Patent Application
20210349077
Present invention relates to the methods to measure permeability of lipid membranes (including cellular membranes) to various substances. The invented method can be used to estimate the effectiveness of treatments to prevent the increase in permeability caused by various means.
As an example of factor increasing permeability of lipid membranes are effects of so-called “misfolded” peptides and proteins. Such proteins usually do not have specific conformation immediately after synthesis and are soluble, but with time they form intra- and inter-molecular hydrogen bonds and create structures called beta-pleated sheets. These structures elongate into protofibrils, which can aggregate, become insoluble, and form clumps. Protein clumps formed in the biological tissues can be easily identified by histological staining. The diseases characterized by accumulation of such clumps are called “amyloid diseases”. The list of amyloid diseases includes but is not limited to Alzheimer's disease (AD), Parkinson's disease, Amyotrophic Lateral Sclerosis, Huntington's disease, diabetes mellitus type II, prion disease, Creutzfeldt-Jakob disease and many others.
It was demonstrated that before forming large insoluble protein clumps (with multiple molecules involved into single aggregate), at the stage of oligomers (only several molecules, as low as 3) these proteins can form a barrel-like structure which penetrates the lipid bilayer and forms membrane channel. This channel allows ions (such as Ca2+, Na+, K+), as well as organic molecules, and even macromolecules (such as dextrans) to go through the membrane. Essentially, if this process occurs in living cell, the cell loses the ability to control internal content. The first cells which are affected are excitable cells, which are dependent on the transmembrane ion gradients creating membrane potential.
There is a limited number of approaches to observe permeability of membranes. Two most wide-spread techniques are planar lipid bilayers (lipid bilayer is formed over small hole in a Teflon disc) or patch (membrane is formed in the opening of small glass pipette). Both approaches are labor-intensive, and difficult to apply to screening applications. Most importantly, each membrane serves as a single target, so it either allows observation of single channels (without an option to observe how many channels are formed) or measuring composite permeability (no way to distinguish few channels with high permeability vs many channels with low permeability). Similarly, in previous research disclosures when liposomes were used as a test object, it was impossible to distinguish few channels with high permeability vs many channels with low permeability.
Our invention creates an alternative way to observe channel formation—we can identify the number of channels formed in the suspension of liposomes, because each liposome is measured independently. Also, the technique allows for its use in high-throughput screening.
Diseases caused by misfolded proteins are very different. However, they have a common feature: immediately after the synthesis the protein has no secondary or tertiary structure and is soluble, but under various conditions it may undergo conformational changes, which ultimately result in the formation of beta-sheets and, after polymerization in the loss of the solubility. Individual molecules with intramolecular beta-sheet structure become linked to other such molecules forming oligomers, then elongate and form protofibrils. Protofibrils tend to aggregate and attract other molecules with relatively low solubility. As a result, insoluble conglomerates become large enough to be visible after histochemical staining of tissue sections. This was how these diseases were identified and grouped as amyloid diseases—various methods of staining reveal amorphous clumps of substance in brain or other tissues. Importantly, there was a correlation between where the clumps could be observed with clinical observations—dopaminergic areas contained such clumps in Parkinson's disease, while cortical areas are prone to the accumulation of clumps in Alzheimer's disease. Appearance of inclusions usually was accompanied by the disappearance of cells, such as dopaminergic neurons (Parkinson's disease) or cortical cells (Alzheimer's disease). Such correlation prompted early theory that the insoluble substance is the cause of the disease.
With time, the observations started to accumulate that clinical severity of disease does not necessarily is dependent on the number or the size of such inclusions. Importantly, the expression of inclusions has much better correlation with the length of disease than with the severity. Even more, the presence of inclusion does not necessarily result in the presence of the disease—there were multiple postmortem observations of highly expressed inclusions in medically healthy patients. However, there was strong correlation between the disappearance of neurons and clinical outcome. This led to the understanding that insoluble protein is a just another consequence of some process which is also responsible for cellular death.
Major promise to finding the cure for this group of diseases is in the comprehension of the process, which underlies the formation of insoluble protein inclusions, and the relationship of this process to the cellular death. Preventing cellular death is the only way to treat, delay the onset or slow down these diseases. Together with preventative screening and/or early diagnosis, such treatment can be a way to eradicate neurodegenerative diseases.
As it was mentioned above, freshly synthesized polypeptides do not have fixed conformation and are water-soluble. Over time, some molecules develop hydrogen bonds which fix specific turns and form beta-sheets, one of major secondary protein structures. Intramolecular hydrogen bonds fix turns within the molecules (label 1 at the
It is now become wide-accepted that cellular or neuronal toxicity is mediated by oligomeric structures, while soluble monomers and formed insoluble large-size fibrils appear mostly non-toxic. The mechanism of cellular toxicity induced by oligomers is intensely studied. Multiple pathways were proposed from increased lipid peroxidation to the release of cytokines by immune-competent cells. Among feature which is characteristic for all studied peptides known to be involved in amyloid diseases is that they affect intracellular electrolyte balance including the increase of intracellular calcium. It was demonstrated that the mechanism involves the formation of protein channels in cell membranes after physical interaction of polypeptide with said membranes. The size of oligomers which are most toxic to cells is estimated to be in low single digit numbers, such as trimers (three molecules per globule which is binding the cell).
To treat the AD, we need to prevent or slow down the processes which ultimately result in neuronal death. Importantly, the very process which initiates cellular toxicity, the insertion of amyloid into the cellular membrane and functioning of ion channels, is not targeted by currently available drugs. We are strongly convinced that the major reason for the absence of such treatments is the absence of techniques which allow high throughput studies of ion disturbances induced by misfolding peptides in general, and by amyloid peptides in particular. In this invention, we claim that proposed technique can be used to study the formation of channels in artificial and cellular membranes, and that this technique can be used to screen chemical entities able to prevent ion disturbances induced by channel-forming peptides.
In this invention we describe the method to identify the formation of functional ion channels in model lipid membranes and the method of high throughput screening of substances which are able to prevent disturbances induced by membrane channel.
Various embodiments of present invention provide the methods of high-throughput testing of peptides which are able to form membrane channels; ways to identify permeability characteristics of membrane channels, as well as methods of screening compounds for potential medical use to treat diseases which are developing due to misfolding of proteins and formation of membrane ion channels.
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.
The formation of ion channels is best observed in unilamellar liposomes because the channels are not formed only in the outer membranes, but not in the internal membranes; therefore, in multilamellar liposomes, ions can not reach the internal volume of the vesicle. Usually, preparatory techniques to form unilamellar liposomes result in the suspension of vesicles with the diameters less than one micrometer, so they do not effectively scatter light. In case, we are interested to estimate the number of formed channels, it is possible to count the number of permeabilized vesicles, for example, using a method of flow cytometry. To identify objects which are smaller than the wavelength in the flow, it is required to use the parameter other than scattering, which is usually used to identify cells that have the size of several micrometers or more. To identify liposomes in the flow, one of possibilities is to add lipid-soluble fluorescent probe (MP, membrane probe), so the vesicle can be identified using intrinsic fluorescence.
The liposomes are prepared in the solution containing ion-sensitive fluorescent probe (ISP) in ion-free medium and are cleared from extravesicular ISP. Membranes are impermeant to the ion, so even after the addition of ion to the medium, ISP remains free of calcium and has typical calcium-free fluorescent properties. If the membrane become permeant to calcium, for example because of channel formation, ions enter the liposomes, bind ISP, so ISP fluorescence has ion-bound properties (either the intensity changes dramatically, or spectra of excitation and/or emission shift in ratiometric probes). The figure represents the situation when the intensity of fluorescence of ion-bound probe increases after binding ion, so impermeable liposomes are non-fluorescent in the measurement channel corresponding to the ISP, while permeable liposomes are intensely fluorescent in the same channel.
Probes are selected in a way that allows for reliable measuring specific fluorescence of each probe through selection of excitation and emission wavelengths (colors)—“Exc Color” and “Em Color”, correspondingly.
Unilamellar liposomes are created with Fluo-3 membrane-impermeant fluorescent dye sensitive to the concentration of calcium (an example of ion sensitive probe, ISP, at the
To identify liposomes in the flow, lipids can be supplemented with a lipophilic dye such as 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD), which serves as membrane probe (MP at the
In the flow cytometer, the appearance of channels is visible as increasing proportion of calcium-loaded liposomes compared with calcium-free liposomes. When every liposome becomes permeant to calcium, all liposomes will be presented as loaded with calcium. This condition is used for normalization and can be achieved in control experiments by the addition of calcium ionophore such as ionomycin.
If such technique is used to screen compounds for the ability to prevent the formation or function of membrane channels, we need to use the ratio of the number of liposomes in status shown at B to the total number of liposomes (both shown at A and B) as an endpoint.
The liposomes are prepared to contain fluorescent probes of various sizes (shown as intravesicular crosshatched circles) and are cleared from extravesicular fluorescent probes. Membranes are impermeant to these probes and can contain membrane probes to identify the liposomes in the flow, because scattering usually cannot be used due to the size of unilamellar liposomes. In this example, the probes of only two sizes are shown—small and large. If liposomal membrane is impermeable, the liposome carries both probes and corresponding event in flow cytometry recording has high intensity in channels corresponding to both large and small probe. If a small channel is formed, small probe is leaking, while large remains trapped intravesicularly. Therefore, the liposome with a small channel will have fluorescence corresponding only to the large probe. Finally, if a large channel is formed, then both probes will be leaking, and liposome will have no fluorescence corresponding to either small or large probe.
The compound set was designed from the fluorescent probes available commercially to be used with a commercial flow cytometer containing four separate lasers with parallel arrangement. One of probes can be efficiently excited by two lasers, so can be detected on two separate channels. Spectra of compounds measurable in each of four channels are shown, the insert presents the distribution of molecular weights of compounds included in the set.
The sets which include compounds with molecular weights of different ranges can be built. Building the sets in the macromolecular range can dramatically benefit from availability of dextrans or other polymers with defined molecular weight, which are custom labeled with appropriate fluorescent moieties.
In this example. liposomes are made of lipid containing red fluorescent probe (such as DiD) and contain membrane-impermeant water-soluble calcium-sensitive dye which has green fluorescence (such as Fluo-3). The intensity of red fluorescence reflects the amount of membrane material in the liposome. The amount of membrane material increases in larger liposomes (elongated vesicles) and with increased number of lipid layers in the liposome (double circles). Liposomes are formed in calcium-free buffer; calcium-sensitive fluorescence probe is non-fluorescent in calcium-free solution (liposome has the same intensity of green fluorescence as one without dye) but becomes highly fluorescent after binding calcium (intensity of green fluorescence increases). Calcium level inside liposome increases when the liposome has ion channels or in the presence of ionophores.
Liposomes (400 nm) were made of phosphatidylcholine with DiD and were extruded in the buffer containing 1 mM Fluo-3 (A-H) or without Fluo-3 (I, J). Addition of extravesicular calcium (A) or chelating agent EGTA (E) did not affect intensity of fluorescence. In the presence of calcium (A-D), the addition of Aβ25-35 in concentrations above 10 μM slightly but reproducibly increased green signal from vesicles (B—20 μM; C—50 μM.) Addition of ionophore ionomycin (permeabilize membranes to calcium) significantly increased fluorescence (D).
The effect of Aβ25-35 was the same in calcium-free medium with EGTA (F,G). As predicted, ionomycin was not affecting Fluo-3 signal in the absence of calcium (H). In liposomes extruded without Fluo-3, Aβ25-35 still shifted the distribution upward (J) with the same magnitude as in liposomes containing calcium-sensitive fluorescent probe (D).
Liposomes (400 nm) were made of phosphatidylserine with DiD and were extruded in the buffer without (A-C) or with (D-I) 1 mM Fluo-3. Addition of extravesicular calcium (A, D) or chelating agent EGTA (G) did not affect intensity of fluorescence. Addition of 5 μM Aβ25-35 increased green signal from vesicles only in the presence of both Fluo-3 and calcium (E) but did not have effect in DiD-only liposomes (B) or in calcium-free buffer (H). Addition of ionomycin did not further increase the Fluo-3 signal from vesicles with low DiD signal but additionally shifted upwards the part of the distribution with high DiD signal (F). As predicted, ionomycin did not produce any effect in DiD-only vesicles (C) and in the absence of calcium (I).
Liposomes made of phosphatidylserine and contained both DiD and Fluo-3. In a calcium-containing medium, membranes are not permeant to calcium, so there is a minimal number of liposomes with increased levels of green fluorescence (A). The addition of 2 μM Aβ25-35 makes some of liposomes permeant to calcium (B). Increasing concentration of amyloid peptide increases the number of permeant liposomes (C, D).
This figure summarizes data from experiments, described at the
Phosphatidylserine liposomes were prepared to contain both DiD and Fluo-3 as described for previous figures. Liposomes were tested to be permeabilized by Aβ25-35.
In this experiment, stock solution of peptides was prepared in DMSO to extend the testing to the peptide which are not soluble in water-based excipients. Peptides were added to create final concentration of 10 μM.
The separation of multilamellar from unilamellar liposomes is important because peptide-formed ion channels cannot be transferred from outer lipid layer to the internal layers, therefore multilamellar liposomes are less sensitive to permeabilization by peptides: only the space between two most peripheral layers would become equilibrated with the medium. It is important that ionophores carry ions across membranes because these molecules are both water and lipid soluble. Therefore, ionophores affect fluorescence of ion-sensitive probes in both unilamellar and multilamellar liposomes.
However, the experiments may require preparations containing relatively large liposomes—not 100-200 nm, but 400 nm—because of low intensity of fluorescence of ion-sensitive dyes. Two-fold increase of diameter results in 8-fold increase of internal volume (increasing the signal for enclosed ion-sensitive probe) and 4-fold increase of membrane surface (less membrane probe can be used, so less effect on the lipid content will be introduced).
Liposomes are created from lipids containing membrane fluorescent probe (Mem, recorded in the channel 1). Extrusion buffer contains ion-sensitive fluorescent probe (ISP, recorded in the channels 2) and volume fluorescent probe (Vol, channel 4). Also, immediately before the experiment, membrane-impermeant surface fluorescence probe (Sur, channel 3) is added.
Liposomes are identified in the flow using thresholds for membrane and volume probe. Using both signals, it is possible to select events reflecting passing liposomes of sufficient size and carrying embedded probe. Liposomes which lost volume label are excluded from the analysis (because same liposomes most likely lost ion-sensitive probe, too).
Added surface probe will bind only to an outer leaflet of membrane, therefore the ratio of membrane fluorescence to surface fluorescence allows to separate multilamellar liposomes from unilamellar ones. Unilamellar liposomes have the highest ratio of surface probe to membrane probe.
The figure simplifies the panel building at the schematics to have each fluorophore identified by using specific pair of excitation and emission wavelengths (laser-filter-detector), but clearly compensation procedure can be used where needed.
In this specific implementation of the technique, following fluorescent probes are used: calcium-sensing probe Fluo-4; membrane probe DiD; volume probe—dextran-tetramethylrhodamine; surface probe—Pacific Blue-labeled Annexin V.
Channels are: for Pacific Blue—violet laser (405 nm)—detector with the filter covering 450 nm; For Fluo-4—blue laser (488 nm)—detector with the filter covering 520 nm; for tetramethylrhodamine—yellow laser (561 nm)—detector with the filter covering 580 nm; for DiD —red laser (637 nm)—detector with the filter covering 665 nm.
Depending on the sequence of adding the components into the reaction, the technique allows to distinguish effect of chemical on formation of peptide oligomers able to form ion channels, the process of incorporation of oligomer into the membrane, and the functioning of already formed channel.
This approach can be used for a purification of channels, or creating liposomal preparation enriched with liposomes carrying membrane channels.
The technique is the extension of fluorescence-activated sorting, which can be performed on commercial flow cytometers. The sample with particles (vesicles or cells) is infused into the constant flow of sheath liquid. The flow of sheath liquid is much higher than the flow of the sample, so in relatively narrow tubing cells separate from each other and become arranged in a line. Each particle passes the beam of laser individually providing the separate event which can be recorded by multiple detectors for the fluorescence and scattering where appropriate. After parameters of fluorescence of each particle are measured, and the particle reaches the outlet of the tube (due to known delay), it can be decided if particular particle should be collected in a specific collecting vessel. Usually, it is done by charging the droplet with the particle with a specific charge, which forces the droplet to change the direction in the electric field created by a pair of electrodes.
Modern flowmetry-based sorters can separate the particles into several subpopulations according to fluorescent properties. In case of liposomes, permeabilized by membrane channels, due to presence of dramatically different profile of fluorescence (such as ion-sensitive probe), the liposomes with channels can be identified in the flow (as shown at the density plot with corresponding gates). Considering that commercial sorters can separate several thousands of droplets per second, it is possible to purify liposomes in the millions. Unfortunately for protein analysis, each channel contains only several molecules of peptide, while every liposome has only one channel. Therefore, even millions of channels represent total amount of peptide below sensitivity of any possible analytical techniques. However, even now, there are applications for such preparations. First, purified liposomes with channels can be used in the testing procedures to remove excessive amounts of peptide in unrelated conformation. The analysis of inhibition of channels will not contain noise from non-permeabilized liposomes. Second, the suspensions can be used to reconstruct purified protein membrane aggregates into larger membranes for electrophysiological or similar studies. Finally, there are techniques which allow antibody generation, based on bacteriophage or similar technologies, which can generate clones of needed antibodies using single copies of separated antibody.
The invention is the method to detect membrane permeability in artificial lipid vesicles (liposomes) for ions and various compounds. The permeabilization of membrane of individual liposome is detected by measuring the fluorescence of intravesicular probe which changes when the ion or the compound of interest become able to pass the membrane of liposome (
The permeabilization to calcium ions will be described first as an example. To detect calcium transmembrane transfer, calcium-sensitive probes are used, such as Fluo-3 or Fluo-4 which dramatically increase their fluorescence upon binding calcium. Liposomes are formed in a calcium-free medium containing calcium-sensitive probe. To make ion-sensitive dye non-fluorescent, the extrusion buffer needs to be calcium-free, that is accomplished by the addition of calcium-chelators such EGTA or EDTA. Liposomes also include membrane probe and volume probes, which have fluorescence that is independent of calcium. Membrane probe is used to identify the liposome in the flow, while volume probe is needed to confirm that there is no non-specific leakage of intravesicular content. Extravesicular probes are washed out (by dialysis, repeated centrifugation etc). An addition of calcium to the suspension of intact liposomes does not result in the increase of fluorescence, because lipid membranes are not permeant to calcium. The suspension of liposomes is subjected to flow cytometric analysis. The identification of the liposome in the flow (passing the particle through laser beam is called “an event”) is performed using fluorescence of the membrane and volume probe. The liposome, that is impermeant to the calcium, does not have fluorescence of calcium-sensitive probe, but the liposome that is permeant (for example due to the presence of membrane channel) has calcium-sensitive probe intensely fluorescing (
The technique can be used to study permeabilization to any ion, for which an appropriate fluorescent ion-sensitive probe can be identified. Lipid membranes are not permeant to sodium, potassium, or protons. Embodiments of this invention describe the measurement of channels translocating potassium, sodium, and protons. Calcium is added to test membrane permeability. To extend the technique to test permeability to other ions, appropriate ion-sensitive probes and ionophores need to be used (Table). Examples of extrusion and incubation buffers that are applicable to technique to detect membrane permeabilization to various ions are also shown in the Table.
To detect non-specific membrane permeabilization to various compounds, the leakage of fluorescent compounds (such as Lucifer Yellow) themselves is studied. In this case, liposomes are formed with enclosed fluorescent compound. Intact liposomes contain the fluorescent label, while permeabilized liposome loses the compound and does not have corresponding fluorescence (
Also, the ability to quench or perform energy transfer can be adopted to study transmembrane transfer of various compounds. For example, permeabilization to manganese can be observed by quenching.
Our main driving force to make this invention was to study molecular mechanisms of cytotoxic effects of amyloidogenic peptides which are mediated, at least in part, by the formation of ion channels in cellular membranes. We claim that the described technique can be used for studying the effectiveness of various treatments to affect the permeability of lipid membranes to ions. We expect that this method will result in the development of high-throughput screening technique to find chemical entities that are able to prevent ion disturbances caused by amyloid-formed ion channels with overarching goal to ameliorate said disturbances and break the biochemical cascade induced by these peptides leading to neuronal death in Alzheimer's disease.
Among embodiments of this invention are the methods to select chemical entities, which are effective in the treatment of amyloid diseases. We claim that the method allows for the distinguishing treatments affecting various steps of amyloid channel formation—the creation of channel-forming units during aggregation of peptides, the incorporation of channel-forming aggregated into the membranes or affecting the function of already formed channels. The embodiments of the technique are possible to make applicable to high-throughput applications.
In another embodiment of this invention, we claim that it is possible to overcome a major limitation of studying membrane channel formation—the need of relatively large liposomes, which makes a significant ratio of vesicle being multilamellar. Considering that peptide-formed channels are formed only in the outer lipid layer, multilammelar liposomes are not an ideal study object. By adding surface probe, such as Annexin V bound to fluorescent label, liposomes can be quantified by the ratio of surface probe to membrane probe. Using only liposomes which have high ratio of surface signal to membrane signal (essentially equal amount of surface probe and membrane probe typical for unilamellar liposomes) allows to separate liposomes made of single lipid layer (unilamellar liposomes). In this way, unilammelar liposomes can be distinguished from multilamellar liposomes, and analyzed separately.
Finally, we claim that by using the extension of analytical method to identify liposomes carrying the membrane channel, the liposomes containing channels can be separated from liposomes without a channel. In this embodiment of the invention, the peptide in the form of the channel can be concentrated. The purified channels can be used not only for basic research, but also for multiple applications such as effective screening technique to identify compounds affecting amyloid membrane channel formation, and the production of macromolecules with the affinity to the channels (such as antibodies etc).
Using the invented method, we found that full-length amyloid peptide Aβ1-42 does not create channels permeabilizing membranes to calcium (
Mixture of liposomes with embedded calcium-sensitive probe is prepared. To do that, liposomes with the diameter 200 or 400 nm are extruded from phosphatidylserine containing membrane probe (i.e. DiD) in a calcium-free buffer containing calcium-sensitive probe (i.e. Fluo-4) and volume probe (i.e. dextran-tetramethylrhodamin with molecular weight 2,000,000 Da). Extravesicular probes are cleared using centrifugation. Solutions of peptides (freshly prepared or aged to allow aggregation) are added to liposomes, followed by surface probe (i.e. Annexin V bound to Pacific Blue). After short incubation, calcium is added, and the mixture is analyzed on flow cytometer. Calcium ionophore ionomycin is used as a positive control, and a vehicle for peptide serves as a negative control.
Liposomes of interest (unilamellar liposomes that retained integrity of internal content) are identified by intense fluorescence of volume probe and corresponding membrane probe. Integrity of content is controlled by the presence of volume probe. Number of lipid layers is estimated by the ratio of intensity fluorescence of membrane probe to surface probe. Unilamellar liposomes have the lowest ratio. In identified liposomes, the concentration of calcium is estimated. Liposomes without channels have low calcium, and corresponding low fluorescent signal of Fluo-4. Liposomes with channels have high calcium and intense fluorescence of Fluo-4. The ratio of the number of liposomes with channels to total number of liposomes (or to the number of liposomes without channels) is the endpoint of test. Peptides which statistically significantly increase the ratio of permeabilized liposomes are considered channel-forming.
Based on previous experimental data, it is reasonable to expect that amyloid membrane channels formed by various peptides are non-selective and can pass various ions (sodium, potassium, or protons).
Mixture of liposomes with embedded ion-sensitive probes is prepared. To do that, liposomes with the diameter 200 or 400 nm are extruded from phosphatidylserine containing membrane probe (i.e. DiD) in a appropriate buffer containing one or several ion-sensitive probe (see the Table in the detailed description of the invention) and volume probe (i.e. dextran-tetramethylrhodamin with molecular weight 2,000,000 Da). Extravesicular probes are cleared using centrifugation. Solutions of peptides (freshly prepared or aged to allow aggregation) are added to liposomes, followed by surface probe (i.e. Annexin V bound to Pacific Blue). After short incubation, test ions are added, and the mixture is analyzed on flow cytometer. Appropriate ionophores are used as a positive control for permeabilization to a specific ion, and a vehicle for peptide serves as a negative control.
The method for screening chemical entities for an ability to prevent membrane permeabilization induced by misfolding peptides through membrane channel formation will be used to find drug candidates to treat neurodegenerative diseases. For example, chemical entities able to prevent channel functioning induced by amyloid peptides can be effective in the prevention or in the treatment of Alzheimer's disease.
A suspension of liposomes with embedded ion-sensitive probes is prepared. To do that, liposomes with the diameter 200 or 400 nm are extruded from phosphatidylserine containing membrane probe (i.e. DiD) in a appropriate buffer containing one or several ion-sensitive probe and volume probe. Extravesicular probes are cleared using centrifugation. Solutions of peptide (freshly prepared or aged to allow aggregation) are added to liposomes, followed by surface probe (i.e. Annexin V bound to Pacific Blue). After short incubation, test ions are added, and the mixture is analyzed on flow cytometer. Appropriate ionophores are used as a positive control for permeabilization to a specific ion, and a vehicle for peptide serves as a negative control.
Chemical entity that significantly decrease the ratio of permeabilized liposomes to the total number of liposomes is considered effective against channel-mediated permeability of membranes.
Tested drug is added to the test system at various stages to dissect which step of channel formation and function is affected by the drug. First, the drug is mixed and pre-incubated with channel-forming peptide. Considering that the drug is present at all stages—formation of channel-forming units in the solution, incorporation of channels into the membrane, and when the membrane channel transports ion, this timing can be applied as a first screen—if there is no effect in this screen, the compound does not prevent permeabilization by any mechanism.
Alternatively, the drug is added and pre-incubated with liposomes. The channel-forming peptide is added in the presence of the drug. In this case, the drug can prevent the incorporation of the channel and the permeability of formed channel. The comparison with the previous timeline, will allow for an identification of drug effect on aggregation of peptide into channel-forming units.
Finally, channel-forming peptide can be added to liposomes first. If drug is added immediately before adding test ion, the drug can only affect the functionality of the formed channel. By comparing three sequences, it is possible to dissect the mechanism of anti-permeabilization effect of the drug. As it was mentioned, for the purposed of high-throughput screening, it will be logical to apply the first sequence (drug is co-incubated with the peptide), which allows to identify drugs which are not effective against membrane permeabilization by misfolding peptide by any mechanism.
The embodiment of the technique which includes flow sorting allows to separate liposomes which have ion channels from ones without channel. Essentially, it is functional purification of the protein in the form of channel. Formed channels are relatively stable, therefore, collected suspension of purified channels incorporated into liposomes can be stored at least for a limited time, and even transported to those who can use them for their own applications. Purified liposomes with channels can serve as a study object. They also can be used as a test object in screening applications if non-purified pool of liposomes contains too many other objects. The excessive number of other objects can be detrimental, for example, in detecting rare events or where total non-specific absorption on lipid could be an issue.
Purified channels incorporated into the liposomes can be used in various applications to develop macromolecules with affinity to channels (such as antibodies). The amount of purified peptide would be most likely not sufficient for typical immunization protocol, because each liposome contains only a single channel (essentially a single macromolecular complex to be targeted by antibody). However, those who are skilled in arts, can apply alternative techniques which can be effective with negligibly small amount of available antigen, such as phage-based technologies to generate affine molecules.
Generated macromolecules with a specific affinity to peptides in a channel form can be a therapeutic in the treatment of degenerative diseases which are caused by said peptides. Also, the antibodies can be a research and/or diagnostic tool to label this pathophysiologically relevant marker in biological samples.
After Aβ25-35 is added to the liposomal preparation, the effects of the peptide develop within the first minute. The incubation of liposomes with the peptide for up to one hour does not change the number of permeabilized liposomes. That means that the interaction of the peptide with the membrane occurs quickly and once inserted into the membrane, a peptide aggregate that already formed a channel is not able to affect other vesicles. It can be concluded that the solutions contain some amount of peptide which is ready to incorporate into membranes and form channels. We named such peptide aggregates “channel-forming units”.
There is a linear relationship between the number of added units (concentration of added peptide) and the number of liposomes permeabilized by the channels. Therefore, each permeabilized liposome carries a single channel, so the number of permeabilized liposomes reflects the number of formed channels and can be used as a test system.
Our core hypothesis of the etiology and pathogenesis of Alzheimer's disease (which we believe is relevant to other degenerative diseases) is that proteolytic enzymes digest long peptides into shorter fragments which are able to form membrane channels. Permeabilization of cellular membranes by channels initiates biochemical processes leading to cell death. In one of embodiments of this invention, the process of membrane channel formation from fragments produced by proteases from longer peptides is monitored.
A suspension of liposomes with embedded ion-sensitive probes is prepared. To do that, liposomes with the diameter 200 or 400 nm are extruded from phosphatidylserine containing membrane probe (i.e. DiD) in an appropriate buffer containing one or several ion-sensitive probe and volume probe. Extravesicular probes are cleared using centrifugation.
Proteases (pure enzymes, their mixtures, or biological samples with proteolytic activity) are added and mixed with the solutions of proteins. During incubation of resulting sample, the aliquots are taken over time. The aliquots are added to the liposomal preparations and the percentage of permeabilized liposomes is estimated. Considering that invented method provides the measurement of the number of channel-forming units in the sample, it is possible to observe the number of channel-forming units produced by fragments produced by proteases from long peptide.
Drugs, which are tested for the ability to modify proteolytic activity, can be added together with proteases to the long peptide. Drug-induced change of the number of permeabilized liposomes produced by products of the proteolytic digestion can be used to screen chemical entities with anti-degenerative properties.
| Number | Date | Country | |
|---|---|---|---|
| 63199982 | Feb 2021 | US | |
| 62704459 | May 2020 | US |
