Functional hole groom control

Abstract

The pore body is a main secretion structure of eukaryotic cells. The present disclosure introduces compositions and methods for controlling and modulating pore structures. Comprising a method for identifying pore-associated proteins and interacting small molecules. The use of small molecules targeting one or more porosomal proteins; compositions and uses of nanobodies conjugated to small molecules; reconstructing a hole body; and creation and use of the artificial pore structure.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application 63 / 396,040, filed on August 8, 2022; U.S. Provisional Patent Application 63 / 431,946, filed on December 12, 2022; U.S. Provisional Patent Application 63 / 444,451, filed on February 9, 2023; U.S. Provisional Patent Application 63 / 447,371, filed on February 22, 2023; U.S. Provisional Patent Application 63 / 458,471, filed on April 11, 2023; U.S. Provisional Patent Application 63 / 459,762, filed on April 17, 2023; U.S. Provisional Patent Application 63 / 472,674, filed on June 13, 2023; and U.S. Patent Application 63 / 523,970, filed on June 29, 2023, the entire contents of each of which are incorporated herein by reference. This application is a further CIP (Continuation - in - Part) of U.S. Patent Application 18 / 222,784, filed on July 17, 2023, the entire contents of which are incorporated herein by reference. Technical field

[0003] The present disclosure provides methods and compositions for modulating, including regulating or altering, the structure and function of porosome organelles and / or their constituent proteins. In some embodiments, porosome modulation methods can alter disease progression. In some embodiments, identified small - molecule modulators, or pharmaceutically acceptable salts or solvates thereof, can be used to modulate the activity of one or more porosome proteins, thereby affecting the structure and function of porosome organelles. Methods for treating porosome - related defects using such targeted molecules are also disclosed. Other embodiments include the functional reconstitution of porosomes or porosome - like structures in target cells to overcome physiological defects. Other embodiments further include the use of humanized nanobodies alone or in combination with small molecules to target one or more porosome proteins, thereby affecting changes in the structure and / or function of the porosome complex. Background art

[0004] Porosome organelles are cup - shaped supramolecular lipoprotein structures located on the cytoplasmic membrane. They are the sites where intracellular secretory vesicles transiently dock, fuse, and secrete their contents extracellularly.

[0005] The typical size range of the pore body structure is from 15 nm in neurons to 100 - 180 nm in endocrine and exocrine cells. The pore body consists of approximately 30 - 40 kinds of proteins, and the composition of the pore body depends on the cell type. Pore body-mediated secretion across the cytoplasmic membrane is a fundamental process for cells to communicate and exchange information with the environment. In a multicellular environment, pore body secretion enables cell communities to communicate and maintain homeostasis, thus maintaining life. The pore body exists in all secretory cells, from pancreatic acinar cells that secrete digestive enzymes to growth hormone and insulin-secreting cells that release hormones, mast cells, chromaffin cells, hair cells in the inner ear, and neurons that secrete neurotransmitters. The pore body has been immunoisolated outside of multiple cells (including pancreatic exocrine β cells that secrete insulin, human airway epithelial cells, and neuronal cells), has been identified biochemically, and has been functionally reconstituted in artificial lipid membranes. There has been a large amount of cumulative evidence regarding the role of pore body-associated proteins in cell secretion and secretion defects, including: neurotransmission and neurological diseases; respiratory diseases; and insulin secretion diseases. Therefore, cell secretion defects caused by dysfunction of the pore body or pore body components are related to many disease mechanisms, including cystic fibrosis, diabetes, Alzheimer's disease, Down syndrome, schizophrenia, digestive and immune system diseases, etc.

[0006] Although the biological "parts list" has been relatively clear and many proteins have been identified in human cells, the systematic-level understanding of disease patterns has only just begun to be fully emphasized. The following statements are no longer correct: Any given disease results from a bad copy of a single protein; or, in the face of epigenetics, the expression of a given protein structure only originates from a single gene. In addition, many proteins are found to be involved in multiple cellular processes together (for example, they are usually described as being visible in different metabolic or enzymatic pathways), making it difficult to target a single protein without altering multiple cellular functions.

[0007] Therefore, targeting a single protein (whether binding or degrading) may treat a disease, but it may also inhibit those basic body functions and / or other biochemical pathways that require that protein. This can lead to adverse side effects. In addition, each protein requires a "groove" or "binding pocket" for the supposed drug to grab and bind. In addition, this binding pocket on the target protein is also affected by multiple neighboring proteins, so a careful understanding of the entire complex is required for proper treatment. In addition, for small molecules, it is estimated that only 20% - 25% of proteins have the grooves required for binding and being modulated, inhibited, or activated.

[0008] Given the great need arising from the nature of diseases caused by secretory function defects, there is an urgent need for methods capable of modulating the structure or function of pore bodies to correct or improve secretory defects; and, as a result, to treat diseases caused by pore body secretory defects. In addition, in cases where the pore body structure is defective or absent, it is necessary to restore the pore body structure or provide a pore body or pore body-like structure to the affected cells. SUMMARY OF THE INVENTION

[0010] The present disclosure provides a method for identifying protein interactions that modulate pore body function or restore the function of specific pore body proteins. In some embodiments, high-throughput chemical screening techniques are employed. In other embodiments, computer simulation techniques are used. The identified proteins are then verified for activity and function in animal models and ultimately in humans. In some embodiments, the method involves: creating a first pore body sample mixture; then incubating the pore body sample mixture with a labeling group to produce a probe-protein complex. The probe-protein complex is then harvested and fragmented to produce protein fragments. The protein fragments are then analyzed by proteomic methods. The proteins in the pore body sample mixture can then be identified, thereby creating a first identified proteome. A value is assigned to each protein in the first identified proteome. In some cases, the assigned value can indicate the absolute amount (e.g., grams or moles) or relative amount (e.g., percentage or ratio) of that protein in the pore body sample mixture. The above steps are repeated for a second sample pore body mixture to obtain a second value for each protein in the second identified proteome. Then the ratio between the values of the paired proteins in the first and second identified proteomes is calculated. In some cases, it is the first value divided by the second value; in other cases, it is the second value divided by the first value. The resulting ratio thus determines the protein-protein interactions inside or near the pore body structure.

[0011] In some embodiments, the first sample pore body mixture is from a standard, control, or wild-type cell sample, while the second sample pore body mixture is from a test cell sample. Additionally, the test cell sample can be a knockout cell line.

[0012] In other embodiments, the first and second pore body cell sample mixtures can be selected from at least one of the following groups: cell samples, cell lysate samples, and isolated pore body proteins.

[0013] In other embodiments of the above method, the probe-protein complex is conjugated to a chromophore during the incubation step. In other embodiments, the probe-protein complex or a sub-sample thereof is separated by electrophoresis and visualized after incubation and before harvesting the probe-protein complex.

[0014] In some other embodiments of the above method, fragmentation is accomplished or effected by at least one selected from mechanical stress, pressure, and chemical fragmentation agents. In still other embodiments, the chemical fragmentation agent is a protease.

[0015] In other embodiments of the above method, proteomic methods include the use of mass spectrometry. In fact, in other embodiments, the proteomic method is at least one method selected from the group consisting of LC, LC-MS, MALDI-TOF, GC-MS, CE-MS, and NMR. In addition, in other embodiments, the values for each protein in the first and second identified proteomes are obtained by mass spectrometry analysis. In other embodiments, the value is the area under the curve of a signal intensity plot (as a function of mass-to-charge ratio). In other embodiments, the values for each protein in the first and second identified proteomes are related to the reactivity of Lys residues within the protein. In other embodiments, the identified protein-protein interactions can be confirmed with small molecules. The confirmation can be carried out by chemical cross-linking followed by confirmation of the linkage by mass spectrometry.

[0016] In some embodiments, the functions of the pore bodies from the first and second samples can be examined in an artificial lipid bilayer membrane.

[0017] In other embodiments, artificial pore bodies can be created and constructed in an artificial lipid bilayer membrane.

[0018] For all of the above, kits can be created to carry out one or more steps of the method.

[0019] The present disclosure includes methods of modulating prosome activity in a subject in need thereof, such as by administering to the subject an effective amount of an identified small molecule.

[0020] In some embodiments, the identified small molecule modulator or a pharmaceutically acceptable salt or solvate thereof is used to modulate the activity of one or more pore body proteins.

[0021] In some embodiments, the pore body protein can be, for example, a neuronal pore body protein; a mucus-secreting pore body protein of the respiratory epithelium; an insulin-secreting protein; a digestive enzyme-secreting protein; or a signal molecule-secreting protein.

[0022] In some embodiments, the present disclosure provides small molecules, such as molecules less than 1000 MW, which can target and regulate the production of one or more pore body constituent proteins by modulating, controlling the expression, methylation status, or interference (such as RNAi, siRNA) of the genomic sequences (DNA, RNA) encoding the constituent proteins. In some embodiments, host cells are harvested from a patient, treated with the small molecule to alter the pore body structure or function, and then returned to the patient.

[0023] In some embodiments, exosome release is controlled via altered structure and / or function of a pore complex or pore-associated protein. In some embodiments, one or more cell types can be "knocked out" of a gene for a putative pore complex or pore-associated protein using CRISPER, RNAi, or other methods known in the art.

[0024] In some embodiments, the proteome and lipidome databases are interrelated to identify candidate interaction sites. Protein-protein or protein-lipid candidates are chemically cross-linked and evaluated by mass spectrometry to confirm the interaction. After confirming the interaction, a multivalent small molecule is created. This multivalent molecule can, for example, contain one or more peptide target sequences attached to the cross-linked molecule. This cross-linked molecule is further configured to keep the peptide target sequence in the correct direction and distance to allow the peptide target sequence to bind to at least two pore body related targets, thereby resulting in modulation of pore body function. The validation of pore body function modulation is then performed through the standard drug validation pathway from animal models to human clinical trials. For example, the validation can be performed by a computer evaluation of the binding affinity of the target pore body protein and lipid, and then by cell culture, organoids and animal studies.

[0025] In some embodiments, pore bodies isolated from some tissues or cell types in artificial lipid bilayer membranes can also be used to screen, optimize dosage, or determine the efficacy of candidate drugs. In some embodiments, isolated pore bodies in artificial lipid bilayer membranes can be administered to subjects suffering from pore body-mediated diseases (e.g., diseases known or implicated in altered secretory function, such as cystic fibrosis, diabetes, Alzheimer's disease, etc.).

[0026] Some embodiments relate to a method of modulating the activity of a protomer in a subject in need thereof. In such embodiments, an effective amount of an identified small molecule is administered to a subject in need thereof.

[0027] In some embodiments, the identified small molecule modulators, or pharmaceutically acceptable salts or solvates thereof, can be used to modulate the activity of one or more poromyosin proteins.

[0028] In some embodiments, the pore protein may be a neuronal pore protein. In further embodiments, the pore protein may be from an airway epithelial cell. In further embodiments, the pore protein may be an insulin secretory protein. In further embodiments, the protein may be a digestive enzyme secretory protein. In further embodiments, the pore protein may be a signaling molecule secretory protein from a signaling molecule secreting cell.

[0029] In some embodiments, one or more small molecules can target and modulate the production of one or more pore body constituent proteins by regulating, modulating, controlling the expression, methylation status, or interfering (e.g., RNAi, siRNA) of one or more amino acid chains (DNA, RNA) encoding the constituent proteins. In some embodiments, host cells can be harvested from a patient, incubated, amplified, purified, treated with small molecules to alter the pore body structure or function, and then returned to the patient.

[0030] In additional embodiments, the present disclosure provides an engineered nanobody that is used alone or in combination with one or more small molecules to alter the function of the pore body. For example, a nanobody targeting a first pore body protein binding site can be used in combination with one or more small molecules that interact with one or more other pore body proteins. In other embodiments, an artificial pore body is crosslinked to a humanized nanobody to bind to one or more domains of one or more pore body or pore body-associated proteins. The crosslinked artificial pore body nanobody can then be delivered to a subject.

[0031] In other embodiments, a composition of at least one crosslinking molecule and at least one small molecule modulator targeting a pore body protein is provided. In some embodiments, the crosslinking molecule is an ELP deblocker. In some other embodiments, the crosslinking molecule is p-acetylphenylalanine. In some other embodiments, the crosslinking molecule is maleimide. In some other embodiments, the small molecule modulator is CDN1163. In some other embodiments, there is at least a second small molecule modulator. In some other embodiments, the second small molecule modulator targets pore body lipids. In some embodiments, the targeted pore body protein is at least one selected from the group consisting of synaptotagmin-1A, SNAP-25, SNAP-23, and actin. In some other embodiments, the small molecule modulator is a modulator targeting the protein identified by the above method. In some other embodiments, a nanobody is provided that is humanized to target and bind to one or more domains of one or more pore body proteins. In some other embodiments, the humanized nanobody contains an artificial cysteine that is constructed to be able to attach an ELP deblocker to the nanobody. In some other embodiments, the nanobody is further linked to one or more small molecules to form a multivalent structure.

[0032] In some other embodiments, the present disclosure provides humanized nanobodies having one or more domains targeting one or more porins and a small molecule with an engineered cysteine. The ELP diblock binds to the cysteine and binds to pAcF. In some embodiments, a drug is attached to pAcF. In some other embodiments, the drug attached to pAcF is doxorubicin. In some other embodiments, one or more domains of one or more porins are identified as interacting with one or more porins found to interact with each other. In still other embodiments, the one or more domains further include at least one selected from the group consisting of: K+ channels, aquaporin water channels, anion exchangers, membrane fusion proteins, sodium bicarbonate transporters, Gαi3, synaptotagmin-1A, SNAP-25, SNAP-23, and actin.

[0033] The present disclosure also provides a method that includes extracting porins from a non-human source. The porins are then reconstituted into human cells. In some embodiments, the porins are extracted from human epithelial cells and / or stem cells. In some other embodiments of the method, the extracted porins are reconstituted into organoids or artificial lipid bilayers. In some other embodiments, the non-human source is a pig or other mammal.

[0034] The present disclosure also provides a method of identifying small molecules that target specific porins to modulate or restore porin function. In some embodiments, single CRISPR knockouts of selected porins are used to determine which other proteins are lost from the porin complex in addition to the knocked-out protein. Those other proteins that are lost from the porin complex are considered to be related to each other and to the knocked-out protein in the complex.

[0035] Some embodiments include methods of modulating porin-mediated insulin secretion in a subject in need thereof. In such embodiments, porins effective to synthesize and secrete insulin under a glucose challenge are induced to be overexpressed in the subject, and / or an effective amount of an identified small molecule for synthesizing and secreting insulin under a glucose challenge is administered to the subject. The porin of this embodiment can be the porin of the pancreatic β cells of the subject.

[0036] In some embodiments, overexpression of the insulin-secreting porin ATP2C1 (ATPase secretory pathway Ca2+ transporter) or APOa1 alone or together increases the expression and secretion of insulin in β cells of the endocrine pancreas.

[0037] In some embodiments, the Ca2+-ATPase activator CDN1163 is used to increase insulin expression and its secretion in β-cells of the endocrine pancreas.

[0038] In some embodiments, the present disclosure provides a method for reconstructing a functional connexon complex into living cells to improve or correct a secretory defect resulting from a dysfunction of one or more connexins. In some embodiments, the reconstruction is performed on neuronal cells or cells from the exocrine and endocrine pancreas. In other embodiments, other cell types include mucin-secreting connexon complexes in the lung epithelium of patients with cystic fibrosis (CF).

[0039] In another embodiment, the present disclosure provides a method for large-scale isolation of mucin-secreting connexon complexes from human lung epithelial cells. In a specific embodiment, the method enables the isolation of mucin-secreting connexon complexes from Calu3 and other epithelial cells used in reconstructive therapy in CF patients. This can prevent immune rejection and can improve secretory defects resulting from the dysfunction of the connexin-related cystic fibrosis transmembrane conductance regulator (“CFTR”) protein.

[0040] In another embodiment, the present disclosure provides a method for identifying protein-protein interactions within a functional connexon complex in a cell. Specifically, the identification of protein-protein interactions within a mucin-secreting connexon complex in airway epithelium with the CFTR protein is disclosed. Accordingly, there are also embodiments that enable the fine-tuning of the regulation of the mucin-secreting connexon secretory mechanism in lung epithelial cells and its precise targeting with small molecule-drug-nanobody complexes.

[0041] In another embodiment, there is a method for identifying one or more modulators of one or more mucin-secreting connexin proteins to optimize mucin production and secretion in airway epithelial cells. These mucin-secreting connexin protein modulators can be used alone or in combination with reconstructed connexon complexes in CF therapy.

[0042] The present disclosure also provides a method for appropriately identifying and matching small molecules to target specific connexin proteins to modulate or restore mucin-secreting connexon function. In some embodiments, CRISPR is used to knock out one connexin protein at a time to determine which other proteins are lost from the connexon complex in addition to the knocked-out protein, particularly the CFTR protein. In some embodiments, those proteins that are lost from the connexon complex when CFTR is knocked out are classified as CFTR-related proteins in the complex. In some embodiments, the identified associated proteins are targeted to modulate and improve secretory function and correct CFTR-mediated secretory defects.

[0043] Some embodiments relate to a method of modulating protic-mediated mucin secretion activity in a subject in need thereof. In such embodiments, effective reconstitution of functional mucin secretion and / or an effective amount of an identified small molecule is administered to airway epithelial cells of a subject in need thereof to assist in the proper secretion of mucin. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The features and advantages of embodiments of the present invention become apparent by reference to the following detailed description when read in conjunction with the accompanying drawings, which are exemplary and not restrictive, wherein:

[0045] FIG. 1 shows examples of porosome structures in exocrine pancreas, neurons, and pituitary growth hormone-secreting cells in different imaging modes.

[0046] Figure 2 A schematic diagram of porosome-mediated secretion is shown.

[0047] Figure 3 A schematic diagram of an embodiment of the present invention is shown; the porosome complex and its precise targeting for therapy.

[0048] FIG. 4 shows a schematic diagram of the predicted interactions between identified proteins within the neuronal porosome proteome and other regulatory proteins.

[0049] Figure 5 is an example of drug targeting specificity, which utilizes small molecule drug combination design and also tissue-specific and porosome-specific multivalent nanobodies.

[0050] Figure 6 Depicts porosome-associated proteins associated with different types of diseases.

[0051] FIG. 7 shows knockout (KO) and overexpression (OE) of porosome proteins ATP2C1 and APOa1 in insulin-secreting Min6 cells.

[0052] Figure 8 Shows glucose-stimulated insulin secretion under knockout (KO) and overexpression (OE) of three porosome proteins ATP2C1, APOa1, and TREK1 in Min6 cells. Overexpression of APOa1 results in increased synthesis and secretion of insulin.

[0053] Figure 9 Shows glucose-stimulated insulin secretion in Min6 cells after exposure to increasing concentrations of the ATP2C1 activator CDN1163 for 2 hours. 10 micromolar CDN1163 was found to be the optimal dose.

[0054] Figure 10 presents representative electron micrographs of cultured Calu-3 cells, confirming the presence of microvilli (MV) and 100-nm cup-shaped pore bodies (P) on the plasma membrane.

[0055] Figure 11 The example Ussing chamber experiment showed that forskolin-stimulated chloride release from Calu-3 cells was inhibited by the presence of the CFTR inhibitor GlyH-101.

[0056] Figure 12 demonstrates that the CFTR inhibitors 172 and GlyH-101 inhibit the forskolin-stimulated secretion of intravesicular mucin from Calu-3 cells.

[0057] The transmission electron micrograph of Figure 13 presents a pore body complex associated with docking secretory vesicles located at the apical end of exocrine pancreatic acinar cells.

[0058] Figure 14 presents transmission electron micrographs of insulin-secreting Min6 cells.

[0059] The electron micrograph of Figure 15 shows a reconstructed exocrine pancreatic pore body complex in liposomes. Note that once reconstructed, cup-shaped pore bodies are formed.

[0060] Figure 16 shows that a pore body complex from the exocrine pancreas reconstructed in a lipid bilayer is functional.

[0061] Figure 17 presents the appearance of immunoreactivity enrichment of TREK-1, Gi3, and Syntaxin-1A in insulin-secreting Min6 cells reconstructed with pore bodies.

[0062] Figure 18 shows a reconstructed insulin-secreting pore body in live Min6 cells, demonstrating increased glucose-stimulated insulin secretion.

[0063] Figure 19 shows that in Min6 cells reconstructed with pore bodies, immunoreactivity enrichment of Gi3 and Syntaxin-1A appears even after 48 hours, and glucose-stimulated insulin secretion increases.

[0064] Figure 20 The schematic diagram shows the delivery of a functional mucin-secreting pore body complex to airway epithelial cells of CF patients and the improvement of CF disease symptoms.

[0065] Figure 21Shows immunoblot analysis of total Calu-3 cell homogenate (CH) and isolated pore body complex (P), demonstrating the presence of several pore body proteins, namely actin, Gαi3, and vimentin. Notably, these proteins are enriched in the pore body complex.

[0066] Figure 22 Shows CFTR-associated pore body complex immunoisolated using CFTR-specific antibodies. Notably, pull-down of pore body-associated proteins such as synaptotagmin-1A (present as a 70 kDa t- / v-SNARE complex), SNAP-25 (present as a 70 kDa t- / v-SNARE complex), SNAP-23 (present as a 68 kDa t- / v-SNARE complex), and actin.

[0067] Figure 23 Shows that in ΔF508-CFTR human CF bronchial epithelial cells, mucus secretion is restored by CDN1163.

[0068] Figure 24 Shows that in ΔF508-CFTR human CF bronchial epithelial cells, mucus secretion is increased by α-CPA.

[0069] Figure 25 The schematic diagram shows some interactions between CFTR and associated proteins. DETAILED DESCRIPTION OF THE INVENTION

[0071] Identification of Pore-Protein Interaction Targets

[0072] Modern drug discovery is the result of the evolution of past technologies; from screening for active substances in biological extracts directly from nature, to the emergence of molecular biology techniques for generating drugs targeting specific molecular targets, to the modern drug R & D paradigm of "rational drug design" created in the 1970s and 1980s, which focuses on small molecules and antibodies (for blocking specific functions of individual protein targets) and recombinant protein-based therapeutic agents. Subsequently, breakthroughs in genomics and gene editing technologies, stem cell biology, patient-derived organoids, cryo-electron microscopy, synthetic biology, and small molecule screening and image analysis using AI machine learning and high-throughput screening have further transformed the structure of drug discovery. In fact, with the help of AlphaFold2 and its ilk of AI models, the final folded structure of a given protein has become predictable and can be easily verified based on a given gene sequence. In addition, such models also allow the development of so-called designer proteins not seen in natural environments.

[0073] Despite these breakthroughs, as the National Cancer Institute of the United States has pointed out, a shocking fact is that 85% of human proteins are not druggable (i.e., these proteins, including disease-causing proteins, cannot be targeted pharmacologically). In fact, it has been pointed out that "the number of new drugs has been declining because it has become increasingly difficult to create new drugs. In other words, we have run out of proteins that can be targeted with drugs. The remaining targets are 'undruggable'. Most of the proteins that drive the disease process are undruggable." (Brent Stockwell, Columbia University). The American Society of Clinical Oncology further points out that "the pharmacological targeting of refractory proteins is now a key challenge in modern drug development and requires innovation and the development of new technologies."

[0074] To address the huge obstacle of "undruggable" proteins to drug development, a few innovative methods have been tried. One approach is protein degradation targeting, which means tagging disease-causing proteins for destruction to prevent their function. The second method is to create a chemical proteomics platform for identifying previously unknown binding pockets in undruggable targets in an attempt to generate various small molecule therapies across indications.

[0075] In sharp contrast to the above two methods, the embodiments of the present invention cover a completely different third method; this method adopts a system-level approach, targeting multiple proteins of the cell communication mechanism, such as the pore body and its ability to alter cell secretion activity. This method solves the problems of specificity and precise targeting, while overcoming the "undruggable" problem, and provides a unique way to address previously difficult-to-treat diseases (including cystic fibrosis, diabetes, Alzheimer's disease), as well as cancers caused by the dysfunction of proteins and lipids involved in cell secretion. The uniqueness of the present invention lies in its pioneering focus on targeting druggable and undruggable proteins and lipids that cause secretion defects and thus lead to diseases such as cystic fibrosis, cancer, neurological, endocrine, and immune diseases. Therefore, the embodiments of the present invention control or alter the course of the disease by targeting multiple proteins within a dysfunctional target functional cell complex (such as the pore body structure). In some embodiments, the target functional cell complex is the pore body. In some other embodiments, multiple proteins in the pore body are targeted. In some other embodiments, one or more additional identified proteins not found in the pore body are also identified and targeted. The advantages of this precise targeting result in the creation of precise therapies, with the expectation of overall reduced treatment side effects and increased efficacy. Figure 1 shows examples of pore body structures in exocrine pancreas, neurons, and neuroendocrine cells under different imaging modalities.

[0076] As shown in Figure 1 (to be described in further detail below), the pore body is a cup-shaped supramolecular lipoprotein structure in the cytoplasmic membrane of eukaryotic cells. Secretory vesicles transiently dock and fuse with the pore body complex during vesicle fusion and content release during secretion. The pore body structure consists of many proteins that are themselves often involved in other cellular complexes. Examples of purified synaptic pore body proteins are shown in Table 1. More examples of complex pore body proteins can be found in the general technical literature in this field. Since the pore body is embedded in the lipid bilayer, pore body proteins can be entirely on one side or the other of the membrane or can span the membrane.

[0077] As Figure 2 shown, the secretory vesicle membrane transiently fuses at the base of the pore body via SNARE proteins to form a fusion pore or continuity for the release of vesicle contents from the cell. After secretion is complete, the fusion pore temporarily formed at the base of the pore body is resealed. The pore body is only a few nanometers in size and contains various different types of proteins, especially channels for chloride and calcium, actin, and SNARE proteins that mediate the docking and fusion of vesicles with the cell membrane. Once the secretory vesicles dock with SNARE proteins, they expand, thereby increasing their internal pressure. Then, they transiently fuse at the base of the pore body, and these compressed contents within the secretory vesicles are expelled from the cell. Examination of the cell after secretion using an electron microscope shows that partially emptied vesicles increase after secretion. This suggests that during secretion, only a portion of the vesicle contents can be expelled from the cell. This is only possible if the vesicles transiently establish continuity with the cytoplasmic membrane, expel a portion of their contents, then detach, reseal, and withdraw into the cytosol (endocytosis). In this way, secretory vesicles can be reused for subsequent rounds of exocytosis-endocytosis until their contents are completely emptied. In rapidly secreting cells such as neurons, neurotransmitter transporters on the secretory vesicle membrane can refill the vesicles after their release during neurotransmission.

[0078] The size of the pore body varies depending on the cell type. For example, the pore body diameter in exocrine pancreas, endocrine, and neuroendocrine cells ranges from 100 nm to 180 nm, while the pore body diameter in neurons ranges from 10 nm to 15 nm (about 1 / 10 the size of pancreatic pore bodies). When secretory vesicles containing v-SNARE dock at the bottom of the pore body containing t-SNARE, a membrane continuity is constructed by forming a t- / v-SNARE loop complex between the two opposing membranes. The size of the t / v-SNARE complex is proportional to the size of the vesicle. Secretory vesicles usually contain dehydrins (inactive), which are activated once they are hydrated. GTP is necessary for transporting water through aquaporins or water channels and for transporting ions through ion channels in order to hydrate the vesicle water before secretion. Once the vesicles fuse at the bottom of the pore body, the contents of the vesicles are expelled from the cell under high pressure.

[0079] Generally, the kinetics of pore body opening to the outside of the cell is regulated by actin and unconventional myosin; however, neurons that require a rapid response have a centrally moving plug that can open and reseal the t- / v-SNARE-induced continuity between synaptic vesicles and the bottom of the pore body, thereby releasing neurotransmitters. The pore body has been shown to be a universal secretory mechanism in various cells. The neuronal pore body proteome and its detailed structure have been resolved by solution x-ray, providing the composition and possible molecular architecture of the said mechanism. (Table 1)

[0080] Table 1: Composition and possible molecular architecture of the said mechanism

[0081]

[0082]

[0083] An example of the pore body structure is shown in Figure 1, which depicts examples of pore bodies in exocrine pancreas, neurons, and pituitary growth hormone-secreting cells. Figure 1A is an electron micrograph of a single pore body on the apical plasma membrane (PM) of a pancreatic acinar cell, showing the pore body membrane (POM, yellow arrow) associated with the membrane of secretory vesicles called zymogen granules (ZGM). The circular structure (blue arrow), most likely actin-myosin, forms the neck of the pore body complex. Figure 1B is an atomic force microscopy (AFM) micrograph of the apical surface topology of a live pancreatic acinar cell, showing four openings or pore bodies (one indicated by the yellow arrow). The diameter of each pore body in the exocrine pancreas ranges from 100 - 180 nm. Figure 1CIs an electron micrograph of a neuronal pore body (red arrow) with a docked synaptic vesicle (SV) at its base, located in the presynaptic membrane (Pre-SM) of a nerve terminal. Note the central plug in the pore body complex. Figure 1D Is an AFM micrograph of a neuronal pore body on the presynaptic membrane in isolated synaptosomes. Note the central plug (red arrow). The neuronal pore body is an order of magnitude smaller (10 - 17 nm in diameter) compared to pore bodies in the exocrine pancreas. Figure 1E Is an electron micrograph of a pore body next to microvilli (MV) on the apical plasma membrane (PM) of a pancreatic acinar cell, with docked secretory vesicles or ZGs. Figure 1F Is an AFM micrograph of the apical surface topology of living GH (growth hormone) cells of the porcine pituitary, showing pore bodies (black circular openings) with diameters of 100 - 180 nm. Images from: Proc Natl Acad Sci 94:316 - 321 (1997); Biophys J 85:2035 - 2043 (2003); Cell Biol Int 28:699 - 708 (2004); J Microscopy 232:106 - 111 (2008); Endocrinology 143:1144 - 1148 (2003), the entire contents of which are incorporated herein by reference for the teachings contained therein.

[0084] As described above, the biological "parts list" is relatively well - defined and many proteins have been identified in human cells. However, a systematic - level understanding of disease patterns has only just begun to be fully appreciated. Many individual proteins are involved in multiple cellular processes (e.g., seen in multiple enzyme - activity pathways), so it is difficult to treat diseases by targeting a single protein type without compromising multiple cellular functions. However, combinations of some proteins (and their associated binding partners, coenzymes, etc.) provide a higher level of specificity for any specifically chosen biological process. Embodiments of the present invention provide the ability to selectively modulate specific biological processes by targeting protein combinations that perform specific biological functions within molecular complexes. This allows for the precise targeting of drugs at the "system - level" physiology, inhibiting or stimulating cellular processes rather than individual proteins or lipid molecules.

[0085] An example of a system-level cellular process is secretion through the pore body complex (the general secretion mechanism of cells). The pore body enables communication (language) between cells in the body by secreting chemical messages (such as neurotransmitters from nerve cells or hormones from endocrine cells, such as insulin from beta cells of the endocrine pancreas). These chemical messages are stored in secretory vesicles within the cell. The pore body secretion mechanism consists of more than 30 proteins that give instructions to the secretory vesicles to appropriately dock at the bottom of the pore body, fuse, expand, and release a certain amount of the vesicle contents to the outside. No single component of the pore body is unique to the pore body. Instead, more than 30 proteins together combine in the appropriate conformation in the complex to provide the function for this structure. Accordingly, embodiments of the present invention relate to correcting cellular function by targeting specific polymers (such as the pore body complex) without altering the activity of other cellular complexes and thus without altering other cellular processes that have one or more individual components with the target structure.

[0086] To be able to precisely target the proteins within the pore body complex, it is crucial to understand the protein-protein interactions within the pore body of a certain tissue or cell type. To achieve this, embodiments of the present invention use a method called "interactomics". Users of the embodiments of the interactomics process described herein can determine the protein-protein interactions within the pore body complex. Then, a person of ordinary skill in the art can verify such interactions by knocking out pore body proteins one by one via CRISPR. Then, those using the skills taught herein as well as standard techniques in the art can determine which other proteins are lost from the pore body complex in addition to the knocked-out proteins. We teach that the proteins additionally lost from the pore body complex are proteins that are associated with each other within the complex. Such systematic studies allow one to decipher the protein distribution within the pore body complex and help target specific proteins within the complex to modulate and improve secretory defects and the resulting diseases.

[0087] In some embodiments of the present invention, a non-volatile computer-readable storage device executes machine learning algorithms, such as algorithms known and trained on a small molecule database, and these small molecules are cross-referenced to target multiple proteins within the pore body complex. Other embodiments utilize existing "bivalent" or "trivalent" forms of small molecules with appropriate nanobody presentation for targeted delivery. Such a drug delivery system will help with precise targeting to modulate protein function only when binding to two or more target protein targets. The nanobody can further be used as the basis for a crosslinking molecule.

[0088] As a non - limiting example, superparamagnetic iron oxide (SPIO) nanoparticles have the ability to rapidly enter and release the cancer drug doxorubicin into cancer cells. Dextran - coated SPIO nanoparticle ferrofluids, which function with red - autofluorescent doxorubicin and the green - fluorescent dye fluorescein isothiocyanate as a reporter, can track intracellular nanoparticle trafficking and drug release. Such engineered nanoparticles increase the rate of drug entry and release in human pancreatic cancer cells by >20 - fold and have therapeutic potential as an advanced drug delivery and imaging platform.

[0089] Just as the letters in an alphabet form meaningful words in the correct combination, it is almost impossible to search for a word using only one of its constituent letters. However, if two or more of the letters that make up the word are used in the search, especially if the letters are arranged in the order in which they are used in the word, the likelihood of identifying the word is greatly increased. Extending the general meaning of this metaphor further, and not being completely limited to any single theory of operation; similarly, as Figure 3 shown in the schematic example of the pore complex, when a drug is engineered to target and bind to multiple protein targets within a protein complex, the probability that the drug will find its specific cellular target complex is greatly increased. For example, in Figure 3 a hypothetical drug that targets multiple pore - body proteins is created by linking different compounds (yellow triangle, red square, blue circle) that interact with two or more adjacent protein targets to each other. Thus, the yellow - triangle / red - square combination can bind to two adjacent yellow - red pore - body proteins without affecting the blue pore - body protein. Similarly, the red - square / blue - circle combination does not affect the yellow protein. Therefore, for the design and creation of such "targeted" molecules, it is important to clarify and understand whether the targeted proteins are adjacent or otherwise interact with each other.

[0090] Each protein in the pore complex (i.e., the "word") is considered a different letter in the alphabet. The correct combination of each protein letter with the others forms the pore - body word. Any mismatch or alteration of a protein letter results in a misspelling of the word "pore complex" and thus leads to disease.

[0091] In one embodiment of the present invention, candidate protein - protein interactions within a cellular functional complex (such as a pore complex) are identified using the continuously updated STRING (Search Tool for the Retrieval of Interacting Genes / Proteins) database. STRING is a database of known and predicted protein - protein interactions. The interactions include direct (physical) and indirect (functional) associations; they result from computational predictions, knowledge transfer between organisms, and interactions aggregated from other (primary) databases. Currently, the STRING database has at least 20 billion known protein - protein interactions involving nearly 68 million proteins.

[0092] For example, FIG. 4 is a schematic diagram depicting the interactions between the identified proteins within the predicted neuronal pore proteome and other regulatory proteins. Due to space limitations, Table 2 below lists the proteins identified by numerical characters in FIG. 4. These interactions were generated using STRING 9.0 from the input of the identified proteins in the neuronal pore. STRING 9.0 is a database of known and predicted protein interactions. These interactions include genomic, high-throughput, conserved co-expression, and previously known direct (physical) and indirect (functional) associations.

[0093] Two clusters of protein-protein interactions identified in the pore complex are exemplified. The cluster on the left, and most likely present at the apex of the pore cup, are cytoskeletal structures and signaling proteins. The cluster on the right represents proteins mainly involved in membrane fusion, including SNARE proteins and calcium channels, and thus their location should be at the bottom of the pore cup, facing the cytoplasm. Interestingly, the heterotrimeric form of the GTP-binding protein and the GTP-binding membrane fission protein dynamin (Dnm2) are present in the left cluster. The presence of dynamin in the left cluster is not too surprising as they are microtubule-associated proteins and intersectin 1 is also known to interact with dynamin. However, their involvement in the fission of the neck of the fused vesicles at the bottom of the pore requires their presence at the bottom of the pore. The confidence of the predicted functional interactions shown is >99%.

[0094] After identifying the protein-protein interactions of interest, mass spectrometry chemical cross-linking is then performed for: experimentally confirming the in silico interactions; further determining the interaction domains in 3D; and identifying possible drug sites. Thus, practitioners of the embodiments of the present invention can design and present small molecules, or small molecules attached to carrier molecules, which are designed to simultaneously bind multiple protein targets in a protein complex and functionally modulate the physiological function of the protein complex.

[0095] Table 2: Proteins referred to by numbers in FIG. 4

[0096]

[0097]

[0098] The small molecules identified as candidate drugs after going through the above process can then be further verified for drug suitability (safety, efficacy, and stability) according to traditional cell culture, organoid, and animal research pathways, and subsequently clinical trials are conducted on the candidate drugs.

[0099] In an example of the above method, mass spectrometry was performed on immunoisolated pore body complexes from a human bronchial epithelial cell line, namely the control WT-CFTR human bronchial epithelial cell line (CFBE41o-6.2), as a control or wild-type cell sample, and the experimental ΔF508-CFTR human CF bronchial epithelial cell line (CFBE41o), a ΔF508del(- / -) homozygous deletion, as a test cell sample. Table 3 below lists the proteins identified as associated with the pore body complex. All quantifications are relative; note, however, that in this example, the ratio is calculated as test / WT. Samples were digested with trypsin and analyzed on an Orbitrap Eclipse MS system. The data were analyzed in Proteome Discoverer 2.4 using the Sequest and Percolator algorithms. The values shown represent quantification based on a plurality of consensus. Note that in the pore body complex from the ΔF508-CFTR cell line (test cell sample), the Ras GTPase-activating-like protein IQGAP1 is absent. This suggests that the IQGAP1 gene product may interact with the CFTR protein in the pore body complex in its native state. Thus, upregulating pore body GTPase activity may be used as a CF (cystic fibrosis) therapy. Similarly, a practitioner taught herein may select a pore body GTPase as a pore body target protein to be acted upon by a humanized nanobody, a small molecule, or both.

[0100] Table 3: Mass spectrometry analysis of pore bodies isolated from the WT-CFTR human bronchial epithelial cell line and the experimental ΔF508-CFTR cell line ΔF508del(- / -)

[0101]

[0102]

[0103] *Note that in the pore body complex from the ΔF508-CFTR cell line, the Ras GTPase-activating-like protein IQGAP1 is absent, suggesting interaction with the CFTR protein within the native pore body complex.

[0104] Figure 6 Depicts pore body proteins that play a role in specific disease categories. Figure 6 Further depicts some of the disease scopes that can be addressed using the embodiments of the pore body-targeted tissue-specific drug design, development, and delivery platform detailed herein. Secretory function defects are addressed by the pore body. Secretory defects can be caused by defects in one or more pore body proteins, resulting in hypersecretion (such as in some cancers) or attributable to a reduction or loss of proper secretion (such as in diabetes or cystic fibrosis).

[0105] Hypersecretion can be treated with specific small molecules that bind to and interact with one or more porins to ameliorate the defect. For example, t-SNARE or v-SNARE nanobodies can be used to modulate the reduction in secretion.

[0106] In cases of insufficient or defective porosome function, an entire functional porosome can be reconstructed in the defective tissue (e.g., lung epithelium in the case of cystic fibrosis). In some embodiments, for reconstruction, porosomes are extracted from porcine or human sources and placed into human cells. In other embodiments, a nanoscale porosome complex for reconstruction is obtained from CALU 3 or other human airway epithelial cells to treat cystic fibrosis. Those skilled in the art can readily envision other cell types suitable for reconstruction upon reviewing porin proteins and their role in disease, at least as disclosed herein. Thus, reconstruction therapy involves reconstructing or introducing a normally functioning CFTR-associated secretory porosome complex (the entire 100 nm porosome complex containing the CFTR protein) onto the plasma membrane of the lung epithelium of CF patients. Reconstruction addresses different CFTR mutations. Reconstruction therapy will improve the mucus secretion defect caused by the mutant dysfunctional CFTR.

[0107] Table 4 below details the classes of porin proteins and their alleged role in disease.

[0108] Table 4: Classes of Porin Proteins and Their Alleged Role in Disease

[0109]

[0110]

[0111] Neuronal Pore-Protein

[0112] As shown in Table 4, in Alzheimer's disease, the proteins 2,3-cyclic nucleotide phosphodiesterase (CNPase) and heat shock protein 70 (HSP70) are thought to play a role in disease pathology. Increased levels of CNPase and HSP70 (both present in the neuronal porosome complex) were found, while the level of porosome-associated dihydropyrimidinase-related protein 2 (DRP-2) was decreased. Similarly, the porin proteins SNAP-25 and synaptophysin were significantly reduced in neurons of Alzheimer's disease patients.

[0113] Reduced CNPase levels have been reported in the frontal and temporal cortices of Alzheimer's disease and Down syndrome patients. Lower CNPase levels have also been detected in the prefrontal cortex of schizophrenia patients. In addition, an allele associated with low levels of CNPase has been reported to be associated with schizophrenia.

[0114] Examples of neuronal pore body proteins can include: tubulin β, myosin 7b, spectrin, creatine kinase, dystrophin, langerin, GTPase-activating protein (GAP), intersectin type 1 (ITSN-1), cytoplasmic actin type 1, sodium / potassium-transporting ATPase subunit α-3, plasma membrane calcium-transporting ATPase 1, plasma membrane calcium-transporting ATPase 2, brain acid-soluble protein 1, adenylate cyclase-associated protein 1, 2′,3′-cyclic nucleotide 3′-phosphodiesterase, dihydropyrimidinase-related protein 2, dihydropyrimidinase-related protein 3, dihydropyrimidinase-related protein 5, glutamine synthetase, guanine nucleotide-binding protein G(o) subunit α, neural cell adhesion molecule 1, vesicular fusion ATPase, Ras-related protein Rab-3A, reticulon-3, reticulon-4, synaptosome-associated protein 25, syntaxin-1A, syntaxin-1B, syntaxin-binding protein 1, synapsin-2, synaptophysin, synaptotagmin-1, tubulin α-1A chain, vesicle-associated membrane protein 1, vesicle-associated membrane protein 2, brain-type V proton ATPase subunit B. Embodiments of the present invention can include one or more identified small molecules that directly act on one or more of the above proteins to affect neuronal pore body structure and / or function.

[0115] Insulin-Secreting Pore-Protein - Diabetes

[0116] According to data from the Centers for Disease Control and Prevention (CDC) in the United States, more than 37 million Americans have diabetes (1 in every 10 people), and approximately 90-95% of them have type 2 diabetes. Although type 2 diabetes typically occurs in people over 45 years old, the number of children developing this disease is increasing. The hormone insulin is produced and secreted by β cells in the endocrine pancreas. Insulin acts on the cells of the body to allow sugar to enter and be used as energy. In type 2 diabetes, the cells do not respond to insulin, a condition known as insulin resistance. As a result, the β cells produce more insulin in an attempt to get the cells to respond. Eventually, the β cells are unable to increase production to keep up with demand, and blood sugar levels rise, setting the stage for type 2 diabetes. High blood sugar levels damage physiological functions and can lead to serious health problems such as cardiovascular disease, blindness, kidney disease, and dementia. Type 2 diabetes can be controlled through a healthy diet and medications, but these methods alone are often not completely successful because current medications often cause adverse side effects.

[0117] After a meal, the exocrine pancreas secretes digestive enzymes to aid in food digestion. As blood glucose rises after digestion, it triggers the β-cells of the endocrine pancreas to secrete insulin. Insulin stored in the secretory vesicles of β-cells is released upon glucose stimulation, which is due to the complete collapse of the vesicle membrane at the plasma membrane or the temporary fusion of the secretory vesicles at the bottom of the plasma membrane-associated pore bodies. In some embodiments of the present invention, the functional reconstruction of the pore body complex that secretes insulin in living β-cells of the endocrine pancreas opens a window for the treatment of diabetes.

[0118] Therefore, there are mainly two problems in type 2 diabetes: one is that the β-cells in the pancreas cannot produce enough insulin; the other is that the cells in the body do not respond well to insulin and cannot allow sugar to enter the body. Currently, type 2 diabetes cannot be cured. It can only be controlled through diet, physical activity, and the following available medications:

[0119] Metformin ( etc.) is usually the first-choice prescription drug for treating type 2 diabetes. Its main function is to reduce glucose production in the liver and increase the body's sensitivity to insulin, thus making more effective use of insulin. It is well known that metformin inhibits insulin secretion in the β-cells of the endocrine pancreas. Patients taking metformin may develop vitamin B-12 deficiency and may need to take supplements. Other possible side effects of metformin include nausea, abdominal pain, diarrhea, and bloating.

[0120] Metformin is sometimes also used in combination with other drugs, including Sulfonylureas, Glinides, Thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, and insulin therapy.

[0121] Sulfonylureas can help the body secrete more insulin. For example, Glyburide Glipizide (Glucotrol ) and Glimepiride Possible side effects include hypoglycemia and weight gain.

[0122] Glinides stimulate the pancreas to secrete more insulin. They act faster than Sulfonylureas but have a shorter duration of action in the body. Examples include repaglinide and nateglinide. Possible side effects include hypoglycemia and weight gain.

[0123] Thiazolidinediones make the body tissues more sensitive to insulin. An example of this drug is Pioglitazone Possible side effects include the risk of congestive heart failure, the risk of bladder cancer (pioglitazone), the risk of fractures, and weight gain.

[0124] DPP-4 inhibitors help lower blood sugar levels, but the effect is often very limited. For example, sitagliptin saxagliptin and linagliptin Possible side effects include the risk of pancreatitis and joint pain.

[0125] GLP-1 receptor agonists are injectable drugs that slow digestion and help lower blood sugar levels. Their use is usually associated with weight loss, and some may reduce the risk of heart attack and stroke. Examples include exenatide ( Bydureon ) liraglutide and semaglutide Possible side effects include the risk of pancreatitis, nausea, vomiting, and diarrhea.

[0126] SGLT2 inhibitors affect the blood filtration function of the kidneys by preventing glucose from returning to the bloodstream. As a result, glucose is excreted in the urine. These drugs may reduce such risks in people at higher risk of heart attack and stroke. Examples are canagliflozin dapagliflozin and empagliflozin Possible side effects include vaginal fungal infections, urinary tract infections, low blood pressure, high cholesterol, the risk of gangrene, the risk of fractures (canagliflozin), and the risk of amputation (canagliflozin).

[0127] Some people with type 2 diabetes require insulin treatment. In the past, insulin treatment was used as a last resort, but now, if blood sugar levels are still not at the target despite lifestyle changes and taking other medications, insulin treatment may be prescribed more quickly. Different types of insulin vary in their onset of action and duration of effect. For example, long-acting insulin is designed to take effect at night or throughout the day to keep blood sugar levels stable. Short-acting insulin is usually used at mealtimes to moderate the blood sugar spike that accompanies meals.

[0128] In summary, the existing drugs for managing type 2 diabetes mainly follow the following strategies: reducing glucose production, increasing the body's sensitivity to insulin, increasing insulin secretion, and increasing the kidneys' excretion of glucose. There is currently no drug that can simultaneously increase insulin production and secretion in the endocrine pancreatic beta cells.

[0129] Examples of insulin-secreting porosome proteins can include: cytoplasmic type 1 actin, tubulin alpha-1A, cofilin-1, calcium-transporting ATPase type 2C, ankyrin repeat domain-containing protein, unconventional myosin-X, Rab11 family-interacting protein 4, Arf-GAP with SH3 domain, transmembrane protein 194A, Rab4,6,33,10,15,35,1,38,27,39, Rab GDP dissociation inhibitor alpha, potassium channel subfamily K member 2, Rho GTPase-activating protein 40, heat shock protein HSP 90, heat shock cognate 71 kDa protein, synaptosomal-associated protein 25, ankyrin repeat domain-containing protein, profilin-1, tubulin beta2A, destrin, guanine nucleotide-binding protein beta-2, Rho GDP-dissociation inhibitor 1, calmodulin, microtubule-associated proteins 1 and 2, ADP-ribosylation factor 5, ADP-ribosylation factor-like protein 3, apolipoprotein A-1, Arf-GAP. Embodiments of the present invention can include one or more identified small molecules that directly act on one or more of the above proteins to affect the structure and / or function of insulin-secreting porosomes. In other embodiments, the small molecules can enhance the ability of porosome constituent proteins to form porosome assemblies.

[0130] In some embodiments, small molecules, such as 17-demethoxy-17(2-propylanilino)geldanamycin, can modulate heat shock proteins, such as HSP90, to affect the formation of porosome structure and subsequent function. In some embodiments, small molecules can disrupt the formation of porosome structure by inhibiting the binding and assembly of constituent proteins.

[0131] Other embodiments of the present invention include a porosome protein that regulates both the expression and secretion of insulin in endocrine pancreatic beta cells. The identified insulin-secreting porosome proteins are ATP2C1 (ATPase secretory pathway Ca+2 transporting 1) and APOa1, which show the expression and secretion of insulin in endocrine pancreatic beta cells. These proteins were identified using CRISPR knockout and overexpression of different insulin-secreting porosome proteins. The above-identified proteins belong to the P-type cation-transporting ATPase family. This magnesium-dependent enzyme catalyzes the hydrolysis of ATP and the transport of calcium ions.

[0132] In another embodiment of the present invention, there is provided a method of identifying one or more modulators of insulin-secretory pore-forming proteins to modulate insulin production and secretion in endocrine pancreatic β-cells. Some embodiments utilize overexpression of ATP2C1 and APOa1 in β-cells induced by mRNA. Similarly, CDN1163, a Ca2+-ATPase (SERCA) activator, has been identified as being capable of increasing both insulin expression and secretion in endocrine pancreatic β-cells. In some embodiments, the activator can be used alone or in combination with modulators.

[0133] The Ca2+-ATPase (SERCA) activator CDN1163 has been identified as being usable alone or in combination with metformin and / or other existing type 2 diabetes drugs mentioned above (including modulators of ATP2C1-related pore-forming proteins within the secretory complex) to increase insulin expression and secretion in endocrine pancreatic β-cells.

[0134] Some embodiments of the present invention use the above method to identify protein-protein interactions within the functional complexes of cells. In particular, protein-protein interactions within the insulin-secretory pore complex in endocrine pancreatic β-cells enable fine-tuning of the insulin-secretory pore secretion mechanism in endocrine pancreatic β-cells and precise targeting thereof with small molecule drug-nanobody complexes.

[0135] As shown in Figure 7, in other embodiments, overexpression of different insulin-secretory pore-forming proteins enables the identification of insulin-secretory pore-forming proteins ATP2C1 (ATPase secretory pathway Ca+2 transport 1) and APOa1, which exhibit increased insulin expression and glucose-stimulated insulin secretion in endocrine pancreatic β-cells (see Figure 8 ). Thus, the Ca2+-ATPase activator CDN1163 can be used to increase insulin expression and glucose-stimulated insulin release in endocrine pancreatic β-cells (see Figure 9 ). In fact, Figure 7 exemplifies the knockout (KO) and overexpression (OE) of three pore-forming proteins: ATP2C1, APOa1, and TREK1 in Min6 cells. Note the Western blots of CRISPR / Cas9 empty (SCRM) and KO and control (CON) and overexpressed proteins. GAPDH is the loading control. Figure 8It exemplifies the glucose-stimulated insulin secretion in the knockout (KO) and overexpression (OE) states of three pore-forming proteins, ATP2C1, APOa1, and TREK1, in Min6 cells. Note that, compared with the other two KOs (i.e., APOa1 and TREK1), the insulin secretion at the 10-minute and 30-minute time points was significantly reduced in ATP2C1 KO. Interestingly, the OE of ATP2C1 and APOa1 led to an increase in both the synthesis and secretion of insulin, while TREK1 showed a decrease in both the synthesis and secretion of insulin. Finally, as Figure 9 shown, the glucose-stimulated insulin secretion in Min6 cells increased after 2 hours of exposure to the ATP2C1 activator CDN1163. Note that 10 μM CDN1163 had the best effect on glucose-stimulated insulin secretion.

[0136] These results suggest that targeting the overexpression of ATP2C1 or APOa1 or both in pancreatic endocrine β-cells using mRNA or other gene therapies can treat type 2 diabetes. Similarly, small molecule activators of the two pore-forming proteins can be used as drugs for treating type 2 diabetes.

[0137] Pore Reconstruction Therapy for Type 1 Diabetes (T1D)

[0138] Current therapies for type 1 diabetes (T1D) are based on insulin injection and cadaveric islet transplantation, which have many drawbacks. Therefore, new methods are being developed to reproduce cells that secrete pancreatic hormones in vitro. The most promising method is to generate stem cell (SC)-derived β-cells, which can provide an unlimited source of insulin. Recent studies have provided methods for generating β-cell-like cell clusters that exhibit glucose-stimulated insulin secretion, which is one of the main characteristics of β-cells. However, compared with native β-cells, SC-derived β-cells do not undergo complete functional maturation; thus, they exhibit limited glucose-stimulated insulin secretion, creating an urgent need to be addressed. The results of ongoing clinical trials suggest that existing protocols for generating SC-islets that can improve glycemic control in human T1D patients need to further enhance insulin secretion that mimics that of the original mature islets. This would reduce the number of cells required for transplantation and make it easier to manufacture sufficient cell numbers for treatment. It has been reported that if the insulin secretion per cell doubles, then the number of cells required to cure patients may be only half of the original. Reducing the graft volume can also simplify the transplantation procedure, reduce the nutritional exchange requirements at the transplantation site, and provide the possibility of alternative transplantation sites. Due to the reduced number of cells required for successful transplantation, the production cost and logistics requirements of cell therapy will be significantly reduced. Reconstructing the insulin-secreting pore complex into SC-islets will be able to enhance glucose-stimulated insulin secretion and address this issue.

[0139] Exosome Release Control

[0140] In addition to secreting neurotransmitters, digestive enzymes, or hormones, cells also communicate with each other by secreting membrane-bound nanostructures called extracellular vesicles (EVs), which were first discovered and reported in 1983 as a mechanism for the selective externalization of transferrin receptors in sheep reticulocytes. In 1985, electron micrographs confirmed the externalization of transferrin receptors in sheep reticulocytes in the form of vesicles. Over the past 35 years, there have been great advances in our understanding of the biology, function, and biomedical applications of EVs. EVs are vesicles that are packed with proteins, DNA, and RNA, which are destined for specific target cells in the body. This intercellular communication via EVs has also been implicated in various pathologies such as cancer, neurological diseases, and inflammation. Although EV cargo includes plasma membrane and endosomal proteins, they may also contain materials from various intracellular compartments such as mitochondria. Studies have reported the presence of mitochondrial DNA within EVs. Although multivesicular bodies may fuse at the plasma membrane to release their cargo, the molecular mechanisms by which EVs and / or their cargo are released from various cell types remain unclear. It is thought that EV release may occur via the exocyst complex.

[0141] In some embodiments, EV release is controlled by structural and / or functional alterations of the exocyst complex or exocyst-associated proteins. In some embodiments, one or more cell types may "knock out" the genes of putative exocyst complex or exocyst-associated proteins by using CRISPER, RNAi, or other methods known in the art.

[0142] For example, in Min6 cells of the rat brain, ATP2C1 was knocked out by using the CRISPER / Cas9 system for genome editing. Comparing such cells with control wild-type (SCRM) cells, it was found that EVs were released via the exocyst complex, indicating that glucose-stimulated insulin secretion was lost. Thus, small molecules that enhance or alter the production of ATP2C1 likely affect insulin secretion.

[0143] Nanobody

[0144] Nanobodies are a class of antibodies consisting of a single polypeptide chain, with a multifunctional molecular binding scaffold found in camelid species, different from the large Y-shaped conventional antibodies found in other mammalian species including humans. In some embodiments of the present invention, engineered nanobodies against different porin targets are used to bind the nanobody to the protein target, thereby precisely targeting a drug to a specific porin and / or altering the structure and function of the porin. In some embodiments, the variable region of the camelid variable domain (VHH) nanobody is humanized to target and bind one or more domains of one or more porins. Nanobodies targeting multiple porins can be used, in addition to facilitating precise targeting of small molecules, to physically or chemically alter the structure and / or function of the porin, thereby altering the course of porin-mediated diseases. Thus, one or more identical or different classes of nanobodies can be linked to one or more small molecules to fine-tune the targeting and response to porin structure-function consequences.

[0145] Figure 5 Drug targeting specificity is exemplified using a combination of small molecule drug design and tissue- and porin-specific multivalent nanobodies (top). Site-specific functionalization of nanobodies is also exemplified by engineering cysteines (middle, bottom). Cysteines are introduced into the nanobody by genetic modification. Maleimide is one of the most widely used thiol-reactive chemical groups. The yellow ellipse represents the backbone of the foreign cysteine. The red sphere represents the functional group (small molecule drug) attached to the maleimide. Finally, an example chemical reaction involved in such a preparation is provided (bottom): the addition of the amino acid para-acetylphenylalanine (pAcF) provides a bioorthogonal ketone for the attachment of the anticancer drug doxorubicin (Dox) when a reactive amino acid is present in the elastin-like polypeptide nanoparticle (ELP diblock) targeted by the nanobody. Diblock copolymers based on elastin-like polypeptides (ELPs) have the potential to undergo specific phase transitions when subjected to thermal stimuli. This ability is particularly suitable for forming carriers, such as micellar structures, for delivering active cargo molecules. Similarly, multivalent nanobodies with both tissue- and porin-specific domains can be generated to precisely target a combination of small molecule drugs. In this context, the term "multivalent" refers to the common operational usage of the term to describe more than one nanobody, or molecule, or other functional element forming part of the combined drug.

[0146] Examples of Neuronal Diseases and Cancer

[0147] As previously mentioned, the pore body structure includes multiple proteins and their associated ligands, molecular chaperones, and other plural molecules (such as lipids). Although it is generally believed that some diseases are caused by mutations / deformations in the structure of a single protein, as described above, it has only recently been recognized that dysfunction and deformities of larger structures (such as pore bodies) can lead to diseases. The following non-limiting examples illustrate pore body protein dysfunction and its contribution to diseases, followed by exemplary single-target small molecules that may affect the disease state.

[0148] Neuronal diseases: (such as Alzheimer's disease, Down syndrome, and schizophrenia): In Alzheimer's disease, increased levels of the pore body protein CNPase (2,3-cyclic nucleotide phosphodiesterase) and heat shock protein 70 (HSP70) present in the neuronal pore body complex have been found, while the level of the pore body-associated dihydropyrimidinase-related protein 2 (DRP-2) is decreased. Similarly, the pore body proteins SNAP-25 and synaptophysin are significantly reduced in the neurons of Alzheimer's disease patients. Similarly, in Down syndrome and schizophrenia: It has been reported that the level of the pore body protein CNPase is also decreased in the frontal and temporal cortices of Alzheimer's disease and Down syndrome patients. Low levels of CNPase have also been detected in the prefrontal cortex of schizophrenia patients. In addition, an allele associated with low levels of CNPase has also been reported to be related to schizophrenia.

[0149] Small molecule inhibitors and stimulants of pore body phosphodiesterase, such as Vinpocetine, BAY 60-7550, Rolipram, Etazolate, Sildenafil, S14, VP1.15, PF-04447943, papaverine, and small molecule inhibitors of HSP70, such as Apoptozole, VER155008, JG98, HA15, and YUM70, as well as the small molecule activator of HSP70, ML346, can be used to treat neuronal diseases, especially Alzheimer's disease.

[0150] Cancer: Cellular secretion is an important mediator of cancer progression. For example, the Ras superfamily of small GTPases present in the pore body is associated with 33% of human cancers. However, the direct pharmacological inhibition of Ras mutants remains challenging. Thus, an alternative strategy is to inhibit the activity of the pore body protein V-ATPase while continuing to screen and design novel small molecules that directly bind to and inhibit Ras GTPase. Similarly, non-small cell lung cancer (NSCLC) has a poor prognosis and remains the most common cause of cancer-related death globally. The pore body protein tubulin β is closely associated with drug-refractory and invasive NSCLC. βIII-tubulin is also associated with the resistance of multiple tumor types, including ovarian, breast, and gastric cancers, to taxanes or vinorelbine. Small molecule drugs that target microtubules, such as Docetaxel, Taxol, Podophyllotoxin, Etoposide, Vinblastine, Vincristine, Vinorelbine, Griseofulvin, Cytocholasin A and E, TN-16, Myoseverin, Nocodazole, Vindesine, Phomopsin A, d-24851, Monastrol, AMP-PNP, Adociasulfate-2, Terpendole-E, Tubacin, Scriptaid, DPD, and C2-8, can be used in the above combination methods.

[0151] Small molecules that target the porous lipids involved in cancer therapy can also be used in the embodiments of the present invention. Research reports that cholesterol on the cytoplasmic membrane is crucial for cellular secretion. Depletion of cholesterol on the cell membrane reduces the entry of phosphatidylserine (PS) into the cytoplasmic membrane, resulting in the loss of secretion. Phosphatidylserine (PS) is normally located in the inner layer of the membrane bilayer of healthy cells, but its expression level is high on the surface of cancer cells. This makes it possible to develop selective therapeutic agents that target cancer cells without affecting healthy cells. For example, SapC-DOPS is a PS-targeting nanovesicle that can effectively target and kill multiple cancer types, including pancreatic, lung, brain, and pediatric tumors. SapC-DOPS selectively induces apoptotic cell death in malignant and metastatic cells, and non-transformed cells are not affected due to low surface PS expression. In another approach, small molecules that deplete plasma membrane cholesterol, such as cyclodextrin, in combination with SapC-DOPS, have potential uses in cancer therapy.

[0152] Table 5 lists other porins and small molecule drugs targeting porins and lipids, and explains as much as possible their potential roles in treating various diseases.

[0153] Table 5: Other porins and small molecule drugs targeting porins

[0154]

[0155]

[0156]

[0157]

[0158] It should be understood that references to pharmaceutically acceptable salts include solvate addition forms, especially solvates. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and can be formed during crystallization with pharmaceutically acceptable solvents (such as water, ethanol, etc.). Hydrates are formed when the solvent is water, and alcoholates are formed when the solvent is alcohol. Solvates of the compounds described herein can be conveniently prepared or formed during the processes described herein. In addition, the compounds provided herein can exist in non-solvated form as well as solvated form. Generally, for the compounds and methods provided herein, the solvated form is considered equivalent to the non-solvated form.

[0159] Functional Mucus-Secreting Pore Reconstruction - Cystic Fibrosis Treatment

[0160] Cystic fibrosis (CF) is a disease in which thick, sticky mucus accumulates in the lungs, digestive tract, and other parts of the body. It is one of the most common life-threatening chronic lung diseases in children and young people. Cystic fibrosis is inherited through families and is caused by a defective gene that causes the body to produce abnormally thick and sticky mucus. The abnormal mucus accumulates in the lung airways and in the pancreas. Mucus accumulation can lead to life-threatening lung infections and severe digestive problems. This disease can also affect the sweat glands and the male reproductive system. Many people carry the CF gene but have no symptoms. This is because people with CF must inherit a defective gene from each parent. Some CF is more common in people of Northern or Central European descent. Most children with CF are diagnosed before the age of 2, especially in cases where newborn screening is carried out throughout the United States. A small number of children are not diagnosed with the disease until they are over 18 years old. The condition of these children is usually less severe.

[0161] Nearly 40,000 children and adults in the United States have cystic fibrosis, and an estimated 105,000 people are diagnosed with CF in 94 countries. For people with CF, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the CFTR protein to malfunction. When the protein doesn't work properly, it can't help chloride ions move to the cell surface. Without chloride ions to attract water to the cell surface, the mucus in various organs becomes thick and sticky. In the lungs, the mucus clogs the airways and traps disease-causing agents (such as bacteria), leading to infections, inflammation, respiratory failure, and other complications. Therefore, avoiding exposure to bacteria is a top concern for people with CF.

[0162] The most common mutation in the CFTR gene is delF508, a trinucleotide deletion that results in the loss of the amino acid phenylalanine (F) at position 508 of the protein. This mutation accounts for two-thirds (66 - 70%) of CF cases globally and 90% of cases in the United States; however, over 1,500 other mutations can cause CF. While most people have two working copies (alleles) of the CFTR gene, only one is needed to prevent cystic fibrosis. CF occurs when neither allele can produce a functional CFTR protein. Therefore, CF is considered an autosomal recessive genetic disease.

[0163] There is currently no cure for cystic fibrosis. Lung infections are treated with antibiotics, which can be administered intravenously, inhaled, or taken orally. Sometimes, the antibiotic azithromycin is used long-term. Inhaling hypertonic saline and salbutmol may also be helpful. If lung function continues to deteriorate, lung transplantation may be an option. Airway clearance techniques such as chest physiotherapy have some short-term benefits, but the long-term effects are unclear. The average life expectancy is 42 - 50 years. Lung problems (infections, loss of capacity) are the cause of death in 80% of people with cystic fibrosis.

[0164] Since there is currently no known cure for cystic fibrosis, the treatment of CF focuses on improving breathing, preventing and treating lung infections, and thinning the mucus in the lung epithelium. Treatment methods include medications, therapies to clear mucus from the lungs, and in some cases, lung transplantation. Pharmacogenomic approaches have led to the development of drugs that target the root cause of the disease. Since different CFTR mutations have different effects on the CFTR protein, these drugs can only be used to treat people with certain CFTR mutations.

[0165] As shown in FIG. 10, the presence of porosomes in human airway epithelia is known. Similarly, the presence of a porosome proteome in these cell types is known (Table 6). In fact, FIG. 10 shows representative electron micrographs of cultured Calu-3 cells (cells originally obtained from a lung adenocarcinoma patient), showing the presence of microvilli (MV) and porosomes (P) on the plasma membrane. (A) Calu-3 cells show the presence of dense microvilli and porosomes on the plasma membrane. (B-D) Note the flask-shaped porosomes, which are ~100 nm in diameter (E), 200-300 nm deep, and open to the cell surface (red arrow). In (C), what appears to be mucus was found at the opening of the porosome leading to the outside of the cell. In the two porosomes depicted in (D), the porosome closer to the center appears to be cut directly from the center of the organelle, while the porosome on the left appears to be cut from the bottom. (E) Similar to the AFM image, the microvilli have an average diameter of 92 nm. The human airway-associated porosome complex is similar to the porosome complexes present in the exocrine and endocrine pancreas shown in FIGS. 13 and 14.

[0166] In fact, Figure 11 Illustrates the use of Ussing chamber experiments showing that forskolin stimulates the release of chloride ions from Calu-3 cells, and that this release is inhibited by the presence of the CFTR inhibitor GlyH-101. Note that two independent experiments showed similar stimulation and inhibition curves.

[0167] For its part, FIG. 12 shows that the CFTR inhibitors 172 and GlyH-101 inhibit the forskolin-stimulated secretion of intravesicular mucin from Calu-3 cells. A and B are from control cells, C and D are from cells exposed to 172, and E and F are from cells exposed to GlyH-101. Vesicles outlined in red are partial / empty vesicles, and vesicles outlined in green are full vesicles. Scale bar = 500 nm.

[0168] Table 6: Major proteins in the Calu-3 cell porosome proteome identified by LC-MS / MS and WESTERN blot [*] analysis

[0169]

[0170]

[0171] *Porosome complexes immunoisolated with SNAP-25 were obtained from 1% Triton-Lubrol-lysed Calu-3 cells.

[0172] It is well known that the cystic fibrosis transmembrane conductance regulator (CFTR) is part of the porosome complex in human airway epithelial cells. Therefore, it was hypothesized that this porosome-associated CFTR regulates the quality of mucus secretion through the porosome complex in the cytoplasmic membrane, and it was later found to be true. These findings have deepened the understanding of the effect of CFTR-associated porosomes on mucus secretion in the lung epithelium and provided important insights into the etiology of CF disease.

[0173] The transmission electron micrograph of Figure 13 presents the porosome complex associated with docked secretory vesicles at the apical end of exocrine pancreatic acinar cells. (a) A part of the apical end of a pancreatic acinar cell, showing a porosome and a docked secretory vesicle called a zymogen granule (ZG) within the square outlined in green. The docked ZG fuses at the bottom of the porosome complex to form a fusion pore (FP). Electron-dense secretory vesicles of the exocrine pancreas. (Scale bar = 400 nm; for Figure 4a only). (b) The area within the green square in panel (a) is magnified, showing apical microvilli (MV) and a cross-section through the porosome and ZG. Note that the bilayer of the ZG membrane (ZGM) is directly connected to the bottom of the porosome cup to form a continuum or FP. (c) An enlarged view of the porosome more details the porosome bilayer and a cross-section across three protein rings, with the thicker ring (blue arrow) closer to the outer opening of the porosome complex. The third ring (the lowest ring) away from the porosome opening docks and fuses with the ZGM. (d) The porosome membrane is outlined with a yellow border for better clarity. The porosome membrane is continuous with the apical plasma membrane (PM) at the apical end of the pancreatic acinar cell facing the lumen (L), and also defines the precise contact and fusion points of the ZGM at the bottom of the porosome membrane, thus forming the FP (12).

[0174] Turning to Figure 14, transmission electron micrographs of Min6 cells (endocrine pancreatic β cells) secreting insulin are shown, indicating the presence of porosomes in the cytoplasmic membrane (a) and porosomes associated with docked secretory vesicles at the apical end of the cell (b). Clathrin-coated vesicles are shown in (c), which are different from the cup-shaped porosome complex. The average size of the isolated porosomes is 91 nm, which is also confirmed by photon correlation spectroscopy (d).

[0175] Therefore, in view of the presence of porosomes in endothelial cell structures, the techniques for altering porosome structure, small molecule targeting, or reconstitution taught herein are applicable to airway cells. Similarly, when airway passages are exposed to air, drug delivery methods known in the art, such as nebulizers, inhalers, atomizers, etc., are contemplated for delivering any treatment taught herein.

[0176] Pore Reconstruction

[0177] As will be described in more detail below, the pore complex has been functionally reconstituted in artificial lipid membranes (Figs. 15 and 16) and in live cells (Figs. 17 and 18). In addition, the pore complex reconstituted in live cells is stable and functional (Fig. 19). This robust pore reconstitution ability provides a therapeutic approach for treating CF disease.

[0178] The electron micrograph of Fig. 15 presents the reconstituted exocrine pancreatic pore complex in liposomes. The pore complex presents a cup-and-basket morphology. (a) shows 500-nm lipid vesicles incorporating pore complexes isolated from the exocrine pancreas. b-d show the reconstituted complexes at higher magnification. Scale bar = 100 nm.

[0179] Turning to Fig. 16, it is exemplified that the lipid bilayer-reconstituted pore complex from the exocrine pancreas is functional. (a) The schematic diagram shows the bilayer setup for electrophysiological measurements. (b) It is exemplified that zymogen granules (ZG) added to the cis side of the bilayer fuse with the reconstituted pore complex (as evidenced by the increase in capacitance and current activity), and the simultaneous time-dependent release of amylase (a major ZG inclusion) to the trans side of the membrane. The movement of amylase from the cis side to the trans side of the chamber is determined by immunoblot analysis of the contents in the cis and trans chambers over time. (c) As evidenced by immunoblot analysis of immunoisolated complexes, electrophysiological measurements in the presence and absence of the chloride channel blocker DIDS demonstrate the presence of chloride channels associated with the complex.

[0180] Fig. 17 exemplifies the enriched presence of TREK-1, Gi3, and synaptotagmin-1A immunoreactivities in insulin-secreting Min6 cells with pore reconstitution. Western blot analysis was performed on 5 μg of Min6 cell homogenates from control and pore-reconstituted cells. Note the enriched presence of all three pore complex proteins: TREK-1, Gi3, and synaptotagmin-1A. No change in insulin immunoreactivity was observed in the homogenates of reconstituted Min6 cells. B, Immunofluorescence microscopy confirmed the increase in SNAP-25 (green) and Gi3 (red) immunoreactivities and the increased co-localization of them in pore-reconstituted Min6 cells. Data represent 1 of 4 similar experiments. Scale bar (insets) a and b, 20 μm (13).

[0181] Figure 18 shows the reconstitution of secretory insulin pore bodies in live Min6 cells, demonstrating increased glucose-stimulated insulin secretion. Note the increased time-dependent insulin release in the reconstituted Min6 cells. A, Representative insulin immunoblots of total Min6 cell homogenates (TH) and glucose-stimulated insulin release at 0, 10, and 30 minutes in control and pore body-reconstituted experimental Min6 cells. The preproinsulin band was only seen in the TH fraction and not in the secretory fraction. B, Bar graph of percent insulin release at 0, 10, and 30 minutes in control and reconstituted experimental Min6 cells. A significant increase in time-dependent insulin release was observed in pore body-reconstituted Min6 cells at the 30-minute time point (n = 6; *P < 0.05). Note that no change in basal insulin release was observed in pore body-reconstituted Min6 cells. C, The calculated insulin secretion rate was 0.062% / min of the total amount in control cells and increased to 0.107% / min of the total amount in pore body-reconstituted cells, a 70% increase in the insulin release rate.

[0182] Figure 19 exemplifies the enrichment of Gαi3 and synaptotagmin-1A immunoreactivity in homogenates of pore body-reconstituted Min6 cells and the consequent glucose-stimulated insulin release observed at 24 and 48 hours after reconstitution. A, Representative Western blots of Min6 cell homogenates from control and pore body-reconstituted (experimental) Min6 cells at 24 and 48 hours, showing the enrichment of pore body proteins Gi3 and synaptotagmin-1A. No change in total insulin immunoreactivity was detected in the experimental homogenates. B, The enrichment of pore body proteins shown in panel A is reflected in elevated levels of glucose-stimulated insulin release in live Min6 cells at 24 and 48 hours after pore body reconstitution. Results are representative of 1 of 3 independent experiments.

[0183] Mucin is stored in the secretory vesicles of mucin-secreting cells of the airway epithelium, and its appearance is either due to complete collapse of the vesicle membrane at the plasma membrane or due to transient fusion of the secretory vesicle at the base of the plasma membrane-associated pore body. Functional reconstitution of the secretory insulin pore body complex in live β cells of the endocrine pancreas opens a window for the treatment of cystic fibrosis and type 1 diabetes.

[0184] Examples of insulin-secreting pore body proteins in human airway epithelium include those listed in Table 7 below. Embodiments of the invention can include one or more small molecules that have been identified to directly act on one or more of the above proteins to affect the structure and / or function of the mucin-secreting pore body.

[0185] Table 7: Major proteins identified in the calu-3 cell porosome proteome by LC-MS / MS and western blot analysis.

[0186]

[0187]

[0188] *The porosome complex isolated by SNAP-25 immunoisolation was obtained from Calu-3 cells lysed with 1% Triton-Lubrol.

[0189] Embodiments of the present invention provide a method for large-scale isolation of mucin-secreting porosome complexes from healthy human airway epithelia not affected by CF, and reconstitution of such porosome complexes into CF-affected airway epithelia to restore porosome function. Figure 20 The example schematic shows a functional mucin-secreting porosome complex entering airway epithelial cells of CF patients and ameliorating CF disease. The situation where the CFTR complex exists as part of the overall porosome structure is represented by a connecting line, not precisely positioned, only to highlight the presence of this particular subassembly of the overall porosome structure. In some embodiments, porosome proteins are knocked out one by one using CRISPR to determine which other proteins in the complex are lost from the porosome complex in addition to the knocked-out protein. Those proteins lost from the porosome complex are proteins associated with each other and the knocked-out protein in the complex. Such systematic studies enable the revelation of the distribution of all proteins in the porosome complex and contribute to targeting specific proteins in the complex to modulate and improve secretory function and correct secretory defects and the diseases caused thereby. In addition, since airway epithelial cells are a terminally differentiated population with an average half-life of 6 months in the trachea and over 18 months in the lung, they are an ideal target for CF porosome complex reconstitution therapy.

[0190] In some embodiments, Calu-3 and other suitable human airway epithelial cell lines are selected to harvest porosomes for therapy: human airway epithelial cell lines are used to isolate porosome complexes that secrete human mucins in CF therapy. For example, Calu-3 cells are grown in Dulbecco's modified Eagle medium: Nutrient Mixture F-12 with 15% fetal bovine serum (DMEM / F-9212). The cells are incubated at 37 °C and 5% CO2 in humidified air of 93%. Calu-3 cells are a source for isolating mucin-secreting porosomes for CF research and therapy. The mucin-secreting porosome complex is used with the protein (Affinity resin for immunoprecipitation and antibody purification procedures) coupled SNAP-25 specific antibody was isolated from Calu-3 cells. Calu-3 cells were lysed with lysis buffer containing 2% Triton X-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, a nonionic surfactant), 1 mM benzamidine, 5 mM Mg-ATP and 5 mM EDTA in PBS (pH 7.4), supplemented with protease inhibitor mixture. Each immunoprecipitation was scaled up and adjusted according to the following protocol as required: 2 mg of Triton-lysed Calu-3 cells, 5 μg of SNAP-25 antibody conjugated to protein beads, incubated on ice for 1 hour, and then washed 3 times with 10 volumes of wash buffer (500 mM NaCl, 10 mM Tris and 2 mM EDTA; pH 7.5). The immunoprecipitated complex associated with the beads was eluted with low pH (pH 3) PBS to dissociate the pore complex from the antibody bound to the beads, and the eluted sample was immediately restored to neutral pH with a total volume of 200 μL. Before animal and / or human clinical trials, various dilutions of the isolated complex were tested to optimally reconstruct into the CF epithelium of human lung organoids. The isolated pore complex suspension was administered and tested in an animal model of CF disease using a nebulizer to evenly distribute it into the airway epithelium and lungs. Such systematic studies enabled the optimization of the functional reconstruction of the isolated pore complex and contributed to the improvement of secretory function and the correction of secretory defects and the resulting CF disease. We further note that when administered by nebulization, no additional steps are required to ensure the correct insertion or orientation of the pore complex into epithelial cells.

[0191] In some embodiments, since Gαi3 is present in the pore complex of Calu-3 cells (as Figure 21 shown), and previous studies have reported that "pertussis toxin, which unbinds GTP-bound G proteins from their receptors, and guanosine 5'-[β-thiophosphate], which blocks the interaction of G proteins with their effector proteins, increase Cl- currents in airway epithelia isolated from CF patients" (PNAS 1992, 89(22): 10623-10627), this suggests that targeting Gαi3 in the pore complex leads to the restoration of cAMP-activated Cl- currents and normal mucin secretion in the airway epithelia of CF patients.

[0192] In some embodiments, vimentin is present in the pore complex of mucin-secreting cells of human epithelia. As Figure 21As shown, the figure depicts an immunoblot analysis of the total homogenate (CH) of Calu-3 cells and the isolated pore complex (P), demonstrating the presence of the pore complex proteins actin, Gαi3, and vimentin. Note the enrichment of these proteins in the pore complex. Since earlier studies reported that "4β-phorbol 12-myristate 13-acetate (PMA)-mediated vimentin phosphorylation appears to be an intermediate step in PKC-stimulated glycoconjugate secretion, which is impaired in CF disease." (Am J Physiol. 1994, 266(3 pt 1) C611-C621), it is suggested that targeting vimentin could modulate vimentin activity in the mucin-secreting pore complex of airway epithelium, thereby restoring normal mucin secretion in the airways of CF patients. Additionally, it is known that CFTR has a regulatory domain that is a substrate for protein kinase A (PKA) and C (PKC).

[0193] Figure 22 Illustrated is the immunoisolation of the CFTR complex with a CFTR-specific antibody, resulting in the pull-down of pore complex-associated proteins such as syntaxin-1A (in the form of a 70 kDa t- / v-SNARE complex), SNAP-25 (in the form of a 70 kDa t- / v-SNARE complex), SNAP-23 (in the form of a 68 kDa t- / v-SNARE complex), and actin. Thus, in some embodiments, since actin is present in the pore complex of mucin-secreting cells of human epithelium and since earlier studies reported that "4β-phorbol 12-myristate 13-acetate (PMA)-mediated vimentin phosphorylation appears to be an intermediate step in PKC-stimulated glycoconjugate secretion, which is impaired in CF disease." (Am J Physiol. 1994, 266(3 pt 1) C611-C621), it is suggested that targeting vimentin to modulate its activity in the mucin-secreting pore complex of airway epithelium will result in the restoration of normal mucin secretion in the airways of CF patients.

[0194] In some embodiments, since CFTR also regulates several other transporters, including K+ channels, aquaporin water channels, anion exchange factors, the membrane fusion protein syntaxin-1A, and sodium bicarbonate transporters, it is suggested that targeting and modulating their activity in the mucin-secreting pore complex of airway epithelium with small molecules will result in the restoration of normal mucin secretion in the airways of CF patients.

[0195] Figure 23It is exemplified that the mucus secretion of ΔF508-CFTR human CF bronchial epithelial cells is restored by CDN1163. For mucus secretion of human airway epithelium, the CFBE41o-6.2WT-CFTR human CF bronchial epithelial cell line and the CFBE41o-human CF bronchial epithelial cell line (ΔF508-CFTR) were exposed to CDN1163 [4-(1-methylethoxy)-N-(2-methyl-8-quinolinyl)-benzamide], an allosteric Ca2+-ATPase (SERCA) activator. Figure 23 It is shown that CDN1163 restores mucus secretion in CFBE41o-human CF bronchial epithelial cells (ΔF508-CFTR) to normal levels within one hour. Note that the basal level of Muc5B secretion in CFBE41o-human CF bronchial epithelial cells (ΔF508-CFTR) is almost half that of normal wild-type (WT) cells. CDN1163, a Ca2+ ATPase (SERCA) activator, restores the secretion of mucus (Muc5B) in CF bronchial epithelial ΔF508 cells within one hour. Thus, CDN1163 [4-(1-methylethoxy)-N-(2-methyl-8-quinolinyl)-benzamide] can be used as a small molecule therapy to restore normal mucus function in CF patients. The dose concentration range can be from 1 nM (nanomolar) to 20 μM (micromolar), as measured by tissue concentration in subjects. Such measurement techniques can include blood sampling followed by analysis, breath analyzer analysis, urine analysis, biopsy, and other techniques known to those skilled in the art.

[0196] In line with Figure 23 similarly, Figure 24Illustrated the increased mucus secretion by α-CPA in ΔF508-CFTR human CF bronchial epithelial cells. The CFBE41o-6.2WT-CFTR human CF bronchial epithelial cell line and the CFBE41o- human CF bronchial epithelial cell line (ΔF508-CFTR) were exposed to Cyclopiezomic Acid (α-cyclopiezomic acid or α-CPA), a Ca2+-ATPase (SERCA) inhibitor. Our results showed that α-CPA stimulated mucus secretion in CFBE41o- human CF bronchial epithelial cells (ΔF508-CFTR) within one hour. Note that the basal level of Muc5B secretion in CFBE41o- human CF bronchial epithelial cells (ΔF508-CFTR) was almost half that of normal wild-type (WT) cells. α-CPA, as a Ca2+ ATPase (SERCA) inhibitor, significantly (more than 10-fold) stimulated mucus (Muc5B) secretion in CF bronchial epithelial ΔF508 cells within one hour. Thus, alpha-CPA and small molecules currently used for different indications with similar binding epitopes, such as leflunomide, teriflunomide, tolvaptan, conivaptan, omeprazole, lansoprazole, rufinamide, prazosin, terazosin, and roflumilast, can be used as small molecule therapies to restore normal mucus function in CF patients. The data are shown below.

[0197] Figure 25 The schematic diagram of... illustrated some interactions between CFTR and related proteins (Nature Reviews Mol. Cell Biol. 2006, 7: 426-436). In addition to mediating Cl- secretion, CFTR also regulates several other transporters, including K+ channels, aquaporin water channels, anion exchangers, the membrane fusion protein synaptotagmin-1A, and sodium bicarbonate transporters. Thus, in some embodiments, since K+ channels are present in the mucin-secreting pore body complex and the hydration of mucus is a problem in CF disease, the small molecules described herein can be used to target the interactions between K+ channels, aquaporin water channels, and CFTR.

[0198] In cystic fibrosis, mucus hypersecretion is caused by bacterial pathogens through the mobilization of intracellular Ca2+, and a Ca2+-ATPase (SERCA) has been found in the pore complex, suggesting that this ion channel plays a role in mucus secretion in human airway epithelium.

[0199] CDN1163 is an allosteric sarcoplasmic / endoplasmic reticulum Ca2+-ATPase (SERCA activator) that improves Ca2+ homeostasis. Its official name is 4-(1-methylethoxy)-N-(2-methyl-8-quinolinyl)-benzamide, CAS number: 892711-75-0; molecular formula: C20H20N2O2; molecular weight: 320.4.

[0200] CDN1163 alleviates diabetes and metabolic disorders, with IC50 and target: SERCA. In vitro: CDN1163 (5.5 - 25 mM; 0 - 8 hours; rat cardiomyocytes) treatment reduces resistin and nuclear NFATc expression induced by high levels of glucose and increases phosphorylation of AMPKα in a time-dependent manner. In vivo: CDN1163 (50 mg / kg; intraperitoneal injection; injected into male ob / ob mice and lean ob / + mice for 5 days) increases SERCA2 Ca2+-ATPase activity, reduces endoplasmic reticulum (ER) stress-induced cell death (in vitro) and improves hepatic Ca2+ transport activity. CDN1163 reduces blood glucose levels and improves metabolic parameters and gluconeogenic gene expression, reverses hepatic steatosis, inhibits ER stress and ER stress-induced apoptosis, and improves mitochondrial efficiency in ob / ob mice.

[0201] The embodiments of the above invention can be used alone or in combination with each other. As a non-limiting example, small molecules targeting the pore complex or parts thereof can be used in combination with the reconstruction of the above pore body. Similarly, patients receiving treatment for the above neurological diseases may also be receiving treatment for similar insulin secretion defects (such as diabetes). Although some embodiments of the present invention are related to the treatment / therapy of cystic fibrosis above, the present invention is not limited thereto. Specifically, the therapies and treatment methods disclosed herein can be used to treat any secretory disease, and cystic fibrosis and type 1 diabetes are just two of them. In one embodiment, the method of the present invention can also be used, for example, to treat chronic obstructive pulmonary disease (COPD).

[0202] Synthesis of Compounds

[0203] In some embodiments, the synthesis of the compounds described herein is accomplished using methods described in the chemical literature, using the methods described herein, or by a combination thereof. In addition, the solvents, temperatures, and other reaction conditions presented herein may vary.

[0204] In some other embodiments, the starting materials and reagents used to synthesize the compounds described herein are synthesized or obtained from commercial sources such as, but not limited to, Sigma-Aldrich, Fisher Scientific (Fisher Chemicals), and Acros Organics. Chemicals can be identified by a variety of names and nomenclatures, including standard IUPAC nomenclature; CAS numbers; written molecular formulas; bond diagrams, etc.

[0205] In still other embodiments, the compounds described herein, as well as other related compounds with different substituents, are synthesized using the techniques and materials described herein and those recognized in the art, such as, see: Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplements (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry Fourth Edition, (Wiley 1992); Carey and Sundberg, Advanced Organic Chemistry Fourth Edition, Volumes A and B (Plenum 2000, 2001), and Green and Wuts, Protective Groups in Organic Synthesis Third Edition, (Wiley 1999) (all of which are incorporated herein by reference for such disclosures). General methods for preparing the compounds disclosed herein can be derived from reactions that can be modified by using appropriate reagents and conditions to introduce the various moieties visible in the formulae provided herein. As a guide, the following synthetic methods can be used.

[0206] In the reaction, it may be necessary to protect those reactive functional groups that are desired in the final product, such as hydroxyl, amino, imino, mercapto or carboxyl groups, to avoid their unnecessary participation in the reaction. Techniques suitable for creating and removing protecting groups are described in detail in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York, N.Y., 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, N.Y., 1994, which are incorporated herein by reference for such disclosures.

[0207] In some embodiments, the identified compounds or small molecules are purchased from various suppliers, including Sigma-Aldrich, Acros, Fisher, Fluka, Santa Cruz, CombiBlocks, BioBlocks, and Matrix Scientific.

[0208] Cells, Analytical Techniques and Instruments

[0209] In some embodiments, methods for profiling a pore body to determine a reactive molecule or a modulating molecule are also described herein. In some cases, the method includes profiling a cell sample containing a pore body or a cell lysate sample containing a pore body. In some embodiments, the cell sample or the cell lysate sample is obtained from an animal cell. In some cases, the animal cell includes cells from a marine invertebrate, a fish, an insect, an amphibian, a reptile, or a mammal. In some cases, the mammalian cell is a primate, an ape, a horse, a cow, a pig, a dog, a cat, or a rodent. In some cases, the mammal is a primate, an ape, a dog, a cat, a rabbit, a ferret, etc. In some cases, the rodent is a mouse, a rat, a hamster, a gerbil, a hamster, a chinchilla, or a guinea pig. In some embodiments, the bird cell is from a canary, a parakeet, or a parrot. In some embodiments, the reptile cell is from a sea turtle, a lizard, or a snake. In some cases, the fish cell is from a tropical fish. In some cases, the fish cell is from a zebrafish (e.g., Danio rerio). In some cases, the worm cell is from a nematode (e.g., Caenorhabditis elegans). In some cases, the amphibian cell is from a frog. In some embodiments, the arthropod cell is from a tarantula or a hermit crab.

[0210] In some embodiments, the pore complex isolated from a cell or cell lysate sample is from mammalian cells. In some cases, the mammalian cells are epithelial cells, connective tissue cells, hormone-secreting cells, nerve cells, skeletal muscle cells, blood cells, or immune system cells.

[0211] Exemplary mammalian cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293H cells, HEK293 cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F TM cells, Flp-In TM T-REx TM 293 cell line, Flp-In TM -293 cell line, Flp-In TM -3T3 cell line, Flp-In TM -BHK cell line, Flp-In TM -CHO cell line, Flp-In TM -CV-1 cell line, Flp-In TM -Jurkat cell line, FreeStyle TM 293-F cells, FreeStyle TM CHO-S cells, GripTite TM 293MSR cell line, GS-CHO cell line, HepaRG TM cells, T-REx TM Jurkat cell line, Per.C6 cells, T-REx TM -293 cell line, T-REx TM -CHO cell line, T-REx TM -HeLa cell line, NC-HIMT cell line and PC12 cell line.

[0212] In some cases, a cell sample or a cell lysate sample containing a pore body is obtained from cells of a tumor cell line. In some cases, the cell sample or the cell lysate sample is obtained from cells of a solid tumor cell line. In some cases, the solid tumor cell line is a sarcoma cell line. In some cases, the solid tumor cell line is a carcinoma cell line. In some embodiments, the sarcoma cell line is derived from a cell line of the following tumors: alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of soft tissue, dedifferentiated liposarcoma, fibroma, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, olfactory neuroblastoma, Ewing sarcoma, extra-renal rhabdoid tumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, infantile fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi sarcoma, leiomyosarcoma of bone, liposarcoma, liposarcoma of bone, malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH) of bone, malignant mesenchymoma, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic sarcoma, tumors with perivascular epithelioid cell differentiation, osteosarcoma, parosteal osteosarcoma, tumors with perivascular epithelioid cell differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET / extraskeletal Ewing tumor, rhabdomyosarcoma, round cell liposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovial sarcoma, telangiectatic osteosarcoma.

[0213] In some embodiments, the carcinoma cell line is from a cell line of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, anal cancer, appendiceal cancer, cholangiocarcinoma (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, carcinoma of unknown primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer or vulvar cancer.

[0214] In some cases, a cell sample or cell lysate sample containing a pore body is obtained from cells of a hematologic malignancy cell line. In some cases, the hematologic malignancy cell line is a T cell line. In some cases, it is a B cell line. In some cases, the hematologic malignancy cell line is obtained from the following T cell lines: peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma, cutaneous T cell lymphoma, adult T cell leukemia / lymphoma (ATLL), blastic NK cell lymphoma, enteropathy-type T cell lymphoma, hepatosplenic γ-δ T cell lymphoma, lymphoblastic lymphoma, nasal NK / T cell lymphoma, or therapy-related T cell lymphoma.

[0215] In some cases, the hematologic malignancy cell line is obtained from the following B cell lines: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk small lymphocytic lymphoma (SLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B cell lymphoma, primary mediastinal B cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma, plasmacytosis, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[0216] In some embodiments, a cell sample or cell lysate sample containing a pore body is obtained from a tumor cell line. Exemplary tumor cell lines include but are not limited to 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB-231, SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460, A2780, SKOV-3 / Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948, DLD-1, SW480, Capan-1, MC / 9, B72.3, B25.2, B6.2, B38.1, DMS153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T, LMH, LMH / 2A, SNU-398, PLHC-1, HepG2 / SF, OCI-Ly1, OCI-Ly2, OCI-Ly3, OCI-Ly4, OCI-Ly6, OCI-Ly7, OCI-Ly10, OCI-Ly18, OCI-Ly19, U2932, DB, HBL-1, RIVA, SUDHL2, TMD8, MEC1, MEC2, 8E5, CCRF-CEM, MOLT-3, TALL-104, AML-193, THP-1, BDCM, HL-60, Jurkat, RPMI 8226, MOLT-4, RS4, K-562, KASUMI-1, Daudi, GA-10, Raji, JeKo-1, NK-92, and Mino.

[0217] In some embodiments, a cell sample or cell lysate sample containing pores is from any tissue or fluid of an individual. Samples include, but are not limited to, tissues (such as connective tissue, muscle tissue, nerve tissue, or epithelial tissue), whole blood, isolated bone marrow, bone marrow aspirate, pleural fluid, peritoneal fluid, central spinal fluid, peritoneal fluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites, pericardial fluid, urine, saliva, bronchoalveolar lavage fluid, sweat, tears, ear fluid, sputum, hydrocele, semen, vaginal fluid, milk, amniotic fluid, and secretions of the respiratory, intestinal, or urogenital tracts. In some embodiments, the cell sample or cell lysate sample is a tissue sample, such as a sample obtained from a biopsy or tumor tissue sample. In some embodiments, the cell sample or cell lysate sample is a serum sample. In some embodiments, the cell sample or cell lysate sample is a blood cell sample containing one or more peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell sample or cell lysate sample contains one or more circulating tumor cells (CTCs). In some embodiments, the cell sample or cell lysate sample contains one or more disseminated tumor cells (DTCs, e.g., in a bone marrow aspirate sample).

[0218] In some embodiments, a cell sample or cell lysate sample containing pores is obtained from an individual by any suitable tool for obtaining such a sample using well-known conventional clinical methods. Procedures for obtaining tissue samples from an individual are well-known. For example, procedures for extracting and processing tissue samples (such as tissue samples obtained from a needle biopsy) are well-known and are utilized to obtain the samples used in the provided methods. Generally, to collect such a tissue sample, a fine hollow needle is inserted into a tissue mass (such as a tumor mass) to obtain a cell sample, which is then examined under a microscope after staining.

[0219] Sample Preparation and Analysis

[0220] In some embodiments, a sample solution containing pores includes a cell sample, a cell lysate sample, or a sample containing isolated proteins. In some cases, the sample solution includes a solution or medium (media) such as a buffer (e.g., phosphate-buffered saline). In some embodiments, the medium is an isotope-labeled medium. In some cases, the sample solution is a cell solution.

[0221] In some embodiments, a solution sample containing pores (e.g., a cell sample, a cell lysate sample, or a sample containing isolated proteins) is incubated with a compound that analyzes protein-probe interactions. In some cases, the solution sample (e.g., a cell sample, a cell lysate sample, or a sample containing isolated proteins) is further incubated in the presence of an additional compound probe. In other cases, the solution sample (e.g., a cell sample, a cell lysate sample, or a sample containing isolated proteins) is also incubated with a ligand. In such cases, the solution sample is incubated with the probe and the ligand for competitive protein profiling.

[0222] In some cases, a cell sample or a cell lysate sample containing pores is compared with a control. In some cases, a difference in a set of probe-protein interactions between the sample and the control is observed. In some cases, this difference is related to the interaction between a small molecule and one or more pore proteins.

[0223] In some embodiments, one or more methods are used to label a solution sample containing pores (e.g., a cell sample, a cell lysate sample, or a sample containing isolated proteins) to analyze probe-protein interactions. In some cases, the method includes labeling the sample (e.g., a cell sample, a cell lysate sample, or a sample containing isolated proteins) with an enriched medium. In some cases, the sample (e.g., a cell sample, a cell lysate sample, or a sample containing isolated proteins) is labeled with isotopically labeled amino acids (e.g., amino acids labeled with 13C or 15N). In some cases, the labeled sample is further compared with an unlabeled sample to detect differences in probe-protein interactions between the two samples. In some cases, this difference is the difference in the target protein and its interaction with a small molecule ligand in the labeled sample compared to the unlabeled sample. In some cases, the difference is an increase, decrease, or absence of protein-probe interactions in the two samples. In some cases, the method of isotopic labeling is called SILAC, i.e., stable isotope labeling with amino acids in cell culture.

[0224] In some embodiments, the method includes incubating a solution sample or a well sample mixture (e.g., a cell sample, a cell lysate sample, or a sample containing isolated proteins) with a labeling group to label one or more target proteins for further analysis. The labeling group can be an isotope-labeled group, such as an amino acid or acid enriched in 13C, 15N, or deuterium, or can include a molecular label, such as biotin, folic acid, luciferase, or an amino acid tag. The label can also include a linker, which can optionally be isotope-labeled. The linker can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more residues in length and may also include a cleavage site, such as a protease cleavage site (e.g., a TEV cleavage site). In some cases, the labeling group is a biotin-linker moiety, which can optionally be isotope-labeled at one or more amino acid residue positions within the linker. In some cases, the biotin-linker moiety is an isotope-labeled TEV-tag.

[0225] In some embodiments, the isotope-coded dimethylation (ReDi) method is used to process the sample. In some cases, the ReDi labeling method includes reacting a peptide with formaldehyde to form a Schiff base, which is then reduced with cyanoborohydride. This reaction dimethylates the free amino groups on the N-terminus and lysine side chains and monomethylates the N-terminal proline. In some cases, the ReDi labeling method includes methylating peptides from a first processed sample that are "lightly" labeled with reagents that carry hydrogen atoms in their natural isotope distribution and methylating peptides from a second processed sample that are "heavily" labeled with deuterated formaldehyde and cyanoborohydride. Subsequent proteomic analysis (e.g., mass spectrometry) based on the relative peptide abundances between the heavy and light peptide versions can be used to analyze probe-protein interactions.

[0226] In some embodiments, the isotope-coded relative and absolute quantification (iTRAQ) method is used to process the sample. In some cases, the iTRAQ method is based on covalently labeling the N-terminus and side-chain amino groups of peptides in the processed sample. In some cases, reagents such as 4-plex or 8-plex are used to label the peptides.

[0227] In some embodiments, the probe-protein complex is further conjugated to a chromophore (e.g., a fluorophore). In some cases, the probe-protein complex or a subsample thereof is separated and visualized using an electrophoresis system (e.g., by gel electrophoresis or capillary electrophoresis). Exemplary gel electrophoresis includes agarose-based gels, polyacrylamide-based gels, or starch-based gels. In some cases, the probe-protein is subjected to native electrophoresis conditions. In some cases, the probe-protein is subjected to denaturing electrophoresis conditions.

[0228] In some embodiments, the probe-protein complex is harvested using standard techniques known in the art and depends on the specific chemical properties of the probe-protein complex. Exemplary techniques can include, at least: tangential flow filtration, chromatography, centrifugation, liquid chromatography, electrophoresis, etc., used alone or in combination, as is commonly used to separate target proteins from mixtures.

[0229] In some cases, the harvested probe-protein complex is further fragmented to produce protein fragments. In some cases, fragmentation is produced by mechanical stress, pressure, or chemical fragmentation. In some cases, the protein from the probe-protein complex is fragmented by chemical fragmentation. In some embodiments, the chemical fragmentation reagent is a protease.

[0230] Exemplary proteases include, but are not limited to, serine proteases such as chymotrypsin A, penicillin G acylase precursor, dipeptidase E, DmpA aminopeptidase, subtilisin, prolyl oligopeptidase, D-Ala-D-Ala peptidase C, signal peptidase I, cytomegalovirus assembly protein, Lon-A peptidase, peptidase C1p, Escherichia coli phage K1F endosialidase CIMCD self-cleaving protein, nucleoporin 145, lactoferrin, muramyl tetrapeptide carboxypeptidase LD-carboxypeptidase, or rhomboid-1; threonine proteases such as ornithine acetyltransferase; cysteine proteases such as TEV protease, amidophosphoribosyltransferase precursor, γ-glutamyl hydrolase (Rattus norvegicus), hedgehog protein, DmpA aminopeptidase, papain, bromelain, cathepsin K, calpain, caspase-1, separase, adenain, pyroglutamyl-peptidase I, sortase A, hepatitis C virus peptidase 2, sindbis virus nsP2-type peptidase, dipeptidyl-peptidase VI, or DeSI-1 peptidase; aspartic proteases such as β-secretase 1 (BACE1), β-secretase 2 (BACE2), cathepsin D, cathepsin E, chymosin, napsin-A, nepenthesin, pepsin, plasmepsin, presenilin, or renin; glutamic proteases such as AfuGprA; and metalloproteases such as peptidase_M48.

[0231] In some cases, fragmentation is random fragmentation. In some cases, fragmentation produces protein fragments of a specific length, or cleavage occurs in an amino acid region of a specific sequence.

[0232] In some cases, the protein fragments are further analyzed by proteomic methods such as liquid chromatography (LC) (e.g., high performance liquid chromatography), liquid chromatography - mass spectrometry (LC - MS), matrix - assisted laser desorption / ionization (MALDI - TOF), gas chromatography - mass spectrometry (GC - MS), capillary electrophoresis - mass spectrometry (CE - MS), or nuclear magnetic resonance imaging (NMR).

[0233] In some embodiments, the LC method is any suitable LC method well - known in the art for separating a sample into its individual components. This separation occurs based on the interaction of the sample with the mobile phase and the stationary phase. Since multiple stationary phase / mobile phase combinations are used for separating mixtures, there are several different types of chromatography, which are classified based on the physical state of those phases. In some embodiments, LC is further divided into normal - phase chromatography, reverse - phase chromatography, size - exclusion chromatography, ion - exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, flash chromatography, chiral chromatography, and aqueous normal - phase chromatography.

[0234] In some embodiments, the LC method is high performance liquid chromatography (HPLC). In some embodiments, the HPLC method is further divided into normal - phase chromatography, reverse - phase chromatography, size - exclusion chromatography, ion - exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, chiral chromatography, and aqueous normal - phase chromatography.

[0235] In some embodiments, the HPLC methods of the present disclosure are carried out by any standard techniques well - known in the art. Exemplary HPLC methods include hydrophilic interaction liquid chromatography (HILIC), electrostatic repulsion - hydrophilic interaction liquid chromatography (ERLIC), and reverse - phase liquid chromatography (RPLC).

[0236] In some embodiments, LC is combined with mass spectrometry into an LC-MS method. In some embodiments, LC-MS methods include ultra-performance liquid chromatography - electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS), ultra-performance liquid chromatography - electrospray ionization tandem mass spectrometry (UPLC-ESI-MS / MS), reversed-phase liquid chromatography - mass spectrometry (RPLC-MS), hydrophilic interaction liquid chromatography - mass spectrometry (HILIC-MS), hydrophilic interaction liquid chromatography - triple quadrupole tandem mass spectrometry (HILIC-QQQ), electrostatic repulsion - hydrophilic interaction liquid chromatography - mass spectrometry (ERLIC-MS), liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS), liquid chromatography - tandem mass spectrometry (LC-MS / MS), multidimensional liquid chromatography combined with tandem mass spectrometry (LC / LC-MS / MS). In some cases, the LC-MS method is LC / LC-MS / MS. In some embodiments, the LC-MS methods herein are performed using standard techniques well known in the art.

[0237] In some embodiments, GC is combined with mass spectrometry as a GC-MS method. In some embodiments, GC-MS methods include two-dimensional gas chromatography time-of-flight mass spectrometry (GC*GC-TOFMS), gas chromatography time-of-flight mass spectrometry (GC-QTOF-MS), and gas chromatography - tandem mass spectrometry (GC-MS / MS).

[0238] In some embodiments, CE is combined with mass spectrometry as a CE-MS method. In some embodiments, CE-MS methods include capillary electrophoresis - negative electrospray ionization - mass spectrometry (CE-ESI-MS), capillary electrophoresis - negative electrospray ionization - quadrupole time-of-flight mass spectrometry (CE-ESI-QTOF-MS), and capillary electrophoresis - quadrupole time-of-flight mass spectrometry (CE-QTOF-MS).

[0239] In some embodiments, the nuclear magnetic resonance (NMR) method is any suitable method well known in the art for detecting one or more binding proteins or protein fragments that interact with the small molecules described herein. In some embodiments, the NMR methods include one-dimensional (1D) NMR methods, two-dimensional (2D) NMR methods, solid-state NMR methods, and NMR chromatography. Exemplary 1D NMR methods include 1H, 13C, 15N, 17O, 19F, 31P, 39K, 23Na, 33S, 87Sr, 27Al, 43Ca, 35Cl, 37Cl, 63Cu, 65Cu, 57Fe, 25Mg, 199Hg, or 67Zn NMR methods, distortionless enhancement by polarization transfer (DEPT) methods, attached proton test (APT) methods, and 1D-natural abundance double quantum special transition experiment (INADEQUATE) methods. Exemplary 2D NMR methods include correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), 2D-INADEQUATE, 2D-adequate double quantum transfer experiment (ADEQUATE), nuclear Overhauser effect spectroscopy (NOESY), rotating frame NOE spectroscopy (ROESY), heteronuclear multiple quantum correlation spectroscopy (HMQC), heteronuclear single quantum coherence spectroscopy (HSQC), short-range coupling and long-range coupling methods. Exemplary solid-state NMR methods include solid-state 13C NMR, high-resolution magic angle spinning (HR-MAS), and cross-polarization magic angle spinning (CP-MAS) NMR methods. Exemplary NMR techniques include diffusion-ordered spectroscopy (DOSY), DOSY-TOCSY, and DOSY-HSQC.

[0240] In some embodiments, the protein fragments are analyzed by the method described by Weerapana et al., “Quantitative reactivity profiling predicts functional cysteines in proteomes,” Nature, 468:790-795 (2010).

[0241] In some embodiments, the results of mass spectrometry are analyzed by an algorithm for protein identification. In some embodiments, the algorithm combines the results of mass spectrometry with a protein sequence database for protein identification.

[0242] In some embodiments, the algorithm includes the ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot.

[0243] In some embodiments, a value is assigned to each protein in the probe - protein complex. In some embodiments, the value assigned to each protein in the probe - protein complex is obtained from mass spectrometry. In some cases, the value is the area under the curve of a plot of signal intensity versus mass - to - charge ratio. In some cases, the value is related to the reactivity of Lys residues within the protein.

[0244] In some cases, the ratio between a first value obtained from a first protein sample and a second value obtained from a second protein sample is calculated. In some cases, the ratio is greater than 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, the ratio is at most 20.

[0245] In some cases, the ratio is calculated based on an average value. In some cases, the average value is the average of at least two, three, or four values of the protein from each cell solution, or the protein is observed at least two, three, or four times in each cell solution, and a value is assigned for each observation time. In some cases, the ratio also has a standard deviation less than 12, 10, or 8.

[0246] For example, in some embodiments of the present invention, a first isolated complex sample of pore bodies is generated from a human epithelial cell line, and a second isolated complex sample of pore bodies is generated from a cystic fibrosis mutant line 508. The ratio can be first:second or second:first, as long as the ratio is consistently performed between all the proteins being compared from each sample mixture. In some cases, any substance with a ratio of 20% or less in the 95% confidence interval is considered a missing protein. Those skilled in the art can understand the need to vary the ratio and the cut - off value of the confidence interval without departing from the scope of the present invention. In some embodiments of the present invention, the second sample mixture can be a "knock - out" cell line, in which one or more pore body proteins are "knocked out" using a technique such as CRISPER.

[0247] Kit / Product

[0248] In some embodiments, kits and articles of manufacture for generating porin adducts or for use in one or more of the methods described herein are disclosed. In some embodiments, kits for detecting porin-ligand interactions are described herein. In some embodiments, such kits include small molecule ligands, small molecule fragments or libraries, compound probes, and / or controls, as well as reagents suitable for performing one or more of the methods described herein. In some cases, the kits further include a sample, such as a cell sample, and a suitable solution, such as a buffer or medium. In some embodiments, the kits further include recombinant porin for one or more of the methods described herein. In some embodiments, additional components of the kits include a carrier, package, or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each container including a separate element for use in the methods described herein. Suitable containers include, for example, bottles, vials, plates, syringes, and test tubes. In one embodiment, the container is formed of a variety of materials, such as glass or plastic.

[0249] Articles of manufacture provided herein contain packaging material. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, bags, containers, and any packaging material suitable for the selected formulation and intended mode of use.

[0250] For example, the container includes a probe, a test compound, and one or more reagents for use in the methods disclosed herein. Such kits optionally include an identifying description or label or instructions related to its use in the methods described herein.

[0251] Kits generally include a label listing the contents and / or instructions for use, as well as a package insert with instructions for use. A set of instructions is usually also included.

[0252] In one embodiment, the label is located on or associated with the container. In one embodiment, the label is located on the container when letters, numbers, or other characters constituting the label are attached, molded, or etched into the container itself; the label is associated with the container when the label is located in a receptacle or carrier that houses the container, such as a package insert. In one embodiment, the label is used to indicate that the contents are for a specific therapeutic application. The label also indicates instructions for use of the contents, such as instructions for use in the methods described herein.

[0253] Some Terms

[0254] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit any claimed subject matter. In this application, unless otherwise expressly stated, the use of the singular includes the plural. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. In this application, unless otherwise indicated, the use of "or" means "and / or". Further, the use of the term "including" and other forms (e.g., "include", "includes", and "included") is not limiting.

[0255] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Thus, "about 5 μL" means "about 5 μL" and "5 μL". Generally, the term "about" includes amounts that are expected to be within experimental error or within the expected error of manufacturing, production, or experimental tolerances.

[0256] Appropriate modifications to the foregoing will be apparent to those of ordinary skill in the art and are naturally covered and expressly contemplated. For example, normal manufacturing tolerances may result in variations from the foregoing formulations without departing from the broader scope of the invention.

[0257] The compounds described herein can be formed into, and / or used as, acceptable salts. Types of acceptable salts include, but are not limited to: (1) acid addition salts, formed by reacting the free base form of the compound with an acceptable inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, metaphosphoric acid, etc.; or an acceptable organic acid, such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, etc.; (2) salts formed when an acidic proton in the parent compound is replaced by a metal ion, such as an alkali metal ion (e.g., lithium, sodium, potassium), an alkaline earth metal ion (e.g., magnesium or calcium), or an aluminum ion. In some cases, the compounds described herein can coordinate with an organic base, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, dicyclohexylamine, tris-(hydroxymethyl)methylamine. In other cases, the compounds described herein can form salts with an amino acid, such as, but not limited to, arginine, lysine, etc. Acceptable inorganic bases for forming salts with compounds containing an acidic proton include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.

[0258] The effective dosage and method of administration of specific embodiments of the present invention may vary depending on the individual patient and the stage of any existing diseases (such as influenza, COVID-19, HIV, other comorbidities), as well as other factors known to those skilled in the art. The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmacological procedures in cell culture or experimental animals, such as ED50 (the dose that has a therapeutic effect on 50% of the population) and LD50 (the dose that has a lethal effect on 50% of the population). The dose ratio of toxicity to therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Pharmaceutical compositions with a large therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used to formulate a dosage range for human use. The dosage of such compounds is preferably within a circulating concentration range that includes the ED50 and has little or no toxicity. The dosage varies within this range depending on the dosage form employed, the sensitivity of the patient, and the route of administration.

[0259] The exact dosage is selected by the individual physician according to the patient to be treated. The dosage and administration are adjusted to provide a sufficient level of the embodiments of the present invention to maintain the desired effect (e.g., eliminating or reducing the enveloped virus particles or activity in the host). Other factors that can be considered include the severity of any disease state, the age, weight, and sex of the patient; diet, time and frequency of administration, drug combination, response sensitivity, and tolerance / response to the treatment.

[0260] The short-acting pharmaceutical composition is administered daily, while the long-acting pharmaceutical composition is administered once every 2, 3 to 4 days, weekly, or every two weeks or longer. Depending on the half-life and clearance rate of the specific formulation, the pharmaceutical composition of the present invention can be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more times a day.

[0261] The normal dosage of the active ingredient may vary from about 1 to 100,000 μg, with a maximum total dosage of about 10 g, depending on the route of administration. Desirable dosages include 250 μg, 500 μg, 1 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, and 10 g.

[0262] More specifically, the dosage of the active ingredient described herein is a dosage that provides a sufficient amount to achieve the desired effect, including the effects described above (e.g., modulating, activating, or interacting with one or more porins and / or achieving an effect on the pore structure). Thus, the dosage of the active ingredient preferably produces a tissue concentration or blood concentration of about 1 to 800 μM. The preferred dosage produces a tissue or blood concentration of more than about 10 μM to about 500 μM. Preferred dosages are, for example, the amounts of the active ingredient required to achieve the following tissue or blood concentrations or both: 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 220 μM, 240 μM, 250 μM, 260 μM, 280 μM, 300 μM, 320 μM, 340 μM, 360 μM, 380 μM, 400 μM, 420 μM, 440 μM, 460 μM, 480 μM, and 500 μM. Although dosages that produce a tissue concentration of more than 800 μM are not necessarily preferred, they are conceivable and can be used in conjunction with some embodiments of the present invention. Embodiments of the present invention can be administered continuously to maintain a stable concentration of the therapeutic agent.

[0263] Finally, the written description uses examples to disclose the invention, including the best mode, and also enables those skilled in the art to practice the invention, including making and using any device or system and performing any incorporated method. The patentable scope of the present invention is defined by the claims and may include other examples that occur to those skilled in the art. If these other examples have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that do not differ materially from the literal language of the claims, then these other examples are intended to fall within the scope of the claims.

[0264] The pharmacologically active compounds of the present invention can be processed according to conventional pharmaceutical methods and good manufacturing practices to produce medicaments for prophylactic administration or as part of a treatment regimen to a patient (e.g., a mammal, including a human).

[0265] As used herein, the term "sequence" expressly encompasses DNA, cDNA, RNA, and the peptide chains encoded thereby (both forward and reverse). Knowledge of one of the sequences implies knowledge of the other sequences through the standard rules of complementarity and codon encoding, as shown in the standardized DNA, RNA, and amino acid codon tables.

[0266] As used herein, an element or step recited in the singular and preceded by the word "a" or "an" should be understood as not excluding a plurality of said elements or steps, unless explicitly stated to the contrary. In addition, a reference to "one embodiment" of the present invention should not be construed as excluding the existence of additional embodiments that also incorporate the recited features. Further, unless explicitly stated to the contrary, an embodiment comprising, including, or having an element or elements with a particular property may include additional elements that do not have that property.

[0267] Since some changes may be made to the above invention without departing from the spirit and scope of the invention herein involved, all of the subject matter described above shown in the drawings should be construed merely as illustrative examples of the inventive concept herein and not as limiting the present invention.

Claims

1. A method for identifying protein-protein interactions in a pore structure, comprising: a) Create a first pore body sample mixture; b) Incubate the pore body sample mixture with a labeling group to generate a probe - protein complex; c) Harvest the probe - protein complex; d) Fragment the probe - protein complex to generate protein fragments; e) Analyze these protein fragments by proteomic methods; f) Identify one or more proteins in the pore body sample mixture to create a first identified proteome; g) Assign a value to each protein in the first identified proteome; h) Perform steps a) - g) on a second sample pore body mixture to obtain second values for each protein in the second identified proteome; i) Calculate the ratio between the values of paired proteins in the first and second identified proteomes; wherein the ratio determines protein - protein interactions inside or adjacent to the pore body structure.

2. The method of claim 1, wherein the first sample pore mixture is from a standard, control or wild-type cell sample, and the second sample pore mixture is from a test cell sample.

3. The method of claim 2, wherein the test cell sample is from a knockout cell line.

4. A kit configured to contain materials required to perform the steps of claim 1.

5. The method of claim 1, wherein the proteomics method is at least one selected from the group consisting of: LC, LC-MS, MALDI-TOF, GC-MS, CE-MS, and NMR.

6. The method of claim 1, wherein the values of each protein in the first and second identification proteomes are related to the reactivity of Lys residues within the protein.

7. The method of claim 1, further comprising: j) Use small molecules to confirm specific protein - protein interactions within the pore body complex.

8. The method of claim 7, wherein the confirmation is performed by chemical cross-linking, and subsequent confirmation of the linkage is performed by mass spectrometry.

9. The method of claim 1, wherein the functions of the first and second sample pore complexes are examined in an artificial lipid bilayer membrane.

10. A composition comprising: Artificial pores in an artificial lipid bilayer membrane.

11. A method comprising: Cross - link an artificial pore body to a humanized nanobody to target and bind one or more domains of one or more pore body or pore body - associated proteins; Deliver the cross - linked pore body - nanobody to a subject.

12. A composition comprising: At least one cross-linking molecule; At least one small molecule regulator targeting pore proteins.

13. The composition of claim 12, wherein the cross-linking molecule is an ELP diblock.

14. The composition of claim 12, further comprising: A nanobody that is humanized to target and bind one or more domains of one or more pore body proteins.

15. A composition comprising: A humanized nanobody having one or more small molecules and an artificial cysteine, wherein the small molecules target multiple domains of one or more pore body proteins; The cysteine binds to an ELP diblock; The ELP diblock binds to pAcF.

16. The composition of claim 15, wherein a drug is attached to pAcF.

17. The composition of claim 16, wherein the drug attached to pAcF is doxorubicin.

18. A method comprising: Extract pore bodies from non - human sources; Reconstitute the extracted pore bodies in human cells.

19. The method of claim 18, wherein the pore bodies are extracted from human epithelial cells and stem cells.

20. The method of claim 18, wherein the isolated pore bodies are reconstructed into organoids or artificial lipid bilayers.

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