Patent Application
20230368862
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 7, 2022, is named 44010111US-PAT.xml and is 14,741 bytes in size.
The ryanodine receptor (RyR) is required for excitation-contraction coupling. Although RyR is tightly regulated, inherited mutations and stress-induced post-translational modifications can result in a Ca2+ leak in skeletal myopathies, heart failure, and exercise-induced sudden death. Compounds known as Rycals® repair the leaky RyR and are effective in preventing and treating disease symptoms and restoring normal RyR function.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
In some embodiments, the present disclosure provides a composition comprising a complex suspended in a solid medium, the solid medium comprising vitreous ice, wherein the complex comprises a protein and a synthetic compound, wherein the protein is a ryanodine receptor 2 protein (RyR2) or a mutant thereof.
In some embodiments, the synthetic compound is
or an ionized form thereof.
In some embodiments, the protein is a mutant RyR2, for example a RyR2 protein containing at least one mutation that is associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the RyR2 protein is post-translationally-modified RyR2 protein, for example a RyR2 protein containing at least one post-translational modification selected from phosphorylation, oxidation and nitrosylation. In some embodiments, the post-translationally modified RyR2 protein is associated with heart failure. In some embodiments, the RyR2 protein is a mutated and post-translationally modified RyR2 protein.
In some embodiments, the present disclosure provides a method for predicting a docked position of a target ligand in a binding site of a biomolecule, the method comprising:
In some embodiments, the present disclosure provides a method of identifying a plurality of potential lead compounds, the method comprising the steps of:
In some embodiments, the present disclosure provides a computer-implemented method of quantifying binding affinity between a ligand and a receptor molecule, the method comprising:
In some embodiments, the present disclosure provides a method comprising:
In some embodiments, the present disclosure provides a method comprising:
In some embodiments, the present disclosure provides a method of identifying a compound having RyR2 modulatory activity, the method comprising:
In some embodiments, the present disclosure provides a method for identifying a compound having RyR2 modulatory activity, comprising:
In some embodiments, the present disclosure provides a method for identifying a compound that preferentially binds to a mutated, post-translationally modified RyR2 or a combination thereof, comprising:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Located on the sarco/endoplasmic reticulum (SR/ER) membrane, the ryanodine receptor (RyR) is the largest known ion channel, at over two megadaltons, and is the primary mediator of the Ca2+ release required for excitation-contraction coupling in cardiac and skeletal muscle. RyR is required for excitation-contraction coupling. RyR1 is the primary isoform in skeletal muscle while RyR2 is the predominant cardiac isoform. RyR1 and RyR2 are also found in neurons. RyR3 is present where RyR1 and RyR2 are each present, but with significantly lower expression levels. Beyond their expression pattern, RyR1 and RyR2 are unique in how each is activated. In skeletal muscle, RyR1 is activated by the direct, mechanical interaction with the dihydropyridine receptor (DHPR). RyR2 is instead activated by Ca2+ in the process termed calcium-induced calcium release (CICR) in which Ca2+ binding to RyR2 creates a cascade effect as the release of Ca2+ through the RyR creates a high local concentration of Ca2+, which can cause neighboring RyR channels to open. RyR, a tetramer, forms tetrads in muscle tissue and under normal conditions, undergoes cooperative activation through the process termed coupled gating.
The correct activation of RyR, and thus activation of the appropriate downstream Ca2+ signaling pathways, is regulated by multiple ligands and protein interactions. Aside from Ca2+, ATP, and caffeine, RyR also binds calmodulin (CaM). CaM is an inhibitor of ryanodine receptor type 2 (RyR2). CaM can act as either an activator of ryanodine receptor type 1 (RyR1) under low Ca2+ conditions (˜150 nM), such as those at rest, or an inhibitor of RyR1 under high Ca2+ conditions (>1 μM). High Ca2+ conditions occur locally following intracellular Ca2+ release. Calstabin, a second accessory protein, also binds the RyR. This interaction stabilizes the closed state of the channel. In disease states, RyR can be nitrosylated, oxidized and/or phosphorylated to cause calstabin to dissociate from the channel. This dissociation results in Ca2+ leaking into the cytosol and inappropriate triggering of downstream Ca2+ signaling pathways.
RyR comprises three major segments, each composed of several domains. The first, the cytosolic shell, consists of the N-terminal domain (NTD) with two segments (A & B) and an N-terminal solenoid, three SPRY domains, two RYR domains (RY1&2 and RY3&4), and the junctional and bridging solenoids (J-Sol and Br-Sol). The cytosolic shell also houses the calstabin binding site, which binds in a pocket formed by the Br-Sol and the SPRY domains, specifically SPRY1, and calmodulin, which binds on the other side of the Br-Sol from calstabin, with the N-terminal domain of CaM binding along the face of the Br-Sol while the C-terminal domain binds a peptide within a pocket of the Br-Sol.
Although RyR is tightly regulated, inherited mutations and stress-induced post-translational modifications (e.g., phosphorylation, nitrosylation and oxidation) can result in a Ca2+ leak. As a key player in Ca2+ signaling, leaky RyR channels are associated with a wide variety of disease states including skeletal muscle myopathies such as RyR-related myopathy (RYR-RM), dystrophies such as muscular dystrophy (e.g., Duchenne Muscular Dystrophy), cardiac diseases such as heart failure and catecholaminergic polymorphic ventricular tachycardia (CPVT), diabetes, and neurological disorders such as post-traumatic stress disorders (PTSD) and Alzheimer's disease.
Compounds known as ryanodine receptor modulators (also known as Rycals®) can repair leaky RyR and are effective in preventing and treating disease symptoms and restoring normal RyR function. Ryanodine receptor modulators can have efficacy in a host of diseases, both in vitro and in vivo using animal models. Ryanodine receptor modulators can repair the Ca2+ leak by preferentially binding to leaky RyR compared to normal RyR, and causing reassociation of calstabin, thus restabilizing the closed state of the channel. Mutations in RyR have been linked to rare genetic forms of cardiac and skeletal muscle disorders and ryanodine receptor modulators be effective in animal models in these disorders.
Given the structure of several ryanodine receptor modulator compounds, which contain aromatics and charged groups, ryanodine receptor modulators were initially hypothesized to bind near the caffeine binding site based on early cryo-electron microscopy (cryo-EM) structures with limited resolution. Advances in cryo-EM, and particularly direct detection cameras and novel processing methods including local refinement, have dramatically improved the resolution of cryo-EM maps, allowing unambiguous identification of ligand binding sites, including identification of a novel ATP binding site as described herein, and binding sites for Ca2+, and caffeine.
In some embodiments, the present disclosure utilizes cryo-EM techniques to generate a high resolution model of RyR2. In some embodiments, a high resolution model of RyR2 includes a ryanodine receptor modulator (e.g., Compound 1) bound to a ryanodine receptor modulator binding site in the RY1&2 domain of RyR2. In some embodiments, a ryanodine receptor modulator compound binds cooperatively with ATP and stabilizes the closed state of RyR2.
Ca2+, ATP, and xanthine are known to bind within the C-terminal domain (CTD) of RyR2. Disclosed herein is the identification of an additional ATP-binding site, in the periphery of the cytosolic shell of the RyR, in the RY1&2 domain that is comprised within the SPRY domain. In some embodiments, this region is also the ryanodine receptor modulator (Rycal) binding site. As demonstrated herein, ryanodine receptor modulator binding to RyR2 can increase in the presence of ATP. In some embodiments, Compound 1 binds in the RY1&2 domain cooperatively with ATP and stabilizes the closed state of the RyR2 channel despite the presence of activating ligands (Ca2+, ATP, and xanthine).
The present disclosure relates to methods and compositions useful for the identification of a binding site for ryanodine receptor modulators (Rycals) in ryanodine receptor type 2 (RyR2). The present disclosure also provides compositions useful for the analysis of the ryanodine receptor modulator binding site in RyR2 via cryo-EM. The present disclosure further provides methods (e.g., computational methods) for identifying compounds that bind to RyR2. The present disclosure further provides methods for screening for compounds that bind to RyR2 by utilizing a cryo-EM model of RyR2.
Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures and embedded in an environment of vitreous water. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in a cryogenic fluid such as liquid ethane or a mixture of liquid ethane and propane.
The structures of the disclosure can be determined using cryo-EM with a sample frozen at a temperature of from about −40° C. to about −280° C. In some embodiments, the cryo-EM used for structural determination uses a sample frozen at a temperature of from about −40° C. to about −100° C., from about −100° C., to about −150° C., from about −150° C. to about −175° C., from about −175° C. to about −200° C., from about −200° C. to about −225° C., from about −225° C. to about −250° C., or from about −250° C. to about −280° C. In some embodiments, the cryo-EM used for structural determination uses a sample frozen at a temperature of from about −40° C. to about −100° C. In some embodiments, the cryo-EM used for structural determination uses a sample frozen at a temperature of from about −150° C. to about −175° C. In some embodiments, the cryo-EM used for structural determination uses a sample frozen at a temperature of from about −175° C. to about −200° C. In some embodiments, the cryo-EM used for structural determination uses a sample frozen at a temperature of from about −250° C. to about −280° C. In some embodiments, the cryo-EM used for structural determination uses a sample frozen at a temperature of about −150° C., about −175° C., about −200° C., about −250° C., or about −280° C. In some embodiments, the cryo-EM used for structural determination uses a sample frozen at a temperature of about −175° C. In some embodiments, the cryo-EM used for structural determination uses a sample frozen at a temperature of about −200° C. In some embodiments, the cryo-EM used for structural determination uses a sample frozen in liquid nitrogen. In some embodiments, the cryo-EM used for structural determination uses a sample frozen in liquid helium. In some embodiments, the cryo-EM used for structural determination uses a sample frozen in liquid ethane. In some embodiments, the cryo-EM used for structural determination uses a sample frozen in liquid propane. In some embodiments, the cryo-EM used for structural determination uses a sample frozen in mixture of liquid nitrogen and liquid propane.
The structures of the disclosure can be determined using a protein concentration of from about 50 nM to about 5 μM. In some embodiments, a structure of the disclosure can be determined using a protein concentration of from about 50 nM to about 250 nM, from about 250 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 μM, or from about 1 μM to about 5 μM. In some embodiments, a structure of the disclosure can be determined using a protein concentration of from about 50 nM to about 250 nM. In some embodiments, a structure of the disclosure can be determined using a protein concentration of from about 250 nM to about 500 nM. In some embodiments, a structure of the disclosure can be determined using a protein concentration of from about 500 nM to about 750 nM. In some embodiments, a structure of the disclosure can be determined using a protein concentration of from about 750 nM to about 1 μM. In some embodiments, a structure of the disclosure can be determined using a protein concentration of from about 1 μM to about 5 μM.
The structures of the disclosure can be determined using a sample solution with a pH of about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, the sample solution has a pH of about 7.0. In some embodiments, the sample solution has a pH of about 7.1. In some embodiments, the sample solution has a pH of about 7.2. In some embodiments, the sample solution has a pH of about 7.3. In some embodiments, the sample solution has a pH of about 7.4. In some embodiments, the sample solution has a pH of about 7.5.
The structures of the disclosure (e.g., compositions comprising RyR2 and a ryanodine receptor modulator such as compound 1 bound to a ryanodine receptor modulator binding site on RyR2, and optionally an ATP molecule bound to an ATP binding site on the RyR2) can be determined at a resolution of from about 15 Å to about 2 Å. In some embodiments, the structures of the disclosure can be determined at a resolution of from about 15 Å to about 12 Å, from about 12 Å to about 9 Å, from about 9 Å to about 6 Å, from about 6 Å to about 5 Å, from about 5 Å to about 4 Å, from about 4 Å to about 3 Å, or from about 3 Å to about 2 Å. In some embodiments, the structures of the disclosure can be determined at a resolution of about 2.45 Å. In some embodiments, the structures of the disclosure is determined at a resolution of about 3.1 Å. In some embodiments, the structures of the disclosure is determined at a resolution from about 2 Å to about 3.5 Å, from about 2 Å to about 3.4 Å, from about 2 Å to about 3.3 Å, from about 2 Å to about 3.2 Å, from about 2 Å to about 3.1 Å, from about 2 Å to about 3 Å, from about 2 Å to about 2.9 Å, from about 2 Å to about 2.8 Å, from about 2 Å to about 2.7 Å, from about 2 Å to about 2.6 Å, from about 2 Å to about 2.5 Å, from about 2.1 Å to about 2.5 Å, from about 2.2 Å to about 2.5 Å, from about 2.3 Å to about 2.5 Å, or from about 2.4 Å to about 2.5 Å.
In some embodiments, the present disclosure provides compositions useful for the determination of the ryanodine receptor modulator binding site in RyR2 via methods such as cryo-EM. In some embodiments, the present disclosure provides a composition comprising a complex suspended in a solid medium, wherein the complex comprises a biomolecule (e.g., a protein) and a synthetic compound, wherein the protein is a ryanodine receptor 2 protein (RyR2) or a mutant thereof.
In some embodiments, the present disclosure provides a composition comprising a complex suspended in a solid medium, the solid medium is or comprises a cryo-electron microscopy grid, wherein the complex comprises a biomolecule (e.g., a protein) and a synthetic compound, wherein the protein is a ryanodine receptor 2 protein (RyR2) or a mutant thereof.
In some embodiments, the present disclosure provides a composition comprising a complex suspended in a non-biological solid medium, wherein the complex comprises a biomolecule (e.g., a protein) and a synthetic compound, wherein the protein is a ryanodine receptor 2 protein (RyR2) or a mutant thereof, optionally with one or more proteins associated with RyR2.
In some embodiments, the composition is prepared by a process, the process comprising vitrifying an aqueous solution that is applied to an electron microscopy grid, wherein the aqueous solution comprises the protein and the synthetic compound.
An electron microscopy grid is a support structure used to insert specimens, for example, for use in an electron microscope. The grid structures can be flat with various suitable materials (e.g., copper, gold, rhodium, nickel, molybdenum, ceramic, etc.) for the grids themselves. In some cases, the grid structure can have plating (e.g., rhodium), coating (e.g., carbon, gold, plastic, silicon nitride, etc.), a suitable thickness (e.g., from 20 to 50 micron), and a suitable diameter (e.g., 3 mm). The grid structures generally have crossing bars and spacings/holes between the bars (e.g., nanometer to micrometer scale holes). The bars can come in various suitable sizes or pitch, patterns (e.g., regular or irregular), and shapes (e.g., numbers or letters built into the grid bars).
In some embodiments, prior to the vitrifying, the aqueous solution is applied to the electron microscopy grid, and excess aqueous solution is removed from the electron microscopy grid by blotting the excess aqueous solution.
In some embodiments, the aqueous solution is dispensed onto the electron microscopy grid from a dispensing apparatus located on the side of the electron microscopy grid opposed to the side abutting blotting material. Once the liquid sample is dispensed onto the cryo-EM grid, the blotting material can pull excess solution through the electron microscopy grid to produce a thin liquid film of the aqueous solution on the electron microscopy grid.
In some embodiments, the vitrifying comprises plunge freezing the aqueous solution applied to the electron microscopy grid into liquid ethane chilled with liquid nitrogen.
In some embodiments, the aqueous solution further comprises a buffering agent. Suitable buffering agents can include, for example, zwitterionic amines, such as TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid), Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid), Tris (2-amino-2-(hydroxymethyl)propane-1,3-diol), and Tricine (N-[tris(hydroxymethyl)methyl]glycine), as well as zwitterionic sulfonic acids, such as TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)), and MES (2-(N-morpholino)ethanesulfonic acid). In some embodiments, the buffering agent is HEPES. In some embodiments, the buffering agent is EGTA.
In some embodiments, the aqueous solution further comprises a phospholipid. In some embodiments, the phospholipid is a phosphatidylcholine, such as, for example, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the phospholipid is a phosphatidylserine, such as, for example, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS), or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS). In some embodiments, the phospholipid is DOPS.
In some embodiments, the aqueous solution further comprises a surfactant. Surfactants can be used in a composition disclosed herein to increase the solubility of a protein (e.g. RyR2). In some embodiments, the surfactant is a zwitterionic surfactant, such as, for example, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO). In some embodiments, the zwitterionic surfactant is CHAPS.
In some embodiments, the aqueous solution further comprises a disulfide-reducing agent, which can be, for example, tris (2-carboxyethyl) phosphine hydrochloride (TCEP), beta-mercaptoethanol (BME), tributylphosphine (TBP). or dithiothreitol (DTT). In some embodiments, the disulfide-reducing agent is TCEP.
In some embodiments, the aqueous solution further comprises a protease inhibitor. Suitable protease inhibitors can include, for example, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), phenylmethylsulfonyl fluoride (PMSF), leupeptin, N-ethylmaleimide, antipain, pepstatin, alpha 2-macro-globulin, EDTA, bestatin, amastatin, and benzamidine. In some embodiments, the protease inhibitor is AEBSF. In some embodiments, the protease inhibitor is benzamidine hydrochloride.
In some embodiments, the aqueous solution further comprises xanthine. The concentration of xanthine in the aqueous solution can be, for example, about 1 mM to about 1000 μM, about 1 μM to about 900 μM, about 1 μM to about 800 μM, about 1 μM to about 700 μM, about 1 μM to about 600 μM, about 100 μM to about 1000 μM, about 100 μM to about 900 μM, about 100 μM to about 800 μM, about 100 μM to about 700 μM, about 100 μM to about 600 μM, about 200 μM to about 1000 μM, about 200 μM to about 900 μM, about 200 μM to about 800 μM, about 200 μM to about 700 μM, or about 200 μM to about 600 μM. In some embodiments, caffeine is present at a concentration of from about 200 μM to about 600 μM.
In some embodiments, xanthine is present at a concentration of about 100 μM, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM, or about 1000 μM. In some embodiments, caffeine is present at a concentration of about 500 μM.
In some embodiments, the aqueous solution further comprises dissolved Ca2+. The concentration of dissolved Ca2+ in the aqueous solution can be, for example, about 1 μM to about 400 μM, about 1 μM to about 350 μM, about 1 μM to about 300 μM, about 1 μM to about 250 μM, about 1 μM to about 200 μM, about 50 μM to about 400 μM, about 50 μM to about 350 μM, about 50 μM to about 300 μM, about 50 μM to about 250 μM, or about 50 μM to about 200 μM. In some embodiments, dissolved Ca2+ is present at a concentration from about 50 μM to about 250 μM. In some embodiments, dissolved Ca2+ is present at a concentration from about 100 μM to about 200 μM.
In some embodiments, dissolved Ca2+ is present at a concentration of about 100 μM, about 110 μM, about 120 μM, about 130 μM, about 140 μM, about 150 μM, about 160 μM, about 170 μM, about 180 μM, about 190 μM, or about 200 μM. In some embodiments, dissolved Ca2+ is present at a concentration of about 150 μM.
The concentration of the protein in the aqueous solution can be, for example about 1 mg/mL to about 20 mg/mL, 2 mg/mL to about 20 mg/mL, 3 mg/mL to about 20 mg/mL, 4 mg/mL to about 20 mg/mL, 4 mg/mL to about 20 mg/mL, 1 mg/mL to about 15 mg/mL, 2 mg/mL to about 15 mg/mL, 3 mg/mL to about 15 mg/mL, 4 mg/mL to about 15 mg/mL, 1 mg/mL to about 10 mg/mL, 2 mg/mL to about 10 mg/mL, or 3 mg/mL to about 10 mg/mL. In some embodiments, the protein is present in the aqueous solution at a concentration from about 1 mg/mL to about 15 mg/mL. In some embodiments, the protein is present in the aqueous solution at a concentration from about 1 mg/mL to about 10 mg/mL. In some embodiments, the protein is present in the aqueous solution at a concentration from about 4 mg/mL to about 8 mg/mL.
In some embodiments, the protein is present in the aqueous solution at a concentration of about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, about 13 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, or about 20 mg/mL.
In some embodiments, the aqueous solution further comprises sodium adenosine triphosphate (NaATP). The concentration of NaATP in the aqueous solution can be, for example, about 1 mM to about 15 mM, about 1 mM to about 50 mM, about 1 mM to about 30 mM, about 1 mM to about 30 mM, about 2 mM to about 30 mM, about 3 mM to about 30 mM, about 4 mM to about 30 mM, about 5 mM to about 30 mM, about 6 mM to about 30 mM, about 7 mM to about 30 mM, about 8 mM to about 30 mM, about 9 mM to about 30 mM, about 10 mM to about 30 mM, 1 mM to about 15 mM, about 2 mM to about 15 mM, about 3 mM to about 15 mM, about 4 mM to about 15 mM, or about 5 mM to about 15 mM. In some embodiments, NaATP is present in the aqueous solution at a concentration from about 3 mM to about 15 nM.
In some embodiments, NaATP is present in the aqueous solution at a concentration of about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of NaATP is about 10 mM.
In some embodiments, the aqueous solution further comprises cAMP (cyclic adenosine monophosphate). The concentration of cAMP in the aqueous solution can be, for example, about 1 μM to about 400 μM, about 1 μM to about 350 μM, about 1 μM to about 300 μM, about 1 μM to about 250 μM, about 1 μM to about 200 μM, about 50 μM to about 400 μM, about 50 μM to about 350 μM, about 50 μM to about 300 μM, or about 50 μM to about 250 μM. In some embodiments, cAMP is present the aqueous solution at a concentration from about 100 μM to about 300 μM. In some embodiments, cAMP is present at the aqueous solution a concentration from about 150 μM to about 250 μM.
In some embodiments, the aqueous solution is substantially free of cellular membrane. Prior to adding protein to the solution, the protein can be separated from cellular membranes by homogenization of cells containing the protein, and subjecting the resulting homogenate to chromatography.
In some embodiments, the aqueous solution further comprises calmodulin. In some embodiments, the calmodulin is human calmodulin. In some embodiments, the calmodulin has a sequence according to SEQ ID NO: 1.
The aqueous solution can be prepared or stored in a vessel. In some embodiments, the vessel is a vial, ampule, test tube, or microwell plate.
In some embodiments, the complex further comprises a nucleoside-containing molecule. In some embodiments, the nucleoside-containing molecule is a purine nucleoside-containing molecule. In some embodiments, the nucleoside-containing molecule is a nucleotide or nucleoside polyphosphate. In some embodiments, the nucleoside-containing molecule is an adenosine triphosphate (ATP) molecule.
In some embodiments, the nucleoside-containing molecule and the synthetic compound bind a RYR domain of the protein. In some embodiments, the RYR domain is a RY1&2 domain. In some embodiments, the RY1&2 domain has a three-dimensional structure according to TABLE 3. In some embodiments, the synthetic compound has a three-dimensional conformation according to TABLE 4. In some embodiments, the ATP molecule has a three-dimensional conformation according to TABLE 5. In some embodiments, the ATP molecule binds the protein and the synthetic compound. In some embodiments, the synthetic compound binds cooperatively with the ATP molecule in the RY 1&2 domain of RyR2. In some embodiments, the synthetic compound is a ryanodine receptor modulator, e.g., Compound 1.
In some embodiments, the complex further comprises a second ATP molecule, wherein both ATP molecules bind a common RYR domain of the protein.
In some embodiments, the complex further comprises a second binding site for a nucleoside-containing molecule. In some embodiments, the complex further comprises a second nucleoside-containing molecule. In some embodiments, the second nucleoside-containing molecule binds a C-terminal domain of the RyR2 protein. In some embodiments, the second nucleoside-containing molecule is a nucleotide or nucleoside polyphosphate. In some embodiments, the second nucleoside-containing molecule is a second ATP molecule.
In some embodiments, the complex further comprises calmodulin. In some embodiments, the calmodulin is human calmodulin.
In some embodiments, the complex further comprises calstabin (i.e., peptidyl-prolyl cis-trans isomerase FKBP1B). In some embodiments, the calstabin is human calstabin. In some embodiments, the calstabin has a sequence according to SEQ ID NO: 2.
In some embodiments, the RyR2 protein is in a resting (closed) state. In some embodiments, the RyR2 protein is in the primed state. In some embodiments, a primed state comprises a higher distribution of open probability (Po) as compared to a RyR2 in a resting (closed) state. In some embodiments, a primed state RyR2 comprises about 30% to about 60% of the RyR channel in an open state. In some embodiments, a primed state RyR2 comprises about 30%, about 35%, about 40%, about 45%, about 50%, about 55% or about 60% of the RyR channel in an open state.
In some embodiments, the complex further comprises a xanthine alkaloid molecule. In some embodiments, the complex further comprises a xanthine molecule, such as, for example, theobromine, theophylline, caffeine, or xanthine. In some embodiments, the complex further comprises a Ca2+ ion.
In some embodiments, the solid medium comprises vitreous ice. In some embodiments, the solid medium is substantially free of crystalline ice.
In some embodiments, the composition is substantially free of cellular membrane. In some embodiments, the RyR2 is a purified RyR2. In some embodiments, the RyR2 is a semi-purified RyR2 that is substantially free of cellular membrane.
In some embodiments, the composition further comprises additional complexes, wherein each of the additional complexes independently comprises the protein and the synthetic compound. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the RyR2 protein in the additional complexes is in a closed state. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the additional complexes is in an open state.
In some embodiments, the RyR2 protein is in a phosphorylated state, wherein the phosphorylated state is prepared by a process comprising contacting RyR2 protein with a phosphorylation reagent. In some embodiments, the phosphorylation reagent comprises protein kinase A. In some embodiments, the phosphorylation reagent further comprises ATP. In some embodiments, the phosphorylation reagent further comprises MgCl2.
In some embodiments, the protein is in a dephosphorylated state, wherein the dephosphorylated state is prepared by a process comprising contacting RyR2 protein with a dephosphorylation reagent. In some embodiments, the dephosphorylation reagent comprises phosphatase lambda. In some embodiments, the dephosphorylation reagent further comprises MnCl2.
In some embodiments, the synthetic compound binds a RYR domain of the protein. In some embodiments, the RYR domain is a RY1&2 domain.
In some embodiments, the protein is wild type RyR2. In some embodiments, the protein is mutant RyR2. In some embodiments, the mutant RyR2 is R2474S RyR2. In some embodiments, the protein is human RyR2. In some embodiments, the protein is a tetramer of RyR2 monomers, wherein each RyR2 monomer is SEQ ID NO: 3. In some embodiments, the protein is a tetramer of RyR2 monomers, wherein each RyR2 monomer is SEQ ID NO: 4. In some embodiments, the RyR2 protein is C4-symmetrical. In some embodiments, the protein comprises four RY1&2 domains, each with a three-dimensional conformation according to TABLE 3.
In some embodiments, the RyR2 protein is in a closed state. In some embodiments, the RyR2 protein is in an open state. In some embodiments, the RyR2 protein is in a primed state, wherein the RyR2 in the primed state has an open probability (Po) that is higher an open probability (Po) of the RyR2 in a closed state, and an open probability (Po) that is lower than an open probability (Po) of the RyR2 protein in an open state. In some embodiments, the protein is wild type RyR2. In some embodiments, the protein is a mutant RyR2. In some embodiments, the mutant RyR2 contains at least one mutation that is associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the mutation is R2474S. In some embodiments, the mutation is RyR2-R420Q. In some embodiments, the mutation is RyR2-R420W.
In some embodiments, the protein is a post-translationally modified RyR2 protein. In some embodiments, the post-translationally modified RyR2 protein is a phosphorylated RyR2 protein. In some embodiments, the post-translationally modified RyR2 is an oxidized RyR2 protein. In some embodiments, the post-translationally modified RyR2 is a nitrosylated RyR2. In some embodiments, the post-translationally modified RyR2 protein is associated with a cardiac disease. In some embodiments, the post-translationally modified RyR2 protein is associated with heart failure. In some embodiments, the post-translationally modified RyR2 protein is associated with a cardiac arrhythmia. In some embodiments, the RyR2 is a mutated and post-translationally modified RyR2
In some embodiments, the mutation destabilizes an interaction between NTD and BSol domains of the RyR2 protein. In some embodiments, the mutation destabilizes a cytosolic shell of the RyR2 protein, wherein the cytosolic shell comprises NTD, SPRY, JSol and BSol domains of the RyR2 proteins.
The relative difference in conformational states between a structure (e.g., a protein structure or protein domain structure) and a reference structure (e.g., a reference protein structure or a reference protein domain structure) can be quantified by calculating the average distance between atoms of the structure and the reference structure when the structure and reference structure are superimposed. A nonlimiting example of a measure of a difference in conformational states between a structure and a reference structure is root mean square deviation of atomic positions (RMSD), which can be defined as:
where δi is the distance between atom i and an analogous atom of a reference structure, or the mean position of the N equivalent atoms.
Root mean square deviation of atomic positions (RMSD) of a protein or protein domain structure relative to a reference protein or protein domain structure can be calculated computationally via software such as, for example, UCSF ChimeraX, SuperPose. LGA (Local-Global Alignment), or PDBeFold. In some embodiments, RMSD is calculated on the basis of Ca atomic coordinates of the structure and reference structure.
In some embodiments, the present disclosure provides a composition comprising a complex suspended in a solid medium, wherein the complex comprises a protein. In some embodiments, the present disclosure provides a composition comprising a complex suspended in vitreous ice, wherein the complex comprises a protein. In some embodiments, the protein is a ryanodine receptor protein or a mutant thereof. In some embodiments, the protein is a ryanodine receptor 2 protein (RyR2) or a mutant thereof. The composition can be, for example, a composition suitable for analysis via single-particle cryogenic electron microscopy, where the solid medium is an amorphous material such as vitreous ice.
In some embodiments, the composition is analyzed in a study, wherein the study comprises: (i) determining a structure of the protein (e.g., RyR2 or a mutant thereof) or a domain of the protein by subjecting the complex to single particle cryogenic electron microscopy analysis; and (ii) calculating RMSD of the protein or domain of the protein relative to a reference structure. In some embodiments, the protein is a RyR2 mutant (e.g, R2474S RyR2, RyR2-R420Q, or RyR2-R420W), and the reference structure is a structure of a wild type RyR2 protein. In some embodiments, the complex further comprises a synthetic compound (e.g., a ryanodine receptor channel modulator).
In some embodiments, the reference structure is obtained by a process comprising analyzing a reference composition via single particle cryogenic electron microscopy, wherein the reference composition comprises a reference complex suspended in a solid medium, wherein the reference complex comprises a reference protein. In some embodiments, the reference composition is prepared by a process comprising vitrifying a reference aqueous solution applied to an electron microscopy grid, wherein the reference aqueous solution comprises the reference complex. The reference aqueous solution can be identical to the aqueous solution used to prepare the composition, except that (a) the reference aqueous solution comprises the reference protein in place of the protein, and/or (b) the aqueous solution comprises a synthetic compound, the reference aqueous solution comprises calmodulin, the reference aqueous solution does not comprise the synthetic compound, and the aqueous solution does not comprise calmodulin. In some embodiments, the reference structure is a structure according to Protein Data Bank entry 7U9Q.
In some embodiments, the protein is R2474S RyR2, wherein if a study is conducted, the study comprising:
In some embodiments, the protein is R2474S RyR2, wherein if a study is conducted, the study comprising:
In some embodiments, the protein is R2474S RyR2, wherein if a study is conducted, the study comprising:
In some embodiments, the protein is R2474S RyR2, wherein if a study is conducted, the study comprising:
In some embodiments, the protein is R2474S RyR2, wherein if a study is conducted, the study comprising:
In some embodiments, the protein is R2474S RyR2, wherein if a study is conducted, the study comprising:
In some embodiments, the protein is R2474S RyR2, wherein if a study is conducted, the study comprising:
In some embodiments, the protein is R2474S RyR2, wherein if a study is conducted, the study comprising:
In some embodiments, the reference protein is the wild type RyR2 protein. In some embodiments, the protein and the reference protein are each independently in a phosphorylated state. In some embodiments, the phosphorylated state of the protein or the reference protein is prepared by a process comprising contacting RyR2 protein or a mutant thereof with a phosphorylation reagent. In some embodiments, the phosphorylation reagent is protein kinase A. In some embodiments, the protein and the reference protein are each independently in a dephosphorylated state. In some embodiments, the dephosphorylated state of the protein or the reference protein is prepared by a process comprising contacting RyR2 protein or a mutant thereof with a dephosphorylation reagent. In some embodiments, the dephosphorylation reagent comprises phosphatase lambda.
In some embodiments, the protein and the reference protein are each independently in an oxidized state. In some embodiments, the protein and the reference protein are each independently in a nitrosylated state.
The synthetic compound in the compositions described herein can be a ryanodine receptor modulator compound, such as a benzothiazepane derivative. Some benzothiazepine compounds are voltage-gated Ca2+ channel blockers, but ryanodine receptor modulator compounds can be free of any channel blocking activity. The inability of certain ryanodine receptor modulator compounds to block Ca2+ channels can be associated with the mechanism of stabilizing the closed state of the RyR without inhibiting the channel. In some embodiments, a ryanodine receptor modulator compounds are modulators of the RyR channel. In some embodiments, ryanodine receptor modulator compounds are allosteric modulators of the RyR channel.
Ryanodine receptor modulator compounds of the disclosure can be used as therapeutics because in some disease states, RyR leaks Ca2+ due to destabilization of the closed state of the channel after post-translational modifications such as nitrosylation, oxidation and phosphorylation. In other disease states, Ca2+ leak is present due to inherited mutations. The genetic mutations can predispose the RyR channel to post-translational modifications such as oxidation and nitrosylation, further exacerbating the leak. These mutations and post-translational modifications cause the stabilizing subunit, calstabin, to dissociate from the channel, increasing the open probability of the channel, resulting in Ca2+ leak. In disease models involving leaky RyR in cells, animals, and patients, treatment with a ryanodine receptor modulator compound can reverse the leak and restore calstabin binding.
In some embodiments, the synthetic compound comprises a benzazepane or benzothiazepane (e.g., 2,3,4,5-tetrahydro-1,4-benzothiazepine) moiety. In some embodiments, the synthetic compound comprises a benzothiazepane moiety. In some embodiments, the synthetic compound comprises a benzothiazepine moiety. In some embodiments, the synthetic compound comprises a 1,4-benzothiazepine moiety.
The term “alkyl” as used herein refers to a linear or branched, saturated hydrocarbon having from 1 to 6 carbon atoms. Representative alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. The term “C1-C4 alkyl” refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and isobutyl.
The term “alkenyl” as used herein refers to a linear or branched hydrocarbon having from 2 to 6 carbon atoms and having at least one carbon-carbon double bond. In one embodiment, the alkenyl has one or two double bonds. The alkenyl moiety may exist in the E or Z conformation and the compounds of the present invention include both conformations.
The term “alkynyl” as used herein refers to a linear or branched hydrocarbon having from 2 to 6 carbon atoms and having at least one carbon-carbon triple bond.
The term “aryl” as used herein refers to an aromatic group containing 1 to 3 aromatic rings, either fused or linked.
The term “cyclic group” as used herein includes a cycloalkyl group and a heterocyclic group.
The term “cycloalkyl” as used herein refers to a three- to seven-membered saturated or partially unsaturated carbon ring. Any suitable ring position of the cycloalkyl group may be covalently linked to the defined chemical structure. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
The term “halogen” as used herein refers to fluorine, chlorine, bromine, and iodine.
The term “heterocyclic group” or “heterocyclic” or “heterocyclyl” or “heterocyclo” as used herein refers to fully saturated, or partially or fully unsaturated, including aromatic (i.e., “heteroaryl”) cyclic groups (for example, 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 16 membered tricyclic ring systems) which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclic group may be attached to the remainder of the molecule at any heteroatom or carbon atom of the ring or ring system. Examples of heterocyclic groups include, but are not limited to, azepanyl, azetidinyl, aziridinyl, dioxolanyl, furanyl, furazanyl, homo piperazinyl, imidazolidinyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, piperazinyl, piperidinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiomorpholinyl, thiophenyl, triazinyl, and triazolyl. Examples of bicyclic heterocyclic groups include indolyl, isoindolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, benzofurazanyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), triazinylazepinyl, tetrahydroquinolinyl and the like. Examples of tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like.
The term “phenyl” as used herein refers to a substituted or unsubstituted phenyl group.
The aforementioned terms “alkyl,” “alkenyl,” “alkynyl,” “aryl,” “phenyl,” “cyclic group,” “cycloalkyl,” “heterocyclyl,” “heterocyclo,” and “heterocycle” can further be optionally substituted with one or more substituents. Examples of substituents include but are not limited to one or more of the following groups: hydrogen, halogen, CF3, OCF3, cyano, nitro, N3, oxo, cycloalkyl, alkenyl, alkynyl, heterocycle, aryl, alkylaryl, heteroaryl, ORa, SRa, S(═O)Re, S(═O)2Re, P(═O)2Re, S(═O)2ORa, P(═O)2ORa, NRbRc, NRbS(═O)2Re, NRbP(═O)2Re, S(═O)2NRbRc, P(═O)2NRbRc, C(═O)ORa, C(═O)Ra, C(═O)NRbRc, OC(═O)Ra, OC(═O)NRbRc, NR C(═O)ORa, NRdC(═O)NRbRc, NRdS(═O)2NRbRc, NRdP(═O)2NRbRc, NRbC(═O)Ra, or NRbP(═O)2Re, wherein Ra is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkylaryl, heteroaryl, heterocycle, or aryl; Rb, Rc and Rd are independently hydrogen, alkyl, cycloalkyl, alkylaryl, heteroaryl, heterocycle, aryl, or said Rb and Rc, together with the N to which Rb and Rc are bonded optionally form a heterocycle; and Rc is alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylaryl, heteroaryl, heterocycle, or aryl. In the aforementioned examples of substituents, groups such as alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, alkylaryl; heteroaryl, heterocycle and aryl can themselves be optionally substituted.
Example substituents can further optionally include at least one labeling group, such as a fluorescent, a bioluminescent, a chemiluminescent, a colorimetric and a radioactive labeling group. A fluorescent labeling group can be selected from bodipy, dansyl, fluorescein, rhodamine, Texas red, cyanine dyes, pyrene, coumarins, Cascade Blue™, Pacific Blue, Marina Blue, Oregon Green, 4′,6-Diamidino-2-phenylindole (DAPI), indopyra dyes, lucifer yellow, propidium iodide, porphyrins, arginine, and variants and derivatives thereof. For example, ARM118 of the present invention contains a labeling group BODIPY, which is a family of fluorophores based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene moiety. For further information on fluorescent label moieties and fluorescence techniques, see, e.g., Handbook of Fluorescent Probes and Research Chemicals, by Richard P. Haughland, Sixth Edition, Molecular Probes, (1996), which is hereby incorporated by reference in its entirety. One of skill in the art can readily select a suitable labeling group, and conjugate such a labeling group to any of the compounds of the invention, without undue experimentation.
The disclosure provides the use of pharmaceutically-acceptable salts of any compound described herein. Pharmaceutically-acceptable salts include, for example, acid-addition salts and base-addition salts. The acid that is added to the compound to form an acid-addition salt can be an organic acid or an inorganic acid. A base that is added to the compound to form a base-addition salt can be an organic base or an inorganic base. In some embodiments, a pharmaceutically-acceptable salt is a metal salt. In some embodiments, a pharmaceutically-acceptable salt is an ammonium salt.
Metal salts can arise from the addition of an inorganic base to a compound of the disclosure. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In some embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.
In some embodiments, a metal salt is a lithium salt, a sodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesium salt, a manganese salt, an iron salt, a calcium salt, a strontium salt, a cobalt salt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt, or a zinc salt.
Ammonium salts can arise from the addition of ammonia or an organic amine to a compound of the present disclosure. In some embodiments, the organic amine is triethyl amine, diisopropyl amine, ethanol amine, diethanol amine, triethanol amine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrazole, imidazole, or pyrazine.
In some embodiments, an ammonium salt is a triethyl amine salt, a trimethyl amine salt, a diisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, a triethanol amine salt, a morpholine salt, an N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt, an N-ethylpiperidine salt, a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrazole salt, a pyridazine salt, a pyrimidine salt, an imidazole salt, or a pyrazine salt.
Acid addition salts can arise from the addition of an acid to a compound of the present disclosure. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. In some embodiments, the acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, gentisic acid, gluconic acid, glucuronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, trifluoroacetic acid, mandelic acid, cinnamic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, propionic acid, butyric acid, fumaric acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid.
In some embodiments, the salt is a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactate salt, a salicylate salt, a tartrate salt, an ascorbate salt, a gentisate salt, a gluconate salt, a glucuronate salt, a saccharate salt, a formate salt, a benzoate salt, a glutamate salt, a pantothenate salt, an acetate salt, a trifluoroacetate salt, a mandelate salt, a cinnamate salt, an aspartate salt, a stearate salt, a palmitate salt, a glycolate salt, a propionate salt, a butyrate salt, a fumarate salt, a hemifumarate salt, a succinate salt, a methanesulfonate salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt, an oxalate salt, or a maleate salt.
In some embodiments, a compound capable of binding RyR2 is a compound of Formula I:
wherein,
In some embodiments, R2 is unsubstituted alkyl.
In some embodiments, the present disclosure provides compounds of Formula I-a:
wherein:
In some embodiments, the present disclosure provides a compound of formula I-a, wherein each R is independently halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1, or 2.
In some embodiments, the present disclosure provides a compound of formula I-a, wherein R2 is —C═O(R5), —C═S(R6), —SO2R7, —P(═O)R8R9, or —(CH2)m—R10.
In some embodiments, the present disclosure provides a compound of formula I-b:
wherein
In some embodiments, the present disclosure provides a compound of formula I-b, wherein R′ and R″ are each independently H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R′ is H or OMe, and R″ is H.
In some embodiments, the present disclosure provides a compound of formula I-b, wherein R2 is —C═O(R5), —C═S(R6), SO2R7, P(═O)R8R9, or —(CH2)m—R10.
In some embodiments, the present disclosure provides a compound formula of I-c:
In some embodiments, the present disclosure provides a compound of formula I-c, wherein each R is independently halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1, or 2.
In some embodiments, the present disclosure provides a compound of formula I-c, wherein R7 is alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl, each of which is independently substituted or unsubstituted; or —OH or —NR15R16.
In some embodiments, the present disclosure provides a compound of formula of I-d:
In some embodiments, the present disclosure provides a compound of formula wherein R′ and R″ are each independently H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R′ is H or OMe, and R″ is H.
In some embodiments, the present disclosure provides a compound of formula I-d, wherein R7 is alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl, each of which is independently substituted or unsubstituted; or —OH, or —NR15R16.
In some embodiments, the present disclosure provides a compound of formula of I-e:
In some embodiments, the present disclosure provides a compound of formula I-e, wherein each R is independently halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1, or 2.
In some embodiments, the present disclosure provides a compound of formula I-e, wherein R5 is alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl, each of which is independently substituted or unsubstituted; or —NR15R16, —NHOH, —OR15, or —CH2X.
In some embodiments, the present disclosure provides a compound of formula of I-f:
In some embodiments, the present disclosure provides a compound of formula I-f, wherein R′ and R″ are each independently H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R′ is H or OMe, and R″ is H.
In some embodiments, the present disclosure provides a compound of formula I-f, wherein R5 is alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl, each of which is independently substituted or unsubstituted; or —NR15R16, —NHOH, —OR15, or —CH2X.
In some embodiments, the present disclosure provides a compound of formula of I-g:
wherein
In some embodiments, the present disclosure provides a compound of formula I-g, wherein each R is independently selected from the group consisting of H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S-C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl and propenyl; and n is 0, 1, or 2.
In some embodiments, the present disclosure provides a compound of formula I-g, wherein R15 and R16 are each independently alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl, each of which is independently substituted or unsubstituted; or H, OH, or NH2; or R15 and R16 together with the N to which they are bonded form a heterocycle that is substituted or unsubstituted.
In some embodiments, the present disclosure provides a compound of formula I-g, wherein W is O or S.
In some embodiments, the present disclosure provides a compound of formula of I-h:
In some embodiments, the present disclosure provides a compound of formula wherein R′ and R″ are each independently H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R′ is H or OMe, and R″ is H.
In some embodiments, the present disclosure provides a compound of formula I-h, wherein R15 and R16 are each independently alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl, each of which is independently substituted or unsubstituted; or H, OH, NH2; or R15 and R16 together with the N to which R15 and R16 are bonded form a heterocycle that is substituted or unsubstituted.
In some embodiments, the present disclosure provides a compound of formula I-g, wherein W is O or S.
In some embodiments, the present disclosure provides a compound of formula of I-i:
wherein
In some embodiments, the present disclosure provides a compound of formula I-i, wherein each R is independently halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1, or 2.
In some embodiments, the present disclosure provides a compound of formula I-i, wherein R17 is —NR15R16 or —OR15. In some embodiments, R17 is —OH, —OMe, —Net, —NHEt, —NHPh, —NH2, or —NHCH2pyridyl.
In some embodiments, the present disclosure provides a compound of formula of I-j:
In some embodiments, the present disclosure provides a compound of formula I-j, wherein R′ and R″ are each independently H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R′ is H or OMe, and R″ is H.
In some embodiments, the present disclosure provides a compound of formula I-j, wherein R17 is NR15R16 or —OR15. In some embodiments, R17 is —OH, —OMe, —Net, —NHEt, —NHPh, —NH2, or —NHCH2pyridyl.
In some embodiments, the present disclosure provides a compound of formula I-k or I-k-1:
In some embodiments, the present disclosure provides a compound of formula I-k, wherein each R is independently H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S-C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R is OMe at position 7 of the benzothiazepine ring.
In some embodiments, the present disclosure provides a compound of formula I-k-1, wherein R′ and R″ are each independently H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S-C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R′ is H or OMe, and R″ is H.
In some embodiments, the present disclosure provides a compound of formula I-k or I-k-1, wherein R18 is —NR15R16, —(C═O)OR15, —OR15, alkyl that is substituted or unsubstituted, or aryl that is substituted or unsubstituted. In some embodiments, m is 1, and R18 is Ph, —C(═O)OMe, C(═O)OH, aminoalkyl, NH2, NHOH, or NHCbz. In other embodiments, m is 0, and R18 is C1-C4 alkyl. In other embodiments, R18 is Me, Et, propyl, and butyl. In some embodiments, m is 2, and R18 is pyrrolidine, piperidine, piperazine, or morpholine. In some embodiments, m is 3, 4, 5, 5, 7, or 8, and R18 is a fluorescent labeling group selected from bodipy, dansyl, fluorescein, rhodamine, Texas red, cyanine dyes, pyrene, coumarins, Cascade Blue™, Pacific Blue, Marina Blue, Oregon Green, 4′,6-Diamidino-2-phenylindole (DAPI), indopyra dyes, lucifer yellow, propidium iodide, porphyrins, arginine, and variants and derivatives thereof.
In some embodiments, the present disclosure provides a compound of formula of I-1 or I-1-1:
wherein
In some embodiments, the present disclosure provides a compound of formula I-1, wherein each R is independently halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R is OMe at position 7 of the benzothiazepine ring.
In some embodiments, the present disclosure provides a compound of formula I-1-1, wherein R′ and R″ are each independently H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R′ is H or OMe, and R″ is H.
In some embodiments, the present disclosure provides a compound of formula I-1 or I-1-1, wherein R6 is acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl, each of which is independently substituted or unsubstituted; or —NR15R16, —NHNR15R16, —OR15, —NHOH, or —CH2X. In some embodiments, R6 is —NR15R16. In some embodiments, R6 is —NHPh, pyrrolidine, piperidine, piperazine, morpholine. In some embodiments, R6 is alkoxyl. In some embodiments, R6 is —O-tBu.
In some embodiments, the present disclosure provides a compound of formula I-m or I-m-1:
wherein
In some embodiments, the present disclosure provides a compound of formula I-m, wherein each R is independently halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R is OMe at position 7 of the benzothiazepine ring.
In some embodiments, the present disclosure provides a compound of formula I-m-1, wherein R′ and R″ are each independently H, halogen, —OH, OMe, —NH2, —NO2, —CN, —CF3, —OCF3, —N3, —S(═O)2C1-C4alkyl, —S(═O)C1-C4alkyl, —S—C1-C4alkyl, —OS(═O)2CF3, Ph, —NHCH2Ph, —C(═O)Me, —OC(═O)Me, morpholinyl, or propenyl; and n is 0, 1 or 2. In some embodiments, R′ is H or OMe, and R″ is H.
In some embodiments, the present disclosure provides a compound of formula I-m or I-m-1, wherein R8 and R9 are each independently alkyl, aryl, OH, alkoxyl, or alkylamino. In some embodiments, R8 is C1-C4alkyl. In some embodiments, R8 is Me, Et, propyl or butyl. In some embodiments, R9 is aryl. In some embodiments, R9 is phenyl.
In some embodiments, the present disclosure provides a compound of formula I-n,
wherein:
In some embodiments, Ra is H.
Representative compounds of Formula I-n include without limitation S101, S102, S103, and S114.
In some embodiments, the present disclosure provides a compound of Formula I-o:
Representative compounds of Formula I-o include without limitation S107, S110, S111, S120, and S121.
In some embodiments, the present disclosure provides a compound of Formula I-p:
wherein:
In some embodiments, the present disclosure provides compounds of Formula II or Formula III:
wherein:
In some embodiments, the compound of formula (I) is selected from:
In some embodiments, the synthetic compound is a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I-k), (I-k-1), (I-1), (I-1-1), (I-m), (I-m-1), (I-n), (I-o), (I-p), (II), or (III). In some embodiments, the synthetic compound is S1, S2, S3, S4, S5, S6, S7, S9, S1, S12, S13, S14, S19, S20, S22, S23, S24, S25, S26, S27, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S68, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, S100, S101, S102, S103, S104, S105, S107, S108, S109, S110, S111, S112, S113, S114, S115, S116, S117, S118, S119, S120, S121, S122, or S123, as herein defined.
In some embodiments, the synthetic compound is:
or a pharmaceutically-acceptable salt thereof or an ionized form thereof.
In some embodiments, the synthetic compound is:
or a pharmaceutically-acceptable salt or an ionized form thereof.
Compounds described herein may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present disclosure.
All stereoisomers of the compounds of the present disclosure (for example, those which may exist due to asymmetric carbons on various substituents), including enantiomeric forms and diastereomeric forms, are contemplated within the scope of this invention. Individual stereoisomers of the compounds of the disclosure may, for example, be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a specified activity), or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers. The chiral centers of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations. The racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.
The present disclosure provides methods for identifying a compound that binds to a biomolecular target (e.g. RyR2). In some embodiments, the methods described herein can include screening a library of three-dimensional compound structures to identify ligands that fit a binding pocket of the biomolecular target such as RyR2.
In some embodiments, provided is a method comprising:
In some embodiments, each of the determining the open probability (Po) of the first RyR2 protein and the determining the open probability (Po) of the second RyR2 protein comprises recording a single channel Ca2+ current. In some embodiments, the method further comprises determining a difference between the Po of the first RyR2 protein and Po of the second RyR2 protein. In some embodiments, the method further comprises identifying the test compound as a target for further analysis based on the difference between the Po of the first RyR2 protein and Po of the second RyR2 protein. In some embodiments, the Po of the first RyR2 protein is lower than the Po of the second RyR2 protein.
In some embodiments, the method further comprises performing an analogous assay wherein another compound is used in place of the test compound, wherein the analogous assay provides a difference between:
In some embodiments, the difference of the Po in (b)(ii) is greater than the difference of the Po in (b)(i). In some embodiments, the RyR2 is a mutant RyR2. In some embodiments, the mutant RyR2 contains at least one mutation that is associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the mutation is R2474S. In some embodiments, the RyR2 protein is a post-translationally modified RyR2 protein. In some embodiments, the post-translationally modified RyR2 protein is a phosphorylated, oxidized or nitrosylated RyR2. In some embodiments, the RyR2 is a mutated and post-translationally modified RyR2.
In some embodiments, provided is a method comprising:
In some embodiments, each of the determining the open probability (Po) of the first RyR2 protein and the determining the open probability (Po) of second RyR2 protein comprises recording a single channel Ca2+ current. In some embodiments, the method further comprises determining a difference between the Po of the first RyR2 protein and the Po of the second RyR2 protein. In some embodiments, the method further comprises identifying the test compound as a target for further analysis based on the difference between the Po of the first RyR2 protein and the Po of the second RyR2 protein. In some embodiments, the Po of the first RyR2 protein is lower than the Po of the second RyR2 protein.
In some embodiments, the method further comprises performing an analogous assay wherein another compound is used in place of the test compound, wherein the analogous assay provides a difference between:
In some embodiments, the difference of the Po in (b)(ii) is greater than the difference of the Po in (b)(i). In some embodiments, the RyR2 is a mutant RyR2. In some embodiments, the mutant RyR2 contains at least one mutation that is associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the mutation is R2474S. In some embodiments, the RyR2 protein is a post-translationally modified RyR2 protein. In some embodiments, the post-translationally modified RyR2 protein is a phosphorylated, oxidized or nitrosylated RyR2. In some embodiments, the RyR2 is a mutated and post-translationally modified RyR2. In some embodiments, the method further comprises: subsequent to the contacting the first RyR2 protein with the reagent and the test compound, fusing a first microsome containing the first RyR2 protein to a first planar lipid bilayer, and subsequent to the contacting the second RyR2 protein with the reagent, fusing a second microsome containing the second RyR2 protein to a second planar lipid bilayer.
In some embodiments, provided is a method of identifying a compound having RyR2 modulatory activity, the method comprising:
In some embodiments, the RyR2 protein is a mutant RyR2 protein. In some embodiments, the mutant RyR2 contains at least one mutation that is associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the mutation is R2474S. In some embodiments, the RyR2 protein is a post-translationally modified RyR2 protein. In some embodiments, the post-translationally modified RyR2 protein is a phosphorylated, oxidized or nitrosylated RyR2. In some embodiments, the RyR2 protein is a mutated and post-translationally modified RyR2 protein. In some embodiments, the test compound preferentially binds to a mutant RyR2 relative to wild-type RyR2. In some embodiments, the test compound preferentially binds to post-translationally modified RyR2 relative to wild-type RyR2. In some embodiments, the test compound preferentially binds to a mutated and post-translationally modified RyR2 relative to a wild-type RyR2. In some embodiments, determining the open probability (Po) of the RyR2 protein comprises recording a single channel Ca2+ current.
In some embodiments, provided is a method for identifying a compound having RyR2 modulatory activity, comprising:
In some embodiments, the ligand is radiolabeled. In some embodiments, determining the ability of the test compound to displace the ligand from the RyR2 protein comprises determining a radioactive signal in the RyR2 protein. In some embodiments, the RyR2 protein is a mutant RyR2 protein. In some embodiments, the mutant RyR2 contains at least one mutation that is associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the mutation is R2474S. In some embodiments, the RyR2 protein is a post-translationally modified RyR2 protein. In some embodiments, the RyR2 protein is a mutated and post-translationally modified RyR2 protein. In some embodiments, the test compound preferentially binds to a mutant RyR2 relative to wild-type RyR2. In some embodiments, the test compound preferentially binds to post-translationally modified RyR2 relative to wild-type RyR2. In some embodiments, the test compound preferentially binds to a mutant and post-translationally modified RyR2 relative to a wild-type RyR2.
In some embodiments, provided is a method for identifying a compound that preferentially binds to a mutated, post-translationally modified RyR2 or a combination thereof, comprising:
In some embodiments, the RyR2 protein is a mutant RyR2 protein. In some embodiments, the mutant RyR2 contains at least one mutation that is associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). In some embodiments, the mutation is R2474S. In some embodiments, the RyR2 protein is a post-translationally modified RyR2 protein. In some embodiments, the RyR2 protein is a mutated and post-translationally modified RyR2 protein. In some embodiments, the test compound preferentially binds to a mutant RyR2 relative to wild-type RyR2. In some embodiments, the test compound preferentially binds to post-translationally modified RyR2 relative to wild-type RyR2. In some embodiments, the test compound preferentially binds to a mutant and post-translationally modified RyR2 relative to a wild-type RyR2. In some embodiments, the test compound contains a benzothiazepane moiety.
In some embodiments, the test compound is a compound of Formula (I):
wherein:
In some embodiments, the test compound is a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I-k), (I-k-1), (I-1), (I-1-1), (I-m), (I-m-1), (I-n), (I-o), (I-p), (II) or (III).
In some embodiments, the test compound is
or an ionized form thereof.
In some embodiments, the test compound is
or an ionized form thereof.
In some embodiments, a cryo-EM model disclosed herein can be used as a tool to screen for ryanodine receptor modulator compounds that bind RyR2. In some embodiments, a cryo-EM model disclosed herein can be used as a tool to screen for ryanodine receptor modulator compounds which preferentially bind leaky RyR2 (e.g., mutated RyR2, or post-translationally modified RyR2 (e.g., phosphorylated, dephosphorylated, oxidized and/or nitrosylated RyR2)) and stabilize the closed state of the RyR channel.
Structures of compounds (e.g., Compound 1) and biomolecular targets (e.g. RyR2) provided herein can be used in computational methods for identifying ligands that bind to a biomolecular target (e.g. RyR2). Such methods can include, for example, screening a library of three-dimensional compound structures to identify ligands that fit a binding pocket of the biomolecular target via a molecular docking system (e.g. Glide, DOCK, AutoDock, AutoDock Vina, FRED, and EnzyDock); de-novo generation of a structure of a ligand that binds the biomolecular target via a ligand structure prediction system (e.g., CHARMM, AMBER, or GROMACS); optimization of known ligands (e.g., Compound 1) by evaluating binding of proposed analogs within the binding cavity of the biomolecular target, and combinations of the preceding.
Structures of compounds (e.g., Compound 1) and biomolecular targets (e.g. RyR2) provided herein can be used in computational methods of predicting a docked position of a target ligand in a binding site of a biomolecule, such as the use of a computer to assist in predicting a docked position of a target ligand in a binding site of a biomolecule that is capable of undergoing an induced fit as disclosed in US20210193273A1, which is incorporated herein by reference in its entirety.
In some embodiments, the present disclosure provides a method for predicting a docked position of a target ligand in a binding site of a biomolecule, the method comprising:
In some embodiments, the RY1&2 domain comprises a structure according to TABLE 3. In some embodiments, the template ligand has a three-dimensional conformation according to TABLE 4. In some embodiments, the RY1&2 domain further comprises second binding site. In some embodiments, the second binding site is an ATP-binding site. In some embodiments, the RY1&2 domain further comprises a nucleoside-containing molecule. In some embodiments, the nucleoside-containing molecule is an ATP molecule. In some embodiments the target ligand cooperatively binds the RY1&2 domain with the ATP molecule. In some embodiments, the ATP molecule has a three-dimensional conformation according to TABLE 5. In some embodiments, the target ligand cooperatively binds the RY1&2 domain with the ATP molecule.
In some embodiments, the target ligand is a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I-k), (I-k-1), (I-1), (I-1-1), (I-m), (I-m-1), (I-n), (I-o), (I-p), (II) or (III). In some embodiments, the target ligand and the template ligand are each independently a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I-k), (I-k-1), (I-1), (I-1-1), (I-m), (I-m-1), (I-n), (I-o), (I-p), (II) or (III).
In some embodiments, the template ligand is
In some embodiments, the template ligand is
In some embodiments, the template ligand-biomolecule structure obtained by single-particle cryogenic electron microscopy analysis has a resolution from about 2 Å to about 3.5 Å, from about 2 Å to about 3.4 Å, from about 2 Å to about 3.3 Å, from about 2 Å to about 3.2 Å, from about 2 Å to about 3.1 Å, from about 2 Å to about 3 Å, from about 2 Å to about 2.9 Å, from about 2 Å to about 2.8 Å, from about 2 Å to about 2.7 Å, from about 2 Å to about 2.6 Å, from about 2 Å to about 2.5 Å, from about 2.1 Å to about 2.5 Å, from about 2.2 Å to about 2.5 Å, from about 2.3 Å to about 2.5 Å, or from about 2.4 Å to about 2.5 Å.
In some embodiments, the method further comprises selecting the target ligand from a plurality of ligand candidates, each of the ligand candidates being different from the template ligand, and wherein selecting the target ligand comprises comparing the pharmacophore model of the template ligand to a pharmacophore model of each respective one of the plurality of ligand candidates.
In some embodiments, the method further comprises receiving a plurality of template ligand-biomolecule structures, each template ligand-biomolecule structure having a different template ligand docked in the binding site of the biomolecule, and generating the pharmacophore model of the template ligand by combining information from each of the template ligands from the plurality of template ligand-biomolecule structures.
In some embodiments, the target ligand has more than one structural conformation in the unbound state, and the docked position of the target ligand in the binding site of the biomolecule is predicted by enumerating a set of potential target ligand conformations and overlapping a respective pharmacophore model of the target ligand for each of the potential target ligand conformations with the pharmacophore model of the template ligand while the template ligand is in the binding site of the biomolecule.
In some embodiments, predicting the docked position of the target ligand in the binding site of the biomolecule comprises ignoring at least one clash between the target ligand conformation's atomic coordinates and the biomolecule's atomic coordinates.
In some embodiments, the method further comprises, for each target ligand conformation, modifying atomic coordinates of the biomolecule to reduce clashes between the docked target ligand conformation's atomic coordinates and the biomolecule's atomic coordinates, thereby creating an altered ligand-biomolecule structure comprising the docked target ligand and an altered biomolecule.
In some embodiments, the method further comprises predicting a re-docked position of each target ligand conformation by predicting each target ligand conformation's position in the binding site of the altered biomolecule; and for each target ligand conformation, modifying atomic coordinates of the altered biomolecule to reduce clashes between the atomic coordinates of the target ligand conformation's re-docked position and the atomic coordinates of the altered biomolecule, thereby creating are-altered ligand-biomolecule structure comprising a re-docked target ligand and a re-altered biomolecule.
In some embodiments, the method further comprises ranking each altered and re-altered ligand-biomolecule structure using a scoring function.
In some embodiments, the method further comprises identifying a subset of high-ranking target ligands corresponding to target ligands having a threshold value for an empirical activity.
Structures of compounds (e.g., Compound 1) and biomolecular targets (e.g. RyR2) provided herein can be used in systems, devices, and methods that can generate lead compounds on the basis of known structure and activity of a lead compound (e.g., Compound 1) and the structure of a binding site for the lead compound, such as the systems, devices, and methods provided in US20210217500A1, which is incorporated herein by reference in its entirety.
In some embodiments, the present disclosure provides a method of identifying a plurality of potential lead compounds, the method comprising the steps of:
In some embodiments, the structure of the biomolecular target obtained by single-particle cryogenic electron microscopy analysis has a resolution from about 2 Å to about 3.5 Å, from about 2 Å to about 3.4 Å, from about 2 Å to about 3.3 Å, from about 2 Å to about 3.2 Å, from about 2 Å to about 3.1 Å, from about 2 Å to about 3 Å, from about 2 Å to about 2.9 Å, from about 2 Å to about 2.8 Å, from about 2 Å to about 2.7 Å, from about 2 Å to about 2.6 Å, from about 2 Å to about 2.5 Å, from about 2.1 Å to about 2.5 Å, from about 2.2 Å to about 2.5 Å, from about 2.3 Å to about 2.5 Å, or from about 2.4 Å to about 2.5 Å.
In some embodiments, the initial lead compound is a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I-k), (I-k-1), (I-1), (I-1-1), (I-m), (I-m-1), (I-n), (I-o), (I-p), (II) or (III).
In some embodiments, the initial lead compound is
In some embodiments, the initial lead compound is
In some embodiments, the RY1&2 domain comprises a structure according to TABLE 3. In some embodiments, the RY1&2 domain contains an ATP molecule. In some embodiments, the ATP molecule has a three-dimensional conformation according to TABLE 5.
In some embodiments, the method further comprises obtaining a synthesized set of at least some of the potential lead compounds predicted to not bind with the biomolecular target to establish a second set of potential lead compounds and empirically determining an activity of each of the second set of synthesized potential lead compounds.
In some embodiments, the method further comprises comparing the empirically determined activity of each of the first set of synthesized potential lead compounds with a threshold activity level.
In some embodiments, the method further comprises comparing the empirically determined activity of each of the second set of synthesized potential lead compounds with a pre-determined activity level.
In some embodiments, the plurality of alternative cores are chosen from a database of synthetically feasible cores.
In some embodiments, the difference in binding free energy is calculated using a free energy perturbation technique.
In some embodiments, the generation of at least one potential lead compound comprises creating an additional covalent bond or annihilating an existing covalent bond, or both creating an additional first covalent bond and annihilating an existing second covalent bond different from the first covalent bond.
In some embodiments, the free energy perturbation technique uses a soft bond potential to calculate a bonded stretch interaction energy of existing covalent bonds for annihilation and additional covalent bonds for creation.
In some embodiments, the present disclosure provides a method for pharmaceutical drug discovery, comprising:
In some embodiments, the structure of the biomolecular target obtained by single-particle cryogenic electron microscopy analysis has a resolution from about 2 Å to about 3.5 Å, from about 2 Å to about 3.4 Å, from about 2 Å to about 3.3 Å, from about 2 Å to about 3.2 Å, from about 2 Å to about 3.1 Å, from about 2 Å to about 3 Å, from about 2 Å to about 2.9 Å, from about 2 Å to about 2.8 Å, from about 2 Å to about 2.7 Å, from about 2 Å to about 2.6 Å, from about 2 Å to about 2.5 Å, from about 2.1 Å to about 2.5 Å, from about 2.2 Å to about 2.5 Å, from about 2.3 Å to about 2.5 Å, or from about 2.4 Å to about 2.5 Å.
In some embodiments, the initial lead compound is a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I-k), (I-k-1), (I-1), (I-1-1), (I-m), (I-m-1), (I-n), (I-o), (I-p), (II) or (III).
In some embodiments, the initial lead compound is
In some embodiments, the initial lead compound is
In some embodiments, the RY1&2 domain comprises a structure according to TABLE 3. In some embodiments, the RY1&2 domain contains an ATP molecule. In some embodiments, the ATP molecule has a three-dimensional conformation according to TABLE 5.
Structures of compounds (e.g., Compound 1) and biomolecular targets (e.g. RyR2) provided herein can be used in methods that estimate binding affinity between a ligand and a receptor molecule, including the systems and methods disclosed in U.S. Pat. No. 8,160,820B2, which is incorporated by reference herein in its entirety.
In some embodiments, the present disclosure provides a computer-implemented method of quantifying binding affinity between a ligand and a receptor molecule domain, the method comprising:
In some embodiments, the structure of the receptor molecule domain obtained by single-particle cryogenic electron microscopy analysis has a resolution from about 2 Å to about 3.5 Å, from about 2 Å to about 3.4 Å, from about 2 Å to about 3.3 Å, from about 2 Å to about 3.2 Å, from about 2 Å to about 3.1 Å, from about 2 Å to about 3 Å, from about 2 Å to about 2.9 Å, from about 2 Å to about 2.8 Å, from about 2 Å to about 2.7 Å, from about 2 Å to about 2.6 Å, from about 2 Å to about 2.5 Å, from about 2.1 Å to about 2.5 Å, from about 2.2 Å to about 2.5 Å, from about 2.3 Å to about 2.5 Å, or from about 2.4 Å to about 2.5 Å.
In some embodiments, ligand molecule is a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I-k), (I-k-1), (I-1), (I-1-1), (I-m), (I-m-1), (I-n), (I-o), (I-p), (II) or
In some embodiments, the ligand molecule is
or a pharmaceutically-acceptable salt or ionized form thereof.
In some embodiments, the ligand molecule is
or a pharmaceutically-acceptable salt or ionized form thereof.
In some embodiments, the complex further comprises a RyR2 protein, wherein the RY1&2 domain is a domain of the RyR2 protein.
In some embodiments, the data representing the receptor molecule domain represents a three-dimensional structure of the receptor molecule according to TABLE 3. In some embodiments, the data representing a ligand molecule represents a three-dimensional structure of the ligand molecule according to TABLE 4.
In some embodiments, the receptor molecule domain contains an ATP molecule. In some embodiments, the data representing the receptor molecule domain further comprises data representing a three-dimensional structure of the ATP molecule according to TABLE 5.
In some embodiments, quantifying the binding affinity includes a step that scores hydrophobic interactions between one or more ligand atoms and one or more receptor atoms by awarding a bonus for the presence of hydrophobic enclosure of one or more atoms of said ligand by the receptor molecule domain, said bonus being indicative of enhanced binding affinity between said ligand and said receptor molecule domain.
In some embodiments, the method further comprises calculating an initial binding affinity and then adjusting the initial binding affinity based on the classification of the ring structure as buried, solvent exposed, or solvent exposed on one face.
In some embodiments, the classification of a ring structure as buried, solvent exposed, or solvent exposed on one surface, includes using a parameter substantially correlated with the number of close contacts on both sides of the ring structure or part thereof with the receptor molecule domain.
In some embodiments, the number of close contacts at different distances between receptor atoms and the two ring faces are determined, an initial classification of the ring is made based on the numbers of these contacts, and this initial classification is then followed by calculation of a scoring function, said scoring function comprising identifying a first ring shell and a second ring shell, and calculating the number of water molecules in the first shell and in the second shell, or calculating the number of water molecules in the first and second shell combined.
In some embodiments, the scoring function for classification of the ring structure as buried, solvent exposed, or solvent exposed on one surface, includes using a parameter substantially correlated with the lipophilic-lipophilic pair score between the ring structure or part thereof and the receptor molecule domain.
In some embodiments, the scoring function used to classify a ring structure as buried, solvent exposed, or solvent exposed on one surface, includes calculating the degree of enclosure of each atom of the ring structure by atoms of the receptor.
In some embodiments, the scoring function used to classify a ring structure as buried, solvent exposed, or solvent exposed on one surface, includes using a parameter that is substantially correlated with the degree of enclosure of each atom of the ring structure by atoms of the receptor.
In some embodiments, the scoring function enabling classification of the ring structure as buried, solvent exposed, or solvent exposed on one surface, includes the use of a parameter corresponding to a hydrophobic interaction of the ring structure or part thereof with the receptor molecule domain.
In some embodiments, the information displayed by computer includes a depiction of at least one of the degree to which the ring structure is enclosed by atoms of the receptor molecule domain; water molecules surrounding the ring structure in a first shell or a second shell or both the first and the second shell of the ligand; a value of a lipophilic-lipophilic pair score of the ring structure; and a number of close contacts of a face of the ring structure with the receptor molecule domain.
In some embodiments, solvent exposed ring structures in the ligand, if any, are substantially ignored in quantifying the component of the binding affinity between the ligand and the receptor molecule domains, other than to recognize hydrogen bonds and other parameters that are independent of the classification of ring structure.
In some embodiments, hydrophobic contribution to binding affinity from ring structures classified as solvent exposed, if any, is substantially ignored in quantifying the component of the binding affinity.
In some embodiments, a ring structure is classified as buried, and the method further comprises identifying a quantity representative of a strain energy induced in the ligand-receptor complex by the buried ring structure, in which the quantification of the component of binding affinity is further based in part on strain energy.
In some embodiments, the method further comprises identifying a quantity representative of a strain energy induced in the ligand-receptor complex by the aggregate of the ring structures identified as buried; identifying a quantity representative of a total neutral-neutral hydrogen bond energy; and quantifying the component of binding affinity between the ligand and the receptor molecule domain based at least in part on the quantity representative of the strain energy induced in the receptor by the aggregate of the buried ring structures, and on the quantity representative of the total neutral-neutral hydrogen bond energy.
In some embodiments, quantifying the component of binding affinity further comprises identifying a hydrogen bond capping energy associated with the entire ligand, and the component of binding affinity is quantified based on a greater of the hydrogen bond capping energy and the quantity representative of the strain energy induced in the receptor by the aggregate of the identified structures.
In some embodiments, the method further comprises identifying a binding motif of the receptor molecule domain with respect to the ligand; identifying a reorganization energy of the receptor molecule domain based on the binding motif, and identifying a first ring structure as contributing to the reorganization energy, the quantity representative of strain energy being identified independently of the classification of the first ring structure.
In some embodiments, the component of binding affinity attributable to strain is quantified using at least one of: molecular dynamics, molecule mechanics, conformational searching and minimization.
In some embodiments, the information displayed by computer includes a depiction of solvent exposure, if any, of the ring structure.
In some embodiments, the information displayed by computer includes a depiction of burial, if any, of the ring structure.
In some embodiments, the information displayed by computer includes a depiction of at least one of: the degree to which the ring structure is enclosed by atoms of the receptor molecule domain; water molecules surrounding the ring structure in a first shell or a second shell or both the first and the second shell of the ligand; a value of a lipophilic-lipophilic pair score of the ring structure; and a number of close contacts of a face of the ring structure with the receptor molecule domain.
In some embodiments, the method further comprises performing a test on a physical sample that includes the ligand and the receptor molecule domain, test components being selected based at least in part on the binding affinity between the ligand or part thereof and the receptor molecule, or on the component of such binding affinity.
Embodiments of the subject matter and the functional operations described in this specification can be implemented in digital electronic circuitry, in tangibly-embodied computer software or firmware, in computer hardware, including the structures disclosed in this specification and their structural equivalents, or in combinations of one or more of them. Embodiments of the subject matter described in this specification can be implemented as one or more computer programs, i.e., one or more modules of computer program instructions encoded on a tangible non-transitory storage medium for execution by, or to control the operation of, data processing apparatus. The computer storage medium can be a machine-readable storage device, a machine-readable storage substrate, a random or serial access memory device, or a combination of one or more of them. Alternatively, or in addition, the program instructions can be encoded on an artificially generated propagated signal, e.g., a machine-generated electrical, optical, or electromagnetic signal, that is generated to encode information for transmission to suitable receiver apparatus for execution by a data processing apparatus.
The term “data processing apparatus” refers to data processing hardware and encompasses all kinds of apparatus, devices, and machines for processing data, including by way of example a programmable processor, a computer, or multiple processors or computers. The apparatus can also be, or further include, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit). The apparatus can optionally include, in addition to hardware, code that creates an execution environment for computer programs, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, or a combination of one or more of them.
A computer program, which is also referred to or described as a program, software, a software application, an app, a module, a software module, a script, or code, can be written in any form of programming language, including compiled or interpreted languages, or declarative or procedural languages; and can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. A program can, but need not, correspond to a file in a file system. A program can be stored in a portion of a file that holds other programs or data, e.g., one or more scripts stored in a markup language document, in a single file dedicated to the program in question, or in multiple coordinated files, e.g., files that store one or more modules, sub programs, or portions of code. A computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a data communication network.
The processes and logic flows described in this specification can be performed by one or more programmable computers executing one or more computer programs to perform functions by operating on input data and generating output. The processes and logic flows can also be performed by special purpose logic circuitry, e.g., an FPGA or an ASIC, or by a combination of special purpose logic circuitry and one or more programmed computers.
Computers suitable for the execution of a computer program can be based on general or special purpose microprocessors or both, or any other kind of central processing unit. Generally, a central processing unit receives instructions and data from a read only memory or a random access memory or both. The essential elements of a computer are a central processing unit for performing or executing instructions and one or more memory devices for storing instructions and data. The central processing unit and the memory can be supplemented by, or incorporated in, special purpose logic circuitry. Generally, a computer can also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto optical disks, or optical disks. However, a computer need not have such devices. Moreover, a computer can be embedded in another device, e.g., a mobile telephone, a personal digital assistant (PDA), a mobile audio or video player, a game console, a Global Positioning System (GPS) receiver, or a portable storage device, e.g., a universal serial bus (USB) flash drive, to name just a few.
Computer readable media suitable for storing computer program instructions and data include all forms of non-volatile memory, media and memory devices, including by way of example semiconductor memory devices, e.g., EPROM, EEPROM, and flash memory devices; magnetic disks, e.g., internal hard disks or removable disks; magneto optical disks; and CD ROM and DVD-ROM disks.
To provide for interaction with a user, embodiments of the subject matter described in this specification can be implemented on a computer having a display device, e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor, for displaying information to the user and a keyboard and a pointing device, e.g., a mouse or a trackball, by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well; for example, feedback provided to the user can be any form of sensory feedback, e.g., visual feedback, auditory feedback, or tactile feedback; and input from the user can be received in any form, including acoustic, speech, or tactile input. In addition, a computer can interact with a user by sending documents to and receiving documents from a device that is used by the user; for example, by sending web pages to a web browser on a user's device in response to requests received from the web browser. Also, a computer can interact with a user by sending text messages or other forms of message to a personal device, e.g., a smartphone that is running a messaging application, and receiving responsive messages from the user in return.
Embodiments of the subject matter described in this specification can be implemented in a computing system that includes a back end component, e.g., as a data server, or that includes a middleware component, e.g., an application server, or that includes a front end component, e.g., a client computer having a graphical user interface, a web browser, or an app through which a user can interact with an implementation of the subject matter described in this specification, or any combination of one or more such back end, middleware, or front end components. The components of the system can be interconnected by any form or medium of digital data communication, e.g., a communication network. Examples of communication networks include a local area network (LAN) and a wide area network (WAN), e.g., the Internet.
The computing system can include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other. In some embodiments, a server transmits data, e.g., an HTML page, to a user device, e.g., for purposes of displaying data to and receiving user input from a user interacting with the device, which acts as a client. Data generated at the user device, e.g., a result of the user interaction, can be received at the server from the device.
The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.
Complementary DNA (cDNA) for human RyR2 was subcloned into the A1.2 vector in two steps, using the Nhe I-Xho I fragment and then the Not I-Nhe I fragment of human RYR2. Human RYR2 was inserted 3′ of a cytomegalovirus (CMV) promoter and followed in 5′ by internal ribosomal entry site (IRES)-green fluorescent protein (GFP). The sequence of the construct was confirmed by Sanger sequencing and restriction digestion with Afl III enzyme. The resulting vector (G418-resistant) was cotransfected into HEK293T cells with a plasmid carrying a puromycin resistance gene using calcium chloride. Cells were maintained in a G418- and puromycin-containing medium for approximately 3 weeks and then underwent two cycles of clonal selection, where the top 0.1% of the most highly fluorescent cells were propagated.
Constructs expressing RyR2-R2474S were formed by introducing the respective mutation into fragments of human RYR2 using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent). Three nucleotide changes were introduced (WT sequence: AGGGTCTAT to R2474S mutant: AGCGTATAC). The first was the mutation R2474S, and the other two were silent mutations that introduced a BstZ171 restriction site (GTATAC) to facilitate screening for mutant clones. Each fragment was subcloned into a full-length human RYR2 construct in pCMV5 vector, confirmed by sequencing, and expressed in 293T/17 cells using Lipofectamine 2000 (Thermo Fisher Scientific). For final expression, HEK293 cells grown in 150-mm dishes with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Invitrogen), penicillin (100 U/ml), streptomycin (100 μg/ml), and 2 mM 1-glutamine were cotransfected with 25 mg per dish of human RYR2-R2474S cDNA using PEI MAX at a 1:5 ratio (Polysciences). Cells were collected 48 hours after transfection.
Recombinant human GST-calstabin-2 was expressed in BL21 (DE3) Escherichia co/i cells with a thrombin protease cleavage site between GST and calstabin-2. Protein expression was induced with 0.8 mM isopropyl-o-d-thiogalactopyranoside (IPTG) added to E. coli at an OD600 (optical density at 600 nm) of 0.8 with overnight incubation at 18° C. before centrifugation for 10 min at 6500 g. The pellets were resuspended in buffer A (phosphate-buffered saline+0.5 mM AEBSF) and lysed using an emulsiflex (Avestin EmulsiFlex-C3). The lysate was pelleted by centrifugation for 10 min at 100,000 g. The supernatant was then loaded into a 5-ml GSTrap HP column (Cytiva) and washed with 5 column volume (CV) of buffer A to remove contaminants before elution with buffer B [tris (pH 8), 2 mM DTT, and 20 mM glutathione]. Fractions containing GST-calstabin-2 were pooled, concentrated, and dialyzed overnight at 4° C. into buffer A. final concentration was determined by spectroscopy using NanoDrop 1000 (Thermo Fisher Scientific) with absorbance at 280 nm and an extinction coefficient of 46,200 M−1 cm−1. GST-calstabin-2 was stored at −80° C.
Recombinant human calmodulin (CaM) was expressed in BL21 (DE3) E. coli cells with an N-terminal 6-histidine tag and a tobacco etch virus (TEV) protease cleavage site. Protein expression was induced with 0.8 mM IPTG added to E. coli at an OD600 of 0.8 with overnight incubation at 18° C. before centrifugation for 10 min at 6500 g and storage at −80° C. CaM was purified using a two-step 5-ml HisTrap HP column (Cytiva) purification. In brief, the pellets were resuspended in buffer A [20 mM Hepes (pH 7.5), 150 mM NaCl, 20 mM imidazole, 5 mM 2-Mercaptoethanol, and 0.5 mM AEBSF] and lysed using an emulsiflex (Avestin EmulsiFlex-C3). The lysate was pelleted by centrifugation for 10 min at 100,000 g. The supernatant was then loaded over a HisTrap column and washed with 5 CV of buffer A to remove contaminants before elution using a linear gradient from buffer A to buffer B (buffer A containing 500 mM imidazole). Fractions containing CaM were pooled, 1 to 2 mg of purified TEV protease was added, and the mixture was dialyzed overnight at 4° C. into buffer C (buffer A with no imidazole). CaM was then loaded onto a HisTrap column with the flowthrough collected and the wash fractionated to retain fractions containing CaM before elution of TEV and any remaining contaminants with a linear gradient from buffer C to buffer B. The flowthrough and any fractions containing CaM were pooled, concentrated to >2 mM, and determined by spectroscopy using NanoDrop 1000 (Thermo Fisher Scientific) with absorbance at 280 nm and the extinction coefficient of CaM (3000 M−1 cm−1). CaM was stored at −20° C. TEV protease was purified in the same manner except for using an uncleavable his-tag and thus ending after the first HisTrap column wherein the purified protease was stored at −80° C. in buffer C with 10% glycerol.
To obtain high-resolution structures of WT and mutant human RyR2, the purification of channels expressed recombinantly in HEK293 cells was optimized. All purification steps were performed on ice unless otherwise stated. HEK293 cells (25 to 50 dishes) expressing human RyR2 or RyR2-R2474S (prepared according to EXAMPLE 1 and EXAMPLE 2) were harvested by centrifugation for 10 min at 1500 g. The pellet fraction was resuspended in tris malate buffer [10 mM tris malate (pH 6.8), 1 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM benzamidine, 0.5 mM 4-benzenesulfonyl fluoride hydrochloride (AEBSF), and protease inhibitor cocktail] and was sonicated with six pulses of 20 s at 35% amplitude. The membrane fraction was precipitated by centrifugation at 100,000 g for 30 min and was resuspended with a glass homogenizer in CHAPS buffer [10 mM Hepes (pH 7.4), 1 M NaCl, 1.5% CHAPS, 0.5% phosphatidylcholine (PC), 1 mM EGTA, 2 mM DTT, 0.5 mM AEBSF, 1 mM benzamidine, and protease inhibitor cocktail]. The sample was diluted 1:4 with the same buffer without NaCl. To achieve stabilization of peripherical domains, 100 nmol of GST-calstabin-2 was added to both tris malate buffer and CHAPS buffer. The remaining insoluble material was separated with a second centrifugation at 100,000 g for 30 min.
The supernatant-containing detergent-solubilized human RyR2—was filtered and loaded into a 5-ml HiTrap Q HP column (Cytiva) previously equilibrated with buffer A [10 mM Hepes (pH 7.4), 0.4% CHAPS, 1 mM EGTA, 0.001% dioleoylphosphatidylcholine (DOPC), 250 mM NaCl, and 0.5 mM TCEP (tris(2-carboxyethyl)phosphine)]. The HiTrap Q HP column was eluted with a linear gradient between 300 and 600 mM NaCl. The fractions containing human RyR2 (300 to 350 mM NaCl) were pooled, and 100 nmol of GST-calstabin-2 (prepared according to EXAMPLE 3) was added. The pooled fractions were loaded into a 1-ml GSTrap HP column (Cytiva), which was left recirculating overnight. The GSTrap HP column was washed with buffer A and eluted with glutathione buffer {10 mM Hepes (pH 8), 0.4% CHAPS, 1 mM EGTA, 0.001% DOPC, 200 mM NaCl, 10 mM GSH [glutathione (reduced form)], and 1 mM DTT}. Immediately after, a second 1-ml HiTrap Q HP column (Cytiva) binding/elution step was applied to separate human RyR2 from the excess of unbound GST-calstabin-2 and GSH.
Six preparations of RyR2 and RyR2-R2474S were prepared:
For phosphorylated samples (P-RyR2, P-RyR2+CaM, P-RyR2-R2474S, P-RyR2-R2474S+Cpd1, and P-RyR2-R2474S+CaM), simultaneous cleavage of GST tag and PKA phosphorylation was performed by addition of 50 U of thrombin and 100 U of PKA (+10 mM EGTA, 8 mM MgCl2, and 100 μM ATP for activity), respectively, for 30 min on ice. For samples requiring dephosphorylation treatment (DeP-RyR2), PKA was replaced by 2000 U of phosphatase lambda (PX from NEB, +1×Protein MetalloPhosphatases (PMP) buffer, and 1 mM MnCl2). Each sample was concentrated to 0.5 ml, and a gel filtration step was run with TSKgel G4SWXL (TOSOH Biosciences) with buffer A. RyR2 fractions were pooled and concentrated to a concentration of 4 to 8 mg/ml (with centrifugal filters of 100-kDa cutoff) and were filtered (with centrifugal filters of 0.22-μm cutoff) to eliminate aggregates.
To resemble exercise diastolic conditions, 10 mM NaATP, 500 μM xanthine, 150 nM Ca2+ free (650 μM total Ca2+), and 200 μM cAMP were added to all samples. 20 μM CaM (prepared according to EXAMPLE 4) was added to PKA-phosphorylated RyR2 (P-RyR2+CaM), 40 μM CaM was added to PKA-phosphorylated RyR2-R2474S (P-RyR2-R2474S+CaM), and 500 μM Compound 1 was added to PKA-phosphorylated RyR2-R2474S (P-RyR2-R2474S+Cpd1). MaxChelator webserver was used to calculate total/free Ca2+ concentrations.
Quality control was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots using anti-RyR-5029 and anti-RyR2-pS2809 antibodies for total and phosphorylated RyR2, respectively.
Each of the final samples (3 μl each) prepared in EXAMPLE 5 was applied to UltrAuFoil holey gold grids (Quantifoil R 0.6/1.0, Au 300) previously cleaned with easiGlow (PELCO). Grids were blotted with ashless filter paper (Whatman) using blot force 10 and blot time 8 s before vitrification by plunge-freezing into liquid ethane chilled with liquid nitrogen using Vitrobot Mark IV (Thermo Fisher Scientific) operated at 4° C. with 100% relative humidity.
Prepared grids were screened in-house on a Glacios Cryo-TEM (Thermo Fisher Scientific) microscope with a 200-kV x-FEG source and a Falcon 3EC direct electron detector (Thermo Fisher Scientific). Microscope operations and data collection were carried out using EPU software (Thermo Fisher Scientific). High-resolution data collection was performed at Columbia University on a Titan Krios 300-kV (Thermo Fisher Scientific) microscope equipped with an energy filter (slit width 20 eV) and a K3 direct electron detector (Gatan). Data were collected using Leginon and at a nominal magnification of ×105,000 in electron counting mode, corresponding to a pixel size of 0.83 Å. The electron dose rate was set to 16 e−/pixel per second with 2.5-s exposures for a total dose of 50 to 60 e/A2.
Cryo-EM data processing was performed in cryoSPARC with image stacks aligned using Patch motion, defocus value estimation by Patch CTF estimation. Particle picking was performed using the template picker with templates created from preexisting cryo-EM maps. Particles were subjected to 2D classification in cryoSPARC with 100 classes. Particles from the highest-resolution classes were pooled for ab initio 3D reconstruction with a single class followed by homogeneous refinement with C4 symmetry imposed. 3D variability and further clustering were performed using a mask comprising the TM domain to separate those particles in the closed and open states followed by heterogeneous refinement with four classes to further select the best particles. C4 symmetry expansion was performed before local refinements. The masks used were TaF+TM+CTD domains (residues 4131 to 4967), calstabin-2+NTD+SPRY domains (residues 1 to 1646), JSol+CSol domains (residues 1700 to 2476, 3590 to 4130), and BSol domain (residues 2400 to 3344). Only the TaF+TM+CTD mask used C4 symmetry. Smaller masks, for a second round of 3D variability analysis, clustering, and local refinement, were RY1&2 (residues 862 to 1076), RY3&4 (residues 2685 to 2909), and BSol2 (residues 3042 to 3344). Local refinements used a dynamical mask with a far distance of 10, 50, and 150 Å for the initial masks, small masks, and expanded RY3&4 mask for comparing the effect of RY3&4 stabilization, respectively. The resulting maps were combined in ChimeraX to generate a composite map before calibration of the pixel size using correlation coefficients with a map generated from the crystal structure of the NTD of RyR2 (PDB ID: 4JKQ). The pixel size was altered by 0.001 Å per step, up to 20 steps in each direction.
An initial model of RYR2 was generated with Phenix tool sculptor from a 2.45-Å structure of RyR1 (PDB ID: 7TZC). Truncated residues were manually corrected to obtain the full side chains. Only domains RY1&2 and RY3&4, which show weak cryo-EM density and confidence in structure, were based on crystallographic structures. The initial model of RyR2 RY1&2 domain was generated with Phenix tool sculptor from the RY1&2 domain of RyR3 with ATP (PDB ID: 6UHIH). The model of RyR2 RY3&4 domain was obtained as is (PDB ID: 4ETV). Calstabin-2 and CaM models were obtained as is (PDB ID: 6JI8). Model building was performed in Coot and refined with Phenix tool RealSpaceRefine. Figures of the final structure were created using ChimeraX. cryo-EM statistics are summarized in TABLE 1A and TABLE 1B.
‡Map resolution range represents the range determined by local refinements in cryoSPARC using the masks described in Methods.
#Value obtained from Phenix validation tool.
‡Map resolution range represents the range determined by local refinements in cryoSPARC using the masks described in Methods.
This analysis determined the conformation of a certain query domain from a model X relative to reference closed and open states of RyR2. To this end, the RMSD of Ca of the query domain was measured between model X and the closed state (RMSDX-closed), and between model X and the open state (RMSDX-open). The normalized difference in RMSD was calculated as RMSDX-closed/(RMSDX-closed+RMSDX-open). A value of 0 means that the conformation of the query domain is identical to the same domain in the closed state. A value of 1 means that the conformation of the query domain is identical to the same domain in the open state. A value of 0.5 means that the conformation of the query domain is equally distant to the closed and open states. The error, considered as the intrinsic variability between atomic models, was calculated as the RMSD between aligned domains of the respective models. Propagation of error was calculated using the webserver uncertaintycalculator.com. Bar graphs were made with GraphPad Prism software. RMSD was measured in ChimeraX. When needed, previous alignment of the atomic models centered on the control domains was performed.
For cryo-EM experiments, conditions that resemble those found during the resting phase of the cardiac cycle known as diastole (150 nM free Ca12 and 10 mM ATP) were used. Under these conditions, wild-type RyR2 channels are tightly closed, but CPVT RyR2 variants are leaky. Wild-type RyR2 and RyR2-R2474S were pretreated with PKA to induce hyperphosphorylation and to mimic exercise-induced β-adrenergic signaling, which can be a trigger for fatal cardiac arrhythmias in patients with CPVT (
Across the six preparations of RyR1 subjected to cryo-EM analysis, 10 structures of human RyR2 were obtained:
˜3-Å resolution was obtained for most structures.
The highest-resolution cryo-EM coulombic potential maps (cryo-EM maps) show well-defined densities of most domains, including the NTD, SPRY1-3, JSol, BSol1, and activation core and channel domains, where near-atomic details including “holes” in aromatic residues were observed.
For the remaining dynamic domains usually missing in previously published structures (RY1&2, RY3&4, and BSol2), the local resolution was reduced but sufficient to follow the backbone and detect bulky side chains. The cryo-EM map quality was sufficient to build atomic models with a high level of confidence for the previously less well-resolved domains of RyR2 and the accessory proteins calstabin-2 (known as PPIase FKBP1B or, alternatively, as FKBP12.6) and CaM. Using this high-resolution model of the human RyR2 isoform, the residue span and nomenclature of domains and subdomains for human RyR2 were updated as shown in TABLE 2.
To separate the particles in the closed state from those in the open states in the same dataset, local three-dimensional (3D) variability, clustering, and heterogeneous refinement were performed, which rendered better results than 3D classification or heterogeneous refinement alone. This improvement was attributed to the dynamic cytosolic shell of RyR2, which, being almost 90% of the mass of the protein, can predominate and result in mixed populations.
The three-dimensional atomic coordinates as determined by cryo-EM for the RY1&2 domain in PKA phosphorylated RyR2-R2474S treated with 20M Compound 1 (P-RyR2-R2474S+Cpd1-C, residues 862-1076), are provided in TABLE 3. The three-dimensional atomic coordinates for Compound 1 and ATP bound to the RY1&2 domain of the P-RyR2-R2474S+Cpd1-C structure are provided in TABLE 4 and TABLE 5, respectively.
1atom site ID number as assigned in mmCIF file for RCSB PBD structure 7UA1;
2element symbol representing atom species;
3atom identifier assigned in mmCIF file for RCSB PBD structure 7UA1;
4encompassing residue type;
5encompassing residue number;
6-8Atom-site coordinates in angstroms specified according to a set of orthogonal Cartesian axes related to the cell axes; Isotropic atomic displacement parameter, or equivalent isotropic atomic displacement parameter, Beq, calculated from the anisotropic displacement parameters, where: Beq = (1/3) sumi[sumj(Bij Ai Aj a*i a*j)]; A = the real space cell lengths; a* = the reciprocal space cell lengths; and Bij = 8 pi2 Uij.
1-3,6-9See description for TABLE 3 above.
1-3,6-9See description for TABLE 3 above.
Comparison of the pore region from all the structures showed no differences in the closed states and no differences in the open states. The comparison suggests that the methodology implemented is reproducible.
Of the three activators present in the sample, only ATP was detected in the closed state structures (
Xanthine occupied the site formed by W4645, I4926, and Y4944. To confirm the functional role of xanthine, single-channel experiments were performed showing that, at presumed physiological concentrations during exercise (10 μM), a ˜25-fold increase was observed in the open probability of RyR2 in the presence of xanthine (see
To understand the mechanism underlying CPVT better, structures of PKA-phosphorylated RyR2 (open, closed), PKA-phosphorylated RyR2-R2474S (“primed”), PKA-phosphorylated RyR2-R2474S bound to Compound 1 (closed), and PKA-phosphorylated RyR2-R2474S bound to CaM (closed) were compared. (
As seen, the cytosolic shell of the PKA-phosphorylated RyR2 was shifted downward and outward when going from the closed state (P-RyR2-C, PDB: 7U9Q) to the open state (P-RyR2-0 PDB: 7U9R) (
The cytosolic shell of primed PKA-phosphorylated RyR2-R2474S (P-RyR2-R2474S-Pr, PDB: 7U9X) was shifted downward and outward compared to closed PKA-phosphorylated RyR2. This observation suggests that the CPVT mutant RyR2-R2474S is in a primed state (
The PKA-phosphorylated RyR2-R2474S in the presence of Compound 1 (P-RyR2-R2474S+Cpd1-C; PDB: 7UA1)—a benzothiazepine derivative that effectively lessens a likelihood of ventricular tachycardiac and sudden cardiac death in murine models of CPVT-exhibited an upward and inward shift of the cytosolic shell compared to primed PKA-phosphorylated RyR2-R2474S (P-RyR2-R2474S-Pr), thus reversing the primed state back toward the closed state of the channel (
Visual inspection of the cryo-EM maps shows conformation changes that suggest a primed state of the PKA-phosphorylated RyR2-R2474S channels (
TABLE 6 shows the differences in normalized RMSD analysis between primed PKA RyR2-R2474S vs. closed/open PKA RyR2.
TABLE 7 shows the differences in normalized RMSD analysis between Closed PKA RyR2-R2474S+Compound 1 vs. closed/open PKA RyR2.
TABLE 8 shows the differences in normalized RMSD analysis between Closed PKA RyR2 vs. closed/open dephosphorylated RyR2.
TABLE 9 shows the differences in normalized RMSD analysis between closed state with stabilized RY3&4 vs. closed state with destabilized RY3&4/open state.
In TABLES 6-9, RMSD difference values close to 0 indicated that the conformation is similar to the closed state, whereas values close to 1 indicated that the conformation is similar to the open state. Normalization allowed the direct comparison between different domains. RMSD analysis showed an average value of ˜0.7, which indicates that the cytosolic domains of the primed PKA-phosphorylated RyR2-R2474S were in nearly open positions, reducing the energetic barrier of the primed state to adopt a fully open state. Thus, the primed PKA-phosphorylated RyR2-R2474S is more readily activated and promotes an SR Ca2+ leak during diastole when the activating [Ca2+]cyt is very low (˜150 nM) and the WT channels are tightly closed.
Analyzing in detail the region of the mutation, the Arg to Ser mutation was readily identified because of the shortened side-chain density for the mutant residue S2474 compared to the WT residue R2474 (
Incubation with Compound 1 partially reversed the primed state of PKA-phosphorylated RyR2-R2474S, placing the channel in a state closer to that of the closed PKA-phosphorylated RyR2 (
Analysis of the rest of the cryo-EM map showed no additional densities that could correspond to Compound 1. Compound 1 density was absent in the particles in the open state. This observation suggests that Compound 1 and congeners thereof act by stabilizing the closed state of RyR2. In the absence of Compound 1, the RY1&2 domain of RyR2 showed a weak density that was attributed to one ATP molecule (
To ascertain the role of CaM in CPVT, the structure of closed PKA-phosphorylated RyR2+CaM (P-RyR2-C, PDB: 7U9T) and closed PKA-phosphorylated RyR2-R2474S+CaM (P-RyR2-R2474S+CaM-C, PDB: 7UA3) was analyzed.
At a free Ca2+ concentration of 150 nM, CaM binds to PKA-phosphorylated RyR2 and PKA-phosphorylated RyR2-R2474S in the extended conformation that corresponds to the apo-CaM state (
In the case of closed PKA-phosphorylated RyR2-R2474S+CaM (P-RyR2-R2474S+CaM-C, PDB: 7UA3), apo-CaM reverses the changes in the BSol2 domain of the closed state introduced by the RyR2-R2474S mutation (
To determine the effect that PKA treatment has on the structures of RyR2 and the phosphorylation RY3&4 domain, WT RyR2 was pretreated with phosphatase X to obtain a completely dephosphorylated control (
Analysis of the global structure showed that the cytosolic shell of the closed PKA-phosphorylated RyR2 (P-RyR2-C, PDB: 7U9Q) showed a small shift outward and downward compared to the closed dephosphorylated RyR2 (DeP-RyR2-C, PDB: 7UA5;
The RY3&4 phosphorylation domain has not been previously resolved in the cryo-EM structures of any RyRs due to the intrinsic dynamic behavior. Under the hypothesis that this dynamic nature of the RY3&4 domain would be necessary for interacting with modulator enzymes such as PKA and protein phosphatase 1 (PP1), 3D variability analysis centered on the BSol containing the RY3&4 domain was performed.
This 3D variability analysis revealed two distinct populations: one where the RY3&4 domain was stabilized and could be resolved, and one where there was no substantial density, suggesting that RY3&4 is detached from the BSol domain (
To improve the local cryo-EM map of the RY3&4 domain, symmetry expanded particles with the stabilized RY3&4 domain were clustered and analyzed. The resulting cryo-EM map with a local resolution of 2.90 Å allowed an atomic model of this domain to be constructed with high confidence (
The interaction between the stabilization of the RY3&4 domain and the surrounding RyR2 structure was also analyzed. Comparison to the destabilized RY3&4 domain in the closed state showed that stabilization of the RY3&4 domain increased the distance between the adjacent BSol1 and SPRY3 domains, adopting a conformation that is closer to the open conformation (
Two intramembrane helices laterally positioned and encircling the TM domain were observed.
Previous cryo-EM analyses of RyR2 attributed these densities to detergent and lipids. These auxiliary helices (Sx) were predicted to exist in the RyR2 sequence upstream of the six transmembrane helices that form the pore (S1 to S6). The Jpred4 algorithm was used to predict the secondary structure of the fragment, which was previously reported to encompass these helices.
Predicted Sx helices were modeled because of the presence of well-resolved densities for large side chains, including W4288 as well as several tyrosine and phenylalanine residues (
In CPVT patients, SR Ca2+ leak occurs via mutant RyR2 channels during diastole when the heart is supposed to be electrically silent, resulting in afterdepolarizations, arrhythmias, and eventually sudden cardiac death. Intense exercise and adrenergic stimulation can cause two independent but synergistic events that affect RyR2. The β-adrenergic response to exercise (i) results in PKA phosphorylation of RyR2 mainly at S2808 and (ii) activates SR Ca2+ uptake via SERCA2a, thus increasing the SR Ca2+ load, and the driving force for Ca2+ leak out of the membrane via RyR2 channels. By virtue of being in a primed state, the CPVT variant RyR2-R2474S is likely more sensitive to channel-activating posttranslational modifications (e.g., phosphorylation, nitrosylation or oxidation), resulting in a diastolic SR Ca2+ leak that can trigger fatal cardiac arrhythmias during intense exercise (
As demonstrated herein, the CPVT mutation R2474S can put the channel into a primed state that is independent of the binding of activators including Ca2+. Thus, the mutant CPVT channel RyR2-R2474S is able to be inappropriately activated during diastole when the [Ca2+]cyt is too low to activate the WT RyR2 channel. This inappropriate activation of the mutant channel results in diastolic SR Ca2+ leak that can trigger fatal cardiac arrhythmias. Stabilizing effects of Compound 1 and CaM through different mechanisms are demonstrated herein. One involves the stabilization of the BSol domain through the interaction with the Compound 1-bound RY1&2 domain, and the other occurs via the stabilization of the CSol and BSol3 domains through the binding of CaM to CAMBD2 and the BSol domain.
As demonstrated herein, the CPVT mutation can disrupt local interactions that destabilize the BSol domain and induces a primed state. This primed state leads to inappropriate opening of RyR2 channels that can be reversed by treatment with the RyR2 stabilizer Compound 1. Compound 1 binds to a cleft in the RY1&2 domain where can stabilize interactions between residues that are required to reduce flexibility between domains, particularly with the BSol1 domain, of the cytosolic shell. The net effect of Compound 1 binding is to stabilize the overall channel structure closer to the closed state. This renders the channel less likely to be inappropriately activated during diastole when the conditions favor the closed state of the channel (e.g., low non-activating [Ca2+]cyt).
To confirm the functional role of xanthine in RyR2, single-channel experiments were performed. ER vesicles from HEK293 cells expressing RyR2 were prepared by homogenizing cell pellets on ice using a Teflon glass homogenizer with two volumes of solution containing 20 mM tris-maleate (pH 7.4), 1 mM EDTA, 1 mM DTT, and protease inhibitors (Roche). Homogenate was then spun by centrifuge at 4000 g for 15 min at 4° C., and the resulting supernatant was spun by centrifuge at 40,000 g for 30 min at 4° C. The final pellet, containing the ER fractions, was resuspended and aliquoted in 250 mM sucrose, 10 mM Mops (pH 7.4), 1 mM EDTA, 1 mM DTT, and protease inhibitors. Samples were frozen in liquid nitrogen and stored at −80° C.
ER vesicles were fused to planar lipid bilayers formed by painting a lipid mixture of phosphatidylethanolamine and PC (Avanti Polar Lipids) in a 5:3 ratio in decane across a 200-am hole in polysulfonate cups (Warner Instruments) separating two chambers. The trans chamber (1.0 ml), representing the intra-SR (luminal) compartment, was connected to the head stage input of a bilayer voltage clamp amplifier. The cis chamber (1.0 ml), representing the cytoplasmic compartment, was held at virtual ground. The following asymmetrical solutions were used: for the cis solution, 1 mM EGTA, 250/125 mM Hepes/tris, 50 mM KCl (pH 7.35); for the trans solution, 53 mM Ca(OH)2, 50 mM KCl, 250 mM Hepes (pH 7.35). The concentration of free Ca2+ in the cis chamber was calculated as previously described. ER vesicles were added to the cis side, and fusion with the lipid bilayer was induced by making the cis side hyperosmotic by the addition of 400 to 500 mM KCl. After the appearance of potassium and chloride channels, the cis side was perfused with the cis solution. At the end of each experiment, 10 μM ryanodine was added to block the RyR2 channel. Single-channel currents were recorded at 0 mV using Bilayer Clamp BC-525D (Warner Instruments), filtered at 1 kHz using Low-Pass Bessel Filter 8 Pole (Warner Instruments), and digitized at 4 kHz. All experiments were performed at room temperature (23° C.). Data acquisition was performed by using Digidata 1322A and Axoscope 10.1 software (Axon Instruments). The recordings were analyzed using Clampfit 10.1 (Molecular Devices) and GraphPad Prism software.
This experiment determined that at presumed physiological concentrations during exercise (10 μM), there was a ˜25-fold increase in the open probability of RyR2 in the presence of xanthine
Ryr2R2474S/WT mice undergo sustained ventricular tachycardia and sudden cardiac death after exercise and epinephrine. This experiment evaluated the effect of Compound 1 treatment on frequency of ventricular tachycardia and sudden cardiac death in Ryr2R2474S/WT mice.
Ryr2R2474S/WT mice were implanted with radio telemetry transmitters (Data Sciences International). Briefly, the transmitter (PhysioTel, ETA-F10 transmitter) was inserted in mice subcutaneously along the back under general anesthesia (5% inhaled isoflurane). Two electrocardiogram (ECG) electrodes were placed hypodermically in the region of the right shoulder (negative pole) and toward the lower left chest (positive pole) to approximate lead II of the Einthoven surface ECG. During the procedure, respiratory and cardiac rhythm, adequacy of anesthetic depth, muscle relaxation, body temperature, and analgesia were monitored to avoid anesthesia-related complications. Postoperative pain was considered during a 1-week post-implantation period, and carprofen (5 mg/kg, subcutaneously) was given. A minimum period of 2 weeks was allowed for recovery from the surgery. Animals were housed in individual stainless-steel cages for telemetry recordings. Environmental parameters were recorded continuously and maintained within a fixed range: room temperature at 15° C. to 21° C. and 45 to 65% relative humidity. The artificial day/night cycle was 12-hour light/12-hour dark with light on at 0700 hours. Drinking water was provided ad libitum. Solid diet (300 g) was given daily in the morning. ECG waveforms were continuously recorded at a sampling rate of 2000 Hz using a signal transmitter-receiver (RPC-1) connected to a data acquisition system (Ponemah system, Data Sciences International). Compound 1 (50 mg/kg per day) was given in drinking water for 2 weeks before ECG recordings. Mice were recorded for 1 hour at baseline and then given epinephrine injection (1 mg/kg, intraperitoneally) and recorded for another 2 hours. The animals used in the study were maintained and studied according to protocols approved by the Institutional Animal Care and Use Committee of Columbia University (reference no. AC-AABP1551).
This experiment determined that treatment with Compound 1 reduced the frequency of ventricular tachycardia and prevented sudden cardiac death in Ryr2R2474S/WT mice.
Ca2+ leak was measured in microsomes from heart lysates isolated from control Ryr2R2474S/WT mice, Ryr2R2474S/WT mice treated with epinephrine, and Ryr2R2474S/WT mice treated with epinephrine and Compound 1 in EXAMPLE 8. 10704.1 Cardiac muscle SR microsomes were prepared by homogenizing heart samples on ice using a Teflon glass homogenizer (50 times) with 2 volumes of 20 mM tris-maleate (pH 7.4), 1 mM EDTA, 1 mM DTT, and protease inhibitors (Roche). Homogenate was then spun by centrifuge at 4000 g for 15 min at 4° C., and the resultant supernatant was spun by centrifuge at 50,000 g for 45 min at 4° C. Pellets were resuspended in lysis buffer containing 300 mM sucrose. Microsomes (5 g/ml) were diluted into a buffer (pH 7.2) containing 8 mM K-phosphocreatine, and creatine kinase (2 U/ml), mixed with 3 μM Fluo-4 and added to multiple wells of a 96-well plate. Ca2+ loading of the microsomes was initiated by adding 1 mM ATP. After Ca2+ uptake (50 s), 3 μM thapsigargin was added to inhibit the Ca2+ reuptake by SERCA. SR Ca2+ leak was measured by the increase in intensity of the Fluo-4 signal (measured in a Tecan fluorescence plate reader). Ca2+ leak was quantified as the difference between the average Fluo-4 signal before and after addition of thapsigargin. Graphs were plotted with GraphPad Prism software.
This experiment determined that Compound 1 treatment prevented SR Ca2+ leak via RyR2-R2474S channels.
Mass spectroscopic analysis of the hyperphosphorylated channels prepared in EXAMPLE 5 was conducted to determine phosphorylation sites.
ER vesicles from HEK293 cells expressing RyR2 were separated on 4 to 12% gradient SDS-PAGE and sent for mass spectrometry analysis to in-house Columbia Proteomics Shared Resource (HICCC). Protein gel slices were excised, and in-gel digestion was performed. Digested peptides were collected and further extracted from gel slices in extraction buffer (1:2 ratio by volume of 5% formic acid:acetonitrile) at high speed, shaking in an air thermostat. Peptides were separated within 80 min at a flow rate of 400 nl/min on a reversed-phase C18 column with an integrated CaptiveSpray Emitter (25 cm×75 m, 1.6 m, IonOpticks). Mobile phases A and B were with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The fraction of B was linearly increased from 2 to 23% within 70 min, followed by an increase to 35% within 10 min and a further increase to 80% before reequilibration. The timsTOF Pro was operated in parallel accumulation-serial fragmentation (PASEF) mode with the following settings: mass range, 100 to 1700 mass/charge ratio (m/z); 1/K0 start, 0.6 V s/cm2; end, 1.6 V s/cm2; ramp time, 100 ms; lock duty cycle to 100%; capillary voltage, 1600 V; dry gas, 3 l/min; and dry temperature, 200° C. PASEF settings: 10 Tandem Mass Spectrometry frames (1.16 s duty cycle); charge range, 0 to 5; active exclusion for 0.4 min; target intensity, 20,000; intensity threshold, 2500; and collision-induced dissociation collision energy, 59 eV. A polygon filter was applied to the m/z and ion mobility plane to select features most likely representing peptide precursors rather than singly charged background ions. Acquired PASEF raw files were analyzed using the MaxQuant environment v.2.0.1.0 and Andromeda for database searches. MaxQuant was configured to search with the reference human proteome database downloaded from UniProt. The following modifications were used for protein identification and quantification: Carbamidomethylation of cysteine residues (+57.021 Da) was set as static modifications, while the oxidation of methionine residues (+15.995 Da), deamidation (+0.984) on asparagine and glutamine, and phosphorylation (+79.966) on serine, threonine, and tyrosine were set as a variable modification. Results obtained from MaxQuant, Phospho (STY) sites table was used for RyR2 phospho-site quantification. Sequence coverage was obtained with the software Scaffold 5.
TABLE 10 and TABLE 11 are mass-spectroscopy tables indicating the phosphopeptides detected before and after PKA phosphorylation of recombinant human RyR2 samples.
2808
The analysis revealed that RyR2-S2808 is the main detected phosphorylated peptide before and after PKA phosphorylation. Other detected residues, with good localization probability and score difference, were considered secondary phosphorylation sites (TABLE 10 and TABLE 11, underlined values). Mass-spectrometry is a semi-quantitative technique, indicating that intensities can be only compared within the same sample (within the same column).
The results showed that RyR2-S2808 was the major and only significantly PKA-phosphorylated site in RyR2 treated with PKA (coverage of 75%;
This application claims the benefit of U.S. Provisional Patent Application No. 63/286,861, filed on Dec. 7, 2021, the content of which is incorporated by reference herein in its entirety.
This present disclosure was made with government support under R01HL1 R01HTL145473, R01DK118240, R01HTL142903, R01HTL140934, R01AR070194, R25HL156002R25, R25NS076445 and T32 HL120826, awarded by the National Institutes of Health (NIH). The government has certain rights in the disclosure.
| Number | Date | Country | |
|---|---|---|---|
| 63286861 | Dec 2021 | US |
