Patent Application
20220152090
The present disclosure relates to a method for treating an amyloidogenic disease, particularly to a method for the clearance of amyloid-beta 42 (Aβ42).
Non-equilibrium states, or more specifically, far-from-equilibrium conditions, have been considered critical for the modulation of biomolecular assembly in living systems. One interesting example is the role of microtubules in the formation of cytoskeleton networks, an assembly process that exhibits a non-equilibrium behavior for its self-assembly and infrequent decay. Another example is the amyloid peptides (Aβ), which undergo self-aggregation to form amyloid plaques in triggering the neurodegenerative cascade of Alzheimer's disease (AD) (E. N. Cline, M A. Bicca, K. L. Viola, W L. Klein, J. Alzheimers Dis. 2018, 64, S567-S610; A. K. Buell, Biochem. J. 2019, 476, 2677-2703; 1 Vaquer-Alicea, M. I. Diamond, Annu. Rev. Biochem. 2019, 88, 785-810). Among the various Aβ isoforms, Aβ40 and Aβ42 are the two most abundant species. Although Aβ40 is more abundant than Aβ42 in cerebrospinal fluid, Aβ42 has a higher self-aggregation potential than Aβ40 in contributing to the amyloid deposits in AD brains. From the perspective of chemical kinetics, the self-propagation of Aβ42 represents a dynamic self-assembly process involving a transition state from the pre-organized protofibrils to a stable form of fibrils (M. Ahmed, J. Davis, D. Aucoin, T Sato, S. Ahuja, S. Aimoto, J. I. Elliott, W E. Van Nostrand, S. O. Smith, Nat. Struct. Mol. Biol. 2010, 17, 561-U556; S. M Butterfield, H. A. Lashuel, Angew. Chem. Int. Ed. 2010, 49, 5628-5654; I. W Hamley, Chem. Rev. 2012, 112, 5147-5192; Z. Fu, D. Aucoin, J. Davis, W E. Van Nostrand, S. Smith, Biochemistry 2015, 54, 4197-4207; J. A. Luiken, P G. Bolhuis, J. Phys. Chem. B 2015, 119, 12568-12579; B. Morel, M P Carrasco, S. Jurado, C. Marco, F Conejero-Lara, Phys. Chem. Chem. Phys. 2018, 20, 20597-20614).
Altered homeostasis between Aβ peptide production and clearance is defined as the pathological basis for the accumulated Aβ fibrils in AD brains. However, efforts aimed at blocking Aβ42 production have not been successful, as evidenced by the fact that more than 200 clinical trials of drugs designed to decrease Aβ42 production have been terminated. There is thus a need in the art for treatment of amyloidogenic diseases.
The present disclosure provides a method for treating or preventing or delaying the onset or progression of an amyloidogenic disease in a subject in need, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of amphiphilic liposaccharide to the subject.
In some embodiments of the disclosure, the amphiphilic liposaccharide is as the sole active pharmaceutical agent or agents present in a therapeutically effective amount in the pharmaceutical composition.
In some embodiments of the disclosure, the amphiphilic liposaccharide comprises a lipid A and oligosaccharide. Examples of the amphiphilic liposaccharide include but are not limited to lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), 2-deoxy-6-O-(2-deoxy-3-O-[(3R)-3-hydroxytetradecanoyl]-2-{[(3R)-3-hydroxytetradecanoyl]amino}-4-O-phosphono-β-D-glucopyranosyl)-3-O-[(3R)-3-hydroxytetradecanoyl]-2-{[(3R)-3-hydroxytetradecanoyl]amino}-1-O-phosphono-α-
In some embodiments of the disclosure, the method is for the clearance of amyloid-beta 42.
In some embodiments of the disclosure, the method is for triggering non-equilibrium co-assembly of amyloid-beta 42.
In some embodiments of the disclosure, the method is for retaining neuronal cell viability.
In some embodiments of the disclosure, the method is for rescuing Aβ42-induced apoptosis.
In some embodiments of the disclosure, the method is for enhancing endo-lysosomal clearance of amyloid-beta 42.
Examples of the amyloidogenic disease include but are not limited to Alzheimer's disease, mild cognitive impairment, Parkinson's disease with dementia, Down's syndrome, diffuse Lewy body (DLB) disease, cerebral amyloid angiopathy (CAA), vascular dementia, and mixed dementia.
The present disclosure also provides a method for selecting an agent for treating or preventing or delaying the onset or progression of an amyloidogenic disease, comprising contacting the agent with a neuronal cell, wherein if the agent enhances clearance of amyloid-beta 42 or triggers non-equilibrium co-assembly of amyloid-beta 42, the agent is a candidate agent for treating or preventing or delaying the onset or progression of an amyloidogenic disease.
In some embodiments, the agent retains neuronal cell viability, rescues Aβ42-induced apoptosis, or enhances endo-lysosomal clearance of amyloid-beta 42.
In some embodiment, the agent is an amphiphilic liposaccharide; particularly, comprising lipid A and an oligosaccharide, such as lipopolysaccharide, monophosphoryl lipid A, 2-deoxy-6-O-(2-deoxy-3-O-[(3R)-3-hydroxytetradecanoyl]-2-{[(3R)-3-hydroxytetra-decanoyl]amino}-4-O-phosphono-3-
The present disclosure also provides a liposaccharide of 2-deoxy-6-O-(2-deoxy-3-O-[(3R)-3-hydroxytetradecanoyl]-2-{[(3R)-3-hydroxytetradecanoyl]amino}-4-O-phosphono-β-
The finding suggested that the self-propagating rate in the presence of LPS is slightly slower than Aβ42 alone.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skills in the art to which this disclosure pertains.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
As used herein, the term “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
As used herein, “treating” or “treatment” of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical or sub-clinical symptoms of the state, disorder or condition developing in a mammal that may be afflicted with or predisposed to the state, disorder or condition but has not yet experienced or displayed clinical or subclinical symptoms of the state, disorder or condition; and/or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; and/or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms; and/or (4) causing a decrease in the severity of one or more symptoms of the disease.
The term “preventing” or “prevention” is recognized in the art, and when used in relation to a condition, it includes administering, prior to onset of the condition, an agent to reduce the frequency or severity of or delay the onset of symptoms of a medical condition in a subject relative to a subject which does not receive the agent.
The term “amyloidogenic disease” includes any disease associated with (or caused by) the formation or deposition of insoluble amyloid fibrils. Exemplary amyloidogenic disease include, but are not limited to Alzheimer's disease (AD), mild cognitive impairment, Parkinson's Disease with dementia, Down's Syndrome, Diffuse Lewy Body (DLB) disease, Cerebral Amyloid Angiopathy (CAA), vascular dementia and mixed dementia (vascular dementia and AD), amyloidosis associated with multiple myeloma, primary systemic amyloidosis (PSA), and secondary systemic amyloidosis with evidence of coexisting previous chronic inflammatory or infectious conditions. Different amyloidogenic diseases are defined or characterized by the nature of the polypeptide component of the fibrils deposited. For example, in subjects or patients having Alzheimer's disease, β-amyloid protein (e.g., wild-type, variant, or truncated β-amyloid protein) is the characterizing polypeptide component of the amyloid deposit. PSA involves the deposition of insoluble monoclonal immunoglobulin (Ig) light (L) chains or L-chain fragments in various tissues, including smooth and striated muscles, connective tissues, blood vessel walls, and peripheral nerves.
As used herein, “onset” means the occurrence in a subject of clinical symptoms associated or consistent with a diagnosis amyloidogenic disease.
As used herein, “delay” in the onset or progression of a phase consistent with amyloidogenic disease means an increase in time from a first time point to onset or worsening of a phase consistent with amyloidogenic disease, such as cognitive impairment of the Alzheimer type. For example, a delay in the onset of amyloidogenic disease means that the onset of amyloidogenic disease, as defined herein, in a subject at risk to develop amyloidogenic disease is delayed from happening at its natural time frame by at least six months, 1 year, 1½ years, 2, years, 2½ years, 3 years, 3½ years, 4 years, 4½ years, 5 years, 5½ years, 6 years, 6½ years, 7 years, 7½ years or 8 years or more, and preferably from 3 years to 8 years and more preferably for 5 years after a normal cognitive subject has been determined to be at high risk to develop amyloidogenic disease. By way of further example, a delay in the progression of cognitive impairment that may progress to amyloidogenic disease or a delay in the progression of dementia means that the rate of cognitive decline is slowed relative to its natural time frame. These determinations are performed by using appropriate statistical analysis.
As used herein, the terms “patient,” “subject,” “individual,” and the like are used interchangeably, and refer to any animal, including any vertebrate or mammal, and, in particular, a human, and can also refer to, e.g., as an individual or patient.
As used herein, the term “in need of treatment” refers to a judgment made by a caregiver (e.g., physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals), and such judgment is that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that include the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the compounds of the present disclosure.
The term “administering” includes routes of administration which allow the agent of the disclosure to perform their intended function.
As used in the present disclosure, the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent administered to an animal, for example a human, for treating or eliminating a particular disease or pathological condition that the animal suffers. In some embodiments of the disclosure, the pharmaceutical composition optionally comprises pharmaceutically acceptable excipients.
The term “effective amount” of an agent as provided herein refers to a sufficient amount of the ingredient to provide the desired regulation of a desired function. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the disease state, physical conditions, age, sex, species and weight of the subject, the specific identity and formulation of the composition, etc. Dosage regimens may be adjusted to induce the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.
The term “pharmaceutically acceptable” as used herein refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (either a human or non-human animal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts.
As used herein, the term “amphiphilicity” refers to the property of one substance having both a hydrophobic site and a hydrophilic site. For example, when the medium is water, a substance having amphiphilicity forms micelle particles and the particles can be observed.
As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and/or agents for pharmaceutical active substances is well known in the art. For example, the pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, lotion, gel, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream, suppository or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally; or (9) nasally.
The present disclosure identifies a non-equilibrium state of interaction between supramolecular liposaccharide and amyloid. Structurally, the polymerized propagation of amyloid presents a specific groove that is recognized by the amphiphilicity of liposaccharide bait in a non-equilibrium manner. Functionally, the transient complex elicits a cellular response to clear extracellular amyloid deposits via an endolysosomal mechanism in neuronal cells. Since the impaired clearance of toxic amyloid deposits correlates with pathology, the non-equilibrium interaction between liposaccharide and amyloid represents a useful target for therapeutics.
Accordingly, the present disclosure provides a method for treating or preventing or delaying the onset or progression of an amyloidogenic disease in a subject in need, comprising administering a therapeutically effective amount of amphiphilic liposaccharide as an active ingredient or a pharmaceutical composition comprising the same to the subject. In another aspect, the present disclosure provides use of amphiphilic liposaccharide as an active ingredient or a pharmaceutical composition comprising the same in the manufacture of a medicament for treating or preventing or delaying the onset or progression of an amyloidogenic disease in a subject in need.
In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of amphiphilic liposaccharide as an active ingredient for treating or preventing or delaying the onset or progression of an amyloidogenic disease in a subject in need.
In some embodiments of the disclosure, the amphiphilic liposaccharide acts as the sole active pharmaceutical agent or agents present in a therapeutically effective amount in the method or pharmaceutical composition as described herein. In other embodiments of the disclosure, the method comprises administering a combination comprising the pharmaceutical composition and a second pharmaceutical composition, and the second pharmaceutical composition comprises a second therapeutically active agent for treating the amyloidogenic disease, such as an antibody.
The liposaccharide disclosed herein refers to a compound comprising lipid, saccharide and lipid-saccharide conjugates. The liposaccharide may be derived from a natural source or artificial. An exemplary embodiment of the lipid is lipid A. Commonly, lipid A comprises two glucosamine (carbohydrate/sugar) units, in an β(1→6) linkage, with attached acyl chains (“fatty acids”), and normally containing one phosphate group on each carbohydrate. The lipid A as described herein may be modified. The term “oligosaccharide” refers to a carbohydrate structure having from 2 to about 7 saccharide units. The particular saccharide units employed are not critical and include, by way of example, all natural and synthetic derivatives of glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose, sialic acid, 3-deoxy-D,L-octulosonic acid, and the like.
Particularly, examples of the amphiphilic liposaccharide include but are not limited to lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), lipid A derivative IX (2-deoxy-6-O-(2-deoxy-3-O-[(3R)-3-hydroxytetradecanoyl]-2-{[(3R)-3-hydroxytetra-decanoyl]amino}-4-O-phosphono-β-
The term “lipopolysaccharide” (LPS) refers to large molecules consisting of a lipid and a polysaccharide (glycophospholipid) joined by a covalent bond. LPS comprises three parts: 1) O antigen; 2) Core oligosaccharide, and 3) Lipid A. The O-antigen is a repetitive glycan polymer attached to the core oligosaccharide, and comprises the outermost domain of the LPS molecule. Core oligosaccharide attaches directly to lipid A and commonly contains sugars such as heptose and 3-deoxy-D-mannooctulosonic acid (also known as KDO, keto-deoxyoctulosonate). Lipid A is a phosphorylated glucosamine disaccharide linked to multiple fatty acids.
According to the disclosure, “monophosphoryl lipid A” is a detoxified endotoxin lipid A fraction, which lacks a saccharide and a phosphate group.
According to the disclosure, “lipid A derivatives IX and XI (denoted as PIX and PXI)” is a detoxified endotoxin lipid A fraction that belongs to one of MPL analogues that lacks two lipid chains.
The term “salts” includes any anionic and cationic complex, such as the complex formed between a cationic lipid disclosed herein and one or more anions. Non-limiting examples of anions include inorganic and organic anions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfate, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate, polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite, bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate, arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate, hydroxide, peroxide, permanganate, and mixtures thereof. In particular embodiments, the salts of the cationic lipids disclosed herein are crystalline salts.
As disclosed herein, the pharmaceutical composition is for the clearance of amyloid-beta 42, for triggering non-equilibrium co-assembly of amyloid-beta 42, for retaining neuronal cell viability, for rescuing Aβ42-induced apoptosis, and/or for enhancing endo-lysosomal clearance of amyloid-beta 42.
As illustrated in the Examples, the amphiphilic groove of Aβ42 protofibrils is important for complexing with amphiphilic liposaccharide through a pattern of non-equilibrium behavior. Liposaccharide may actually act as a bait in attracting the Aβ42 intermediates that sometimes deviate from their supposed thermodynamic self-assembly process. With respect to functionality, such a transient supramolecule-supramolecule interaction can potently stimulate a strong cellular response toward autophagy-mediated protein degradation for Aβ42 peptides in neuronal cells. Moreover, since the oscillation of the non-equilibrium state appears to sustainably maintain the far-from-equilibrium behavior during the interaction between these two supramolecules (that is, liposaccharide and Aβ42), Aβ42 peptides are found to be persistently imported into the cells for degradation. Consequently, the extracellular Aβ42 protofibrils are eventually diminished over a prolonged incubation time with the cells. The structural-functional theory regarding the non-equilibrium state of supramolecule-supramolecule binding indicates a target for the treatment of the amyloidogenic disease.
The present disclosure also provides a method for selecting an agent for treating or preventing or delaying the onset or progression of an amyloidogenic disease, comprising contacting the agent with a neuronal cell, wherein if the agent enhances clearance of amyloid-beta 42 or triggers non-equilibrium co-assembly of amyloid-beta 42, the agent is a candidate agent for treating or preventing or delaying the onset or progression of an amyloidogenic disease.
The present disclosure also provides a liposaccharide of 2-deoxy-6-O-(2-deoxy-3-O-[(3R)-3-hydroxytetradecanoyl]-2-{[(3R)-3-hydroxytetradecanoyl]amino}-4-O-phosphono-β-
The following examples are provided to aid those skilled in the art in practicing the present disclosure.
Methods
The general procedures used for preparing the transient LPS-Aβ42 complex and the validation of cell cytotoxicity were as follows. First, Aβ42 (100 μM) and LPS (1000 nM, derived from Escherichia coli O111:B4, Sigma-Aldrich) were added to each group medium containing 2.5% dimethyl sulfoxide (DMSO) and then sterilized by using UV light. The medium was then replaced and the remaining mixture was incubated for 72 hours at 37° C. in 5% CO2 humidified air. Cell viability was measured using the CCK8 (Sigma-Aldrich 96992) assay according to the manufacturer's protocol. A 0.22-μm filter (non-pyogenic, Millex®-GV) was used to remove unbound LPS, if needed, in the LPS-Aβ42 solution.
Materials and Methods
An LPS-coated plate for Aβ42 and Aβ40 binding assay. 100 mM Na2CO3, 20 mM EDTA, and 0.1 mL of 30 μg/mL LPS solution were added to a 96-well immunoassay plate (Costar 9018, Corning Corporation), which was then incubated at 37° C. for 3 hours. The coated plate was then washed with deionized water and dried for one day. PBS solution containing 1% BSA was then added to block the coated plate at 37° C. for 30 minutes. Finally, the coated plate was washed three times with PBS solution containing 0.1% BSA. Subsequently, different concentrations of Aβ42 protofibrils (with or without added SAuM or colistin) were added to the coated wells and incubated at 37° C. for 16 hours, before being washed three times with PBS. Bio-red protein staining dye was then added into the coated wells to assay the Aβ42 or Aβ40 concentrations of each well and measure the absorption value at 625 nm using an ELISA reader. The values were substituted into the calibration curve to calculate the concentrations of Aβ42 or Aβ40 in each well.
Polymerization rate of Aβ42. The methods for the liquid type were similar to those used for the LPS-coated plate, but a filter was used to remove Aβ42 fibers during the sample preparation. In brief, a mixture of Aβ42 and LPS was incubated at 37° C. in a time-dependent manner. Then, the Aβ42 fibers were removed using a 0.1-um filter of MWCO (Millipore MILLEX-HIP) to avoid interference. The Aβ42 filtrate was collected and dried before adding bio-red protein staining dye, and the absorption value of the filtrate was then measured at 625 nm using an ELISA reader. The values were substituted into the calibration curve to calculate the concentrations of Aβ42 in each well.
Identification of the non-equilibrium steady state between LPS and Aβ42. A RPMI medium containing Aβ42 (100 μM) and LPS (1.0 nM) was incubated at 37° C. At different time points, a small proportion of the solution was taken out to stain with a fluorescent dye, bis-ANS (1.0 μM, 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt, Sigma-Aldrich), and was immediately measured using a fluorescence spectrophotometer (Varian, Cary Eclipse, excitation wavelength at 390 nm).
Transmission electron microscopy (TEM). The mixtures of LPS and individual Aβ42 were prepared in deionized water. Samples were mounted on a 400-mesh Cu grid with carbon supporting film and stained with 2% phosphotungstic acid. Excess staining reagent was removed using filter paper, and the grid was dried prior to transmission electron microscopy measurements (Hitachi H-7650, Japan) at 100 kV.
Cell viability. Neural cells were maintained in MEM (Gibco 11095-080) supplemented with 10% FBS and 1×MEM NEAA (Gibco 11140-050). Neural SH-SY5Y cells were plated at a density of 8000 cells per well in 96-well plates (Costar 3599) and allowed to attach overnight at 37° C. in 5% CO2 humidified air. Aβ42 (100 μM), LPS (1000 nM), SAuM (1000 nM), or colistin (1000 nM) were then added to each group medium containing 2.5% dimethyl sulfoxide (DMSO) and sterilized by using UV light. The medium was then replaced every day and the remaining mixture was incubated for 72 hours at 37° C. in 5% CO2 humidified air. Cell viability was measured using the CCK8 (Sigma-Aldrich 96992) assay according to the manufacturer's protocol. A 0.22-μm filter (non-pyogenic, Millex®-GV) was used to remove unbound LPS, if needed, in the LPS-Aβ42 solution.
Western Blots. To assess the level of Aβ42 in the cell culture medium, Aβ42 peptides were collected from the entire volume of the cell culture medium and total cell lysates and mixed with protein sample buffer (final 0.1 M Tris-HCl, pH6.8, 10% glycerol, 2% SDS, 1% 0-mercaptoethanol, and 0.01% bromophenol blue) for Western blot analysis. The samples of cell mixture were analyzed by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Millipore). After blocking with blocking buffer containing 5% w/v nonfat dry milk in PBST (1× phosphate-buffered saline and 0.1% Tween 20) at room temperature for 1 h, the membrane was incubated with a primary antibody diluted in the blocking buffer at 4° C. overnight. After hybridization with primary antibody, the membrane was washed three times with PBST before the addition of an HRP-conjugated secondary antibody against the primary antibody. The membrane was then washed three times with PBST before immunoreactive bands were detected by chemiluminescence (PerkinElmer) or Ponceau S staining (Bersting Technology). Primary antibodies used in this study included the following: anti-Aβ (#8243, Cell Signaling Technology), anti-LC3 (#4108, Cell Signaling Technology), anti-cathepsin B (sc-13985, Santa Cruz Biotech), anti-cathepsin D (sc-6486, Santa Cruz Biotech), and anti-GAPDH (GTX100118, GeneTex). Culture medium containing 2.5% dimethyl sulfoxide (DMSO) was used for each group.
Statistical analyses. For biologic assays, we used GraphPad Prism (v7.02) to perform one-way ANOVAs. All data were expressed as mean±SEM, and P-values of less than 0.05 were considered to be statistically significant and showed asterisks (**, P<0.01; ***, P<0.001).
The measurement of critical aggregation concentration (CAC). The CAC of LPS was measured by small-angle X-ray scattering (SAXS). The SAXS data for the sample solutions, which were collected using a Pilatus 1M-F detector, were used to extract the zero-angle intensity Io (q=0) and radius of gyration Rg of the LPS micelles on the basis of Guinier approximation. The value of CAC was then extracted from the intercept of the linear regression fitting of the concentration-dependent zero-angle intensity Io (q=0), as shown in
We proposed that these two supramolecules might form an intermolecular interaction in a non-equilibrium state that could subsequently impact the survival of neural cells (
To test this hypothesis, we investigated whether LPS could form a transient complex with Aβ42 through a non-equilibrium co-assembly process that subsequently leads to dissociation. To do so, bis-ANS, a specific fluorescent dye sensitive to Aβ42 hydrophobicity (N. D. Younan, J. H. Viles, Biochemistry 2015, 54, 4297-4306), was used to assess the degree of Aβ42 hydrophobicity during the process of amyloid polymerization upon the administration of LPS. The results revealed a repeated oscillating pattern of association and dissociation between the two molecules (
Next, it was of interest to investigate whether the transient association between LPS and Aβ42 protofibrils impacts cytotoxicity. Surprisingly, cell viability assays suggested that SH-SY5Y neuronal cells retained more than 95% cell viability after co-treatment of Aβ42 with LPS. In contrast, less than 20% cell viability was retained in cells treated with Aβ42 alone (
To understand the underlying mechanisms that direct this cellular process, we first tested whether matrix metalloproteinases (MMPs) secreted from cultured cells might mediate the destruction of Aβ42 peptides in the extracellular milieu. However, the treatment of neuronal cells with pan-MMP inhibitors showed no changes in Aβ42 levels in cells co-treated with LPS (
Furthermore, it was essential to clarify (i) why LPS can only bind with Aβ42 but not with Aβ40 (
Prompted by the finding that Aβ42 formed a complex with amphiphilic LPS, we speculated that the soluble Aβ42 protofibrils might possess a specific groove that acts as a kinetic trap and enables the docking of amphiphilic LPS. To test this possibility, we used a unique LPS sequester, an atomic sheet-like gold nanocluster (identified as SAuM) with a specific dock for the lipid A of the hydrophobic domain, that has been demonstrated in our previous work (F. H. Liao, T. H. Wu, C. N. Yao, S. C. Kuo, C. J. Su, U. S. Jeng, S. Y. Lin, Angew. Chem. Int. Ed. 2020, 59, 1430-1434; P. Pristovsek, J. Kidric, J. Med. Chem. 1999, 42, 4604-4613). Indeed, the binding efficiency of Aβ42 protofibrils and LPS was found to be significantly decreased in the presence of the SAuM (
We then integrated these results into a model of the structural-functional interaction between LPS and Aβ42 in modulating the endocytic clearance of Aβ42 in neuronal cells (
Although LPS reveals an efficient degradation of Aβ42 in SH-SY5Y neural cells, it still has a biosafety concern from its intricately cytotoxic immunity. We then found “Monophosphoryl lipid A (MPL, CAS 1246298-63-4, Sigma-Aldrich)” is a detoxified endotoxin lipid A fraction that belongs to one of LPS analogues, which lacks a saccharide and a phosphate group. Compared to LPS, MPL that has a controllable proinflammatory response was currently approved for clinical use in vaccine adjuvant.
We further found “diphosphoryl or monophosphoryl lipid Aderivatives with four lipid chains (structures of IX and XI listed in
Synthesis of PIX and PXI
The synthetic routes for lipid A derivatives IX and XI are shown in
Specifically, the regioselective reductive ring opening reaction of 4,6-O-benzylidene acetal of a laboratory prepared compound (I) with triethylsilane (Et3SiH) and dichlorophenylborane (PhBCl2) gives a desired glucosamine 6-OH acceptor (II). The imidate donor (IV) can be prepared from Compound I through a stepwise deallylation followed by trichloroacetimidate formation. Trimethylsilyl triflate (TMSOTf)-promoted glycosylation of acceptor (II) with donor (IV) provides β-(1→6)-linked disaccharide (V), which is sequentially converted into Compound VI with four lipid chains in 3 steps: (1) removal of the O-acyl groups with NaOMe liberates two hydroxy groups, (2) cleavage of the N-2,2,2-trichloroethoxycarbonyl (Troc) groups with Zn/HOAc furnishes two free amino groups, and (3) acylation of the generated hydroxy and amino groups with prepared (R)-3-benzyloxytetradecanoic acid. Compound VI is subjected to regioselective ring opening with Et3SiH/Boron trifluoride diethyl etherate (BF3.Et2O) to afford the 4′-OH disaccharide VII. Subsequently, the allyl group at the anomeric position is removed, and the simultaneous phosphitylation is achieved with phosphoramidite in the presence of 1H-tetrazole, followed by oxidation with meta-chloroperoxybenzoic acid (mCPBA) to provide 1,4′-O-diphosphorylated compound VIII. Finally, deprotection of the benzyl ether is accomplished by hydrogenolysis with Pd(OH)2 under H2(g), delivering the target molecule IX. On the other hand, the lipid A derivative XI can be synthesized from intermediate VII through phosphorylation, deallylation, and hydrogenolysis.
While the present disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives thereto and modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are regarded as falling within the scope of the present disclosure.
This patent application claims the benefit of U.S. Prov. Ser. No. 63/113,578 filed 13 Nov. 2020. The application is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63113578 | Nov 2020 | US |
