EPH-ephrin tetramerization inhibitors that target a previously unknown and very high-affinity chelator-type intermolecular protein-protein interaction

Abstract

Compositions and methods for inhibiting EPH-EPHRIN tetramerization are provided. The compounds and methods herein further comprise methods for administering novel EPH-EPHRIN tetramerization inhibitors to a subject in need thereof to treat conditions caused or worsened by abnormal, defective, or excessive EPH-EPHRIN expression and / or signaling.

Description

EPH-EPHRIN TETRAMERIZATION INHIBITORS THAT TARGET A PREVIOUSLY UNKNOWN AND VERY HIGH-AFFINITY CHELATOR-TYPE INTERMOLECULAR PROTEIN-PROTEIN INTERACTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority of provisional patent application numbers 63 / 638,913 entitled “EPH-EPHRIN TETRAMERIZATION INHIBITORS THAT TARGET A PREVIOUSLY UNKNOWN AND VERY HIGH AFFINITY CHELATOR-TYPE INTERMOLECULAR PROTEIN-PROTEIN INTERACTION” and 63 / 713,479 entitled “EPH- EPHRIN TETRAMERIZATION INHIBITORS THAT TARGET A PREVIOUSLY UNKNOWN AND VERY HIGH AFFINITY CHELATOR-TYPE INTERMOLECULAR PROTEIN-PROTEIN INTERACTION” filed on April 25, 2024 and October 29, 2024 respectively, the entire contents of which are incorporated herein by reference.ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under grant number DK128819 awarded by the National Institutes of Health and grant numbers W81XWH-20- CPMRP-IIRA, CP200270 and DAMD 11115013 awarded by the United States Department of Defense. The government has certain rights in this invention.BACKGROUND

[0003] 1. Field

[0004] The present inventive concept is directed to compounds capable of inhibiting EPH- EPHRIN tetramerization and their use in treatments for conditions caused or worsened by disrupted, abnormal, defective, or excessive expression and / or interactions that affect EPH forward and / or EPHRIN reverse (bidirectional) cell-cell signaling mediated by this large family of evolutionarily conserved receptor-ligand molecules. As described in PCT / US2023 / 077841 (final application filed 10 / 26 / 23 and incorporated herein by reference in its entirety), EPH- EPHRIN tetramers are very high affinity macromolecular complexes that we showed can be disrupted by small MW chemicals.

[0005] 2. Discussion of Related Art

[0006] Chronic pain is a major problem that is poorly treated using drugs with efficacy, safety, and addiction issues. A further drawback of the current painkillers available today isthat they do not target the key synaptic machinery in the spinal cord that responds to peripheral nerve damage and directly causes the pathological long-lasting changes in neurons that leads to central sensitization and chronic pain. Specifically, central sensitization is due to the strengthening of synaptic connections formed by the pain-sensing peripheral nociceptor neurons, the C-fibers, which terminate superficially onto the spinal cord dorsal horn (DH) neurons. The plasticity of these synapses leads to enhanced transmission of pain impulses from the spinal cord to produce a heightened sensitivity to heat and touch stimuli (hyperalgesia and allodynia). The highly conserved EphB1 and EphB2 receptor tyrosine kinases interacting with their cognate transmembrane EphrinBI , EphrinB2, and EphrinB3 ligands are key trans-synaptic players thought to generate this plasticity and cause central sensitization and thus form a key conserved receptor-ligand system important for pain signaling.

[0007] The present disclosure is based on, in part, the development of highly sensitive biochemical and biophysical assays that can be run in small volumes and used for high- throughput screens (HTS) to search for chemicals that will disrupt the interactions and activities of the EPH and EPHRIN molecules. Exemplary examples herein describe how EphrinB reverse signaling may contribute to chronic conditions and show that compounds disclosed herein block both forward and reverse signaling, and target both EphB1 and EphB2. Accordingly, the present disclosure provides for novel therapeutics aimed at decreasing the ability of an Eph receptor (e.g., EphB1 and / or EphB2) to bind with an associated Ephrin ligand (e.g., EphrinBI and / or EphrinB2).BRIEF SUMMARY

[0008] In an aspect, disclosed is a method of inhibiting formation of an EPH-EPHRIN tetramer, the method comprising: contacting an EPH or EPHRIN with an EPH-EPHRIN tetramerization antagonist that interferes with formation or stability of an EPH-EPHRIN tetramer by competing with a copper chelating or copper-binding like function of the EPH or EPHRIN molecule. In an aspect, the EPH-EPHRIN tetramer comprises EphB1 , EphB2, EphrinBI (Efnbl), EphrinB2 (Efnb2), EphrinB3 (Efnb3), or EphrinA5 (EfnA5) or any combination thereof. In an aspect, the formation of the EPH-EPHRIN tetramer is inhibited in vivo.

[0009] In another aspect, disclosed is a method of alleviating or relieving pain in a subject in need thereof, the method comprising administering an affective amount of an EPH- EPHRIN tetramerization inhibitor to the subject. In an aspect, the pain comprises chronic neuropathic pain.

[0010] In another aspect, disclosed is a method of treating a synaptopathy in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRINtetramerization inhibitor to the subject. In an aspect, the synaptopathy comprises abnormal, defective, or excessive EPH-EPHRIN signaling and optionally comprises disrupted NMDA receptor signaling. In an aspect, the synaptopathy is associated with anxiety, epilepsy, or chronic pain.

[0011] In another aspect, disclosed is a method of treating addiction or opioid dependency in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

[0012] In another aspect, disclosed is a method of treating a fibrotic and / or an inflammatory disease or condition in a subject in need thereof, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject. In an aspect, the fibrotic and / or inflammatory disease or condition comprises abnormal, defective, or excessive EPH-EPHRIN signaling and optionally comprises NASH / MASH liver disease, chronic kidney disease, scleroderma, systemic sclerosis, heart fibrosis, lung fibrosis, intestinal inflammatory fibrotic disorders, neuroinflammation, inflammation and / or fibrosis of another organ, and / or abnormal wound healing optionally selected from keloids, hypertrophic scarring, and / or heterotopic ossification.

[0013] In another aspect, disclosed is a method of treating a metabolic disease in a subject in need thereof, the method comprising administering an effective amount of an EPH- EPHRIN tetramerization inhibitor to the subject, optionally wherein the metabolic disorder comprises diabetes, obesity or a cardiovascular pathology associated with a metabolic disease.

[0014] In another aspect, disclosed is a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject. In an aspect, the cancer comprises abnormal, defective, or excessive EPH-EPHRIN signaling and optionally comprises GBM (glioblastoma), pancreatic cancer, and / or colon cancer.

[0015] In another aspect, disclosed is a method of treating a viral infection in a subject in need thereof, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject. In an aspect, the viral infection comprises abnormal, defective, or excessive EPH-EPHRIN signaling and optionally comprises an infection by henipavirus and / or human immunodeficiency virus (HIV). In an aspect, the tetramerization inhibitor is further envisioned to disrupt viral recognition of receptor molecules expressed on a cell and / or impede viral entry into a cell to reduce / block infection.

[0016] In an aspect, any of the methods disclosed herein, comprise administering a pharmaceutical composition comprising the EPH-EPHRIN tetramerization inhibitor and oneor more pharmaceutical excipients to the subject. In an aspect, the pharmaceutical composition is administered intravenously, subcutaneously, intraperitoneally, orally, and / or topically. In an aspect, the subject is a human, a livestock animal, a companion animal, a lab animal, or a zoological animal.

[0017] In an aspect, the EPH-EPHRIN tetramerization inhibitor of any of the methods disclosed herein, comprises any one of the compounds disclosed herein.

[0018] In an aspect, the EPH-EPHRIN tetramerization inhibitor of any of the methods disclosed herein, comprises any one or more of the compounds disclosed herein.BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1A depicts the electropositive “copper-like” and copper-binding “chelator-like” chemistry associated with the tetramer interfaces described herein on a schematic of the crystal structure of dimers formed between the ectodomains of the EphB2 receptor and one of its ligands, EphrinB2. The cartoon drawings of EphB2 and EphrinB2 are originally from Himanen et al., 2001 “Crystal structure of an Eph receptor-ephrin complex”. Nature 414: 933-938 (2001).

[0020] FIG. 1 B depicts crystal structures of the EphB2-EphrinB2 circular tetramer formed by the high-affinity association of two EphB2-EphrinB2 dimers through their respective tetramer interfaces, and subsequent association of individual tetramers into higher order tetramer clusters involved in activation of bidirectional cell-cell signaling.

[0021] FIG. 1C depicts metal chelators A20 and 8-hydroxyquinoline (8-HQ) binding to the electropositive copper-like region of EphB2 to disrupt its tetramer interface from interacting with the cognate chelator-like tetramer interface of EphrinB2.

[0022] FIG. 1D depicts use of a primary amine A20 / 8-HQ like chelator chemical (1° amine) that can be immobilized onto an AR2G BLI Octet biosensor chip to biophysically measure the kinetics of its ability to bind to the electropositive copper-like region of the EphB2 tetramer interface.

[0023] FIG. 1 E depicts metal chelators EDTA and EGTA binding to the electropositive copper-like tetramer interface region of EphB2 in a similar fashion to compete with formation of EphB2-EphrinB2 circular tetramers.

[0024] FIG. 1F depicts SW117057 (L08) acting as an EphB2 chelator to disrupt formation of the EphB2-EphrinB2 circular tetramer.

[0025] FIG. 1G depicts SW036148 (P08) acting as an EphB2 chelator to disrupt formation of the EphB2-EphrinB2 circular tetramer.

[0026] FIG. 1 H depicts modifications of A20 / 8-HQ chemicals in the search to find those that retain potent ability to specifically chelate EPH molecules and compete with formation of EPH-EPHRIN tetramers, but have lost or greatly reduced their ability to chelate metals (yellow highlights).

[0027] FIG. 11 is a schematic visualizing that proteins can exhibit “hot spots” for binding of small molecule antagonists and that these can disrupt protein-protein interactions, and a schematic of various changes around A20 chemical that alter its ability to disrupt EPH- EPHRIN tetramerization.

[0028] FIG. 1J is a schematic showing stages of EPH-EPHRIN protein interactions at sites of cell-cell contact that leads to formation of dimers, tetramers, and higher order tetramer clusters between the EPH (blue) and EPHRIN (pink) ectodomains. Formation of tetramers and tetramer clusters results in robust activation of the intracellular domains of both molecules by tyrosine phosphorylation (small yellow circles), which then leads to the transduction of bidirectional signals into both the EPH-expressing cell (forward signaling) and EPHRIN-expressing cell (reverse signaling). EPH-EPHRIN bidirectional signaling controls diverse cellular behaviors, especially during development, and is known to become unhinged in many pathological situations.

[0029] FIG. 2A-2C depict examples of Octet RED384 biophysical kinetic analysis sensorgram protein-protein interaction data showing that A20-I, Clioquinol, EDTA, or EGTA disrupt ability of EphrinB2 (EB2) to form stable tetramers with EphB1 , EphB2, or EphB4 during the association step compared to control runs where no compound was included. Human EB2-Fc ectodomain was loaded on AHC Octet sensorchips and after baseline measurements, the immobilized protein was exposed to 25 nM soluble human EphB1-His, EphB2-His, or EphB4-His ectodomain proteins for an 80” association step followed by a 500” dissociation step with either no compound, 10 pM A20-I (2xHCI salt, as max control), 3.2 pM Clioquinol, 3.2 pM EDTA, or 3.2 pM EGTA in PBSTD (PBS with 0.05% Tween-20 and 1% DMSO).

[0030] FIG. 2D depicts Octet RED384 kinetic analysis used to calculate the IC50 of Clioquinol on disrupting the EphrinB2-EphB2 tetramer. Immobilized human EB2-Fc binding to 25 nM soluble human EphB2-His with 0, 0.1 , 0.2, 0.4, 0.8, 1.6, or 3.2 pM of Clioquinol or 2 pM A20-I (2xHCI, as max control) was tested in PBSTD. The IC50 values shown in red here for Clioquinol and below for other compounds tested represent the relative area under the curve (AUC) that leads to a 50% reduction calculated from the dose-response sensorgram traces and compared to the sensorgram results using the (1) no compound trace and (2) the max control trace. This relative analysis provides the most accurate IC50information concerning effectiveness of any one compound to disrupt the Eph-Ephrin tetramer under analysis.

[0031] FIG. 2E depicts Octet RED384 kinetic analysis used to calculate the IC50 of 8-HQ on disrupting the EphrinB2-EphB2 tetramer. Immobilized human EB2-Fc binding to 25 nM soluble human EphB2-His with 0, 1, 2, 4, 8, 16, or 32 pM of 8-HQ (1xHCI salt) or 2 pM A20- I (2xHCI, as max control) was tested in PBSTD.

[0032] FIG. 2F depicts Octet RED384 kinetic analysis used to calculate the IC50 of EDTA on disrupting the EphrinB2-EphB2 tetramer. Immobilized human EB2-Fc binding to 25 nM soluble human EphB2-His with 0, 0.1 , 0.2, 0.4, 0.8, 1.6, or 3.2 pM of EDTA or 2 pM A20-I (2xHCI, as max control) was tested in PBSTD.

[0033] FIG. 2G depicts Octet RED384 kinetic analysis used to calculate the IC50 of EGTA on disrupting the EphrinB2-EphB2 tetramer. Immobilized human EB2-Fc binding to 25 nM soluble human EphB2-His with 0, 0.1 , 0.2, 0.4, 0.8, 1.6, or 3.2 pM of EGTA or 2 pM A20-I (2xHCI, as max control) was tested in PBSTD.

[0034] FIG. 2H depicts Octet RED384 kinetic analysis data showing that A20 is a reversible antagonist of EphB1 binding to EphrinB2. Immobilized rat EphB1-Fc ectodomain protein pretreated with 100 pM A20 did not affect its subsequent ability to bind 30 nM soluble mouse EB2-His ectodomain protein (red sensorgram) and interacted with similar kinetics as the immobilized EphB1-Fc protein not pre-exposed to the compound (green sensorgram). Addition of A20 during the association step did compete with EB2 binding to the immobilized EphB1 protein (purple sensorgram). Binding assays were conducted in PBSTD, association time was for 400” and dissociation time was for 500”.

[0035] FIG. 2I depicts Octet RED384 kinetic analysis sensorgram data showing that A20-I, EDTA, and EGTA are all reversible antagonists of EphrinB2 binding to EphB2. Immobilized human EB2-Fc ectodomain pre-treated with 10 pM of A20-I, EDTA, or EGTA retained ability to subsequently form tetramers with soluble EphB2-His ectodomain protein, whereas presence of these chemicals at 10 pM during the association step strongly prevented formation of the circular tetramer.

[0036] FIG. 3A-3F depict Octet RED384 kinetic analysis sensorgram data showing that copper at high concentrations simultaneously accelerates EphrinB2-EphB1, EphrinB2- EphB2, or EphrinB2-EphB4 dimer formation and impedes formation of the tetramer compared to A20-I. Immobilized human EB2-Fc ectodomain binding to 25 nM of the indicated soluble human EphB-His ectodomain in the presence of 0, 1 , 10, 100 pM CuSO4 in PBSTD was tested.

[0037] FIG. 3G-3I depict Octet RED384 kinetic analysis sensorgram data showing cobalt, nickel, or magnesium / calcium do not significantly affect EphrinB2-EphB2 dimer or tetramer formation. Immobilized human EB2-Fc binding to 25 nM soluble human EphB2-His in the presence of 10-100 pM C0CI2, 10-100 pM NiCl2, or 500 pM MgCl2+ 900 pM CaCl2in PBSTD was tested.

[0038] FIG. 3J-3L depict Octet RED384 kinetic analysis sensorgram data showing copper presented as CuCl2at 100 pM also affects the EphrinB2-EphB1 , EphrinB2-EphB2, and EphrinB2-EphB4 interactions. Immobilized human EB2-Fc binding to 25 nM indicated soluble human EphB-His, in the presence of 0, 1 , 10, 100 pM CuCl2in PBSTD was tested.

[0039] FIG. 3M-3N depict Octet RED384 kinetic analysis sensorgram data showing copper presented as CuCl2at 20 pM, 40 pM, or 80 pM also affects EphrinB2-EphB1 and EphrinB2- EphB2 interactions. Immobilized human EB2-Fc binding to 25 nM indicated soluble human EphB-His in the presence CuCl2in PBSTD was tested.

[0040] FIG. 3O-3P depict Octet RED384 kinetic analysis sensorgram data showing zinc presented as ZnCl2 at 20 pM, 40 pM, or 80 pM affects EphrinB2-EphB1 and EphrinB2-EphB2 interactions. Immobilized human EB2-Fc binding to 25 nM indicated soluble human EphB- His in the presence ZnCl2in PBSTD was tested.

[0041] FIG. 3Q depict Octet RED384 kinetic analysis sensorgram data showing copper presented as CuCl2at 20 and 80 pM affects the EphrinB2-EphB4 interaction. Immobilized human EB2-Fc binding to 25 nM soluble human EphB4-His in the presence of CuCl2in PBSTD was tested.

[0042] FIG. 3R depicts Octet RED384 kinetic analysis sensorgram data showing zinc presented as ZnCl2at 20, 40, or 80 pM affects the EphrinB2-EphB4 interaction. Immobilized human EB2-Fc binding to 25 nM soluble human EphB4-His in the presence of ZnCl2in PBSTD was tested.

[0043] FIG. 3S-3U depict Octet RED384 kinetic analysis sensorgram data showing the effect of copper, zinc, and iron compared with nickel and cobalt presented as sulfates (SO4) on EphrinB2-EphB1 , EphrinB2-EphB2, and EphrinB2-EphB4 interactions. Immobilized human EB2-Fc binding to 25 nM soluble human EphB1-His, EphB2-His, or EphB4-His in PBSTD with 100 pM of either CUSO4, ZnSO4, FeSO4, COSO4, or NiSO4 in PBSTD was tested.

[0044] FIG. 4A is a photograph of an in vitro copper chelation assay plate showing A20 and 8-HQ are copper chelators. In the standard chelation assay, increasing amounts of an experimental chemical are combined with a set amount of CuSCk, pyrocatechol violet (PV), and DMSO (if necessary depending on compound solubility) in 1X AOX buffer (5 mM potassium phosphate, pH 6.1 with 0.9% sodium chloride) in triplicate wells of an assay plate.This colorimetric assay is based on the metal chelator activity of PV that when complexed with copper is bluish-violet in color, and that addition of a competing chelator will remove metal from PV and lead to loss of blue-violet color. As the assay is only accurate if the compound under study dissolves well, DMSO may be included to increase solubility and achieve clear and transparent solutions. Negative control wells contain PV without CuSO4, positive control wells have PV+CuSO4 but no added A20 / 8-HQ experimental chemical. Absorbance was measured at 632 nm.

[0045] FIG. 4B is a line graph of the data in Fig. 4A showing the percentage of copper chelation over various concentrations of 8-HQ, 8-HQ-like, A20, and A20-l-OMe. A20 and 8- HQ exhibit significantly greater ability to chelate copper at high concentrations compared to the other two compounds tested in this experiment.

[0046] FIG. 4C is a bar graph of the data in FIG. 4A showing the percentage of copper chelation over various concentrations of 8-HQ, 8-HQ-like, A20, and A20-l-QMe.

[0047] FIG. 5A depicts chemical structures of 8-HQ and analogs of A20, including the A20-2xHCI and A20-l 2xHCI salts. In example, the hydroxy group of the 8-HQ ring structure can be modified by adding either an ether linkage (e.g., A20-l-QMe) or an ester linkage in attempts to disrupt metal chelator activity.

[0048] FIG. 5B is a photograph of in vitro copper chelation assay plate showing effect of increasing amounts of 8-HQ, A20-2xHCI salt, A20-l 2xHCI salt, and A20-l-QMe (top) and resulting graphs of the data after scanning the plate for absorbance at 632 nm (bottom). The ether modified A20-l-QMe shows reduced copper chelator activity compared to the three compounds that have an intact 8-HQ ring. AOX buffer containing 30% DMSO was used.

[0049] FIG. 6A shows the chemical structures of chelators 8-HQ, EDTA, and EGTA.

[0050] FIG. 6B shows the chemical structures of 8-HQ or EDTA bound to metals like copper, zinc, or aluminum.

[0051] FIG. 6C is a photograph of an in vitro copper chelation assay plate showing effect of increasing amounts of EDTA, 8-HQ, and 3511-l-2xHCI salt, and comparing a store-bought copper chelation assay kit with one made in Henkemeyer laboratory for a fraction of the cost (top), with the actual numerical data after scanning the plate for absorbance at 632 nm shown (middle), and the resulting graphs of the data (bottom). The home-made assay is more robust, detecting copper chelation activity for all three chemicals as expected, with 3511-1 showing less activity than EDTA or 8-HQ. AOX buffer containing 30% DMSO was used.

[0052] FIG. 6D depict graphs of percentage of copper chelation over various concentrations of two 8-HQs, A20 and A20-I, and compared to EDTA. Two independent experiments areshown. Similar to 3511-1 shown in FIG. 6C, the assay results shown here indicate that A20 and A20-I exhibit somewhat lower copper chelator activity compared to EDTA.

[0053] FIG. 7A shows the chemical structures of A20 and BQPB4 (From: PCT / US2023 / 077841 , final application filed 10 / 26 / 23) with a number of different FDA approved 8-HQ-containing drugs and compounds in development, including Clioquinol, lodoquinol, Nitroxoline, PBT2 / PBT-1033, and Alzheimer’s 151 and 152. All are presumed to be metal chelators as they contain an intact 8-HQ ring structure.

[0054] FIG. 7B is a photograph of an in vitro copper chelation assay plate showing effect of increasing amounts of 8-HQ, PBT2 / PBT-1033, Nitroxoline, A20-2xHCI, A20-l-2xHCI, and 3511-l 2xHCI (top), with the actual numerical data after scanning the plate for absorbance at 632 nm shown (middle), and the resulting graphs of the data (bottom). All chemicals as expected showed copper chelation activity, with PBT2 / PBT-1033 exhibiting somewhat greater activity and 3511-1 showing slightly less activity compared to the others.

[0055] FIG. 8A shows a unique 8-HQ chemical that contains a Primary Amine and can be coupled / immobilized to AR2G Octet biosensor chips.

[0056] FIG. 8B depict Octet RED384 kinetic analysis sensorgram data that shows the Primary Amine compound exhibits a dose-dependent ability to reduce formation of both EphrinB2-EphB1 and EphrinB2-EphB2 tetramers. Immobilized human EB2-Fc binding to 25 nM of the indicated soluble human EphB-His, in the presence of 0, 1 , 10, 100 pM Primary Amine in PBSTD was tested. The two compounds tested are different synthesis of the same chemical, the “B” preparation is more purified and shows better ability to reduce tetramer formation.

[0057] FIG. 9A depict Octet RED384 kinetic analysis sensorgram data that show when Primary Amine B is loaded on AR2G biosensor chips it is able to bind 300 nM soluble EphB1 , and to a greater extent EphB2 with clear 1 : 1 binding dynamics, but shows no binding to the EphB4 or EphrinB2 ectodomains. This data shows that an 8-HQ containing chelator molecule is able to interact with EphB2 and EphB1 ectodomains, presumably through the electropositive copper-like property schematized above for EphB2 in FIG. 1. Primary Amine B was loaded onto activated AR2G biosensors for 4 minutes in pH 5 acetate buffer, quenched with BSA, and then after baseline measurements were tested for binding to 300 nM of the indicated soluble human EphB1-His, EphB2-His, EphB4-His, and EphrinB2-His ectodomains for 80” association and 100” dissociation steps in PBST.

[0058] FIG. 9B depict additional Octet RED384 kinetic analysis of Primary Amine B binding to increasing concentrations of soluble human EphB2-His ectodomain. Binding of EphB2 to immobilized Primary Amine shows a clear dose-response with no binding at 12.5 nM EphB2,and gradually increasing in binding response as concentration was increased to 25, 50, 300, and 400 nM of the ectodomain protein.

[0059] FIG. 9C identifies the electropositive arginine-rich residues in the ligand binding ectodomain F’-G and K-L loops of EphB2 that form a key part of the tetramer interface on a protein alignment of the of all the different Eph receptors. Highlighted are the Eph residues involved in dimerization with EphrinB2 (pink), tetramerization with EphrinB2 (yellow), and those involved in Eph-Eph homotypic interactions that aids crystal packing and clustering of circular tetramers (green).

[0060] FIG. 9D focuses on the sequence of electropositive arginine residues in the F’-G and K-L tetramerization loops of EphB2 that form a portion of the tetramer interface, compared to the amino acid sequences of other Eph receptors. Note that the tetramerization interface regions of the EphB2 and EphB1 ectodomains are very similar to each other, and share clusters of electropositive arginines in the F’-G loop (RRR in EphB2 and NRR in EphB1) and K-L loop (RN in both EphB2 and EphB1) that fold together at a distinct location on the surface of the receptor to interact with the C-D loop of the EphrinB2 tetramerization interface that contains a negative charged aspartic acid. The other EphB receptors are less electropositive, EphB4 for instance has F’-G loop (PRR) the proline likely introducing a kink to disrupt the loop, and K-L loop (KA). As lysine is less electropositive than arginine, it seems EphB2 is most electropositive with 4 arginines, followed by EphB1 with 3 arginines, and then EphB4 with only 2 arginines and a lysine (plus the kink in F’-G loop which likely interferes with a confirmation favorable to tetramerization). It should be noted that the structure of EphB4 bound to EphrinB2 only indicates a dimer is formed as the crystals showed no evidence of tetramers, unlike what is observed for EphB2-EphrinB2 crystals, and is consistent with our biophysical data that shows A20 compounds have almost no effect on the EphB4-EphrinB2 interaction. It should also be noted that the eight EphA class receptors are different from EphB2 and EphB1, their F’-G loops have either zero or 1 arginine / lysine and the K-L loops are either KK, KR, or RK, so any tetramers formed by EphA molecules will probably be of relatively lower affinity, and with most of it driven by the K-L loop.

[0061] FIG. 10A outlines how the top hits from the original AlphaScreen chemiluminescent HTS conducted by Henkemeyer laboratory for compounds that disrupted the EphB1- EphrinB2 interaction were sorted and re-evaluated using Octet biophysical kinetic methods. The original HTS resulted in recovery of 1 ,246 primary hits that reduced the chemiluminescent signal >10%, and which after additional Alpha, ELISA, and pulldown analysis ultimately led to a single lead chemical, A20. Subsequent deeper analysis of the 1 ,246 primary hits determined that 77 of them contain an 8-HQ ring structure (including A20) or a modified 8-HQ ring structure that lacks the OH group (termed 8-HQ-like). These 8-HQsand 8-HQ-like compounds and a subset of the other top hits from the original HTS (those that reduced Alpha chemiluminescent signal >25%) were re-evaluated using Octet technology.

[0062] FIG. 10B-10F depict example Octet RED384 kinetic analysis sensorgram data that show how 245 top hit compounds from the original primary HTS screen were re-evaluated for ability to disrupt the human EphrinB2-EphB1 interaction, initially by testing all selected hits at 3.2 pM (in PBSTD). 10B shows the set-up for the re-assessment, with biosensor chips to obtain control sensorgrams with (1) no added compound, (2) max inhibition with 2 pM A20- I, and a % max condition with 0.5 pM A20-I, which is essentially the IC50 of this compound and thus gives a trace of approximately 50% inhibition of the tetramer. 10C shows potent hit compounds with tetramer inhibitory activity below max control (rank 3), 10D with similar strong hit compounds with inhibitor activity at the max (rank 2), 10E with poor inhibitor activity (rank 1), and 10F with no inhibitor activity (rank 0). Almost all of the hit compounds that ranked 2 or 3 in this analysis were determined to be additional chemicals that contained an 8-HQ ring structure.

[0063] FIG. 11 is a table listing the 73 top hits with an intact 8-HQ ring structure that were re-evaluated at 3.2 pM in Octet and ranked for their ability inhibit the EphrinB2-EphB1 tetramer. Most of the 8-HQ re-evaluated here showed potent ability to disrupt the EphrinB2- EphB1 tetramer, ranking either a 3 or 2, including yellow highlighted compounds A20 and 3511-0013 described in PCT / US2023 / 077841 (final application filed 10 / 26 / 23).

[0064] FIG. 12A depicts the chemical structure of a particularly potent 8-HQ identified after re-evaluation of the top HTS hits, G15 (SW160569), a sulfur-containing free base compound.

[0065] FIG. 12B-12C depicts Octet RED384 kinetic analysis sensorgram data testing G15 at 0, 0.1 , 0.2, 0.4, 0.8, 1.6, and 3.2 pM for ability to disrupt formation of the human EphrinB2- EphB1 and EphrinB2-EphB2 tetramers and to calculate IC50 values.

[0066] FIG. 13A depicts the structure of a non-8-HQ chemical identified after re-evaluation of the top HTS hits from the primary AlphaScreen, P08 (SW036148), the free base and a 2xHCI salt form generated herein. While P08 is similar to 8-HQ in that it has a quinoline ring structure and an oxygen at position 8, the hydrogen is replaced with a cyclic linkage to connect the 8 oxygen back to the quinoline ring to create a 3 ring structure. As the free OH group on 8-HQs is thought to provide electrons needed for metal chelation, modifications of this oxygen like that in P08 may impair metal binding.

[0067] FIG. 13B-13C depicts Octet RED384 kinetic analysis sensorgram data testing P08 free base at various concentrations for ability to disrupt formation of the human EphrinB2- EphB1 and EphrinB2-EphB2 tetramers and to calculate IC50 values.

[0068] FIG. 13D-13F depicts Octet RED384 kinetic analysis sensorgram data testing P08 free base at various concentrations for ability to disrupt formation of the human EphrinBI- EphB2, EphrinB2-EphB2, and EphrinB3-EphB2 tetramers and to calculate IC50 values. Compared to EphrinB2-EphB2, the P08 compound seems particularly effective at disrupting tetramers formed between EphrinB1-EphB2 and EphrinB3-EphB2 with IC50s at or below 100 nM.

[0069] FIG. 13G is a photograph of in vitro copper chelation assay plate showing effect of increasing amounts of P08 free base compared to 8-HQ (top), with the resulting graphs of the data (bottom). P08 showed little copper chelation activity.

[0070] FIG. 14A depicts the structure of an extremely potent non-8-HQ chemical identified after re-evaluation of the top HTS hits from the primary AlphaScreen, L08 (SW117057), the free base and 2xHCI and 4xHCI salt forms generated herein. This non-8-HQ compound from the HTS library forms a potent new pharmacophore that disrupts EPH-EPHRIN tetramers.

[0071] FIG. 14B-14F depicts Octet RED384 kinetic analysis sensorgram data testing L08 free base (14B-14D) and 2xHCI salt (14E-14F) at various concentrations for ability to disrupt formation of the human EphrinB2-EphB1 , EphrinB2-EphB2, and EphrinB2-EphB4 tetramers and to calculate IC50 values.

[0072] FIG. 14G are photographs of various stages in setting up a modified in vitro copper chelation assay that show L08 is a strong copper chelator. L08 which presents in solution as a red colored compound, turns dark blue when copper is added to the solution, indicating it can bind copper and in doing so changes color, similar to PV used in the standard chelation assay.

[0073] FIG. 14H a modified in vitro copper chelation assay, L08 was used as the color indicator (replacing PV), and increasing concentrations of EDTA and EGTA chelators were added to determine the amount needed to strip the copper from L08 and return the solution to a red color. It was determined that equal molar amounts of EDTA or EGTA was required to strip the copper from L08. This indicates L08 is as strong a metal chelator as EDTA or EGTA.

[0074] FIG. 15A depicts the structures of A20-I, a classic 8-HQ compound, with a modified A20-I similar to P08 that contains a cyclic linkage to generate a 3 ring structure and protect the oxygen and is viewed from two different orientations. Potential modifications of the R group are indicated.

[0075] FIG. 15B depicts the structures of P08 and four other chemicals from the HTS library that have the same modification of the oxygen to generate a cyclic linkage and a 3 ring molecule. The level of AlphaScreen signal reduction achieved by these five P08-likecompounds against the EphB1-EphrinB2 interaction in the original HTS screen is shown, with P08 exhibiting 13.2% reduction. Relative areas under the curve IC50 values are shown for Octet studies using the human EphrinB2-EphB2 interaction.

[0076] FIG. 15C depicts the structures of other available cyclic linked P08-like chemicals and IC50 results using Octet RED384 kinetic analysis for ability to disrupt formation of the human EphrinB2-EphB2 tetramer. All cyclic P08-like compounds tested in Octet were able to disrupt formation of the human EphrinB2-EphB2 circular tetramer and exhibited decent IC50 values near or below 1 pM.

[0077] FIG. 16 show modifications of A20 chemicals to disrupt the hydroxy group of the 8- HQ involved in copper binding and metal chelation. Multiple options are employed to modify the oxygen, including a cyclic linkage (option 1), ether linkage (option 2), ester linkage (option 3), or carbamate linkage (option 4). Potential modifications of R group are indicated.

[0078] FIG. 17 show modifications of A20 chemicals that disrupt the hydroxy group of the 8-HQ with a cyclic linkage (option 1)

[0079] FIG. 18 show modifications of A20 chemicals that disrupt the hydroxy group of the 8-HQ with an ether linkage (option 2).

[0080] FIG. 19 show modifications of A20 chemicals that disrupt the hydroxy group of the 8-HQ with an ester linkage (option 3).

[0081] FIG. 20 show modifications of A20 chemicals that disrupt the hydroxy group of the 8-HQ with a carbamate linkage (option 4).

[0082] FIG. 21 A structures of ether protected compound A20-I-QMP, free base and 3xHCI salt forms, and another interesting available ether protected 8-HQ-like compound, #7892274.

[0083] FIG. 21 B is a photograph of in vitro copper chelation assay plate showing effect of increasing amounts of A20-I-QMP free base compared to 8-HQ (top). A20-I-QMP showed little if any copper chelation activity.

[0084] FIG. 21C show Octet sensorgram data testing ether protected compound A20-I- OMP.3xHCI for ability to disrupt the human EphrinB2-EphB2 circular tetramer. As A20-I-OMP shows fairly weak tetramer inhibitor activity, the concentration range used in this run included 0, 1 , 2, 4, 8, 16, and 32 pM compound.

[0085] FIG. 21 D show Octet sensorgram data testing ether protected compound A20-I- OMP.3xHCI for ability to disrupt the human EphrinB1-EphB2 circular tetramer. As A20-I-OMP shows fairly weak tetramer inhibitor activity, the concentration range used in this run included 0, 1 , 10, and 100 pM compound. Compared to the EphrinB2-EphB2 interaction, A20-I-OMP was better able to disrupt the EphrinB1-EphB2 interaction with a lower IC50 value of 3 pM.

[0086] FIG. 21 E show Octet sensorgram data testing ether protected compound A20-I- OMP-3xHCI for ability to disrupt the human EphrinB3-EphB2 circular tetramer. Compared to the EphrinB2-EphB2 and EphrinB1-EphB2 interactions, A20-I-OMP was better able to disrupt the EphrinB3-EphB2 interaction with a lower IC50 value below 1 pM.

[0087] FIG. 21 F show Octet sensorgram data testing ether protected compound #7892274 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. As #7892274 shows quite weak tetramer inhibitor activity, the concentration range used in this run included 0, 1 , 10, and 100 pM compound.

[0088] FIG. 22A structures of interesting available ester protected compounds.

[0089] FIG. 22B show Octet sensorgram data testing ester protected compound #7489299 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. The concentration range used in this run included 0, 1 , 10, and 100 pM compound.

[0090] FIG. 22C show Octet sensorgram data testing ester protected compound #7135823 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. The concentration range used in this run included 0, 1 , 10, and 100 pM compound.

[0091] FIG. 22D show Octet sensorgram data testing ester protected compound #7514277 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. The concentration range used in this run included 0, 1 , 10, and 100 pM compound.

[0092] FIG. 22E show Octet sensorgram data testing ester protected compound #7526470 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. The concentration range used in this run included 0, 1 , 10, and 100 pM compound.

[0093] FIG. 22F show Octet sensorgram data testing ester protected compound #7509057 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. The concentration range used in this run included 0, 1 , 10, and 100 pM compound.

[0094] FIG. 22G show Octet sensorgram data testing ester protected compound #5253340 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. The concentration range used in this run included 0, 1 , 10, and 100 pM compound.

[0095] FIG. 23A structures of interesting available carbamate protected compounds.

[0096] FIG. 23B show Octet sensorgram data testing carbamate protected compound #5121792 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. The concentration range used in this run included 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 pM compound.

[0097] FIG. 23C show Octet sensorgram data testing carbamate protected compound #7735411 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. The concentration range used in this run included 0, 1 , 10, and 100 pM compound.

[0098] FIG. 23D show Octet sensorgram data testing carbamate protected compound #5216461 for ability to disrupt the human EphrinB2-EphB2 circular tetramer. The concentration range used in this run included 0, 1 , 10, and 100 pM compound.

[0099] FIG. 24A is a scanned image of an anti-EphB2 immunoblot with chemiluminescent techniques to detect the receptor protein in various phosphotyrosine immunoprecipitations (IP) from whole E14.5 day mouse embryo protein lysates from wild-type (WT) and EphB2 knockout (KO) specimens, and used to quantify the phosphorylation of the EphB2 receptor as a readout of its activation state. IP1 lanes are immunoprecipitations of phosphotyrosine containing proteins using either (1) a phospho-pEphB1 / B2 specific antibody, (2) the general pTyrlOOO antibody, (3) the general 4G10 antibody that also detects pTyr proteins, or (4) a combination of all three pTyr antibodies. IP2 lanes are subsequent immunoprecipitations of the phosphotyrosine cleared lysates using a specific pan anti-EphB2 antibody to precipitate the remaining unphosphorylated EphB2 to provide a readout of total tEphB2 present in the lysates (minus the small amount of pEphB2). 100% of the pTyr precipitated proteins in IP1 were loaded on the gel, whereas only 2% of the pan tEphB2 in IP2 was loaded. Unphosphorylated tEphB2, which comprises the bulk of EphB2 protein in the lysate migrates in the SDS-PAGE gel as a single species just below the 130 KDa protein standard marker (leftmost lane), whereas pEphB2 migration is slightly retarded in the gel at or just above the 130 KDa marker, consistent with its phosphorylation affecting its movement in the gel. Note the complete absence of any pEphB2 or tEphB2 protein in the lanes from KO mutant embryos, demonstrating the specificity of the antibody detection methods used in these experiments. As only 2% of the tEphB2 was loaded and the pEphB2 signal is still less intense than that, it is clear that the percentage / level of active pEphB2 in the E14.5 embryo is less than 2% of the total tEphB2 present in the lysate.

[0100] FIG. 24B shows the bands selected for volume analysis to quantify the intensity of pEphB2 and tEphB2 signals.

[0101] FIG. 24C shows the analysis used to quantify the percentage of pEphB2 present in the lysates. All three of the phosphotyrosine antibodies were each able to precipitate in IP1 comparable amounts of pEphB2, and combining all three only increased the signal slightly. This indicates any one of these antibodies can more or less precipitate the majority of pEphB2 in the lysates. The data further shows that in the E14.5 day embryo no more than 1% of EphB2 is in the active, pEphB2, state.

[0102] FIG. 25A-25B are two exposures of an anti-EphB2 immunoblot analyzing 3-4 month old adult brain protein lysates for IP1 (pEphB2) and IP2 (tEphB2) from three different WT mice and from an EphB2 KO, and K661 R (kinase-dead) and F620D (kinase-overactive) point mutant mice. No pEphB2 is detected in any of the adult brains.

[0103] FIG. 26A-26C are three exposures of an anti-EphB2 immunoblot analyzing WT brain protein lysates at different developmental stages for IP1 (pEphB2) and IP2 (tEphB2). While pEphB2 is readily detected in the E16.5 day embryonic brain and at early postnatal time points, little if any is detected at later stages.

[0104] FIG. 26D shows the bands selected for volume analysis to quantify the intensity of pEphB2 and tEphB2 signals.

[0105] FIG. 26E shows the analysis used to quantify the percentage of pEphB2 present in the lysates. Even in the E16.5 day embryonic brain, the pEphB2 signal represents only 1.39% of the total EphB2, and this level decreases to a barely detectable <0.1% in the adult brain.

[0106] FIG. 27A is a scanned image of an anti-EphB2 immunoblot analyzing intestine lysates collected at postnatal day 8 (P8) after four intraperitoneal dosings with A20.2xHCI salt (vehicle is 100% PBS) over two days, assessing three different V and three A20 treated animals for IP1 (pEphB2) and IP2 (tEphB2). While pEphB2 is readily detected in the three V treated intestines, less pEphB2 and tEphB2 is detected in the A20 treated samples.

[0107] FIG. 27B shows the volume analysis to quantify the intensity of pEphB2 and tEphB2 signals in the three V and A20 intestines. A20 resulted in a significant reduction in the percentage of pEphB2 (% pEphB2), as well as in overall the levels of pEphB2 and tEphB2 protein.

[0108] FIG. 27C is a scanned image of an anti-EphB2 immunoblot analyzing intestine lysates collected at postnatal day 8 (P8) after four intraperitoneal dosings with EDTA (vehicle is 100% PBS) over two days, assessing three different V and three EDTA treated animals for IP1 (pEphB2) and IP2 (tEphB2). While pEphB2 is readily detected in the three V treated intestines, less pEphB2 and tEphB2 is detected in the EDTA treated samples.

[0109] FIG. 27D shows the volume analysis to quantify the intensity of pEphB2 and tEphB2 signals in the three V and EDTA intestines. EDTA treatment lead to a trend for a lower percentage of pEphB2 (% pEphB2), as well as lower levels of pEphB2 and somewhat less tEphB2 protein.

[0110] FIG. 27E shows the combined analysis of pEphB2 (% pEphB2) in six V, three A20, and three EDTA intestine samples as the animal dosings and biochemical experiments shown here were conducted at the same time. Combined, the data indicates A20 and EDTA are each able to significantly reduce the percentage of pEphB2 in the intestines.

[0111] FIG. 28A is a scanned image of an anti-EphB2 immunoblot analyzing kidney lysates collected at postnatal day 8 (P8) after four intraperitoneal dosings with A20.2xHCI salt (vehicle is 100% PBS) over two days, assessing three different V and three A20 treated animals for IP1 (pEphB2) and IP2 (tEphB2). While pEphB2 and tEphB2 is readily detectedin the three V treated kidneys, less pEphB2 and tEphB2 is detected in the A20 treated samples.

[0112] FIG. 28B shows the volume analysis to quantify the intensity of pEphB2 and tEphB2 signals in the three V and A20 kidneys. While A20 treatment may result in a slight reduction in the level of pEphB2, what is most significant here is a great reduction in level of tEphB2 protein.

[0113] FIG. 29A shows our development of a highly sensitive biochemical assay that measures amount of active, reverse signaling tyrosine phosphorylated EphrinBI (pEphrinB1 / pEB1) protein present in lysates from various tissues. Shown is a scanned image of an anti-EphrinB1 immunoblot of protein lysates prepared from E14.5 day mouse embryos, postnatal day 3 (P3) mouse brain, and adult lungs, liver, and kidney that were first immunoprecipitated with anti-pTyr1000 beads (IP) to purify all the phosphotyrosinecontaining proteins including any pEphrinBI protein (100% of the IP was loaded), and run adjacent to the remaining phosphotyrosine cleared lysates (1% of the lysate was loaded). The lysate lanes show that EphrinBI was expressed in all tissues analyzed, with relatively high levels detected in the embryo, brain, and lung tissues. The IP lanes showed strong levels of activated pEphrinBI in the embryo, with little present in the postnatal brain and adult lungs, though very strong relative amounts of pEphrinBI were detected in the liver and kidney samples.

[0114] FIG. 29B shows the volume analysis to quantify the intensity of pEphrinBI and tEphrinBI signals in the different samples. The adult liver and kidney samples contained the greatest levels of pEphrinBI, at 2% and 0.4% of the total tEphrinBI present in those respective lysates.

[0115] FIG. 30A is a schematic outlining the carbon tetrachloride (CCI4) chronic liver injury model and intraperitoneal dosing of A20 chemicals (3511-1 and BQPB4, both at 20 mg / kg) at the end of the study.

[0116] FIG. 30B is a scanned image of an anti-EphrinB1 immunoblot analyzing pEphrinBI levels in adult liver lysates collected from normal controls, chronic diseased / injured livers (using either CCI4 injury model or non-obesogenic diet fed model for 12 weeks), and from CCI4 injured mice that also received six intraperitoneal dosings with 3511-l.2xHCI or BQPB43511-l.2xHCI salts over the final three days.

[0117] FIG. 30C shows the volume analysis to quantify the intensity of pEphrinBI and tEphrinBI signals in the livers. While the injured livers showed a trend towards increased levels of % pEphrinBI, the most important data here is the strong significant reduction observed for % pEphrinBI levels in livers subjected to treatment with A20 chemicals. Thisdata shows that injection of A20 chemicals results in a drastic reduction in pEphrinBI levels in the liver, indicating the compounds work as expected to reduce EphrinBI reverse signaling in vivo.

[0118] FIG. 31 A shows the structure of an interesting available P08-like cyclic protected compound, Z56835953, that is provided as a salt compound.

[0119] FIG. 31 B is a photograph of an in vitro copper chelation assay plate showing effect of increasing amounts of Z56835953 compared to 8-HQ. Z56835953 showed greatly reduced copper chelator activity.

[0120] FIG. 31 C depicts Octet RED384 kinetic analysis sensorgram data testing Z56835953 at various concentrations for ability to disrupt formation of the human EphrinBI- EphB2, EphrinB2-EphB2, and EphrinB3-EphB2 tetramers and to calculate IC50 values. Compared to EphrinB2-EphB2, the Z56835953 compound seems particularly effective at disrupting tetramers formed between EphrinB1-EphB2 and EphrinB3-EphB2 with IC50s below 100 nM.

[0121] FIG. 31D is a scanned image of an anti-EphrinB1 immunoblot analyzing adult kidney lysates collected after eight intraperitoneal dosings with Z56835953 over a four day period. Two different vehicle treated kidneys and one Z56835953 treated kidney were assessed for pEphrinBI using pTyr1000 beads for immunoprecipitation. While high levels of pEphrinBI is readily detected in the two vehicle treated kidneys (8 and 16%), very little (0.3%) is detected in the Z56835953 treated kidney.

[0122] FIG. 32A depicts a proton NMR spectrum of compound A20-Br-OMe.

[0123] FIG. 32B depicts a proton NMR spectrum of compound A20-l-OMe.

[0124] FIG. 32C depicts a proton NMR spectrum of compound A20-I-OMP.

[0125] FIG. 33A outline of additional experiments that further assess the ability of A20 andEDTA to reduce level of active, tyrosine phosphorylated pEphB2 in vivo after dosing into animals, here using newly available SH2 superbinder domain beads from Precision Proteomics (Ontario, Canada) to immunoprecipitate phosphotyrosine proteins from whole cell lysates. As these SH2 superbinder beads show very little background in our experiments, we have found them to be a vastly superior reagent to detect the extremely low in vivo levels of active phospho-EphB2 and phospho-EphrinB that we aim to detect in native tissues collected directly from animals and not subjected to any manipulation other than dosing with a tetramer inhibitor compound.

[0126] FIG. 33B are chemiluminescent images of two anti-EphB2 immunoblots done at the same time analyzing SH2 superbinder bead IPs of intestine whole protein lysates collectedat postnatal day 8 (P8) after four intraperitoneal dosings with A20 or EDTA salts (vehicle is 100% PBS) over two days. Two different V lysates were analyzed, one on each gel, with duplicate lysates of the three A20 and three EDTA treated animals on the two gels.

[0127] FIG. 33C shows the bands selected for volume analysis to quantify the intensity of pEphB2 (green) and tEphB2 (red) signals in the P8 intestines. Note that the green pEphB2 band in the IP lanes is most intense in the two V treated intestines, whereas less pEphB2 is detected in the A20 and EDTA treated lysates.

[0128] FIG. 33D band volume analysis of pEphB2 and tEphB2 in the indicated lanes and calculation of % pEphB2 levels.

[0129] FIG. 33E graphs of % pEphB2, pEphB2, and tEphB2 levels for the V, A20, and EDTA treated intestines, with the each duplicate A20 and EDTA sample averaged. Quantification of the data shows both A20 and EDTA led to significant reductions in % pEphB2, highly significant reductions in pEphB2 levels, with no change in tEphB2 levels.

[0130] FIG. 33F graphs of % pEphB2, pEphB2, and tEphB2 levels for the V, A20, and EDTA treated intestines, with each duplicate A20 and EDTA sample plotted separately. The quantification here indicates both A20 and EDTA led to highly significant reductions in % pEphB2 and pEphB2 levels, again with affecting tEphB2 levels.

[0131] FIG. 34A outlines experiments to detect in vivo levels of tyrosine phosphorylated EphrinB2 (pEphrinB2) in protein lysates made from E14.5 day mouse embryos, here comparing EphrinB2 + / + wild-type (WT) animals to EphrinB Iz / lz mutant (LZ) specimens which do not make a WT sized protein. The lysates were immunoprecipitated with either SH2 superbinder beads, pTyrlOOO antibody beads, or 4G10 antibody + Sepharose beads, and after washing, 100% of the immunoprecipitate and 1 % of the lysate of each sample were run on a gel and immunoblotted with anti-EphrinB2 antibodies.

[0132] FIG. 34B shows two exposures of the anti-EphrinB2 immunoblot done, note that EphrinB2 protein is only detected in the lanes containing WT lysates, no protein is present in the LZ mutant lanes as expected. Also note that in the WT samples, considerably more tEphrinB2 protein is detected in the lysate lanes compared to the pEphrinB2 detected in the IP lanes. This shows that the % level of pEphrinB2 in the mouse embryo is much less than 1 %.

[0133] FIG. 34C shows the bands selected for volume analysis to quantify the intensity of pEphrinB2 (green) and tEphrinB2 (red) signals in E14.5 day WT embryos. As expected, the LZ mutants show no EphrinB2 protein expression (stippled lines). Quantification of the band analysis data in the WT samples shows that the SH2 superbinder beads and pTyrlOOO beads both indicate a very low 0.14% and 0.13% level of % pEphrinB2 in the E14.5 day mouseembryo (the strong background band in the 4G10 IP gives a less accurate level of % pEphrinB2).

[0134] FIG. 35A outlines experiments using SH2 superbinder beads to detect in vivo levels of tyrosine phosphorylated EphrinB2 (pEphrinB2) in protein lysates made from P8 mouse intestines after four intraperitoneal dosings with EDTA salt (vehicle is 100% PBS) over two days.

[0135] FIG. 35B shows two exposures of the anti-EphrinB2 immunoblot, note that much more EphrinB2 protein is detected in all the lysate lanes (L, only 1% loaded) compared to the IP lanes (100% loaded), indicating significantly less than 1% of EphrinB2 protein is in the active tyrosine phosphorylated pEphrinB2 state in P8 intestines. The light exposure confirms relatively equal amounts of tEphrinB2 protein is present in the lysates of the seven different samples analyzed.

[0136] FIG. 35C shows the bands selected for volume analysis to quantify the intensity of pEphrinB2 (green) and tEphrinB2 (red) signals in the four V and three EDTA treated P8 intestines. Quantification of the band analysis data shows that the SH2 superbinder beads indicate a very low ~0.1% level of % pEphrinB2 in the V control intestines that is significantly reduced to ~0.05% pEphrinB2 in EDTA treated samples. Consistent with this, the EDTA treated intestines showed a significant >50% reduction in the pEphrinB2 band compared to the vehicle controls, and with no significant difference detected in total tEphrinB2 band protein levels between the two treatment groups.DETAILED DESCRIPTION

[0137] In some aspects, the current disclosure is based on the surprising discovery and characterization of low molecular weight compounds that selectively inhibit EPH-EPHRIN receptor-ligand tetramerization and that they do so by interfering with a previously undescribed copper chelator-like intermolecular protein-protein interaction which drives EPH-EPHRIN tetramerization. These compounds may be used to inhibit Eph forward signaling and Ephrin reverse signaling (bidirectional signaling) and have potential use as a novel group of therapeutics to combat all sorts of unmet medical conditions. Accordingly, various EPH-EPHRIN tetramerization inhibitor chemicals are described herein along with methods of treatment thereof.I. Definitions

[0138] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the termsused in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nded. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5thEd., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0139] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of” the disclosed elements, in which additional elements other than the listed elements are not included.

[0140] “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual that includes a pharmaceutical agent. As used herein a pharmaceutical composition comprises one or more of receptors, vectors, cells disclosed herein compounded with suitable pharmaceuticals carriers or excipients.

[0141] “Treatment” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease.

[0142] As used herein, the term “patient”, “subject”, or “test subject” refers to any organism to which provided compound or compounds described herein are administered in accordance with the present invention e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, dogs, cats, horses (and other farm animals), non-human primates, humans). In an aspect, a subject is a human. In some aspects, a subject may be suffering from, and / or susceptible to a disease, disorder, and / or condition (e.g., a condition caused or worsened by abnormal, defective, or excessive EPH-EPHRIN expression and / or signaling).

[0143] The term “effective amount” as used herein is defined as the amount of the molecules of the present invention that are necessary to result in the desired physiological change in the cell or tissue to which it is administered. The term “therapeutically effective amount” as used herein is defined as the amount of the molecules of the present invention that achieves a desired effect with respect to whatever condition is being treated. For example, a desired effect in a method for treating pain could be a reduction or ameliorationof pain in the subject while a desired effect in a method of treating cancer could be a reduction in tumor size or an arrest in tumor growth or metastasis. A skilled artisan readily recognizes that in many cases the molecules may not provide a cure but may provide a partial benefit, such as alleviation or improvement of at least one symptom or parameter. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of molecules that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount.”

[0144] As used herein, the term “alkyl” to saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.), cyclic alkyl groups (or “cycloalkyl” or “alicyclic” or “carbocyclic” groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups). The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogous to alkyls, but which contain at least one double or triple carbon-carbon bond respectively.

[0145] As used herein, the term “alkoxy” refers to alkyl groups linked to the remainder of the molecule through an oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. The alkoxy groups can be straight-chain or branched.

[0146] As used herein, the term “Amino” refers to unsubstituted or substituted moiety of the formula — NRaRb, in which Ra and Rb are each independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, taken together with the nitrogen atom to which they are attached, form a cyclic moiety having from 3 to 8 atoms in the ring. Thus, the term amino includes cyclic amino moieties such as piperidinyl or pyrrolidinyl groups, unless otherwise stated.

[0147] As used herein, the term “heterocyclic group” refers to closed ring structures analogous to carbocyclic groups in which one or more of the carbon atoms in the ring is an element other than carbon, for example, nitrogen, sulfur, or oxygen. Heterocyclic groups may be saturated or unsaturated. Additionally, heterocyclic groups (such as pyrrolyl, pyridyl, isoquinolyl, quinolyl, purinyl, and furyl) may have aromatic character, in which case they may be referred to as “heteroaryl” or “heteroaromatic” groups. Exemplary heterocyclic groups include, but are not limited to pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, quinoline, piperazine, pyridine, pyrazine, pyridazine, pyrimidine, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine.

[0148] As used herein, the term “acceptable salt” refers to salts of the compounds of the invention which are acceptable for the methods of the invention.

[0149] As used herein, the term “haloalkyl” refers to an alkyl group, as defined herein, substituted with one or more halogen. For example, trifluoromethyl is a haloalkyl group.

[0150] As used herein, the term “cyano” refers to a moiety comprising a carbon connected to a nitrogen with a triple bond (e.g., -CN).

[0151] As used herein, the term “azido” refers a linear, polyatomic anion with the formula N-3 and structure -N=N +=N - three nitrogens (e.g., -N3).

[0152] As used herein, the term “amido” refers to a functional group comprising a primary, secondary or tertiary amide. The “amido” group may further comprise additional R groups (e.g., further alkyls, cycloalkyls, aryls etc).

[0153] As used herein, the term “carbonyl” refers to a functional group comprising at least one -CO moiety. It may further comprise additional R groups (e.g., further alkyls, cycloalkyls, aryls etc).

[0154] As used herein, the term C4-C10 aryl refers to an aromatic ring comprising 4 to 10 carbons and no heteroatoms. As used herein, the term C4-C10 heteroaryl refers to an aromatic ring of 4 to 10 carbons wherein at least one carbon has been replaced with a heteroatom (e.g., N, O or S). As used herein, a C4-C10 cycloalkyl refers to a non-aromatic cyclic moiety having 4 to 10 carbons. As used herein, a C4-C10 heterocycloalkyl refers to a non-aromatic cyclic moiety having 4 to 6 carbon atoms, wherein at least one carbon atom has been replaced with a heteroatom (e.g, N, O or S). Any of the C4-C10 aryls, heteroaryls, cycloalkyls or heterocycloalkyls used herein may optionally be substituted, unless otherwise stated. As used herein, the term C4-C10 when associated with a ring (e.g., a cycloalkyl, an aryl, a heterocycloalkyl or a heteroaryl as described above) explicitly and implicitly contemplates any intervening ring size (e.g., a 4-member ring, a 5-member ring, a 6- member ring, a 7- member ring, a 8- member ring, a 9 member ring, or a 10 member ring).

[0155] As used herein, the term “substituted” refers to a compound where one or more hydrogen atoms have been replaced by a group of atoms or an atom. A substituted alkyl may be substituted with one or more bromine or methoxy groups.

[0156] As used herein, the term “may form a fused ring with”, means that the referenced functional groups (e.g., R groups) connected to a first ring may be connected to form a second ring fused to the first ring. A fused ring may be a fused aryl ring.

[0157] As used herein, the term EPH-EPHRIN tetramerization refers to the formation of an Eph receptor - Ephrin ligand macromolecular complex, between any of the known 14 EPHreceptors and 8 EPHRIN ligands, A and B classes. As described in PCT / US2023 / 077841 (final application filed 10 / 26 / 23 and incorporated herein by reference in its entirety), Eph- Ephrin dimers first associate / bind together and then two of these dimers associate / bind to form the EPH-EPHRIN tetramer, which is a very high-affinity stable circular structure that brings together the four components to activate the receptors and ligands to transduce their forward and reverse signals into the cells they are expressed on to mediate bidirectional signaling. EPH-EPHRIN tetramers once formed then assemble into much larger tetramer clusters which leads to greatly enhanced bidirectional signaling. Without tetramers, the formation of these larger tetramer clusters also cannot occur, and so compounds that affect tetramerization will also be envisioned to affect the formation of these larger tetramer clusters. EPH-EPHRIN tetramerization and tetramer clustering is distinct from dimerization which may or may not occur earlier. The formation of EPH-EPHRIN tetramers and tetramer clusters, as well as accurate measurements of Eph forward signaling and Ephrin reverse signaling, may be determined according to methods in the art and as described in the Examples herein below.

[0158] As used herein, the disclosure of numerical ranges by numerical endpoints includes all numbers encompassed by that range (e.g., “1 to 5” includes but is not limited to 1 , 1.25, 1.5, 1.75, 2, 2.3, 2. 5, 2.8, 3, 3.1, 3.3, 3.8, 3.9, 4, 4.25, 4.5, 4.75 and 5). Unless otherwise indicated, all numbers used herein to express quantities, amounts, dimensions, measurements, and the like should be understood as encompassing the specific quantities, amounts, dimensions, measurements and so on, and also as encompassing such instances modified by the term “about.” Accordingly, unless indicated to the contrary, the numerical descriptions set forth herein may vary while remaining well within the teachings of the present disclosure. At the very least, each numerical value should be construed in view of the number of significant digits and by applying routine rounding techniques. As various changes could be made in the above-described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense. As various changes could be made in the above-described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.I. Methods of Treatment

[0159] Provided herein are methods for inhibiting formation of an EPH-EPHRIN tetramer, the method comprising contacting an EPH or EPHRIN with an EPH-EPHRIN tetramerization inhibitor as provided herein below. In an aspect, the EPH-EPHRIN tetramerization inhibitorblocks or interferes with a copper chelating or copper-binding like function of the EPH or EPHRIN.

[0160] In an aspect, the EPH-EPHRIN tetramer may comprise EphB1 , EphB2, EphB3, EphrinB2, EphrinBI , EphrinB3, or EphrinA5, or any combination thereof. As discussed further herein below, EPH-EPHRIN signaling contributes to many physiological conditions. Accordingly, the present disclosure provides for methods of treating, attenuating, and preventing a condition related to EPH-EPHRIN signaling in a subject in need thereof. In various aspects, disrupted Eph-Ephrin signaling leads to, exacerbates or directly contributes to the condition to be treated. For example, Eph-Ephrin tetramerization contributes to pain signaling as well as synaptic plasticity. Accordingly, the present disclosure provides for methods of treating, attenuating, and preventing pain in a subject in need thereof. In certain aspects, the pain may be chronic, neuropathic pain. In other aspects, the present disclosure provides for methods of treating, attenuating, and preventing a synaptopathy in a subject in need thereof. In some aspects, the synaptopathy is caused by disrupted NMDA receptor signaling. In various aspects, the condition may be anxiety or epilepsy. In still further aspects, a method for treating, attenuating, and / or preventing an addiction (e.g., an opioid addiction) in a subject in need thereof is provided.

[0161] In further aspects, methods of treating, attenuating, and preventing a condition selected from the group consisting of pain (e.g., chronic, neuropathic pain), addiction and dependency (e.g., opioid addiction and dependency), other neurological disorders (e.g., anxiety, epilepsy, seizures), fibrotic and inflammatory diseases (e.g., NASH / MASH liver fibrosis, chronic kidney disease, scleroderma and systemic sclerosis (skin fibrosis), inflammation and / or fibrosis of other organs (e.g., lung fibrosis or heart fibrosis), neuroinflammation, intestinal disorders, and abnormal wound healing (e.g., keloids, hypertrophic scarring, heterotopic ossification), metabolic disorders (e.g., diabetes, obesity), cancer (e.g GBM (glioblastoma), pancreatic cancer, colon cancer), viral infections (e.g. henipavirus and HIV infections) is provided. In any of the methods provided herein, the condition to be treated (e.g., chronic pain, acute pain, cancer, a fibrotic and / or inflammatory disease, a viral infection, a metabolic disorder) may comprise abnormal, defective or excessive EPH-EPHRIN expression and / or signaling. That is, any of the methods herein may involve treating a condition caused or worsened by abnormal, defective, or excessive EPH- EPHRIN interactions (tetramerization and tetramer clustering) and cell signaling.

[0162] In any of the foregoing methods, an EPH-EPHRIN tetramerization inhibitor as provided herein or other metal chelator acting moiety or any pharmaceutically acceptable salt thereof, may be administered to the subject. In some aspects, the compound may be administered in a pharmaceutical composition or formulation as described herein, alone oralongside another suitable therapy for the condition. Additional aspects related to formulations, dosing and other aspects related to administration are described further below.

[0163] In various embodiments, a subject in need thereof can be having, suspected of having, or at risk of being in pain, or having a synaptic disorder. For example, the subject in need thereof can be having, suspected of having, or at risk of having neuropathic pain. For example, the subject in need thereof can be having, suspected of having, or at risk of having a synaptic disorder.

[0164] A suitable subject includes a human, a livestock animal, a companion animal, a lab animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g., a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non- limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In yet another embodiment, the subject may be a companion animal. Non- limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a specific embodiment, the animal is a laboratory animal. Non-limiting examples of a laboratory animal may include rodents, canines, felines, and non-human primates. In certain embodiments, the animal is a rodent. Non-limiting examples of rodents may include mice, rats, guinea pigs, etc. In preferred embodiments, the subject is a human.II. EPH-EPHRIN Tetramerization Inhibitors

[0165] In some aspects the methods provided herein involve use of a tetramerization inhibitor that comprises an 8-hydroxyquinoline compound of Formulaor a pharmaceutically appropriate salt thereof, wherein: R is hydrogen, an alkyl, a substituted or unsubstituted alkyl aryl, a substituted or unsubstituted alkyl heteroaryl, a substituted or unsubstituted carbonyl, or a substituted or unsubstituted amide, and each Ri to R6is independently hydrogen, a halo, a nitro, an alkyl, an ester, a substituted fused heterocycle, a substituted fused heteroaryl,SO2N(Ri5)(Ri6), wherein: A is -0-, SO2, NR17, or CRis; R7 to R12 are each independently hydrogen, a nitro, a halo, a substituted or unsubstituted carbonyl, a substituted or unsubstituted ketone, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted fused heterocycloalkyl, a substituted or unsubstituted fused heteroaryl, a substituted or unsubstituted fused cycloalkyl, a substituted or unsubstituted fused aryl, a substituted or unsubstituted alkyl aryl, or a substituted or unsubstituted alkyl cycloalkyl; one of R13 and R14 is hydrogen and the other is a nitro, a substituted or unsubstituted carbonyl, a substituted or unsubstituted ketone, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted fused heterocycloalkyl, a substituted or unsubstituted fused heteroaryl, a substituted or unsubstituted fused cycloalkyl, a substituted or unsubstituted fused aryl, a substituted or unsubstituted alkyl aryl, a substituted or unsubstituted alkyl cycloalkyl, or R13 and R14 are each a substituted or unsubstituted alkyl that together form a heterocyloalkyl, wherein the heterocycloalkyl is substituted or unsubstituted; R15and Ri6are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, a substituted or unsubstituted alkyl aryl, a substituted or unsubstituted alkyl cycloalkyl, a substituted or unsubstituted fused heterocycloalkyl, a substituted or unsubstituted fused heteroaryl, or R15 and RI6are each a substituted or unsubstituted alkyl that together form a substitute or unsubstituted heterocyloalkyl; and R17 or Ri8are each independently hydrogen, a substituted or unsubstituted aryl, a substituted or unsubstituted alkyl aryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted alkyl cycloalkyl; and at least one of R, R1, R2, R3 , R4, R5, and R6is not hydrogen.

[0166] In an aspect, R is hydrogen, a C1-C4 alkyl, a substituted or unsubstituted alkylaryl,unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted alkylaryl, a substituted or unsubstituted alkyl heteroaryl, and wherein each R20 and R21 is independently hydrogen, alkyl, a substituted or unsubstituted aryl, or wherein each R20 and R21 are a substituted or unsubstituted alkyl so that together form a heterocyloalkyl, wherein the heterocycloalkyl is substituted or unsubstituted, with the proviso that at least one of R20 and R21 is not hydrogen.

[0167] For example, in an aspect R is hydrogen. In an aspect, the EPH-EPHRIN inhibitor, wherein Ri, R3, Re, R12, R13 and R14are as previously defined.

[0168] In another aspect, R is a C1-C4 alkyl, a substituted or unsubstituted alkylaryl,For an aspect, EPH-PHRIN inhibitor comprises a structure

[0169] In an aspect, the EPH-EPHRIN inhibitor comprises a structure of Formula l-C-a, I-C-b, or l-C-c:

[0170] In an aspect, R isand R19 is as previously defined.

[0171] For instance, in an aspect, the EPH-EPHRIN tetramerization inhibitor comprises a compound of Formulawherein Ri, R3, and Re are as previously defined and R19 is selected from the group consisting of methyl, ethyl, substituted or unsubstituted phenyl, or a substituted or unsubstituted benzyl, wherein the phenyl and benzyl are each optionally substituted with 1 to 3 substituents selected from an alkoxy, a nitro, a halogen, or an alkyl.

[0172] In another aspect, R can are as previously defined.

[0173] For instance, in an aspect, the EPH-EPHRIN tetramerization inhibitor can comprise a compound of Formulawherein R1 and R3 are as defined previously and R20and R2I are each independently selected from hydrogen, a C1 to C4 alkyl, a substituted or unsubstituted phenyl, or wherein R20and R2I together with the nitrogen form a morpholine ring, with the proviso that at least one R20and R2I is not hydrogen.

[0174] In any of the foregoing aspects, R7, Rs and R9can be each independently hydrogen or an unsubstituted aryl. For instance, in an aspect, R7, Rs and R9can each independently hydrogen or phenyl.

[0175] In another aspect, R10can be nitro or a halo.

[0176] In another aspect, R11can be an alkyl, a substituted or unsubstituted aryl or substituted or unsubstituted alkylaryl. For instance, in an aspect R11is methyl, ethyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, wherein the phenyl and benzyl are each optionally substituted with a halogen, an alkoxy, or an alkyl.

[0177] In an aspect, each of RI2, R13, and R11is independently hydrogen, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted fused heteroaryl, a substituted or unsubstituted fused heterocycloalkyl, or a substituted or unsubstituted carbonyl. For instance, each of RI2, R13, and R14 can be a hydrogen, asubstitute or unsubstituted phenyl, a substituted or unsubstituted pyridinyl, a substituted or unsubstituted furanyl, a substituted or unsubstituted thiophenyl, a benzo[1 ,3]dioxole, a C1- C4 unsubstituted ketone, or a C1 to C4 aryl substituted ketone.

[0178] For example, in an aspect, each of R12, R13, and R14 is independently: hydrogen, anR22, R23, R24, and R25are each independently hydrogen, a halo, an alkyl, a nitro, a substituted or unsubstituted alkoxy, an amino, an ester, a hydroxy, or a thioether.

[0179] In further aspects, R22, R23, R24, and R25 can each independently be selected from hydrogen, a methyl, an ethyl, an isopropyl, a chloro, a bromo, a fluoro, a nitro, -OH, - N(CH3)2, - SCH3,, -COOCH3 and - OCH(R26)(R2?), and wherein R26 and R27 are each independently hydrogen, a C1-C3 alkyl, a C1-C3 alkenyl, a halo, or an aryl.

[0180] In another aspect, R13 and R14 together form an unsubstituted piperidine ring.

[0181] In another aspect, R15 and RI6are each independently hydrogen, an alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted fused heterocycloaryl.

[0182] For instance, in an aspect, R15 and R16 are each independently hydrogen, a C1 toC6 alkyl, benzyl, an unsubstituted cycloheptane,

[0183] In an aspect, R15 and R16 together form a substituted piperazine ring. In some aspects, the piperazine ring is substituted with a substituted aryl. For example, in some aspects, the piperazine ring may be substituted with a methoxy substituted phenyl.

[0184] In accord with the foregoing aspects, each of R1 to R6can independently be hydrogen, a bromo, a chloro, an iodo, a methyl, an ethyl, -OCH3, -NO2,

[0185] In further aspects, R3may be selected from

[0186] In still further aspects, R can be selected from hydrogen,alkyl or aryl, R29is hydrogen, alkyl, or alkoxy, R30is hydrogen, an alkyl, or an alkoxy.

[0187] In any of the foregoing aspects, R3can be selected fromwherein A2is O, SO2, CH2, or NR3I, and R3I is a substituted or unsubstituted aryl.

[0188] In any of the foregoing aspects, R1 can be H, nitro, or a halogen.

[0189] In still further aspects, the EPH-EPHRIN tetramerization inhibitor may be selected,an wherein n is 0-3, Ai is CH or O, B is N or CH, Bi is NH or CH2, R28 is alkyl or aryl, R29 is hydrogen, alkyl, or alkoxy, R30 is hydrogen, an alkyl, or an alkoxy; and R33 is selected from:NR31 , and R31 is a substituted or unsubstituted aryl; and X is H, NO2, or a halogen.

[0190] In various aspects, B can be N. In still further aspects, R29 is methyl or methoxy.

[0192] In still further aspects, R34 can be hydrogen.

[0193] Further to the foregoing aspects, R2, R4, and Rs can each be hydrogen.

[0194] In various aspects, the compound of Formula I (including, for instance, a compound of Formulas l-A, l-B, l-C, l-D, l-E, l-F, l-G and l-H), may be selected from:A16 104G13salt thereof.

[0196] In some aspects, the EPH-EPHRIN tetramerization inhibitor is an 8-HQ compound selected from:any pharmaceutically appropriate salt thereof.

[0197] In another aspect, the EPH-EPHRIN tetramerization inhibitor may be a compound selected from a 8-hydroxyquinoline-like compound of Formulapharmaceutically appropriate salt thereof.

[0198] In some aspects, m is 0 or 1.

[0199] In some aspects, R33 is Br, I, or Cl.

[0200] In some aspects, m is 0 and R33 is Cl and the compound comprises Formula Ilawherein R32 is C3-C6cycloalkyl, C1-C6unsubstituted alkyl, or C1-C6substituted alkyl. In some embodiments, the C3-C6 cycloalkyl is a cyclopentane or cyclohexane. In some embodiments, the Ci-Ce unsubstituted alkyl is butyl. In some embodiments, the C1-C6substituted alkyl is substituted methyl or substituted ethyl. The methyl or ethyl may be substituted with one or more pyridine, furan, benzene, C3-C6 cycloalkene, or a substituted cycloheteroalkyl.

[0201] The compounds of Formula Ila may be a compound selected from

[0202] In another aspect, the EPH-EPHRIN tetramerization inhibitor may be selected from a 8-hydroxyquinoline-like compound of Formula (lib)pharmaceutically appropriate salt thereof, wherein: R32 is selected from the group consistingwherein n is 0-3, A1 is CH or O, B is N or CH, Bi is NH or CH2, R28 is alkyl or aryl, R29 is hydrogen, alkyl, or alkoxy, R30 is hydrogen, an alkyl, or an alkoxy, R33is selected from:wherein A2is O, SO2, CH2, or NR31, and R31 is a substituted or unsubstituted aryl.

[0203] In some aspects, B can be N. In some aspects, R29 is methyl or methoxy.For instance, in some aspects, the EPH-EPHRIN tetramerization inhibitor is selected fromany pharmaceutically appropriate salt thereof.

[0206] aspect, A2is O, SO2or NR31. In another aspect, R31 is a substituted phenyl. For example, R31 can be a halo or alkoxy substituted phenyl.

[0207] In an example,

[0208] In various aspects, the EPH-EPHRIN tetramerization inhibitor is selected from:

[0209] In another aspect, R33 isIn some aspects, the EPH-EPHRIN

[0210] In another aspect, R33 canFor instance, in an aspect, the EPH-EPHRIN tetramerization inhibitor can be selected from:any pharmaceutically appropriate salt thereof.

[0212] Other EPH-EPHRIN tetramerization inhibitors not encompassed by Formulas I and II herein are contemplated and can be used in the methods provided herein. For instance, in one aspect, an EPH-EPHRIN tetramerization inhibitor for use in the methods herein can

[0213] In another aspect, the EPH-EPHRIN tetramerization inhibitor is an FDA approved agent that may have copper chelating activity. For instance, the EPH-EPHRINa pharmaceutically appropriate salt thereof.

[0214] In another aspect, the EPH-EPHRIN tetramerization inhibitor is a metal chelatorand any pharmaceutically appropriate salt thereof.

[0215] In another aspect, the EPH-EPHRIN tetramerization inhibitor is a transition metal compound selected from CuSC>4, FeSO4, ZnSO4, NiSO4, COSO4, CuCl2, ZnCl2, NiCl2, C0CI2,MgCl2and CaCL.

[0216] In preferred aspects, the EPH-EPHRIN tetramerization inhibitor is selected from any of the compounds listed in groups (a), (b), (c), (d), (e), (f), or (g) below:appropriate salt thereof;pharmaceutically appropriate salt thereof.

[0217] As described in more detail in the Examples below, it was surprisingly found that some pharmaceutically appropriate salt versions of some of the EPH-EPHRIN tetramerization inhibitors showed improved properties (e.g., improved ability to inhibit Eph / Ephrin tetramerization) compared to their non-salt (e.g., free base) forms. Accordingly, in some aspects, the compound of EPH-EPHRIN tetramerization inhibitor is a pharmaceutically appropriate salt (e.g., an HCI salt).

[0218] In some aspects, the pharmaceutically appropriate salt may be selected from the group consisting of:

[0219] As noted, any of the EPH-EPHRIN tetramerization inhibitors described herein (i.e., those of Formulas I and II and others) may inhibit EPH-EPHRIN tetramerization by interfering with a heretofore unknown electropositive copper-like binding site on the surface of the EPH receptor molecule. In an aspect, the inhibitor may interact and bind (chelate) this electropositive copper-like binding site on EPH molecules, preventing the EPHRIN ligand molecule from binding. In this fashion, the inhibitor acts as a competitive antagonist to the EPHRIN, which is shown herein below to be a copper chelator and whose binding to EPH molecules is influenced by copper and other transition metals.

[0220] In any of the foregoing aspects, the compound may have an IC50 of less than 2 pM, less than 1.6 pM, less than 1 pM or less than 0.5 pM. In some aspects, the compound may have an IC50 greater than about 1.6 pM. In other aspects, the compound may have an IC50 of about 1 .0 to 1.6 pM. In still other aspects, the compound may have an IC50 of about 0.4 to about 1.0 pM. In still other aspects, the IC50 may be less than 0.4 pM. IC50 and EPH- EPHRIN inhibition may be measured according to known methods in the art, including those described in the Examples herein.III. Pharmaceutical Formulations and Treatment Regimens

[0221] The compounds disclosed herein for use according to the methods herein described may be provided per se or as part of a pharmaceutical composition, where the compounds can be mixed with suitable carriers or excipients.

[0222] As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0223] Herein the term “active ingredient” refers to a compound, such as those described herein, that inhibits EPH-EPHRIN tetramerization.Pharmaceutically acceptable carriers and excipients

[0224] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0225] In various embodiments, compositions disclosed herein may further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, anti- oxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, and combinations thereof. Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0226] In various embodiments, pharmaceutical compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In other embodiments, any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art.

[0227] In various embodiments, pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise pharmaceutical compositions described herein include: water- soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water- insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of polymers as suspending agent(s) by total weight of the composition.

[0228] In various embodiments, pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of the composition may be increased by the addition of one or more gelling or thickening agents. In other aspects, compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In still other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of gelling or thickening agent(s) by total weight of the composition. In yet other aspects, suitable thickening agents can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or combinations thereof. In some embodiments, suitable thickening agent may be carboxymethylcellulose.

[0229] In various embodiments, pharmaceutical compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more agents by total weight of the composition. In other aspects, one or more of these agents may be added to improve the performance, efficacy, safety, shelf-life and / or other property of the muscarinic antagonist composition of the present disclosure. In some aspects, additives will be biocompatible, and will not be harsh, abrasive, or allergenic.

[0230] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more acidifying agents by total weight of the composition.

[0231] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more alkalizing agents by total weight of the composition.

[0232] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidativeprocess. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite and other materials known to one of ordinary skill in the art. In some aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more antioxidants by total weight of the composition.

[0233] In other embodiments, pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In another aspect, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more buffering agents by total weight of the composition. In other aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some embodiments, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In other embodiments, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6. In a preferred embodiment, compositions disclosed herein may have a pH greater than about 6.8.

[0234] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more preservatives by total weight of the composition.

[0235] In other embodiments, pharmaceutical compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In other aspects, compositionsdisclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In still other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more surface- acting reagents or detergents by total weight of the composition.

[0236] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more stabilizers by total weight of the composition.

[0237] In other embodiments, pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose and others known to those or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolarity may be measured using methods commonly known in the art. In preferred embodiments, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L or about 250 mOsm / L to about 320 mOsm / L. In other aspects, a composition herein may have an osmolality ranging from about 100 mOsm / kg to about 1000 mOsm / kg, from about 200 mOsm / kg to about 800 mOsm / kg, from about 250 mOsm / kg to about 500 mOsm / kg, or from about 250 mOsm / kg to about 320 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg or from about 280 mOsm / kg to about 320 mOsm / kg. In some embodiments, a pharmaceutical composition described herein has an osmolarity of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320mOsm / L, or about 280 mOsm / L to about 320 mOsm / L. In still other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more tonicity modifiers by total weight of the composition.Dosage formulations

[0238] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially trans-nasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as, intravenous, intraperitoneal, intranasal injections, intraocular (e.g., via eye drops) or topical (e.g., creams or ointments).

[0239] One may administer the pharmaceutical composition in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some embodiments, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra- cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, a pharmaceutical composition disclosed herein can administered to the human patient via at least two administration routes. In some examples, the combination of administration routes by be intracerebroventricular injection and intravenous injection; intrathecal injection and intravenous injection; intra-cisterna magna injection and intravenous injection; and intra-parenchymal injection and intravenous injection.

[0240] Pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0241] Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0242] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

[0243] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally,an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0244] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0245] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0246] Pharmaceutical compositions suitable for use in context of the present disclosure include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients (i.e., a compound disclosed herein) effective to prevent, slow, alleviate or ameliorate symptoms of a disorder (e.g., a pain disorder, a psychiatric disorder, or other neurological / cognitive disorder brought about by disrupted synaptic activity) or prolong the survival of the subject being treated.

[0247] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0248] For any preparation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0249] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).

[0250] Dosage amount and interval may be adjusted individually to brain or blood levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0251] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Effective doses may be extrapolated from dose- responsive curves derived from in vitro or in vivo test systems.V. Kits

[0252] The present disclosure provides kits for use in treating or alleviating a target condition, such as neuropathic pain, as described herein. In some embodiments, the kit can include instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of a composition containing a compound (e.g., EPH-EPHRIN tetramerization inhibitor such as a compound of Formula I or Formula II or any others described herein) disclosed herein and to treat, delay the onset, or alleviate a target disease as those described herein. The kit may further include a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease. In still other embodiments, the instructions can include a description of administering a compound to an individual at risk of the target disease.

[0253] The instructions relating to the use of a composition containing a compound inhibiting EPH-EPHRIN tetramerization (e.g., a compound of Formula I or Formula II or any others described herein) generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0254] The label or package insert indicates that the composition is used for treating, delaying the onset and / or alleviating the disease. Instructions may be provided for practicing any of the methods described herein.

[0255] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some examples, at least one active agent in the composition can be a compound (e.g., a CDK8 inhibitor) as described herein.

[0256] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.EXAMPLES

[0257] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0258] The publications discussed throughout are provided solely for their disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0259] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0260] Unless specified, reagents employed in the examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art. The examples illustrate various aspects of the invention and practice of the methods of the invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Thus, although the invention has been described in some detail by way of illustration and example for purposes of clarityof understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.Summary of Examples

[0261] The following Examples detail how it was discovered that EPH and EPHRIN proteins have a heretofore unknown copper-like binding property which enabled the discovery of additional compounds that inhibit tetramerization of the EPH-EPHRIN complex. These inhibitor compounds act to antagonize a previously unidentified copper chelating or copper binding-like property of the interacting EPH-EPHRIN signaling molecules which drives formation of the circular tetramer. Simply stated, the tetramer interface of EPH receptors contain a number of electropositive amino acids that we propose mimic copper and are recognized and bound by the cognate tetramer interface of EPHRIN ligands which share structural features of copper binding proteins (Cupredoxins) and we propose mimic metal chelators. Thus, the high affinity EPH-EPHRIN circular tetramer is formed through the EPHRIN proteinaceous chelator activity binding to the electropositive copper-like region of the EPH, in a tandem fashion, with two such precisely aligned dimers to lock in the high affinity circular macromolecular structure. This concept is graphically shown in FIG. 1A, with more details about the tetrameric and dimeric interface between EphrinB2 and EphB2 illustrated in FIG. 1 B. As schematized in FIG. 1C-1G, certain metal chelators were shown in the Examples below to disrupt formation of the EPH-EPHRIN circular tetramer. This new information paved the way forward to conceive a series of compounds that act as specific EPH-EPHRIN chelators to selectively disrupt the circular tetramer (FIG. 1H). As such, this schematic illustrates various small molecular weight compounds discussed herein in a search for inhibitors of the EPH-EPHRIN tetramer that may exhibit reduced general metal chelator activity but still retain potent ability to bind to the copper-like region of EPH molecules and compete with EPHRIN binding. These compounds are different from the earlier compounds summarized in FIG. 11 which are all 8-hydroxyquinoline (8-HQ) containing small molecules that likely have potent general metal chelator activity. The schematic shown in FIG. 11 shows earlier efforts at modifying the A20 molecule to improve tetramerization inhibition and is expanded upon herein. FIG. 1J shows a schematic of full length Eph and Ephrin proteins illustrating the different protein-protein interactions that EPH and EPHRIN molecules participate in on the cell surface, starting with initial interactions to form dimers, followed by recruitment of two dimers into a circular tetramer (which leads to tyrosine phosphorylation of both the EPH and EPHRIN proteins on intracellular residues and activation of bidirectional signaling), and then higher-order tetramer-tetramer interactions which leads to massive clustering of the molecules and supercharged cell-cell signaling.Compounds that interfere with formation of the EPH-EPHRIN circular tetramer are thus envisioned to exert a powerful brake on both forward and reverse cell signaling.Example 1

[0262] In a first set of Experiments, a biophysical protein-protein interaction assay using an Octet RED384 and as described in PCT / US2023 / 077841 (final application filed 10 / 26 / 23) (incorporated herein by reference in its entirety) was used to examine the EPH-EPHRIN ectodomain binding kinetics and measure the effect of selected chelators on the dimerization and tetramerization dynamics. As described extensively in PCT / US2023 / 07784 and shown in the below examples, in the absence of any small molecule chelator, the Eph and Ephrin ectodomain proteins exhibit complex, heterologous protein-protein interactions involving relatively low affinity fast-0 N / fast-OFF dimer formation followed by more slower formation and accumulation of the circular tetramer species, which is a very stable, high affinity macromolecular structure with low / sub-nanomolar dissociation constants (KD). As shown in FIG. 2A-2C, known copper chelators (clioquinol, EDTA, and EGTA) were all shown to inhibit tetramer formation when immobilized human EphrinB2 was exposed to 25 nM soluble human EphB1 , EphB2, or EphB4 ectodomains and did so as well as a gold standard (A20-I, as discussed in PCT / US2023 / 077841). Note that all of the copper chelators showed greatest ability to disrupt the EphrinB2-EphB2 interaction during the association step, followed by EphrinB2-EphB1 , and with very little effect on the EphrinB2-EphB4 interaction, which is primarily dimer-driven (as discussed in PCT / US2023 / 077841). We then focused on the EphrinB2-EphB2 interaction to determine the half-maximal inhibitory concentration (IC50) of selected metal chelators (FIG. 2D-2G). While 8-HQ itself showed only weak ability to interfere with the tetramer (IC50 = 4.7 pM), A20-I and clioquinol which both contain an 8-HQ ring structure were potent inhibitors with IC50 < 0.5 pM, indicating that the 8-HQ base chemical can be modified to enhance the desired tetramer antagonist activity. EDTA and EGTA exhibited even stronger ability to disrupt tetramer formation with IC50 values < 0.1 pM, consistent with this these chelators being hexadentate metal binders versus the relatively weaker 8-HQ compounds which are bidentate chelators. It was further found that pretreating the EPHB protein (FIG. 2H) or EPHRINB protein (FIG. 2I) with a selected chelator compound did not inhibit subsequent ability to form EPH-EPHRIN tetramers. This shows the compound needs to be present with the EPH and EPHRIN proteins during the kinetic binding assay to markedly inhibit tetramerization.

[0263] We then thought to test whether copper or other transition metals could interfere with EPH-EPHRIN tetramerization. As shown in FIG. 3A-3F it was surprisingly found that copper itself (i.e. , CuSO4) could inhibit tetramerization in a dose dependent manner and did so better than the gold standard (A20-I), though at much higher concentrations (up to 100pM). It was further noted that, unlike the A20 / clioquinol 8-HQ-containing compounds or EDTA / EGTA chelators, copper again at high concentrations was also able to strongly increase the EPH-EPHRIN dimer interaction, as the Octet sensorgram traces with copper included showed an initial much higher slope of dimer binding within the first 10 seconds of the association step. This effect of copper on dimerization and tetramerization binding kinetics was observed for all three interactions analyzed, EphrinB2-EphB1 , EphrinB2-EphB2, and EphrinB2-EphB4. The ability of copper to alter both dimer and tetramer binding kinetics is consistent with the distant evolutionarily ancestry of the EPHRIN extracellular domain to known copper binding proteins, including the Cupredoxins. While no one has previously shown direct binding of copper to EPHRIN proteins, our data shows copper has a strong ability to influence EPH-EPHRIN binding kinetics, presumably by interacting with the EPHRIN ectodomain and thereby modifying its ability to bind EPH molecules. Other transition metals were also tested to see if they would have an effect on EPH-EPHRIN binding dynamics, including cobalt, nickel, magnesium+calcium, zinc, and iron (FIG. 3G-3U). It was found that some metals did not affect the interactions (cobalt, nickel, magnesium+calcium) while others had a very strong inhibitory effect similar to copper (zinc and iron).

[0264] To this end, a series of experiments were undertaken to determine whether certain EPH-EPHRIN tetramerization inhibitors (identified by HTS and ongoing medchem efforts) are also copper chelators. These experiments are shown in FIGS. 4-7 and indicate all 8-HQ- containing chemicals tested as expected are potent chelators (8-HQ, Nitroxoline, PBT-1033, A20, A20-I, and 3511-1) as is EDTA. Curiously, novel compound 3511-1 may exhibit somewhat weaker copper chelator activity compared to the other 8-HQ-containing chemicals assessed, even though this is a very potent inhibitor of the EPH-EPHRIN tetramer. This suggests potent EPH-EPHRIN tetramer inhibitors that also show reduced general metal chelator activity are possible.

[0265] The above data leads to the novel idea that the extracellular regions of EPH proteins involved in tetramerization look like copper and the extracellular domain of cognate EPHRIN proteins look like they bind copper and act as special proteinaceous chelators to bind the electropositive copper-like region of the receptor and this drives the interaction to form the high-affinity circular tetramer (FIG. 1A). If EPH looks like copper, then it could be predicted that 8-HQ-containing small molecules like the A20 chemicals (or other metal chelators like EDTA or EGTA) act to chelate this copper-like region of EPH and will directly bind to the receptor protein to cage the copper-like region and in doing so block / prevent / compete for the ability of the EPHRIN to bind and form tetramers. Furthermore, given that the EphrinB2- EphB2 interaction is most strongly tetramer-driven, followed by the EphrinB2-EphB1 interaction, and with the EphrinB2-EphB4 interaction mainly dimer-driven (see PCT / US2023 / 077841 for more details), it could be predicted that small molecule chelatorswill bind most strongly to EphB2, followed by EphB1 , and with EphB4 exhibiting only weak binding. To address these ideas, a novel 8-HQ compound was synthesized that contains a primary amine (1°) which will enable its coupling to AR2G Octet biosensor chips and be used for biophysical measurements to study for binding to various EPH proteins (FIG. 8A). The primary amine compound was first tested to determine if it can inhibit the EphrinB2-EphB1 and EphrinB2-EphB1 tetramers, with data indicating it has weak, but detectable activity with IC50 of 2-4 pM similar to 8-HQ base chemical (FIG. 8B). Primary amine compound was then coupled / immobilized to AR2G biosensor chips and then 300 nM of soluble human EphB1 , EphB2, EphB4, and EphrinB2 ectodomains were passed over the chips for association and dissociation steps. This revealed that as predicted the EphB2 ectodomain showed strong binding to primary amine compound with simple 1:1 dynamics, followed by somewhat less binding of EphB1 , and no binding of either EphB4 or EphrinB2 (FIG. 9A). In an additional test, increasing concentrations of soluble human EphB2 ectodomain from 12.5 to 400 nM resulted in a dose-dependent increase in binding response to the immobilized primary amine compound (FIG. 9B). This data show EphB2 and EphB1 do indeed look like copper and are directly bound by small molecule chelators. Scrutiny of EPH receptor ectodomain amino acid sequences shows the two loops F’-G and K-L that converge to the same region of the tetramer interface are both electropositive to provide a cluster of positively charged surface- exposed residues, with the prototype EphB2 having three arginines and EphB1 with two (FIG. 9C-9D). It is noteworthy that the EphB4 receptor, which exhibits a more dimer-driven interaction with EphrinB2, is less electropositive here with a lysine replacing the arginine in the K-L loop and the F’-G loop having a proline residue which likely introduces a kink that reduces this protein’s ability to tetramerize. The EphA receptors as a group are slightly different in these loops, with the F’-G look retaining the conserved though buried arginine present in most Eph receptors (Arg 88 in EphB2) but not the exposed Arg 87 and 89 residues as in EphB2. The K-L loop of all the EphA receptors with conserved positioning of two electropositive arginine / lysine residues, however, is more electropositive than the EphB group receptors. Nevertheless, with both A and B group tetramer interactions, there appears to be a conserved electropositive feature of these two loops that (1) are normally recognized by the proteinaceous Ephrin “chelators” to assist in the formation of circular tetramers, and (2) can also be recognized by small molecular weight chelator chemicals like A20 which will bind to the Eph molecule and and act as an antagonist to compete away the ability of Ephrins to bind and form tetramers.Example 2

[0266] In additional analysis, we re-examined the data / results from our original HTS that led to discovery of the A20 lead chemical (see PCT / US2023 / 077841 for more details). In 2017, approximately 240,000 chemicals were screened in high-throughput to identify thosethat could disrupt the EphrinB2-EphB1 protein-protein interaction using AlphaScreen technology. A total of 1 ,246 primary hits were initially identified that reduced the Alpha signal >10%, in a really tight assay with Z’ value of approximately 0.95. Ultimately, after secondary and tertiary screenings, only one single compound was identified, the lead chemical was called A20. Interestingly, upon going back and looking more carefully at these 1,246 initial hits, it was realized that 77 of them were chemicals that contained either an intact 8-HQ ring structure or a modified 8-HQ structure (see below). This information was eye opening and indicated 6% of the top 1 ,246 primary hits were 8-HQs. As a total of 1 ,084 chemicals in the library screened contain 8-HQ (<0.5% of the library), it became obvious that a disproportionate number of the primary hits in our HTS were indeed this type of metal chelator. It was decided to revisit the top hits from the HTS, hoping to identify additional small molecule inhibitors missed in our original AlphaScreen analysis as the more powerful Octet system was now available to relatively easily assess their effect on the EPH-EPHRIN interaction (FIG. 10A).

[0267] The selected 245 primary hits from the original HTS were cherry-picked at the UT Southwestern HTS core and provided at 320 pM in DMSO. All these cherry-picked hits were first assessed at 3.2 pM in the Octet for ability to disrupt the binding of immobilized human EB2-Fc to 25 nM soluble human EphB1-His protein (FIG. 10B-10F). Initial rankings for tetramer inhibitor activity are: Rank 3 = very strong inhibitor activity below A20-I Max control, Rank 2 = strong tetramer inhibitors at A20-I Max control, Rank 1 = low tetramer inhibitor activity at 1 / 2 A20-I Max control, Rank 0 = no tetramer inhibitor activity. Strong compounds that ranked 3 or 2 were then tested at 0.5 pM for both inhibition of the EB2-EphB1 and EB2- EphB2 interactions, and many were selected for IC50 determination using 0.1 - 3.2 pM concentrations. Potential interesting but weak compounds were also tested at higher concentrations, 3.2, 6.4, 9.6, and 19.2 pM. Tables 1-12 below show structures, IC50 values, and additional data / information for the various chemicals and transition metals studied herein.

[0268] There were three classes of interesting compounds identified:

[0269] A) 8-HQ compounds

[0270] 67 of the 245 HTS hits assessed contain an intact 8-HQ ring structure, with 25 of them ranked inhibitor 3 (including A20), 37 ranked inhibitor 2, and the remaining 5 with inhibitor rankings of 1 or 0 (FIG. 11). This demonstrates how robust our HTS screen of 240,000 chemicals actually was, and how powerful 8-HQ chemicals are at interfering with EPH-EPHRIN tetramerization. An example of one particularly potent new 8-HQ chemical is compound termed G15 (SW160569) which is a sulfur-containing 8-HQ that shows strong ability to target EphrinB2-EphB1 and EphrinB2-EphB2 tetramers (FIG. 12A-12C).

[0271] B) 8-HQ-like compounds

[0272] 10 of the 245 HTS hits assessed are similar to an 8-HQ, described as 8-HQ-like, but they do not have the OH group (it is “protected”) and thus may exhibit weak or no metal chelator activity. Of these 8-HQ-like compounds, P08 (SW036148), exhibited strong ability to inhibit formation of EphrinB2-EphB1 and EphrinB2-EphB2 tetramers with IC50 values of approximately 0.5 pM (FIG. 13A-13C). Interestingly, when immobilized EphrinBI or EphrinB3 ectodomains were tested for binding to soluble EphB2, compound P08 exhibited even more potent ability to inhibit EphrinB1-EphB2 and EphrinB3-EphB2 tetramers with IC50 values of 50-100 nM (FIG. 13D-13F). P08 looks very interesting as its OH group is protected by a cyclic ring structure (see below), and it may not exhibit strong general metal chelator activity (FIG. 13G).

[0273] C) Non-8-HQ compounds.

[0274] The remaining 168 cherry-picked primary hits are not related to 8-HQ. Of these 168 hits, one interesting new compound was identified, L08 (SW117057), that exhibited very strong ability to inhibit formation of EphrinB2-EphB1 and EphrinB2-EphB2 tetramers and forms a new pharmacophore unrelated to A20 chemicals (FIG. 14A-14F). Additional studies indicate L08 is also a strong copper binding molecule (FIG. 14G-14H), and so is another class of metal chelator that can affect EPH-EPHRIN tetramerization.

[0275] Table 1. IC50 determination of 8-HQ and 8-HQ protected compounds from the original HTS screen.Table 2. IC50 determination of G15-based 8-HQ and 8-HQ protected compounds.Table 3. IC50 determination of P08 protected compounds.Table 4. IC50 determination of carbamate protected compounds.Table 5. IC50 determination of ester protected compounds.Table 6a. IC50 determination of ether protected compounds.Table 6b. IC50 determination of ether compounds.Table 7. IC50 determination of primary amine compounds.Table 8. IC50 determination of store bought 8-HQ’s.Table 9. IC50 determination of FDA approved drugs.Table 10. IC50 determination of store bought chelators.Table 11. Effect of transition metals on EPH-EPHRIN dimer and tetramer formation.Table 12. IC50 determination of store-bought L08 free base and salts generated herein.Example 3

[0276] Our re-evaluation of the HTS screen revealed the existence of “protected” 8-HQ-like chemicals in the library that retains ability to disrupt the EPH-EPHRIN tetramer, but may have lost general metal chelator activity, such as the compound P08 described above. We further identified four other P08-like cyclic protected 8-HQ-like compounds in the top 1 ,246 hits from the original AlphaScreen HTS. Small amounts of these four compounds were obtained from the HTS core and Octet analysis revealed that they too have decent tetramer inhibitor activity with IC50 values <2 pM (FIG. 15A-15B). Additional available P08-like chemicals were also obtained and tested in Octet, revealing they too exhibit IC50 values -0.5-1 pM (FIG. 15C).

[0277] FIG. 16 shows the general plan for making additional “protected” A20 related chemicals that target the key oxygen in the 8-HQ ring structure as schematized (FIG. 16). Inaddition to P08-like cyclic protected compounds (option 1 , FIG. 17), other modifications to the oxygen include protecting it with an ether linkage (option 2, FIG. 18), ester linkage (option 3, FIG. 19), or carbamate linkage (option 4, FIG. 20). Using available compounds and those synthesized in house, we demonstrate that ether modifications (FIG. 21), ester modifications (FIG. 22), and carbamate modifications (FIG. 23) can all lead to chemicals capable of inhibiting the EPH-EPHRIN tetramer.Example 4

[0278] In another set of experiments, selected EPH and EPHRIN proteins were studied for their endogenous expression levels and activation status in vivo using newly developed and highly sensitive immunoprecipitation and western blot detection methods of protein lysates obtained from various tissues / organs and following various treatment conditions with tetramer inhibitor compounds. These studies focus on the EphB2, EphrinBI, and EphrinB2 proteins as excellent antibody reagents are available which are necessary to obtain strong convincing data from endogenous freshly collected tissues. The first point here is to show we can accurately determine both the total amount / level of EphB2, EphrinBI , and EphrinB2 protein that is expressed in any one tissue / sample (tEphB2, tEphrinBI , and tEphrinB2), and we can determine their respective activation state / level which is measured by assessing how many of the molecules are tyrosine phosphorylated (pEphB2, pEphrinBI , and pEphrinB2) and by quantifying the percentage of each protein that is in a phosphorylated state. Determining the percentage of active tyrosine phosphorylated protein is done by calculating ratios of pEphB2 / tEphB2, pEphrinB1 / tEphrinB1 , and pEphrinB2 / tEphrinB2 from these experiments. The second point here is to demonstrate that administration of tetramer inhibitors in vivo can significantly reduce the level of pEphB2, pEphrinBI, and pEphrinB2 in target tissues. These data demonstrate that tetramerization inhibitor compounds described herein when dosed into an animal are able to reduce the tyrosine phosphorylation and activation of both EPH-mediated forward signaling and EPHRIN-mediated reverse signaling. These data are shown in FIGS. 24-28, 33 (focus on EphB2), FIGS. 29-31 (focus on EphrinBI), and FIGS. 34-35 (focus on EphrinB2).Methods

[0279] In general, key aspects of the methods utilized here are detailed in the figures and accompanying legends, with additional information in PCT / US2023 / 077841. Chemical synthesis methods are as follows:

[0282] Synthesis of Exemplary Compounds

[0283] The following A20-related compounds were synthesized according to schematics depicted above. Additional related compounds could be envisioned by slight modifications of the methods described herein. A method for generating a salt of one of these compounds is depicted A20-Br-OMe and is described further below. Salts of other compounds synthesized below can be easily envisioned based on the methods above.

[0284] Compound A20-Br-OMe (4-((7-bromo-8-methoxyquinolin-5- yl)methyl)morpholine, A20-Br-OMe)

[0286] Synthetic Procedure for compound A20-Br-OMe: In a 25 mL round bottom flask, A20 (124.4 mg, 0.3849 mmol) was dissolved in acetone (15 mL). Then methyl iodide (Mel) (218.5 mg, 1.5396 mmol) and potassium carbonate (K2CO3) (106.2 mg, 0.7698 mmol) were added to the solution and the reaction mixture was refluxed for 2-4 hours. After cooling to room temperature, the acetone was removed, and the crude product was purified bycolumn chromatography (petroleum ether : ethyl acetate = 4: 1). Obtained A20-Br-OMe as an oil (89.6 mg, 69%).

[0287] Compound A20-Br-OMe-2HCI (4-((7-bromo-8-methoxyquinolin-5- yl)methyl)morpholine hydrochloride)

[0289] Synthetic Procedure for compound A20-Br-OMe 2HCI: In a 25 mL round bottom flask, A20-Br-OMe (80.0 mg, 0.2372 mmol) was dissolved in dichloromethane (DCM) (3 mL). After cooling down in an ice bath, 2M HCI in diethyl ether (0.24 mL, 0.4757 mmol) was added dropwise to the solution. A pale-yellow solid was immediately formed. The reaction mixture was stirred for 2-4 hrs, and then was filtered and washed with large amount of dichloromethane (DCM). The product was dried under vacuum and obtained as a white solid (92.4 mg, 95%).

[0290] Compound A20-l-OMe (4-((7-iodo-8-methoxyquinolin-5-yl)methyl)morpholine, A20-l-OMe)

[0291]

[0292] Synthetic Procedure for A20-l-OMe: In a 25 mL round bottom flask, A20-I (100.0 mg, 0.2701 mmol) was dissolved in acetone (15 mL). Then Mel (153.4 mg, 1.081 mmol) and K2CO3 (74.5 mg, 0.5402 mmol) were added to the solution and the reaction mixture was refluxed for 2-4 hours. After cooling down to room temperature, the acetone was removed, and the crude product was purified by column chromatography (hexane : ethyl acetate = 2:1). Obtained A20-l-OMe as an oil (63.3 mg, 61%).

[0293] Compound A20-l-OMe-2HCI (4-((7-iodo-8-methoxyquinolin-5- yl)methyl)morpholine hydrochloride)

[0294]

[0295] Synthetic Procedure for A20-l-OMe 2HCI: In a 25 mL round bottom flask, A20-I-OMe (50.0 mg, 0.1301 mmol) was dissolved in DCM (3 mL). After cooling down in an ice bath, 2M HCI in diethyl ether (0.13 mL, 0.2603 mmol) was added dropwise to the solution.A pale-yellow solid was immediately formed. The reaction mixture was stirred for 2-4 hrs, and then was filtered and washed with large amount of DCM. The product was dried under vacuum and obtained as a white solid (50.9 mg, 93%).

[0296] Compound A20-I-OMP (4-((7-iodo-8-(pyridin-4-ylmethoxy)quinolin-5- yl)methyl)morpholine, A20-l-OMe)

[0297] Synthetic Procedure for A20-I-OMP: In a 25 mL round bottom flask, 4- (bromomethyl)pyridine hydrobromide (82.0 mg, 0.3242 mmol), K2CO3 (149.1 mg, 1.0804 mmol), and acetone (10 mL) were added. The mixture was stirred at room temperature for 15-30 min, and then A20-I (100.0 mg, 0.2701 mmol) was added. The reaction mixture was reflux for 2-4 hours. After cooling down to room temperature, the acetone was removed, and the crude product was purified by column chromatography (dichloromethane : methanol = 20:1). Obtained A20-I-OMP as a pale-yellow solid (104.7 mg, 84%).

[0298] Compound A20-I-OMP-3HCI (4-((7-iodo-8-(pyridin-4-ylmethoxy)quinolin-5- yl)methyl)morpholine hydrochloride, A20-l-OMe-3HCI)

[0299] Synthetic Procedure A20-l-OMe-3HCI: In a 25 mL round bottom flask, A20-I- OMP (80.3 mg, 0.1741 mmol) was dissolved in dichloromethane (DCM) (10 mL). After cooling down in an ice bath, 2M HCI in diethyl ether (0.26 mL, 0.5222 mmol) was added dropwise to the solution. A pale-yellow solid was immediately formed. The reaction mixture was stirred for 2-4 hrs, and then was filtered and washed with large amount of dichloromethane (DCM). The product was dried under vacuum and obtained as a white solid (91.4 mg, 92%).

[0300] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present inventive concept. Additionally, a number of well- known processes and elements have not been described in order to avoid unnecessarily obscuring the present inventive concept. Accordingly, this description should not be taken as limiting the scope of the present inventive concept.

[0301] Those skilled in the art will appreciate that the presently disclosed embodiments teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.

Claims

CLAIMS1. A compound according to Formula (lib):or a pharmaceutically appropriate salt thereof, wherein:R32 is selected from the group consisting of:wherein n is 0-3;A1 is CH or O;B is N or CH;Bi is NH or CH2;R28 is alkyl or aryl;R29 is hydrogen, alkyl, or alkoxy;R30 is hydrogen, alkyl, or alkoxy,R33 is selected from:wherein A2is O, SO2, CH2, or NR31 , andR31 is a substituted or unsubstituted aryl.

2. The compound of claim 1 , or a pharmaceutically appropriate salt thereof, wherein:B is N.

3. The compound of claim 1 , or a pharmaceutically appropriate salt thereof, wherein:B is CH.

4. The compound of claim 1 , or a pharmaceutically appropriate salt thereof, wherein:R29 is methyl or methoxy.

5. The compound of claim 1 , or a pharmaceutically appropriate salt thereof, wherein:R30 is hydrogen, methyl, or methoxy.

6. The compound of claim 1 , or a pharmaceutically appropriate salt thereof, wherein:

8. The compound according to claim 1 , or a pharmaceutically appropriate salt thereof, wherein: the compound is selected from:or a pharmaceutically appropriate salt thereof.A compound according to Formula l-F, lor a pharmaceutically appropriate salt thereof, wherein:wherein n is 0-3;Ai is CH or O;B is N or CH;Bi is NH or CH2;R28 is hydrogen, alkyl or aryl;R29 is hydrogen, alkyl, or alkoxy;R30 is hydrogen, alkyl, or alkoxy;R33 is selected from:whereinA2is O, SO2, CH2, or NR31, andR31 is a substituted or unsubstituted aryl; andX is H, NO2, or a halogen.

10. The compound of claim 9, or a pharmaceutically appropriate salt thereof, wherein:B is N.

11. The compound of claim 9, or a pharmaceutically appropriate salt thereof, wherein:B is CH.

12. The method compound of claim 9, or a pharmaceutically appropriate salt thereof, wherein:R2g is methyl or methoxy.

13. The compound of claim 9, or a pharmaceutically appropriate salt thereof, wherein:R30 is hydrogen, methyl or methoxy.

14. The compound of claim 9, or a pharmaceutically appropriate salt thereof, wherein:X is Br, I, or Cl.

15. The compound of claim 9, or a pharmaceutically appropriate salt thereof, wherein:R32is selected from: butyl, tert-butyl,16. The compound of claim 9, or a pharmaceutically appropriate salt thereof, wherein:R34 is hydrogen.

17. The compound of claim 9, or a pharmaceutically appropriate salt thereof, wherein:the compound is selected from:or a pharmaceutically appropriate salt thereof.

18. A method of inhibiting formation of an EPH-EPHRIN tetramer, the method comprising: contacting an EPH or EPH RIN with an EPH-EPHRIN tetramerization inhibitor that antagonizes formation of an EPH-EPHRIN tetramer by binding to a copper-like region of an EPH protein to prevent / compete with the copper-chelating-like or copper-binding-like activity of the EPHRIN protein.

19. The method of claim 18, wherein the EPH-EPHRIN tetramer comprises EphB1 , EphB2, EphrinBI , EphrinB2, EphrinB3, or EphrinA5 or any combination thereof.

20. The method of any one of claims 18 to 19, wherein formation of the EPH-EPHRIN tetramer is inhibited in vivo.

21. A method of alleviating or relieving pain in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

22. The method of claim 21 , wherein the pain comprises chronic neuropathic pain.

23. A method of treating a synaptopathy in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

24. The method of claim 23, wherein the synaptopathy comprises abnormal, defective, or excessive EPH-EPHRIN expression and / or signaling and optionally comprises disrupted NMDA receptor signaling.

25. The method of claim 23 or 24, wherein the synaptopathy is associated with anxiety or epilepsy.

26. A method of treating addiction or opioid dependency in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

27. A method of treating a fibrotic and / or an inflammatory disease or condition in a subject in need thereof, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

28. The method of claim 27, wherein the fibrotic and / or inflammatory disease or condition comprises abnormal, defective, or excessive EPH-EPHRIN expression and / or signaling and optionally comprises NASH liver fibrosis, chronic kidney disease, scleroderma, systemic sclerosis, heart fibrosis, lung fibrosis, inflammation and / or fibrosis of another organ, neuroinflammation, and / or abnormal wound healing optionally selected from keloids, hypertrophic scarring, and / or heterotopic ossification.

29. A method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

30. The method of claim 29, wherein the cancer comprises abnormal, defective, or excessive EPH-EPHRIN expression and / or signaling and optionally comprises GBM (glioblastoma), pancreatic cancer, and / or colon cancer.

31. A method of treating a viral infection in a subject in need thereof, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

32. The method of claim 31 , wherein the viral infection comprises abnormal, defective, or excessive EPH-EPHRIN expression and / or signaling and optionally comprises an infection by henipavirus and / or human immunodeficiency virus (HIV). In an aspect, the tetramerization inhibitor is further envisioned to disrupt viral recognition of receptor molecules expressed on a cell and / or impede viral entry into a cell to reduce / block infection.

33. A method of treating a metabolic disease in a subject in need thereof, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject, optionally wherein the metabolic disease comprises diabetes, obesity or a cardiovascular pathology associated with a metabolic disease.

34. The method of any one of claims 21 to 33, comprising administering a pharmaceutical composition comprising the EPH-EPHRIN tetramerization inhibitor and one or more pharmaceutical excipients to the subject.

35. The method of claim 34, wherein the pharmaceutical composition is administered intravenously, subcutaneously, intraperitoneally, orally, and / or topically.

36. The method of any one of claims 22 to 35, wherein the subject is a human, a livestock animal, a companion animal, a lab animal, or a zoological animal.

37. The method of any one of claims 18 to 36, wherein the EPH-EPHRIN tetramerization inhibitor comprises an 8-hydroxyquinoline or 8-hydroxyquinoline-like compound of Formula Ior a pharmaceutically appropriate salt thereof, wherein:R is hydrogen, an alkyl, a substituted or unsubstituted alkyl aryl, a substituted or unsubstituted alkyl heteroaryl, a substituted or unsubstituted carbonyl, or a substituted or unsubstituted amide, andeach R1to R6is independently hydrogen, a halo, a nitro, an alkyl, an ester, a substituted fused- SO2N(Rl5)(Rl6), wherein: A is -O-, SO2, NR17, or CRis.R7to R12 are each independently hydrogen, a nitro, a halo, a substituted or unsubstituted carbonyl, a substituted or unsubstituted ketone, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted fused heterocycloalkyl, a substituted or unsubstituted fused heteroaryl, a substituted or unsubstituted fused cycloalkyl, a substituted or unsubstituted fused aryl, a substituted or unsubstituted alkyl aryl, or a substituted or unsubstituted alkyl cycloalkyl; one of R13 and R14 is hydrogen and the other is a nitro, a substituted or unsubstituted carbonyl, a substituted or unsubstituted ketone, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted fused heterocycloalkyl, a substituted or unsubstituted fused heteroaryl, a substituted or unsubstituted fused cycloalkyl, a substituted or unsubstituted fused aryl, a substituted or unsubstituted alkyl aryl, a substituted or unsubstituted alkyl cycloalkyl, or R13 and R14 are each a substituted or unsubstituted alkyl that together form a heterocyloalkyl, wherein the heterocycloalkyl is substituted or unsubstituted;R15 and R-ie are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, a substituted or unsubstituted alkyl aryl, a substituted or unsubstituted alkyl cycloalkyl, a substituted or unsubstituted fused heterocycloalkyl, a substituted or unsubstituted fused heteroaryl, or R15 and RI6are each a substituted or unsubstituted alkyl that together form a substitute or unsubstituted heterocyloalkyl; andR17 or Ris are each independently hydrogen, a substituted or unsubstituted aryl, a substituted or unsubstituted alkyl aryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted alkyl cycloalkyl; and at least one of R, R1, R2, R3, R4, Rs, and Re is not hydrogen.

38. The method of claim 37, wherein R is hydrogen, a C1-C4 alkyl, a substituted or unsubstituted alkylaryl,or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted alkylaryl, a substituted or unsubstituted alkyl heteroaryl, and wherein each R20and R21 is independently hydrogen, alkyl, a substituted or unsubstituted aryl, or wherein each R2o and R21are a substituted or unsubstituted alkyl so that together form a heterocyloalkyl, wherein the heterocycloalkyl is substituted or unsubstituted, with the proviso that at least one of R20and R21is not hydrogen.

39. The method of claim 37 or 38, wherein R is hydrogen.

40. The method of claim 39, wherein the EPH-EPHRIN inhibitor comprises a structure of Formula l-A or l-B:wherein R1, R3, Rs, R12, R13 and R14 are as previously defined.

41. The method of claim 37 or 38, wherein R is a C1-C4 alkyl, a substituted or unsubstituted alkylaryl,42. The method of claim 40 or 41 , wherein the EPH-EPHRIN inhibitor comprises a structure of Formula l-C:wherein Ri, R3, R4, Rs and R6are as previously defined and R is a C1-C4 alkyl, a substituted or unsubstituted alkylaryl,43. The method of claim 42, wherein the EPH-EPHRIN inhibitor comprises a structure of Formula l-C-a, l-C-b, or l-C-c:

44. The method of claim 37 or 38, wherein R isand R19 is as previously defined.

45. The method of claim 44, wherein the EPH-EPHRIN tetramerization inhibitor comprises a compound of Formula l-D:, wherein R1, R3, and R6are as previously defined and R19is selected from the group consisting of methyl, ethyl, substituted or unsubstituted phenyl, or a substituted or unsubstituted benzyl, wherein the phenyl and benzyl are each optionally substituted with 1 to 3 substituents selected from an alkoxy, a nitro, a halogen, or an alkyl.

46. The method of claim 37 or 38 whereindefined.

47. The method of claim 46, wherein the EPH-EPHRIN tetramerization inhibitor comprises a compound of Formula l-E:, wherein Ri and R3 are as defined previously and R20 and R21 are each independently selected from hydrogen, a C1 to C4 alkyl, a substituted or unsubstituted phenyl, or wherein R2o and R21 together with the nitrogen form a morpholine ring, with the proviso that at least one R20 and R21 is not hydrogen.

48. The method of any one of claims 37 to 47, wherein R7, Rs and R9are each independently hydrogen or an unsubstituted aryl.

49. The method of any one of claims 37 to 48, wherein R7, Rs and R9are each independently hydrogen or phenyl.

50. The method of any one of claims 37 to 49, wherein Rw is nitro or a halo.

51. The method of any one of claims 37 to 50, wherein R11is an alkyl, a substituted or unsubstituted aryl or substituted or unsubstituted alkylaryl.

52. The method of any one of claims 37 to 51 , wherein R11is methyl, ethyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, wherein the phenyl and benzyl are each optionally substituted with a halogen, an alkoxy, or an alkyl.

53. The method of any one of claims 37 to 52, wherein each of R12, R13, and RI4is independently hydrogen, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted fused heteroaryl, a substituted or unsubstituted fused heterocycloalkyl, or a substituted or unsubstituted carbonyl.

54. The method of any one of claims 37 to 53, wherein each of R12, R13, and RI4is hydrogen, a substitute or unsubstituted phenyl, a substituted or unsubstituted pyridinyl, a substituted or unsubstituted furanyl, a substituted or unsubstituted thiophenyl, a benzo[1,3]dioxole, a C1-C4 unsubstituted ketone, or a C1 to C4 aryl substituted ketone.

55. The method of any one of claims 37 to 54, wherein each of R12, R13, and RI4is independently: hydrogen, an alkyl,and wherein R22, R23, R24, and R25are each independently hydrogen, a halo, an alkyl, a nitro, a substituted or unsubstituted alkoxy, an amino, an ester, a hydroxy, or a thioether.

56. The method of claim 55, wherein R22, R23, R24, and R25 are each independently hydrogen, a methyl, an ethyl, an isopropyl, a chloro, a bromo, a fluoro, a nitro, -OH, - N(CH3)2, - SCH3,, - COOCH3 and - OCH(R26)(R27), and wherein R26 and R27 are each independently hydrogen, a C1- C3 alkyl, a C1-C3 alkenyl, a halo, or an aryl.

57. The method of any one of claims 37 to 56, wherein R13 and R14 together form an unsubstituted piperidine ring.

58. The method of any one of claims 37 to 57, wherein R15 and R16 are each independently hydrogen, an alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted fused heterocycloaryl.

59. The method of any one of claims 37 to 58, wherein R15 and R16 are each independently hydrogen, a C1 to C6 alkyl, benzyl, an unsubstituted cycloheptane,60. The method of any one of claims 37 to 59, wherein R15 and R16 together form a substituted piperazine ring.

61. The method of claim 60, wherein the piperazine ring is substituted with a substituted aryl.

62. The method of claim 61 , wherein the substituted aryl comprises a methoxy substituted phenyl.

63. The method of any one of claims 37 to 62, wherein R1 to Re are each independently hydrogen, a bromo, a chloro, an iodo, a methyl, an ethyl, -OCH3, -NO2,64. The method of any one of claims 37 to 63, wherein R3is selected from65. The method of any one of claims 37 to 64, wherein R is selected from hydrogen,,and wherein n is 0-3, Ai is H or , B is N or H, Bi is NH or H2, R28 is alkyl or aryl, R29 is hydrogen, alkyl, or alkoxy, R30 is hydrogen, an alkyl, or an alkoxy.

66. The method of claim 65, wherein R3is selected fromA2 is O, SO2, CH2, or NR31, and R31 is a substituted or unsubstituted aryl.

67. The method of claim 65 or 66, wherein Ri is H, nitro, or a halogen.

68. The method of claim 65 or 67, wherein the EPH-EPHRIN tetramerization inhibitor is selected from a compound of Formula l-F, l-G, or l-H:, whereinR32 and R34are each independently selected from hydrogen,wherein n is 0-3, A1 is CH or O, B is N or CH, B1is NH or CH2, R28 is alkyl or aryl, R29is hydrogen, alkyl, or or alkoxy, R30 is hydrogen, an alkyl, or an alkoxy;R33 is selected from:wherein A2is O, SO2, CH2, or NR31, and R31 is a substituted or unsubstituted aryl; and X is H, NO2, or a halogen.

69. The method of any one of claims 65 or 68, wherein B is N.

70. The method of any one of claims 65 to 69, wherein R29 is methyl or methoxy.

71. The method of any one of claims 68 to 70, wherein R32 is selected from: butyl, tert-butyl,72. The method of any one of claims 68 to 71 , wherein R34 is hydrogen.

73. The method of any one of claims 37 to 72, wherein R2, R4and R5are each hydrogen.

74. The method of any one of claims 37 to 73, wherein the compound of Formula I is selected from the group consisting of:

75. The method of any one of claims 18-36, wherein the EPH-EPHRIN tetramerization inhibitor is an 8-hydroxyquinoline-like compound of:Formulaor a pharmaceutically appropriate salt thereof, wherein R32 is C3-C6 cycloalkyl, C1-C6unsubstituted alkyl, or C1-C6substituted alkyl.

76. The method of any one of claims 18-36, wherein the EPH-EPHRIN tetramerization inhibitor is an 8-hydroxyquinoline-like compound of:or a pharmaceutically appropriate salt thereof, wherein:wherein n is 0-3, Ai is CH or O, B is N or CH, Bi is NH or CH2, R28 is alkyl or aryl, R29 is hydrogen, alkyl, or alkoxy, R30 is hydrogen, an alkyl, or an alkoxy,R33 is selected from:, wherein A2is O, SO2, CH2, or NR31, and R31 is a substituted or unsubstituted aryl.

77. The method of claim 76, wherein B is N.

78. The method of claim 76 or 77, wherein R29 is methyl or methoxy.

79. The method of any one of claims 77 to 78, wherein R32 is selected from: butyl, tert-butyl,80. The method of any one of claims 76 to 79, wherein81. The method of claim 80, wherein the compound is selected from the group consisting of:

82. The method of any one of claims 76 to 79, wherein R33 is83. The method of claim 82, wherein A2is O, SO2or NR31.

84. The method of claim 82, wherein R31 is a substituted phenyl.

85. The method of claim 83, wherein R31 is a halo or alkoxy substituted phenyl.

86. The method of any one of claims 76 to 79, wherein R33 is87. The method of claim 86, wherein the compound is selected from the group consisting of:

89. The method of claim 88, wherein the compound is selected from:

90. The method of any one of claims 76 to 79, wherein R33 is91. The method of claim 90, wherein the compound is selected from the group consisting of:

92. The method of any one of claim 76 to 79, wherein93. The method of claim 92, wherein the compound is selected from the group consisting of:

94. The method of any one of claims 18 to 36, wherein the EPH-EPHRIN tetramerization95. The method of any one of claims 18 to 36, wherein the EPH-EPHRIN tetramerization inhibitor is:pharmaceutically appropriate salt thereof.

96. The method of any one of claims 18 to 36, wherein the EPH-EPHRIN tetramerization inhibitor is selected from:pharmaceutically appropriate salt thereof.

97. The method of any one of claims 18 to 36, wherein the EPH-EPHRIN tetramerization inhibitor is a copper chelator comprising:

98. The method of any one of claims 18 to 36, wherein the EPH-EPHRIN tetramerization inhibitor is a transition metal selected from C11SO4, FeSO4, ZnSO4, NiSO4, COSO4, CuCI2, ZnCl2, NiCl2, CoCI2, and MgCl2, and CaCl2.

99. The method of any one of claims 18 to 76, wherein the EPH-EPHRIN tetramerization inhibitor is selected from the group consisting of:(a)and any pharmaceutically appropriate salt thereof;, and any pharmaceutically appropriate salt thereof;

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