Method of treatment of degenerative diseases caused by membrane channel-forming peptides fragments

Abstract

The present invention provides the method to prevent or slow down the progression of degenerative diseases caused by membrane channel-forming peptides. For many of these diseases, there is no known treatment based on the etiology and pathogenesis of the corresponding disease. Until recently, there was no integrative theory explaining multiple symptoms and observations associated with such diseases. In response to this challenge, we developed the amyloid degradation toxicity theory of Alzheimer's disease (AD). Within this concept, the etiology of the disease is the formation of beta-amyloid fragments which form membrane channels. We claim that the stopping the production of toxic fragments by inhibiting biochemical pathways producing channel-forming fragments (for example, by protease inhibitors) will prevent or slow down the progression AD. Also, we claim that the same molecular mechanism is involved in multiple neurodegenerative diseases and diabetes type II, so the invented method can be used to treat them.

Claims

1. The method of treatment of degenerative diseases using an inhibition of enzymes degrading peptides or proteins into fragments which are able to form membrane channels in cellular membranes.

2. The method of claim 1, wherein said inhibition is achieved by an introduction of a molecule with inhibitory activity (such as giving the patient a medicine).

3. The method of claim 1, wherein said inhibition is achieved by a modification of synthesis of such enzymes.

4. The method of claim 1, wherein said inhibition is made by a macromolecule or a mix of macromolecules which can bind such enzymes (such as antibodies or affibodies).

5. The method of claim 1, wherein said inhibition of said enzymes is achieved by increased synthesis of endogenous inhibitor or inhibitors.

6. The method of claim 1, wherein the inhibition of such enzymes is achieved by the introduction of a synthesis of inhibitor or inhibitors in the organism (such as viral delivery of nucleic acid sequence for the peptide inhibitor).

7. The method of claim 1, wherein the disease is Alzheimer disease or other degenerative disease caused by peptides from the family of beta-amyloid (full-length or its fragments, native or including mutations, occurring naturally, or induced artificially).

8. The method of claim 1, wherein the disease is Parkinson's disease or other degenerative disease, caused by peptides from the family of alpha-synuclein (full-length or its fragments, native or including mutations, occurring naturally, or induced artificially).

9. The method of claim 1, wherein the disease is ALS (amyotrophic lateral sclerosis) or other degenerative disease, caused by peptides from the family of superoxide dismutase (full-length or its fragments, native or including mutations, occurring naturally, or induced artificially).

10. The method of claim 1, wherein the disease is ALS (amyotrophic lateral sclerosis) or other degenerative disease, caused by peptides from the family of TDP-43 (full-length or its fragments, native or including mutations, occurring naturally, or induced artificially).

11. The method of claim 1, wherein the disease is Alzheimer's disease or other degenerative disease, caused by peptides from the family of tau protein (full-length or its fragments, native or including mutations, occurring naturally or induced artificially, phosphorylated or not).

12. The method of claim 1, wherein the disease is diabetes mellitus type II or other degenerative disease, caused by peptides from the family of amylin (full-length or its fragments, native or including mutations, occurring naturally, or induced artificially).

13. The method of claim 1, wherein the disease is Huntington's disease or other degenerative disease, caused by peptides from the family of Huntingtin's protein (full-length or its fragments, native or including mutations, occurring naturally, or induced artificially).

14. The method of claim 1, wherein the disease is prion disease or other disease, caused by peptides from the family of prions (full-length or its fragments, native or including mutations, occurring naturally or induced artificially).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0048] FIG. 1. Correlation of diagnosis of Alzheimer's disease with values of two major biomarkers of AD.

[0049] The data on concentrations of soluble Aβ42 in cerebrospinal fluid (CSF-Aβ42) and the density of amyloid depositions in the brain measured by PET using appropriate .sup.18F-conjugated label were obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database (adni.loni.usc.edu). The investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or making any conclusions relevant to this invention. A complete listing of ADNI investigators can be found at: http://adni.loni.usc.edu/wp-content/uploads/how to_apply/ADNI_Acknowledgement_List.pdf [0050] A. Scatter plot of CSF-Aβ42 (in ng/ml) vs beta-amyloid load (in centiloids) in patients with Alzheimer's disease (AD) and subject with normal cognition (NC). Most patients with AD have significant amyloid accumulation (located to the right of the line at 50 CL), while data points for most patients with normal cognition are located to the left of the line at 20 CL (no noticeable accumulation of amyloid depositions in the brain). However, some AD patients have low levels of amyloid depositions, while some subjects with normal cognition have significant accumulation of amyloid depositions. [0051] B. Due a strong negative correlation with CSF-Aβ42, depositions of amyloid in the brain are frequently considered interchangeable methods of AD diagnostics. However, CSF-Aβ42 provide independent diagnostic information. Within a subgroup with high or low amyloid load, the level of soluble beta-amyloid in the CSF correlates with the probability of AD diagnosis: the subjects with high level of soluble beta-amyloid are almost free of disease, while subjects with low levels are prone to the development of AD.

[0052] Such distribution of two biomarkers is defined by dramatically increased cellular uptake of soluble beta-amyloid (the analysis is shown at the FIGS. 2&3), which is the molecular mechanism of Alzheimer's disease according to recently proposed by us amyloid degradation toxicity hypothesis.

[0053] FIG. 2. A schematic of the single compartment model of beta-amyloid turnover used to describe the mathematical relationship between CSF-Aβ42 and amyloid load in the brain.

[0054] To analyze the data shown at the FIG. 1A, we used mathematical model describing the schematic shown at this Figure. The concentration of soluble amyloid in the interstitial fluid (ISF, the liquid between cells and neurons in the brain), which we denote as [ISF], is defined by several processes: 1) synthesis by cells, 2) filtration of the protein into the CSF, 3) aggregation into non-soluble plaques, and 4) uptake by cells. The model is based on several assumptions:

[0055] Synthesis rate (SYN) is independent of both interstitial Aβ42 and the density of plaques.

[0056] The rate of removal of the protein through the CSF is a product of the CSF removal rate (FLOW.sub.CSF) and CSF-Aβ42 ([CSF]): FLOW.sub.CSF.Math.[CSF].

[0057] The concentrations of the soluble beta-amyloid in the ISF and the CSF have a similar order of magnitude and are correlated. The model assumes a linear relationship between the concentrations of soluble Aβ42 in the ISF and the CSF with a coefficient of transfer K.sub.T: [CSF]=K.sub.T.Math.[ISF].

[0058] Existing plaques serve as seeds for aggregation of soluble Aβ42 in the ISF. The rate of loss of soluble Aβ42 in the ISF due to aggregation is the product Aβ42 concentration in the ISF, the concentration of plaques ([PET], calculated from the intensity of the PET signal), and the coefficient of aggregation K.sub.a: K.sub.a.Math.[PET].Math.[ISF].

[0059] The rate of cellular uptake of soluble Aβ42 is proportional to the interstitial concentration [ISF] with a coefficient of uptake K.sub.u: K.sub.u.Math.[ISF].

[0060] FIG. 3. Beta-amyloid turnover parameters in subjects with normal cognition (NC), patients with Alzheimer's disease (AD), patients with late-onset mild cognitive impairment (LMCI) and early-onset mild cognitive impairment (EMCI).

[0061] The parameters were inferred from two major AD biomarkers (CSF-Aβ42 and beta-amyloid density) in research subjects from the ADNI database. The data on concentrations of soluble Aβ42 in cerebrospinal fluid (CSF-Aβ42) and the density of amyloid depositions in the brain measured by PET using appropriate 18F-conjugated label were obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database (adni.loni.usc.edu). The investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or making any conclusions relevant to this invention. A complete listing of ADNI investigators can be found at: http://adni.loni.usc.edu/wp-content/uploads/how to_apply/ADNI_Acknowledgement_List.pdf [0062] A. Scatter plot of CSF-Aβ42 vs beta-amyloid load for subjects with NC and AD patients showing the lines representing best fits by Equation (FIG. 2) for each group. [0063] B. The 95% confidence regions of the parameters characterizing beta-amyloid turnover in the three groups (NC, AD, LMCI). All three groups are different if both KF and SYN are considered. [0064] C. A comparison of beta-amyloid turnover parameters in subjects with normal cognition (NC), patients with either early-onset and late-onset mild cognitive impairment (EMCI and LMCI), and patients with Alzheimer's disease (AD). The inferred values of the beta-amyloid synthesis rate (SYN) and the removal rate (KF) for all studied groups. * Only the values of KF for the NC and AD groups are statistically different (z-test, p<0.05). [0065] D. The 95% confidence regions of the parameters characterizing beta-amyloid turnover in the NC, EMCI, and LMCI groups. The confidence regions for the NC and LMCI groups do not overlap, while the confidence regions for the NC and EMCI groups do. [0066] E. The difference in the aggregation-independent amyloid removal rate (parameter KF) between NC subjects and AD patients translates into a much greater relative difference in cellular amyloid uptake rate. CSF flow in AD patients is either the same or lower than in NC subjects while the removal with the CSF is a prevalent mechanism defining interstitial concentration of soluble amyloid. The dark-gray and light-gray parts of the bars represent the cellular amyloid uptake rate and the rate of removal through the CSF (the components of the rate of amyloid removal), respectively. If the ratio of the two components is 50/50 in the NC group, the cellular uptake rate is 2.5 times greater in the AD group than in the NC group. If the ratio is 75/25, the difference is 4-fold.

[0067] FIG. 4. The schematic of polymerization of amyloidogenic proteins.

[0068] Amyloid peptides are initially soluble without secondary or tertiary structure. With time, they are stabilized by intra- and intermolecular hydrogen bonds (1 and 2, correspondingly) forming beta-pleated sheets (one of major secondary structures in proteins). Elongation of these supramolecular structures results in formation of protofibrils which have 3-sheet core with polypeptide tails looking to the sides of the protofibril (3). Protofibrils stick to each other through interaction between side polypeptide chains (4) and may involve other proteins (5), which may or may not be containing carbohydrate and lipid components (glyco- and lipoproteins). At oligomeric stage, beta-sheet can form barrel-like structures (6), which can incorporate into lipid membranes and serve as ion channels.

[0069] FIG. 5. Aβ.sub.25-35, unlike Aβ.sub.22-35 and Aβ.sub.1-42, makes negatively charged liposomes permeable to calcium.

[0070] The figure is a composite of data which was presented and analyzed in detail in (Zaretsky and Zaretskaia 2020, Zaretsky and Zaretskaia 2020). Liposomes (400 nm) were made of either phosphatidylserine (A-E, negatively charged lipid) or phosphatidylcholine (F-H, neutral lipid) with added DiD and were extruded in the Ca-free buffer containing 1 mM Fluo-3. After the addition of calcium, aliquots of peptide were added. Ionomycin was used as a positive control. Fluorescence of liposomes was analyzed using a flow cytometer. Each dot at the graph represent a single liposome registered in the flow. The intensity of DiD fluorescence characterizes the amount of membrane material in the liposome, while the fluorescence of Fluo-3 depends on the intraliposomal presence of calcium. Permeabilized liposomes (Fluo-3 is saturated with calcium entering through damaged membrane) appear at the graph above the rest of liposomes. The liposomes inside rectangle (area R1) are permeabilized, and the number of liposomes which were counted to be within the area is shown. Total number of analyzed events represented at each graph is approximately 100,000. [0071] A. Control phosphatidylserine liposomes (negatively charged) are not permeable to calcium; therefore, the number of permeable liposomes is low. [0072] B. The addition of ionomycin permeabilizes liposomes, so thousands of liposomes become permeable to calcium. [0073] C. The addition of Aβ.sub.25-35 which carries positive charge creates multiple liposomes permeable to calcium. This number depends on the concentration of added peptide (see the data in the (Zaretsky and Zaretskaia 2020). [0074] D. Longer peptide Aβ.sub.22-35 which carries overall negative charge does not permeabilize liposomes. [0075] E. Full-length amyloid peptide Aβ.sub.1-42 also does not permeabilize liposomal membranes. [0076] F. Control phosphatidylcholine liposomes (non-charged) are not permeable to calcium; similar to observations in negatively charged liposomes (Panel A), the number of permeable liposomes is low. [0077] G. As in case of negatively charged liposomes (Panel B), ionomycin permeabilizes thousands of liposomes. [0078] H. In contrast to negatively charged liposomes, neutral liposomes are not permeabilized by Aβ.sub.25-35 (the number of permeabilized liposomes is not more than in control, Panel F).

[0079] FIG. 6. Distribution of amyloid channel conductances and of corresponding theoretically predicted molecular weight cut-offs. The Figures are adapted from (Zaretsky, Zaretskaia et al. 2021)

[0080] Amyloid channels were formed by 20 μM Aβ.sub.25-35 in planar lipid bilayers at pH 5.0 (to mimic the conditions inside lysosomes). The conductances of membrane channels formed by beta-amyloid have a wide range of values, from below 100 nS to above 1 nS. Most of channels (around 90% at pH 7.4) are relatively small (below 200 pS), but at acidic pH more than 30% of channels had a conductance exceeding 200 nS. Channels with giant conductance up to 1 nS are rare. [0081] A. To look at the channel conductance in the context of molecular weight cut-off, we calculated molecular weights of compounds of spherical shape which the density of globular proteins which can come through circular pores in lipid membrane filled with saline using formulas used to calculate conductance of pores (Bode, Baker et al. 2017) and the size of proteins (Erickson 2009). In these ideal conditions, passage of 50 kDa protein requires only 2.4 nm pore with conductance of 2.2 nS. [0082] B. The percentage of channels with a conductance which in model conditions is sufficient to pass globular proteins of various molecular mass based on the distribution shown at the Panel A. Log-log scale was used to estimate the shape of the dependence as a power function. Linear fitting of distributions was performed for conductances which correspond to MWCO exceeding 500 Da. Insert: Extrapolation of probability of giant channels based on fitting with a power function for MW up to 50 kDa. For comparison, lysosomal cathepsins (proteases) have MW of 20-30 kDa, so there is biologically significant probability that they can pass lysosomal membranes if the membranes are permeabilized by beta-amyloid fragments.

[0083] FIG. 7. The pathway of beta-amyloid metabolism in healthy cells and potential membrane targets containing negative charge required for amyloid channel formation.

[0084] Negative charge exists in several subtypes of cellular membranes: in lysosomes, inner mitochondrial membrane, and inner leaflet of plasma membrane. There is no known mechanism for channel-forming amyloid fragments to access inner leaflet of plasma membrane and mitochondria (both are considered targets based on known pathophysiology of Alzheimer's disease), unless the fragments leak from lysosomes. Giant amyloid channels can be a mechanism for such leakage. Even if channel-forming fragments can leak to the cytoplasm, the delivery to the inner mitochondrial membrane still requires yet unknown mechanism. However, mitochondrial disfunction can be explained without such transport by assuming lysosomal disfunction as shown at the FIG. 8.

[0085] FIG. 8. Amyloid degradation toxicity hypothesis (adapted from (Zaretsky and Zaretskaia 2021)).

[0086] The endocytic vesicle containing the amyloid peptide is merged with a lysosome. Endopeptidases produce various short fragments, which are mostly degraded by acidic exopeptidases. Short fragments can form non-selective membrane channels, dissipating the pH gradient. The neutralization inhibits acidic proteases with exopeptidases being inhibited more than endopeptidases. Lysosomal failure leads to cell death through several pathways. 1. Channel-forming fragments leak to the cytoplasm through the permeabilized membrane and target other membranes, including the plasma membrane. 2. Lysosomal enzymes leak to the cytoplasm and cause necrosis or activate apoptosis. 3. Dysfunctional lysosomes accumulate, and the recycling of organelles fails. Damaged mitochondria are not recycled and produce reactive oxygen species, damaging other organelles.

[0087] FIG. 9. The sequence of events resulting in neuronal death and the progression of Alzheimer's disease as suggested by the amyloid degradation toxicity hypothesis.

[0088] Beta-amyloid which is internalized by cells through endocytosis meets lysosomal degradation enzymes after endosomes merge with lysosomes. Degradation of any protein occurs through either cutting it into large fragments by endopeptidases or by cutting mono-, di- or tri-peptides from the ends of polypeptide chain by exopeptidases (which could be appropriately named di- or tri-peptidases). It is logical that after the fragments are formed, they are further degraded by exopeptidases.

[0089] Only some beta-amyloid fragments are channel-forming (and toxic), their appearance depends on the activity of endopeptidases (circle with a digit 1). It is likely that most of formed fragments are non-toxic products. Also, toxic fragments are mostly quickly degraded into non-toxic products (by other exopeptidases and endopeptidases, circle with a digit 2). All that process occurs inside lysosomes and is promoted by intralysosomal acidic conditions (pH<5). Only minor number of toxic fragments aggregate into channel-forming units and incorporate into lysosomal membranes. If this happens, it results in lysosomal disfunction which in turn produce multiple sequela associated with Alzheimer's disease, such as mitochondrial disfunction, increased production of reactive oxygen species, low brain metabolism, accumulation of large vacuoles (former endosomes) carrying non-digested cargo. Some giant channels allow for the leakage of lysosomal proteases into the cytoplasm. Leaked proteases either digest intracellular proteins (necrosis) or activate apoptosis-related cytoplasmic proteases, which initiate “programmed” cell death. In healthy individuals, the probability of such leakage is extremely low, so induced neuronal death is not reaching critical level, which is needed for the development of significant neuronal loss as is observed in patients with Alzheimer's disease.

[0090] FIG. 10. Part of amino acid sequence of beta-amyloid including channel-forming fragment Aβ.sub.25-35 and non-channel-forming fragment Aβ.sub.22-35.

[0091] Channel-forming fragment has mostly non-polar amino acids and only one charged amino acid lysin (K), so the overall charge of the peptide is positive. In contrast, longer peptide Aβ.sub.22-35 has two additional charged amino acids—both negatively charged. All genetic mutations which affect this region and are known cause familial types of Alzheimer's disease include the removal of at least of one negative charge. Removal of one negatively charged amino acid increases the probability of the formation of fragments which carry prerequisites of channel-formation: made mostly of non-polar amino acids, able to form beta-sheet structure, and carrying one positively charged amino acid, promoting the interaction with negatively charged membranes.

[0092] FIG. 11. Endogenous proteases easily produce channel-forming peptides in mutation-carrying patients.

[0093] C-terminal part of full-length peptide Aβ.sub.1-42 is shown. The peptide is synthesized by digestion of Amyloid Precursor Protein (APP) by β- and γ-secretase. However, there is an alternative processing pathway which includes α-secretase. [0094] A. The sites where APP is cut by α- and γ-secretases are shown. In case of Uppsala deletion, six amino acids appear to be absent (positions 19-24 in the Aβ.sub.1-42 peptide, or 690-695 in the APP). The location of the deletion is shown. [0095] B. The fragment which is formed after complete digestion by α- and γ-secretases is compared with channel-forming fragment Aβ.sub.22-35. Both are made mostly of non-polar amino acids, able to form beta-sheet structure, and carry one positively charged amino acid, promoting the interaction with negatively charged membranes.

[0096] FIG. 12. Classification of Alzheimer's disease based on biochemical process which leads to the beta-amyloid-induced activation of proteolytic damage of neurons and the development and the progression of Alzheimer's disease.

[0097] As shown at the FIG. 9, terminal cellular phenomenon defining Alzheimer's disease is neuronal death through apoptosis or necrosis. Four steps leading to the appearance of active proteases in the cytoplasm are the basis for this classification.

[0098] Alzheimer's disease Type I. Increased uptake of Aβ due to the intense cellular uptake is the first option which is the etiology of. Due to dramatically increased availability of beta-amyloid to degradation enzymes, increased production of channel-forming fragments leads to the faster neuronal death. The comparison of distributions of major biomarkers data in patients with cognitive impairments (including early- and late-onset AD) and subjects with NC demonstrates that typical late-onset AD can be considered type I (see FIG. 3). In contrast, patients with early-onset AD do not have increased uptake, so they can belong to one of next three types within this classification.

[0099] Alzheimer's disease Type II. In contrast to AD type I, the uptake is not increased. However, lysosomal proteolytic activity is disbalanced in favor of endoproteases due to activation of rate of production of endoproteolytic products. Such disbalance results in increased production of channel-forming fragments and leads to faster neuronal death. As was discussed at the FIG. 10, familial types of AD which are associated with mutations in the gene coding amyloid precursor protein, have amino acid substitutions in very specific areas of this protein—removing negatively charged amino acids just outside beta-sheet-forming region increase the production of peptides which 1) can form beta-sheet, 2) carries positive charge, 3) contain mostly hydrophobic amino acids (see FIG. 11)—all features required for the formation of amyloid channels. Increased of production of channel-forming fragments is the feature of AD Type II.

[0100] Alzheimer's Type DD (degradation deficiency). The concentration of channel-forming fragments depends on the rate of their formation, but also is influenced by the rate of their farther degradation. If the activity of exoproteases, which are responsible for the digestion of short fragments is decreased, the concentration of toxic proteolytic intermediates increases, and so does the rate of neuronal death. Essentially, this type is about disbalance of proteolytic activity in favor of endoproteases, but unlike AD Type II, due to the decrease of exoproteolytic activity. The example of this phenomenon is shown at the FIG. 15—in low concentrations, anti-chymotrypsin mostly inhibits endoproteolytic activity and ameliorates beta-amyloid toxicity. However, in higher concentration, same inhibitor becomes active towards exoproteases, and its protective action against beta-amyloid toxicity disappears. Similarly, unlike amyloid-protecting aprotinin, which inhibits mostly serine proteases (such as trypsin) which exhibit significant endoproteolytic activity (see FIG. 14), leupeptin, which is more active against cysteine proteases (such as cathepsin B) which exhibit mostly exoproteolytic activity, promotes beta-amyloid toxicity (see FIG. 14).

[0101] Alzheimer's Type DS (damage sensitivity). It is obvious that channels are formed with some frequency. However, most likely, not every channel formation event results in host cell death. Firstly, formed channel could be too small for leaking proteases. Without leaking digestive enzymes, disfunctional lysosome can affect normal function of the neuron, but does not necessarily kill the host cell. Secondly, damaged lysosome can be repaired or recycled. Thirdly, leaking lysosomal proteases are inhibited by cytoplasmic inhibitors, such as cystatins, and therefore, the cell could be protected from fatal outcome. According to common sense, same number of formed channels can have no tissue-level consequences in subjects with high resistance, while result in noticeable neuronal death in subjects with low resistance to this outcome. While we cannot offer examples of this mechanism, it should be considered as potentially possible.

[0102] It is important to acknowledge, that the progression of AD in a specific patient can be the result of a combination of various mechanisms.

[0103] FIG. 13. Alzheimer's disease of various etiologies can be treated by one class of agents.

[0104] A: The summary of the mechanisms mediating different types of AD. Please, note that insufficiency of exoproteolytic activity will allow more beta-amyloid to be digested into toxic fragments (Type DD connects to two arrows at the graph).

[0105] B: All four described types of AD can be prevented or treated by the inhibition of exoproteases responsible for the formation of toxic channel-forming beta-amyloid fragments, because all degradation of endocytosed beta-amyloid results in the formation of non-toxic products without toxic intermediaries. Due to a significant overlap in activity of different enzymes (the redundancy is needed for effective digestion of various nutrients), we expect that it is possible to achieve medical progress without jeopardizing normal catabolism in the brain, as well as at the system level.

[0106] FIG. 14. Selective inhibition of protease prevents apoptosis induced by beta-amyloid (adapted from (Frautschy, Horn et al. 1998)).

[0107] Rats were chronically infused intracerebroventricularly with beta-amyloid. Animals were treated either with aprotinin (A) or leupeptin (B), inhibitors of proteases. After the sacrifice, the brains were sectioned and stained for TUNEL, the marker of apoptosis. Aprotinin is more specific towards serine proteases, which are known to have significant endo-proteolytic activity, while leupeptine inhibits wider range of proteases, including cathepsins (such as cathepsin B) with characteristic exopeptidase activity.

[0108] Leupeptin tends to increase apoptosis by itself, while aprotinin tends to decrease even spontaneously occurring apoptosis. Chronic infusion of Aβ increases the expression of apoptotic marker TUNEL in the brain. Non-selective inhibition of proteolytic activity with simultaneous i.c.v. infusion of leupeptin promoted apoptosis and an accumulation of extracellular and intracellular Aβ immunoreactivity. In contrast, more selective serine protease inhibitor aprotinin prevented Aβ-induced apoptosis and did not exacerbate intracellular accumulation of amyloid. Protective effect of aprotinin in the absence of promotion of amyloid accumulation demonstrates the possibility to provide the protection without promoting accumulation of amyloid deposits (even though we believe that amyloid deposits themselves are harmless, this point of view is not universally shared).

[0109] In the manuscript, which contains this data, the effects are considered in the context of how each inhibitor promotes accumulation of amyloid deposits (leupeptin increases deposits, so promotes the toxicity, while aprotinin does not promote depositions, so does not cause toxicity). There was no connection to toxicity mechanisms related to the formation of channel-forming fragments.

[0110] FIG. 15. α1-Anti-chymotrypsin (ACT) dose-dependently inhibit cellular toxicity of beta-amyloid (adapted from (Schubert 1997)).

[0111] Two cell lines (rat primary cortical cultures and 15 cells derived from pancreatic islets of Langerhans) were exposed to various concentrations of beta-amyloid in the presence of various concentrations of α1-anti-chymotrypsin, natural serine peptidase inhibitor (serpin). Cell survival was measured using MTT assay.

[0112] Beta-amyloid induced drop in cell viability, which was prevented by α1-anti-chymotrypsin. In some conditions, the protection could reach 100%. The optimal dose for inhibition of amyloid toxicity was similar in tests with different concentrations of Aβ but depended on the cell strain: in 15 cells it was 10.sup.−7M, in primary cortical cultures—10.sup.−8M. However, further increase of ACT concentration leads to disappearance of protection (as we discuss in this text, it is due to non-specific inhibition of wider range of proteases). In this manuscript, the author directly challenges the question that anti-proteolytic action is important for the protection, but can not find mechanistic interpretation, and therefore, is not able to reach conclusions about pharmaceutical prospects of new class of pharmacological agents discovered by us.

[0113] FIG. 16. Death of pancreatic beta-cells in diabetes Type II can be mediated by a mechanism mediating amyloid degradation toxicity.

[0114] Diabetes Type II is characterized by progressive death of beta-cells of islets of Langerhans. These cells are best known due their production of insulin—blood sugar controlling hormone. However, same cells also produce amylin or islet amyloid polypeptide (IAPP). Amylin is amyloidogenic protein and is found in pancreatic tissue of patients with diabetes Type II. It progressively replaces beta-cells in the tissues. Importantly, it is known that amylin can form membrane channels, but unlike Alzheimer's disease, this phenomenon did not catch any significant interest as a druggable process, yet.

[0115] The similarity between potentially channel-forming fragments of beta-amyloid and amylin is shown as a table. Each amino acid is labeled according to the physical properties and involvement into beta-sheet formation. It is clear that amylin contains amino acid sequence which is ready to form amyloid membrane channel after appropriate digestion.

[0116] FIG. 17. α1-Anti-chymotrypsin (ACT) dose-dependently inhibit cellular toxicity of amylin (adapted from (Schubert 1997)).

[0117] 15 cells derived from pancreatic islets of Langerhans were exposed to amylin in the presence of various concentrations of α1-anti-chymotrypsin, natural serine peptidase inhibitor (serpin). Cell survival was measured using MTT assay.

[0118] Amylin induced drop in cell viability, which was prevented by α1-anti-chymotrypsin. Unlike effects against beta-amyloid (see FIG. 15), the protection against amylin was not reaching 100% (based on the amount of presented information in the manuscript, it could be suggested that the author actually placed much less attention to this part of the study). Similar to the case of anti-beta-amyloid protection, ACT demonstrated optimal dose, further increase of ACT concentration significantly decreased the protective effect.

[0119] FIG. 18. Amino acid sequence of superoxide dismutase (SOD) which is associated with the development of ALS (amyotrophic lateral sclerosis).

[0120] Like Alzheimer's disease and diabetes type II, ALS is characterized by the death of specific cell type. Dying cells in all degenerative diseases exhibit several important similarities such as lysosomal disfunction, increased production of reactive oxygen species linked to improper recycling of mitochondria. One of hypothetical mechanisms involved into the development of ALS includes superoxide dismutase, key enzyme needed for protection against damaging intermediaries formed by active oxygen. SOD is the protein which is considered misfolding (forming aggregates despite being soluble initially).

[0121] The schematic of biochemical events described at the FIGS. 8 and 9 (amyloid degradation toxicity) can be traced to the ALS when amino acid sequence of superoxide dismutase, the protein which is believed to be involved into the pathogenesis of ALS, is considered. The molecule of SOD contains 154 amino acids, which form two pairs of beta-sheets—top and bottom beta-sheets. Each beta-sheet is formed by four strands. The amino acid sequence is shown, the sequences involved into each strand of beta-sheet and the charges of amino acids which carry either positive or negative charge.

[0122] FIG. 19. Superoxide dismutase (SOD) contains amino acid sequences which are potentially membrane channel-forming.

[0123] At least two fragments of amino acid sequence of SOD resemble the structure of membrane channel-forming fragment of beta-amyloid. Both fragments consist mostly by non-polar amino acids, form beta-sheet, and have a single positively charged amino acid in the sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0124] According to our amyloid degradation toxicity theory, the cytotoxic effects of amyloidogenic peptides are mediated, at least in part, by the formation of membrane channels in cellular membranes. Unlike full-length amyloid peptides which are not effective in forming channels, some degradation products of said peptides are. Therefore, we claim that the prevention of proteolytic degradation of said peptides is the method to prevent or slow down the development of diseases caused by said peptides. Said prevention is the therapeutic method, which is clearly distinct from other treatment options known so far, such as the decrease of production of amyloid peptides from pro-peptides, inactivation of full-length peptides by antibodies, or ameliorating consequences of ion transport disturbance.

[0125] We claim that the use of chemical entities which selectively inhibit proteases digesting amyloid peptides into fragments which form membrane channels can be achieved without significant effect on overall metabolism of amyloid peptide and without excessive production of extracellular deposits of said peptides which can be histochemically stained as amyloid in postmortem specimens in patients. Main embodiment of this invention is a method to treat neurodegenerative disease, such as Alzheimer's disease using inhibitors of proteases.

[0126] Among embodiments of this invention is the method to select chemical entities, which are effective in the treatment of said diseases using the method invented by us previously ((Zaretsky and Zaretskaia 2020), patent pending). The etiology of neurodegenerative diseases such as Alzheimer's disease is in the formation of peptide fragments which are able to form channels. The method considers mixing biological samples (such as purified enzymes or homogenates of tissues) with protein of interest (such as beta-amyloid peptide in studies of Alzheimer's disease or superoxide dismutase in studies of amyotrophic lateral sclerosis) and collecting samples from the mixture after desired times. Degradation of amyloid protein by proteases in the sample results in the production of fragments. If fragments are able to form membrane channels, the presence of channel-forming units is tested using test liposomes and flowmetric technique to measure permeabilization of membranes (Zaretsky and Zaretskaia 2020).

[0127] We expect that high-throughput testing will reveal multiple chemical entities which can be used to treat amyloidogenic diseases using invented method, with special interest that some of these effective chemical entities may be already approved by FDA or other regulatory agency as the drugs with other indications (such as aprotinin). Availability of medicines, which are effective with off-label use, may be a fast pathway to deliver life-saving treatments to patients.

[0128] Various ways to suppress the enzymatic proteolytic activity can be suggested, such as the use of neutralizing anti-enzymatic antibodies, genetic modification of synthesis of appropriate enzymes, the modification of synthesis of endogenous inhibitors or the delivery of exogenous synthetic mechanisms to produce appropriate inhibitors in situ.

EXAMPLES OF HOW THE INVENTION WILL BE USED

Example 1

[0129] New pharmacologic class of drugs to treat neurodegenerative diseases is established. The criterion to belong to this new class is the drug's ability to prevent the degradation of beta-amyloid into fragments which are able to form membrane ion channels.

Example 2

[0130] The method for studying degradation of proteins into fragments which are able to form membrane channels will be used to study molecular mechanisms involved in the progression of neurodegenerative diseases, such as Alzheimer's disease. One of applications is to screen enzymes responsible for said degradation.

[0131] Liposomes with embedded ion-sensitive probe are used as a test system. Liposomes are extruded from phosphatidylserine containing membrane probe (i.e. DiD) in a calcium-free buffer containing ion-sensitive probe (i.e. Fluo-4) and volume probe (i.e. dextran-tetramethylrhodamin) or membrane probe (DiD). Extravesicular probes are cleared using either centrifugation, or dialysis. After the addition of calcium, the intravesicular calcium-sensing probe remain non-fluorescent because membranes are not permeable to calcium. If membrane channels are formed, calcium enters permeable liposomes, saturates the calcium-sensing dye, so the liposome becomes fluorescent. The number of fluorescent liposomes is an estimate of the number of channel-forming units in the solution.

[0132] To assay channel-forming unit formation, full-length beta-amyloid is mixed with sample that possesses protein-degrading activity. After incubation in desired conditions, the mixture is added to the suspension of liposomes, and analyzed on the flow cytometer. The number of formed channels is estimated from the number of permeabilized liposomes.

[0133] The sample with protein-degrading activity could be a solution of protease (recombinant or purified from a natural source), homogenate of a tissue, lysate of organelle preparation (for example, isolated lysosomes or mitochondria).

Example 3

[0134] The method for high throughput testing of chemical entities for an ability to inhibit enzymes degrading proteins into fragments which are able to form membrane channels will be used to find drug candidates to treat neurodegenerative diseases, such as Alzheimer's disease.

[0135] This is an extension of the technique described in the Example 2 using a particular enzyme that was validated as a key player involved in the peptide channel-mediated cellular toxicity.

[0136] Chemical entity that significantly decreases the formation of membrane channels is considered effective against channel-mediated permeabilization of membranes.

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