characteristics of a-sheet: (1) circular dichroism near zero and (2) appropriate NMR signature (Shea et al., PNAS USA 2019, 118, 8895-900; Shea and Daggett, Biophysica 2022, 2, 91-110; Prosswimmer and Daggett, Open Biol. 2022, 12, 220-61). Importantly, molecular dynamics simulation reveals that single-polypeptide chains in A-sheet conformation are bent to avoid side chain clashes (Hayward and Milner- White, Proteins 2008, 71, 415-425), a situation anticipated by Pauling and Cory (Pauling and Corey, PNAS USA 1951, 37, 251-56; Pauling and Corey, PNAS USA 1951, 37, 256-61). The existence of the steric clashes is the probable reason that a-sheet is relatively unknown. A reasonable opinion is that further exploration should be made of the possible linkage of AD to the bent fibers seen in AD-brain by electron microscopy.

[0033] A second possible reason for the obscurity of ci-sheet is that empirical detection of flsheet has occurred only for 4-6 amino acid long stretches, e.g., in ATP -binding sites, metal binding sites, and phosphate binding sites. In these cases, a-sheet is bent (Milner-White et al., Biol. Direct 2008, 3, 3; Milner-White and Russell, Genes (Basel) 2011, 2, 671-688). Longer flsheet has been seen only by molecular dynamics simulation (Shea and Daggett, Biophysica 2022, 2, 91-110; Prosswimmer and Daggett, Open Biol. 2022, 12, 220-61). Pauling and Corey hadproposed a-sheet bending to be the reason that bird feathers are bent, although they did not use the term “a-sheet” (Pauling and Corey, PNAS USA 1951, 37, 256-61).

[0034] Inter-conversion of a-sheet and fl-sheet polypeptide occurs by rotations around C-N and C-C bonds that generate approximately 180° rotation of every other amino acid of a polypeptide projected to two dimensions (Prosswimmer and Daggett, Open Biol. 2022, 12, 220261; Pauling and Corey, PNAS USA 1951, 37, 251-56; Hayward and Milner-White, Proteins 2008, 71, 415-25). In addition to bending, a second a-sheet characteristic is the presence of all a- carboxyl groups on one edge and presence of all a-amino groups on the opposing edge of a polypeptide chain. At physiological pH, this implies net separation of negative and positive charges along the surface of the polypeptide. In contrast, along the surface of P-sheet, a-carboxyl and a-amino groups alternate so that this charge separation does not occur (Prosswimmer and Daggett, Open Biol. 2022, 12, 220261; Pauling and Corey, PNAS USA 1951, 37, 251-56; Hayward and Milner- White, Proteins 2008, 71, 415-25).

[0035] Viruses, a-sheel, and Alzheimer ’s Disease. Independently, a previous study (Serwer and Wright, Viruses 2017, 9, 119) revealed a phage T3 capsid with incompletely packaged DNA and a hyper-expanded capsid outer shell. The hyper-expansion implied thinning of the outer shell that strongly suggested either P-sheet or a-sheet structure of the subunits of the shell (Server and Wright, Viruses 2017, 9, 119; Server and Wright, Viruses 2018, 10, 307). However, this capsid also had unusually high negative charge on its surface, suggesting the presence of the charge separation characteristic of a-sheet (Server and Wright, Viruses 2018, 10, 307). Of course, the surfaces of viral capsids are also bent, which supports the assumption of a-sheet structured subunits.

[0036] Furthermore, electron microscopy of thin sections has revealed that these hyperexpanded capsids are enhanced in infected cells by (1) embedding in agarose gel during infection and (2) adding proflavine, a dye known to inhibit DNA packaging, to the agarose-embedded, infected cells. Thus, the hyper-expanded capsids are thought to be products of DNA packaging (Server et al., Pharmaceuticals 2020, 13, 18).

[0037] It has been proposed that these observations are linked to the observations of others that (1) herpesvirus infections are precursors of AD, and (2) this precursor-product relationship is dependent on the presence of the ApoE-e4 allele (Itzhaki, Vaccines 2021, 9, 679; Itzhaki, J.Alzheimer’s Dis. 2020, 76, 85-87; Itzhaki and Lathe, J. Alzheimer’s Dis. 2018, 64, 363-66; Piotrowski et al., Neurolmmune Pharm. Ther. 2023, 2, 253-66). The proposed linkage is via (1) the sequence homology and structural homology-based conclusion that phages and herpesviruses have the same DNA packaging pathway (Serwer et al., Pharmaceuticals 2020, 13, 18), and (2) the finding of effects of the ApoE-s4 allele on the composition of branched, gel-promoting proteins in the basement membrane, a gel that surrounds neuronal cells (Salloway et al., J. Neurol. Sci. 2001, 203-04, 183-87). The hypothesis is that the a-sheet-generating effect of gelembedding of phage T3-infected bacteria is mimicked (details not proposed) by alteration of the basement membrane around cells in which herpesviruses are propagating.

[0038] The studies of virus infection-association of AD do not, to the best of our knowledge, consider either of the following possibilities. (1) A virus assembly-associated version of the viral capsid (not the virus) is the trigger. (2) The triggering of symptoms is protein conformationbased, specifically, a-sheet-based.

[0039] Integration of Data to Develop a Disease Model. The above observations were integrated for the purpose of progressing towards deducing a detailed pathway for both AD and other amyloid associated, neurodegenerative diseases. The first possible pathway deductionassist is evidence supporting the proposal that these diseases are caused by anti-insult activity of an innate immune system. To the best of our knowledge, this evidence originated in and is, primarily, the observation that amyloid proteins are related to viral proteins (Bandea, bioR iv 2013). The integration continues via the deduction that the likely origins of amyloid proteins are previous virus infections, with selection for cellular retention of the proteins via the proteins’ capacity to counter future virus infections, a protein level CRISPR, so to speak (Wang and Doudna, Science 2023, 379, eadd8643; Sauvagere and Siatka, Genes 2023, 14, 1542).

[0040] The second possible pathway deduction-assist is evidence supporting the proposal that AD is correlated with previous virus infection, especially with herpesviruses. This evidence is the observation that, that since -1995, age normalized AD frequency has decreased in the United States and Western Europe, but not China and Japan (Wolters et al., Neurology 2020, 95, e519— e531), where anti-herpes virus vaccination was less emphasized. These results and others demonstrate correlation of lower AD incidence with introduction of anti-herpesvirus and otheranti-virus measures (Bukhbinder et al., Hum. Vaccin. Immunother. 2023, 19, 2216625; Wu et al., Front. Immunol. 2022, 13, 872542).

[0041] The third possible pathway deduction-assist is the evidence for (1) similarity of phage to herpesvirus DNA packaging, and (2) in-gel propagation enhanced, DNA packaging associated phage capsid hyper-expansion, which is likely to include conversion of capsid subunits to flsheet conformation. It is proposed that herpesviruses (1) form the DNA packaging intermediates that phages form, including the hyper-expanded capsid for which a-sheet structured subunits is proposed, (2) stimulate the innate immune-conversion to a-sheet structure of amyloid protein, and (3) are triggers of neurodegenerative diseases, including AD.

[0042] The proposed pathway, as deduced, is simple. (1) Virus infection stimulates the conversion of amyloid-forming protein to a-sheet conformation for the evolved purpose of coassembling with a-sheet structured viral assembly intermediates and, thus, inhibiting infection (left pathway 4 at the bottom of FIG. 1). (2) Amyloid protein associated diseases are caused by loss of control of the amount of a-sheet conversion that occurs, resulting in excessive and toxic production of a-sheet (right pathway 5 at the bottom of FIG. 1).

[0043] This proposed mechanism (1) does not include mis-folding of amyloid-forming protein, as other a-sheet-based hypothesis do, and (2) includes an anti-viral function for conversion to a-sheet, as originally suggested in Bandea, hioR / v (2013) without structural details. Also, this mechanism provides the following explanations for hereditary ataxias caused by over-repeating, sometimes by < 10%, of a normally repeated amino acid sequence within a protein that also has non-repeated amino acid sequence: (1) The reason that these repeats are primarily poly G and poly Q is formation of a-sheet. Both poly G and poly Q have been shown to have a-sheet potential (Armen et al., PNAS USA 2005, 102, 13433-38; Babin et al., Proteins 2011, 79, 937-46). (2) The reason for existence of these amino acid repeats is innate immune function. This poly G / poly Q presence does not have, to our knowledge, a previously published explanation. (3) The conversion to toxicity by minor increase in repeat length (Armen et al., PNAS USA 2005, 102, 13433-38; Babin et al., Proteins 2011, 79, 937-46) occurs because of length-dependence of the stability of a-sheet conformation.

[0044] Finally, this latter scenario is supported by the following facts. Poly G is the only poly-amino acid without side chains and therefore is readily a-sheet-forming (Milner- White andRussell, Biol. Direct 2008, 3, 3; Milner-White and Russell, Genes (Basel) 2011, 2, 671-88). Poly Q is the uncharged poly-amino acid with the most hydrophilic side chain (Kyte and Doolittle, J. Mol. Biol. 1982, 757, 105-32). Side chain hydrophilicity has been found to be a factor that encourages P-sheet to a-sheet conversion of peptides (Prosswimmer and Daggett, Open Biol. 2022, 12, 220-61). Electrical charge of side chains confers hydrophilicity but might disrupt hydrophobic interactions observed by modeling of the formation of a-sheet by poly Q (Babin et al., Proteins 2011, 79, 937-46).

[0045] The failure of the above drug trials does not imply that all drug trials have been complete failures. Partial successes are possible. One way to find the “thin edge of the anti-AD drug wedge” is to search among the trials for relevant, possibly useful, partial successes. Such relevance would be a linkage to the data and model discussed herein. Searching did not have to go far to find partial success that has the potential to be linked to the above model. Methylene blue (MB) is (1) the oldest synthetic dye, (2) usable in humans up to about 2 mg / kg body weight, and (3) a partial AD-suppressor in both humans and animals (Howland, J. Psychosoc. Nurs. Ment. Health Serv. 2016, 54, 21-26; Bums et al., Antioxidants 2022, 11, 2402; Hashmi et al., Cureus 2023, 75, e46732). The AD-suppression is thought to originate primarily via enhancement of respiration in mitochondria (Bums et al., Antioxidants 2022, 11, 2402), an effect that MB has been shown to have (Xue et al., Cells 2021, 10, 3379). It is also possible that MB binds and detoxifies the toxic conformation of amyloid-forming proteins.

[0046] While testing the gated loading of anti-cancer compounds in bacteriophage (phage) T4, an interesting observation was made. The idea was to use T4, a phage that has been found highly persistent in murine blood, as a gated drug delivery vehicle (Serwer and Wright, Biophysica 2022, 2, 366-80). The inventors had already successfully achieved gated T4 loading with ethidium and bleomycin without any detected disturbance of the phage capsid (Serwer and Wright, Biophysica 2022, 2, 366-80). However, when the T4-loading of methylene blue was tested, a surprising, possibly AD model-related discovery was made. MB induced a transformation of T4 capsid subunits to an apparent (not proven) a-sheet related structure. These observations supported the model of FIG. 1 and suggested a path for finding anti-AD drugs more effective than MB.

[0047] The methods described herein are used to probe for anti-AD drugs with a model that accounts for data on AD. Previous probing was not sufficiently successful apparently because the previous probes simply ignored most of the key data (Shea et al., PNAS USA 2019, 116, 8895- 900). Methods described herein use procedures that are more cost effective and time efficient than other procedures known to us.I. Capsid Expansion Assays

[0048] Currently efforts are being directed toward determining the structure of the capsid shells of methylene blue expanded T4 by use of (1) atomic force microscopy, to determine surface charge, and, most critically, (2) cryo-electron microscopy with three-dimensional reconstruction, to determine the polypeptide chain conformation of the shell subunits. The cryo- EM analysis will determine directly if the subunits have alpha-sheet structure.

[0049] Compounds related to methylene blue, including derivatives and structurally similar compounds, together with other anti-AD candidates, are being tested using the methods described herein. Examples of structurally similar compounds: acridine orange, new methylene blue, leucomethylene blue, toluidine blue, and the like.

[0050] The objective is to identify anti-neurodegenerative disease compounds with an increased effectiveness compared to methylene blue and its derivative (including leucomethylene blue) that have been shown by others to have marginal anti-Alzheimer’s disease (AD) activity (Bums et al., Antioxidants 2022, 11, 2402). These compounds (many, like methylene blue, are tricyclic phenothiazines) are not effective enough to be used in general practice for therapy of neurodegenerative diseases, although methylene blue is used in humans for other purposes (Bums et al., Antioxidants 2022, 11, 2402). The methods described herein can be used to screen for compound(s) with more potent anti-AD activity and / or activity against other amyloid- associated neurodegenerative diseases.

[0051] In previous publications, others have found evidence that the neurotoxicity in AD is generated by an amyloid protein conformation called alpha-sheet (Shea et al., PNAS USA 2019, 116, 8895-900). However, current drug probes, as reviewed in Bums et al., Antioxidants 2022,II, 2402 do not rely on this observation. The inventors have found evidence that alpha-sheet is also a likely protein conformation associated with the assembly of bacteriophages. Based on homology, this conformation is also likely to be present during assembly of human-infectingviruses, such as herpesviruses (Serwer and Wright, Biophysica 2021, 7, 15-21; Serwer et al., J. Alzheimer's Dis. 2022, 88, 429-38). The basis of the methods described herein is that bacteriophages include an alpha-sheet conformation in subunits of the DNA-containing shells (capsids) that have been found to hyper-expand shells during phage DNA packaging (Serwer and Wright, Biophysica 2021, 7, 15-21; Serwer et al., J. Alzheimer's Dis. 2022, 88, 429-38). This is proposed to be linked to the following observation: incubation with methylene blue or other potential alpha-sheet interacting compounds causes hyper-expansion of the shell of bacteriophage, e.g., phage T4 or related phages. It is noted that other dyes, such as ethidium and bleomycin, have not been found to cause hyper-expansion. Thus, the inventors deduced as likely that at least part of the anti-AD activity of methylene blue is associated with its selective binding to alpha-sheet. To test for additional anti-AD compounds, the methods described herein use detection of hyper-expansion-inducing binding of a compound to bacteriophage. The change in conformation of the phage is detected by a microscopy observed change in size or by size fractionation of bacteriophage in a treated sample. In certain aspects, size fractionation is carried out by native agarose gel electrophoresis (AGE) followed by staining for a bacteriophage component, e.g., DNA.

[0052] The methods include (1) incubating a test molecule with an appropriate bacteriophage for an appropriate amount of time and at an appropriate temperature / condition, forming an incubated test sample, (2) followed by size fractionation of the test sample to identify a test sample contained aggregated or expanded bacteriophage. In certain aspects, the size fractionation is performed under conditions in which expanded or aggregated bacteriophage condition is maintained.

[0053] Size fractionation of bacteriophage by native gel electrophoresis is a technique that uses an electric field to migrate phage particles through a gel matrix that introduces sieving that separates the particles based on their size. A summary of the process includes the following: (a) Preparation of Samples. Bacteriophage samples are typically mixed with a loading buffer containing a dense agent like sucrose and a tracking dye for visualization. The sucrose or equivalent compound in this mixture provides density for the samples to sink into the gel wells before gel electrophoresis, (b) Gel Preparation. Agarose or, relatively rarely, polyacrylamide gels are the gels typically used for size fractionation of phages. The concentration of the gel is chosen based on the size range of phages being studied. Higher concentrations of the gel are used forsieving-based resolving of smaller phages, while lower concentrations are used for sieving-based resolving of larger phages, (c) Loading Samples. The prepared phage samples are loaded, using a pipette, into wells formed in the gel. (d) Electrophoresis. An electrical field is applied across the gel, causing the (typically) negatively charged phage particles to migrate through the gel matrix towards the positively charged electrode, (e) Visualization. After electrophoresis, the gel can be stained with a dye that binds to nucleic acids, such as ethidium bromide or SYBR Green, to visualize the DNA contained within the phage particles. The gel is then illuminated with UV light to visualize the separated bands corresponding to different phages, (f) Analysis. The separated bands on the gel are formed by phage particles in different size / charge states. Typical settings used include but are not limited to: (i) Agarose Concentration. The concentration of agarose in the gel is usually between 0.2% to 2.0%. Higher concentrations are used for separating smaller particles or compounds, while lower concentrations are used for larger particles, (ii) Buffer. The choice of buffer depends on the specific requirements of the study. Commonly used buffers include Tris-acetate-magnesium (TAM) buffer and Tris-borate- magnesium (TBM) buffer, (iii) pH. The pH of the running buffer is typically around 8.0, which helps maintain the stability during electrophoresis, (iv) Voltage per distance (electrical field). The electrical field applied across the gel depends on the size of the particles / fragments and the desired resolution. Typically, fields range from 1-5 volts / cm. Higher fields lead to faster migration but may result in heating of the gel. (v) Duration. The duration of electrophoresis varies based on the size and surface charge density of the particles / fragments and the desired separation. Generally, it can range from 30 minutes to 24 hours, but can be longer.

[0054] Other non-denaturing methods for size and charge fractionating bacteriophage typically involve techniques that do not disrupt the structure or integrity of the phage particles. These methods are important for identifying conformational changes in phage when exposed to potential therapeutic agents. Some common non-denaturing methods for size fractionating bacteriophages include: (i) Size Exclusion Chromatography (SEC). SEC separates particles based on their size as they pass through a porous stationary phase. Larger particles elute earlier than smaller ones. A test sample is loaded onto the column, and as the sample moves through the column, smaller phages are retarded more by the stationary phase, resulting in separation based on size, (ii) Ultrafiltration. Ultrafiltration involves the use of membranes with defined pore sizes to separate particles based on their size. Phage-containing solutions are passed through themembrane, where phage particles larger than the pore size are retained while smaller particles pass through. By using membranes with different pore sizes, phages in specific size ranges can be separated, (iii) Differential Centrifugation. This method exploits the differences in either sedimentation rates or densities (or both) of particles in a centrifugal field. By subjecting phagecontaining solutions to sequential rounds of centrifugation at different conditions, larger phages can be pelleted at lower speeds, while smaller phages remain in the supernatant. Variations include rate zonal centrifugation in sucrose density gradients and buoyant density centrifugation in cesium chloride density gradients, (iv) Field-Flow Fractionation (FFF). FFF is a chromatographic technique that separates particles based on differences in their diffusion rates within a flowing carrier fluid. It can separate particles ranging from a few nanometers to several micrometers in size, including bacteriophages. FFF can achieve high resolution and is suitable for isolatingphages in specific size ranges, (v) Buoyant Density Centrifugation. This technique separates particles based on their buoyant densities. A density gradient is formed during centrifugation. Phage-containing samples are mixed with the density gradient forming compound and subjected to centrifugation, resulting in the formation of bands containing particles of different densities in the density gradient generated by the centrifugation.

[0055] In one example, bacteriophage T4 is used. Bacteriophage T4, is a member of the Myoviridae family, which is characterized by viruses with a long, contractile tail. The T4 phage is specifically part of the Tevenvirinae subfamily, which includes viruses that are lytic for Escherichia coli and closely related bacteria. T4-like phages are a group of phages that share genetic and morphological characteristics with the T4 phage. They are known for their complex structure, including an elongated head with icosahedral caps and a contractile tail, which they use to infect their host bacteria, typically E. coli. Any phage that interacts with compounds resulting in hyper-expansion, shell thinning (and likely alpha-sheet structured subunits) and possibly aggregates can be used in the described methods. The family of phages related to T4 includes a variety of subgroups and individual phages, some of which are: T-even phages: This includes T2, T4, and T6 phages, which are related and share a high degree of genetic homology. They are distinguished by their host range and minor morphological differences. The classification and nomenclature of bacteriophages are continually evolving as new phages are discovered and as genomic and proteomic analyses reveal more about their relationships.

[0056] The phage / compound incubation can be carried out at 5, 10, 20, 30, 40, 50, 60, 70, to 80 °C for 5, 10, 20, 30, 40, 50, 60 seconds, minutes, or hours (including all values and ranges there between). In one example Phage T4 was incubated at 54 °C in the presence of methylene blue. As the concentration of methylene blue increased above 0.25 mM, the bands of monomeric, infective phage T4 and T4 without its tail (called a head) decreased in intensity. As this occurred, DNA staining at the origin increased, as the phages and heads were aggregating and / or growing larger. The lowest aggregation-inducing concentration was ~2.5 mM. The aggregation is associated with capsid hyper-expansion, presumably associated with compound-induced conversion of capsid subunits to alpha-sheet conformation. A candidate for improved anti-AD drug is projected to cause phage and head aggregation at a drug concentration lower than 2.5 mM.

[0057] In vitro methods, such as those proposed here, are more practical than in vivo assays in model animals. The methods described herein can be used as an in vitro screen followed by more complex animal studies with compounds identified as drug candidates. In vitro, one can assay several drugs per day at minimal cost in supplies and equipment.

[0058] A proof of principle for the screening methods includes incubating phage T4 with methylene blue followed by analysis by agarose gel electrophoresis (AGE). Previous dyes (including ethidium and bleomycin) had entered the T4 capsid when the temperature was raised to 54 °C. However, in the case of methylene blue, the results were different. By electron microscopy of T4, after incubation at 54 °C with 10.0 mM methylene blue, aggregates were observed. In addition, T4 phages with a hyper-expanded and less elongated outer shell were observed, an appearance not previously seen. The particles with a hyper-expanded outer shell also had to have shells that were thinned, by the law of conservation of mass. The shells and packaged DNA of some phages appeared to have started a hyper-expansion related transition, but not to have completed this transition. This was concluded from the decreased density of the DNA, relative to the DNA in the intact phage particle. The DNA appeared to be bulging out of the capsid for the particles. However, the DNA was never seen to leave the capsid.

[0059] Thus, there is a MB-induced transition that thins and hyper-expands the capsid’s shell. Thinning is probably possible only for a sheet structure. Two sheet structures exist, alphasheet and beta-sheet. The evidence indicates an alpha-sheet structure because alpha-sheetstructure has separation of alpha-amino group charges from alpha-carboxyl group charges. This separation can cause an increase in average surface charge density seen by electrophoresis for phage T3. Beta-sheet does not have this charge separation. Charge-charge interaction is part of the rationale for methylene blue (positively charged) selectively binding and promoting an alphasheet conformation of the subunits of the capsid’s shell.A. The Effect of Methylene Blue (MB) on Phage T4

[0060] The phage T4 gate is opened by raising temperature to 50-58 °C and closed by lowering temperature. The loading is in the interior of the DNA-containing protein shell (head) of T4. The profde of loaded T4 during native agarose gel electrophoresis (AGE) is not changed by the loading of 7.1 mM bleomycin (Serwer and Wright, Biophysica 2022, 2, 366-380), which is also true at 10.7 mM bleomycin. The reasons for the absence of loading associated change are the following. (1) Migration during AGE is determined only by the surface of a particle (Shaw, Electrophoresis, Academic Press, London 1969, p. 4-26). (2) Loading does not change the surface enough to alter migration during AGE. Both ethidium and bleomycin remain loaded when the gate is closed by lowering temperature to 42 °C or lower (Serwer and Wright, Biophysica 2022, 2, 366-380). For example, after AGE at 25 °C, phage T4 does not stain with the dyes typically used to stain nucleic acids, even though the T4 head contains a dsDNA genome. The reason is dye-impermeability of the head’s protein shell in a gate-closed state. Impermeability is confirmed by the restoring of phage T4 DNA staining that occurs after incubating the gel in conditions that cause expulsion of packaged T4 DNA (Serwer and Wright, Biophysica 2022, 2, 366-380).

[0061] In contrast, the following was observed after attempts to MB-load phage T4 and analysis by AGE followed by GelStar staining (FIG. 2a) and then by GelStar staining after expulsion of DNA from phage capsids (FIG. 2b). As MB concentration (indicated above a lane in mM) increased above 2.5 mM, a band of T4, seen in FIG. 2b, but not in FIG. 2a (because of phage impermeability to GelStar), was progressively lost. At 5 mM MB, some lost phage band intensity was at the origin, even before the phage DNA was expelled (FIG. 2a). At higher MB concentration, the origin-associated fluorescence was also lost.

[0062] The origin associated DNA is explained by phage aggregation. Because of the possibility of aggregation, electron microscopy, rather than solution chemistry, was the optimal way to learn more about what the MB was doing to phage T4.

[0063] Electron Microscopy (EM) by Negative Staining. EM confirmed the aggregation for T4 phage that had been incubated with 10,0 mM MB and then negatively stained. Scanning of the specimen revealed that the dominant feature was the presence of aggregates. The aggregates were not present before incubation with methylene blue. These observations confirmed what was seen by AGE.

[0064] The aggregates had primarily bacteriophages and phage heads. Some phages had atypical heads (e g., arrows #1 in FIG. 3). These heads appeared rounder and larger than the heads of a typical phage particle (arrow #2 in FIG. 3), some of which had expelled the DNA genome (arrow #3 in FIG. 3). For the particles indicated by arrows #1 in FIG. 3, the exclusion of stain by packaged DNA was less than it was for particles with an apparently intact head (arrow #2 in FIG. 3). The likely explanation is that these particles had a hyper-expanded outer shell, as confirmed by increase in the size of the images of these particles. Particle flattening increases the size, but is not known to alter the shape, of negatively stained phages (Serwer, P. Flattening and shrinkage of bacteriophage T7 after preparation for electron microscopy by negative staining. J, Ultrastrnct. Res. 1977, 58, 235-243). However, the increase of size for arrow #1 particles in FIG. 2a is great enough to favor assumption of a real increase in size. This assumption is tested below in

[0059] ,

[0065] Particles indicated by arrows #4 in FIG 3 appear to have started a hyper-expansion related transition, but not to have completed this transition with the result that a bulge was present. This was concluded from the decreased density of the DNA and the fact that the DNA was not expelled. Both total and incomplete hyper-expansion required thinning of a head’s outer protein shell. Thinning was suggested at the positions of arrows #1. However, the use of negative staining limited resolution to about 2 nm, which makes this observation at the limit of reliability. Both shell thinning and shell size increase were further pursued by cryo-electron microscopy (cryo-EM), which does not cause either particle flattening change in shape.

[0066] Cryo-Electron Microscopy (Cryo-EM). Cryo-EM confirmed the presence of (1) aggregates similar to those seen by negative staining, (2) separately observable particles at theedges of aggregates, and (3) apparent reduced DNA density and hyper-expansion for some, but not all, particles. The hyper-expanded particles (e.g., arrows #la and lb in FIG. 4) had a more isometric profile than the oblong phages, one of the latter indicated by arrow #2 in FIG. 4. The DNA-containing shell diameter for arrow #1 -particles was 8-20% larger than the length, 115-120 nm, of the long axis of phage heads, such as the one indicated by arrow #2 in FIG. 4. The short axis of the arrow #2-shells was 84-88 nm. Thus, the arrow #la, Ib-particles were hyperexpanded, assuming that shell breakage was not the source of an apparent hyper-expansion.

[0067] Three observations indicated that many, if not all, of the arrow #1 -particles did not have broken shells. First, no sign of breakage existed, and the outer shell was thinned to 1-2 nm (e.g., particle indicated by arrow la in FIG. 3) in relation to the ~ 3 nm thick shells of capsids that had lost their DNA (arrow #3 in FIG. 3). Shell thinning is a necessary consequence of hyperexpansion, by conservation of mass. Second, when DNA appeared to be no longer present, the capsid appeared unbroken (arrow #3 in FIG. 3). Third, some particles had a bulge where DNA was pushing against the capsid’s shell. However, the DNA appeared to still be restrained, as though a protein shell still existed to restrain the DNA (arrow #4 in FIG. 3). If the shell had broken, the DNA would have been expelled.

[0068] However, shells at a bulge were expected to be thinned and more difficult to observe. Indeed, the bulge of particle 4 in FIG. 3 appeared to have a barely visible shell of about 1 nm in thickness, at the tip of arrow #4. The opposing region of this shell (top of particle indicated by arrow #4) appeared to be thickened. The thickening can be explained by binding of MB, positively charged (Howland, J. Psychosoc. Nurs. Ment. Health Serv. 2016, 54, 21-26), to the shell, which, in turn, can be explained by repeated negative charge on shell as a consequence of a-sheet structure. This explanation includes thinning of the opposing shell region via MB association and subsequent dissociation.

[0069] Model and Implied Path to Improved Diagnosis. The above thoughts suggest adjusting the theoretical framework for developing therapy of both AD and other amyloid associated neurodegenerative diseases. Specifically, the past strategy of removing or inactivating amyloid proteins is (1) anti-logical in the context of the above, although the dominant current strategy, and (2) basically makes use of theory that had been constructed without use of data significantly beyond what Alzheimer (Alzheimer, Allg. Z. Psychiat. 1907, 64, 146-48) andFischer (Fischer, Psychiat. Neurol. 1907, 22, 361-372) published over 100 years ago (reviewed in Goedert, Brain 2009, 132, 1102-111). In more general terms, “It can scarcely be denied that the supreme goal of all theory is to make the irreducible basic elements as simple and as few as possible without having to surrender the adequate representation of a single datum of experience.” (Albert Einstein). The latter part of this quotation is key here.

[0070] The proposed strategy is based on an updated model (FIG. 1) that has been constructed from data identified by searching current publications. This model has several aspects that can be tested.

[0071] First, the structure of subunits of the MB-altered, hyper-expanded, DNA-containing T4 shells can be determined via 3-D reconstruction by cryo-electron microscopy. Unaggregated particles of this type are needed for this study. Unaggregated versions of the MB -transformed capsids exist and show evidence that these capsids are hyper-expanded. If the subunits have a version of a-sheet structure, it will probably be longer than 4-6 amino acids which would be longer than what has been seen by procedures other than molecular dynamics simulation.

[0072] Second, given the existence of appropriate human samples in brain banks, more extensive analysis can be performed of lipofuscin-associated and other neuronal fibers in AD- brain. The data cited herein constitute preliminary evidence in favor of the idea that the key diagnostic criterion and key therapeutic target is a-sheet, not P-sheet. The past sparsity of EM of AD brain is a limitation that should be overcome.

[0073] Third, if MB is found to be a probe for the toxic form of amyloid proteins, then work on MB-amyloid binding might converge with whole body fluorescence imaging to produce a live-patient assay for AD. MB exhibits near infrared fluorescence emission that is the basis of whole-body fluorescence imaging (Cwalinski et al., J. Clin. Med. 2020, 9, 3538). This was part of the original motivation for testing the loading of MB in phage T4. Finally, in our opinion, the key remaining challenge for determining the pathway of AD disease is to track the disease process in vivo at its sub-clinical stages. This challenge includes developing tracking procedures that are sensitive to the dynamics of protein conformation. If such an in vivo assay were developed, it could also be used to determine the amount of a-sheet vs. the use of various therapies, i.e., to determine the effectiveness of a therapy. If MB was the therapeutic used, success would be indicated by progressive decrease in the fluorescence signal from MB.

[0074] Path to Improved Therapy. If MB affinity for T4 is an a-sheet affinity, the following would explain at least part of the observed AD-therapeutic effect of MB. This effect occurs via blocking of the toxic activities of a-sheet structured amyloid protein. Evidence exists that these toxic activities include formation of membrane channels (Quist et al., PNAS USA 2005, 102, 10427-432). However, if such binding stabilizes a-sheet, the model of FIG. 1 indicates that this binding could aggravate the disease. Also, even if MB is always net therapeutic, MB might be far from the most potent molecule.

[0075] Thus, rapid initial screening for more effective, MB-related drugs is appropriate. If MB-binding to T4 is specific for a-sheet, the procedure in FIG. 2 can be adapted to screen for such compounds. Additional screening would be used if other therapeutic effects, such as increasing mitochondrial oxidation by acting as a terminal electron receptor (already observed for MB (Wen et al., J. Biol. Chem. 2011, 286, 16504-15)), are needed. Modifying MB and conducting the above screening would be based on the model of FIG. 1, which is projected to be more success-promoting than trying to eliminate amyloid-forming proteins.

[0076] An alternative is the use of either phage T4 itself, or MB-bound T4 as therapeutic. The therapeutic concept involved is binding of these particles to toxic, a-sheet structured amyloid protein, accompanied by inactivation of the toxic protein. To simplify discussion, when protein assembly occurs via self-assembling protein conformation, such as the assembly to form either -sheet or a-sheet, the term, conformation-derived assembly is used below.

[0077] The foreign character of an a-sheet based, conformation-derived assembly, therapeutic concept decreases when one considers the following. (1) A filamentous phage is found to have anti-amyloid protein assembly effects in murine models of Parkinson’s disease (Dimant et al., Biochem. Biophys. Res. Commun. 2009, 383, 491-96; Frenkel et al., A4S USA 2000, 97, 11455-59). (2) A protein at the tip of the filament, gp3, is the active component Frenkel et al., PNAS USA 2000, 97, 11455-59). This work was, however, done in the context immunotherapy, assuming misfolding of the amyloid protein, and without considering either the potential anti-viral activity or the potential a-sheet structure of amyloid-forming proteins (Krishnan et al., J. Mol. Biol. 2014, 426, 2500-2519) (reviewed in Messing, Gene 2016, 583, 85- 89). Therapeutic activity was apparently not part of these studies. If therapeutically active, (1) the activity-promoting characteristic of gp3 is likely to be conformation-derived assembly withamyloid-forming proteins. The discussion above suggests that the conformation involved would be a-sheet.

[0078] Relationship to Development of Phage T4 as Anti-Tumor Drug Delivery Vehicle. High murine blood persistence of phage T4, coupled with gated T4 loading of drug (Serwer and Wright, Biophysica 2022, 2, 366-80), have promoted interest in the use of phage T4 as an antitumor drug delivery vehicle (DDV). The concept is that the drug is (1) DDV-loaded with the gate open, (2) DDV-transported and inoculated with the gate closed and (3) released in a tumor via gate opening. Means to do (1) and (2) with phage T4 have been developed (Serwer and Wright, Biophysica 2022, 2, 366-80). However, tumor-specific drug releasing is still an open topic.B . Anti -Tumor therapies

[0079] Phage can be used as drug delivery vehicles (DDV) that unload selectively in tumors. The phage can be loaded with an anticancer compound or therapy. Also, the loaded phage can be modified or include permeabilization compounds or precursors thereof that are activated in the tumor microenvironment. Certain phage can be used as a DDV without modification. For example, tumors are typically bacteria-rich and the tumor-associated bacteria might cause drug release by causing T4 DNA injection. Tumor regression with bleomycin-loaded T4 has been observed under conditions where no steps were taken to release drug. In certain embodiments a therapeutic phage can be loaded with an anti-cancer compound, wherein the phage is activated in a tumor microenvironment releasing the anti-cancer compound from the therapeutic phage into the tumor microenvironment. The anti-cancer compound can be a chemotherapeutic or a radiotherapeutic or a sensitizer to anti-cancer therapy. In certain aspects the anti-cancer compound is a precursor compound that is activated in the tumor microenvironment.

[0080] Other aspects are directed to methods for treating cancer comprising administering (i) a therapeutic phage, (2) a loaded therapeutic phage, and / or (3) a loaded therapeutic phage with activatable permeabilization to a subject having cancer or directly to a tumor or vasculature providing blood to a tumor. The method can include administering a second therapy to the subject or the tumor. Administration can be co-admini strati on or administration within 1, 2, 3, 4, 5, 6 hours, days, or weeks of a first administration. In certain aspects the second therapy is a checkpoint inhibitor or T-cell therapy; or is a radiation therapy or light therapy.

[0081] Certain embodiments are directed to methods for treating cancer comprising administering a bacteriophage to a subject having cancer or directly to a tumor or vasculature providing blood to a tumor.

[0082] In certain embodiments, MB or other a-sheet interacting / binding molecules identified using the currently described methods can be used to promote intra-tumor gate-opening (i.e., induced permeability or uncapping of a phage vehicle) during the use of a bacteriophage (e.g., T4 phage) as a drug delivery vehicle. A drug-loaded phage (e.g., gated phage) can be administered that localizes at or in a tumor. The localized phage is then exposed to an agent that permeabilizes or uncaps the loaded phage (e.g., open-gated or damaged phage), delivering a localized dose of a therapeutic compound to the area in which the drug-loaded phage is localized. The following are the details for use of phage as gated or reversibly sealed drug delivery vehicle: (1) Drug is loaded by opening the gate (permeabilizing or uncapped phage). (2) Transportation and inoculation are performed after closing the gate. (3) Drug release in a tumor is achieved by re-opening the gate (e.g., exposing the drug loading phage to a permeabilizing or gate-opening compound such as those identified in the screening methods described). Means for (1) and (2) with phage have been developed.

[0083] Certain embodiments are directed methods of drug delivery by drug delivery vehicles that are (1) produced by biological systems in a form that is capable of being loaded and sealed (i.e., gated) and (2) are capable of genome derived optimization for specificity. In certain aspects, optimization can include mutation and / or selection of the drug delivery vehicle. The drug delivery vehicles can include rapidly propagating, biological entities such as bacterial viruses (bacteriophages or phages). In certain embodiments the drug delivery vehicles are double- stranded DNA phages. In certain aspects the drug delivery vehicles are capsids of a double- stranded DNA phage, which are highly stable (protease-resistant) with a low- permeability. In certain aspects the capsid comprises a phage T3 capsid shell comprising gplO proteins and a portal or gate comprising gp8 proteins. The portal of the capsid can control permeability. In certain aspects the drug delivery vehicle can be conditionally permeable to compounds with a molecular weight above at least 314 Daltons (Da). Extension of this range is feasible via genetic alteration of the gp8 protein or gplO protein. Under certain conditions, a capsid can be impermeable to, for example, Metrizamide, which has a molecular weight of 789 Da. Thus, after loading, a drug can be sealed or localized in the cavity as a highly stable, drug-loaded capsid. In certain aspects, capsids can be sealed after loading by modifying temperature, solution conditions, or adding a capping moiety.

[0084] In further aspects, the capsid shells can be selected for a particular specificity in binding or localization or property, e.g., in certain aspects a capsid can be selected after mutation of a nucleic acid encoding a capsid component. For example, when part of a phage particle, a capsid can be selected for increased persistence, preferential binding or localization to a target cell or target organ, or for a favorable packaging parameter such as permeability or efficiency relative to a particular drug. A selected capsid typically has the advantages of having needed permeability characteristics, high stability, and simplicity / low expense of purification in 10-100 milligram quantities or greater. The use of mutation / sel ection enables flexibility and adaptability to targeted cells that have evolved to vehicle-resistance.

[0085] In certain embodiments, MB or other a-sheet interacting / binding molecules identified using the currently described methods can be used to promote intra-tumor gate-opening (i.e., induced permeability or uncapping of a phage vehicle) during the use of a bacteriophage (e.g., T4 phage) as a drug delivery vehicle. A drug-loaded phage (e.g., gated phage) can be administered that localizes at or in a tumor. The localized phage is then exposed to an agent that permeabilizes or uncaps the loaded phage (e.g., open-gated or damaged phage), delivering a localized dose of a therapeutic compound to the area in which the drug-loaded phage is localized. The following are the details for use of phage as gated or reversibly sealed drug delivery vehicle: (1) Drug is loaded by opening the gate (permeabilizing or uncapped phage). (2) Transportation and inoculation are performed after closing the gate. (3) Drug release in a tumor is achieved by re-opening the gate (e.g., exposing the drug loading phage to a permeabilizing or gate-opening compound such as those identified in the screening methods described). Means for (1) and (2) with phage have been developed.

[0086] In certain aspects a subject can be administered an effective amount of a drug delivery vehicle described herein. The term “effective amount” means an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. For example, an “effective amount” of an anti-cancer agent can result in a decrease in cancer cell growth or, to some extent, the growth of some cancer or tumor cells. The termincludes an amount capable of invoking a growth inhibitory, cytostatic and / or cytotoxic effect, including apoptosis of cancer or tumor cells.

[0087] The compositions and methods described can be used in treating pathological conditions such as cancer. Treating or treatment in reference to cancer, means an amount capable of invoking one or more of the following effects: (1) inhibition, to some extent, of cancer or tumor growth, including slowing down growth or complete growth arrest; (2) reduction in the number of cancer or tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down, or complete stopping) of metastasis; (5) enhancement of anti-tumor immune response, which may, but is not required to, result in the regression or rejection of the tumor, or (6) relief, to some extent, of one or more symptoms associated with a cancer or tumor. The therapeutically effective amount may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of one or more anti-cancer agents to elicit a desired response in the individual. A “therapeutically effective amount” is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.

[0088] In one example a phage can be loaded MB that has been modified by attachment to a molecular group that blocks phage-permeabilization activity. The concept is to have the blocking group sensitive to removal specifically in the tumor environment. This environment includes relatively low pH, high lactate concentration, low pH induced proteolytic activity (Warburg Effect: reviewed in Jaworska et al., Pharmacol. Rep. 2023, 75, 876-90) and high glutamate concentration (reviewed in Jin et al., Exp. Mol. Med. 2023, 55, 706-15).

[0089] As previously mentioned in Messing, Gene 2016, 583, 85-89, work on biomedicine has the potential to introduce therapies that were not anticipated when the work started. The reason is the complexity of biology and the associated limitation of our knowledge of how biology works. Thus, as we test models for non-infectious diseases, an open mind is critical for developing therapies.II. Examples

[0090] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE 1APPLYING CHEMISTRY TO A MODEL AND IMPLIED THERAPY FOR ALZHEIMER’S DISEASE

[0091] A study is described to (i) test the effectiveness of substituting for MB several MB related and other compounds; (ii) test for dye-binding conditions such that the hyper-expanded capsid of phage T4 is susceptible to high resolution 3D reconstruction; and (iii) (a) correlate the data obtained with the structures of the various dyes tested, and (b) project the structures of compounds optimized for both high effectiveness in binding hyper-expanded phage T4 and low toxicity. These compounds will be candidates for a superior diagnostics I therapeutics for AD.

[0092] Preliminary Data

[0093] The problem of MB-induced aggregation of partially purified phage T4 was solved by further purifying phage T4 before binding of MB. Further purification was by rate zonal centrifugation in a sucrose gradient, as in

[0041] , The threshold for MB-induced aggregation was now high enough to use native (intact, infective phage) agarose gel electrophoresis (AGE) to detect MB-incubated phage T4. However, observation of hyper-expansion now required a post- MB-incubation step.

[0094] Specifically, phage T4 was incubated at 58 °C with MB and then subjected to AGE, followed by staining with Gel Star, a dye that has thus far been nucleic acid specific

[0042] , As previously found for T4 without MB-incubation

[0041] , MB-incubated phage T4 particles, were impermeable to Gel Star, and, thus, did not stain until the DNA genome was expelled by incubating the agarose gel in a low-ionic strength, chelating solution (0.002 M EDTA, pH 7.4; see

[0041] ).

[0095] As the concentration of MB rose from 0.0 to 0.4 pM, GelStar staining of phage T4 ( j> 4 in FIG. 5) slightly decreased; MB concentration [pM] is above a lane. As [MB] increased above 0.4 pM, staining progressively increased, eventually to over lOx the DNA-specific staining at 0.0 pM MB. Protein-specific Coomassie staining (same gel) was unchanged by increase in [MB] (not shown). Thus, the phage’s protein capsid was fluorescently GelStarstained, starting at about 0.8 pM MB. The capsid region involved was the phage without its tail (head; H4 band in FIG. 6).

[0096] GelStar does not stain DNA-free T4 capsids before MB incubation (not shown). Thus, MB-treated capsid subunits probably acquire an unusual structure that, like RNA and DNA, has a string of negative charges on one surface. This is a characteristic of a-sheet, but not of any other known structure that the capsid subunits of phage T4 might have. Presumably, the capsids are also hyper-expanded. Hyperexpansion is confirmed by electron microscopy, but only after stabilization described in the next paragraph.

[0097] This stabilization was achieved during the following test of the conformation of capsid subunits. After MB-incubation and before AGE and electron microscopy, phages were incubated with an antibody, called Al l [16, 43, 44], that is specific for a non-P-sheet, possibly toxic conformation of amyloid proteins, including A[3. The hypothesis is that Al l antibody specifically recognizes a- sheet, mostly independently of the amino acid sequence

[0016] ,

[0098] The result was that, as the concentration of the Al l antibody increased, the fluorescence of phage T4 (heads) further increased ( 4 and H4 bands in FIG. 6; the dilution of the Al 1 antibody is indicated above a lane). This increase could only occur in the capsid-induced (not the DNA induced) fluorescence of GelStar because (1) the antibody was specific for protein, and (2) the increase did not occur when T4 was not incubated with MB.

[0099] Furthermore, in a negatively stained specimen, the MB-, Al l antibody-incubated (without DNA expulsion) T4 was 15-25% hyper-expanded and had a less elongated capsid (FIG. 7). Thus, (1) a potential road to hyper-expanded T4 specimens for high resolution cryo-EM reconstruction has been determined, and (2) the evidence suggests that cr-sheet structured capsid subunits will be identified.

[0100] Preliminary data indicate that MB-related dyes have the same specificity and a binding constant / specificity spectrum exist with some dyes more specific than MB.

[0101] Studies have been designed to use the procedures of FIG. 5-7 to determine the activity of MB related compounds (mostly dyes) and other dyes in phage T4 hyper-expansion. The MB-related dyes are all commercially available.

[0102] Other studies have been designed to test for dye / antibody-binding conditions such that the capsid of phage T4 is more symmetric and, therefore, analyzable by high resolution symmetric 3D reconstruction. This sets the stage for 3D cryo-EM reconstruction of what could be the first > 6 amino acid long, naturally occurring a -sheet structured protein region.

[0103] Additional studies are designed to (1) correlate the data with the structures of the various dyes tested, and (2) project the structure of compounds optimized for low toxicity and high effectiveness in hyper-expanding the capsid of phage T4. These compounds are candidates for superior diagnostics / therapeutics for AD.

[0104] Proj ect Approach

[0105] Phage T4 is purified in large amounts by differential centrifugal pelleting, followed by rate zonal ultracentrifugation in a sucrose gradient (details

[0041] ). This will be used to repeat the experiments of FIG. 5 and 6 using the following compounds substituted for MB, which is a thionine dye: toluidine blue, acridine orange, new methylene blue and leucomethylene blue, neutral red, Nile blue, cresyl violet and 1,9-dimethyl methylene blue, all MB-related [44-46], This work can be extended to compounds unrelated to MB. For each compound, we will determine the following [MB] values: (1) initiating fluorescence increase and (2) generating half- maximal fluorescence increase.

[0106] Preliminary data (FIG. 9) indicate that acridine orange (1) causes the T4 hyperexpansion at concentration over 5x lower than MB and (2) results in T4 aggregation and band loss at the highest concentrations. These determinations can be determined for the above dyes, with at least three repetitions for each dye, to increase accuracy.

[0107] It is anticipated that the above data generate a spectrum of hyper-expansion promoting effectiveness. This spectrum will be used to improve diagnosis and therapy of AD and possibly other amyloid associated neurodegenerative diseases.

[0108] Electron microscopy of phage T4 that has been hyper-expanded by incubation with each of the dyes that test positive can be performed. This can be done with specimen preparation by negative staining

[0015] of samples dye-modified in two ways. First, we will perform the incubation used for FIG. 7 which includes the incubation with Al l antibody. Second, we willomit the incubation with Al 1 antibody but will expel DNA by incubation in the presence of EDTA, as done for FIG. 5. The DNA can be digested before electron microscopy.

[0109] Electron microscopy can be performed at dye concentrations known from work to generate hyper-expansion. We will begin with the dye type / concentration that works at the lowest dye concentration. We will continue the negative stain-electron microscopy of the above specimens to identify conditions that reproducibly generate hyper-expansion with an outer T4 capsid shell that appears symmetric for over 80% of the T4 phages. If the symmetry is confirmed by cryo-EM below, this will produce enough hyper-expanded phages for 3D reconstruction.

[0110] To further test for symmetry of T4 in the most promising preparations, we will use cryo-EM, as previously done in

[0015] , We will perform a low-resolution (~ 2.5 nm) reconstruction. Conditions for symmetrization and reconstruction of hyper-expanded phage T4 capsids can be identified.

[0111] For several dyes, data obtained will be used to compare dye structure with dye effectiveness. Dye effectiveness will be assumed to increase as the critical [MB] values decrease.References[1] De Ture and Dickson, Mol. Neurodegener. 2019, 14, 32.[2] Dubois et al., Lancet Neurol. 2021, 20, 484-496.[3] 2024 Alzheimer's disease facts and figures. Alzheimer s Dement. 2024, 20, 3708-3821.[4] Kumar et al., Alzheimer Disease. 2024. In: StatPearls, Treasure Island (FL): StatPearls Publishing 2024 Jan.[5] Hardy and Higgins, Science 1992, 256, 184-185.[6] Zhang et al., Sig. Transduct. Target Ther. 2023, 8, 248.[7] Ma et al., Molecules 2022, 27, 1210.[8] Thawabteh et al., Molecules 2024, 29, 5131.[9] Herrup, Nat. Neurosci. 2015, 18, 794-799.

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Claims

CLAIMS1. A method for identifying compounds comprising:(i) incubating a phage with a test compound forming a test mixture; and(ii) subjecting the test mixture to size fraction identifying aggregated phage / test compound complexes, the aggregated phage being indicative of a conformational change in the phage.

2. The method of claim 1, wherein size fractionation is by agarose gel electrophoresis.

3. The method of claim 2, wherein the agarose gel electrophoresis is native gel electrophoresis.

4. The method of claim 2, wherein the agarose gel is a 0.5% agarose gel.

5. The method of claim 2, wherein the agarose gel electrophoresis is carried out at a voltage of 50 volts for an hour.

6. The method of claim 1, wherein the phage is a bacteriophage T4.

7. The method of claim 1, wherein the test compound and the phage are incubated for 30 minutes to an hour.

8. The method of claim 7, wherein the test compound and the phage are incubated at a temperature of 50 °C.

9. The method of claim 1, further comprising staining fractionated phage with a DNA stain.

10. A compound identified using the methods of any one of claim 1 to claim 8.

11. A method of treating a neurodegenerative disease by administering a compound of claim 10.

12. The method of claim 11, wherein the neurodegenerative disease is Alzheimer’s disease.

13. A therapeutic phage loaded with an anti -cancer compound and a precursor phage- permeabilization compound, wherein the precursor phage permeabilization compound is configured to be activated in a tumor microenvironment releasing the anti -cancer compound from the therapeutic phage into the tumor microenvironment.

14. The therapeutic phage of claim 13, wherein the anti-cancer compound is a chemotherapeutic or a radiotherapeutic or a sensitizer to anti -cancer therapy.

15. The therapeutic phage of claim 13, wherein the anti-cancer compound is a precursor compound that is activated in the tumor microenvironment.

16. The therapeutic phage of claim 13, wherein the precursor phage permeabilization compound is precursor methylene blue compound that is activated in the tumormicroenvironment releasing methylene blue which in turn permeabilizes the therapeutic phage releasing the anti -cancer compound to the tumor microenvironment.

17. A method for treating cancer comprising administering a therapeutic phage of claim 13 to a subject having cancer or directly to a tumor or vasculature providing blood to a tumor.

18. The method of claim 17, further comprising administering a second therapy to the subject or the tumor.

19. The method of claim 18, wherein the second therapy is a checkpoint inhibitor or T-cell therapy.

20. The method of claim 18, wherein the second therapy is an electromagnetic wave therapy.

21. A therapeutic phage loaded with an anti -cancer compound, wherein the phage is activated in a tumor microenvironment releasing the anti -cancer compound from the therapeutic phage into the tumor microenvironment.

22. The therapeutic phage of claim 21, wherein the anti-cancer compound is a chemotherapeutic or a radiotherapeutic or a sensitizer to anti -cancer therapy.

23. The therapeutic phage of claim 21, wherein the anti-cancer compound is a precursor compound that is activated in the tumor microenvironment.

24. A method for treating cancer comprising administering a therapeutic phage of claim 21 to a subject having cancer or directly to a tumor or vasculature providing blood to a tumor.

25. The method of claim 24, further comprising administering a second therapy to the subject or the tumor.

26. The method of claim 25, wherein the second therapy is a checkpoint inhibitor or T-cell therapy.

27. The method of claim 25, wherein the second therapy is a radiation therapy or light therapy.

28. A method for treating cancer comprising administering a bacteriophage to a subject having cancer or directly to a tumor or vasculature providing blood to a tumor.