Functional porosome engineering

Abstract

Porosomes are the primary secretory structures of eukaryotic cells. Provided herein are compositions and methods for the control and modulation of porosome structure, including methods for identifying porosome-associated proteins and interacting small molecules, the use of small molecules that target one or more porosome proteins, compositions and uses of nanobodies conjugated with small molecules, reconstitution of porosomes, and the creation and use of artificial porosome structures.

Description

[Technical Field]

[0001] The present disclosure provides methods and compositions for modulating, e.g., regulating or altering, the structure and function of porosome organelles and / or their constituent proteins. In certain 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 and affect the structure and function of porosome organelles. Methods of using such targeting molecules for the treatment of porosome-associated disorders are also disclosed. Further embodiments include functional reconstitution of porosomes or porosome-like structures in target cells to overcome physiological defects. Further embodiments further include the use of humanized nanobodies, alone or in combination with small molecules, that target one or more porosome proteins to alter the structure and / or function of the porosome complex. [Background technology]

[0002] Porosomes are cup-shaped supramolecular lipoprotein structures located in the plasma membrane, where intracellular secretory vesicles transiently dock, fuse, and secrete their contents extracellularly.

[0003] Typical porosome structures range in size from 15 nm in neurons to 100–180 nm in endocrine and exocrine cells. Porosomes are composed of approximately 30–40 proteins, and porosome composition varies among cell types. Porosome-mediated secretion across the plasma membrane is a fundamental process by which cells communicate and exchange information with the extracellular environment. In multicellular systems, porosome-mediated secretion allows cell populations to communicate with each other, maintain homeostasis, and thus sustain life. Porosomes are present 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 of the inner ear, and neurons that secrete neurotransmitters. Porosomes have been immunoisolated from a variety of cells, including insulin-secreting β cells of the exocrine pancreas, human airway epithelial cells, and neurons, biochemically characterized, and functionally reconstituted into artificial lipid membranes. Much evidence is accumulating regarding the role of porosome-associated proteins in cellular secretion and secretory disorders, such as neurotransmission and neurological disorders, respiratory disorders, insulin secretion disorders, etc. Thus, impaired cellular secretion due to dysfunction of porosomes or porosome components has been implicated in the mechanistic underpinnings of numerous diseases, including cystic fibrosis, diabetes, Alzheimer's disease, Down's syndrome, schizophrenia, digestive disorders, and immune disorders, among others.

[0004] Although the "parts list" of biology is relatively well defined and many proteins have been identified in human cells, our systems-level understanding of disease modalities is only just beginning to be fully appreciated. It is no longer true that any given disease results from a faulty copy of a single protein. Furthermore, given epigenetics, it is no longer true that the expression of a given protein structure results solely from a single gene. Additionally, it is becoming clear that many proteins collectively participate in multiple cellular processes (e.g., they are typically expressed as found in different metabolic or enzymatic pathways). Therefore, targeting a single protein without altering multiple cellular functions is challenging.

[0005] Therefore, targeting a single protein for either binding or degradation may treat a disease but may also inhibit important bodily functions and / or alternative biochemical pathways that require the same protein, resulting in adverse side effects. Furthermore, each protein requires a "groove" or "binding pocket" for a putative drug to capture and bind. Furthermore, the binding pocket on the surface of a target protein is also influenced by neighboring proteins, requiring a careful understanding of the entire complex to develop an appropriate treatment. Furthermore, it is estimated that only 20-25% of proteins possess the groove necessary for small molecule binding, modulation, inhibition, or activation.

[0006] Given the significant demands posed by the nature of diseases caused by secretory dysfunction, there is a great need for methods to modulate porosome structure or function in order to correct or otherwise alter the secretory disorder, thereby treating diseases resulting from impaired porosome secretion. Furthermore, when porosome structure is defective or absent, there is a need to restore porosome structure or to provide porosomes or porosome-like structures to affected cells. Summary of the Invention

[0007] The present disclosure provides methods for identifying protein interactions that modulate porosome function or restore the function of specific porosome proteins. In some embodiments, high-throughput chemical screening techniques are used. In yet other embodiments, in silico techniques are used. The identified proteins are then validated for activity and function in animal models and ultimately in humans. In certain embodiments, the method involves creating a first porosome sample mixture and then incubating the porosome sample mixture with a labeling group to generate probe-protein complexes. The probe-protein complexes are then recovered and fragmented to obtain protein fragments. The protein fragments are then analyzed by proteomic methods. Proteins in the porosome sample mixture can then be identified to create a first identified protein set. A value is assigned to each protein in the first identified protein set. In some examples, the assigned value can represent the amount of the protein in the porosome sample mixture in either absolute terms (e.g., grams or moles) or relative terms (e.g., percentage or ratio). The above steps are repeated for a second sample porosome mixture to obtain a second value for each protein in the second identified protein set. The ratio of the values ​​for paired proteins in the first and second identified protein sets is then calculated. In some instances, the first value is expressed as a ratio to the second value. In other instances, the second value is expressed as a ratio to the first value. The resulting ratio is thus a measure of protein-protein interactions within or adjacent to the porosome structure.

[0008] In certain embodiments, the first sample porosome mixture is derived from a standard, control, or wild-type cell sample, and the second sample porosome mixture is derived from a test cell sample. Additionally, the test cell sample may be a knockout cell line.

[0009] In yet other embodiments, the first and second porosomal cell sample mixtures may be at least one selected from a cell sample, a cell lysate sample, and isolated porosomal proteins.

[0010] In yet other embodiments of the above method, the probe-protein complex is conjugated to a chromophore during the incubation step, hi yet other embodiments, the probe-protein complex or a subsample thereof is separated and visualized by electrophoresis after incubation and prior to recovery of the probe-protein complex.

[0011] In certain other embodiments of the above methods, the fragmentation is completed or achieved by at least one selected from mechanical stress, pressure, and a chemical fragmentation agent, hi yet other embodiments, the chemical fragmentation agent is a protease.

[0012] In yet another embodiment of the above method, the proteomic method comprises the use of mass spectrometry. Indeed, in yet another embodiment, the proteomic method is at least one selected from LC, LC-MS, MALDI-TOF, GC-MS, CE-MS, and NMR. Additionally, in a further embodiment, the value for each protein in the first and second identified protein sets is obtained from mass spectrometry. In a further embodiment, the value is the area under the curve of a plot of signal intensity as a function of mass-to-charge ratio. In a further embodiment, the value for each protein in the first and second identified protein sets correlates with the reactivity of Lys residues within the protein. In a further embodiment, the identified protein-protein interactions can be confirmed using small molecules. Confirmation can be achieved through chemical cross-linking followed by confirmation of binding by mass spectrometry.

[0013] In certain embodiments, the function of porosomes from the first and second samples can be tested in artificial lipid bilayer membranes. In yet another embodiment, artificial porosomes can be made and assembled in artificial lipid bilayer membranes.

[0014] For all of the above, kits can be prepared to accomplish one or more steps of the method. The present disclosure includes methods of modulating prosomal activity in a subject in need thereof, for example, by administering to the subject an effective amount of an identified small molecule.

[0015] In some embodiments, the identified small molecule modulators, or pharmaceutically acceptable salts or solvates thereof, are used to modulate the activity of one or more porosomal proteins.

[0016] In some embodiments, the porosomal protein can be, for example, a neuronal porosomal protein, a mucus secretory porosomal protein of airway epithelium, an insulin secretory protein, a digestive enzyme secretory protein, or a signal molecule secretory protein.

[0017] In some embodiments, the present disclosure provides small molecules, e.g., molecules less than 1000 MW, that can target and modulate the production of one or more porosome component proteins by modulating, regulating, or controlling the expression, methylation status, or interference (e.g., RNAi, siRNA) of the genomic sequences (DNA, RNA) encoding the component proteins. In some embodiments, host cells are removed from a patient, treated with a small molecule to alter porosome structure or function, and then returned to the patient.

[0018] In some embodiments, exosome release is controlled by altered structure and / or function of porosome complexes or porosome-associated proteins. In certain embodiments, genes for putative porosome complexes or porosome-associated proteins may be "knocked out" in one or more cell types using CRISPER, RNAi, or other methods known in the art.

[0019] In certain embodiments, proteome and lipidome databases are cross-correlated to identify candidate interaction sites. Protein-protein or protein-lipid candidates are chemically cross-linked and evaluated by mass spectrometry to confirm the interaction. Once the interaction is confirmed, multivalent small molecules are generated. Such multivalent molecules may contain, for example, one or more peptide target sequences bound to a bridge molecule. Such bridge molecules are further configured to hold the peptide target sequences in the correct orientation and distance, allowing the peptide target sequences to bind to at least two porosome-associated targets and modulate porosome function. The modulation of porosome function is then validated through standard drug validation pathways, from animal models to human clinical trials. For example, validation can be achieved by first assessing binding affinity to target porosome proteins and lipids in silico, followed by cell culture, organoid, and animal studies.

[0020] In certain embodiments, porosomes isolated from specific tissues or cell types in artificial lipid bilayer membranes can also be used to screen candidate drugs, optimize dosage, or determine efficacy. In certain embodiments, isolated porosomes in artificial lipid bilayer membranes can be administered to subjects suffering from porosome-mediated diseases (e.g., diseases in which alterations in secretory function are known or implicated, such as cystic fibrosis, diabetes, Alzheimer's disease, etc.).

[0021] Some embodiments relate to methods of modulating prosomal activity in a subject in need thereof, in which an effective amount of the identified small molecule is administered to a subject in need thereof.

[0022] In some embodiments, the identified small molecule modulators, or pharmaceutically acceptable salts or solvates thereof, may be used to modulate the activity of one or more porosomal proteins.

[0023] In some embodiments, the porosomal protein can be a neuronal porosomal protein. In yet other embodiments, the porosomal protein can be derived from airway epithelial cells. In yet other embodiments, the porosomal protein can be an insulin-secreting protein. In yet other embodiments, the protein can be a digestive enzyme-secreting protein. In yet other embodiments, the porosomal protein can be a signal molecule-secreting protein derived from signal molecule-secreting cells.

[0024] In some embodiments, one or more small molecules can target and modulate the production of one or more porosome-constituting proteins by modulating, regulating, or controlling the expression, methylation status, or interference (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, grown, purified, treated with small molecules to alter porosome structure or function, and then returned to the patient.

[0025] In yet other embodiments, the present disclosure provides engineered nanobodies that are used alone or in combination with one or more small molecules to alter porosome function. For example, a nanobody that targets a first porosomal protein binding site can be used in combination with one or more small molecules that interact with one or more additional porosomal proteins. In yet other embodiments, an artificial porosome is crosslinked to a humanized nanobody that binds to one or more domains of one or more porosomes or porosome-associated proteins. The crosslinked artificial porosome nanobody can then be delivered to a subject.

[0026] In yet another embodiment, a composition is provided comprising at least one bridge molecule and at least one small molecule modulator that targets a porosomal protein. In certain embodiments, the bridge molecule is an ELP deblock. In still other embodiments, the bridge molecule is p-acetylphenylalanine. In still other embodiments, the bridge molecule is maleimide. In still other embodiments, the small molecule modulator is CDN1163. In still other embodiments, at least a second small molecule modulator is present. In still other embodiments, the second small molecule modulator targets a porosomal lipid. In certain embodiments, the target porosomal protein is at least one selected from syntaxin-1A, SNAP-25, SNAP-23, and actin. In still other embodiments, the small molecule modulator targets a protein identified using the above method. In still other embodiments, a humanized Nanobody is provided that targets and binds to one or more domains of one or more porosomal proteins. In still other embodiments, the humanized Nanobody contains an artificial cysteine ​​configured to allow attachment of an ELP deblock to the Nanobody. In yet other embodiments, the Nanobody is further conjugated to one or more small molecules to form a multivalent structure.

[0027] In yet another embodiment, the present disclosure provides humanized Nanobodies comprising one or more small molecules targeting a domain of one or more porosomal proteins and an artificial cysteine. The ELP diblock binds to the cysteine ​​and pAcF. In certain embodiments, a drug is conjugated to pAcF. In yet another embodiment, the drug conjugated to pAcF is doxorubicin. In yet another embodiment, one or more domains of one or more porosomal proteins are identified that interact with one or more porosomal proteins that are known to interact with each other. In yet another embodiment, the one or more domains further comprise at least one selected from a K+ channel, an aquaporin water channel, an anion exchanger, a membrane fusion protein, a sodium bicarbonate transporter, Gαi3, syntaxin-1A, SNAP-25, SNAP-23, and actin.

[0028] The present disclosure further provides methods comprising extracting porosomes from a non-human source. The porosomes are then reconstituted into human cells. In some embodiments, the porosomes are extracted from human epithelial cells and / or stem cells. In yet other embodiments of the method, the extracted porosomes are reconstituted into organoids or artificial lipid bilayer membranes. In yet other embodiments, the non-human source is a pig or other mammal.

[0029] The present disclosure also provides methods for identifying small molecules that target specific porosome proteins to modulate or restore porosome function. In some embodiments, a single CRISPR knockout of a selected porosome protein is used to determine additional proteins that are lost from the porosome complex in addition to the knocked-out protein. These additional proteins are believed to be associated with each other and with the knocked-out protein within the complex.

[0030] Some embodiments include a method for modulating porosome-mediated insulin secretion in a subject in need thereof. In such embodiments, overexpression of a porosomal protein effective to synthesize and secrete insulin upon glucose loading is induced in the subject, and / or an effective amount of an identified small molecule to synthesize and secrete insulin upon glucose loading is administered to the subject. The porosomal protein in this embodiment can be a porosomal protein in pancreatic beta cells of the subject.

[0031] In some embodiments, overexpression of either or both of the insulin-secreting porosomal proteins ATP2C1 (ATPase secretory pathway Ca2+ transport) or APOa1 can increase both insulin expression and secretion in beta cells of the endocrine pancreas.

[0032] In some embodiments, the Ca2+-ATPase activator CDN1163 is used to increase both insulin expression and secretion in beta cells of the endocrine pancreas.

[0033] In certain embodiments, the present disclosure provides methods for reconstituting functional porosome complexes into living cells to ameliorate or correct secretory disorders resulting in dysfunction of one or more porosome proteins. In certain embodiments, reconstitution is performed on neurons or cells from the exocrine and endocrine pancreas. In yet other embodiments, reconstitution includes mucin-secreting porosome complexes in other cell types, such as the lung epithelium of cystic fibrosis (CF) patients.

[0034] In another embodiment, the present disclosure provides a method for the scale-up isolation of mucin-secreting porosome complexes from human lung epithelial cells. In certain embodiments, the method enables the isolation of mucin-secreting porosome complexes from Calu3 and other epithelial cells for reconstitution therapy in CF patients, thereby preventing immune rejection and ameliorating the secretory defect caused by dysfunction of the porosome-associated cystic fibrosis transmembrane conductance regulator ("CFTR") protein.

[0035] In a further embodiment, the present disclosure provides a method for identifying protein-protein interactions in functional porosome complexes within cells. In particular, the identification of protein-protein interactions with the CFTR protein within mucin-secreting porosome complexes in airway epithelia is disclosed. Thus, in a further embodiment, small molecule drug-nanobody conjugates can be used to fine-tune and precisely target the regulation of the mucin-secreting porosome secretion machinery in lung epithelial cells.

[0036] In a further embodiment, there is a method for identifying one or more modulators of mucin secretion porosomal proteins to optimize mucin production and secretion from airway epithelial cells, and these mucin secretion porosomal protein modulators are used alone or in combination with reconstituted porosome complexes in CF therapy.

[0037] The present disclosure further provides an approach for appropriately identifying and tailoring small molecules that target specific porosome proteins to regulate or restore mucin secretory porosome function. In some embodiments, CRISPR knockout of one porosome protein at a time is used to determine which other proteins in the complex are lost from the porosome complex in addition to the knocked-out protein, particularly the CFTR protein. In certain embodiments, proteins lost from the porosome complex when CFTR is knocked out are classified as being related to CFTR in the complex. In certain embodiments, the identified related proteins are targeted to regulate and improve secretory function and correct CFTR-mediated secretory disorders.

[0038] Some embodiments relate to methods of modulating prosome-mediated mucin secretion activity in a subject in need thereof, in which an 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 facilitate proper secretion of mucins. [Brief explanation of the drawings]

[0039] The features and advantages of embodiments of the present invention will become apparent from a reading of the following detailed description with reference to the drawings, which are illustrative but non-limiting. [Figure 1] FIG. 1 shows examples of porosome structures in the exocrine pancreas, neurons, and somatotrophs of the pituitary gland by different imaging modalities. [Figure 2] FIG. 1 is a schematic diagram of porosome-mediated secretion. [Figure 3] FIG. 1 is a schematic diagram of an embodiment of the present invention (porosome complexes and precise targeting of porosome complexes for therapy). [Figure 4A] FIG. 1 is a schematic diagram showing predicted interactions between identified proteins and other regulatory proteins within the neuronal porosome proteome. [Figure 4B]FIG. 1 is a schematic diagram showing predicted interactions between identified proteins and other regulatory proteins within the neuronal porosome proteome. [Figure 5] 1 is an example of drug target specificity through combinatorial small molecule drug design using tissue-specific and porosome-specific multivalent nanobodies. [Figure 6] 1 is a depiction of porosome-associated proteins involved in different disease classes. [Figure 7] FIG. 1 shows knockout (KO) and overexpression (OE) of porosomal proteins ATP2C1 and APOa1 in insulin-secreting Min6 cells. [Figure 8] Figure 1 shows glucose-stimulated insulin secretion in Min6 cells in knockout (KO) and overexpression (OE) of three porosomal proteins, ATP2C1, APOa1, and TREK1. Overexpression of APOa1 leads to increased insulin synthesis and secretion. [Figure 9] 1 shows glucose-stimulated insulin secretion in Min6 cells after a 2-hour exposure to increasing concentrations of the ATP2C1 activator CDN1163, where 10 micromolar CDN1163 was found to be the optimal dose. [Figure 10] Representative electron micrographs of Calu-3 cells in culture showing the presence of microvilli (MV) and 100 nm cup-shaped porosomes (P) at the plasma membrane. [Figure 11] FIG. 1 illustrates the use of Ussing chamber experiments to demonstrate that forskolin-stimulated chloride release from Calu-3 cells is inhibited in the presence of the CFTR inhibitor GlyH-101. [Figure 12] FIG. 1 shows that the CFTR inhibitors 172 and GlyH-101 inhibit forskolin-stimulated secretion of intravesicular mucin from Calu-3 cells. [Figure 13] Transmission electron micrograph of porosome complexes associated with docked secretory vesicles at the apex of exocrine pancreatic acinar cells. [Figure 14]Transmission electron micrographs of insulin-secreting Min6 cells. [Figure 15] Electron micrographs of porosome complexes in liposomes in reconstituted exocrine pancreas. Note the formation of reconstituted cup-shaped porosomes. [Figure 16] FIG. 1 shows that lipid bilayer-reconstituted porosome complexes from the exocrine pancreas are functional. [Figure 17] FIG. 1 shows the abundant presence of TREK-1, Gi3, and syntaxin-1A immunoreactivity in porosome-reconstituted insulin-secreting Min6 cells. [Figure 18] FIG. 1 shows insulin secreting porosomes reconstituted in live Min6 cells, demonstrating increased glucose-stimulated insulin secretion. [Figure 19] FIG. 10 shows the abundant presence of G″i3 and syntaxin-1A immunoreactivity and increased glucose-stimulated insulin secretion in porosome-reconstituted Min6 cells even after 48 hours. [Figure 20] FIG. 1 is a schematic diagram of delivery of functional mucin-secreting porosome complexes to airway epithelial cells of CF patients and amelioration of CF disease symptoms. [Figure 21] Immunoblot analysis of whole Calu-3 cell homogenates (CH) and isolated porosome complexes (P) shows the presence of the porosome proteins actin, Gαi3, and vimentin. Note the abundant presence of these proteins in the porosome complexes. [Figure 22] (Figure 1) Immunoisolated CFTR-associated polosome complexes using a CFTR-specific antibody. Note the pull-down of polosome-associated proteins such as syntaxin-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. [Figure 23]FIG. 1 shows the restoration of mucus secretion in ΔF508-CFTR human CF bronchial epithelial cells by CDN1163. [Figure 24] FIG. 1 shows that α-CPA increases mucus secretion in ΔF508-CFTR human CF bronchial epithelial cells. [Figure 25] FIG. 1 is a schematic diagram showing some of the interactions between CFTR and associated proteins. DETAILED DESCRIPTION OF THE INVENTION

[0040] Identification of porosome protein interaction targets Modern drug discovery is the result of an evolution from past technologies: screening biological extracts directly from nature for active substances, the use of molecular biology techniques to generate drugs directed at specific molecular targets, and the creation of the modern drug development paradigm of "rational drug design" in the 1970s and 1980s, which focused on small molecules, antibodies (used to block the specific function of a single target protein), and recombinant protein-based therapeutics. Subsequent technological innovations, such as genomics and gene editing, stem cell biology, patient-derived organoids, cryo-electron microscopy, synthetic biology, small molecule screening using AI machine learning and high-throughput screening, and image analysis, have further transformed the landscape of drug discovery. Indeed, with the aid of AI models such as AlphaFold2 and similar, the final folding structure of a given protein can now be predicted and easily verified from a given gene sequence. Furthermore, such models enable the development of so-called engineered proteins, which do not exist in nature.

[0041] Despite these advances, the National Cancer Institute (NCI) points out that 85% of human proteins are not druggable (i.e., these proteins, including those that cause disease, cannot be pharmacologically targeted). In fact, "The number of new drugs is declining because it is becoming increasingly difficult to develop new drugs. In other words, we are running out of proteins that can be drugged. What remains are 'undruggable.' Most proteins that drive disease processes are undruggable." (Brent Stockwell, Columbia University) The American Society of Clinical Oncology further points out that "pharmacological targeting of recalcitrant proteins currently represents a significant challenge in modern drug development, necessitating innovation and the development of new technologies."

[0042] Attempts to address the formidable barriers of "undruggable" proteins to drug development include a select few innovative approaches. One approach is targeted proteolysis, which tags and destroys disease-causing proteins to block their function. A second approach is the creation of chemoproteomic platforms used to identify unknown binding pockets in undruggable targets in order to produce diverse small molecule therapeutics across indications.

[0043] In stark contrast to the two approaches outlined above, embodiments of the present invention embrace a third, entirely different approach. This takes a systems-level approach to targeting multiple proteins in the cellular communication machinery, embodied by porosomes and their ability to alter cellular secretory activity. This approach addresses specificity and precise targeting while overcoming the "undruggability" problem, providing a unique route to address previously difficult-to-treat diseases caused by dysfunction of proteins and lipids involved in cellular secretion, ranging from cystic fibrosis to diabetes, Alzheimer's disease, and cancer. The present invention is unique in its pioneering efforts to target both druggable and undruggable proteins and lipids that cause secretory disorders and consequently lead to cystic fibrosis, cancer, neurological, endocrine, and immune disorders, among others. Thus, embodiments of the present invention control or alter disease progression by targeting multiple proteins within dysfunctional target functional cellular complexes (e.g., porosome structures). In certain embodiments, the target functional cellular complexes are porosomes. In yet other embodiments, multiple proteins within porosomes are targeted. In yet another embodiment, one or more additional proteins not present in porosomes are also identified and targeted. The benefits of such precise targeting can lead to the creation of precision therapies, which are expected to reduce overall side effects and increase therapeutic efficacy. Figure 1 shows examples of porosome structures in the exocrine pancreas, neurons, and neuroendocrine cells using different imaging modalities.

[0044] As shown in Figure 1 (described in more detail below), porosomes are cup-shaped supramolecular lipoprotein structures in the plasma membrane of eukaryotic cells. Secretory vesicles transiently dock and fuse with porosome complexes during the process of vesicle fusion and content release during secretion. The porosome structure is composed of many proteins, which themselves are often involved in other cellular complexes. Examples of purified neuronal porosome proteins are listed in Table 1. Further examples of proteins that constitute porosomes can be obtained by those skilled in the art from the literature. Because porosomes are embedded in the lipid bilayer, porosomal proteins may reside entirely on one side of the membrane or the other, or may be transmembrane.

[0045] As shown in Figure 2, transient fusion of secretory vesicle membranes at the base of the porosome via SNARE proteins forms a fusion pore or continuum for the release of vesicle contents from the cell. After secretion is complete, the fusion pore temporarily formed at the base of the porosome reseals. Porosomes are a few nanometers in size and contain many different proteins, particularly chloride and calcium channels, actin, and SNARE proteins, which mediate the docking and fusion of vesicles with the plasma membrane. When secretory vesicles dock with SNARE proteins, the vesicles expand, increasing their internal pressure. The vesicles then transiently fuse at the base of the porosome, releasing the pressurized contents from the cell. Examination of cells after secretion using electron microscopy has shown an increased presence of partially empty vesicles after secretion, suggesting that only a portion of the vesicle contents can be released outside the cell during secretion. This may only be possible if the vesicles temporarily establish continuity with the plasma membrane, release a portion of their contents, then detach, reseal, and retreat into the cytosol (endocytosis). In this way, secretory vesicles can be reused in subsequent cycles of exocytosis and endocytosis until their contents are completely released. In fast-secreting cells, such as neurons, neurotransmitter transporters present in the secretory vesicle membrane can refill vesicles after release during neurotransmission.

[0046] The size of porosomes varies depending on the cell type. For example, the diameter of porosomes in the exocrine pancreas, as well as in endocrine and neuroendocrine cells, ranges from 100 to 180 nm, whereas the diameter of porosomes in neurons ranges from 10 to 15 nm (approximately 1 / 10 the size of pancreatic porosomes). When secretory vesicles containing v-SNAREs dock at the base of porosomes containing t-SNAREs, membrane continuity is established by the formation of a t- / v-SNARE ring complex between the two opposing membranes. The size of the t / v-SNARE complex is directly proportional to the size of the secretory vesicle. Secretory vesicles typically contain dehydrated (inactive) proteins that are activated upon hydration. To hydrate the vesicles before secretion, GTP is required for water transport through water channels or aquaporins and ion transport through ion channels. When the vesicles fuse at the base of the porosome, the pressurized vesicle contents are released from the cell.

[0047] Generally, the dynamics of porosome opening to the extracellular space are regulated by actin and non-classical myosin. However, neurons requiring a rapid response possess a vertically moving central plug, allowing the t- / v-SNARE-induced opening and resealing of the continuum between synaptic vesicles and the base of the porosome to release neurotransmitters. Porosomes have been demonstrated to be a universal secretory mechanism in cells. Solution X-ray analysis has been used to elucidate the neuronal porosome proteome and its detailed structure, revealing the composition and possible molecular architecture of this mechanism (Table 1). JPEG2025530654000002.jpg139170

[0048] Examples of porosome structures are shown in Figure 1, which shows examples of porosomes in the exocrine pancreas, neurons, and somatotroph cells of the pituitary gland. Figure 1A is an electron micrograph of a single porosome in the apical plasma membrane (PM) of a pancreatic acinar cell, showing the porosome membrane (POM, yellow arrow) integrated with the membrane of a secretory vesicle called a zymogen granule (ZGM). An actin-myosin-like circular ring structure (blue arrow) forms the neck of the porosome complex. Figure 1B is an atomic force microscopy (AFM) micrograph of the apical surface topology of a living pancreatic acinar cell, showing the presence of four openings or porosomes (one indicated by a yellow arrow). The size of porosomes in the exocrine pancreas ranges from 100 to 180 nm in diameter. Figure 1C is an electron micrograph of a neuronal porosome (red arrow) with a synaptic vesicle (SV) docked at its base in the presynaptic membrane (Pre-SM) of a nerve terminal. Note the central plug of the porosome complex. Figure 1D is an AFM micrograph of a neuronal porosome in the presynaptic membrane of an isolated synapse. Note the central plug (red arrow). Neuronal porosomes are 10 times smaller (10-17 nm in diameter) than porosomes in the exocrine pancreas. Figure 1E is an electron micrograph of a porosome adjacent to a microvilli (MV) at 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 a live GH cell from a pig pituitary, showing the presence of porosomes (black circular openings) with diameters of 100-180 nm. Images taken from the following website. 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 teachings of which are incorporated by reference in their entireties.

[0049] As discussed above, the "parts list" of biology is relatively well-defined, and many proteins have been identified in human cells. However, our systems-level understanding of disease modalities is only just beginning to be fully appreciated. Because many individual proteins are involved in multiple cellular processes (e.g., found in multiple enzymatic pathways), targeting a single protein type for disease treatment without damaging multiple cellular functions is challenging. However, protein combinations (with binding partners, coenzymes, etc.) offer a higher level of specificity for any particular selected biological process. Embodiments of the present invention provide the ability to selectively modulate specific biological processes by targeting combinations of proteins within molecular complexes that perform specific biological functions. This allows for precise targeting of drugs at the "systems level" of physiology to inhibit or stimulate cellular processes, rather than individual proteins or lipid molecules.

[0050] One such example of a systems-level cellular process is secretion via the porosome complex, the universal secretory machinery of cells. Porosomes enable communication (the language) between cells in the body by secreting chemical messages, such as neurotransmitters from neurons or hormones from endocrine cells (e.g., insulin from beta cells in the endocrine pancreas). These chemical messages are stored in secretory vesicles within the cell. The porosome secretory machinery, comprised of more than 30 proteins, instructs secretory vesicles to properly dock at the base of the porosome, fuse, swell, and release precise amounts of their contents. None of the individual components of the porosome are unique to the porosome. Rather, the proper conformational assembly of the more than 30 proteins within the complex confers functionality to the structure. Thus, embodiments of the present invention involve targeting specific multimers, such as the porosome complex, to modify cellular function without altering the activity of other cellular complexes and, therefore, other cellular processes involving one or more of the individual components of the targeted structure.

[0051] To be able to precisely target proteins within the porosome complex, it is important to gain knowledge of protein-protein interactions within the porosomes of a particular tissue or cell type. To accomplish this, embodiments of the present invention use an approach called "interactomics." Users of embodiments of the interactomics process described herein can determine protein-protein interactions within the porosome complex. One skilled in the art can then validate these interactions by CRISPR knockout of porosome proteins one by one. Using the skills taught herein, along with standard techniques in the art, one can then determine which other proteins are lost within the porosome complex beyond the knockout protein. The inventors suggest that these additional proteins lost from the porosome complex are proteins that associate with each other within the complex. Such systematic studies can decipher the distribution of proteins within the porosome complex and aid in targeting specific proteins within the complex to regulate and ameliorate secretory disorders and resulting diseases.

[0052] In certain embodiments of the present invention, a non-volatile computer-readable memory device runs a known trained machine learning algorithm on a database of small molecules that are cross-referenced to target multiple proteins within the porosome complex. Further embodiments utilize "bivalent" or "trivalent" forms of existing small molecules that are presented using appropriate nanobodies for targeted delivery. Such drug delivery systems aid in precise targeting to modulate protein function only when bound to two or more target proteins. The nanobodies can also serve as the basis for bridge molecules.

[0053] As a non-limiting example, superparamagnetic iron oxide (SPIO) nanoparticles have the ability to rapidly penetrate cancer cells and release the anticancer drug doxorubicin. Dextran-coated SPIO nanoparticle ferrofluids functionalized with red autofluorescent doxorubicin and the green fluorescent dye fluorescein isothiocyanate as a reporter enable tracking of intracellular nanoparticle trafficking and drug release. These modified nanoparticles enable over 20-fold faster penetration and drug release in human pancreatic cancer cells, and hold therapeutic potential as an advanced drug delivery and imaging platform.

[0054] Just as the letters of the alphabet, when arranged in the correct combination, form a meaningful word, searching for a word using only one of its constituent letters is nearly impossible. However, when two or more of the constituent letters of a word are used in a search, the likelihood of identifying the word increases significantly, especially when the letters are arranged in the order in which they are used in the word. Extending this analogy further in a general sense and without being entirely bound to any single theory of operation, similarly, if a drug is engineered to target and bind multiple target proteins within a protein complex, as shown schematically in Figure 3 for an exemplary porosome, the probability of the drug finding a specific cellular target complex increases significantly. In Figure 3, for example, a hypothetical drug targeting multiple porosome proteins is created by combining compounds (yellow triangle, red square, blue circle) that interact with two or more adjacent target proteins. Thus, the yellow triangle-red square combination can bind to two adjacent yellow-red porosome proteins without affecting the blue porosome protein. Similarly, the red square-blue circle combination has no effect on the yellow protein. Therefore, for the design and creation of such "targeted" molecules, it is important to elucidate and understand whether the target proteins are adjacent to or otherwise interact with each other.

[0055] Each protein in the porosome complex (i.e., "word") is considered a different letter of the alphabet. When each protein letter correctly associates with the other letters, the word porosome is constructed. An incorrect combination of protein letters or a change in any protein letter can result in a misspelling of the word porosome, causing disease.

[0056] In one embodiment of the present invention, candidate protein-protein interactions within cellular functional complexes, such as the porosome complex, are identified using the continuously updated STRING (Search Tool for Finding Interacting Genes / Proteins) database. STRING is a database of known and predicted protein-protein interactions. The interactions include direct (physical) and indirect (functional) associations. These are derived from computational predictions, knowledge transfer between organisms, and interactions aggregated from other (primary) databases. Currently, the STRING database contains at least 20 billion known protein-protein interactions between nearly 68 million proteins.

[0057] For example, Figure 4 is a schematic diagram showing predicted interactions between identified proteins in the neuronal porosome proteome and other regulatory proteins. Due to space limitations, Table 2 below lists the proteins in Figure 4 numerically. These interactions were generated using STRING9.0 from the input of identified proteins in the neuronal porosome. STRING9.0 is a database of known and predicted protein interactions. The interactions include direct (physical) and indirect (functional) associations derived from genomic, high-throughput, conserved co-expression, and prior knowledge.

[0058] Two clusters of protein-protein interactions identified in the porosome complex are shown. The cluster on the left, most likely present at the apical end of the porosome cup, contains cytoskeletal structure and signaling proteins. The cluster on the right represents proteins primarily involved in membrane fusion, including SNARE proteins and calcium channels; therefore, their location is likely at the bottom of the porosome cup, facing the cytosol. Interestingly, the heterotrimeric 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 particularly surprising, as dynamin is a microtubule-associated protein and intersectin 1 is also known to interact with dynamin. However, its presence at the bottom of the porosome is required for dynamin to participate in the fission of the neck of fused vesicles at the bottom of the porosome. The confidence level of the predicted functional interactions shown is over 99%.

[0059] Identified protein-protein interactions of interest are then followed by chemical crosslinking, and the in silico interactions are experimentally confirmed using mass spectrometry, followed by 3D determination of the interaction domains and identification of potentially druggable sites. Thus, practitioners of embodiments of the present invention can design and present small molecules, or small molecules conjugated to carrier molecules, designed to simultaneously bind to multiple target proteins within a protein complex and functionally modulate the physiological function of the protein complex. JPEG2025530654000003.jpg185170

[0060] Small molecules identified as drug candidates through the above process are then further validated using conventional research pathways in cell culture, organoids, and animals to determine drug suitability (safety, efficacy, and stability), after which the candidate drugs undergo clinical trials.

[0061] In one example of the above method, mass spectrometry analysis was performed on porosome complexes immunoisolated from two human bronchial epithelial cell lines: the control WT-CFTR human bronchial epithelial cell line (CFBE41o-6.2), which served as a control or wild-type cell sample, and the ΔF508del(- / -) homozygous deletion strain, the experimental ΔF508-CFTR human CF bronchial epithelial cell line (CFBE41o), which served as a test cell sample. Proteins identified and associated with the porosome complexes are listed in Table 3 below. All quantifications are relative. However, please note that in this example, the ratio was calculated as test / WT. Samples were digested with trypsin and analyzed on an OrbitrapEclipse MS system. Data were analyzed in ProteomeDiscoverer 2.4 using the Sequest and Percolator algorithms. Values ​​shown represent quantification based on multiple consensus. Note that the RasGTPase-activating protein IQGAP1 was absent from the porosome complex (test cell sample) derived from the ΔF508-CFTR cell line. This suggests that the IQGAP1 gene product may interact with the CFTR protein in the native porosome complex. Therefore, upregulation of porosome GTPase activity could potentially be used as a treatment for CF. Similarly, practitioners of the teachings herein may select porosome GTPase as the porosome-targeted protein to be acted upon by either humanized nanobodies, small molecules, or both. JPEG2025530654000004.jpg208170

[0062] *Note that the RasGTPase-activating-like protein IQGAP1 is absent in porosome complexes from the ΔF508-CFTR cell line, suggesting that it interacts with CFTR protein within the native porosome complex.

[0063] Figure 6 depicts porosomal proteins found to act in specific disease classes. Figure 6 further depicts some of the broad range of diseases that can be addressed using embodiments of the porosome-targeted tissue-specific drug design, development, and delivery platform described in detail herein. Defects in secretory function are addressed via porosomes. Secretory disorders can be caused by defects in one or more porosomal proteins, resulting in excessive secretion, such as in certain cancers, or causing a reduction or loss of proper secretion, such as in diabetes and cystic fibrosis.

[0064] Hypersecretion can be treated by specific small molecules that bind and interact with one or more porosomal proteins to ameliorate the defect. For example, t-SNARE or v-SNARE nanobodies can be used to regulate decreased secretion.

[0065] In cases where porosomes are impaired or defective, functional whole porosomes can be reconstituted in defective tissues, such as the lung epithelium in cystic fibrosis. In some embodiments, porosomes from porcine or human sources are extracted and introduced into human cells for reconstitution. In yet other embodiments, nanoscale porosome complexes for reconstitution are obtained from CALU3 or other human airway epithelial cells to address cystic fibrosis. Those skilled in the art can readily envision additional cell types suitable for reconstitution, given at least the porosomal proteins disclosed herein and their role in disease. Thus, reconstitution therapy involves reconstituting or introducing normal, functional CFTR-associated secretory porosome complexes (whole 100-nanometer porosome complexes containing CFTR protein) into the plasma membrane of lung epithelium from CF patients. Reconstitution addresses the problems associated with various CFTR mutations. Reconstitution therapy improves the mucus secretion defects caused by mutated, dysfunctional CFTR.

[0066] Further detailed below in Table 4 are classes of porosomal proteins and their intended roles in disease. JPEG2025530654000005.jpg79170

[0067] Neuronal porosome proteins In Alzheimer's disease, the proteins 2,3-cyclic nucleotide phosphodiesterase (CNPase) and heat shock protein 70 (HSP70) have been suggested to play a role in disease pathology, as shown in Table 4. Levels of CNPase and HSP70, both present in neuronal porosome complexes, have been found to be elevated, while levels of porosome-associated dihydropyrimidinase-related protein-2 (DRP-2) have been found to be decreased. Similarly, the porosomal proteins SNAP-25 and synaptophysin are significantly decreased in neurons of Alzheimer's disease patients.

[0068] Decreased levels of CNPase have also been reported in the frontal and temporal cortex of patients with Alzheimer's disease and Down's syndrome. Low CNPase levels have also been detected in the prefrontal cortex of patients with schizophrenia. Furthermore, alleles associated with low CNPase levels have also been reported to be associated with schizophrenia.

[0069] Examples of neuronal porosome proteins include tubulin β, myosin 7b, spectrin, creatine kinase, dystrophin, langerin, GTPase-activating protein (GAP), intersectin 1 isoform (ITSN-1), actin, cytoplasmic 1, sodium / potassium transporting ATPase subunit α3, plasma membrane calcium transporting ATPase 1, plasma membrane calcium transporting ATPase 2, brain acid-soluble protein 1, adenylyl cyclase-related protein 1, 2′,3′-cyclic nucleotide 3′-phosphodiesterase, dihydropyrimidinase-related protein 2, and dihydropyrimidinase-related protein. Protein 3, dihydropyrimidinase-related protein 5, glutamine synthetase, guanine nucleotide-binding protein G(o) subunit alpha, neural cell adhesion molecule 1, vesicle-fusion ATPase, Ras-related protein Rab-3A, reticulon-3, reticulon-4, synaptosomal-associated protein 25, syntaxin-1A, syntaxin-1B, syntaxin-binding protein 1, synapsin-2, synaptophysin, synaptotagmin-1, tubulin alpha-1A chain, vesicle-associated membrane protein 1, vesicle-associated membrane protein 2, V-type proton ATPase subunit B, brain isoform. Embodiments of the present invention may include one or more identified small molecules that act directly on one or more of the above proteins to affect neuronal porosome structure and / or function.

[0070] Insulin-secreting porosome protein - diabetes According to the Centers for Disease Control and Prevention (CDC), over 37 million Americans have diabetes (1 in 10), and approximately 90–95% of these have type 2 diabetes. Type 2 diabetes typically develops in people over the age of 45, but the number of children developing type 2 diabetes is also increasing. The hormone insulin is produced and secreted by beta cells in the endocrine pancreas. Insulin directs the body's cells to take up glucose for energy. In type 2 diabetes, cells become insensitive to insulin, a condition known as insulin resistance. As a result, beta cells produce more insulin to stimulate their response. Eventually, beta cells are no longer able to continue increasing production, leading to elevated blood glucose levels and the onset of type 2 diabetes. Hyperglycemia is detrimental to physiological function and can lead to serious health problems, including cardiovascular disease, blindness, kidney disease, and dementia. Type 2 diabetes can be managed with a healthy diet and medication, but these alone are often ineffective, and current medications often cause unwanted side effects.

[0071] After a meal, secretion of digestive enzymes from the exocrine pancreas aids in food digestion. The resulting rise in blood glucose after digestion stimulates the secretion of insulin from beta cells in the endocrine pancreas. Glucose-stimulated release of insulin stored in secretory vesicles in beta cells occurs either through complete disruption of the vesicle membrane at the plasma membrane or through transient fusion of secretory vesicles at the base of plasma membrane-associated porosomes. In some embodiments of the present invention, functional reconstitution of insulin-secreting porosome complexes in living beta cells of the endocrine pancreas opens new possibilities for the treatment of diabetes.

[0072] Therefore, there are two main problems with type 2 diabetes. One is that the beta cells in the pancreas do not produce enough insulin, and the other is that the cells in the body do not respond well to insulin and are unable to absorb sugar. Currently, there is no cure for type 2 diabetes. Type 2 diabetes can only be managed through diet and exercise therapy, combined with the following medications:

[0073] Metformin (Fortamet®, Glumetza®, and others) is typically the first medication prescribed to patients with type 2 diabetes. Metformin works primarily by reducing glucose production in the liver and improving the body's sensitivity to insulin, allowing it to be used more effectively. Metformin is known to inhibit insulin secretion from beta cells in the endocrine pancreas. Patients taking metformin may develop vitamin B12 deficiency and need to take supplements. Other possible side effects of metformin include nausea, abdominal pain, diarrhea, and abdominal bloating.

[0074] Metformin may also be used in combination with other medications, including sulfonylureas, glinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, and insulin therapy.

[0075] Sulfonylureas help the body secrete insulin. Examples include glyburide (DiaBeta®, Glynase®), glipizide (Glucotrol XL®), and glimepiride (Amaryl®). Possible side effects include hypoglycemia and weight gain.

[0076] Glinides stimulate the pancreas to increase insulin secretion. Glinides are more rapid acting 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.

[0077] Thiazolidinediones make body tissues more sensitive to insulin. An example of this drug is pioglitazone (Actos®). Possible side effects include risk of congestive heart failure, risk of bladder cancer (pioglitazone), risk of bone fractures, and weight gain.

[0078] DPP-4 inhibitors help lower blood sugar levels, but tend to do so only marginally. Examples include sitagliptin (Januvia®), saxagliptin (Onglyza®), and linagliptin (Tradjenta®). Possible side effects include a risk of pancreatitis and joint pain.

[0079] GLP-1 receptor agonists are injectable medications that slow digestion and help lower blood sugar levels. The use of GLP-1 receptor agonists is often accompanied by weight loss, and some may reduce the risk of heart attack and stroke. Examples include exenatide (Byetta®, Bydureon Bcise®), liraglutide (Saxenda®, Victoza®), and semaglutide (Rybelsus®, Ozempic®, Wegovy®). Possible side effects include the risk of pancreatitis, nausea, vomiting, and diarrhea.

[0080] SGLT2 inhibitors affect the kidney's blood-filtering function by blocking glucose from returning to the bloodstream, which results in glucose being excreted in the urine. These drugs may reduce the risk of heart attack and stroke in people at high risk. Examples include canagliflozin (Invokana®), dapagliflozin (Farxiga®), and empagliflozin (Jardiance®). Possible side effects include vaginal candidiasis, urinary tract infections, low blood pressure, high cholesterol, risk of gangrene, risk of fractures (canagliflozin), and risk of amputation (canagliflozin).

[0081] Some people with type 2 diabetes require insulin therapy. In the past, insulin treatment was used as a last resort, but now it may be prescribed earlier if lifestyle changes and other medications do not achieve blood glucose targets. Different types of insulin vary in how quickly they begin to work and how long they last. For example, long-acting insulin is designed to work overnight or throughout the day to stabilize blood glucose levels. Short-acting insulin is typically used with meals to moderate blood glucose spikes associated with meals.

[0082] In summary, currently available treatments for type 2 diabetes follow the following strategy: reducing glucose production, improving the body's sensitivity to insulin, increasing insulin secretion, and increasing glucose excretion by the kidneys. Currently, there are no drugs that increase both insulin production and secretion by the beta cells of the endocrine pancreas.

[0083] Examples of insulin-secreting porosome proteins include actin, cytoplasmic 1, tubulin α-1A, cofilin-1, calcium-transporting ATPase type 2C, ankyrin repeat domain-containing protein, non-classical 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, RabGDP dissociation inhibitor α, potassium channel subfamily K member 2, and RhoGTPase-activating protein. Proteins that are specifically targeted to insulin-secreting porosomes include 40, heat shock protein HSP90, heat shock cognate 71 kDa protein, synaptosomal-associated protein 25, ankyrin repeat domain-containing protein, profilin-1, tubulin β2A, destrin, guanine nucleotide-binding protein β-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, and Arf-GAP. Embodiments of the present invention may include one or more identified small molecules that directly affect the structure and / or function of insulin-secreting porosomes by acting on one or more of the above proteins. In yet other embodiments, the small molecule can enhance the ability of porosome-constituting proteins to form porosome assemblies.

[0084] In some embodiments, small molecules such as 17-demethoxy-17(2-propenylamino)geldanamycin can modulate heat shock proteins such as HSP90 to affect the formation and subsequent function of porosome structures. In some embodiments, small molecules may disrupt the formation of porosome structures through inhibition of the binding and assembly of constituent proteins.

[0085] Further embodiments of the present invention include porosomal proteins that regulate both insulin expression and secretion in endocrine pancreatic beta cells. The identified insulin-secreting porosomal proteins are ATP2C1 (ATPase secretory pathway Ca+2 transport 1) and APOa1, which are responsible for both insulin expression and secretion in endocrine pancreatic beta cells. These proteins were identified using CRISPR knockout and overexpression of different insulin-secreting porosomal proteins. The identified proteins belong to the family of P-type cation-transporting ATPases. This magnesium-dependent enzyme catalyzes the hydrolysis of ATP accompanied by the transport of calcium ions.

[0086] In yet another embodiment of the present invention, there is provided a method for identifying one or more modulators of insulin secretory porosome proteins that regulate insulin production and secretion from endocrine pancreatic beta cells. In a specific embodiment, mRNA-induced overexpression of ATP2C1 and APOa1 in beta cells is used. Similarly, it has been determined that the Ca2+-ATPase (SERCA) activator CDN1163 can be used to increase both insulin expression and secretion in endocrine pancreatic beta cells. In some embodiments, this activator can be used alone or in combination with a modulator.

[0087] It has been determined that the Ca2+-ATPase (SERCA) activator CDN1163 can be used alone or in combination with other existing type 2 diabetes treatments described above, including metformin and / or modulators of the ATP2C1-associated porosomal protein in the secretory complex, to increase both insulin expression and secretion in beta cells of the endocrine pancreas.

[0088] Certain embodiments of the present invention use the above methods to identify protein-protein interactions within functional complexes in cells, particularly within the insulin porosome complex in endocrine pancreatic beta cells, allowing for fine-tuning of the regulation of the insulin porosome secretion machinery in endocrine pancreatic beta cells and its precise targeting using small molecule drug-nanobody conjugates.

[0089] As shown in Figure 7, in yet another embodiment, overexpression of different insulin-secreting porosomal proteins allowed the identification of the insulin-secreting porosomal proteins ATP2C1 (ATPase secretory pathway Ca+2 transport 1) and APOa1, which showed increased insulin expression and glucose-stimulated secretion in endocrine pancreatic beta cells (shown in Figure 8). Therefore, the Ca2+-ATPase activator CDN1163 can be used to increase both insulin expression and glucose-stimulated insulin release in endocrine pancreatic beta cells (see Figure 9). Indeed, Figure 7 shows knockout (KO) and overexpression (OE) of three porosomal proteins, ATP2C1, APOa1, and TREK1, in Min6 cells. Note the Western blots for CRISPR / Cas9 empty (SCRM), KO, control (CON), and overexpressed proteins. GAPDH is a loading control. Meanwhile, Figure 8 shows glucose-stimulated insulin secretion in Min6 cells in knockout (KO) and overexpression (OE) of three porosomal proteins: ATP2C1, APOa1, and TREK1. Note the significant loss of insulin secretion at 10 and 30 min in the ATP2C1 KO compared to the other two KOs (i.e., APOa1 and TREK1). Interestingly, OE of ATP2C1 and APOa1 results in an increase in both insulin synthesis and secretion, whereas TREK1 shows a decrease in both insulin synthesis and secretion. Finally, as seen in Figure 9, glucose-stimulated insulin secretion in Min6 cells is increased after 2 h of exposure to the ATP2C1 activator CDN1163. Note that 10 μM CDN1163 is optimal for glucose-stimulated insulin secretion.

[0090] These results suggest that targeted overexpression of ATP2C1 or APOa1, or both, in endocrine pancreatic beta cells using mRNA or other gene therapy may be therapeutic for type 2 diabetes. Similarly, small molecule activators of both porosomal proteins may function as therapeutic agents for type 2 diabetes.

[0091] Porosome reconstitution therapy for type 1 diabetes (T1D) Current treatments for T1D are based on insulin injections and cadaveric islet transplantation, which have many drawbacks. As a result, new methods are being developed to regenerate pancreatic hormone-producing cells in vitro. The most promising approach is the generation of stem cell (SC)-derived β cells, which can serve as an unlimited source of insulin. Recent studies have provided methods to produce β cell-like cell clusters that exhibit glucose-stimulated insulin secretion, one of the key characteristics of β cells. However, compared to native β cells, SC-derived β cells do not undergo complete functional maturation and therefore exhibit limited glucose-stimulated insulin secretion, raising an urgent need for improved treatment. Results from ongoing clinical trials suggest that current protocols for generating SC-derived islets that can improve glycemic control in human T1D patients require further enhancement of insulin secretion to mimic native adult islets. This would reduce the number of cells required for transplantation and facilitate the production of sufficient cell numbers for treatment. It has been reported that doubling the insulin secretion per cell could potentially halve the number of cells required to cure patients. Reducing graft volume further facilitates the transplantation procedure, reduces the requirement for nutrient exchange at the transplant site, and opens the possibility of alternative transplantation sites. Reducing the number of cells required for successful transplantation significantly reduces both the manufacturing costs and logistical requirements for cell-based therapies. Reconstitution of insulin-secreting porosomes into SC-derived islets solves this problem by enabling enhanced glucose-stimulated insulin secretion.

[0092] Exosome release control In addition to secreting neurotransmitters, digestive enzymes, and hormones, cells communicate with each other through secreted, membrane-bound, nanostructured extracellular vesicles (exosomes). Exosomes were first discovered and described in 1983 as a mechanism for selective extracellular transport of transferrin receptors in sheep reticulocytes. Electron microscopic evidence of the extracellular transport of transferrin receptors in the form of vesicles from sheep reticulocytes was demonstrated in 1985. Over the past 35 years, significant advances have been made in our understanding of exosome biology, function, and biomedical applications. Exosomes contain packages of proteins, DNA, and RNA that are delivered to specific target cells in the body. This exosome-mediated intercellular communication has also been implicated in various pathologies, including cancer, neurological disorders, and inflammatory diseases. Extracellular vesicle cargo includes plasma membrane and endosomal proteins, but may also contain material from various intracellular compartments, such as mitochondria. Studies have reported the presence of mitochondrial DNA within extracellular vesicles. Although multivesicular bodies can fuse with the plasma membrane and release their cargo, the molecular mechanisms of exosome release and / or their cargo from various cell types remain unclear. Exosome release is thought to occur via the porosome complex.

[0093] In some embodiments, exosome release is controlled by altered structure and / or function of porosome complexes or porosome-associated proteins. In certain embodiments, genes for putative porosome complexes or porosome-associated proteins may be "knocked out" in one or more cell types using CRISPER, RNAi, or other methods known in the art.

[0094] For example, in rat brain Min6 cells, a knockout of ATP2C1 was generated by genome editing using the CRISPER / Cas9 system. Comparing these cells with control wild-type (SCRM) cells showed exosome release via porosome complexes and loss of insulin secretion upon both glucose stimulation. Therefore, small molecules that enhance or alter ATP2C1 production are likely to affect insulin secretion.

[0095] Nanobodies Nanobodies are a subclass of antibodies found in camelids, composed of a single polypeptide chain with a versatile molecular binding scaffold, in contrast to the large, Y-shaped, traditional antibodies found in other mammalian species, including humans. In certain embodiments of the present invention, engineered nanobodies targeting different porosomal proteins are used to bind the nanobodies to target proteins, thereby enabling precise drug binding to specific porosomal proteins and / or altering porosome structure and function. In some embodiments, the variable domains of camelid variable domain (VHH) nanobodies are humanized to target and bind one or more domains of one or more porosomal proteins. Nanobodies targeting multiple porosomal proteins not only aid in the precise targeting of small molecules, but may also be used to physically or chemically alter porosomal structure and / or function, thereby altering the course of porosome-mediated diseases. Thus, one or more nanobodies of the same or different classes can be conjugated to one or more small molecules to fine-tune targeting and response to porosomal structure-function outcomes.

[0096] Figure 5 shows the drug target specificity achieved by combinatorial small molecule drug design in combination with tissue-specific and porosome-specific multivalent nanobodies (top). Site-specific functionalization of nanobodies, which can be achieved via engineered cysteines, is further illustrated (center, bottom). Cysteines are introduced into nanobodies by genetic engineering. Maleimides are one of the most widely used sulfhydryl-reactive chemical groups. The yellow oval indicates the backbone of the exogenous cysteine. The red sphere indicates the functional group (small molecule drug) attached to the maleimide. Finally, an example of the chemical reaction involved in such a preparation is shown (bottom). Incorporation of the amino acid p-acetylphenylalanine (pAcF) provides a bioorthogonal ketone for conjugating the anticancer drug doxorubicin (Dox) in the presence of reactive amino acids in nanobody-targeted elastin-like polypeptide nanoparticles (ELP diblocks). Elastin-like polypeptide (ELP)-based diblock copolymers can undergo specific phase transitions upon thermal stimulation. This ability makes them particularly suitable for forming carriers, such as micelles, to deliver active cargo molecules. Similarly, multivalent nanobodies with both tissue-specific and porosome-specific domains can be engineered for precise targeting of combinatorial small molecule drugs. In this case, the term "multivalent" refers to the general usage of the term to describe two or more types of nanobodies, molecules, or other functional elements formed as part of a combinatorial drug.

[0097] Examples of neurological disorders and cancer As previously mentioned, porosome structures contain multiple proteins bound by other associated molecules, such as ligands, chaperones, and lipids. While it has long been understood that some diseases are caused by mutations / abnormalities in the structure of single proteins, as noted above, it is only recently that it has become clear that dysfunction or abnormalities in larger structures, such as porosomes, can contribute to disease. The following non-limiting examples illustrate examples of porosomal protein dysfunction and its contribution to disease, followed by exemplary single-target small molecules that can affect the disease state.

[0098] Neurological diseases: (e.g., Alzheimer's disease, Down's syndrome, and schizophrenia). In Alzheimer's disease, the levels of the porosomal protein CNPase (2,3-cyclic nucleotide phosphodiesterase) and heat shock protein 70 (HSP70), both present in the neuronal porosome complex, have been found to be elevated, while the levels of porosome-associated dihydropyrimidinase-related protein-2 (DRP-2) have been found to be reduced. Similarly, the porosomal proteins SNAP-25 and synaptophysin are significantly reduced in neurons of Alzheimer's disease patients. Similarly, in Down's syndrome and schizophrenia, reduced levels of the porosomal protein CNPase have also been reported in the frontal and temporal cortices of Alzheimer's disease and Down's syndrome patients. Low CNPase levels have also been detected in the prefrontal cortex of schizophrenia patients. Furthermore, alleles associated with low CNPase levels have also been reported to be associated with schizophrenia.

[0099] Small molecule inhibitors and stimulators of porosomal phosphodiesterases, such as vinpocetine, BAY60-7550, rolipram, etazolate, sildenafil, S14, VP1.15, PF-04447943, papaverine, and the small molecule inhibitors of HSP70, apoptozole, VER155008, JG98, HA15, and YUM70, and the small molecule activator of HSP70, ML346, can all be used to treat neurological disorders, particularly Alzheimer's disease.

[0100] Cancer: Cellular secretion is a key mediator of cancer progression. For example, small GTPases of the Ras superfamily present in porosomes are involved in 33% of human cancers. However, direct pharmacological inhibition of Ras mutants remains challenging. Therefore, an alternative strategy is to continue screening and designing novel small molecules that directly bind and inhibit Ras GTPases while inhibiting the activity of the porosomal protein V-ATPase. Similarly, non-small cell lung cancer (NSCLC) has a poor prognosis and remains the leading cause of cancer-related deaths worldwide. The porosomal protein tubulin β is strongly associated with drug-resistant and aggressive NSCLC. βIII-tubulin has also been linked to resistance to taxanes and vincristine in various tumor types, including ovarian, breast, and gastric cancers. Small molecule drugs that target microtubules, such as docetaxel, taxol, podophyllotoxin, etoposide, vinblastine, vincristine, vinorelbine, griseofulvin, cytochalasin A and E, TN-16, myoseverin, nocodazole, vindesine, fomopsin A, d-24851, monastrol, AMP-PNP, adriamycin sulfate-2, terpendol-E, tubacin, scriptaid, DPD, and C2-8, can be used in the combinatorial approaches outlined above.

[0101] Small molecules that target porosomal lipids, which are involved in cancer therapy, may also be useful in embodiments of the present invention. Studies have shown that cholesterol in the cell membrane is important for cellular secretion. Depletion of cholesterol from the cell membrane reduces phosphatidylserine (PS) incorporation into the cytoplasmic membrane, impairing secretion. While PS is normally located in the inner layer of the membrane bilayer of healthy cells, it is expressed at high levels on the surface of cancer cells. This has enabled the development of therapeutic agents that are selective for cancer cells (without affecting healthy cells). For example, SaPC-DOPS is a PS-targeted nanovesicle that effectively targets and kills several types of cancer, including pancreatic, lung, brain, and pediatric tumors. SaPC-DOPS selectively induces apoptotic cell death in malignant and metastatic cells, while unaffected cells due to their low surface PS expression. As yet another approach, the combination of SaPC-DOPS with cell membrane cholesterol-removing small molecules, such as cyclodextrins, may be useful for cancer therapy.

[0102] Table 5 lists additional porosomal proteins and small molecule drugs that target porosomal proteins and lipids, and where possible, describes their potential role in the treatment of various diseases. JPEG2025530654000006.jpg230170

[0103] JPEG2025530654000007.jpg213170

[0104] It should be understood that reference to a pharmaceutically acceptable salt includes solvent addition forms, particularly solvates. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and may be formed during crystallization with a pharmaceutically acceptable solvent 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 easily prepared or formed during the processes described herein. In addition, the compounds described herein can exist in unsolvated and solvated forms. Generally, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods described herein.

[0105] Reconstitution of functional mucus-secreting porosomes - a treatment for cystic fibrosis Cystic fibrosis (CF) is a disease that causes the accumulation of thick, sticky mucus 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 adults. Cystic fibrosis is an inherited disease caused by a genetic defect that causes the body to produce abnormally thick, sticky mucus. Abnormal mucus accumulates in the respiratory passages of the lungs and pancreas. The mucus buildup can lead to life-threatening lung infections and serious digestive problems. The disease can also affect the sweat glands and male reproductive system. Many people carry the CF gene but do not have symptoms. This is due to the fact that a CF patient must inherit two defective genes, one from each parent. Some types of CF are more common in people of Northern and Central European descent. Especially due to the widespread use of newborn screening across the United States, most children with CF are diagnosed by the age of two. In a small percentage of patients, the disease is not detected until they are 18 years of age or older. These children often have milder symptoms.

[0106] Nearly 40,000 children and adults in the United States have cystic fibrosis, and an estimated 105,000 people across 94 countries have been diagnosed with CF. In CF patients, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the CFTR protein to malfunction. When this protein isn't functioning properly, it can't help move chloride ions to the cell surface. Without chloride, which attracts water to the cell surface, mucus in various organs becomes thick and sticky. In the lungs, mucus clogs airways and traps pathogens, including bacteria, leading to infection, inflammation, respiratory failure, and other complications. For this reason, avoiding exposure to bacteria is a primary concern for CF patients.

[0107] The most common mutation in the CFTR gene is delF508, a three-nucleotide 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 worldwide and 90% of cases in the United States. However, more than 1,500 other mutations can also cause CF. Most people have two normal copies (alleles) of the CFTR gene, but only one is required to prevent cystic fibrosis. CF develops when neither allele can produce functional CFTR protein. Therefore, CF is considered an autosomal recessive genetic disorder.

[0108] There is no known cure for cystic fibrosis. Lung infections are treated with antibiotics, which may be administered intravenously, inhaled, or orally. Occasionally, the antibiotic azithromycin is used long-term. Hypertonic saline inhalation and salbutamol may also be helpful. If lung function continues to deteriorate, lung transplantation may be an option. Airway clearance techniques, such as chest physical therapy, have some short-term benefits, but their long-term effectiveness is unknown. Life expectancy is 42 to 50 years. Lung problems (infections, reduced lung capacity) are the cause of death in 80% of cystic fibrosis patients.

[0109] Because there is no known cure for cystic fibrosis, treatment for CF focuses on improving breathing, preventing and treating lung infections, and thinning the mucus in the lung epithelium. Treatments include medications, therapies to remove mucus from the lungs, and, in some cases, lung transplantation. Pharmacogenomic approaches have led to the development of drugs that target the underlying cause of the disease. Because different mutations in CFTR affect the CFTR protein differently, these drugs can only be used to treat patients with specific CFTR mutations.

[0110] The presence of porosomes in human airway epithelia is known, as shown in Figure 10. Similarly, the porosome proteome in these cell types is also known (Table 6). Indeed, Figure 10 shows representative electron micrographs of Calu-3 cells in culture (cells originally obtained from a patient with lung adenocarcinoma), demonstrating the presence of microvilli (MVs) and porosomes (Ps) at the plasma membrane. (A) Calu-3 cells show dense microvilli and porosomes at the plasma membrane. (B-D) Note the flask-shaped porosomes, approximately 100 nm in diameter (E) and 200-300 nm deep, with an opening toward the cell surface (red arrow). In (C), what appears to be mucus can be seen at the opening of the porosome to the outside of the cell. Of the two porosomes shown in (D), the middle one appears to have precisely cut the center of the organelle, while the porosome on the left appears to have been cut at the base. (E) As in the AFM image, the diameter of the microvilli averages 92 nm. The human airway-associated porosome complexes are similar to those present in the exocrine and endocrine pancreas shown in Figures 13 and 14.

[0111] Indeed, Figure 11 demonstrates the use of Ussing chamber experiments to show that forskolin-stimulated chloride release from Calu-3 cells is inhibited in the presence of the CFTR inhibitor GlyH-101. Note that two separate experiments show similar stimulation and inhibition profiles.

[0112] On the other hand, Figure 12 shows that the CFTR inhibitors 172 and GlyH-101 inhibit forskolin-stimulated secretion of intravesicular mucin from Calu-3 cells. A and B are for control cells, C and D are for cells exposed to 172, and E and F are for cells exposed to GlyH-101. Vesicles circled in red are partial / empty vesicles, and vesicles circled in green are filled vesicles. Scale bar = 500 nm. JPEG2025530654000008.jpg181170

[0113] *SNAP-25 immunoisolated porosome complexes were obtained using Calu-3 cells solubilized with 1% Triton-Lubrol. The presence of cystic fibrosis transmembrane conductance regulator (CFTR) as a component of porosome complexes in human airway epithelia was also known. Therefore, it was hypothesized that porosome-associated CFTR regulates the quality of mucus secretion via porosome complexes in the plasma membrane, which subsequently proved to be the case. These findings have deepened our understanding of CFTR-associated porosomes that influence mucus secretion in lung epithelia and provided important insights into the pathogenesis of CF disease.

[0114] Figure 13 shows a transmission electron micrograph of a porosome complex associated with a docked secretory vesicle at the apex of an exocrine pancreatic acinar cell. (a) The green-framed box shows the presence of a porosome and a docked secretory vesicle called a zymogen granule (ZG) in the apical portion of a pancreatic acinar cell. Fusion of the docked ZG at the base of the porosome complex forms a fusion pore (FP). Electron-dense secretory vesicles in the exocrine pancreas. (Bar = 400 nm, Figure 4a only). (b) A magnification of the green boxed area in (a) shows the apical microvilli (MV) and a cross-section of the porosome and ZG. Note that the ZG membrane (ZGM) bilayer is directly attached to the bottom of the porosome cup, forming a continuum or FP. (c) A higher-magnification image of a porosome further details the cross-section of the porosome bilayer and three protein rings, with a thick ring (blue arrow) located near the exterior opening of the porosome complex. The third lowest ring from the porosome opening docks with and fuses with the ZGM. (d) For further clarity, the outline of the porosome membrane is shown in yellow. 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 defines the precise contact and fusion point for the ZGM at the base of the porosome membrane to form the FP (12).

[0115] Referring to Figure 14, transmission electron micrographs of insulin-secreting Min6 cells (pancreatic endocrine beta cells) are shown, demonstrating the presence of porosomes at the plasma membrane (a) and porosomes associated with docked secretory vesicles at the apical end of the cell (b). Clathrin-coated vesicles distinct from the cup-shaped porosome complex are also shown (c). The size of isolated porosomes averages 91 nm, as demonstrated by photon correlation spectroscopy (d).

[0116] Thus, given the presence of porosomes in endothelial cell structure, the techniques of porosome structural alteration, small molecule targeting, or reconstitution taught herein also apply to airway epithelial cells. Similarly, because the airways are in contact with air, drug delivery methods such as nebulizers, inhalers, and atomizers, as known in the art, are contemplated for delivery of any of the therapies taught herein.

[0117] Porosome reconstitution As described in further detail below, porosomes have been functionally reconstituted into both artificial lipid membranes (Figures 15 and 16) and living cells (Figures 17 and 18). Furthermore, porosomes reconstituted into living cells are stable and functional (Figure 19). This powerful ability to reconstitute porosomes offers a therapeutic approach for the treatment of CF disease.

[0118] Figure 15 shows electron micrographs of porosome complexes in reconstituted liposomes from the exocrine pancreas. The porosomes exhibit a cup-like, basket-like morphology. (a) Shown are 500 nm lipid vesicles incorporating porosomes isolated from the exocrine pancreas. (b)–(d) show the reconstituted complexes at higher magnification. Bar = 100 nm.

[0119] Referring to Figure 16, lipid bilayer-reconstituted porosome complexes from the exocrine pancreas have been shown to be functional. (a) Schematic of the bilayer setup for electrophysiological measurements is shown. (b) Zymogen granules (ZG) added to the cis side of the bilayer fuse with the reconstituted porosomes, as evidenced by an increase in capacitance and current activity and the accompanying time-dependent release of amylase (the major ZG content) to the trans side of the membrane. The movement of amylase from the cis side of the chamber to the trans side was determined by immunoblot analysis of the contents of the cis and trans chambers over time. (c) Electrical measurements in the presence and absence of the chloride ion channel blocker DIDS indicate the presence of chloride channels associated with the complexes, as shown by immunoblot analysis of immunoisolated complexes.

[0120] Figure 17 shows the abundant presence of TREK-1, Gi3, and syntaxin-1A immunoreactivity in porosome-reconstituted insulin-secreting Min6 cells. A: Western blot analysis of 5 μg of Min6 cell homogenate from control and porosome-reconstituted cells. Note the abundant presence of all three porosomal proteins: TREK-1, Gi3, and syntaxin-1A. No changes in insulin immunoreactivity were observed in the reconstituted Min6 cell homogenate. B: Immunofluorescence microscopy shows increased immunoreactivity of SNAP-25 (green) and Gi3 (red) in porosome-reconstituted Min6 cells, as well as increased colocalization of these proteins. Data represent one of four similar experiments. Scale bars (insets) a and b, 20 μm (13).

[0121] Figure 18 shows insulin-secreting porosomes reconstituted in live Min6 cells, demonstrating increased glucose-stimulated insulin secretion. Note the time-dependent increase in insulin release from the reconstituted Min6 cells. A: Representative insulin immunoblots of total Min6 cell homogenate (TH) and glucose-stimulated insulin release at 0, 10, and 30 minutes in control and porosome-reconstituted Min6 cells. The preproinsulin band is present only in the TH fraction, not in the secreted fraction. B: Bar graph of insulin release rate at 0, 10, and 30 minutes in control and reconstituted Min6 cells. A significant time-dependent increase in insulin release from porosome-reconstituted Min6 cells is observed at 30 minutes (n = 6, *P < 0.05). Note that no change in basal insulin release is observed in porosome-reconstituted Min6 cells. C: The minute insulin secretion rate was calculated to be 0.062% / min of the total in control cells, but increased to 0.107% / min of the total in porosome-reconstituted cells, representing a 70% increase in insulin release rate.

[0122] Figure 19 shows the abundant presence of Gαi3 and syntaxin-1A immunoreactivity in homogenates of porosome-reconstituted Min6 cells and the resulting glucose-stimulated insulin release observed 24 and 48 hours after reconstitution. A: Representative Western blots of Min6 cell homogenates from control and porosome-reconstituted (experimental) Min6 cells after 24 and 48 hours, respectively, show the abundant presence of porosomal proteins Gi3 and syntaxin-1A. No change in total insulin immunoreactivity was detected in the experimental homogenates. B: The abundant presence of porosomal proteins in A is reflected in increased levels of glucose-stimulated insulin release both 24 and 48 hours after reconstitution of porosomes into live Min6 cells. Results represent one of three separate experiments.

[0123] Mucin storage in secretory vesicles in mucin-secreting cells of the airway epithelium occurs either through complete disruption of the vesicle membrane at the plasma membrane or through transient fusion of secretory vesicles at the base of plasma membrane-associated porosomes. Functional reconstitution of the insulin-secreting porosome complex in living beta cells of the endocrine pancreas opens new possibilities for the treatment of cystic fibrosis and type 1 diabetes.

[0124] Examples of insulin-secreting porosome proteins in human airway epithelia include those listed below in Table 7. Embodiments of the present invention may include one or more identified small molecules that act directly on one or more of the above proteins to affect the structure and / or function of mucin-secreting porosomes. JPEG2025530654000009.jpg183170

[0125] *SNAP-25 immunoisolated porosome complexes were obtained using Calu-3 cells solubilized with 1% Triton-Lubrol. Embodiments of the present invention provide an approach for large-scale isolation of mucin-secreting porosome complexes from normal human airway epithelium in non-CF patients and reconstituting the porosome complexes in the airway epithelium of CF patients to restore porosome function. Figure 20 shows an exemplary schematic diagram of the delivery of functional mucin-secreting porosome complexes to airway epithelial cells of CF patients and amelioration of CF disease. The presence of the CFTR complex as part of the overall porosome structure is indicated by a connecting line. This is not positionally accurate but simply emphasizes the presence of this specific subcomponent within the overall porosome structure. In some embodiments, CRISPR knockout of one porosome protein at a time is used to determine which other proteins within the complex are lost from the porosome complex in addition to the knocked-out protein. These proteins are associated with each other and with the knocked-out protein within the complex. Such systematic studies can decipher the distribution of all proteins within the porosome complex, helping to target specific proteins within the complex to regulate and improve secretory function and correct secretory disorders and resulting diseases. Furthermore, 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 lungs, making them ideal targets for CF-porosome reconstitution therapy.

[0126] In some embodiments, Calu-3 and other suitable human airway epithelial cell lines are selected for therapeutic porosome collection. Human airway epithelial cell lines are used to isolate human mucin-secreting porosome complexes for CF treatment. For example, Calu-3 cells were grown in Dulbecco's Modified Eagle's Medium:Nutrient Mixture F-12 (DMEM / F-92 12) containing 15% fetal bovine serum. Cells were incubated at 37°C in a humidified 93% CO atmosphere with 5% CO2. Calu-3 cells were the source of isolated mucin-secreting porosomes for CF research and treatment. Mucin-secreting porosome complexes were isolated from Calu-3 cells using a SNAP-25-specific antibody conjugated to protein A-Sepharose® (an affinity resin for immunoprecipitation and antibody purification procedures). Calu-3 cells were solubilized using a solubilization buffer consisting of 2% Triton X-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, a non-ionic surfactant), 1 mM benzamidine, 5 mM Mg-ATP, and 5 mM EDTA in PBS (pH 7.4) supplemented with a protease inhibitor mix. For each immunoisolation, the following protocol was used, scaled up and modified as necessary: ​​2 mg of Triton-solubilized Calu-3 cells and 5 μg of SNAP-25 antibody conjugated to protein A-Sepharose® beads were incubated on ice for 1 h, followed by three washes with 10 volumes of wash buffer (500 mM NaCl, 10 mM Tris, and 2 mM EDTA, pH 7.5). The immune pull-down complexes bound to immune-Sepharose® beads were eluted with low-pH (pH 3) PBS to dissociate the porosome complexes from the bead-bound antibodies, and the eluted sample was immediately returned to neutral pH in a total volume of 200 μL. Various dilutions of the isolated complexes were tested for optimal reconstitution with CF epithelium in human lung organoids prior to animal and / or human clinical trials. The isolated porosome suspension was administered to an animal model of CF disease using a nebulizer to ensure uniform distribution to the airway epithelium and lungs.Such systematic studies have enabled us to optimize the functional reconstitution of isolated porosome complexes, improving their secretory function and helping to correct the secretory defect and resulting CF disease. We further note that no additional steps were required to ensure correct insertion or orientation of the porosome complexes into epithelial cells when administered by nebulization.

[0127] In some embodiments, Gαi3 is present in the porosome complex (illustrated in FIG. 21) in Calu-3 cells, and previous studies have reported that "pertussis toxin, which dissociates GTP-bound G proteins from their receptors, and guanosine 5'-[β-phosphate thio], which prevents G proteins from interacting with their effector proteins, increase Cl- currents in airway epithelial cells isolated from CF patients" (PNAS 1992, 89(22): 10623-10627), suggesting that targeting Gαi3 in the porosome complex results in the restoration of cAMP-activated Cl- currents and normal mucin secretion in airway epithelial cells from CF patients.

[0128] In some embodiments, vimentin is present in porosome complexes in mucin-secreting human epithelial cells. This is shown in Figure 21, which shows immunoblot analysis of whole Calu-3 cell homogenate (CH) and isolated porosome complexes (P), demonstrating the presence of the porosome proteins actin, Gαi3, and vimentin. Note the abundant presence of these proteins in the porosome complexes. Previous studies have reported that "4β-phorbol 12-myristate 13-acetate (PMA)-mediated phosphorylation of vimentin, which appears to be an intermediate step in the stimulation of glycoconjugate secretion by PKC, is impaired in CF disease" (Am J Physiol. 1994, 266(3pt1) C611-C621), suggesting that targeting vimentin to regulate its activity in mucin-secreting porosome complexes in airway epithelia could restore normal mucin secretion in the airways of CF patients. Furthermore, CFTR is known to have a regulatory domain that is a substrate for both protein kinases A (PKA) and C (PKC).

[0129] Figure 22 shows immunoisolated CFTR complexes using CFTR-specific antibodies, resulting in pulldown of polosome-associated proteins such as syntaxin-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. Thus, in some embodiments, actin is present in porosome complexes in mucin-secreting cells of human epithelia, and previous studies have reported that "4β-phorbol 12-myristate 13-acetate (PMA)-mediated phosphorylation of vimentin, which appears to be an intermediate step in the stimulation of glycoconjugate secretion by PKC, is impaired in CF disease" (Am J Physiol. 1994, 266(3pt1) C611-C621), suggesting that targeting vimentin to regulate its activity in mucin-secreting porosome complexes in airway epithelia may result in the restoration of normal mucin secretion in the airways of CF patients.

[0130] In some embodiments, CFTR also regulates several other transport proteins, including K+ channels, aquaporin water channels, anion exchangers, the membrane fusion protein syntaxin-1A, and sodium bicarbonate transporters, suggesting that using small molecules to target and modulate the activity of these proteins in the mucin-secreting porosome complexes of airway epithelia may lead to the restoration of normal mucin secretion in the airways of CF patients.

[0131] Figure 23 shows the restoration of mucus secretion in ΔF508-CFTR human CF bronchial epithelial cells by CDN1163. To investigate mucus secretion in human airway epithelia, 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 shows that CDN1163 restores mucus secretion to normal levels in CFBE41o-human CF bronchial epithelial cells (ΔF508-CFTR) within 1 hour. Note that the basal level of Muc5B secretion in CFBE41o-human CF bronchial epithelial cells (ΔF508-CFTR) is approximately half that of normal wild-type (WT) cells. CDN1163, a Ca2+ ATPase (SERCA) activator, restores mucus (Muc5B) secretion in CF bronchial epithelial ΔF508 cells within 1 hour. Therefore, CDN1163 [4-(1-methylethoxy)-N-(2-methyl-8-quinolinyl)-benzamide] can be used as a small molecule therapeutic to restore normal mucus function in CF patients. Dosage concentrations, as measured by intrasubject tissue concentrations, can range from 1 nM (nanomolar) to 20 μM (micromolar). Such measurement techniques include blood sampling and subsequent analysis, breathalyzer analysis, urinalysis, biopsy, and other techniques known to those skilled in the art.

[0132] Similar to Figure 23, Figure 24 shows that α-CPA increases mucus secretion 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 the Ca2+-ATPase (SERCA) inhibitor cyclopiezominic acid (α-cycloprazonic acid or α-CPA). Our results show that α-CPA stimulates mucus secretion in CFBE41o-human CF bronchial epithelial cells (ΔF508-CFTR) within 1 hour. Note that the basal level of Muc5B secretion in CFBE41o-human CF bronchial epithelial cells (ΔF508-CFTR) is approximately half that of normal wild-type (WT) cells. α-CPA, a Ca2+ ATPase (SERCA) inhibitor, dramatically stimulates mucus (Muc5B) secretion (more than 10-fold) in CF bronchial epithelial ΔF508 cells within 1 hour. Therefore, α-CPA and small molecules with similar binding epitopes currently used for different indications, such as leflunomide, teriflunomide, tolvaptan, conivaptan, omeprazole, lansoprazole, rufinamide, prazosin, terazosin, and roflumilast, could be used as small molecule therapeutics to restore normal mucus function in CF patients. Data are presented below.

[0133] Figure 25 shows a schematic diagram illustrating some of the interactions between CFTR and related proteins (Nature Reviews Mol. Cell Biol. 2006, 7: 426-436). CFTR not only mediates Cl secretion, but also regulates several other transport proteins, including K channels, aquaporin water channels, anion exchangers, the membrane fusion protein syntaxin-1A, and sodium bicarbonate transporters. Thus, in some embodiments, because K channels are present in mucin secretory porosome complexes and mucus hydration is a problem in CF disease, small molecules such as those described herein can be used to target the interactions between K channels, aquaporin water channels, and CFTR.

[0134] In cystic fibrosis, bacterial pathogens contribute to mucus hypersecretion through mobilization of intracellular Ca2+, and Ca2+-ATPase (SERCA) has been identified in insulin-secreting porosome complexes, suggesting a role for this ion channel in mucus secretion in human airway epithelia.

[0135] 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, and molecular weight: 320.4.

[0136] CDN1163 alleviates diabetes and metabolic diseases. IC50 and target: SERCA. In vitro: CDN1163 (5.5-25 mM; 0-8 hours; rat cardiomyocytes) treatment time-dependently reduced high glucose-induced resistin and nuclear NFATc expression and increased AMPKα phosphorylation. In vivo: CDN1163 (50 mg / kg; intraperitoneal injection; administered for 5 days to male ob / ob and lean ob / + mice) increased SERCA2 Ca2+-ATPase activity, reduced endoplasmic reticulum (ER) stress-induced cell death in vitro, and improved hepatic Ca2+ transport activity. In vivo, CDN1163 reduced blood glucose levels, improved metabolic parameters and gluconeogenic gene expression, reversed hepatic steatosis, inhibited ER stress and ER stress-induced apoptosis, and improved mitochondrial efficiency in ob / ob mice.

[0137] The above-described embodiments of the invention can be used alone or in combination with each other. As a non-limiting example, small molecules targeting the porosome complex or portions thereof can be used in combination with porosome reconstitution, as described above. Similarly, patients undergoing treatment for neurological disorders such as those described above may also be undergoing similar treatment for insulin secretion disorders, such as diabetes. While certain embodiments of the invention are disclosed above in the context of cystic fibrosis treatment / therapy, the invention is not intended to be so limited in this regard. In particular, it is intended that the therapies and treatments disclosed herein can be utilized in the treatment of any secretory disease, of which cystic fibrosis and type 1 diabetes are only two. In one embodiment, the methods of the invention can also be utilized, for example, in the treatment of chronic obstructive pulmonary disease (COPD).

[0138] Compound synthesis In some embodiments, synthesis of the compounds described herein is achieved using means described in the chemical literature, using methods described herein, or a combination thereof. In addition, solvents, temperatures, and other reaction conditions presented herein may be varied.

[0139] In other embodiments, the starting materials and reagents used for the synthesis of 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. Chemical substances can be identified by multiple names and nomenclatures, including standard IUPAC nomenclature, CAS numbers, chemical formulas, bond diagrams, etc.

[0140] In further embodiments, the compounds described herein, and other related compounds with different substituents, are synthesized using techniques and materials described herein as well as art-recognized techniques and materials, for example, those described in the following documents: 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 Supplementals (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 4th Ed., (Wiley 1992), Carey and Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000, 2001), and Green and Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for preparing the compounds disclosed herein may be derived from reactions or may be modified using appropriate reagents and conditions for the introduction of various moieties shown in the formulas provided herein. The following synthetic methods may be used as a guide.

[0141] In the reactions described, it may be necessary to protect reactive functional groups, such as hydroxy, amino, imino, thio, or carboxy groups, to avoid their undesired participation in the reaction, if these reactive groups are desired in the final product. A detailed description of the techniques applicable to the formation and removal of protecting groups is provided in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, the disclosures of which are incorporated herein by reference.

[0142] In some embodiments, the identified compounds or small molecules are purchased from a variety of suppliers, including Sigma-Aldrich, Acros, Fisher, Fluka, Santa Cruz, CombiBlocks, BioBlocks, and Matrix Scientific.

[0143] Cells, analytical techniques, and equipment Also described herein, in certain embodiments, are methods for profiling porosomes to determine reactive or regulatory molecules. In some instances, the methods include profiling a porosome-containing cell sample or a porosome-containing cell lysate sample. In some embodiments, the cell sample or cell lysate sample is obtained from an animal cell. In some instances, the animal cell includes a cell from a marine invertebrate, fish, insect, amphibian, reptile, or mammal. In some instances, the mammalian cell is a primate, ape, horse, cow, pig, dog, cat, or rodent. In some instances, the mammal is a primate, ape, dog, cat, rabbit, ferret, or the like. In some instances, the rodent is a mouse, rat, hamster, gerbil, chinchilla, or guinea pig. In some embodiments, the avian cell is from a canary, parakeet, or parrot. In some embodiments, the reptilian cell is from a turtle, lizard, or snake. Optionally, the fish cells are from a tropical fish. Optionally, the fish cells are from a zebrafish (e.g., Danino rerio). Optionally, the worm cells are from a nematode (e.g., C. elegans). Optionally, the amphibian cells are from a frog. In some embodiments, the arthropod cells are from a tarantula or a hermit crab.

[0144] In some embodiments, the porosome complexes isolated from the cell or cell lysate sample are obtained from mammalian cells, in some instances, the mammalian cells are epithelial cells, connective tissue cells, hormone-secreting cells, neuronal cells, skeletal muscle cells, blood cells, or immune system cells.

[0145] Exemplary mammalian cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cell, 293H cell, HEK293 cell, CHO DG44 cell, CHO-S cell, CHO-K1 cell, Expi293F (trademark) cell, Flp-In (trademark) T-REx (trademark) 293 cell line, Flp-In (trademark)-293 cell line, Flp-In (trademark)-3T3 cell line, Flp-In (trademark)-BHK cell line, Flp-In (trademark)-CHO cell line, Flp-In (trademark)-CV-1 cell line, Flp-In (trademark)-Jurkat cell line, FreeStyle (trademark) 293-F cell, FreeStyle (trademark) CHO-S cell, GripTite (trademark) 293MSR cell line, GS-CHO cell line, HepaRG (trademark) cell, T-REx (trademark) Jurkat cell line, Per.C6 cell, T-REx (trademark)-293 cell line, T-REx (trademark)-CHO cell line, T-REx (trademark)-HeLa cell line, NC-HIMT cell line, and PC12 cell line.

[0146] In some instances, the porosome-containing cell sample or cell lysate sample is obtained from cells of a tumor cell line. In some instances, the cell sample or cell lysate sample is obtained from cells of a solid tumor cell line. In some instances, the solid tumor cell line is a sarcoma cell line. In some instances, the solid tumor cell line is a carcinoma cell line. In some embodiments, the sarcoma cell line is selected from the group consisting of alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, soft tissue clear cell sarcoma, dedifferentiated liposarcoma, desmoid tumor, desmoplastic small round cell tumor, embryonal rhabdoid tumor, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, neuroepithelioma, Ewing's sarcoma, extrarenal rhabdoid tumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, childhood fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi's sarcoma, leiomyosarcoma of bone, liposarcoma, osteolipoma, and osteolipoma. Cell lines derived from liposarcoma, malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma of bone (MFH), malignant mesenchymoma, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic sarcoma, neoplasm with perivascular epithelial-like differentiation, osteosarcoma, parosteal osteosarcoma, neoplasm with perivascular epithelial-like differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET / extraskeletal Ewing's tumor, rhabdomyosarcoma, round cell liposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovial sarcoma, and telangiectatic osteosarcoma.

[0147] In some embodiments, the cancer cell line is derived from an adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, cancer of unknown primary site (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 cell line.

[0148] In some examples, the porosome-containing cell sample or cell lysate sample is obtained from cells of a hematological malignant cell line. In some examples, the hematological malignant cell line is a T cell cell line. In some examples, the hematological malignant cell line is a B cell cell line. In some examples, the hematological malignant cell line is obtained from a T cell cell line of 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, enteropathic T cell lymphoma, hepatosplenic gamma delta T cell lymphoma, lymphoblastic lymphoma, nasal NK / T cell lymphoma, or therapy-related T cell lymphoma.

[0149] In some examples, the hematological malignant cell line is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous 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's macroglobulinemia, multiple myeloma, extranodal marginal zone B, The B-cell cells may be derived from B-cell cell lines of: large cell lymphoma, extranodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell precursor lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[0150] In some embodiments, the porosome-containing cell sample or cell lysate sample is obtained from a tumor cell line, including 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, and SKOV-3 / Lu c, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948, DLD-1, SW480, Capan-1, MC / 9, B72.3, B 25.2, B6.2, B38.1, DMS153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs817 These include, but are not limited to, .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, RPMI8226, MOLT-4, RS4, K-562, KASUMI-1, Daudi, GA-10, Raji, JeKo-1, NK-92, and Mino.

[0151] In some embodiments, the porosome-containing cell sample or cell lysate sample is derived from any tissue or fluid from an individual. Samples include, but are not limited to, tissue (e.g., connective tissue, muscle tissue, nervous tissue, or epithelial tissue), whole blood, separated bone marrow, bone marrow aspirate, pleural fluid, peritoneal fluid, cerebrospinal fluid, peritoneal fluid, pancreatic fluid, spinal fluid, brain fluid, ascites, pericardial fluid, urine, saliva, bronchial lavage fluid, sweat, tears, ear flow, sputum, hydrocele fluid, semen, vaginal fluid, milk, amniotic fluid, and respiratory, intestinal, or genitourinary secretions. 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., those in a bone marrow aspirate sample).

[0152] In some embodiments, a porosome-containing cell sample or cell lysate sample is obtained from an individual by any suitable means of obtaining a sample using well-known and routine clinical methods. Procedures for obtaining tissue samples from individuals are well known. For example, procedures for obtaining and processing tissue samples, such as by fine needle aspiration biopsy, are well known and can be used to obtain samples for use in the provided methods. Typically, such tissue samples are obtained by inserting a thin, hollow needle into a mass, such as a tumor mass, to sample cells, which are then stained and examined under a microscope.

[0153] Sample preparation and analysis In some embodiments, the porosome-containing sample solution comprises a cell sample, a cell lysate sample, or a sample containing isolated proteins. In some instances, the sample solution comprises a solution such as a buffer (e.g., phosphate-buffered saline) or a medium. In some embodiments, the medium is an isotope-labeled medium. In some instances, the sample solution is a cell solution.

[0154] In some embodiments, a porosome-containing solution sample (e.g., a cell sample, a cell lysate sample, or an isolated protein) is incubated with a compound for analysis of protein-probe interactions. In some examples, the solution sample (e.g., a cell sample, a cell lysate sample, or an isolated protein) is further incubated in the presence of an additional compound probe. In other examples, the solution sample (e.g., a cell sample, a cell lysate sample, or an isolated protein) is further incubated with a ligand. In such cases, the solution sample is incubated with a probe and a ligand for competitive protein profiling analysis.

[0155] In some cases, the porosome-containing cell sample or cell lysate sample is compared to a control. In some cases, differences are observed between the set of probe-protein interactions in the sample and the control. In some instances, the differences correlate with interactions between small molecules and one or more porosome proteins.

[0156] In some embodiments, one or more methods are utilized to label a porosome-containing solution sample (e.g., a cell sample, a cell lysate sample, or an isolated protein) for analysis of probe-protein interactions. In some instances, the method includes labeling the sample (e.g., a cell sample, a cell lysate sample, or an isolated protein) using an enrichment medium. In some instances, the sample (e.g., a cell sample, a cell lysate sample, or an isolated protein) is labeled with an isotope-labeled amino acid, such as a C- or N-labeled amino acid. In some instances, the labeled sample is further compared to an unlabeled sample to detect differences in probe-protein interactions between the two samples. In some instances, this difference is a difference in the interaction of the target protein and the protein with a small molecule ligand in the labeled sample versus the unlabeled sample. In some instances, this difference is an increase, decrease, or absence of protein-probe interaction in the two samples. In some instances, the isotope labeling method is referred to as SILAC (Stable Isotope Labeling Using Amino Acids in Cell Culture).

[0157] In some embodiments, the method includes incubating a solution sample or porosome sample mixture (e.g., a cell sample, a cell lysate sample, or containing isolated proteins) with a labeling group that tags one or more proteins of interest for further analysis. The labeling group can be an isotopic labeling group, such as an amino acid or an acid enriched with C, N, or deuterium, or can include a molecular label, such as biotin, folate, luciferase, or an amino acid tag. The label can further include an isotopically labeled linker. The linker can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more residues in length and can further 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 that is optionally isotopically labeled with C, N, and / or deuterium atoms at one or more amino acid residue positions within the linker. In some cases, the biotin-linker moiety is an isotopically labeled TEV tag.

[0158] In some embodiments, the sample is treated using the isotope reduction dimethylation (ReDi) method. In some cases, the ReDi labeling method involves reacting a peptide with formaldehyde to form a Schiff base, followed by reduction with cyanoborohydride. This reaction dimethylates the free amino groups at the N-terminus and lysine side chains, and monomethylates the N-terminal proline. In some cases, the ReDi labeling method involves methylating peptides from a first treated sample with a "light" label using a reagent with a natural isotope distribution of hydrogen atoms, and peptides from a second treated sample with a "heavy" label using deuterated formaldehyde and cyanoborohydride. Subsequent proteomic analysis (e.g., mass spectrometry) based on the relative peptide abundance of the heavy and light peptide versions can be used to analyze probe-protein interactions.

[0159] In some embodiments, samples are processed using isobaric tagging for relative and absolute quantification (iTRAQ) technology. In some cases, iTRAQ technology is based on covalent labeling of N-terminal and side-chain amines of peptides from processed samples. In some cases, reagents such as 4-plex or 8-plex are used to label peptides.

[0160] In some embodiments, the probe-protein complex is further conjugated to a chromophore, such as a fluorophore. In some instances, the probe-protein complex or a subsample thereof is separated and visualized using an electrophoresis system, such as by gel electrophoresis or capillary electrophoresis. Exemplary gel electrophoresis systems include agarose-based gels, polyacrylamide-based gels, or starch-based gels. In some instances, the probe-protein is subjected to native electrophoresis conditions. In some instances, the probe-protein is subjected to denaturing electrophoresis conditions.

[0161] In certain embodiments, the probe-protein complex is recovered using standard techniques known in the art and depending on the particular chemistry of the probe-protein complex. Exemplary techniques can include at least tangential flow filtration, chromatography, centrifugation, liquid chromatography, electrophoresis, and the like, alone or in combination, as commonly used to separate proteins of interest from mixtures.

[0162] In some instances, the recovered probe-protein complex is further fragmented to generate protein fragments. In some instances, the fragmentation is generated by mechanical stress, pressure, or chemical fragmentation. In some instances, the protein from the probe-protein complex is fragmented by chemical fragmentation. In some embodiments, the chemical fragmentation agent is a protease.

[0163] Exemplary proteases include 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 assemblage, Lon-A peptidase, peptidase C1p, Escherichia coli phage K1F endosialidase CIMCD self-cleaving protein, nucleoporin 145, lactoferrin, murein tetrapeptidase 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, adenine, pyroglutamyl-peptidase I, sortase A, hepatitis C virus peptidase 2, Sindbis virus type nsP2 peptidase, dipeptidyl-peptidase VI, or DeSI-1 peptidase; aspartic acid 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 acid proteases, such as AfuGprA; and metalloproteases, such as peptidase_M48.

[0164] In some instances, the fragmentation is random fragmentation. In some instances, the fragmentation generates protein fragments of specific lengths or results in shearing at specific sequences of amino acid regions.

[0165] In some examples, 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 time of flight (MALDI-TOF), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or nuclear magnetic resonance imaging (NMR).

[0166] In some embodiments, the LC method is any suitable LC method known in the art for separating a sample into individual fractions. This separation occurs based on the interaction of the sample with a mobile phase and a stationary phase. Because there are many stationary / mobile phase combinations that can be used to separate mixtures, there are several different types of chromatography that are classified based on the physical state of the phases. In some embodiments, LC is further classified as normal-phase chromatography, reversed-phase chromatography, size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, flash chromatography, chiral chromatography, and aqueous normal-phase chromatography.

[0167] In some embodiments, the LC method is a high performance liquid chromatography (HPLC) method, which in some embodiments is further classified as 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.

[0168] In some embodiments, the HPLC methods of the present disclosure are performed by any standard technique known in the art. Exemplary HPLC methods include hydrophilic interaction liquid chromatography (HILIC), electrostatic repulsion-hydrophilic interaction liquid chromatography (ERLIC), and reversed-phase liquid chromatography (RPLC).

[0169] In some embodiments, LC is combined with mass spectrometry as an LC-MS method. In some embodiments, the LC-MS method includes ultra-high performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS), ultra-high 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), and multidimensional liquid chromatography-tandem mass spectrometry (LC / LC-MS / MS). In some examples, the LC-MS method is LC / LC-MS / MS. In some embodiments, the LC-MS method of the present disclosure is performed by standard techniques well known in the art.

[0170] In some embodiments, GC is combined with mass spectrometry as a GC-MS method, which in some embodiments includes 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).

[0171] In some embodiments, CE is combined with mass spectrometry as a CE-MS method, which in some embodiments includes 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).

[0172] In some embodiments, nuclear magnetic resonance (NMR) methods are any suitable method 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, NMR methods include one-dimensional (1D) NMR, two-dimensional (2D) NMR, solid-state NMR, and NMR chromatography. Exemplary 1D NMR methods include 1 hydrogen, 13 carbon, 15 nitrogen, 17 oxygen, 19 fluorine, 31 phosphorus, 39 potassium, 23 sodium, 33 sulfur, 87 strontium, 27 aluminum, 43 calcium, 35 chlorine, 37 chlorine, 63 copper, 65 copper, 57 iron, 25 magnesium, 199 mercury, or 67 zinc NMR, distortion-free enhancement by polarization transfer (DEPT), attached proton test (APT), and 1D-incredible natural abundance double quantum transition experiment (INADEQUATE). Exemplary 2D NMR methods include correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), 2D-INADEQUATE, 2D-adequate double quantum transition experiment (ADEQUATE), nuclear Overhauser effect spectroscopy (NOSEY), 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 carbon-13 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.

[0173] In some embodiments, protein fragments are analyzed by the method described in Weerapana et al., "Quantitative reactivity profiling predicts functional cysteines in proteomes," Nature, 468: 790-795 (2010).

[0174] In some embodiments, the mass spectrometry results are analyzed by an algorithm for protein identification. In some embodiments, the algorithm combines the mass spectrometry results with a protein sequence database for protein identification.

[0175] In some embodiments, the algorithm comprises the ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot. In some embodiments, each protein from probe-protein complex is assigned a value.In some embodiments, the value assigned to each protein from probe-protein complex is obtained from mass spectrometry.In some instances, this value is the area under the curve of the plot of signal intensity as a function of mass-to-charge ratio.In some instances, this value is correlated with the reactivity of Lys residue in protein.

[0176] In some instances, a 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 instances, 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 instances, the ratio is at most 20.

[0177] 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 further has a standard deviation of less than 12, 10, or 8.

[0178] By way of example, in certain embodiments of the present invention, the first isolated porosome sample mixture is made from a human epithelial cell line, and the second isolated porosome sample mixture is made from the 508 cystic fibrosis mutant strain. The ratio can be 1:2 or 2:1, as long as the ratio is consistently expressed across all compared proteins in each sample mixture. In some instances, a ratio of 20% or less at a 95% confidence interval is considered an absent protein. Those skilled in the art may understand the need to modify the cutoff values ​​for the ratio and confidence interval without departing from the scope of the present invention. In certain embodiments of the present invention, the second sample mixture may be a "knockout" cell line in which one or more porosome proteins have been "knocked out" using technology such as CRISPER.

[0179] Kits / manufactured articles In certain embodiments, kits and articles of manufacture are disclosed herein for generating porosomal protein adducts or for use with one or more of the methods described herein. In some embodiments, kits for detecting porosomal protein-ligand interactions are described herein. In some embodiments, such kits include small molecule ligands, small molecule fragments or libraries, compound probes, and / or controls, and reagents suitable for performing one or more of the methods described herein. In some examples, the kits further include a sample, such as a cell sample, and an appropriate solution, such as a buffer or medium. In some embodiments, the kits further include recombinant porosomal protein(s) for use in one or more of the methods described herein. In some embodiments, additional components of the kit include a carrier, package, or container compartmentalized to receive one or more containers, such as vials, tubes, etc., each container containing one of the distinct elements used in the methods described herein. Suitable containers include, for example, bottles, vials, plates, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials, such as glass or plastic.

[0180] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, bags, containers, and any packaging material appropriate for the selected formulation and intended mode of use.

[0181] For example, the container may include a probe, a test compound, and one or more reagents for use in the methods disclosed herein. Such kits optionally include identifying descriptions, labels, or instructions for using the kit in the methods described herein.

[0182] Kits typically include a label listing the contents and / or instructions, as well as an instructional package insert. A set of instructions is also typically included. In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when letters, numbers, or other symbols forming the label are attached, molded, or etched onto the container itself, and the label is associated with the container when the label is present in a receptacle or carrier that also holds the container, for example, as a package insert. In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates instructions for using the contents, such as in the methods described herein.

[0183] Specific Terms 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 is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0184] As used herein, ranges and amounts may be expressed as "about" a particular value or range. "About" includes the exact amount. Thus, "about 5 μL" also 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 from manufacturing, production, or experimental tolerances.

[0185] Suitable variations to the above will be apparent to those skilled in the art and are naturally encompassed and expressly intended. For example, normal manufacturing tolerances may induce variations from the formulations presented above without departing from the broader scope of this invention.

[0186] The compounds described herein may be formed and / or used as acceptable salts. Types of acceptable salts include, but are not limited to: (1) acid addition salts formed by reacting a compound in free base form with an acceptable base, where the salt is an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, metaphosphoric acid, or an 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[4.1.2.1]sulfonic acid, or the like. -[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, lauryl sulfuric 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 present in the parent compound is replaced by a metal ion, such as an alkali metal ion (e.g., lithium, sodium, potassium), an alkaline earth ion (e.g., magnesium or calcium), or an aluminum ion. In some cases, the compounds described herein may be coordinated with organic bases, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, dicyclohexylamine, and tris(hydroxymethyl)methylamine. In other cases, the compounds described herein may form salts with amino acids, such as, but not limited to, arginine, lysine, etc. Acceptable inorganic bases used to form salts with compounds containing acidic protons include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.

[0187] The effective dosage and administration method of specific embodiments of the present invention may vary based on the individual patient and the stage of any indicated disease (e.g., influenza, COVID, HIV, other comorbidities), as well as other factors known to those of skill in the art. The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., using the ED50 (the dose therapeutically effective in 50% of a population) and the LD50 (the dose lethal to 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Pharmaceutical compositions with large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate various dosages for human use. The dosage of such compounds preferably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages vary within this range depending on the dosage form used, the patient's sensitivity, and the route of administration.

[0188] The exact dosage will be selected by the individual physician in consideration of the patient being treated. Dosage and administration will be adjusted to provide a sufficient level of the embodiments of the present invention to maintain the desired effect (e.g., elimination or reduction of enveloped virus particles or activity in the host). Additional factors that may be considered include the severity of any disease state, the patient's age, weight, and sex, diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to treatment.

[0189] Short-acting pharmaceutical compositions are administered daily, whereas long-acting pharmaceutical compositions are administered every 2, 3 to 4 days, every week, or once every 2 or more weeks. Depending on the half-life and clearance rate of the particular formulation, pharmaceutical compositions of the present invention may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times per day.

[0190] Typical dosages of active ingredient can vary from about 1 to 100,000 micrograms to about 10 grams total, 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.

[0191] More specifically, the dosage of the active ingredient described herein provides an amount sufficient to achieve the desired effect, including the effects described above (e.g., modulation, activation, or interaction of one or more porosomal proteins and / or achieving an effect on porosomal structure). Accordingly, the dosage of the active ingredient preferably achieves tissue or blood concentrations, or both, of about 1 to 800 μM. Preferred dosages achieve tissue or blood concentrations of greater than about 10 μM to about 500 μM. Preferred doses include, for example, 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, The amount of active ingredient required to achieve tissue or blood concentrations, or both, of 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. Doses resulting in tissue concentrations greater than 800 μM are not necessarily preferred, but are contemplated and may be used in some embodiments of the invention. Constant infusions of embodiments of the invention may be used to maintain a steady concentration of therapeutic agent.

[0192] Finally, the written description uses examples to disclose the invention, including the best mode, and also enables any person skilled in the art to practice the invention, including making and using any devices or systems, and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal words of the claims, or if they contain equivalent structural elements that do not differ substantially from the literal words of the claims.

[0193] The pharmacologically active compounds of this invention can be processed in accordance with conventional methods of pharmacy and good manufacturing practice to produce medicinal products for administration to patients (e.g., mammals, including humans) prophylactically or as part of a treatment regimen.

[0194] As used herein, the term "sequence" expressly contemplates DNA, cDNA, RNA, and the resulting peptide chains encoded thereby, in both sense and antisense orientations. Knowing one entails knowing the other, according to the standard rules of complementarity and codon encoding as exemplified in standardized DNA, RNA, and amino acid codon tables.

[0195] As used herein, elements or steps described in the singular followed by "a" or "an" should be understood as not excluding a plural of that element or step, unless the exclusion is expressly stated. Furthermore, references to "one embodiment" of the invention are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Furthermore, unless expressly stated otherwise, embodiments "comprising," "including," or "having" an element or elements having a particular characteristic may include additional such elements that do not have that characteristic.

[0196] Since certain changes can be made in the above-described invention without departing from the spirit and scope of the invention contained herein, it is intended that all of the subject matter of the above description shown in the accompanying drawings should be interpreted only as examples illustrating the inventive concepts herein and not as limiting the invention.

Claims

1. 1. A method for identifying protein-protein interactions in a porosome structure, comprising: a) creating a first porosome sample mixture; b) incubating the porosome sample mixture with a labeling group to form a probe-protein complex; c) recovering the probe-protein complex; and d) fragmenting the probe-protein complex to obtain protein fragments; e) analyzing said protein fragments by proteomics methods; f) identifying one or more proteins in the porosome sample mixture to generate a first set of identified proteins; g) assigning a value to each protein in the first identified protein set; h) performing steps a) through g) on ​​a second sample porosome mixture to obtain a second value for each protein in the second identified protein set; i) calculating a ratio of values ​​between paired proteins in said first and second identified protein sets; wherein the ratio determines protein-protein interactions within or adjacent to a porosome structure.

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

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

4. A kit configured to contain materials necessary to carry out each step of claim 1.

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

6. 2. The method of claim 1, wherein the value for each protein in the first and second identified protein sets correlates with the reactivity of Lys residues within the protein.

7. 10. The method of claim 1, further comprising: j) identifying specific protein-protein interactions within the porosome complex using small molecules.

8. 8. The method of claim 7, wherein said confirmation is achieved via chemical cross-linking and subsequent confirmation of binding by mass spectrometry.

9. 2. The method of claim 1, wherein the first and second sample porosome complex function is tested in an artificial lipid bilayer membrane.

10. A composition comprising an artificial porosome in an artificial lipid bilayer membrane.

11. crosslinking the artificial porosome with a nanobody that has been humanized to target and bind to one or more domains of one or more porosomes or porosome-associated proteins; delivering the crosslinked artificial porosome-nanobody to a target; A method comprising:

12. A composition comprising at least one cross-linking molecule and at least one small molecule modulator that targets a porosomal protein.

13. The composition of claim 12 , wherein the bridging molecule is an ELP diblock.

14. The composition of claim 12, further comprising a nanobody humanized to target and bind to one or more domains of one or more porosomal proteins.

15. 1. A composition comprising one or more small molecules that target one or more domains of a polosomal protein and a humanized Nanobody with an artificial cysteine, the cysteine ​​is attached to an ELP diblock; The composition, wherein the ELP diblock is conjugated to pAcF.

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

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

18. Extracting porosomes from a non-human source; reconstituting the extracted porosomes into human cells; A method comprising:

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

20. The method of claim 18, wherein the isolated porosomes are reconstituted into organoids or artificial lipid bilayer membranes.

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