Method for analyzing drug molecules in mammalian tissue - Patents.com

Abstract

The present invention provides novel methods for analyzing (eg, imaging) drugs in mammalian tissues, as well as methods for identifying drug targets and analyzing drug-target interactions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 321,294, filed March 18, 2022, currently pending. The entire disclosure of the priority application is incorporated herein by reference in its entirety for all purposes.

[0002] Government support statement This invention was made with Government support under Grant Nos. DK128800, DK114165 and DK124731 awarded by the National Institutes of Health. The Government has certain rights in the invention. [Background technology]

[0003] 2. Background of the Invention Small molecule target identification and validation has been a long-standing challenge in chemical biology and drug discovery. Although significant progress has been made to explore drug-target interactions at the molecular scale, these approaches are generally not well suited to such relationships at the tissue level in vivo. Traditional pharmacokinetics and pharmacodynamics often quantify drug concentrations or drug-target interactions in homogenized organs, losing spatial resolution that is crucial for understanding target binding in vivo. Imaging-based methods such as positron emission tomography (PET) are widely used to characterize small molecule distribution in vivo, but lack sufficient resolution to distinguish drug binding states at the cellular level to accurately identify drug-target interactions. An ideal method should allow in situ visualization of drugs bound to their targets at single-cell resolution while simultaneously being compatible with multiplexed molecular characterization of those drug-target interactions. These goals are particularly important for drugs targeting the central nervous system, where cellular composition and spatial structure are significantly heterogeneous.

[0004] Fluorescence optical microscopy has revolutionized high-resolution in situ imaging of endogenous biomolecules such as proteins and nucleic acids. However, exogenous small molecules are more challenging to image because the addition of a fluorescent tag alters the size and chemical properties of the parent compound, potentially distorting drug distribution as well as on- and off-target binding. Biorthogonal reactions, including the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction, can partially address these issues by using small, inert alkyne / azide handles to allow the incorporation of larger tags after the drug has reached its target. This click chemistry (CC) strategy coupled with chemical proteomics methods such as activity-based protein profiling (CC-ABPP) has proven effective in identifying drug targets in a variety of biological contexts, including animal models, but such approaches have not yet enabled high-resolution spatial imaging of drug-target interactions in vivo due to the low signal-to-noise ratio (SNR) in intact tissues.

[0005] There is a need in the art for more effective tools to observe drug-target interactions at cellular resolution, especially in intact tissues. The present invention aims to address these and other unmet needs. Summary of the Invention

[0006] In one aspect, the present invention provides methods for analyzing drug molecules or their metabolites in mammalian tissue. These methods involve (a) obtaining a tissue sample from a mammalian subject administered a drug molecule, (b) clearing the tissue sample with a delipidizing agent, (c) performing a CuAAC reaction with the cleared tissue sample to label the drug molecule, and (d) examining the drug molecule in the tissue sample with an analytical means. In some methods, the drug molecule to be analyzed is a small molecule. In some methods, the drug molecule to be analyzed is a covalent drug. In different embodiments, the delipidizing agent used can be a hydrogel-based tissue clearing agent, a hydrophobic tissue clearing agent, a hydrophilic tissue clearing agent, or a combination thereof.

[0007] In some preferred embodiments, the cleared tissue sample is subjected to a CuAAC click reaction to label the drug molecule with a fluorophore. In some embodiments, the CuAAC reaction used in the methods of the invention is optimized. In some of these embodiments, the optimized CuAAC reaction uses a CuAAC concentration of about 100 μM to about 150 μM. 2+ Use a concentration. In some embodiments, the CuAAC reaction is optimized by including pre-incubation of cleared tissue before starting the click reaction. In some embodiments, the CuAAC reaction is optimized with a click reaction ligand that improves the signal-to-noise ratio. In various embodiments, the click reaction ligand used can be, for example, BTTP, THPTA or BTTAA.

[0008] Typically, the drug molecule or its metabolite to be analyzed by the method of the present invention contains an alkyne group or an alkyne analogue suitable for click reaction.In some preferred embodiments, the analytical means used to examine the drug molecule in the cleared and click reaction labeled tissue sample is an imaging means.For example, some preferred methods of the present invention can use confocal microscopy to examine the labeled drug molecule in the tissue sample.

[0009] In addition to testing for a drug molecule in a cleared and click-reaction labeled tissue sample, some methods of the invention can further include staining the tissue sample with an agent against a cell type known or suspected to express the target of the drug molecule. In some of these embodiments, the stain used is an antibody or nucleic acid probe specific for the cell type.

[0010] In a related aspect, the present invention provides methods for identifying targets of drug molecules in mammalian tissue. These methods include (a) obtaining a tissue sample from a mammalian subject administered a drug molecule, (b) clearing the tissue sample with a delipidizing agent, (c) performing a CuAAC reaction with the cleared tissue sample to label the drug molecule, and (d) examining the drug molecule in the tissue sample with an analytical means to identify targets of the drug molecule in the tissue. Some of these methods are directed to identifying targets of small molecule drug molecules. Some of these methods are directed to identifying targets of covalently bound drug molecules. In various embodiments, the tissue clearing agent used is a hydrogel-based tissue clearing agent, a hydrophobic tissue clearing agent, or a hydrophilic tissue clearing agent. In some preferred embodiments, the CuAAC reaction for labeling the drug molecule is optimized. In various embodiments, the binding targets in the tissue sample can be identified, for example, by immunostaining, RNA hybridization, or spatially-resolved molecular characterization means.

[0011] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. [Brief description of the drawings]

[0012] [Figure 1]Optimization of click reaction ligands using CLARITY brain slices. TBTA is at 90 μM. For all other ligands, the copper-ligand ratio is 1:2. Reactions under CC-ABPP conditions (TBTA) produce high background in vehicle controls. At 50 μM CuSO4, only BTTP gives faint labeling. Increasing CuSO4 concentration to 100 and 150 μM gives robust labeling without significant background labeling. At least 100 μM CuSO4 is required. 150 μM CuSO4, 300 μM BTTP were used in all CATCH studies unless otherwise stated. Images represent primary somatosensory cortex (S1). Scale bar, 20 μm. [Diagram 2] CATCH labeling in tissues PFA-fixed and cleared by hydrogel-based clearing techniques (CLARITY and SHIELD). Images represent S1. Scale bar, 20 μm. [Diagram 3] For efficient and uniform drug labeling, CATCH requires preincubation of tissue samples overnight in click incubation buffer (5 μM Alexa647-picolyl-azide, 150 μM CuSO4, 300 μM BTTP, 10% DMSO in PBS) before the reaction begins. The tissue samples are then transferred to new incubation buffer. Sodium ascorbate (NaAsb) is added to the incubation buffer to obtain click reaction buffer (5 μM Alexa647-picolyl-azide, 150 μM CuSO4, 300 μM BTTP, 2.5 mM NaAsb, 10% DMSO in PBS). Images represent S1. Scale bar, 20 μm. [Figure 4] CATCH is compatible with drug imaging in the liver and small intestine. Scale bar, 50 μm. [Diagram 5] CATCH is compatible with molecular marker staining for cell type identification. The figure shows PF7845-yne in S1-bound NeuN-positive neurons, but not in lectin-positive blood vessels. Images represent S1. Scale bar, 10 μm. [Figure 6]CATCH is compatible with reversible molecular marker staining. The drug-fluorophore conjugate is resistant to antibody elution (A). After primary antibody staining, NeuN staining can be removed without compromising click-labeled pargyline-in positive cells. The tissue is restained with TH antibody for reversible, multiplexed cell type identification (B). Images represent bridges. Scale bar, 10 μm. [Figure 7] CATCH can resolve drug binding with subcellular resolution. Shown is MJN110-yne binding in presynaptic terminals marked by synpasin staining and MJN110-yne binding to monoacylglycerol lipase (MAGL). Figure represents hippocampal CA1. Scale bar, 10 μm. [Figure 8] CATCH can enable spatially resolved high resolution pharmacology. Schematic of dose-dependent in vivo blockade study. At low doses, the parent drug does not completely block the target, leaving room for subsequent alkyne drug binding. At high doses, the parent drug saturates the target, thereby abolishing alkyne drug binding (A). Zoomed-out representation of PF7845 blockade at the indicated doses followed by PF7845-in injection (1 mg / kg, 1 h, i.p.) in the hippocampus and cortex. PF7845-in in the cortex is completely blocked with 0.02 mg / kg PF7845. However, there is still residual unoccupied FAAH in the hippocampus. Scale bar, 500 μm (B). Schematic of dose-dependent direct alkyne drug mapping. Higher target saturation should be associated with higher CATCH labeling intensity (C). Zoomed-out representation of PF7845-in binding kinetics in the hippocampus and cortex (4 h, i.p.). PF7845-in saturates FAAH in the cortex but not in the hippocampus at 0.1 mg / kg. Scale bar, 500 μm (D). [Figure 9]CATCH can resolve potential drug off-target binding regions. PF7845 is considered a safe FAAH inhibitor, whereas BIA10-2474 is a FAAH inhibitor with clinical neurotoxicity. A dose of 5 mg / kg was used for both drugs to maximize contrast for target visualization. Full coronal section at bregma-4.4 with zoom views in the periaqueductal gray (PAG) and pontine reticular tegmental nucleus (RtTg). BIA10-2474-in (5 mg / kg, 4 h, i.p.) binds to large cellular structures in wild-type but not FAAH- / - mouse PAG, suggesting primarily on-target binding in the PAG. Cytoarchitectures can be found in the PAG of FAAH- / - mice treated with BIA10-2474-in, but not in the RtTg of FAAH- / - mice treated with PF7845-in, indicating off-target binding. For better visualization, image brightness / contrast is optimized for each brain region. Scale bar, 100 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Detailed Description I. Overview A comprehensive understanding of in vivo efficacy and toxicity with high spatial resolution remains a major unmet challenge to realizing the full potential of chemical probes and drugs. Currently, it is not possible to determine drug binding across the whole animal (multiple organ systems) at cellular and molecular resolution in mammals. Biochemical methods such as chemical proteomics provide excellent molecular details of drug-target interactions but do not have the spatial resolution to identify tissue and cellular targets. Currently, imaging methods such as PET resolve larger tissue distributions but are insufficient to assess molecular and cellular specificity.

[0014] The present invention is based in part on the research carried out by the inventors to develop a method for optically imaging covalent drug targets in intact mammalian tissues. The method, called Clearing Assisted Tissue Click Chemistry (CATCH), allows specific and robust in situ fluorescence imaging of drug molecules bound to targets at subcellular resolution, allowing identification of target cell types. As exemplified herein, using well-established inhibitors of endocannabinoid hydrolase and monoamine oxidase, direct or competitive CATCH not only reveals the distinct anatomical distribution and primary cellular targets of different drug compounds in mouse brain, but also reveals unexpected differences in drug binding across and within brain regions, reflecting rare cell types and dose-dependent target shifts across tissues, cells and subcellular compartments that are inaccessible by conventional methods. CATCH is a valuable and differentiated platform for investigating small molecule-target interactions in vivo.

[0015] Thus, the present invention provides a novel method for analyzing drug molecules bound to targets, and related methods for identifying drug targets in tissues. Detailed steps and protocols for carrying out these methods are described in detail herein. As demonstrated herein, the methods of the present invention can be used to analyze (e.g., image) drugs at subcellular resolution in native tissues. Binding targets can then be identified using immunostaining, RNA hybridization or other spatially resolved molecular characterization approaches. The same platform will be able to confirm the in vivo tissue distribution and target identity of drugs to obtain comprehensive on-target and off-target maps of candidate drugs.

[0016] The present invention may employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology that are within the skill of those in the art, and such techniques are fully explained in the literature. (For example, Sambrook et al, ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al, ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DNGlover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press)(1986);Perbal(1984)A Practical Guide To Molecular Cloning;the treatise,Methods In Enzymology(Academic Press,Inc.,NY);Miller and Calos eds.(1987)Gene Transfer Vectors For Mammalian Cells,(Cold Spring Harbor Laboratory);Wu et al,eds.,Methods In Enzymology,Vols.154 and 155;Mayer and Walker,eds.(1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); and in Ausubel et al. (1989) Current Protocols in Molecular Biology (see John Wiley and Sons, Baltimore, Md.). .

[0017] II. Definition The following sections provide more detailed guidance for making and using the compositions of the present invention, as well as for carrying out the methods of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in the present invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1999); st ed.,1992);Oxford Dictionary of Biochemistry and Molecular Biology,Smith et al.(Eds.),Oxford University Press(revised ed.,2000);Encyclopaedic Dictionary of Chemistry,Kumar(Ed.),Anmol Publications Pvt.Ltd.(2002);Dictionary of Microbiology and Molecular Biology,Singleton et al. al.(Eds.),John Wiley&Sons(3 rded.,2002);Dictionary of Chemistry,Hunt(Ed.),Routledge(1 st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4 th ed., 2000). Additionally, the following definitions are provided to assist the reader in the practice of the present invention.

[0018] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude elements that may be optionally present. As such, this statement is intended to serve as a prelude to the use of exclusive terminology, such as "solely," "only," and the like, or for the use of a "negative" limitation in connection with the recitation of claim elements.

[0019] As used herein, the term "alkyne-based compound" refers to a small molecule drug or polymeric molecule that contains at least one reactive alkynyl group. An "alkynyl group" is an unsaturated, linear or branched or cyclic hydrocarbon group consisting of at least one carbon-carbon triple bond. In one embodiment, the alkyne-based substrate preferably contains at least one terminal alkynyl group (-C≡CH).

[0020] As used herein, the term "azide-based substrate" refers to a small molecule or macromolecule that contains at least one azide group. Substrates contemplated in the present invention may include soluble or solid-immobilized, such as surface-immobilized, reagents.

[0021] The terms "specific binding" or "specifically binds to" or "specific for" refer to binding of a binding moiety to a binding target, such as binding of an immunoglobulin to an epitope on a target antigen, e.g., a particular polypeptide, peptide or other target (e.g., a glycoprotein target), and mean binding that is measurably different from a non-specific interaction (e.g., a non-specific interaction can be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining binding of a binding moiety or immunoglobulin to a target molecule compared to binding to a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., excess unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. As used herein, the terms "specific binding" or "specifically binds to" or "specific for" an epitope on a particular polypeptide or particular polypeptide target refers to a K for the target of, for example, at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM or more. d In one example, the term "specific binding" refers to binding by a molecule to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0022] The terms "conjugate," "conjugated," and "conjugation" refer to any form of covalent or non-covalent binding, including, but not limited to, direct genetic or chemical fusion, coupling via a linker or cross-linker, and non-covalent association.

[0023] As used herein, the term "tissue clearing" refers to a process that has the effect of adjusting, aligning, or homogenizing the refractive index (RI) of tissue, generally resulting in increased tissue transparency. Tissue transparency can be quantitatively determined by measuring light transmission through the tissue or by optical absorption spectroscopy, such as confocal microscopy.

[0024] The term "refractive index" or "RI" as used herein refers to the ratio of the velocity of radiation (such as electromagnetic radiation or light) in one medium (such as air, glass or vacuum) to the velocity of the radiation in another medium.

[0025] The term "derivative" refers to a chemical compound / moiety that is similar in structure to the parent compound / moiety, but has a structure that differs from the parent compound / moiety with respect to one or more components, functional groups, atoms, etc. A derivative may be formed from a parent compound / moiety by chemical reaction(s). The difference between a derivative and a parent compound / moiety may include, but is not limited to, the replacement of one or more functional groups with one or more different functional groups, or the introduction or removal of one or more substituents of a hydrogen atom. In some forms, a derivative may also differ from a parent compound / moiety with respect to protonation state. In some forms, a derivative may be derived from a parent compound / moiety via an acid-base reaction. Preferably, a derivative retains the biological activity of the parent compound / moiety, such as at least 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65% and 60% of the biological activity of the parent compound / moiety. In some forms, a derivative has a higher activity compared to the parent compound / moiety.

[0026] The term "target" or "binding target" is used in the broadest sense and specifically includes, but is not limited to, polypeptides, nucleic acids, carbohydrates, lipids, cells and other molecules, with or without their naturally occurring biological function. In some preferred embodiments herein, the target is a cell surface antigen or receptor.

[0027] The term "antigen" refers to an entity or fragment thereof that can bind to an immunoglobulin or trigger a cellular immune response. Immunogen refers to an antigen that can elicit an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term antigen includes regions known as antigenic determinants or epitopes, as defined above.

[0028] III. Tissue clearing and defatting The present invention provides methods for analyzing (e.g., imaging in situ) drug molecules and for detecting drug targets in mammalian tissues with cellular resolution. Drug molecules suitable for analysis in the methods of the present invention can be of any chemical nature. These include, for example, small molecule compounds, peptides, proteins, antibodies, nucleic acids, lipids, polysaccharides, or any combination thereof. In some embodiments, the methods of the present invention relate to the analysis of small molecule drugs. In some preferred embodiments, the drug molecules are alkyne-based compounds that contain an alkyne group or its analogues suitable for click chemistry reactions. In some preferred embodiments, the drug molecules to be analyzed in the methods of the present invention are covalent drugs. Covalent drugs interfere with protein function by forming specific bonds between a ligand and a target protein. Covalent mechanisms of action can offer many pharmacological advantages compared to reversible mechanisms of action, including enhanced potency, selectivity, and long duration of action.

[0029] According to the method of the present invention, first, tissue sample is obtained from mammalian subject administered with drug molecule.Then, tissue sample is cleared with delipidizing agent.The cleared tissue sample is then subjected to CuAAC reaction to label drug molecule.Further analysis, such as imaging, can be carried out to analyze drug molecule (e.g., its spatial distribution) in situ and / or to identify the target of drug molecule at cellular or subcellular resolution.

[0030] Delipidation of tissue samples isolated from a subject can be performed using a variety of techniques well known and routinely practiced in the art. In general, these include hydrogel-based tissue clearing, hydrophobic-based tissue clearing, hydrophilic tissue clearing, and other hybrid tissue clearing methods that combine, for example, hydrogel-based and hydrophobic-based tissue clearing. In some embodiments, delipidation to clear tissue can be performed using methods specifically demonstrated herein, such as HYBRiD, CLARITY, and SHIELD. Hydrogel-based tissue clearing involves embedding tissue samples on a hydrogel matrix to create crosslinked tissue / gel hybrids containing fixed proteins and RNA. These methods use ionic detergents for delipidation, which can be enhanced by electrophoresis without causing significant loss of biomolecules. For example, Chung et al.,Nature 497,332-337,2013;Murray et al.,Cell 163,1500-1514,2015;Ku et al.,Nat.Biotechnol.34,973-981,2016;Woo et al. al.,Exp.Mol.Med.48,e274,2016;Woo et al.,Biochem.Biophys.Res.Commun.524,346-353,2020;Woo et al.,Int.J.Mol.Sci.22,2892,2021;Gradinaru et al. al., Annu. Rev. Biophys.47, 355-376, 2018; and Choi et al., Cell 184, 4115-4136, 2021. Hydrogel-based methods have the advantages of preserving endogenous FP signals, retaining a higher proportion of biomolecules, and better preserving the anatomical morphology and ultrastructure of the brain. As a drawback, hydrogel-based methods usually involve longer and more complicated protocols (compared to hydrophobic and hydrophilic methods) that require in situ hydrogel polymerization. Hydrogel-based methods may also require active (electrophoretic) labeling and clearing.

[0031] The methods of the present invention can also utilize hydrophobic (organic solvent-based) clearing protocols. These methods rely on tissue dehydration, delipidation and permeabilization using organic solvents such as ethanol, methanol, tetrahydrofuran (THF) and tert-butanol. See, for example, Dodt et al., Nat. Methods 4, 331-336, 2007; Renier et al., Cell 159, 896-910, 2014; Erturk et al., Nat. Protoc. 7, 1983-1995, 2012; and Pan et al., Nat. Methods 13, 859-867, 2016. Most of the known protocols for hydrophobic clearing involve simple tissue immersion in a stepwise series of increasing solvent concentrations (passive clearing). However, active clearing protocols, in which the solvent is perfused through the circulatory system, allow for the clearing of entire rodent bodies and large organs such as the human brain. Further incubation in dichloromethane (DCM) may improve tissue delipidation in large samples at the expense of reducing fluorescence from fluorescent proteins (FPs). After dehydration, samples are immersed in a clearing solution consisting of a mixture of benzyl alcohol and benzyl benzoate (BABB) or dibenzyl ether (DBE) to achieve RI homogenization and, consequently, transparency. Hydrophobic-based tissue clearing offers an extremely rapid and reproducible protocol that requires simple immersion. Hydrophobic-based tissue clearing has higher clearing performance (transparency). However, in hydrophobic-based tissue clearing, endogenous FP signals are lost. There is also the possibility of complete loss of lipids and associated biomolecules as well as loss of proteins and nucleic acids. Furthermore, hydrophobic-based tissue clearing may require solvent-resistant materials and optics and involves the use of volatile and toxic solvents.

[0032] Tissue clearing in the method of the present invention may also utilize hydrophilic reagents. Examples of suitable hydrophilic reagents include various detergents for permeabilization and delipidation (e.g., Triton-X100, saponin, sodium dodecyl sulfate (SDS), etc.), as well as high refractive index aqueous solutions containing sugars (fructose, sucrose, sorbitol), urea, amino alcohols, or different combinations thereof. See, for example, Ke et al., Nat. Neurosci. 16, 1154-1161, 2013; Hama et al., Nat. Neurosci. 14, 1481-1488, 2011; Susaki et al., Cell 157, 726-739, 2014; Kuwajima et al., Development 140, 1364-1368, 2013; Yu et al., Sci. Rep. 8, 1964, 2018; and Zhu et al., Proc. Natl. Acad. Sci. USA 116, 11480-11489, 2019. Hydrophilic tissue clearing protocols have the advantages of preservation of endogenous FP signals, use of safer reagents, and compatibility with standard materials and optics. The drawbacks are that hydrophilic tissue clearing may require longer incubation times and protocols using high detergent concentrations may result in loss of lipids and associated biomolecules, proteins and nucleic acids.

[0033] In some embodiments of the present invention, delipidation to make tissues transparent can be performed using certain tissue clearing protocols exemplified herein. For example, delipidation of some tissue samples can be performed using CLARITY (clear lipid-exchanged acrylamide-hybridized rigid imaging-compatible tissue-hydrogel), a hydrogel-based tissue clearing technology exemplified herein (see, e.g., Chung et al., Nature 497, 332-337, 2013). CLARITY converts intact tissues into a fully assembled but optically transparent and polymer-permeable nanoporous hydrogel-hybridized (crosslinked into a three-dimensional network of hydrophilic polymers) form. CLARITY allows in situ hybridization, immunohistochemistry and antibody labeling of intact tissues. This allows for microstructural analysis of clinical samples in a form suitable for probing the basis of physiological function and disease. With CLARITY, as specifically exemplified herein, fixed tissues (e.g., brain tissue samples) undergo incubation and agitation with A1P4 hydrogel. This is followed by degassing and washing prior to the labeling reaction. In some embodiments, another hydrogel-based tissue clearing method described herein, SHIELD (stabilization under harsh conditions via intramolecular epoxide linkages to prevent degradation), can be used. This delipidation method uses epoxide monomers to form controlled intra- and intermolecular crosslinks with biomolecules. SHIELD preserves protein fluorescence and antigenicity, transcripts and tissue structure under a wide range of harsh conditions. See, e.g., Park et al., Nat. Biotechnol. 37, 73-83, 2019.In some other embodiments, hydrophobic tissues can be cleared by fDISCO and iDISCO, exemplified herein, which use THF and methanol to remove lipid components. More detailed guidance for these two tissue clearing protocols is provided in published literature, e.g., Renier et al., Cell 159, 896-910, 2014; Qi et al., Sci.Adv.5, eaau8355, 2019; and Erturk et al., Nat.Protoc.7, 1983-1995, 2012. In yet some other embodiments, hydrophilic tissue clearing can be performed using CUBIC3.0 to remove lipids using water-soluble detergents. This tissue clearing method can be performed as described herein or in published literature (e.g., Tainaka et al., Cell Rep 24, 2196-2210, 2018).

[0034] IV. Labeling drug molecules in cleared tissue samples via click reaction Once the tissue sample has been cleared and delipidated with any of the above protocols, the tissue sample is then subjected to labeling of drug molecules or their metabolites present in the tissue sample. Labeling is accomplished by attaching or immobilizing a detectable label (e.g., a fluorescent agent) to the drug molecule or its metabolite. In some preferred embodiments, the drug molecule or its metabolite is labeled with a fluorophore via a click chemistry reaction (or click reaction). Click chemistry is defined as any chemical reaction that allows high yields, produces no by-products or by-products that are easily removed, is stereospecific, gives a physiologically stable product, exhibits a large thermodynamic driving force, and has simple reaction conditions. See, for example, Kolb et al., Angew. Chem., Int. Ed. 2001, 40, 2004-2021; Kolb et al., Drug Discovery Today 2003, 8, 1128-1137. Unless otherwise stated, the click reaction used in the practice of the present invention is a CuAAC reaction. The CuAAC click reaction is well known and routinely performed in the art. The CuAAC click reaction is a type of Huisgen 1,3-dipolar cycloaddition based on the formation of 1,4-disubstituted [1,2,3] triazoles between terminal alkynes and aliphatic azides in the presence of copper. This reaction has found increasing use in all aspects of drug discovery in medicinal chemistry, including for generating lead compounds through combinatorial methods. Bioconjugation via click chemistry has been rigorously used in proteomics and nucleic acid research. The click reaction is used to label and detect molecules in samples that are compromised by direct labeling or antibody-based secondary detection techniques. The click labels are small enough to easily penetrate complex samples, and the selectivity and stability of the click reaction provide high sensitivity and low background signal. Labeling cleared tissue samples in the practice of the methods of the invention can be easily performed following protocols well known and routinely performed in the art.

[0035] In some embodiments, the click reaction used in the methods of the invention to label drug molecules or metabolites in cleared tissue samples is an optimized or modified CuAAC reaction that provides improved imaging quality. In some embodiments, the modified click reaction uses non-conventional CuAAC reaction conditions that allow for sufficient signal-to-noise ratio (SNR) while maintaining low background signal in the vehicle control. In some embodiments, the modified click reaction uses newer generation click reaction ligands or azide-based substrates. Examples of such newer click reaction ligands include, for example, THPTA, BTTAA, and BTTP, as exemplified herein. In some embodiments, the modified click reaction uses Cu concentrations that are not commonly used in conventional CuAAC reactions. As exemplified herein, some methods of the invention can use Cu concentrations in the click reaction that are about 100 μM to about 300 μM. In some embodiments, Cu concentrations of about 100 μM to about 150 μM can be used. In a preferred embodiment, the click reaction ligand used is BTTP, and as exemplified herein, a Cu concentration of 150 μM is used.

[0036] In some embodiments, the optimized click reaction includes a pre-incubation step of the cleared tissue sample prior to the initiation of the click reaction. In this step, the cleared tissue sample is pre-incubated in an incubation buffer for a period sufficient to condition the tissue for homogenous labeling throughout the tissue. In various embodiments, the pre-incubation period can last at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours or longer. In some embodiments, the incubation buffer can contain Alexa647-picolyl-azide, CuSO4, BTTP and DMSO at appropriate concentrations as detailed herein. After pre-incubation, the treated tissue sample is then transferred to a click reaction buffer to initiate the click reaction. As exemplified herein, the click reaction buffer can contain fresh incubation buffer supplemented with sodium ascorbate (NaAsb).

[0037] As a specific exemplified embodiment of the optimized click reaction, the incubation buffer can contain 5 μM Alexa647-picolyl-azide, 150 μM CuSO4, 300 μM BTTP, 10% DMSO in PBS. The pre-incubation period can last for about 1-16 hours. The complete click reaction buffer contains 5 μM AF647-picolyl-azide, 150 μM CuSO4, 300 μM BTTP, 2.5 mM sodium ascorbate, and 10% DMSO in PBS. A detailed protocol for carrying out the optimized click reaction is exemplified herein.

[0038] V. Clearing and Analysis of Labeled Tissues The cleared and labeled tissue is then analyzed by appropriate means to confirm the presence of the drug molecule and / or to identify the target of the drug molecule in the tissue obtained from the subject to which the drug was administered. Depending on the tissue sample and detectable label used in the click reaction, various physical, optical, immunochemical or biological approaches can be used. These include, for example, confocal microscopy, fluorescence imaging, mass spectrometry imaging, multiphoton microscopy, fluorescence optical microscopy and positron emission tomography (PET). In various embodiments, the analysis of drugs in situ in biological samples can be easily performed using these and other tools that are routinely implemented in the art. See, for example, Diaspro et al., Microsc. Res. Tech. 1999; 47: 163-4; Denk et al., Science 1990; 248: 73-6; Imbert et al., Methods Mol. Biol. 1999; 122: 341-55; Tadrous et al., J. Pathol. 2000; 191: 345-54; Xue et al., Sci. Adv. 2018; 4: eaat 9039; McMahon et al., Proc. SPIE Int. Soc. Opt. Eng. 2020 February; 11219; and Bian et al., STAR Protocols 2022; 3: 101600. In some preferred embodiments, the spatial distribution and target interaction of drug molecules in cleared tissues can be examined first via imaging means. In addition to the confocal microscopy exemplified herein, other suitable imaging means, such as fluorescence lifetime imaging, Raman imaging and autoradiography, may also be used in the practice of the invention.

[0039] As mentioned above, drug molecules or metabolites are labeled with detectable labels such as fluorophores via CuAAC reaction. The cleared and labeled tissue sample can then be examined via appropriate means to analyze the labeled drug molecules in the tissue. In some preferred embodiments, the presence of drug molecules in the tissue and / or their interaction with their targets is analyzed with confocal microscopy as exemplified herein. Confocal microscopy is based on the illumination of a biological specimen (e.g., tissue) with excitation light followed by detection of the emitted light. This means that only fluorescent objects can be examined. In most cases, biological specimens are made fluorescent by the use of labels directed to specific intracellular or intratissue structures (confocal epifluorescence). For many years, the spontaneous autofluorescence of many biological molecules (confocal epitransmission) or their ability to reflect light off opaque surfaces (confocal reflectance) have been exploited.

[0040] In addition to imaging, the cleared and click-labeled tissue sample can be further subjected to cell type registration. In some of these embodiments, the tissue sample can be stained with an agent for a cell type that is known or suspected to express the target of the drug molecule. Cells expressing a particular target molecule or marker of interest can be identified through various assays. These include, for example, flow cytometry, immunohistochemistry and next-generation sequencing. In some embodiments, the tissue sample can be stained with an antibody or nucleic acid probe specific for one or more marker molecules of the cell type.

[0041] Cell markers for numerous cell types, including pathological cells (e.g., tumor cells), are well known and have been extensively characterized in the art. By way of example, when using human immune cells, some well-characterized cell markers include, for example, CD34+, CD38-, CD45RA-, CD49+, CD90 / Thy1+ for hematopoietic stem cells (HSC); CD34+, CD38-, CD45RA-, CD90 / Thy1- for multipotent progenitor cells (MPP); CD34+, CD38+, CD10+, CD45RA+ for common lymphoid progenitor cells (CLP); CD34+, CD38+, CD7-, CD10-, CD45RA-, CD90 / Thy1-, CD135+ for common lymphoid progenitors (CLP); CD34+, CD38+, CD7-, CD10-, CD45RA-, CD135-, IL3Rα- for megakaryocytic and erythroid progenitors (MEP); CD34+, CD38+, CD10-, CD45RA+, CD123+, CD135+ for granulocytic and monocytic progenitors (GMP); CD3-, CD56+, CD94+, NKp46+ for NK cells; CD3+ for T cells; CD19+ for B cells; CD14+ for monocytes; CD11b+, CD68+, CD163+ for macrophages; CD11c+ and HLA-DR+ for dendritic cells; CD11b+, CD16+, CD18+, CD32+, CD44+, CD55+ for neutrophils; CD45+, CD12 for eosinophils. 5+, CD193+, F4 / 80+, Siglec-8+; for basophils, CD19-, CD22+, CD45low, CD123+; for mast cells, CD32+, CD33+, CD117+, CD203c+, FcεRI+; for erythrocytes, CD235a+; for megakaryocytes, CD41b+, CD42a+, CD42b+, CD61+; and for platelets, CD41+, CD42a+, CD42b+, CD61+. For any of these cell types, any one or combination of these known markers can be searched for to identify the cell type in the tissue that is targeted by the drug molecule.

[0042] In some embodiments, the cell marker to be detected from tissue sample is specific to deceased cells.In some of these embodiments, the cell marker is a tumor-specific marker.Examples of such markers include, but are not limited to, HER2 (ERBB2), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), CA 125, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), CA 19-9, CA 15-3, CA 27-29, lactate dehydrogenase (LDH) and neurospecific enolase (NSE).

[0043] [Example] The following examples are provided to further illustrate the present invention, but not to limit its scope.

[0044] Example 1. CATCH enables specific labeling and imaging of drugs in situ. Over the past decade, there has been a renewed interest in irreversible binding of protein targets by chemical probes and drugs that operate by a covalent mechanism, as these chemical probes and drugs can improve the efficacy of binding to difficult targets and provide superior pharmacodynamic properties in vivo. Irreversibility also makes it particularly crucial to understand the cellular targets of covalent drugs with high precision to minimize toxicity. To establish a model system in which visualization of drug targets can be evaluated, we investigated a series of well-characterized covalent inhibitors of endocannabinoid hydrolases that have the potential to treat a wide range of human neurological disorders. Initial experiments were performed using PF-04457845 (or PF7845), a fatty acid amide hydrolase (FAAH) inhibitor, whose urea backbone forms a covalent bond with the active serine residue of FAAH to irreversibly inhibit the enzyme activity. Previous studies have shown that the alkyne-modified analogue PF7845-yne retains similar selectivity as PF7845 and can selectively detect FAAH in whole brain lysates by CC-ABPP combined with in-gel fluorescence readout (SDS-PAGE) (Niphakis et al., ACS Chem Neurosci 3, 418-426, 2012). However, attempts to directly image drug-bound FAAH in brain sections from mice treated with azido-Alexa647-tagged PF7845-yne under CC-ABPP conditions failed to reveal cellular targets due to poor SNR.

[0045] We reasoned that the complex composition of tissues, especially the dense lipid membranes, might interfere with the CuAAC reaction. Therefore, we tested whether CLARITY, a polyacrylamide-based aqueous clearing method, could improve the SNR of CuAAC labeling. After applying CC-ABPP click conditions to brain slices from mice treated with PF7845-in, CLARITY treatment led to a membrane-like fluorescent signal in the primary somatosensory cortex (S1) after clicking with Alexa-647-picolyl-azide, consistent with the expected FAAH binding. However, background nuclear signals were observed in vehicle-treated samples, indicating the occurrence of significant side reactions under these conditions (Figure 1).

[0046] Ligands are used to protect the catalytic Cu(I) from excessive reactive oxygen species (ROS) generation and side reactions with CuAAC. We found that the initial CC-ABPP protocol using 1 mM CuSO4 and the first generation ligand TBTA gave satisfactory results in homogenized systems, but the results were not satisfactory at high concentrations of Cu. 2+ We speculated that Cu may cause significant side effects in untreated tissues. Therefore, we investigated the effect of lower concentrations of Cu. 2+ We compared a wide range of newer generation ligands that allow for the detection of PF7845-in. THPTA, BTTAA and BTTP (Li and Zhang, 2016) significantly eliminated background signals in vehicle-treated samples. Furthermore, BTTP resulted in a weak but clear membrane-like signal in PF7845-in treated tissues using only 50 μM CuSO4 (Figure 1). 2+By gradually increasing the concentration, we found that 150 μM CuSO4 resulted in robust fluorescent labeling with minimal background staining in controls (Figure 1). As expected, fluorescent labeling was strictly dependent on the presence of each component of the CuAAC reaction. Removal of any of the reaction components (CuSO4, BTTP, Alexa-647-picolyl azide) abolished click labeling. Importantly, making tissue optically clear using reflectance matching alone was not sufficient to generate a satisfactory SNR, suggesting that delipidation associated with tissue clearing is necessary to promote CuAAC labeling in situ. We found that direct labeling of samples in the complete click reaction cocktail (5 μM Alexa647-picolyl-azide, 150 μM CuSO4, 300 μM BTTP, 2.5 mM NaAsb, 10% DMSO in PBS) after tissue clearing resulted in only faint labeling on the tissue surface (Figure 3). Insufficient click labeling depth could be overcome by pre-incubating samples in click incubation buffer (5 μM Alexa647-picolyl-azide, 150 μM CuSO4, 300 μM BTTP, 10% DMSO in PBS). In the absence of NaAsb, the Cu-BTTP complex is able to diffuse into the tissue before initiating the reaction, thereby ensuring a uniform catalytic species distribution throughout the tissue depth (Figure 3). Similar results were achieved with different drug delivery routes including oral, intraperitoneal and subcutaneous after tissue clearing. We demonstrated that CATCH can be further applied to peripheral tissues other than the brain (Figure 4). In addition to the liver and small intestine, we demonstrated drug mapping in the large intestine, heart, spleen, colon, lung, kidney and skeletal muscle. We demonstrated that CATCH is compatible with two hydrogel-based tissue clearing technologies, CLARITY and SHIELD (Park et al., Nat Biotechnol 37, 73-83, 2019) (Figure 2).Furthermore, we have demonstrated that tissues cleared by hydrophobic tissue clearing methods such as DISCO (Erturk et al., Nat Protoc 7, 1983-1995, 2012; Qi et al., Sci. Adv. 5, eaau8355, 2019; Renier et al., Cell 159, 896-910, 2014), hydrophilic tissue clearing techniques such as CUBIC3.0 (Tainaka et al., Cell Rep 24, 2196-2210, 2018), and other hybrid tissue clearing techniques such as HYBRiD (Nudell et al., Nat. Methods 19, 479-485, 2022) all give similar labeling profiles after in situ click reactions (Figure 2). For simplicity, we chose to employ the combination of CLARITY, 150 μM CuSO4 and 300 μM BTTP as the standard CATCH protocol for all subsequent experiments.

[0047] Next, we sought to determine whether the CATCH signal was specific to in vivo PF7845-in binding of FAAH by first performing pharmacological blocking experiments. Pretreatment with the parent compound PF7845 completely abolished the CATCH signal generated by PF7845-in throughout the brain. We also compared the PF7845-in CATCH signal in wild-type (FAAH+ / +) mice with FAAH- / - mice. In wild-type mice, robust PF7845-in labeling was observed throughout the brain, showing high overlap with FAAH immunostaining and mRNA HCR (Choi et al., 2018; Sylwestrak et al., Cell 164, 792-804, 2016). In contrast, negligible PF7845-in signal was detected in FAAH- / - mice. Taken together, the pharmacological and genetic results demonstrated that CATCH can visualize target binding with high specificity in tissues from drug-treated mice. Furthermore, we demonstrated that multiple click reactions can visualize drug binding in large tissue volumes for 3D characterization.

[0048] Example 2. CATCH Reveals Brain-Wide Drug-Target Interactions and Detects Rare Targets Illustratively, CATCH uncovers brain-wide drug-target interactions and detects rare targets. Having confirmed the ability of CATCH to detect interactions between PF7845-in and FAAH, we assessed the broader utility of CATCH by examining two other covalent drug-target combinations: the structurally distinct FAAH inhibitor BIA-10-2474 (Kiss et al., 2018) and the monoamine oxidase (MAO) inhibitor pargyline (Krysiak et al., 2012). Similar to PF7845, BIA10-2474 has a urea scaffold and inhibits FAAH via a nucleophilic substitution reaction with its active serine residue (Kiss et al., 2018). In contrast, pargyline inhibits MAO by forming a covalent adduct via an oxidative addition reaction (Krysiak et al., 2012). Alkyne analogues of both drugs have been developed in previous studies (Huang et al., 2019; Krysiak et al., 2012). CATCH imaging revealed similar labeling patterns after in vivo administration of PF7845-yne and BIA10-2474-yne, characterized by strong signals in the neocortex, thalamus and hippocampus. In contrast, pargyline-yne ​​administration resulted in a distinct pattern of labeling enriched in the hypothalamus, pons and lateral ventricles. CATCH allowed immunostaining of FAAH after CuAAC reaction, confirming that the signals of PF7845-yne and BIA10-2474-yne were primarily associated with FAAH (R 2 = 0.95 and 0.84, respectively, for simple linear regression, R 2 The same for the values. No such correlation was observed in pargyline-in treated animals (R 2 = 0.03). Conversely, MAO-A staining showed no correlation for PF7845-in (R 2 =0.005), but showed a better correlation for pargyline-in (R 2= 0.43). Imaging at higher resolution revealed that the FAAH inhibitors targeted neuronal-like structures primarily within the neocortex and hippocampus. In contrast, pargyline-ine bound primarily to vasculature-like structures throughout the brain, with the exception of sparse but specific labeling of neuronal-like structures in the hypothalamus and pons.

[0049] Unexpected toxicity of BIA-10-2474 has been reported in humans, likely due to greater off-target activity compared to PF7845 (Kerbrat et al., 2016; Van Esbroeck et al., 2017). Accordingly, we observed a lower correlation between FAAH expression and BIA-10-2474-in CATCH labeling than PF7845-in (R 2 = 0.84 vs. 0.95). Furthermore, the majority of PF7845-in and BIA-10-2474-in CATCH signals were lost in FAAH- / - mice, including in S1, CA1, and periaqueductal gray (PAG), although, surprisingly, some BIA-10-2474-in labeling remained in the pontine reticular tegmental nucleus (RtTg) (Figure 9). These data showed that BIA10-2474-in binds to off-target sites in RtTg and also demonstrated the ability of CATCH to reveal drug targets with cellular resolution.

[0050] Example 3. Use of CATCH and fluorescent labeling to identify target cell types. As a further illustration of the present invention, we have found that CATCH can be multiplexed with fluorescent labeling to identify target cell types. Understanding the cell types affected by drug treatment in vivo is crucial to accurately interpret pharmacological mechanisms of action, especially for drugs targeting the CNS, which has a diverse range of neural substrates and cell types. Having established that CATCH can visualize drug targets at single-cell resolution, we investigated whether CATCH can be combined with molecular markers of cell identity to characterize and register cell target identity. We demonstrated that PF7845-in binds to neurons, but not blood vessels, in S1, as revealed by NeuN and lectin staining (Figure 5). We performed similar antibody and mRNA staining in a wide range of brain regions of interest, including but not limited to the cortex, hippocampus, hypothalamus, striatum, thalamus, basolateral amygdala, pons, cerebellum and brainstem, to characterize PF7845, BIA-10-2474 and pargyline-binding cell types. In addition to neuronal and vascular staining, we identified long-range projection neurons via Ctip2 antibody staining; Parvalbumin (PV) interneurons via PV antibody staining; Somatostatin (SST) interneurons via SST mRNA hybridization chain reaction (HCR); Astrocytes via Glial Fibrillary Acidic Protein (GFAP) antibody staining; FAAH expression via FAAH antibody staining and FAAH mRNA HCR; MAO-A expression via MAO-A antibody staining; Noradrenergic (NA) neurons via tyrosine hydroxylase (TH) antibody staining, inhibitory neurons via VGAT-Ai14 transgenic mice; neuronal cell bodies via MAP2 antibody staining; and presynaptic terminals of neurons via synapsin antibody staining.

[0051] Using a stable polyacrylamide hydrogel network, we tested the possibility of reversible multiplexed marker staining (Figure 6, A). We identified a small group of neuronal-like structures in the pons that were NeuN negative but targeted by pargyline-yne ​​(Figure 6, B). Based on MAO gene expression data from the Allen Brain Atlas, we suspected that these cells might represent NA neurons in the locus coeruleus (LC) in the pons, commonly identified by TH staining (Schmidt et al., 2019). We tested this by removing NeuN immunostaining from brain sections and re-staining with TH antibody on the same samples. The CATCH signal resulting from CuAAC conjugation of Alexa647 was resistant to the high temperature and SDS elution protocols used to remove NeuN staining, revealing colocalization of pargyline-yne ​​and TH in the same cells (Figure 6, B).

[0052] Example 4. CATCH can reveal drug binding with subcellular resolution. In another illustration, the claimed invention was able to reveal drug binding at subcellular resolution. Specifically, we asked whether CATCH could image drug-target interactions in intracellular compartments within tissues. To this end, we targeted monoacylglycerol lipase (MAGL), a key enzyme involved in terminating the signaling function of the endocannabinoid 2-archidonoyl glycerol (2-AG) (Di Marzo, 2018), using a well-characterized alkyne analog of MJN110, a MAGL inhibitor (Chang et al., ACS Chem Biol 8, 1590-1599, 2013; Niphakis et al., ACS Chem Neurosci 4, 1322-1332, 2013). We confirmed the specificity of the MJN110-in signal generated by CATCH using both pharmacological pretreatment of the parent compound MJN110 and MAGL- / - mice.

[0053] As previously reported, we found that MJN110-yne selectively bound MAGL at 1 mg / kg and showed significant off-target binding of FAAH at 20 mg / kg in gel-based CC-ABPP of whole brain lysates. Since MAGL is restricted to localization to presynaptic axon terminals, whereas FAAH is expressed in neuronal somas, we sought to determine whether CATCH could detect on-target and off-target MJN110-yne binding across subcellular compartments. Since it was possible to observe both axon terminals and somas in the same field of view, we focused on the well-characterized CA3 to CA1 projection in the hippocampus. As expected, we observed an overall higher fluorescence intensity in hippocampi treated with 20 mg / kg MJN110-yne. Higher resolution imaging combined with synapsin and microtubule-associated protein 2 (MAP2) immunostaining revealed that MJN110-in signals were restricted to synapsin-positive axon terminals at low doses (1 mg / kg, Figure 7) and spread to neuronal somas at higher doses (20 mg / kg). Pretreatment of animals with PF7845 significantly inhibited MJN110-in binding in somas but not in axon terminals, indicating that these signals originated from off-target FAAH binding and on-target MAGL binding, respectively. Thus, these data demonstrate that CATCH can visualize drug binding to distinct protein targets with subcellular resolution.

[0054] Example 5. CATCH can measure dose-dependent drug-target binding. As a further illustration, the inventors have found that the claimed method can be used to measure dose-dependent drug-target binding. Finally, after establishing the specificity, scalability, suitability and resolution of CATCH, the inventors tested whether CATCH can be used to quantitatively resolve dose-dependent target binding after in vivo drug administration. Recognizing that the alkyne modifications made to drugs to create CATCH probes have the potential to alter pharmacokinetic (PK) properties, a result that the inventors have confirmed for both FAAH and MAGL alkyne probes, which share similar blood-brain barrier (BBB) ​​permeability and brain distribution with the parent inhibitors but exhibited altered in vivo half-lives, the inventors adopted a competitive strategy to visualize the dose-dependence of drug-target interactions in vivo. In this competitive CATCH approach, animals are first treated with a range of parent drugs, followed by a single high dose of the alkyne probe. Dose-dependent target binding by the parent drug can be recorded based on competitive blockade of the signal in an anatomically specific manner (FIG. 8, A).

[0055] We treated mice with 0.01-1 mg / kg parent PF7845 followed by 1 mg / kg PF7845-in probe to read out dose-dependent brain targeting of PF7845 by competitive CATCH. Consistent with previous in-gel analysis (Niphakis et al., ACS Chem Neurosci, 3, 418-426, 2012), no target binding was detected by CATCH at 0.01 mg / kg, whereas complete FAAH inhibition was achieved at 0.05 mg / kg. Interestingly, we found that at an intermediate dose (0.02 mg / kg), FAAH was completely inhibited in the cortex but not in the hippocampus (Figure 8, B). Specifically, a heterogeneous striped pattern was observed in the CA1 pyramidal cell layer and DG granule cell layer of the hippocampus, suggesting submaximal drug binding in these regions. Next, we stained brain sections with lectin to mark the vasculature, revealing that regions with low probe intensity (i.e., cells previously blocked by parent PF7845) were associated with invading blood vessels. Conversely, we employed a direct approach to visualize dose-dependent alkyne drug PK properties (Figure 8, C). Interestingly, when we directly visualized an intermediate dose of alkyne PF7845-in (0.1 mg / kg; Figure 8, D) by CATCH in the hippocampus, we observed a mirror image intensity pattern, with higher probe intensity in close proximity to the vasculature.

[0056] Taken together, both competitive and direct labeling suggested that PF7845-FAAH interaction at intermediate doses may be limited by the distance between FAAH-expressing neurons and the surrounding capillaries. This effectively creates a drug gradient that results in submaximal binding in neurons more distal to the vasculature. This heterogeneous distribution is unique to the hippocampus, suggesting that this may be due to particularly high levels of FAAH expression or relatively low vasculature volume in this region.

[0057] Finally, because covalent inhibitors typically manage long-term target binding, we found persistent FAAH blockade of competitive probe binding even 24 h after PF7845 administration. Collectively, by complementing competitive and direct labeling strategies, CATCH can reveal dose-dependent, quantitative target binding across heterogeneous brain regions that are not easily accessible by traditional lysate-based methods.

[0058] Example 6. Exemplary Materials and Methods Provided below is detailed information on some of the protocols and materials that can be used in the practice of the present invention.

[0059] Mouse model: Mice were group-housed with a 12-h light / dark cycle and fed standard rodent chow. Both male and female 6-9 week-old WT C57BL6J, FAAH- / -, MAGL- / - and VGAT-Ai14 mice were used. VGAT-Ai14 mice were obtained by crossing VGAT-ires-cre mice with Ai14 mice. All experimental protocols were approved by the Scripps Research Institute Institutional Animal Care and Use Committee and followed NIH guidelines.

[0060] Sample collection: For intraperitoneal (ip) injection, inhibitors were administered to mice in a vehicle of 10% DMSO, 2% Tween-80 in saline. After drug injection at the indicated dose and time, mice were deeply anesthetized with isoflurane and then transcardially perfused with ice-cold PBS followed by ice-cold 4% PFA in PBS containing sucrose. Mouse brains or other peripheral tissues were then dissected and post-fixed in 4% PFA overnight at 4°C. Tissues were washed with PBS, embedded in 2% agarose, and sectioned by a vibratome (Leica VT1000S) as 100- or 500-micron tissue sections. Tissue sections were stored at 4°C in PBS containing 0.02% sodium azide for further processing.

[0061] For in-gel ABPP analysis, mice were decapitated after anesthesia, and target tissues were collected and snap frozen in liquid N2 without perfusion or fixation. Each mouse hemisphere and liver were washed with ice-cold PBS (2 × 1 mL) on ice to remove excess blood. Tissues were homogenized in 1 mL PBS and sonicated for 10 min in ice-cold water. Tissues were centrifuged (1000 g, 10 min, 4 °C), and then the supernatant was centrifuged at high speed (100,000 g, 45 min, 4 °C). The supernatant was discarded and the remaining pellet was gently washed with ice-cold PBS (2 × 0.5 mL). The pellet was resuspended by gentle pipetting, and protein concentration was quantified by Bio-Rad DC Protein Assay Kit. The proteome was then diluted to 1.0 mg / kg for immediate use or aliquoted and stored at -80 °C.

[0062] For quantitative PK studies, blood was collected by cardiac puncture into EDTA microtubes at each collection time point (1 h, 2 h, 4 h, and 24 h) and mixed. Exactly 100 μL of blood was added to 400 μL of ice-cold acetonitrile to immediately inactivate blood esterase activity. Mice were then perfused with ice-cold PBS at 5 mL / min for 2 min until the perfusate was clear. Brains were harvested in four sections (forebrain, cerebellum, brainstem, and spare forebrain) and flash frozen in liquid nitrogen. All acetonitrile-extracted blood samples were centrifuged at 17,000×G for 3 min and the supernatant was transferred to a new tube and frozen at −80°C until bioanalysis.

[0063] In-gel click chemistry ABPP assay: Click chemistry labeling of alkyne inhibitors was performed as previously reported (Niphakis et al., ACS Chem Neurosci, 3, 418-426, 2012). Click reaction buffer was prepared by mixing CuSO4 (50 mM in HO, 1.0 μL / reaction), TBTA (1.7 mM in 1:4 DMSO / t-BuOH, 3.0 μL / reaction), freshly prepared TCEP (50 mM in HO, 1.0 μL / reaction) and 5-TAMRA azide (1.25 mM in DMSO, 1.0 μL / reaction). For every 50 μL of tissue proteome (1.0 mg / kg), 6 μL of click buffer was added. The reaction mixture was gently mixed and left in a dark drawer at room temperature for 1 h. Reactions were quenched by the addition of SDS loading buffer (4x, 18 μL) and run on SDS-PAGE.

[0064] Tissue transparency: CLARITY: For CLARITY embedding, PFA-fixed tissues were incubated in A1P4 hydrogel (1% acrylamide, 0.125% Bis, 4% PFA, 0.025% VA-044 initiator (w / v) in 1x PBS at 4 °C as previously published (Sylwestrak et al., Cell 164, 792-804, 2016). Samples were left overnight with gentle agitation to allow sufficient monomer diffusion. Samples were flushed with nitrogen and degassed for 15 min at room temperature. After degassing, samples were polymerized for 4 h at 37 °C with gentle agitation. Samples were removed from the hydrogel and washed with 8% PBS-SDS (pH = 7.0) for 2 days at 40 °C. After clearing, samples were washed with PBST (pH = 7.0, 0.2% SDS) for 3 × 10 min at room temperature. The samples were washed with Triton-X100 (same below) to remove residual SDS. The samples were washed briefly with PBS and then stored at 4°C in PBS containing 0.02% sodium azide.

[0065] SHIELD: SHIELD processing was performed as previously reported (Park et al., Nat. Biotechnol. 37, 73-83, 2019). Tissue samples were incubated in SHIELD-OFF solution (25% dH2O, 25% SHIELD BUFFER solution, 50% SHIELD epoxy solution) for 1 day at 4°C with gentle agitation. Samples were then transferred to an equal volume of SHIELD-ON buffer and incubated at 37°C for 24 hours with shaking. Samples were washed with PBS for 3×10 min at room temperature to remove residual SHIELD-ON buffer, then cleared with 8% PBS-SDS (pH=7.0) overnight at 40°C. Samples were washed with PBST 3×10 min at room temperature to remove residual SDS. Samples were washed briefly with PBS and then stored at 4°C in PBS containing 0.02% sodium azide.

[0066] iDISCO: iDISCO was modified from a published protocol (Renier et al., Cell 159, 896-910, 2014). All organic solvent washes were performed at 4°C with shaking. Fixed samples were washed in 20%, 40%, 60%, 80% methanol / 0.1% Triton X-100 / 0.3M glycine in H2O (B1N buffer, pH 7), then washed twice with 100% methanol. Samples were then degreased with 100% dichloromethane (DCM), washed three times in 100% methanol, then washed in 80%, 60%, 40%, 20% methanol in B1N buffer. Each of the above washes was for 15 min. Samples were then washed in PBST 3×10 min at room temperature to remove residual organic solvents. Samples were washed briefly with PBS and then stored at 4°C in PBS containing 0.02% sodium azide.

[0067] fDISCO: fDISCO was modified from a published protocol (Qi et al., Sci. Adv. 5, eaau8355, 2019). Samples were washed at room temperature with shaking. Fixed samples were washed in 50%, 70%, 80% tetrahydrofuran (THF) in 25% Quadrol (in 1x PBS to adjust to pH 9), then twice in 100% THF. Samples were dewaxed with 100% DCM, then washed three times with 100% THF, followed by washing with 80%, 70%, 50% THF. Each of the above washes was for 15 min. Samples were then washed with PBST 3x10 min at room temperature to remove residual organic solvents. Samples were washed briefly with PBS, then stored at 4°C in PBS containing 0.02% sodium azide.

[0068] CUBIC3.0: The CUBIC protocol was modified from the recent CUBIC3.0 protocol (Tainaka et al., Cell Rep 24, 2196-2210, 2018). Samples were cleared in CUBIC-L buffer (10 wt% N-butyldiethanolamine, 10 wt% TritonX-100 in dH2O) for 4 h at 37 °C. Samples were then washed in PBST 3 × 10 min at room temperature to remove residual detergent. Samples were washed briefly in PBS and then stored at 4 °C in PBS containing 0.02% sodium azide.

[0069] CATCH labeling: For 100 micron section labeling, the complete CATCH reaction buffer contains 5 μM AF647-picolyl azide, 150 μM CuSO4, 300 μM BTTP, 2.5 mM sodium ascorbate, and 10% DMSO in PBS. Tissue sections were incubated in click incubation buffer (without sodium ascorbate) overnight at room temperature. The tissue was then transferred to freshly prepared incubation buffer, after which the reaction was initiated by adding 100 mM freshly prepared sodium ascorbate. After 1 h at room temperature in the dark with gentle agitation, the reaction was quenched by the addition of 4 mM EDTA in PBS (pH=8.0) and the samples were washed 3×10 min with PBST. After removing the click reaction cocktail, the samples were stained with DAPI (1:3000 dilution in PBS from 10 μM stock solution) for 15 min at room temperature and were ready for RI matching and imaging. Samples can also undergo further staining for cell type registration. For 500 micron sections, 300 μM CuSO4 and 600 μM BTTP were used for incubation and reaction. To achieve complete labeling, the tissue was incubated at 37°C for 2 days and then subjected to four rounds of 1-h click reaction at room temperature.

[0070] Immunostaining: Unless otherwise stated, samples were incubated with primary antibodies in PBST overnight at 4°C. Samples were then washed with PBST for 3×30 min at room temperature and transferred to secondary antibodies diluted in PBST. Samples were incubated with secondary antibodies overnight at room temperature and washed with PBST for 3×30 min. For antibody elution, imaged slides were removed and tissues were washed with 8% PBS-SDS (pH=7.0) for 8 h at 60°C. Samples were then washed with PBST for 3×10 min at room temperature to remove residual SDS and were ready for the next round of staining.

[0071] Hybridization Chain Reaction (HCR): Samples were preincubated in probe hybridization buffer for 30 min at 37°C. Samples were then transferred to fresh probe hybridization buffer containing 16 nM FAAH-B1 or 4 nM SST-B1 probe and incubated overnight at 37°C. Samples were washed with probe wash buffer for 3 x 30 min at 37°C, then washed with 5 x SSCT (750 mM NaCl, 75 mM sodium citrate, 0.1% Tween-20 in H2O) for 2 x 30 min at room temperature. Samples were preincubated in amplification buffer for 30 min at room temperature. HCR hairpins were typically stored as 3 μM, 12 μL aliquots. To each hairpin aliquot, 4 μL of 20 x SSC (VWR, 10128-690) was added. Hairpins were heated to 95°C for 90 s and cooled to room temperature in a dark drawer. The hairpin was then added to fresh amplification buffer to give a final hairpin concentration of 360 nM. The sample was then transferred to the hairpin-containing amplification buffer and incubated overnight at room temperature. The sample was washed 3x for 30 min with 5x SSCT and ready for imaging.

[0072] Confocal microscopy: Labeled samples were immersed in RI matching medium (RapiClear, 1.45RI) and then mounted on glass microscope slides. Tissues were then imaged with an Olympus FV3000 confocal microscope equipped with a 10x, 0.6NA, water immersion objective (XLUMPlanFI, Olympus) for gross characterization, or a 40x, 1.25NA, silicone oil immersion (UPlanSApo, Olympus) for detailed cell type, mRNA expression, and subcellular compartment identification.

[0073] Drug in vivo concentration analysis: Compound concentrations in blood and brain regions (forebrain and brainstem) were determined by LC / MS / MS. Acetonitrile-extracted blood samples were prepared as described above, and blood calibration curves were generated using blood from naïve mice quenched according to the same protocol spiked with serial dilutions of the test article ranging from 0.1 to 5000 ng / mL.

[0074] To each frozen brain tissue, 3 volumes of ice-cold acetonitrile:water 3:1 (vol:vol) per tissue weight (in μL) were added for compound extraction. The tissue was then homogenized in a tissue disrupter (Tissue Lyser II, Qiagen) with one steel bead (5 mm diameter) at 30 Hz for 1 min. The samples were incubated on ice for 45 min. Afterwards, the samples were centrifuged at 2.400 × g for 15 min at 4 °C and the supernatant was transferred to a separate tube. Brain homogenates from naive mice (extracted following the same protocol as the samples) spiked with serial dilutions of the test substances ranging from 0.1 to 5000 ng / mL were used to generate calibration curves for the forebrain and brainstem.

[0075] 10 μL of the extract was injected onto an Agilent 1290 UPLC system equipped with a G7120A pump, a G7129B autocollection device, and a G1170A column manager (Agilent Technologies). Chromatographic separation was achieved using an Acquity UPLC BEH C18 column (2.1×50 mm, 1.7 μm particle size, 130 Å) coupled to an Acquity UPLC BEH C18 Column Guard (2.1×5 mm, 1.7 μm particle size, 130 Å) (Waters Corporation). Mobile phase A consisted of 95:5 water / acetonitrile by volume, and mobile phase B consisted of 95:5 acetonitrile / water by volume. Both mobile phases were supplemented with 0.1% formic acid. The gradient started at 0% B, which was maintained for 0.5 min, and then increased linearly to 100% B in 4 min. Solvent B was then maintained at 100% for 1 min before switching to the initial conditions at 0.1 min. The system was allowed to equilibrate for 1.4 min before the next sample injection. The flow rate was maintained at 0.6 mL / min. The column was heated at 50°C.

[0076] Analytes were quantified using a 6460 triple quadrupole mass spectrometer (Agilent Technologies) equipped with an electrospray Jet Stream source operated in dynamic multiple reaction monitoring (dMRM) mode. Quantitative and qualitative transitions for each compound were optimized using authentic standards in the Optimizer software (Agilent Technologies): MJN110 (quantitative: 462.1 → 235, CE = 10; qualitative: 462.1 → 165, CE = 45), MJN110-in (quantitative: 452.1 → 225, CE = 10; qualitative: 452.1 → 189.6, CE = 45), PF7845 (quantitative: 456.2 → 335.1, CE = 22; qualitative: 456.2 → 122, CE = 30) and PF7845-in (quantitative: 470.2 → 349.2, CE = 18; qualitative: 470.2 → 320.2, CE = 30). The following parameters were kept constant for all transitions: fragmentor = 140, cell acceleration voltage = 4, polarity = positive. Total cycle time was 500 ms. Source parameters were maintained as follows: drying gas temperature = 350°C, drying gas flow rate = 11 L / min, sheath gas temperature = 350°C, sheath gas flow rate = 11 L / min, nebulizer = 50 psi, nozzle (Noozle) voltage = 1500 V (positive) and capillary = 3500 V (positive).

[0077] Compound concentrations were calculated by extrapolating the integrated area under the curve using a calibration curve for each specific compound prepared in the same matrix as the samples, using Masshunter Quantitative Analysis Software (Agilent Technologies).

[0078] Quantification and statistical analysis: Images were typically acquired at 10 μm intervals and analyzed using Fiji-ImageJ for two-dimensional quantification. 3D drug binding was visualized by Imaris 9.2.1. All images were saved and processed as TIFF format.

[0079] Signal profile analysis: As previously reported (Pan et al., Nat. Methods 13, 859-867, 2016), a 40 micron line was drawn across pyramidal cells in cortical layer 5 on the tissue surface. The signal profile was plotted along this line and data was recorded including background, drug-positive membrane pixels and nuclei. The plotted data was then normalized by dividing each value by the average intensity of all intensity values ​​on the same line.

[0080] Click labeling signal analysis: Individual drug-positive cell intensity was quantified as previously reported (Pan et al., Nat. Methods 13, 859-867, 2016; Qi et al., Sci. Adv. 5, eaau8355, 2019). ROIs of 150 × 150 pixels (pixel size 0.414 μm) were generated on the tissue surface in cortical layer 5 and an automatic threshold was applied to calculate the mean drug-positive pixel intensity as I シグナル The average intensity of the remaining pixels was measured as I バックグラウンド is used as background intensity. The average labeling intensity is

number

[0081] Regional drug abundance analysis: Images were stacked in maximum z-projection covering the entire coronal brain section. Based on the mouse brain atlas (The Mouse Brain in Stereotaxic Coordinates, Second Edition), different ROIs were drawn to outline individual brain regions and their average intensities were recorded. Intensity values ​​were then normalized to the average intensity of the vehicle sample in the respective brain region.

[0082] Fluorescence Intensity Correlation Analysis: To quantify the correlation between FAAH and MAO-A immunostaining signals and drug signals, images were first stacked by maximum intensity projection (MIP) to obtain a single-plane image. Images were then compressed 10 times to obtain a final pixel size of 24.9 μm. The entire tissue was then contoured by applying a threshold to the immunostaining channel, and each XY coordinate intensity was preserved in both channels. The intensity value of each pixel was then normalized to the average intensity of each measurement and plotted. A simple linear regression was applied to analyze pixel-by-pixel signal correlation.

[0083] Drug-bound capillary analysis: To quantify drug-bound abundance with respect to proximity to the nearest blood vessel, we first cropped the pyramidal cell layer in CA1 and the granule cell layer in DG. A threshold was applied to quantify the mean nuclear (drug-negative) pixel intensity as background intensity. We then cropped different ROIs based on distance to the nearest blood vessel and applied the same threshold to quantify the mean intensity in non-nuclear pixels. The mean labeling intensity was then obtained by subtracting the background intensity from the mean non-nuclear pixel intensity.

[0084] Subcellular compartment intensity analysis: For MJN110-in characterization, images were acquired at 5 μm from the tissue surface where the highest immunostaining signal could be observed. Different cellular compartments were identified by applying a threshold in the immunostaining signal, and the average drug labeling intensity was measured in the selected threshold region. Three random squares were drawn in the nucleus in different cells, and their average intensity was used as background. Then, for background adjustment, the intensity in the nucleus was subtracted from the recorded intensity values ​​in the soma or axon terminals. The data were then normalized to the average intensity of the control group, as indicated in the legend of each figure.

[0085] Unless otherwise specified, statistical analysis was evaluated using Graphpad Prism 9 using Tukey's multiple comparison test, Sidak's multiple comparison test or Dunnett's multiple comparison test after regular one-way and two-way ANOVA (*p<0.05; **p<0.01; *** p<0.001; **** p<0.0001). All data are presented as mean ± standard deviation and N is indicated in figure legends as biological replicates unless otherwise stated. Correlations between immunostaining and drug intensity across the whole brain were assessed with a simple linear regression model and R-squared was calculated for each drug. For intensity ratios across different subcellular compartments, unpaired Mann-Whitney (Wilcoxon rank sum) tests were used. Drug PK analysis was performed with t-tests.

[0086] *** Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made therein without departing from the spirit or scope of the appended claims.

[0087] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method for analyzing a drug molecule or a metabolite thereof in mammalian tissue, the method comprising: (a) obtaining a tissue sample from a mammalian subject administered the drug molecule; (b) clearing the tissue sample with a delipidizing agent; (c) performing a CuAAC reaction with the cleared tissue sample to label the drug molecule; and (d) examining the drug molecule in the tissue sample with an analytical means.

2. The method of claim 1 , wherein the drug molecule is a small molecule.

3. The method of claim 1 , wherein the drug molecule is a covalent drug.

4. 2. The method of claim 1, wherein the defatting agent is a hydrogel-based tissue clearing agent, a hydrophobic tissue clearing agent, or a hydrophilic tissue clearing agent.

5. 2. The method of claim 1, wherein the drug molecule is labeled with a fluorophore via the CuAAC reaction.

6. The method of claim 1 , wherein the CuAAC reaction is optimized.

7. The optimized CuAAC reaction is about 100 μM to about 150 μM of Cu used in the reaction. ２＋ The method of claim 6, comprising a concentration.

8. 7. The method of claim 6, wherein the CuAAC reaction is optimized by including a pre-incubation of the cleared tissue prior to initiation of the click reaction.

9. 7. The method of claim 6, wherein the CuAAC reaction is optimized using a click reaction ligand that improves the signal-to-noise ratio.

10. The method of claim 9 , wherein the Click reaction ligand is BTTP, THPTA, or BTTAA.

11. The method of claim 1 , wherein the drug molecule or metabolite thereof contains an alkyne group or an analog thereof.

12. The method of claim 1 , wherein the analytical means is an imaging means.

13. The method of claim 13 , wherein the imaging means comprises confocal microscopy.

14. The method of claim 1, further comprising staining the tissue sample with an agent against a cell type known or suspected to express the target of the drug molecule.

15. The method of claim 14, wherein the agent is an antibody or a nucleic acid probe specific for the cell type.

16. 1. A method for identifying a target of a drug molecule in a mammalian tissue, the method comprising: (a) obtaining a tissue sample from a mammalian subject administered the drug molecule; (b) clearing the tissue sample with a delipidating agent; (c) performing a CuAAC reaction with the cleared tissue sample to label the drug molecule; and (d) examining the drug molecule in the tissue sample with an analytical means to identify the target of the drug molecule in the tissue, thereby identifying the target of the drug molecule.

17. The method of claim 16 , wherein the drug molecule is a small molecule.

18. The method of claim 16 , wherein the drug molecule is a covalent drug.

19. 17. The method of claim 16, wherein the defatting agent is a hydrogel-based tissue clearing agent, a hydrophobic tissue clearing agent, or a hydrophilic tissue clearing agent.

20. 17. The method of claim 16, wherein the CuAAC reaction is optimized.

21. 17. The method of claim 16, wherein the binding targets in the tissue sample are identified via immunostaining, RNA hybridization, or spatially resolved molecular characterization means.

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