Compounds and methods for characterizing binding to the human constitutive androstane receptor and multiple kinases

Abstract

The present disclosure relates to relates to compositions useful for probing activity and function of constitutive androstane receptor and kinases using fluorescence assays as disclosed herein. The present disclosure further relates to methods of using the disclosed compositions in methods for probing activity and function of constitutive androstane receptor and kinases. The present disclosure also pertains to kits comprising the disclosed compositions and instructions for using same. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present disclosure.

Description

ATTORNEY DOCKET NO.331904-2010 COMPOUNDS AND METHODS FOR CHARACTERIZING BINDING TO THE HUMAN CONSTITUTIVE ANDROSTANE RECEPTOR AND MULTIPLE KINASES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of and priority to U.S. Provisional Application No. 63 / 701,442, filed on September 30, 2024 which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant GM118041 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Human constitutive androstane receptor (hCAR) is one of master regulators of endobiotic and xenobiotic metabolism and disposition. Its relatively large and flexible ligand binding pocket can accommodate many structurally diverse compounds. hCAR transcriptionally regulates many proteins involved in drug metabolism and disposition. Therefore, binding of ligands to hCAR alters the metabolism and disposition of endobiotics and xenobiotics. Additionally, protein kinases are an important class of drug targets, and the identification of compounds that bind to protein kinases is one necessary step in the process of kinase drug discovery. Despite previous identification and development of compounds that can act as hCAR activators (agonists) and inhibitors (inverse agonists, deactivators, or antagonists), there remains a need for compounds and assays that can be used to characterize the binding of ligands to the hCAR ligand binding domain. Additionally, there is a need for compounds and assays that can be used to characterize binding to multiple protein kinases. These needs and other needs are satisfied by the present disclosure. SUMMARY

[0004] In accordance with the purpose(s) of the disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to compositions useful for probing activity and function of constitutive androstane receptor and kinases using fluorescence assays as disclosed herein. The present disclosure further relates to methods of using the disclosed compositions in methods for probing activity and function of constitutive androstane receptor and kinases. In a further aspect, the present disclosure pertains to kits comprising the disclosed compositions and instructions for using same.

[0005] Disclosed are compounds having a structure represented by a formula:ATTORNEY DOCKET NO.331904-2010 , wherein Ln is a linking q is an integerselected from 0 and 1.

[0006] Also disclosed are methods to determine binding affinity of a compound interacting with a constitutive androstane receptor or a kinase, the method comprising the steps of: (1) providing a solution comprising: (i) a test compound; (ii) a polypeptide comprising a domain, or a ligand binding fragment polypeptide thereof, that can bind a moiety of at least one compound of any one of claims 1-19 having a structure given by the formula: ; (iii) a fluorescent donor comprising adisclosed compound; (2) fluorescence in the fluorescent donor molecule; (3) measuring fluorescence emission from the fluorescent donor molecule and the fluorescent acceptor molecule; and (4) comparing the fluorescence emission to the fluorescence emission of steps (a)-(c) carried out in the absence of the test compound.

[0007] Also disclosed are kits for measuring the binding activity of a test compound to human constitutive androstane receptor (hCAR) and / or a kinase comprising: at least one disclosed compound, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; and optionally, one or more of: (1) instructions for carrying out an in vivo fluorescence assay using at least one disclosed compound; (2) instructions for carrying out an in vitro fluorescence assay using at least one disclosed compound; (3) at least one polypeptide that can bind the at least one disclosed compound; (4) at least one compound that binds to hCAR and / or a kinase; (5) one or more test compounds; and / or (6) at least one buffer and / or salt.

[0008] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described aspects are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described aspects are combinable and interchangeable with one another.ATTORNEY DOCKET NO.331904-2010 BRIEF DESCRIPTION OF THE FIGURES

[0009] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0010] FIG.1 shows chemical structures of a panel of representative hCAR ligands.

[0011] FIG. 2A shows representative results from an HTRF hCAR coactivator recruitment assay.

[0012] FIG.2B shows a representative design of BODIPY FL SNS 032 (10) as a high affinity hCAR fluorescent probe.

[0013] FIGS.3A-3F show representative TR-FRET binding activity of BODIPY FL SNS 032 (10) to hCAR (FIG.3A), hGSK3A (FIG.3B), hCDK9 / CycT1 (FIG.3C), hMAPK15 (FIG.3D), hCASK (FIG.3E), and hCAMKK2 (FIG.3F). Lines connecting solid circles represent Tb-anti- tag antibody and a corresponding tagged-target in the presence of DMSO; lines connecting solid squares represent Tb-anti-tag and a corresponding tagged-target in the presence of a target specific ligand as indicated; and lines connecting solid diamonds represent Tb-anti-tag antibody in the presence of DMSO without the target protein.

[0014] FIGS. 4A-4F show representative selections of BODIPY FL SNS 032 (10) concentration for hCAR (FIG. 4A), hGSK3A (FIG. 4B), hCDK9 / CycT1 (FIG. 4C), hMAPK15 (FIG. 4D), hCASK (FIG. 4E), and hCAMKK2 (FIG. 4F), to be validated based on the Kddetermination experiments.

[0015] FIGS.4G-4L show representative raw TR-FRET signals of 3 sample groups with the selected BODIPY FL SNS 032 (10) concentration for hCAR (FIG. 4G), hGSK3A (FIG. 4H), hCDK9 / CycT1 (FIG.4I), hMAPK15 (FIG.4J), hCASK (FIG.4K), and hCAMKK2 (FIG.4L).

[0016] FIGS.4M-4R show representative TR-FRET signal fold changes of 3 sample groups to respective positive controls for hCAR (FIG.4M), hGSK3A (FIG.4N), hCDK9 / CycT1 (FIG. 4O), hMAPK15 (FIG.4P), hCASK (FIG.4Q), and hCAMKK2 (FIG.4R).

[0017] FIGS.5A-5F show representative dose response inhibition curves of selected ligands against the BODIPY FL SNS 032 (10)-based TR-FRET binding assays for hCAR (FIG.5A), hGSK3 (FIG. 5B), hCDK9 / CycT1 (FIG. 5C), hMAPK15 (FIG. 5D), hCASK (FIG. 5E), and hCAMKK2 (FIG.5F).

[0018] FIG.6 shows a representative1H NMR spectrum of BODIPY FL SNS 032.

[0019] FIG.7 shows a representative13C NMR spectrum of BODIPY FL SNS 032.

[0020] FIG.8 shows a representative high resolution mass spectrum of BODIPY FL SNS 032.

[0021] FIG.9A-9B shows a representative HPLC spectrum of BODIPY FL SNS 032 (FIG.9A) and corresponding peak integration results (FIG.9B).

[0022] FIG.10A-10J shows representative TR-FRET longitudinal signal stability of BODIPYATTORNEY DOCKET NO.331904-2010 FL SNS 032 binding to hCAR. TR-FRET signlans were measured at 30 minutes (FIG.10A), 60 minutes (FIG. 10B), 90 minutes (FIG. 10C), 120 minutes (FIG. 10D), 150 minutes (FIG. 10E), 180 minutes (FIG.10F), 210 minutes (FIG.10G), 240 minutes (FIG.10H), 270 minutes (FIG.10I), and 300 minutes (FIG.10J). Curves connecting solid circles represent Tb-anti-GST and GST-hCAR-LBD in the presence of DMSO; curves connecting solid squares represent Tb-anti-GST and GST-hCAR-LBD in the presence of a target specific ligand as indicated; and curves connecting solid triangles represent Tb-anti-GST in the presence of DMSO without GST-hCAR-LBD protein.

[0023] FIG.11 shows a representative longitudinal signal stability of BODIPY FL SNS 032- mediated hCAR TR-FRET binding assay in response to the inhibition of the hCAR positive control ligand CITCO.

[0024] Additional advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the disclosure. The advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed. DETAILED DESCRIPTION

[0025] Many modifications and other aspects disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific aspects disclosed and that modifications and other aspects are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0026] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0027] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure.

[0028] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no wayATTORNEY DOCKET NO.331904-2010 intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0029] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0030] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0031] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0032] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure. A. DEFINITIONS

[0033] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0034] As used herein, the term “and / or” includes any and all combinations of one or more ofATTORNEY DOCKET NO.331904-2010 the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0035] As used herein, nomenclature for compounds, including organic compounds, can be given using common names, IUPAC, IUBMB, or CAS recommendations for nomenclature. When one or more stereochemical features are present, Cahn-Ingold-Prelog rules for stereochemistry can be employed to designate stereochemical priority, E / Z specification, and the like. One of skill in the art can readily ascertain the structure of a compound if given a name, either by systemic reduction of the compound structure using naming conventions, or by commercially available software, such as CHEMDRAWTM(Cambridgesoft Corporation, U.S.A.).

[0036] Reference to "a" chemical compound refers to one or more molecules of the chemical compound rather than being limited to a single molecule of the chemical compound. Furthermore, the one or more molecules may or may not be identical, so long as they fall under the category of the chemical compound. Thus, for example, "a" chemical compound is interpreted to include one or more molecules of the chemical, where the molecules may or may not be identical (e.g., different isotopic ratios, enantiomers, and the like).

[0037] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polypeptide,” “a residue,” or “a molecule,” includes, but is not limited to, two or more such polypeptides, residues, or molecules, and the like.

[0038] Reference to "a / an" protein and antibody each refers to one or more molecules of the protein and antibody rather than being limited to a single molecule of the protein and antibody. Furthermore, the one or more molecules may or may not be identical, so long as they fall under the category of the protein, and antibody. Thus, for example, "an" antibody is interpreted to include one or more antibody molecules of the antibody, where the antibody molecules may or may not be identical (e.g., different isotypes and / or different antigen binding sites as may be found in a polyclonal antibody).

[0039] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.ATTORNEY DOCKET NO.331904-2010

[0040] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0041] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0042] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0043] As used herein, a “polypeptide” refers to a polymer composed of amino acid residues, structural variants, related naturally-occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides can be prepared,ATTORNEY DOCKET NO.331904-2010 for example, using an automated polypeptide synthesizer. The term “protein” typically refers to large polypeptides. The term “peptide” typically refers to short polypeptides.

[0044] As used herein, a “fragment” of a polypeptide is meant to refer to any portion of a polypeptide or protein smaller than the full-length polypeptide or protein expression product.

[0045] As used herein, the term “variant” refers to a polypeptide, protein or analog thereof that is modified to comprise additional chemical moieties not normally a part of the molecule. Such moieties may modulate the molecule's solubility, absorption, biological half-life, etc. The moieties may alternatively decrease the toxicity of the molecule and eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedure for coupling such moieties to a molecule are well known in the art.

[0046] As used herein, a “tag” is an amino acid sequence fused to a heterologous protein that facilitates the detection or isolation of the heterologous protein. Tags contemplated for use with the compositions and methods described herein include, but are not limited to epitope tags, affinity tags and fluorescent proteins. An epitope tag is typically a short amino acid sequence that can be detected using antibodies that specifically recognize the tag. An affinity tag is a polypeptide sequence that specifically binds a substrate (for example, a histidine tag has affinity for nickel). Fluorescent proteins include, for example, GFP. Although tags are often grouped into the aforementioned categories, one of skill in the art will recognize that some tags can be members of more than one group. For example, specific antibodies are available for some types of affinity tags (e.g., a histidine tag), therefore these types of tags can be considered both affinity and epitope tags. In some embodiments, the nucleic acid modules disclosed herein encode an epitope tag, such as T7, FLAG, hemagglutinin (HA) VSV-G, V5 or c-myc. Antibodies to these and other epitope tags are commercially available for a variety of sources. In some embodiments, the tag is an affinity tag, such as a histidine tag (e.g., His6), MBP, CBP or GST. In some embodiments, the tag is a fluorescent protein, such as GFP or enhanced GFP.

[0047] As used herein, the term “tagging” refers to the process of recombinantly attaching a tag to a protein of interest, such as to facilitate detection or isolation of the protein.

[0048] As used herein, the term “BODIPY FL SNS 032” refers to a compound having a structure represented by a formula: .

[0049] As usedthe subsequentlyATTORNEY DOCKET NO.331904-2010 described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0050] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.

[0051] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0052] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0053] The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain aATTORNEY DOCKET NO.331904-2010 heteroatom. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0054] The term “heterocycle,” as used herein refers to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Heterocycle includes azetidine, dioxane, furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole, including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4- oxadiazole, piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran, tetrazine, including 1,2,4,5-tetrazine, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, thiadiazole, including, 1,2,3- thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, thiazole, thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine, triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like.

[0055] A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more -OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more -CO(CH2)8CO- moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

[0056] As used herein, the term “moiety” refers to one or more parts of the chemical structure of a compound or molecule. A moiety can be used to refer to a single functional group or to a larger portion of a compound or molecule (e.g., a portion of a molecule that contains two or more functional groups). A moiety can be referred to by all or part of its chemical structure and / or by one or more functions or chemical behaviors related to that portion of the molecule. For example, in the structure provided below, the moiety surrounded by the dashed-line box (−CH2CH2OH) can be referred to as, for example, a hydroxy moiety or an ethanol moiety .

[0057] The term “organiccontaining residue, i.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove. Organic residues can contain variousATTORNEY DOCKET NO.331904-2010 heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen, nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc. Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

[0058] A very close synonym of the term “residue” is the term “radical,” which as used in the specification and concluding claims, refers to a fragment, group, or substructure of a molecule described herein, regardless of how the molecule is prepared. For example, a 2,4- thiazolidinedione radical in a particular compound has the structure: , regardless of whether to prepare the compound. In someembodiments the radical (for example an alkyl) can be further modified (i.e., substituted alkyl) by having bonded thereto one or more “substituent radicals.” The number of atoms in a given radical is not critical to the present invention unless it is indicated to the contrary elsewhere herein.

[0059] Generally, the nomenclature used herein and many of the fluorescence, luminescence, computer, detection, chemistry, and laboratory procedures described herein are commonly employed in the art. Standard techniques are generally used for chemical synthesis, fluorescence or luminescence monitoring and detection, optics, molecular biology, and computer software and integration. Chemical reactions, cell assays, and enzymatic reactions are typically performed according to the manufacturer's specifications where appropriate. See, generally, Lakowicz, J. R. Topics in Fluorescence Spectroscopy, (3 volumes) New York: Plenum Press (1991), and Lakowicz, J. R. Emerging applications of florescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi photon excitation and light quenching, Scanning Microsc. Suppl. Vol. 10 (1996) pages 213-24, for fluorescence techniques; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., for molecular biology methods; Cells: A Laboratory Manual, 1st edition (1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., for cell biology methods; and Optics Guide 5 Melles Griot® Irvine Calif., and Optical Waveguide Theory, Snyder & Love (published by Chapman & Hall) for general optical methods, all of which are incorporated herein by reference.

[0060] General methods for performing a variety of fluorescent or luminescent assays onATTORNEY DOCKET NO.331904-2010 luminescent materials are known in the art and are described in, e.g., Lakowicz, J. R., Topics in Fluorescence Spectroscopy, volumes 1 to 3, New York: Plenum Press (1991); Herman, B., Resonance Energy Transfer Microscopy, in Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol.30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp.219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361; and Bernard Valeur, “Molecular Fluorescence: Principles and Applications” Wiley VCH, 2002. Guidance in the selection and use of specific resonance acceptor moieties is available at, for example, Berlman, I. B., Energy transfer parameters of aromatic compounds, Academic Press, New York and London (1973), which contains tables of spectral overlap integrals for the selection of resonance energy transfer pairs. Additional information sources include the Molecular Probes Catalog (2003) and website; and Tsien et al., 1990 Handbook of Biological Confocal Microscopy, pp. 169-178. Instruments useful for performing FP and / or RET and TR-RET applications are available from Tecan Group Ltd. (Switzerland) (Ultra, Ultra 384, Ultra Evolution); Perkin-Elmer (Boston, Mass.) (Fusion, EnVision, Victor V, and ViewLux), Amersham Bioscience (Piscataway, N.J.) (LeadSeeker); and Molecular Devices Corporation (Sunnyvale, Calif.) (Analyst AD, GT, and HT).

[0061] The term “FRET” means “fluorescence resonance energy transfer” or “Forster resonance energy transfer”, and refers to the radiationless transmission of an energy quantum from its site of absorption (the donor) to the site of its utilization (the acceptor) in a molecule, or system of molecules, by resonance interaction between donor and acceptor species, over distances considerably greater than interatomic, without substantial conversion to thermal energy, and without the donor and acceptor coming into kinetic collision. A donor is a moiety that initially absorbs energy (e.g., optical energy or electronic energy). A luminescent metal complex as described herein can comprise two donors: 1) an organic antenna moiety, which absorbs optical energy (e.g., from a photon); and 2) a lanthanide metal ion, which absorbs electronic energy (e.g., transferred from an organic antenna moiety).

[0062] The term “acceptor” refers to a chemical or biological moiety that accepts energy via resonance energy transfer. In FRET applications, acceptors may re-emit energy transferred from a donor fluorescent or luminescent moiety as fluorescence and are “fluorescent acceptor moieties.” As used herein, such a donor fluorescent or luminescent moiety and an acceptor fluorescent moiety are referred to as a “FRET pair.” Examples of acceptors include coumarins and related fluorophores; xanthenes such as fluoresceins and fluorescein derivatives; fluorescent proteins such as GFP and GFP derivatives; rhodols, rhodamines, and derivatives thereof; resorufins; cyanines; difluoroboradiazaindacenes; and phthalocyanines Acceptors, including fluorescent acceptor moieties, can also be useful as fluorescent probes in fluorescence polarization assays.

[0063] The terms “label” or “labeled” refer to the inclusion of a luminescent metal complex orATTORNEY DOCKET NO.331904-2010 a fluorescent acceptor moiety on a molecule or substance.

[0064] The term “covalent” refers to a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds. Covalent attachment of biotin occurs on a particular lysine residue of a peptide sequence. Biotinylation can occur either “in vivo”, whereby intracellular biotinylation enzymes are able to recognize the amino acid sequence to attach the biotin molecule, or “in vitro” whereby biotinylation occurs through the use of the enzyme biotin ligase (BirA) and ATP.

[0065] “Kit” as used herein means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0066] “Instruction(s)” as used herein means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents and are meant to include future updates.

[0067] Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically-labelled or isotopically-substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorous, fluorine and chlorine, such as 2H,3H,13C,14C,15N,18O,17O,35S,18F and36Cl, respectively. Compounds further comprise prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e.,3H, and carbon-14, i.e.,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavierATTORNEY DOCKET NO.331904-2010 isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labelled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labelled reagent for a non- isotopically labelled reagent.

[0068] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0069] Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

[0070] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the sameATTORNEY DOCKET NO.331904-2010 function that are related to the disclosed structures, and that these structures will typically achieve the same result.

[0071] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere). B. ABBREVIATIONS

[0072] DCM dichloromethane

[0073] EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0074] FP fluorescence polarization

[0075] hCAMKK2 human Calcium / calmodulin-dependent protein kinase kinase 2

[0076] hCAR human constitutive androstane receptor

[0077] hCASK human calcium / calmodulin-dependent serine protein kinase

[0078] hCDK9 / CycT1 human cyclin-dependent kinase 9 and cyclin T1 complex

[0079] hGSK3A human glycogen synthase kinase 3 alpha

[0080] hMAPK15 human mitogen-activated protein kinase 15

[0081] HTRF Homogeneous Time Resolved Fluorescence

[0082] ITC isothermal titration calorimetry

[0083] LBD ligand binding domain

[0084] MDR1 multi-drug resistance protein 1

[0085] MRPs multidrug resistance-associated proteins

[0086] SPR surface plasmon resonance

[0087] TR-FRET time-resolved fluorescence energy transfer C. INTRODUCTION

[0088] Human constitutive androstane receptor (hCAR, NR1I3) is a member of the nuclear receptor superfamily and one of master regulators of endobiotic and xenobiotic metabolism and disposition.1hCAR has a relatively large and flexible ligand binding domain (LBD)2that can be bound by many structurally diverse compounds.3, 4hCAR transcriptionally regulates many proteins involved in drug metabolism and disposition, such as cytochrome P450 (CYP) enzymes CYP2Bs, CYP2Cs, CYP3As and drug transporters multi-drug resistance protein 1 (MDR1) and multidrug resistance-associated proteins (MRPs).5Therefore binding of ligands to hCAR alters the metabolism and disposition of endobiotics and xenobiotics.6Among multiple hCAR splicing variants, hCAR1 is the most abundant and constitutively active isoform7and is one of the focuses discussed herein.

[0089] Many compounds3, 8have been identified or developed as hCAR activators (agonists) and inhibitors (inverse agonists, deactivators, or antagonists) with representatives summarized in FIG.1. CITCO (1) is a potent hCAR agonist.9CINPA1 (2),1072 (3),8T0901317 (4),11PK 11195 (5),12clotrimazole (6),13and S07662 (7)14are hCAR inverse agonists. Steroids androstenol (12) and androstanol (13) are mouse CAR inverse agonists,40and androstenolATTORNEY DOCKET NO.331904-2010 (12) also deactivates hCAR.14,41Because hCAR is constitutively active,15hCAR inhibition could provide therapeutic benefit by increasing drug efficacy or reducing unwanted drug-drug interactions.16On the other hand, hCAR agonists have the potential for therapeutic application in diabetes, obesity, and liver protection.16CITCO also activates PXR.42The efforts in developing CITCO analogue as hCAR-specific agonists led to DL5050 (14)43and 39 (15).44

[0090] To characterize its ligands and study the function of hCAR, many assays have been developed,17such as radioligand-based scintillation proximity assay and fluorescent peptide- based time-resolved fluorescence energy transfer (TR-FRET) coactivator peptide recruitment / repression assay, thermal shift assay, surface plasmon resonance (SPR) assay and isothermal titration calorimetry (ITC) assay, as well as luciferase-based reporter assays, mammalian two-hybrid assays and assays using primary human hepatocytes. Among these reported assays, fluorescence polarization (FP) and TR-FRET assays have the advantage of being suitable for high throughput large scale screens.18However, the coactivator peptide- based TR-FRET hCAR assay measures the effect of ligand binding on coactivator recruitment to the C-terminal activation function 2 (AF2) domain of hCAR, instead of competitive binding of the ligand to the LBD.17, 19A fluorescence-based assay capable of measuring ligand binding to the hCAR LBD is more relevant for determining ligand binding to hCAR, and advantageous for high throughput applications. However, although many structurally diverse hCAR ligands have been identified (FIG. 1) with some of them co-crystalized with hCAR ,15, 19, 20a high throughput assay for evaluating ligand interaction with hCAR LBD has not yet been reported.

[0091] Additionally, protein kinases are an important drug target class, and identifying compounds (probes) that bind to protein kinase is a necessary step in kinase drug discovery. Currently, there is no small molecule-based fluorescent probe that can be used to develop hCAR binding assay, and no small molecule-based fluorescent probe that can be used for multiple protein kinases. In one aspect, particular protein kinases of interest include GSK3A, NEK6, CDC2L5, ICK, CDKL5, CSNK1E, GSK3B, DYRK1B, CDK9, CSNK1D, ERK8, CSNK1A1, CDK7, DYRK1A, PCTK2, CLK2, RIOK2, CASK, CTK, NIK, YSK4, LKB1, RIPK5, MTOR, BTK, CDK4-cyclinD1, MAP3K15, CDKL2, BMPR1B, BMX, CSF1R-autoinhibited, STK39, ADCK3, and CAMKK2. In a further aspect, particular protein kinases of interest include hGSK3A, hNEK6, hCDC2L5, hICK, hCDKL5, hCSNK1E, hGSK3B, hDYRK1B, hCDK9, hCSNK1D, hERK8, hCSNK1A1, hCDK7, hDYRK1A, hPCTK2, hCLK2, hRIOK2, hCASK, hCTK, hNIK, hYSK4, hLKB1, hRIPK5, hMTOR, hBTK, hCDK4-cyclinD1, hMAP3K15, hCDKL2, hBMPR1B, hBMX, hCSF1R-autoinhibited, hSTK39, hADCK3, and hCAMKK2.

[0092] In one aspect, the disclosure relates to small molecule compounds that has a relatively high binding affinity for hCAR and other various kinases. In a further aspect, the compounds can be used in binding assays such as TR-FRET and FP to characterize hCAR ligands for their competitive binding activities.ATTORNEY DOCKET NO.331904-2010 D. COMPOUNDS

[0093] In one aspect, the disclosure relates to a compound having a structure represented by a formula: , wherein Ln can be and q can be an integer selected from 0can have a structure represented by a formula: . In another further represented by aformula: .

[0094] The linking; wherein, G can befrom O, NH, CO, CH2, (CH2)2, NHC(O), and C(O)NH; Z1and Z2can be independently selected from CH2and OCH2CH2; R1and R2can be independently selected from a cycloalkyl group, a heterocycloalkyl group, an aryl group, and a heteroaryl group; m1can be an integer selected from 0, 1, 2, and 3; m2can be an integer selected from 0, 1, 2, and 3; n can be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; q1can be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and q2can be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0095] In a further aspect, R1and R2can be independently selected from a C4-C10 cycloalkylATTORNEY DOCKET NO.331904-2010 group, a C4-C10 heterocycloalkyl group, a C4-C10 aryl group, and a C4-C10 heteroaryl group. In another aspect, R1and R2can be independently selected from a C4-C8 cycloalkyl group, a C4-C8 heterocycloalkyl group, a C4-C8 aryl group, and a C4-C8 heteroaryl group. In another aspect, R1and R2can be independently selected from a C4-C6 cycloalkyl group, a C4-C6 heterocycloalkyl group, a C4-C6 aryl group, and a C4-C6 heteroaryl group. The heterocycloalkyl group (including the C4-C10 heterocycloalkyl group and the C4-C8 heterocycloalkyl group) can comprise at least one nitrogen heteroatom, such as pyrrolidine, piperidine, piperazine, a triazinane, or a derivative thereof. The heteroaryl group (including the C4-C8 heteroaryl group and the C4-C6 heteroaryl group) can include at least one nitrogen heteroatom, such as triazole, pyridine, pyridazine, pyrimidine, pyrazine, a triazine, or a derivative thereof.

[0096] The fluorophore moiety can have an excitation maximum in the range of about 460 nm to about 650 nm, about 460 nm to about 600 nm, about 460 nm to about 550 nm, about 460 nm to about 500 nm, about 500 nm to about 650 nm, about 550 nm to about 650 nm, about 600 nm to about 650 nm, or about 500 nm to about 600 nm and / or an emission maximum in the range of about 510 nm to about 670 nm, about 510 nm to about 600 nm, about 510 nm to about 550 nm, about 550 nm to about 670 nm, about 600 nm to about 670 nm, or about 550 nm to about 600 nm. In another aspect, the fluorophore moiety can be derived from a structure represented by a formula: ,ATTORNEY DOCKET NO.331904-2010 ,ATTORNEY DOCKET NO.331904-2010 .

[0097] The fluorophore can attach to the linking moiety (when q is 1) or the nitrogen of the piperidine ring (when q is 0) via one of an amine linking group, an ether linking group, or a carbonyl linking group. E. METHODS

[0098] Also disclosed herein is a method to determine binding affinity of a compound interacting with a constitutive androstane receptor or a kinase, the method comprising the steps of: (a) providing a solution comprising: (i) a test compound; (ii) a polypeptide comprising a domain, or a ligand binding fragment polypeptide thereof, that can bind a moiety of at least one compound disclosed herein that has a structure given by the formula:ATTORNEY DOCKET NO.331904-2010 (where the wavy line indicates that the compound extends beyond that point, such as, for example, being bonded to a linking moiety or a fluorophore moiety); (iii) a fluorescent donor molecule; and (iv) a fluorescent acceptor molecule comprising at least one disclosed compound; (b) illuminating the solution, thereby causing fluorescence in the fluorescent donor molecule; (c) measuring fluorescence emission from the fluorescent donor molecule and the fluorescent acceptor molecule; and (d) comparing the fluorescence emission to the fluorescence emission of steps (a)-(c) carried out in the absence of the test compound.

[0099] In another aspect, the fluorophore can be derived from a structure represented by a formula: .

[0100] In another aspect, the can include chelated terbium andone or more moieties capable of The chelated terbium can include at least one diethylenetriaminepentaacetic acid residue and Tb+3.

[0101] In another aspect, the one or more moieties capable of binding the polypeptide can include an antibody. The antibody can be a monoclonal or a polyclonal antibody. In one aspect, the antibody recognizes an epitope on the polypeptide. In a further aspect, the epitope on the polypeptide can include one or more affinity tags or epitope tags. The affinity tag can be selected from histidine tag, maltose binding protein tag, chitin binding protein tag, and glutathione-S-transferase tag. The epitope tag can be selected from T7 tag, FLAG tag, HA tag, VSV-G tag, V5 tag, and c-myc tag. The at least one polypeptide epitope can be a native sequence of the at least one polypeptide.

[0102] In a further aspect, the polypeptide can be selected from constitutive androstane receptor 1 polypeptide, encoded by a CAR1 gene; glycogen synthase kinase-3 alpha polypeptide, encoded by a GSK3A gene; cyclin-dependent kinase 9 polypeptide, encoded by a CDK9 gene; mitogen-Activated protein kinase 15 polypeptide, encoded by a MAPK15 gene; calcium / calmodulin dependent serine protein kinase polypeptide, encoded by a CASK gene; calcium / calmodulin-dependent protein kinase polypeptide, encoded by a CAMKK2 gene; a splice variant thereof; and a ligand binding fragment polypeptide thereof.

[0103] In a further aspect, the polypeptide can be encoded by the human isoform of the corresponding gene. The polypeptide encoded can be selected from the constitutive androstane receptor 1 polypeptide corresponding to UniProt identifier Q14994; the glycogen synthase kinase-3 alpha polypeptide corresponding to UniProt identifier P49840; the cyclin- dependent kinase 9 polypeptide corresponding to UniProt identifier P50750; the mitogen- Activated protein kinase 15 polypeptide corresponding to UniProt identifier Q8TD08; theATTORNEY DOCKET NO.331904-2010 calcium / calmodulin dependent serine protein kinase polypeptide corresponding to UniProt identifier O14936; and the calcium / calmodulin-dependent protein kinase polypeptide corresponding to UniProt identifier Q96RR4. F. KITS

[0104] Also disclosed herein is a kit for measuring the binding activity of a test compound to human constitutive androstane receptor (hCAR) and / or a kinase comprising: at least one compound disclosed herein, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; and optionally, one or more of: (a) instructions for carrying out an in vivo fluorescence assay using at least one compound disclosed herein; (b) instructions for carrying out an in vitro fluorescence assay using at least one compound disclosed herein; (c) at least one polypeptide that can bind the at least one compound disclosed herein, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; (d) at least one compound that binds to hCAR and / or a kinase; (e) one or more test compounds; and / or (f) at least one buffer and / or salt.

[0105] The disclosed compounds and / or pharmaceutically acceptable salt, solvate, or polymorph thereof can conveniently be presented as a kit, whereby two or more components, can be provided with instructions for measuring the binding activity of a test compound to human constitutive androstane receptor (hCAR) and / or a kinase. Such kits may be provided with all necessary materials and ingredients contained therein, or they may contain instructions for using or making materials or components that must be obtained independently by the person using the kit. It is contemplated that the disclosed kits can be used in connection with the disclosed methods of determining binding affinity and / or the disclosed compounds.

[0106] In one aspect, the in vivo fluorescence assay can be a homogeneous time resolved fluorescence (HTRF) assay. In another aspect, the in vivo fluorescence assay can be a fluorescence polarization assay. In another aspect, the in vitro fluorescence assay can be a homogeneous time resolved fluorescence (HTRF) assay. In another aspect, the in vitro fluorescence assay can be a fluorescence polarization assay.

[0107] The kit can further include an hCAR co-activator. The hCAR co-activator can include a Cy5-PGC1α peptide, a Cy5-SRC peptide, a PGC1α peptide, or a combination thereof.

[0108] The kit can also include instructions for carrying out an in vitro fluorescence assay; and further include the at least one polypeptide that binds a moiety of the least one compound disclosed herein having a structure given by the formula: . The in vivocan be a time-resolved fluorescence energy transfer (TR-FRET) assay.

[0109] In a further aspect, the kit can include a fluorescent donor molecule comprising aATTORNEY DOCKET NO.331904-2010 fluorescence donor molecule bound to one or more moieties capable of binding the at least one polypeptide. The fluorescence donor molecule can be a chelated terbium. The chelated terbium can include at least one diethylenetriaminepentaacetic acid residue and Tb+3.

[0110] The at least one polypeptide can include an antibody. In one aspect, the antibody recognizes an epitope on the at least one polypeptide. The epitope on the at least one polypeptide can include one or more affinity tags or epitope tags. In one aspect, the affinity tag is selected from histidine tag, maltose binding protein tag, chitin binding protein tag, and glutathione-S-transferase tag. In another aspect, the epitope tag is selected from T7 tag, FLAG tag, HA tag, VSV-G tag, V5 tag, and c-myc tag. The at least one polypeptide epitope can be a native sequence of the at least one polypeptide. G. REFERENCES

[0111] References are cited herein throughout using the format of superscripted reference number(s) corresponding to one or more of the following numbered references. For example, citation of references numbers 1 and 2 immediately herein below would be indicated in the disclosure as1,2.

[0112] Ref.1 Wang, Y. M.; Ong, S. S.; Chai, S. C.; Chen, T. Role of CAR and PXR in xenobiotic sensing and metabolism. Expert Opin Drug Metab Toxicol 2012, 8 (7), 803-817.

[0113] Ref.2 Buchman, C. D.; Chai, S. C.; Chen, T. A current structural perspective on PXR and CAR in drug metabolism. Expert Opin Drug Metab Toxicol 2018, 14 (6), 635-647.

[0114] Ref.3 Chai, S. C.; Cherian, M. T.; Wang, Y. M.; Chen, T. Small-molecule modulators of PXR and CAR. Biochim Biophys Acta 2016, 1859 (9), 1141-1154.

[0115] Ref.4 Poudel, S.; Huber, A. D.; Chen, T. Regulation of Nuclear Receptors PXR and CAR by Small Molecules and Signal Crosstalk: Roles in Drug Metabolism and Beyond. Drug Metab Dispos 2023, 51 (2), 228-236.

[0116] Ref.5 Lamba, J. K. Pharmacogenetics of the constitutive androstane receptor. Pharmacogenomics 2008, 9 (1), 71-83.

[0117] Ref.6 Kobayashi, K.; Hashimoto, M.; Honkakoski, P.; Negishi, M. Regulation of gene expression by CAR: an update. Arch Toxicol 2015, 89 (7), 1045-1055.

[0118] Ref.7 Jinno, H.; Tanaka-Kagawa, T.; Hanioka, N.; Ishida, S.; Saeki, M.; Soyama, A.; Itoda, M.; Nishimura, T.; Saito, Y.; Ozawa, S., et al. Identification of novel alternative splice variants of human constitutive androstane receptor and characterization of their expression in the liver. Mol Pharmacol 2004, 65 (3), 496-502.

[0119] Ref.8 Lin, W.; Yang, L.; Chai, S. C.; Lu, Y.; Chen, T. Development of CINPA1 analogs as novel and potent inverse agonists of constitutive androstane receptor. Eur J Med Chem 2016, 108, 505-528.

[0120] Ref.9 Maglich, J. M.; Parks, D. J.; Moore, L. B.; Collins, J. L.; Goodwin, B.; Billin, A. N.; Stoltz, C. A.; Kliewer, S. A.; Lambert, M. H.; Willson, T. M., et al. Identification ofATTORNEY DOCKET NO.331904-2010 a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes. J Biol Chem 2003, 278 (19), 17277-17283.

[0121] Ref.10 Cherian, M. T.; Yang, L.; Chai, S. C.; Lin, W.; Chen, T. Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor. Drug Metab Dispos 2016, 44 (11), 1759-1770.

[0122] Ref.11 Kanno, Y.; Tanuma, N.; Takahashi, A.; Inouye, Y. TO901317, a potent LXR agonist, is an inverse agonist of CAR. J Toxicol Sci 2013, 38 (3), 309-315.

[0123] Ref.12 Li, L.; Chen, T.; Stanton, J. D.; Sueyoshi, T.; Negishi, M.; Wang, H. The peripheral benzodiazepine receptor ligand 1-(2-chlorophenyl-methylpropyl)-3-isoquinoline- carboxamide is a novel antagonist of human constitutive androstane receptor. Mol Pharmacol 2008, 74 (2), 443-453.

[0124] Ref.13 Moore, L. B.; Parks, D. J.; Jones, S. A.; Bledsoe, R. K.; Consler, T. G.; Stimmel, J. B.; Goodwin, B.; Liddle, C.; Blanchard, S. G.; Willson, T. M., et al. Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J Biol Chem 2000, 275 (20), 15122-15127.

[0125] Ref.14 Küblbeck, J.; Jyrkkärinne, J.; Molnár, F.; Kuningas, T.; Patel, J.; Windshügel, B.; Nevalainen, T.; Laitinen, T.; Sippl, W.; Poso, A., et al. New in vitro tools to study human constitutive androstane receptor (CAR) biology: discovery and comparison of human CAR inverse agonists. Mol Pharm 2011, 8 (6), 2424-2433.

[0126] Ref.15 Suino, K.; Peng, L.; Reynolds, R.; Li, Y.; Cha, J. Y.; Repa, J. J.; Kliewer, S. A.; Xu, H. E. The nuclear xenobiotic receptor CAR: structural determinants of constitutive activation and heterodimerization. Mol Cell 2004, 16 (6), 893-905.

[0127] Ref.16 Chai, X.; Zeng, S.; Xie, W. Nuclear receptors PXR and CAR: implications for drug metabolism regulation, pharmacogenomics and beyond. Expert Opin Drug Metab Toxicol 2013, 9 (3), 253-266.

[0128] Ref.17 Chai, S. C.; Lin, W.; Li, Y.; Chen, T. Drug discovery technologies to identify and characterize modulators of the pregnane X receptor and the constitutive androstane receptor. Drug Discov Today 2019, 24 (3), 906-915.

[0129] Ref.18 Raucy, J. L.; Lasker, J. M. Current in vitro high throughput screening approaches to assess nuclear receptor activation. Curr Drug Metab 2010, 11 (9), 806-814.

[0130] Ref.19 Xu, R. X.; Lambert, M. H.; Wisely, B. B.; Warren, E. N.; Weinert, E. E.; Waitt, G. M.; Williams, J. D.; Collins, J. L.; Moore, L. B.; Willson, T. M., et al. A structural basis for constitutive activity in the human CAR / RXRalpha heterodimer. Mol Cell 2004, 16 (6), 919- 928.

[0131] Ref.20 Shan, L.; Vincent, J.; Brunzelle, J. S.; Dussault, I.; Lin, M.; Ianculescu, I.; Sherman, M. A.; Forman, B. M.; Fernandez, E. J. Structure of the murine constitutive androstane receptor complexed to androstenol: a molecular basis for inverse agonism. Mol Cell 2004, 16 (6), 907-917.ATTORNEY DOCKET NO.331904-2010

[0132] Ref.21 Fabian, M. A.; Biggs, W. H., 3rd; Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M., et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 2005, 23 (3), 329-336.

[0133] Ref.22 Olson, C. M.; Jiang, B.; Erb, M. A.; Liang, Y.; Doctor, Z. M.; Zhang, Z.; Zhang, T.; Kwiatkowski, N.; Boukhali, M.; Green, J. L., et al. Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation. Nat Chem Biol 2018, 14 (2), 163-170.

[0134] Ref.23 Chen, R.; Wierda, W. G.; Chubb, S.; Hawtin, R. E.; Fox, J. A.; Keating, M. J.; Gandhi, V.; Plunkett, W. Mechanism of action of SNS-032, a novel cyclin-dependent kinase inhibitor, in chronic lymphocytic leukemia. Blood 2009, 113 (19), 4637-4645.

[0135] Ref.24 Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. M.; Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P. Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol 2011, 29 (11), 1046-1051.

[0136] Ref.25 Conroy, A.; Stockett, D. E.; Walker, D.; Arkin, M. R.; Hoch, U.; Fox, J. A.; Hawtin, R. E. SNS-032 is a potent and selective CDK 2, 7 and 9 inhibitor that drives target modulation in patient samples. Cancer Chemother Pharmacol 2009, 64 (4), 723-732.

[0137] Ref.26 Klevernic, I. V.; Stafford, M. J.; Morrice, N.; Peggie, M.; Morton, S.; Cohen, P. Characterization of the reversible phosphorylation and activation of ERK8. Biochem J 2006, 394 (Pt 1), 365-373.

[0138] Ref.27 Valenciano, A. L.; Knudsen, G. M.; Mackey, Z. B. Extracellular-signal regulated kinase 8 of Trypanosoma brucei uniquely phosphorylates its proliferating cell nuclear antigen homolog and reveals exploitable properties. Cell Cycle 2016, 15 (20), 2827- 2841.

[0139] Ref.28 Russ, N.; Schröder, M.; Berger, B. T.; Mandel, S.; Aydogan, Y.; Mauer, S.; Pohl, C.; Drewry, D. H.; Chaikuad, A.; Müller, S., et al. Design and Development of a Chemical Probe for Pseudokinase Ca(2+) / calmodulin-Dependent Ser / Thr Kinase. J Med Chem 2021, 64 (19), 14358-14376.

[0140] Ref.29 Ramsden, N.; Perrin, J.; Ren, Z.; Lee, B. D.; Zinn, N.; Dawson, V. L.; Tam, D.; Bova, M.; Lang, M.; Drewes, G., et al. Chemoproteomics-based design of potent LRRK2-selective lead compounds that attenuate Parkinson's disease-related toxicity in human neurons. ACS Chem Biol 2011, 6 (10), 1021-1028.

[0141] Ref.30 Wu, Q.; Lin, W.; Li, Z. M.; Rankovic, Z.; White, S. W.; Chen, T.; Yang, J. A protocol for high-throughput screening of histone lysine demethylase 4 inhibitors using TR-FRET assay. STAR Protoc 2021, 2 (3), 100702.

[0142] Ref.31 Lin, W.; Li, Y.; Yang, L.; Chen, T. Development of BODIPY FL VH032 as a High-Affinity and Selective von Hippel-Lindau E3 Ligase Fluorescent Probe and Its Application in a Time-Resolved Fluorescence Resonance Energy-Transfer Assay. ACS Omega 2021, 6 (1), 680-695.ATTORNEY DOCKET NO.331904-2010

[0143] Ref.32 Lin, W.; Li, Y.; Min, J.; Liu, J.; Yang, L.; Lee, R. E.; Chen, T. Development of BODIPY FL Thalidomide As a High-Affinity Fluorescent Probe for Cereblon in a Time-Resolved Fluorescence Resonance Energy Transfer Assay. Bioconjug Chem 2020, 31 (11), 2564-2575.

[0144] Ref.33 Lin, W.; Chen, T. Using TR-FRET to Investigate Protein-Protein Interactions: A Case Study of PXR-Coregulator Interaction. Adv Protein Chem Struct Biol 2018, 110, 31-63.

[0145] Ref.34 Lin, W.; Liu, J.; Jeffries, C.; Yang, L.; Lu, Y.; Lee, R. E.; Chen, T. Development of BODIPY FL vindoline as a novel and high-affinity pregnane X receptor fluorescent probe. Bioconjug Chem 2014, 25 (9), 1664-1677.

[0146] Ref.35 Lin, W.; Chen, T. A vinblastine fluorescent probe for pregnane X receptor in a time-resolved fluorescence resonance energy transfer assay. Anal Biochem 2013, 443 (2), 252-260.

[0147] Ref.36 Yu, D. D.; Lin, W.; Chen, T.; Forman, B. M. Development of time resolved fluorescence resonance energy transfer-based assay for FXR antagonist discovery. Bioorg Med Chem 2013, 21 (14), 4266-4278.

[0148] Ref.37 Shi, Z.; Tian, L.; Qiang, T.; Li, J.; Xing, Y.; Ren, X.; Liu, C.; Liang, C. From Structure Modification to Drug Launch: A Systematic Review of the Ongoing Development of Cyclin-Dependent Kinase Inhibitors for Multiple Cancer Therapy. J Med Chem 2022, 65 (9), 6390-6418.

[0149] Ref.38 Tsakos, M.; Schaffert, E. S.; Clement, L. L.; Villadsen, N. L.; Poulsen, T. B. Ester coupling reactions--an enduring challenge in the chemical synthesis of bioactive natural products. Nat Prod Rep 2015, 32 (4), 605-632.

[0150] Ref.39 Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 1973, 22 (23), 3099-3108.

[0151] Ref.40 Forman, B. M.; Tzameli, I.; Choi, H. S.; Chen, J.; Simha, D.; Seol, W.; Evans, R. M.; Moore, D. D. Androstane metabolites bind to and deactivate the nuclear receptor CAR-beta. Nature 1998, 395 (6702), 612−615.

[0152] Ref.41 Carazo, A.; Pávek, P. The Use of the LanthaScreen TR-FRET CAR Coactivator Assay in the Characterization of Constitutive Androstane Receptor (CAR) Inverse Agonists. Sensors (Basel) 2015, 15 (4), 9265−9276.

[0153] Ref.42 Lin, W.; Bwayi, M.; Wu, J.; Li, Y.; Chai, S. C.; Huber, A. D.; Chen, T. CITCO Directly Binds to and Activates Human Pregnane X Receptor. Mol. Pharmacol.2020, 97 (3), 180−190.

[0154] Ref.43 Liang, D.; Li, L.; Lynch, C.; Diethelm-Varela, B.; Xia, M.; Xue, F.; Wang, H. DL5050, a Selective Agonist for the Human Constitutive Androstane Receptor. ACS Med. Chem. Lett.2019, 10 (7), 1039−1044.ATTORNEY DOCKET NO.331904-2010

[0155] Ref.44 Mejdrová, I.; Dušek, J.; Škach, K.; Stefela, A.; Skoda, J.; Chalupský, K.; Dohnalová, K.; Pavkova, I.; Kronenberger, T.; Rashidian, A.; et al. Discovery of Novel Human Constitutive Androstane Receptor Agonists with the Imidazo[1,2-a]pyridine Structure. J. Med. Chem.2023, 66 (4), 2422−2456. H. ASPECTS

[0156] The following glisting of exemplary aspects supports and is supported by the disclosure provided herein.

[0157] Aspect 1. A compound having a structure represented by a formula wherein Ln is a wherein q is an integer selected from 0 and

[0158] Aspect 2. The compound of aspect 1, wherein q is 1; and wherein the compound has a structure represented by a formula .

[0159] Aspect 3. Themoiety has a structure represented by a formula ; wherein, G is selectedO, NH, CO, CH2, (CH2)2, NHC(O), and C(O)NH; Z1and Z2are independently selected from CH2and OCH2CH2; R1and R2are independently selected from a cycloalkyl group, a heterocycloalkyl group, an aryl group, and a heteroaryl group; m1is an integer selected from 0, 1, 2, and 3; m2is an integer selected from 0, 1, 2, and 3; n is an integer selected from1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; q1is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and q2is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.ATTORNEY DOCKET NO.331904-2010

[0160] Aspect 4. The compound of any one of aspects 1-3, wherein G is selected from NH and CH2.

[0161] Aspect 5. The compound of any one of aspects 1-4, wherein L is selected from NH, CO, CH2, NHC(O), and C(O)NH.

[0162] Aspect 6. The compound of any one of aspects 1-5, wherein R1and R2are independently selected from a C4-C10 cycloalkyl group, a C4-C10 heterocycloalkyl group, a C4-C10 aryl group, and a C4-C10 heteroaryl group.

[0163] Aspect 7. The compound of any one of aspects 1-6, wherein R1and R2are independently selected from a C4-C8 cycloalkyl group, a C4-C8 heterocycloalkyl group, a C4- C8 aryl group, and a C4-C8 heteroaryl group.

[0164] Aspect 8. The compound of any one of aspects 1-7, wherein R1and R2are independently selected from a C4-C6 cycloalkyl group, a C4-C6 heterocycloalkyl group, a C4- C6 aryl group, and a C4-C6 heteroaryl group.

[0165] Aspect 9. The compound of any one of aspects 1-8, wherein the heterocycloalkyl group comprises at least one nitrogen heteroatom.

[0166] Aspect 10. The compound of any one of aspects 1-9, wherein the heteroaryl group comprises at least one nitrogen heteroatom.

[0167] Aspect 11. The compound of any one of aspects 1-10, wherein m1is an integer selected from 0 and 1.

[0168] Aspect 12. The compound of any one of aspects 1-11, wherein m2is an integer selected from 0 and 1.

[0169] Aspect 13. The compound of any one of aspects 1-12, wherein n is an integer selected from 1, 2, 3, 4, 5, and 6.

[0170] Aspect 14. The compound of any one of aspects 1-13, wherein q1is an integer selected from 1, 2, 3, 4, 5, and 6.

[0171] Aspect 15. The compound of any one of aspects 1-14, wherein q2is an integer selected from 1, 2, 3, 4, 5, and 6.

[0172] Aspect 16. The compound of aspect 1, wherein q is 0; and wherein the compound has a structure represented by a formula .

[0173] Aspect 17. Thethe fluorophore moiety has an excitation maximum in the range of about 460 nm to about 650 nm and an emission maximum in the range of about 510 nm to about 670 nm.

[0174] Aspect 18. The compound of any one of aspects 1-17, wherein the fluorophore is derived from a structure represented by a formulaATTORNEY DOCKET NO.331904-2010 , ,ATTORNEY DOCKET NO.331904-2010 , ,ATTORNEY DOCKET NO.331904-2010 . the fluorophoreor one of an amine linking group, an ether linking group, or a carbonyl linking group.

[0176] Aspect 20. A method to determine binding affinity of a compound interacting with a constitutive androstane receptor or a kinase, the method comprising the steps of a) providing a solution comprising: i. a test compound; ii. a polypeptide comprising a domain, or a ligand binding fragment polypeptide thereof, that can bind a moiety of at least one compound of any one of aspects 1-20 having a structure given by the formula; iii. a fluorescent donor molecule comprising the atleast one compound of any one of aspects 1-20; b) illuminating the solution, thereby causing fluorescence in the fluorescent donor molecule; c) measuring fluorescence emission from the fluorescent donor molecule and the fluorescent acceptor molecule; and d) comparing the fluorescence emission to the fluorescence emission of steps (a)-(c) carried out in the absence of the test compound.

[0177] Aspect 21. The method of aspect 20, wherein the fluorophore is derived from a structure represented by a formula .

[0178] Aspect 22. The method21, wherein the fluorescent donor molecule comprises chelated terbium and one or more moieties capable of binding the polypeptide.

[0179] Aspect 23. The method of aspect 22, wherein the chelated terbium comprises at least one diethylenetriaminepentaacetic acid residue and Tb+3.ATTORNEY DOCKET NO.331904-2010

[0180] Aspect 24. The method of aspect 22 or aspect 23, wherein the one or more moieties capable of binding the polypeptide comprises an antibody.

[0181] Aspect 25. The method of aspect 24, wherein the antibody recognizes an epitope on the polypeptide.

[0182] Aspect 26. The method of aspect 25, wherein the epitope on the polypeptide comprises one or more affinity tags or epitope tags.

[0183] Aspect 27. The method of aspect 26, wherein the affinity tag is selected from histidine tag, maltose binding protein tag, chitin binding protein tag, and glutathione-S-transferase tag.

[0184] Aspect 28. The method of aspect 26, wherein the epitope tag is selected from T7 tag, FLAG tag, HA tag, VSV-G tag, V5 tag, and c-myc tag.

[0185] Aspect 29. The method of aspect 25, wherein the at least one polypeptide epitope is a native sequence of the at least one polypeptide.

[0186] Aspect 30. The method of any one of aspects 24-29, wherein the antibody is a monoclonal antibody.

[0187] Aspect 31. The method of any one of aspects 24-29, wherein the antibody is a polyclonal antibody.

[0188] Aspect 32. The method of any one of aspects 20-31, wherein the polypeptide is selected from constitutive androstane receptor 1 polypeptide, encoded by a CAR1 gene; glycogen synthase kinase-3 alpha polypeptide, encoded by a GSK3A gene; cyclin-dependent kinase 9 polypeptide, encoded by a CDK9 gene; mitogen-Activated protein kinase 15 polypeptide, encoded by a MAPK15 gene; calcium / calmodulin dependent serine protein kinase polypeptide, encoded by a CASK gene; calcium / calmodulin-dependent protein kinase polypeptide, encoded by a CAMKK2 gene; a splice variant thereof; and a ligand binding fragment polypeptide thereof.

[0189] Aspect 33. The method of aspect 32, wherein the polypeptide is encoded by the human isoform of the corresponding gene.

[0190] Aspect 34. The method of aspect 33, wherein the polypeptide encoded is selected from the constitutive androstane receptor 1 polypeptide corresponding to UniProt identifier Q14994; the glycogen synthase kinase-3 alpha polypeptide corresponding to UniProt identifier P49840; the cyclin-dependent kinase 9 polypeptide corresponding to UniProt identifier P50750; the mitogen-Activated protein kinase 15 polypeptide corresponding to UniProt identifier Q8TD08; the calcium / calmodulin dependent serine protein kinase polypeptide corresponding to UniProt identifier O14936; and the calcium / calmodulin-dependent protein kinase polypeptide corresponding to UniProt identifier Q96RR4.

[0191] Aspect 35. A kit for measuring the binding activity of a test compound to human constitutive androstane receptor (hCAR) and / or a kinase comprising at least one compound of any of aspects 1-19, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; and optionally, one or more of a) instructions for carrying out an in vivo fluorescence assayATTORNEY DOCKET NO.331904-2010 using at least one compound of any of aspects 1-19; b) instructions for carrying out an in vitro fluorescence assay using at least one compound of any of aspects 1-19; c) at least one polypeptide that can bind the at least one compound of any of aspects 1-19, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; d) at least one compound that binds to hCAR and / or a kinase; e) one or more test compounds; and / or f) at least one buffer and / or salt.

[0192] Aspect 36. The kit of aspect 35, wherein the in vivo fluorescence assay is a homogeneous time resolved fluorescence (HTRF) assay.

[0193] Aspect 37. The kit of aspect 35, wherein the in vivo fluorescence assay is a fluorescence polarization assay.

[0194] Aspect 38. The kit of aspect 35, wherein the in vitro fluorescence assay is a homogeneous time resolved fluorescence (HTRF) assay.

[0195] Aspect 39. The kit of aspect 35, wherein the in vitro fluorescence assay is a fluorescence polarization assay.

[0196] Aspect 40. The kit of any one of aspects 35-39, wherein the kit further comprises an hCAR co-activator.

[0197] Aspect 41. The kit of aspect 40, wherein the hCAR co-activator comprises a Cy5- PGC1α peptide, a Cy5-SRC peptide, a PGC1α peptide, or a combination thereof.

[0198] Aspect 42. The kit of any one of aspects 35-39, wherein the kit comprises instructions for carrying out an in vitro fluorescence assay; and further comprises the at least one polypeptide that binds a moiety of the least one compound of any of aspects 1-19 having a structure given by the formula .

[0199] Aspect 43. Theassay or the in vitro fluorescence assay is a time-resolved fluorescence energy transfer (TR-FRET) assay.

[0200] Aspect 44. The kit of aspect 42 or aspect 43, wherein the kit further comprises a fluorescent donor molecule comprising a fluorescence donor molecule bound to one or more moieties capable of binding the at least one polypeptide.

[0201] Aspect 45. The kit of aspect 44, wherein the fluorescence donor molecule is a chelated terbium.

[0202] Aspect 46. The kit of aspect 45, wherein the chelated terbium comprises at least one diethylenetriaminepentaacetic acid residue and Tb+3.

[0203] Aspect 47. The kit of any one of aspects 44-46, wherein the one or more moieties capable of binding the at least one polypeptide comprises an antibody.

[0204] Aspect 48. The kit of aspect 47, wherein the antibody recognizes an epitope on the atATTORNEY DOCKET NO.331904-2010 least one polypeptide.

[0205] Aspect 49. The kit of aspect 48, wherein the epitope on the at least one polypeptide comprises one or more affinity tags or epitope tags.

[0206] Aspect 50. The kit of aspect 49, wherein the affinity tag is selected from histidine tag, maltose binding protein tag, chitin binding protein tag, and glutathione-S-transferase tag.

[0207] Aspect 51. The kit of aspect 49, wherein the epitope tag is selected from T7 tag, FLAG tag, HA tag, VSV-G tag, V5 tag, and c-myc tag.

[0208] Aspect 52. The kit of aspect 48, wherein the at least one polypeptide epitope is a native sequence of the at least one polypeptide.

[0209] From the foregoing, it will be seen that aspects herein are well adapted to attain all the ends and objects hereinabove set forth together with other advantages which are obvious and which are inherent to the structure.

[0210] While specific elements and steps are discussed in connection to one another, it is understood that any element and / or steps provided herein is contemplated as being combinable with any other elements and / or steps regardless of explicit provision of the same while still being within the scope provided herein.

[0211] It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.

[0212] Since many possible aspects may be made without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings and detailed description is to be interpreted as illustrative and not in a limiting sense.

[0213] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0214] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure. I. EXAMPLES

[0215] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to beATTORNEY DOCKET NO.331904-2010 purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ^C or is at ambient temperature, and pressure is at or near atmospheric. 1. BODIPY FL SNS 032 AS A VERSATILE PROBE FOR CONSTITUTIVE ANDROSTANE RECEPTOR AND MULTIPLE KINASES

[0216] Herein, in an effort to identify ligands suitable for developing hCAR probe, a homogeneous time resolved fluorescence (HTRF) hCAR coactivator recruitment assay was used, with a Cy5-PGC1α peptide as the fluorescent coactivator peptide. It was found that SNS-032 (8 in FIG.1, maximal hCAR inhibition: 26.4 ± 4.5% at 90 µM, an IC50value could not be calculated due to its low activity) (FIG. 2A and Table 1) acts as a weak hCAR inverse agonist. However, conjugation of a bulky group (Thalidomide PEG3) at the SNS-032’s terminal piperazine nitrogen generated THAL-SNS-032 (9) (FIG. 2B) which is a more potent hCAR inverse agonist (maximal hCAR inhibition: 75.4 ± 3.4% at 90 µM and IC50of 6.6 ± 0.9 µM) (FIG.2A and Table 1). hCAR activity for SNS-032 (8) and THAL-SNS-032 (9) has not been reported before. Replacing the bulky Thalidomide PEG3 group of the THAL-SNS-032 (9) with a BODIPY FL group to generate BODIPY FL SNS 032 (10) (FIG.2B) can provide a high affinity fluorescent ligand for hCAR.

[0217] After BODIPY FL SNS 032 (10) was prepared, the HTRF hCAR coactivator recruitment assay was used (with Cy5-PGC1α) (FIG.2A and Table 1) to test its activity, with CITCO as the agonistic control (observed EC50value of 3.6 ± 0.4 nM) and CINPA1 as the inverse agonistic control (observed maximal inhibition: 100.0 ± 1.9% at 30 µM and IC50value of 258.0 ± 11.0 nM). Indeed, BODIPY FL SNS 032 (10) displayed more potent inverse agonistic activityATTORNEY DOCKET NO.331904-2010 (10, maximal inhibition: 95.9 ± 1.6%, IC50of 224.2 ± 18.9 nM) than that of THAL-SNS-032 (9) (FIG. 2A and Table 1). In the HTRF assay, androstenol (12) was an hCAR inverse agonist (IC50of 249.8 nM, Table 1), consistent with published data.41Table 1. hCAR HTRF coactivator recruitment and TR-FRET binding assay results of selected hCAR ligands. Ligand hCAR Coactivator Recruitment hCAR Binding CITCO (1) Max: *100.0 ± 1.1% at 3 µM, 3.6 ± 0.4 nM (EC50) 2.6 ± 0.4 nM (IC50) CINPA1 (2) Max:#-100.0 ± 1.9% at 30 µM, 258.0 ± 11.0 nM (IC50) 162.2 ± 9.0 nM (IC50) Androstenol (12) Max: −84.7% ± 4.0% at 100 μM, 249.8 ± 7.2 nM (IC50) 338.7 ± 9.8 nM (IC50) SNS-032 (8) Max: -26.4 ± 4.5% at 90 µM max: 50.8 ± 4.4% at 100 µM, 51.1 ± 2.8 µM (&half max) THAL-SNS-032 Max: -75.4 ± 3.4% at 90 µM, 6.6 ±± 0.5 (9) 0.9 µM (IC50) µM (half max) BODIPY FL SNS Max: -95.9 ± 1.6% at 3.3 µM, 032 (10) 224.2 ± 18.9 nM (IC50) Not Tested 72 (3) Not Tested 12.8 ± 0.7 nM (IC50) T0901317 (4) Not Tested 650.8 ± 70.7 nM (IC50) PK11195 (5) Not Tested 55.8 ± 2.4 nM (IC50) Clotrimazole (6) Not Tested 32.6 ± 0.9 nM (IC50) S07662 (7) Not Tested 260.0 ± 11.6 nM (IC50) *Positive value means hCAR activation. #Negative value means hCAR inhibition. &A half maximum was calculated for comparison as a reliable IC50could not be calculated.

[0218] BODIPY FL SNS 032 (10, 2 µM) was evaluated using KINOMEscan21against a panel of 468 kinases (DiscoveRx Corporation). Similar to the reported SNS-032 profiling data,22, 23,24GSK3A, CDKL15, CDK7, and CDK9 were also identified as top hits for the BODIPY FL SNS 032 (10) (see Table 14). Using the KINOMEscan result, 6 kinases were selected to represent high-to-low inhibition by BODIPY FL SNS 032 (10) for further evaluation in a TR-FRET binding assay: GSK3A, CDK9, MAPK15, CASK, CAMKK2, and DYRK2 (Table 2, highlighted in bold). Among the top hits, GSK3A was selected because it was the most inhibited kinase by BODIPY FL SNS 032 (10) (see Table 14). CDK9 was selected because it is an intended target of parental compounds SNS-032 (8) and THAL-SNS-032 (9) of BODIPY FL SNS 032 (10). Table 2. Top *41 KINOMEscan hits against BODIPY FL SNS 032. KINOMEscan DiscoveRx Entrez Gene**KINOMEscan TR-FRET Kdin Rank Gene Symbol Symbol Percent Control this reportATTORNEY DOCKET NO.331904-2010#1 GSK3A GSK3A 2.3 4.5 ± 0.2 nM 2 NEK6 NEK6 7.1 Not Tested 3 CDC2L5 CDK13 12 Not Tested 4 ICK ICK 12 Not Tested 5 CDKL5 CDKL5 18 Not Tested 6 CSNK1E CSNK1E 20 Not Tested 7 GSK3B GSK3B 21 Not Tested 8 DYRK1B DYRK1B 23 Not Tested 9 CDK9 CDK9 24 5.1 ± 0.6 nM 10 CSNK1D CSNK1D 29 Not Tested 11 ERK8 MAPK15 31 339.9 ± 17.4 nM 12 CSNK1A1 CSNK1A1 33 Not Tested 13 CDK7 CDK7 35 Not Tested 14 DYRK1A DYRK1A 42 Not Tested 15 PCTK2 CDK17 44 Not Tested 16 CLK2 CLK2 46 Not Tested 17 RIOK2 RIOK2 48 Not Tested 18 CASK CASK 55 551.5 ± 25.3 nM 19 CTK MATK 55 Not Tested 20 NIK MAP3K14 56 Not Tested 21 YSK4 MAP3K19 56 Not Tested 22 LKB1 STK11 57 Not Tested 23 RIPK5 DSTYK 58 Not Tested 24 MTOR MTOR 62 Not Tested 25 BTK BTK 63 Not Tested 26 CDK4- cyclinD1 CDK4 63 Not Tested 27 MAP3K15 MAP3K15 63 Not Tested 28 CDKL2 CDKL2 64 Not Tested 29 BMPR1B BMPR1B 65 Not Tested 30 BMX BMX 65 Not Tested 31 CSF1R- autoinhibited CSF1R 65 Not Tested 32 STK39 STK39 65 Not Tested 33 ADCK3 CABC1 66 Not Tested 34 CAMKK2 CAMKK2 66 533.2 ± 39.7 nM 35 IRAK3 IRAK3 66 Not Tested 36 PCTK3 CDK18 67 Not Tested 37 CSNK2A1 CSNK2A1 69 Not TestedATTORNEY DOCKET NO.331904-2010 38 GCN2(Kin.Do m.2,S808G) EIF2AK4 69 Not Tested HUNK 69 Not Tested STK17B 70 Not TestedDYRK2 70 Not Active *Top 41 KINOMEscan hits (**KINOMEscan Percent Control ≤ 70%; the lower in percentage, the better inhibition against the kinase). #Bold Highlighted: Saturation TR-FRET binding assays evaluated in this report. Note that hDYRK2 failed to bind BODIPY FL SNS 032.

[0219] The potent hCAR inverse agonistic activity and multiple kinase inhibitory activities of BODIPY FL SNS 032 (10) prompted the use of TR-FRET assays to assess its direct binding activity to hCAR and the 6 selected kinases (FIGS.3A-3F and Table 2). Saturation TR-FRET binging experiments were performed in which the optimized dilutions of BODIPY FL SNS 032 (10) were incubated with Tb-anti-tag antibody and corresponding tagged-protein in the presence of DMSO (Group 1) to reveal the binding of BODIPY FL SNS 032 (10) to its protein target from which the corresponding binding dissociation constants (Kd) were derived (lines connecting solid circles, FIGS.3A-3F; Group 1). In FIGS.3A-3F, the TR-FRET signals at a 90-min incubation time are shown. 90-min incubation time was previously shown to be sufficient.30-36Data from the TR-FRET assay displayed the various binding affinity of BODIPY FL SNS 032 (10) to different targets, and for the kinases, consistent with the KINOMEscan data (Table 2). hDYRK2 failed to bind BODIPY FL SNS 032 (Table 2).

[0220] When BODIPY FL SNS 032 (10) was incubated with Tb-anti-tag antibody without the target protein in the presence of DMSO (pink curves; Group 3), the assay signals were contributed by the background interaction between BODIPY FL SNS 032 (10) and the Tb-anti- tag antibody. The assay signals from BODIPY FL SNS 032 (10) incubated with Tb-anti-tag antibody and corresponding tagged-protein in the presence of a target specific ligand (lines connecting solid squares, FIGS. 3A-3F; Group 2) were contributed from non-specific interactions between the BODIPY FL SNS 032 (10) and the tagged protein and the Tb-anti- tag antibody. The specific ligand CITCO,9SNS-032,24SNS-032,25Ro 31-8220,26, 27NR 162,28and CZC-2514629were chosen for hCAR, hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK, and hCAMKK2, respectively. A marginal difference between the background interaction (FIGS.3A- 3F, lines connecting solid diamonds) and the non-specific interactions (FIGS. 3A-3F, lines connecting solid squares) indicated that the non-specific interaction is almost non-existing. In

[0221] Once BODIPY FL SNS 032 (10) was confirmed to bind to hCAR and hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK, and hCAMKK2, BODIPY FL SNS 032 (10) was investigated for its use as a fluorescent probe in a TR-FRET assay to evaluate competitive ligand binding of compounds to these proteins.

[0222] To determine an optimal BODIPY FL SNS 032 (10) concentration for each target, theATTORNEY DOCKET NO.331904-2010 TR-FRET signal fold change of Group 1 curve / Group 2 curve was calculated (used for further assay development), along with the signal fold change of Group 1 curve / Group 3 curve (as a reference), for the saturation binging experiments (FIGS.3A-F) in order to gain insight into the most sensitive BODIPY FL SNS 032 (10) concentration for each target. The normalized TR- FRET signal fold change curves were bell-shaped for all targets (FIGS.4A-F) with the optimal or most sensitive BODIPY FL SNS 032 (10) concentrations corresponding to the peak of the curves (indicated with a vertical “black solid line” for each assay). In comparison, the Kdconcentration of each target was labeled with a vertical “green broken line” for each target, along with its corresponding signal fold change.

[0223] The Kdvalue was used to guide selection of probe concentration, taking into consideration the signal window (the signal fold change) generated by the probe and the need to characterize weak ligands (therefore a lower probe concentration might be needed). To start, BODIPY FL SNS 032 (10, 125 nM) was chosen for hCAR (9.7-fold), hMAPK15 (4.7- fold), hCASK (3.7-fold), and hCAMKK2 (3.0-fold) (FIGS. 4A and 4D-4F) because highest signal fold changes were observed at 125 nM BODIPY FL SNS 032 (10), although it is lower than its Kdconcentration. For hGSK3A (FIG.4B), the highest signal fold change (26.9-fold) was observed with the BODIPY FL SNS 032 (10) concentration at 3.9 nM which was similar to the signal fold change observed at its Kdconcentration (4.5 nM). For hCDK9 / CycT1 (FIG. 4C), the highest signal fold change (18.1-fold) was observed with the BODIPY FL SNS 032 (10) concentration at 15.6 nM which was close to the signal fold change (15.4-fold) observed at its Kdconcentration (5.1 nM). The Kdconcentrations of BODIPY FL SNS 032 (10) for hGSK3A (4.5 nM) and hCDK9 / CycT1 (5.1 nM) were selected.

[0224] Experiments were then performed to test if the selected probe concentration can deliver reasonable assay sensitivities. A similar 3 groups of samples were prepared for each target, as in the binding affinity (Kd) determination experiments, but only with the one selected BODIPY FL SNS 032 (10) concentration. Group 1: Tb-anti-tag antibody, corresponding tagged-protein and DMSO; Group 2. Tb-anti-tag antibody and corresponding tagged-protein in the presence of corresponding positive control ligand (with an optimized concentration) for the protein; or Group 3: Tb-anti-tag antibody and DMSO, with the selected BODIPY FL SNS 032 (10) concentration included for all the 3 groups (FIGS.4G-4L). After the signals of the 3 groups were normalized to that of Group 2 for each target, reasonable signal fold change between the negative control group (Group 1) and the positive control group (Group 2) was observed, 6.5 ± 0.3-fold, 14.2 ± 0.1-fold, 14.9 ± 0.9-fold, 4.7 ± 0.1-fold, 3.1 ± 0.1-fold, and 2.6 ± 0.1-fold for hCAR, hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK, and hCAMKK2, respectively (FIG. 4M-4R). This validated the selection of BODIPY FL SNS 032 (10) concentration for each target.

[0225] The established TR-FRET binding assay conditions to characterize inhibitory activities of ligands against a given target are summarized in Table 3 with all assay incubation timesATTORNEY DOCKET NO.331904-2010 set at 90-min. Table 3. TR-FRET binding assay conditions for hCAR, hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK and hCAMKK2 to characterize inhibitory activities of their ligands. TR-FRET Assay Protein BODIPY FL BMS Positive Negative Components 032 (10) Control Control 2 nM Tb-anti-GST, hCAR Binding 3 µM 2 nM GST-hCAR- 125 nM CITCO DMSO LBD 2 nM Tb-anti-GST, hGSK3A Binding 10 µM SNS 2 nM GST- 4.5 nM - 032 DMSO hGSK3A 2 nM Tb-anti-His, hCDK9 / CycT1B 2 nM Hi 5.1 nM 1 µ S- inding s- 032 DMSO CDK9 / CycT1 2 nM Tb-anti-His,hMAPK15 125 n 1 µM Ro 31- Binding 2 nM Flag- M 8220 DMSO hMARPK15-His 2 nM Tb-anti-GST,hCASK Binding 125 nM NR 2 nM GST 162 DMSO -hCASK hCAMKK2Binding 2 nM GST- 125 nM 30 µM CZC- 25146 DMSO hCAMKK2

[0226] With the BODIPY FL SNS 032 (10) TR-FRET binding assays established for hCAR, hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK, and hCAMKK2, a panel of reported ligands for each target were selected and tested.

[0227] First, ligands were tested for hCAR. CITCO, CINPA1, 72, T0901317, PK11195, Clotrimazole, S07662, SNS-032, and THAL-SNS-032 were chosen as the hCAR ligands and tested for their binding inhibitory activities with the BODIPY FL SNS 032 (10) as the fluorescent probe in the established hCAR TR-FRET binding assay. CINPA1, 72, T0901317, PK11195, Clotrimazole, and S07662 achieved 100% inhibition at their respective high concentrations with corresponding IC50values of 162.2 ± 9.0 nM, 12.8 ± 0.7 nM, 650.8 ± 70.7 nM, 55.8 ± 2.4 nM, 32.6 ± 0.9 nM, and 260.0 ± 11.6 nM, compared to the positive control CITCO (3 µM, 100% inhibition, IC50of 2.6 ± 0.4 nM) (Table 1 and FIG.5A). Although SNS-032 and THAL-SNS-032 could not achieve a complete inhibition within their concentration range tested (up to 100 µM), with respective half max values of 3.1 ± 0.5 µM and 51.1 ± 2.8 µM, THAL-SNS-032 was more potent than SNS-032 as expected (FIG. 5A). For ligands tested by both the Cy5-PGC1α peptide-based hCAR HTRF coactivator recruitment assay and the BODIPY FL SNS 032 (10)- based hCAR TR-FRET binding assay, similar binding activity rank order was observed (Table 1).ATTORNEY DOCKET NO.331904-2010

[0228] A panel of representative kinase inhibitors was then selected: SNS-032, THAL-SNS- 032, Ro 31-8220, NR 162, CZC-25146, Staurosporine, Flavopiridol, Atuveciclib, Fadraciclib, and Riviciclib, to characterize the established BODIPY FL SNS 032 (10)-based TR-FRET binding assays against hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK, and hCAMKK2 under an optimized concentration range for each inhibitor against each kinase. FIGS.5B-5F show the dose response inhibition curves and Table 4 and Table 13 summarize the inhibitory IC50values. SNS-032 was selected because it’s a potent inhibitor against hGSK3A and hCDK9 / CycT124and served as a positive control for the 2 kinases. THAL-SNS-032 was included because it’s a conjugate of SNS-032 and maintained potent inhibitory activities for multiple kinases.22Most importantly, it was based on to develop BODIPY FL SNS 032 (10) as a high affinity hCAR fluorescent probe. Ro 31-8220,26, 27NR 162,28and CZC-2514629were selected because of their potent inhibitory activity against hMAPK15, hCASK, and hCAMKK2, respectively, and served as corresponding controls for the 3 kinases. Additional kinase inhibitors Staurosporine, Flavopiridol, Atuveciclib, Fadraciclib, and Riviciclib were chosen because of their promiscuous kinase inhibition nature.24, 37In this example, with the BODIPY FL SNS 032 (10) as the fluorescent probe under established TR-FRET assay conditions, SNS- 032 had IC50values of 21.2 nM, 2.2 nM, 59.1 nM, 224.4 nM and 4.3 µM against hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK, and hCAMKK2 (Table 4), respectively. Compared to reported corresponding KINOMEscan Kdvalues of 28 nM, 76 nM, 126 nM, 400 nM, and > 10 µM for hGSK3A, hCDK9, hMAPK15, hCASK and hCAMKK2,24the general activity rank order was maintained. SNS-032 displayed more potent inhibitory activity against hCDK9 / CycT1 than that against hGSK3A, likely because of the presence of the CycT1. THAL-SNS-032 had corresponding IC50values of 8.2 nM, 5.6 nM, 79.1 nM, 439.7 nM, and 2.7 µM against hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK and hCAMKK2, respectively (Table 4). Ro 31-8220 had a reported IC50value of 5 to 10 nM against MAPK15 in a phosphorylation assay.26, 27In the BODIPY FL SNS 032 (10)-based TR-FRET kinase binding assays, Ro 31-8220 had an IC50value of 0.9 nM against hMAPK15, along with IC50values of 0.7 nM, 1.0 nM, 1.2 µM and 161.4 nM against hGSK3A, hCDK9 / CycT1, hCASK and hCAMKK2, respectively (Table 4). NR 162 was a designed hCASK inhibitor with high selectivity and potency.28In the BODIPY FL SNS 032 (10)-based TR-FRET kinase binding assays, NR 162 was found to be a potent and selective hCASK inhibitor with an IC50 value of 8.1 nM and without significant inhibitory activities against hGSK3A, hCDK9 / CycT1, hMAPK15, and hCAMKK2 (Table 4). Through a screening approach, CZC-25146 was identified as a potent hCAMKK2 inhibitor with high selectivity.29In the BODIPY FL SNS 032 (10)-based TR-FRET kinase binding assays, CZC- 25146 was a potent hCAMKK2 inhibitor with an IC50value of 51.6 nM (Table 4). It displayed weak inhibitions against hGSK3A (IC50of 20.2 µM), hMAPK15 (IC50of 2.8 µM), and hCASK (maximal inhibition of 40.7% at 30 µM) and no significant inhibition against hCDK9 / CycT1. In the BODIPY FL SNS 032 (10)-based TR-FRET kinase binding assays, Staurosporine andATTORNEY DOCKET NO.331904-2010 Flavopiridol had respective IC50values of 2.1 nM, 3.3 nM, 2.6 nM, 6.3 nM, 0.3 nM and 163.8 nM, 1.4 nM, 163.7 nM, not active, 296.4 nM for hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK, and hCAMKK2, respectively. Compared to the reported KINOMEscan corresponding Kdvalues 140 nM, 100 nM, 35 nM, 19 nM, 0.16 nM and 1300 nM, 6.4 nM, 330 nM, > 10 µM, 430 nM,24the BODIPY FL SNS 032 (10)-based TR-FRET kinase binding assays may have higher assay sensitivity. Additional promiscuous kinase inhibitors Atuveciclib, Fadraciclib, and Riviciclib37were also evaluated with the BODIPY FL SNS 032 (10)-based TR-FRET kinase binding assays with expected inhibitory activities (IC50values) observed (Table 4) except for Atuveciclib against hCASK with low inhibitory activity (maximal inhibition of 27.3% at 90 µM) observed, mostly due to Atuveciclib’s inherent low hCASK inhibition nature. Table 4. Inhibitory activities (IC50values) of a panel of selected kinase inhibitors obtained from the BODIPY FL SNS 032 (10)-based TR-FRET binding assays against hGSK3, hCDK9 / CycT1, hMAPK15, hCASK and hCAMKK2. Kinase Inhibitor hGSK3A hCDK9 / CycT1 hMAPK15 hCASK hCAMKK2 SNS-032 21.2 ± 1.7 nM 2.2 ± 0.1 nM 59.1 ± 3.9 nM 224.4 ± 15.2 4.3 ± 0.2 nM µM THAL-SNS- 439.7 ± 22.4 2.7 ± 0.1 032 8.2 ± 0.8 nM 5.6 ± 0.2 nM 79.1 ± 3.9 nM nM µM 1.2 ± 0.1 Ro 31-8220 0.7 ± 0.03 nM 1.0 ± 0.1 nM 0.9 ± 0.1 nM ± 3.3 µM8.1 ± 0.5 NR 162 NA* NA* NA* NA* nM 40.7 ± CZC-25146 20.2 ± 1.6 µM NA* 2.8 ± 0.2 µM 2.9%@30 51.6 ± 5.7 µM#nM 6.3 ± 0.5 Staurosporine 2.1 ± 0.1 nM 3.3 ± 0.1 nM 2.6 ± 0.1 nM 0.3 ± 0.04 nM nM Flavopiridol 163.8 ± 16.4 nM 1.4 ± 0.1 nM 163.7 ± 16.4 nM NA* 296.4 ± 17.4 nM 27.3 ± Atuveciclib 29.1 ± 2.6 nM 6.8 ± 0.9 nM 2.6 ± 0.1 µM 3.6%@90 1.2 ± 0.1 µM#µM Fadraciclib 11.1 ± 0.9 µM 13.4 ± 1.0 nM 385.9 ± 21.1 nM 20.4 ± 2.2 13.2 ± 0.8Riviciclib 269.6 ± 11.8 nM 0.6 ± 0.06 nM 706.2 ± 43.1 nM 13.2 ± 1.1 6.5 ± 0.2 nM µM *Not Active (maximal inhibitory activity less than 20%) #The maximal %Inhibition was reported along with the corresponding concentration when the maximal %Inhibition < 50%

[0229] In conclusion, BODIPY FL SNS 032 (10) proved to be a high affinity hCAR fluorescent probe (Kdof 298.2 ± 26.2 nM) and was successfully applied in a TR-FRET assay toATTORNEY DOCKET NO.331904-2010 characterize hCAR ligands for their binding inhibitory activities. BODIPY FL SNS 032 (10) is the first hCAR small molecule fluorescent probe. Additionally, due to the promiscuous kinase inhibition nature of SNS-032, BODIPY FL SNS 032 (10) displayed high binding affinities to hGSK3A (Kd of 4.5 ± 0.2 nM), hCDK9 / CycT1 (Kd of 5.1 ± 0.6 nM), hMAPK15 (Kd of 339.9 ± 17.4 nM), hCASK (Kdof 551.5 ± 25.3 nM), and hCAMKK2 (Kdof 533.2 ± 39.7 nM) and was successfully used in TR-FRET assays to characterized activities of inhibitors against these kinases with the potential to be a fluorescent probe for additional kinases. 2. EXPERIMENTAL PROCEDURES FOR BODIPY FL SNS 032 SYNTHESIS AND ANALYSIS

[0230] The synthesis of BODIPY FL SNS 032 (10) is summarized in Scheme 1 with a 1-Ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDCI)-mediated coupling reaction.38In the presence of EDCI, SNS-032 (8, a secondary amine) was reacted to BODIPDY FL carboxylic acid (11, an acid) for 12 h at room temperature (R.T.) with dichloromethane (DCM) as the solvent to generate BODIPY FL SNS 032 (10, yield: 70.9%; purity: 98.4%).

[0231] Chemistry. SNS 032, BODIPY FL carboxylic acid, 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI), dichloromethane (DCM) and all relevant chemical reagents were purchased from LabNetwork. Methanol-d4was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). Reported protocols8were followed to monitor or determine reaction progress, product purity, and product identity; to record1H and13C NMR spectra (FIG.6 and FIG.7, respectively); and to determine high resolution mass spectra (FIG. 8).

[0232] N-(5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)-1-(3-(5,5-difluoro-7,9- dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)propanoyl)piperidine-4- carboxamide (10, BODIPY FL SNS 032). Under room temperature (ca.25 °C), 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI, 302 mg, 1.58 mmol, 2.00 eq) was added into aATTORNEY DOCKET NO.331904-2010 solution of SNS 032 (8, 230 mg, 788 μmol, 1.00 eq) and BODIPY FL carboxylic acid (11, 300 mg, 788 μmol, 1.00 eq) in 2 mL dichloromethane (DCM). The reaction mixture was stirred under room temperature for 12 hr. UPLC-MS monitoring demonstrated the desirable product BODIPY FL SNS 032 (10) was the predominant component in the reaction with minimal starting materials remaining. The reaction mixture was concentrated under reduced pressure. The obtained crude product was purified by reversed-phase HPLC (FIGS. 9A-9B) to yield BODIPY FL SNS 032 (10, 370 mg, 559 μmol, 70.9% yield, 98.4% purity) as an orange solid.1H NMR (400 MHz, Methanol-d4) δ 1.24 (s, 9 H) 1.52-1.71 (m, 2 H) 1.79-1.95 (m, 2 H) 2.28 (s, 3 H) 2.51 (s, 3 H) 2.66-2.92 (m, 4 H) 3.09- 3.18 (m, 1 H) 3.18-3.26 (m, 2 H) 3.99 (s, 2 H) 4.06 (br d, J = 13.88 Hz, 1 H) 4.54 (br d, J = 13.26 Hz, 1 H) 6.21 (s, 1 H) 6.34 (d, J = 3.88 Hz, 1 H) 6.69 (s, 1 H) 7.01 (d, J = 4.00 Hz, 1 H) 7.32 (s, 1 H) 7.43 (s, 1 H).13C NMR (100 MHz, Methanol-d4) δ 173.32, 171.29, 162.12, 161.89, 160.19, 160.04, 159.90, 156.90, 145.17, 144.51, 128.25, 124.42, 120.00, 119.99, 119.67, 119.15, 44.78, 41.75, 40.89, 33.72, 32.13, 31.05, 28.35, 27.77, 27.46, 24.35, 13.47, 9.78. ESI-TOF HRMS m / z: [M + H]+Calcd for C31H38BF2N6O3S2+655.2502; Found 655.2501. [M + H - HF]+Calcd for C31H37BFN6O3S2+635.2440; Found 635.2441.

[0233] Biology. CITCO, S07662, THAL-SNS-032 and NR 162 were purchased from Bio- Techne Corporation (Minneapolis, MN). T0901317 and PK11195 were purchased from Cayman Chemical Company (Ann Arbor, MI). SNS-032 was purchased from MedKoo Biosciences, Inc. (Durham, NC). Ro 31-8220, CZC-25146, GSK-626616, fadraciclib, flavopiridol, atuveciclib and riviciclib were purchased from MedChemExpress USA (Monmouth Junction, NJ). Staurosporine was purchased from LC Laboratories (Woburn, MA). Clotrimazole, Androstenol, DTT, Triton X-100, KCl (1 M) and BSA (30%) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Tb-anti-GST, Tb-anti-His, GST-hCAR-LBD, GST- hGSK3A, His-CDK9 / CycT1, Flag-hMARPK15-His, GST-hCASK, GST-hCAMKK2, GST- hDYRK2, DMSO, Tris pH 7.5 (1 M), Pluronic F-68 (10%) and Pluronic F-127 (10%) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Distilled water to make all assay buffers was purchased from Research Products International (Mount Prospect, IL).384-well Echo LDV plates were purchased from Beckman Coulter, Inc. (Indianapolis, IN). Black 384- well shallow assay plates were purchased from Revvity (Waltham, MA). CINPA1 and 72 were prepared in house.2Cy5-PGC1α (Cy5-EAEEPSLLKKLLLAPANTQ-NH2) was synthesized by the Hartwell Center Macromolecular Synthesis Section at St. Jude Children's Hospital.

[0234] Assay Buffer Compositions. CAR buffer for hCAR HTRF coactivator recruitment assay and hCAR TR-FRET binding assay: 50 mM Tris pH 7.5, 0.1% Pluronic F-68, 5 mM DTT and 0.01% BSA.

[0235] Kinase buffer 1 for TR-FRET hGSK3A and hCDK9 / CycT1 binding assay: 50 mM Tris pH 7.5, 0.01% Triton X-100, 1 mM DTT and 0.01% BSA.

[0236] Kinase buffer 2 for TR-FRET hMAPK15, hCASK and hCAMKK2 binding assay: 50 mMATTORNEY DOCKET NO.331904-2010 Tris pH 7.5, 0.1% Pluronic F-127, 100 mM KCl, 1 mM DTT and 0.01% BSA.

[0237] General Method. All assays were performed at room temperature (approximately 25°C) in black 384-well shallow assay plates using an assay volume of 15 μL per well and with an assay buffer composition specified for each assay which is always freshly prepared from commercial reagents before each assay.

[0238] The reagents were always dispensed in the below sequence to each assay wells: Step 1. Buffer with DMSO or buffer with appropriate probe was first dispensed at 7.5 μL per well (contributing 0.1% DMSO to the final assay mixture). Step 2. An Echo 655T Acoustic Liquid Handler (Beckman Coulter, Inc., Indianapolis, IN) dispensed appropriate chemical DMSO stock or DMSO at 15 nL / well (contributing 0.1% DMSO to the final assay mixture). Step 3. Appropriate protein mixture (7.5 μL per well) was dispensed last.

[0239] Once all assay components were dispensed, the assay plates were shaken for 1 min on an IKA MTS 2 / 4 digital microtiter plate shaker (IKA Works, Wilmington, NC) at 900 RPM to ensure that all assay components were thoroughly mixed. The assay plates were then briefly centrifuged at 201×g (1000 rpm) for 30 s in an Eppendorf 5810 centrifuge with an A-4-62 swing-bucket rotor (Eppendorf AG, Hamburg, Germany). The assay plates were next incubated for 90 min, protected from light exposure. The HTRF or TR-FRET signal for each assay well was determined with a PHERAstar FS plate reader (BMG Labtech, Durham, NC) to measure the fluorescence emission ratio (10,000 × 665 nm / 620 nm for HTRF signals or 10,000 × 520 nm / 490 nm for TR-FRET signals), using a 340-nm excitation filter, a 100-μs delay, and a 200-μs integration time. The TR-FRET signals were measured every 30 minutes from 30 to 300 minutes.

[0240] DMSO Concentration. The final DMSO concentration was 0.2% in all assays with 0.1% from fluorescent probes and 0.1% from either DMSO or tested ligand DMSO stocks.

[0241] Data Analysis. In the BODIPY FL SNS 032 fluorescent probe binding dissociation constant (Kd) determination experiments, raw TR-FRET data for the group 1, group 2 and group 3 (specified in each assay) were graphed (FIG.10A-10J) with the curve-fitting software GraphPad Prism 9.5.1 (GraphPad Software, La Jolla, CA) using the one site-total Nonlinear regression curve fit equation. The binding dissociation constant (Kd) value for the fluorescent probe BODIPY FL SNS 032 against a specific protein was derived from the group 1 data. Group 2 and group 3 data were served as respective non-specific interaction group and background interaction group.

[0242] In the ligand inhibitory activity tests (ligand IC50value determination experiments with BODIPY FL SNS 032 as the fluorescent probe), raw TR-FRET data of tested chemicals were normalized to that of the positive control of each assay and the negative control (DMSO) with Eq.1 to derive %Inhibition: %^^^^ℎ^^^^^^^^^^^^^^ ൌ 100%  െ  100%  ൈ ^ௌ^^^^^^^ೞ^^^ ^^^ೌ^^ିௌ^^^^^ು^ೞ^^^ೡ^ ^^^^^^^^ Eq.1ATTORNEY DOCKET NO.331904-2010

[0243] The normalized %Inhibition values for each ligand were graphed (FIG. 11) with the curve-fitting software GraphPad Prism 9.5.1 using inhibitor versus response (three parameters). Nonlinear regression curve fit equation to generate dose response curves and to determine IC50 values, when applicable.

[0244] The Kivalue for each ligand was then calculated from the corresponding IC50value (Table 12) using the Cheng-Prusoff equation39(Eq. 2) where S is the fluorescent probe BODIPY FL SNS 032 concentration used in each binding assay against hCAR, hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK or hCAMKK2 and Kdis the corresponding binding affinities of the fluorescent probe BODIPY FL SNS 032 to hCAR, hGSK3A, hCDK9 / CycT1, hMAPK15, hCASK and hCAMKK2. ^^ூ^ఱ^ൌబ^ ା ௌ⁄ ^^Eq.2

[0245] All assays were performed in as the mean ± standard deviation (SD) from

[0246] Chemical preparation. The chemicals tested in all assays were solubilized in DMSO to prepare 1,000 × stock solutions in Echo LDV plates at 10 µL / well. An Echo 655T Acoustic Liquid Handler transferred 15 nL corresponding chemical DMSO stock solution to a final 15 µL / well assay volume to make a 1-to-1,000 dilution. The assay specific stock probe, positive control, negative control and ligand concentrations were summarized in Tables 5-11.

[0247] hCAR HTRF coactivator recruitment assay. In the hCAR HTRF coactivator recruitment assay, black 384-well assay plates were first dispensed with Cy5-PGC1α (200 nM with 0.2% DMSO, 7.5 μL / well) in CAR buffer. Dilutions of CITCO, CINPA1, Androstenol, SNS- 032, THAL-SNS-032, BODIPY FL SNS 032, or control CITCO, CINPA1 or DMSO were dispensed (15 nL / well) by an Echo 655T Acoustic Liquid Handler with stock and final ligand concentrations defined in Tables 7-9. Protein mixture of Tb-anti-GST (4 nM) and GST-hCAR- LBD (4 nM) was then dispensed at 7.5 μL / well. After HTRF signal for each well was measured, Eq.3 was employed to calculate %Activity for the CITCO group and Eq.4 was employed to calculate %Activity for the groups of CINPA1, SNS-032, THAL-SNS-032 and BODIPY FL SNS 032. The normalized %Activities for each ligand were plotted with the curve-fitting software GraphPad Prism 9.5.1 using Sigmoidal dose-response fitting equation to derive EC50or IC50, when applicable. %^^^^^^^^^^^^^^^^ ൌ 100%  ൈ ^ௌ^^^^^^^^^ೀିௌ^^^^^ವಾೄೀ^ Eq.3

[0248] TR-against hCAR. Black 384-well assay plates were first dispensed with CAR buffer at 7.5 µL / well. Dilutions of BODIPY FL SNS 032 (15 nL / well), along with DMSO (15 nL / well) or 3 mM CITCO / DMSO (15 nL / well) were dispensed by an Echo 655T Acoustic Liquid Handler. 7.5 µL / well of Tb-anti-GST (4 nM) and GST-hCAR-LBD (4 nM) or Tb-anti-GST (4 nM) along wasATTORNEY DOCKET NO.331904-2010 then dispensed to give 3 groups of samples with final concentrations specified (Table 6) and the final DMSO concentration was 0.2% for all wells with 0.1% from BODIPY FL SNS 032 and 0.1% from stock CITCO in DMSO or DMSO. Group 1. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM), GST-hCAR-LBD (2 nM) and DMSO Group 2. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM), GST-hCAR-LBD (2 nM) and CITCO (3 µM) Group 3. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM) and DMSO

[0249] TR-FRET BODIPY FL SNS 032 binding dissociation constant (Kd) determination against hGSK3A. Black 384-well assay plates were first dispensed with Kinase buffer 1 at 7.5 µL / well. Dilutions of BODIPY FL SNS 032 (15 nL / well), along with DMSO (15 nL / well) or 20 mM SNS-032 / DMSO (15 nL / well) were dispensed by an Echo 655T Acoustic Liquid Handler.7.5 µL / well of Tb-anti-GST (4 nM) and GST-hGSK3A (4 nM) or Tb-anti-GST (4 nM) along was then dispensed to give 3 groups of samples with final concentrations specified (Table 6) and the final DMSO concentration was 0.2% for all wells with 0.1% from BODIPY FL SNS 032 and 0.1% from stock SNS-032 in DMSO or DMSO. Group 1. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM), GST- hGSK3A (2 nM) and DMSO Group 2. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM), GST- hGSK3A (2 nM) and SNS-032 (20 µM) Group 3. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM) and DMSO

[0250] TR-FRET BODIPY FL SNS 032 binding dissociation constant (Kd) determination against hCDK9 / CycT1. Black 384-well assay plates were first dispensed with Kinase buffer 1 at 7.5 µL / well. Dilutions of BODIPY FL SNS 032 (15 nL / well), along with DMSO (15 nL / well) or 20 mM SNS-032 / DMSO (15 nL / well) were dispensed by an Echo 655T Acoustic Liquid Handler.7.5 µL / well of Tb-anti-His (4 nM) and His-hCDK9 / CycT1 (4 nM) or Tb-anti-His (4 nM) along was then dispensed to give 3 groups of samples with final concentrations specified (Table 6) and the final DMSO concentration was 0.2% for all wells with 0.1% from BODIPY FL SNS 032 and 0.1% from stock SNS-032 in DMSO or DMSO. Group 1. Dilutions of BODIPY FL SNS 032 with Tb-anti-His (2 nM), His-hCDK9 / CycT1 (2 nM) and DMSO Group 2. Dilutions of BODIPY FL SNS 032 with Tb-anti-His (2 nM), His-hCDK9 / CycT1 (2 nM) and SNS-032 (20 µM) Group 3. Dilutions of BODIPY FL SNS 032 with Tb-anti-His (2 nM) and DMSO

[0251] TR-FRET BODIPY FL SNS 032 binding dissociation constant (Kd) determination against hMAPK15. Black 384-well assay plates were first dispensed with Kinase buffer 2 at 7.5 µL / well. Dilutions of BODIPY FL SNS 032 (15 nL / well), along with DMSO (15 nL / well) or 2 mM Ro 31-8220 / DMSO (15 nL / well) were dispensed by an Echo 655T Acoustic LiquidATTORNEY DOCKET NO.331904-2010 Handler.7.5 µL / well of Tb-anti-His (4 nM) and Flag-hMARPK15-His (4 nM) or Tb-anti-His (4 nM) along was then dispensed to give 3 groups of samples with final concentrations specified (Table 6) and the final DMSO concentration was 0.2% for all wells with 0.1% from BODIPY FL SNS 032 and 0.1% from stock Ro 31-8220 in DMSO or DMSO. Group 1. Dilutions of BODIPY FL SNS 032 with Tb-anti-His (2 nM), Flag-hMARPK15-His (2 nM) and DMSO Group 2. Dilutions of BODIPY FL SNS 032 with Tb-anti-His (2 nM), Flag-hMARPK15-His (2 nM) and Ro 31-8220 (2 µM) Group 3. Dilutions of BODIPY FL SNS 032 with Tb-anti-His (2 nM) and DMSO

[0252] TR-FRET BODIPY FL SNS 032 binding dissociation constant (Kd) determination against hCASK. Black 384-well assay plates were first dispensed with Kinase buffer 2 at 7.5 µL / well. Dilutions of BODIPY FL SNS 032 (15 nL / well), along with DMSO (15 nL / well) or 5 mM NR 162 / DMSO (15 nL / well) were dispensed by an Echo 655T Acoustic Liquid Handler.7.5 µL / well of Tb-anti-GST (4 nM) and GST-hCASK (4 nM) or Tb-anti-GST (4 nM) along was then dispensed to give 3 groups of samples with final concentrations specified (Table 6) and the final DMSO concentration was 0.2% for all wells with 0.1% from BODIPY FL SNS 032 and 0.1% from stock NR 162 in DMSO or DMSO. Group 1. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM), GST- hCASK (2 nM) and DMSO Group 2. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM), GST- hCASK (2 nM) and NR 162 (5 µM) Group 3. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM) and DMSO

[0253] TR-FRET BODIPY FL SNS 032 binding dissociation constant (Kd) determination against hCAMKK2. Black 384-well assay plates were first dispensed with Kinase buffer 2 at 7.5 µL / well. Dilutions of BODIPY FL SNS 032 (15 nL / well), along with DMSO (15 nL / well) or 30 mM CZC-25146 / DMSO (15 nL / well) were dispensed by an Echo 655T Acoustic Liquid Handler.7.5 µL / well of Tb-anti-GST (4 nM) and GST-hCAMKK2 (4 nM) or Tb-anti-GST (4 nM) along was then dispensed to give 3 groups of samples with final concentrations specified (Table 6) and the final DMSO concentration was 0.2% for all wells with 0.1% from BODIPY FL SNS 032 and 0.1% from stock CZC-25146 in DMSO or DMSO. Group 1. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM), GST- hCAMKK2 (2 nM) and DMSO Group 2. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM), GST- hCAMKK2 (2 nM) and CZC-25146 (30 µM) Group 3. Dilutions of BODIPY FL SNS 032 with Tb-anti-GST (2 nM) and DMSO

[0254] TR-FRET hCAR ligand binding inhibitory activity test with BODIPY FL SNS 032 as the fluorescent probe. Black 384-well assay plates were first dispensed with 250 nM BODIPY FL SNS 032 (7.5 µL / well with 0.2% DMSO, 2 ×) in CAR buffer. Dilutions of CITCO,ATTORNEY DOCKET NO.331904-2010 CINPA1, SNS-032, THAL-SNS-032, 72, T0901317, PK11195, Clotrimazole, S07662, Androstenol or CITCO (3 mM / DMSO, positive control) or DMSO (negative control) were dispensed (15 nL / well) by an Echo 655T Acoustic Liquid Handler with stock and final ligand concentrations defined in Tables 7-9. Protein mixture of Tb-anti-GST (4 nM, 2 ×) and GST- hCAR-LBD (4 nM, 2 ×) was then dispensed at 7.5 μL / well.

[0255] TR-FRET hGSK3A ligand binding inhibitory activity test with BODIPY FL SNS 032 as the fluorescent probe. Black 384-well assay plates were first dispensed with 9 nM BODIPY FL SNS 032 (7.5 µL / well with 0.2% DMSO, 2 ×) in Kinase buffer 1. Dilutions of SNS- 032, THAL-SNS-032, Ro 31-8220, NR 162, CZC-25146, Staurosporine, Flavopiridol, Atuveciclib, Fadraciclib, Riviciclib or SNS-032 (10 mM / DMSO, positive control) or DMSO (negative control) were dispensed (15 nL / well) by an Echo 655T Acoustic Liquid Handler with stock and final ligand concentrations defined in Tables 7, 10 and 11. Protein mixture of Tb- anti-GST (4 nM, 2 ×) and GST-hGSK3A (4 nM, 2 ×) was then dispensed at 7.5 μL / well.

[0256] TR-FRET hCDK9 / CycT1 ligand binding inhibitory activity test with BODIPY FL SNS 032 as the fluorescent probe. Black 384-well assay plates were first dispensed with 10.2 nM BODIPY FL SNS 032 (7.5 µL / well with 0.2% DMSO, 2 ×) in Kinase buffer 1. Dilutions of SNS-032, THAL-SNS-032, Ro 31-8220, NR 162, CZC-25146, Staurosporine, Flavopiridol, Atuveciclib, Fadraciclib, Riviciclib or SNS-032 (1 mM / DMSO, positive control) or DMSO (negative control) were dispensed (15 nL / well) by an Echo 655T Acoustic Liquid Handler with stock and final ligand concentrations defined in Tables 7, 10 and 11. Protein mixture of Tb- anti-His (4 nM, 2 ×) and His-CDK9 / CycT1 (4 nM, 2 ×) was then dispensed at 7.5 μL / well.

[0257] TR-FRET hMAPK15 ligand binding inhibitory activity test with BODIPY FL SNS 032 as the fluorescent probe. Black 384-well assay plates were first dispensed with 250 nM BODIPY FL SNS 032 (7.5 µL / well with 0.2% DMSO, 2 ×) in Kinase buffer 1. Dilutions of SNS- 032, THAL-SNS-032, Ro 31-8220, NR 162, CZC-25146, Staurosporine, Flavopiridol, Atuveciclib, Fadraciclib, Riviciclib or Ro 31-8220 (1 mM / DMSO, positive control) or DMSO (negative control) were dispensed (15 nL / well) by an Echo 655T Acoustic Liquid Handler with stock and final ligand concentrations defined in Tables 7, 10 and 11. Protein mixture of Tb- anti-His (4 nM, 2 ×) and Flag-hMARPK15-His (4 nM, 2 ×) was then dispensed at 7.5 μL / well.

[0258] TR-FRET hCASK ligand binding inhibitory activity test with BODIPY FL SNS 032 as the fluorescent probe. Black 384-well assay plates were first dispensed with 250 nM BODIPY FL SNS 032 (7.5 µL / well with 0.2% DMSO, 2 ×) in Kinase buffer 1. Dilutions of SNS- 032, THAL-SNS-032, Ro 31-8220, NR 162, CZC-25146, Staurosporine, Flavopiridol, Atuveciclib, Fadraciclib, Riviciclib or NR 162 (3 mM / DMSO, positive control) or DMSO (negative control) were dispensed (15 nL / well) by an Echo 655T Acoustic Liquid Handler with stock and final ligand concentrations defined in Tables 7, 10 and 11. Protein mixture of Tb- anti-GST (4 nM, 2 ×) and GST-hCASK (4 nM, 2 ×) was then dispensed at 7.5 μL / well.

[0259] TR-FRET hCAMKK2 ligand binding inhibitory activity test with BODIPY FL SNSATTORNEY DOCKET NO.331904-2010 032 as the fluorescent probe. Black 384-well assay plates were first dispensed with 250 nM BODIPY FL SNS 032 (7.5 µL / well with 0.2% DMSO, 2 ×) in Kinase buffer 1. Dilutions of SNS- 032, THAL-SNS-032, Ro 31-8220, NR 162, CZC-25146, Staurosporine, Flavopiridol, Atuveciclib, Fadraciclib, Riviciclib or CZC-25146 (30 mM / DMSO, positive control) or DMSO (negative control) were dispensed (15 nL / well) by an Echo 655T Acoustic Liquid Handler with stock and final ligand concentrations defined in Tables 7, 10 and 11. Protein mixture of Tb- anti-GST (4 nM, 2 ×) and GST-hCAMKK2 (4 nM, 2 ×) was then dispensed at 7.5 μL / well. Table 5. Stock BODIPY FL SNS 032 concentration ranges and control concentrations in Echo plates in the Kddetermination experiments. TR-FRET Assay BODIPY FL SNS 032 Positive control Negative control hCAR Binding 122.0 nM-to-4 mM 3 mM CITCO / DMSO DMSOhGSK3A Binding 15.3 nM-to-500 µM 20 mM SNS- 032 / DMSO DMSO hCDK9 / CycT1 Binding 30.5 nM-to-1 mMDMSO hMAPK15 Binding 61.0 nM-to-2 mM 2 mM Ro 31- 8220 / DMSO DMSO hCASK Binding 61.0 nM-to-2 mM 5 mM NR 162 / DMSO DMSO hCAMKK2 Binding 61.0 nM-to-2 mM 30 mM CZC- 25146 / DMSO DMSO

[0260] Regarding Table 5, BODIPY FL SNS16 concentration levels for all targets. Table 6. Final BODIPY FL SNS 032 concentration range and final control concentration for each assay in the Kddetermination experiments. TR-FRET Assay BODIPY FL SNS 032* Positive control Negative control hCAR Binding 122.0 pM-to-4,000 3 CITCO 0.1% DMSO hGSK3A BindinghCDK9 / CycT1 30.5 pM-to-1,000 Binding nM 20 µM SNS-032 0.1% DMSO 61.0 pM-to-2,00hMAPK15 Binding 0 nM 0.1% DMSO hCASK Binding 61.0 pM-to-2,000 nM 5 µM NR 162 0.1% DMSO hCAMKK2 Binding 61.0 pM-to-2,000 nM 30 µM CZC-25146 0.1% DMSO

[0261] Regarding Table 6, BODIPY FL SNS 032 was tested at 1-to-2 dilutions for 16ATTORNEY DOCKET NO.331904-2010 concentration levels for all targets and contributed 0.1% DMSO to each assay. Additionally, positive or negative control contributed the other 0.1% DMSO to each assay. Table 7. Stock and final control concentrations for each assay in ligand binding activity tests. ##Positive control Negative control HTRF or TRFRET Assay Name In Echo In Assay In Echo In Assay Plate Plate Name Plate Plate hCAR Coactivator CITCO 3 3 µM with mM / D DMSOMSO 0.1% DMSO(agonistic test) hCAR Coactivator 30 30 µM with CINPA1 DMSO 100% 0.1% (inverse mM / DMSO 0.1% DMSO agonistic test) 3 3 µM with hCAR Binding CITCO DMSO 100% 0.1% mM / DMSO 0.1% DMSO 10 hGSK3A Binding SNS-032mM / DMSO hCDK9 / CycT1 1 SNS-032 DMSO 100% 0.1%Binding mM / DMSO hMAPK15 Ro 31- 1 1 µM with DMSO 100% 0.1% Binding 8220 mM / DMSO 0.1% DMSO 3 3 µM with hCASK Binding NR 162 DMSO 100% 0.1% mM / DMSO 0.1% DMSO hCAMKK2 CZC- 30 30 µM with DMSO 100% 0.1% Binding 25146 mM / DMSO 0.1% DMSO

[0262] Regarding Table 7, positive or negative control contributed 0.1% DMSO to each assay. Table 8. Stock ligand concentration ranges and dilution factors in Echo plates in hCAR coactivator recruitment and binding tests. Ligand hCAR Coactivator hCAR Binding 16.9 nM-to-3 mM, 1-to-3 dilutions 16.9 nM-to-3 mM, 1-to-3 dilutions CITCO for 12 levels for 12 levels 508.1 nM-to-30 mM, 1-to-3 169.3 nM-to-30 mM, 1-to-3 CINPA1 dilutions for 11 levels dilutions for 12 levels 0.6 µM-to-100 mM, 1-to-3 0.6 µM-to-100 mM, 1-to-3 dilutions Androstenol dilutions for 12 levels for 12 levels 508.1 nM-to-90 mM, 1-to-3 48.8 µM-to-100 mM, 1-to-2 dilutions for 12 levels dilutions for 12 levels508.1 nM-to-90 mM, 1-to-3 48.8 µM-to-100 mM, 1-to-2 THAL-SNS-032 dilutions for 12 levels dilutions for 12 levels BODIPY FL 508.1 nM-to-30 mM, 1-to-3 Not Tested SNS 032 dilutions for 11 levelsATTORNEY DOCKET NO.331904-2010 72 Not Tested 5.6 nM-to-1 mM, 1-to-3 dilutions for 12 levels T0901317 Not Tested 508.1 nM-to-90 mM, 1-to-3 dilutions for 12 levels PK11195 Not Tested 56.5 nM-to-10 mM, 1-to-3 dilutions for 12 levels Clotrimazole Not Tested 16.9 nM-to-3 mM, 1-to-3 dilutions for 12 levels S07662 Not Tested 56.5 nM-to-10 mM, 1-to-3 dilutions for 12 levels Table 9. Ligand final concentration ranges and dilution factors in hCAR coactivator and binding tests. #Ligand hCAR Coactivator hCAR Binding CITCO 16.9 pM-to-3 µM, 1-to-3 dilutions 16.9 pM-to-3 µM, 1-to-3 dilutions for 12 levels for 12 levels CINPA1 508.1 pM-to-30 µM, 1-to-3 169.3 pM-to-30 µM, 1-to-3 dilutions dilutions for 11 levels for 12 levels Androstenol 0.6 nM-to-3 dilutions 0.6 nM-to-100 µM, 1-to-3 dilutions for 12 levels for 12 levels 508.1 pM-to-90 µM, 1-to-3 48.8 nM-to-100 µM, 1-to-2 dilutions dilutions for 12 levels for 12 levels 508.1 pM-to-90 µM, 1-to-3 48.8 nM-to-100 µM, 1-to-2 dilutions dilutions for 12 levels for 12 levels508.1 pM-to-30 µM, 1-to-3 SNS 032 dilutions for 11 levels Not Tested 72 Not Tested 5.6 pM-3 dilutions for 12 levels T0901317 Not Tested 508.1 pM-to-90 µM, 1-to-3 dilutions for 12 levels PK11195 Not Tested 56.5 pM-to-10 µM, 1-to-3 dilutions for 12 levels Clotrimazole Not Tested 16.9 pM-to-3 µM, 1-to-3 dilutions for 12 levels S07662 Not Tested 56.5 pM-to-10 µM, 1-to-3 dilutions for 12 levels

[0263] Regarding Table 9, the ligand contributed 0.1% DMSO to each assay. Table 10. Stock ligand concentration ranges in Echo plates in kinase binding tests with all ligands at 1-to-3 dilutions for 12 concentration levels. Ligand hGSK3A hCDK9 / CycT1 hMAPK15 hCASK hCAMKK2 SNS-032 56.5 nM-to- 5.7 nM-to-1 56.5 nM-to- 169.4 nM- 508.1 nM-to- 10 mM mM 10 mM to-30 mM 90 mMATTORNEY DOCKET NO.331904-2010 THAL-SNS- 56.5 nM-to- 5.7 nM-to-1 56.5 nM-to- 169.4 nM- 169.4 nM-to- 032 10 mM mM 10 mM to-30 mM 30 mM Ro 31-8220 0.6 nM-to- 5.7 nM-to-1 5.7 nM-to-1 169.4 nM- 56.5 nM-to- 100 nM mM mM to-30 mM 10 mM NR 162 16.9 nM-to- 16.9 nM-to-3 16.9 nM-to-3 16.9 nM-to- 16.9 nM-to-3 3 mM mM mM 3 mM mM CZC-25146 508.1 nM- 169.4 nM-to- 169.4 nM-to- 169.4 nM- 169.4 nM-to- to-90 mM 30 mM 30 mM to-30 mM 30 mM 1.7 nM-to- 5.7 nM-to-1 16.9 nM-to-3 56.5 nM-to- 5.7 nM-to-1 300 nM mM mM 10 mM mM Flavopiridol 169.4 nM- 5.7 nM-to-1 169.4 nM-to- 169.4 nM- 169.4 nM-to- to-30 mM mM 30 mM to-30 mM 30 mM Atuveciclib 56.5 nM-to- 16.9 nM-to-3 508.1 nM-to- 508.1 nM- 508.1 nM-to- 10 mM mM 90 mM to-90 mM 90 mM Fadraciclib 508.1 nM- 16.9 nM-to-3 169.4 nM-to- 508.1 nM- 508.1 nM-to- to-90 mM mM 30 mM to-90 mM 90 mM Riviciclib 169.4 nM- 5.7 nM-to-1 169.4 nM-to- 169.4 nM- 169.4 nM-to- to-30 mM mM 30 mM to-30 mM 30 mM Table 11. Ligand concentration ranges in kinase binding tests with all ligands at 1-to-3 dilutions for 12 concentration levels. #Ligand hGSK3A hCDK9 / CycT1 hMAPK15 hCASK hCAMKK2 SNS-032 56.5 pM-to- 5.7 pM-to-1 56.5 pM-to- 169.4 pM- 508.1 pM-to- 10 µM µM 10 µM to-30 µM 90 µM THAL-SNS- 56.5 pM-to- 5.7 pM-to-1 56.5 pM-to- 169.4 pM- 169.4 pM-to- 032 10 µM µM 10 µM to-30 µM 30 µM Ro 31-8220 0.6 pM-to- 5.7 pM-to-1 5.7 pM-to-1 169.4 pM- 56.5 pM-to- 100 nM µM µM to-30 µM 10 µM NR 162 16.9 pM-to- 16.9 pM-to-3 16.9 pM-to-3 16.9 pM-to- 16.9 pM-to-3 3 µM µM µM 3 µM µM 508.1 pM- 169.4 pM-to- 169.4 pM-to- 169.4 pM- 169.4 pM-to- to-90 µM 30 µM 30 µM to-30 µM 30 µM Staurosporine 1.7 pM-to- 5.7 pM-to-1 16.9 pM-to-3 56.5 pM-to- 5.7 pM-to-1 300 nM µM µM 10 µM µM Flavopiridol 169.4 pM- 5.7 pM-to-1 169.4 pM-to- 169.4 pM- 169.4 pM-to- to-30 µM µM 30 µM to-30 µM 30 µM Atuveciclib 56.5 pM-to- 16.9 pM-to-3 508.1 pM-to- 508.1 pM- 508.1 pM-to- 10 µM µM 90 µM to-90 µM 90 µM Fadraciclib 508.1 pM- 16.9 pM-to-3 169.4 pM-to- 508.1 pM- 508.1 pM-to- to-90 µM µM 30 µM to-90 µM 90 µM Riviciclib 169.4 pM- 5.7 pM-to-1 169.4 pM-to- 169.4 pM- 169.4 pM-to- to-30 µM µM 30 µM to-30 µM 30 µM

[0264] Regarding Table 11, the ligand contributed 0.1% DMSO to each assay.ATTORNEY DOCKET NO.331904-2010 Table 12. IC50and Kivalues of ligands tested by the BODIPY FL SNS 032-based hCAR TR- FRET binding assay. NApp stands for Not Applicable. Ligand hCAR TR-FRET Binding Assay IC50hCAR TR-FRET Binding Assay Kivalue value CITCO 2.6 ± 0.4 nM 1.8 ± 0.3 nM nM nMBODIPY FL SNS 032 (10) Not Tested NApp 72 (3) 12.8 ± 0.7 nM (IC50) 9.0 ± 0.5 nM T0901317 (4) 650.8 ± 70.7 nM (IC50) 458.3 ± 49.8 nM PK11195 (5) 55.8 ± 2.4 nM (IC50) 39.3 ± 1.7 nM Clotrimazole (6) 32.6 ± 0.9 nM (IC50) 23.0 ± 0.6 nM S07662 (7) 260.0 ± 11.6 nM (IC50) 183.1 ± 8.2 nM Table 13. IC50and Kivalues of ligands tested by the BODIPY FL SNS 032-based TR-FRET binding assays against hGSK3, hCDK9 / CycT1, hMAPK15, hCASK and hCAMKK2. NApp stands for not applicable. Kinase hGSK3A hCDK9 / CycT1 hMAPK15 hCASK hCAMKK2 Inhibitor / SNS-032 10.6 ± 0.9 nM 1.1 ± 0.1 nM 43.1 ± 2.8 nM 182.4 ± 12.4 3.5 ± 0.2 µM nM 439.7 ± 22.4 THAL- 8.2 ± 0.8 nM / 5.6 ± 0.2 nM / 79.1 ± 3.9 nM / nM / 2.7 ± 0.1 µM / SNS-032 4.1 ± 0.4 nM 2.8 ± 0.1 nM 57.7 ± 2.8 nM 357.5 ± 18.2 2.2 ± 0.1 µM nM 161.4 ± 3.3 Ro 31- 0.7 ± 0.03 nM / 1.0 ± 0.1 nM / 0.9 ± 0.1 nM / 1.2 ± 0.1 µM nM / 8220 / 1.0 ± 0.1 0.4 ± 0.02 nM 0.5 ± 0.1 nM 0.7 ± 0.1 nM µM 130.2 ± 2.7 nM NA* / NA* / NA* / 8.1 ± 0.5 nM 1 2 / NA* / NR 6 NApp NApp NApp 6.6 ± 0.4 nM NApp 20.2 ± 1.6 µM / NA* / 2.8 ± 0.2 µM / 40.7 ± 51.6 ± 5.7 nM CZC-25146 2.9%@30 / 41.6 ± 4.6 10.1 ± 0.8 µM NApp 2.0 ± 0.1 µM µM# / NApp nMATTORNEY DOCKET NO.331904-2010 Staurospor 2.1 ± 0.1 nM / 3.3 ± 0.1 nM / 2.6 ± 0.1 nM / 6.3 ± 0.5 nM 0.3 ± 0.04 nM -ine / 5.1 ± 0.4 / 0.2 ± 0.03 1.1 ± 0.1 nM 1.7 ± 0.1 nM 1.9 ± 0.1 nM nM nM 163.7 ± 16.4 296.4 ± 17.4 1.4 ± 0.1 nM / nM / NA* / nM / Flavopiridol 163.8 ± 16.4 nM / 81.9 ± 8.2 nM 0.7 ± 0.1 nM 119.5 ± 12.0 NApp 239.0 ± 14.0 nM nM29.1 ± 2.6 nM / ± / 2.6 ± 0.1 µM / ± 1.2 ± 0.1 µM / Atuveciclib 3.6%@90 14.6 ± 1.3 nM 3.4 ± 0.5 nM 1.9 ± 0.1 µM µM# / NApp 1.0 ± 0.1 µM / 3. ADDITIONAL DATA

[0265] In this example, a table of KINOMEscan results for evaluation of BODIPY FL SNS 032 (10) against a panel of 468 kinases is provided. The BODIPY FL SNS 032 concentration in all evaluations was 2000 nM. Table 14. KINOMEscan hits against BODIPY FL SNS 032. DiscoveRx Gene Symbol Entrez Gene Percent Symbol Control GSK3A GSK3A 2.3 NEK6 NEK6 7.1 CDC2L5 CDK13 12 ICK ICK 12 CDKL5 CDKL5 18 CSNK1E CSNK1E 20 GSK3B GSK3B 21 DYRK1B DYRK1B 23 CDK9 CDK9 24 CSNK1D CSNK1D 29 ERK8 MAPK15 31 CSNK1A1 CSNK1A1 33 CDK7 CDK7 35 DYRK1A DYRK1A 42 PCTK2 CDK17 44ATTORNEY DOCKET NO.331904-2010 CLK2 CLK2 46 RIOK2 RIOK2 48 CASK CASK 55 CTK MATK 55 NIK MAP3K14 56 YSK4 MAP3K19 56 LKB1 STK11 57 RIPK5 DSTYK 58 MTOR MTOR 62 BTK BTK 63 CDK4-cyclinD1 CDK4 63 MAP3K15 MAP3K15 63 CDKL2 CDKL2 64 BMPR1B BMPR1B 65 BMX BMX 65 CSF1R-autoinhibited CSF1R 65 STK39 STK39 65 ADCK3 CABC1 66 CAMKK2 CAMKK2 66 IRAK3 IRAK3 66 PCTK3 CDK18 67 CSNK2A1 CSNK2A1 69 GCN2(Kin.Dom.2,S808G) EIF2AK4 69 HUNK HUNK 69 DRAK2 STK17B 70 DYRK2 DYRK2 70 PLK2 PLK2 71 TAOK3 TAOK3 71 JNK3 MAPK10 72 PHKG2 PHKG2 72 PLK3 PLK3 72 TAOK2 TAOK2 72 DRAK1 STK17A 73 FLT3(D835H) FLT3 73 MYLK MYLK 73 CAMK2A CAMK2A 74 KIT(D816H) KIT 74 PIP5K2B PIP4K2B 74 SRPK1 SRPK1 74 VRK2 VRK2 74 AURKA AURKA 75 MAK MAK 75 MAP3K1 MAP3K1 75 SRMS SRMS 75 ABL1(E255K)- phosphorylated ABL1 76 BRAF BRAF 76 CDK8 CDK8 76ATTORNEY DOCKET NO.331904-2010 FLT3(D835Y) FLT3 76 PAK2 PAK2 76 ACVR1B ACVR1B 77 CDK2 CDK2 77 ERN1 ERN1 77 GRK2 ADRBK1 77 ABL1(Q252H)- nonphosphorylated ABL1 78 ACVR2A ACVR2A 78 DAPK2 DAPK2 78 DDR2 DDR2 78 PCTK1 CDK16 78 TYK2(JH2domain- pseudokinase) TYK2 78 CDK3 CDK3 79 ERBB4 ERBB4 79 PAK7 PAK7 79 ALK ALK 80 BRAF(V600E) BRAF 80 FLT4 FLT4 80 HIPK3 HIPK3 80 ERK3 MAPK6 81 JNK1 MAPK8 81 MEK3 MAP2K3 81 NDR2 STK38L 81 RIOK3 RIOK3 81 STK16 STK16 81 ABL1(H396P)- phosphorylated ABL1 82 CDK11 CDK19 83 DAPK3 DAPK3 83 DCAMKL1 DCLK1 83 FLT3(D835V) FLT3 83 IRAK1 IRAK1 83 LTK LTK 83 PKNB(M.tuberculosis) pknB 83 PLK4 PLK4 83 RPS6KA5(Kin.Dom.2-C- terminal) RPS6KA5 83 TIE2 TEK 83 ABL1(T315I)- phosphorylated ABL1 84 ARK5 NUAK1 84 DCAMKL3 DCLK3 84 EGFR(G719S) EGFR 84 EPHB6 EPHB6 84 INSR INSR 84 KIT-autoinhibited KIT 84 LATS1 LATS1 84ATTORNEY DOCKET NO.331904-2010 MAP3K3 MAP3K3 84 MAP4K5 MAP4K5 84 MST1 STK4 84 PAK4 PAK4 84 RET(V804L) RET 84 RSK2(Kin.Dom.2-C- terminal) RPS6KA3 84 VEGFR2 KDR 84 BUB1 BUB1 85 EPHA2 EPHA2 85 EPHA3 EPHA3 85 FGFR4 FGFR4 85 HIPK1 HIPK1 85 MKNK1 MKNK1 85 MYO3A MYO3A 85 PFCDPK1(P.falciparum) CDPK1 85 PKN2 PKN2 85 PLK1 PLK1 85 TAOK1 TAOK1 85 ABL1(H396P)- nonphosphorylated ABL1 86 AURKC AURKC 86 DMPK2 CDC42BPG 86 IKK-epsilon IKBKE 86 IRAK4 IRAK4 86 PRKCI PRKCI 86 SGK2 SGK2 86 TIE1 TIE1 86 CAMK1B PNCK 87 CSNK2A2 CSNK2A2 87 EGFR(L858R) EGFR 87 FLT3(ITD) FLT3 87 FLT3(K663Q) FLT3 87 FLT3-autoinhibited FLT3 87 JAK3(JH1domain-catalytic) JAK3 87 PIK3CA(I800L) PIK3CA 87 RIPK4 RIPK4 87 SGK3 SGK3 87 TBK1 TBK1 87 CDK4 CDK4 88 GAK GAK 88 GRK3 ADRBK2 88 IKK-beta IKBKB 88 KIT(A829P) KIT 88 MEK5 MAP2K5 88 NIM1 MGC42105 88 p38-gamma MAPK12 88 PFPK5(P.falciparum) MAL13P1.279 88 PIK3CA PIK3CA 88ATTORNEY DOCKET NO.331904-2010 PIP5K2C PIP4K2C 88 RET(M918T) RET 88 SIK SIK1 88 TESK1 TESK1 88 TNIK TNIK 88 TRKC NTRK3 88 WNK4 WNK4 88 ABL1(Q252H)- phosphorylated ABL1 89 ABL1-phosphorylated ABL1 89 BLK BLK 89 BMPR2 BMPR2 89 CAMK1D CAMK1D 89 CAMK2B CAMK2B 89 CDK4-cyclinD3 CDK4 89 EGFR EGFR 89 EPHA1 EPHA1 89 ERBB2 ERBB2 89 FLT3(ITD,F691L) FLT3 89 JAK1(JH2domain- pseudokinase) JAK1 89 JAK2(JH1domain-catalytic) JAK2 89 LIMK1 LIMK1 89 MAP3K2 MAP3K2 89 PIM2 PIM2 89 PRKCE PRKCE 89 RIOK1 RIOK1 89 ASK1 MAP3K5 90 AURKB AURKB 90 BRK PTK6 90 CAMK1 CAMK1 90 CSNK1A1L CSNK1A1L 90 FLT3(N841I) FLT3 90 FYN FYN 90 MARK2 MARK2 90 PAK3 PAK3 90 PFTK1 CDK14 90 PIM3 PIM3 90 RPS6KA4(Kin.Dom.2-C- terminal) RPS6KA4 90 ABL1(F317L)- phosphorylated ABL1 91 ADCK4 ADCK4 91 AKT1 AKT1 91 DDR1 DDR1 91 EGFR(L861Q) EGFR 91 MYLK2 MYLK2 91 p38-delta MAPK13 91 PFTAIRE2 CDK15 91ATTORNEY DOCKET NO.331904-2010 PIK3CG PIK3CG 91 TGFBR2 TGFBR2 91 TNK1 TNK1 91 ABL1(Y253F)- phosphorylated ABL1 92 AXL AXL 92 BMPR1A BMPR1A 92 CSNK1G2 CSNK1G2 92 ERK2 MAPK1 92 FLT1 FLT1 92 p38-beta MAPK11 92 PAK1 PAK1 92 PIK3CA(H1047Y) PIK3CA 92 TYRO3 TYRO3 92 ABL1(M351T)- phosphorylated ABL1 93 ABL1(T315I)- nonphosphorylated ABL1 93 ALK(L1196M) ALK 93 CHEK1 CHEK1 93 EGFR(G719C) EGFR 93 EGFR(S752-I759del) EGFR 93 EPHA6 EPHA6 93 ERK1 MAPK3 93 FLT3 FLT3 93 MELK MELK 93 MYO3B MYO3B 93 NEK3 NEK3 93 NEK7 NEK7 93 PIP5K1C PIP5K1C 93 PRKD3 PRKD3 93 SBK1 SBK1 93 TNNI3K TNNI3K 93 ABL1-nonphosphorylated ABL1 94 ACVR1 ACVR1 94 CHEK2 CHEK2 94 CLK1 CLK1 94 DLK MAP3K12 94 EGFR(L747-T751del,Sins) EGFR 94 HCK HCK 94 ITK ITK 94 LYN LYN 94 MEK1 MAP2K1 94 MEK4 MAP2K4 94 PIK3CA(M1043I) PIK3CA 94 PKAC-alpha PRKACA 94 QSK KIAA0999 94 RET RET 94 SGK SGK1 94ATTORNEY DOCKET NO.331904-2010 STK33 STK33 94 STK36 STK36 94 TLK1 TLK1 94 WNK3 WNK3 94 ACVR2B ACVR2B 95 ALK(C1156Y) ALK 95 BIKE BMP2K 95 BRSK1 BRSK1 95 CDK5 CDK5 95 CIT CIT 95 CLK4 CLK4 95 CSK CSK 95 EGFR(L858R,T790M) EGFR 95 FLT3(ITD,D835V) FLT3 95 FRK FRK 95 JNK2 MAPK9 95 MEK2 MAP2K2 95 NEK4 NEK4 95 PIK3CA(C420R) PIK3CA 95 PIK3CA(E545A) PIK3CA 95 PIK3CA(E545K) PIK3CA 95 PRKG2 PRKG2 95 RET(V804M) RET 95 RSK4(Kin.Dom.1-N- terminal) RPS6KA6 95 ABL1(F317I)- phosphorylated ABL1 96 CAMK4 CAMK4 96 DAPK1 DAPK1 96 EGFR(L747-E749del, A750P) EGFR 96 EGFR(T790M) EGFR 96 EPHB2 EPHB2 96 FGFR2 FGFR2 96 LATS2 LATS2 96 MAST1 MAST1 96 MINK MINK1 96 MLK2 MAP3K10 96 MRCKA CDC42BPA 96 PIK3C2B PIK3C2B 96 ULK1 ULK1 96 WNK1 WNK1 96 YSK1 STK25 96 ZAK ZAK 96 ZAP70 ZAP70 96 ABL1(F317I)- nonphosphorylated ABL1 97 ASK2 MAP3K6 97 CDC2L1 CDK11B 97ATTORNEY DOCKET NO.331904-2010 EIF2AK1 EIF2AK1 97 EPHA4 EPHA4 97 EPHA8 EPHA8 97 FES FES 97 GRK1 GRK1 97 GRK4 GRK4 97 HPK1 MAP4K1 97 LOK STK10 97 MAP4K4 MAP4K4 97 MKK7 MAP2K7 97 NDR1 STK38 97 OSR1 OXSR1 97 PRKD1 PRKD1 97 SgK110 SgK110 97 SNRK SNRK 97 TTK TTK 97 VPS34 PIK3C3 97 YES YES1 97 ACVRL1 ACVRL1 98 AMPK-alpha1 PRKAA1 98 ERK4 MAPK4 98 FGFR3 FGFR3 98 GRK7 GRK7 98 IGF1R IGF1R 98 JAK1(JH1domain-catalytic) JAK1 98 KIT(D816V) KIT 98 KIT(V559D,V654A) KIT 98 MET MET 98 MLK1 MAP3K9 98 MRCKB CDC42BPB 98 MST1R MST1R 98 MST4 MST4 98 NEK2 NEK2 98 PAK6 PAK6 98 PIK3CA(E542K) PIK3CA 98 RSK4(Kin.Dom.2-C- terminal) RPS6KA6 98 TNK2 TNK2 98 ULK2 ULK2 98 WNK2 WNK2 98 YANK1 STK32A 98 CDC2L2 CDC2L2 99 EPHB1 EPHB1 99 ERBB3 ERBB3 99 HASPIN GSG2 99 LZK MAP3K13 99 NEK10 NEK10 99 NEK5 NEK5 99ATTORNEY DOCKET NO.331904-2010 PIK3C2G PIK3C2G 99 RPS6KA5(Kin.Dom.1-N- terminal) RPS6KA5 99 SLK SLK 99 SRPK2 SRPK2 99 SRPK3 SRPK3 99 TSSK3 TSSK3 99 WEE2 WEE2 99 AAK1 AAK1 100 ABL1(F317L)- nonphosphorylated ABL1 100 ABL2 ABL2 100 AKT2 AKT2 100 AKT3 AKT3 100 AMPK-alpha2 PRKAA2 100 ANKK1 ANKK1 100 BRSK2 BRSK2 100 CAMK1G CAMK1G 100 CAMK2D CAMK2D 100 CAMK2G CAMK2G 100 CAMKK1 CAMKK1 100 CDKL1 CDKL1 100 CDKL3 CDKL3 100 CLK3 CLK3 100 CSF1R CSF1R 100 CSNK1G1 CSNK1G1 100 CSNK1G3 CSNK1G3 100 DCAMKL2 DCLK2 100 DMPK DMPK 100 EGFR(E746-A750del) EGFR 100 EGFR(L747-S752del, P753S) EGFR 100 EPHA5 EPHA5 100 EPHA7 EPHA7 100 EPHB3 EPHB3 100 EPHB4 EPHB4 100 ERK5 MAPK7 100 FAK PTK2 100 FER FER 100 FGFR1 FGFR1 100 FGFR3(G697C) FGFR3 100 FGR FGR 100 FLT3(R834Q) FLT3 100 HIPK2 HIPK2 100 HIPK4 HIPK4 100 IKK-alpha CHUK 100 INSRR INSRR 100 KIT KIT 100 KIT(L576P) KIT 100ATTORNEY DOCKET NO.331904-2010 KIT(V559D) KIT 100 KIT(V559D,T670I) KIT 100 LCK LCK 100 LIMK2 LIMK2 100 LRRK2 LRRK2 100 LRRK2(G2019S) LRRK2 100 MAP3K4 MAP3K4 100 MAP4K2 MAP4K2 100 MAP4K3 MAP4K3 100 MAPKAPK2 MAPKAPK2 100 MAPKAPK5 MAPKAPK5 100 MARK1 MARK1 100 MARK3 MARK3 100 MARK4 MARK4 100 MEK6 MAP2K6 100 MERTK MERTK 100 MET(M1250T) MET 100 MET(Y1235D) MET 100 MKNK2 MKNK2 100 MLCK MYLK3 100 MLK3 MAP3K11 100 MST2 STK3 100 MST3 STK24 100 MUSK MUSK 100 MYLK4 MYLK4 100 NEK1 NEK1 100 NEK11 NEK11 100 NEK9 NEK9 100 NLK NLK 100 p38-alpha MAPK14 100 PDGFRA PDGFRA 100 PDGFRB PDGFRB 100 PDPK1 PDPK1 100 PHKG1 PHKG1 100 PIK3CA(H1047L) PIK3CA 100 PIK3CA(Q546K) PIK3CA 100 PIK3CB PIK3CB 100 PIK3CD PIK3CD 100 PIK4CB PI4KB 100 PIKFYVE PIKFYVE 100 PIM1 PIM1 100 PIP5K1A PIP5K1A 100 PKAC-beta PRKACB 100 PKMYT1 PKMYT1 100 PKN1 PKN1 100 PRKCD PRKCD 100 PRKCH PRKCH 100 PRKCQ PRKCQ 100ATTORNEY DOCKET NO.331904-2010 PRKD2 PRKD2 100 PRKG1 PRKG1 100 PRKR EIF2AK2 100 PRKX PRKX 100 PRP4 PRPF4B 100 PYK2 PTK2B 100 RAF1 RAF1 100 RIPK1 RIPK1 100 RIPK2 RIPK2 100 ROCK1 ROCK1 100 ROCK2 ROCK2 100 ROS1 ROS1 100 RPS6KA4(Kin.Dom.1-N- terminal) RPS6KA4 100 RSK1(Kin.Dom.1-N- terminal) RPS6KA1 100 RSK1(Kin.Dom.2-C- terminal) RPS6KA1 100 RSK2(Kin.Dom.1-N- terminal) RPS6KA3 100 RSK3(Kin.Dom.1-N- terminal) RPS6KA2 100 RSK3(Kin.Dom.2-C- terminal) RPS6KA2 100 S6K1 RPS6KB1 100 SIK2 SIK2 100 SNARK NUAK2 100 SRC SRC 100 STK35 STK35 100 SYK SYK 100 TAK1 MAP3K7 100 TEC TEC 100 TGFBR1 TGFBR1 100 TLK2 TLK2 100 TRKA NTRK1 100 TRKB NTRK2 100 TRPM6 TRPM6 100 TSSK1B TSSK1B 100 TXK TXK 100 TYK2(JH1domain-catalytic) TYK2 100 ULK3 ULK3 100 WEE1 WEE1 100 YANK2 STK32B 100 YANK3 STK32C 100

[0266] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the disclosure. Other aspects of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intendedATTORNEY DOCKET NO.331904-2010 that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.

Claims

ATTORNEY DOCKET NO.331904-2010 CLAIMS What is claimed is:

1. A compound having a structure represented by a formula: , wherein Ln is a linkingwherein FP is fluorophore moiety; and wherein q is an integer selected from 0 and 1.

2. The compound of claim 1, wherein q is 1; and wherein the compound has a structure represented by a formula: .

3. The compound ofa structure represented by a formula: ; wherein,G is selected from O, NH, and CH2; L is selected from O, NH, CO, CH2, (CH2)2, NHC(O), and C(O)NH; Z1and Z2are independently selected from CH2and OCH2CH2; R1and R2are independently selected from a cycloalkyl group, a heterocycloalkyl group, an aryl group, and a heteroaryl group;ATTORNEY DOCKET NO.331904-2010 m1is an integer selected from 0, 1, 2, and 3; m2is an integer selected from 0, 1, 2, and 3; n is an integer selected from1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; q1is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and q2is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

4. The compound of any one of claims 1-3, wherein G is selected from NH and CH2.

5. The compound of any one of claims 1-4, wherein L is selected from NH, CO, CH2, NHC(O), and C(O)NH.

6. The compound of any one of claims 1-5, wherein R1and R2are independently selected from a C4-C10 cycloalkyl group, a C4-C10 heterocycloalkyl group, a C4-C10 aryl group, and a C4-C10 heteroaryl group.

7. The compound of any one of claims 1-6, wherein R1and R2are independently selected from a C4-C8 cycloalkyl group, a C4-C8 heterocycloalkyl group, a C4-C8 aryl group, and a C4-C8 heteroaryl group.

8. The compound of any one of claims 1-7, wherein R1and R2are independently selected from a C4-C6 cycloalkyl group, a C4-C6 heterocycloalkyl group, a C4-C6 aryl group, and a C4-C6 heteroaryl group.

9. The compound of any one of claims 1-8, wherein the heterocycloalkyl group comprises at least one nitrogen heteroatom.

10. The compound of any one of claims 1-9, wherein the heteroaryl group comprises at least one nitrogen heteroatom.

11. The compound of any one of claims 1-10, wherein m1is an integer selected from 0 and 1.

12. The compound of any one of claims 1-11, wherein m2is an integer selected from 0 and 1.

13. The compound of any one of claims 1-12, wherein n is an integer selected from 1, 2, 3, 4, 5, and 6.

14. The compound of any one of claims 1-13, wherein q1is an integer selected from 1, 2, 3, 4, 5, and 6.

15. The compound of any one of claims 1-14, wherein q2is an integer selected from 1, 2, 3, 4, 5, and 6.

16. The compound of claim 1, wherein q is 0; and wherein the compound has a structureATTORNEY DOCKET NO.331904-2010 represented by a formula: .

17. The compound of any moiety has anexcitation maximum in the range of about 460 nm to about 650 nm and an emission maximum in the range of about 510 nm to about 670 nm.

18. The compound of any one of claims 1-17, wherein the fluorophore is derived from a structure represented by a formula: ,ATTORNEY DOCKET NO.331904-2010 ,ATTORNEY DOCKET NO.331904-2010 .

19. The compound of any one of claims 1-18, wherein the fluorophore attaches to the linking moiety or the nitrogen of the piperidine ring via one of an amine linking group, an ether linking group, or a carbonyl linking group.

20. A method to determine binding affinity of a compound interacting with a constitutive androstane receptor or a kinase, the method comprising the steps of: a) providing a solution comprising: i. a test compound; ii. a polypeptide comprising a domain, or a ligand binding fragment polypeptide thereof, that can bind a moiety of at least one compound of any one of claims 1- 19 having a structure given by the formula:ATTORNEY DOCKET NO.331904-2010 ; iii. a fluorescent iv. a fluorescentone compound of any one of claims 1-19; b) illuminating the solution, thereby causing fluorescence in the fluorescent donor molecule; c) measuring fluorescence emission from the fluorescent donor molecule and the fluorescent acceptor molecule; and d) comparing the fluorescence emission to the fluorescence emission of steps (a)-(c) carried out in the absence of the test compound.

21. The method of claim 20, wherein the fluorophore is derived from a structure represented by a formula: .

22. The method of claim 20 ordonor molecule comprises chelated terbium and one or more moieties capable of binding the polypeptide.

23. The method of claim 22, wherein the chelated terbium comprises at least one diethylenetriaminepentaacetic acid residue and Tb+3.

24. The method of claim 22 or claim 23, wherein the one or more moieties capable of binding the polypeptide comprises an antibody.

25. The method of claim 24, wherein the antibody recognizes an epitope on the polypeptide.

26. The method of claim 25, wherein the epitope on the polypeptide comprises one or more affinity tags or epitope tags.

27. The method of claim 26, wherein the affinity tag is selected from histidine tag, maltose binding protein tag, chitin binding protein tag, and glutathione-S-transferase tag.

28. The method of claim 26, wherein the epitope tag is selected from T7 tag, FLAG tag, HA tag, VSV-G tag, V5 tag, and c-myc tag.

29. The method of claim 25, wherein the at least one polypeptide epitope is a nativeATTORNEY DOCKET NO.331904-2010 sequence of the at least one polypeptide.

30. The method of any one of claims 24-29, wherein the antibody is a monoclonal antibody.

31. The method of any one of claims 24-29, wherein the antibody is a polyclonal antibody.

32. The method of any one of claims 20-31, wherein the polypeptide is selected from constitutive androstane receptor 1 polypeptide, encoded by a CAR1 gene; glycogen synthase kinase-3 alpha polypeptide, encoded by a GSK3A gene; cyclin-dependent kinase 9 polypeptide, encoded by a CDK9 gene; mitogen-Activated protein kinase 15 polypeptide, encoded by a MAPK15 gene; calcium / calmodulin dependent serine protein kinase polypeptide, encoded by a CASK gene; and calcium / calmodulin-dependent protein kinase polypeptide, encoded by a CAMKK2 gene; a splice variant thereof; and a ligand binding fragment polypeptide thereof.

33. The method of claim 32, wherein the polypeptide is encoded by the human isoform of the corresponding gene.

34. The method of claim 33, wherein the polypeptide encoded is selected from the constitutive androstane receptor 1 polypeptide corresponding to UniProt identifier Q14994; the glycogen synthase kinase-3 alpha polypeptide corresponding to UniProt identifier P49840; the cyclin-dependent kinase 9 polypeptide corresponding to UniProt identifier P50750; the mitogen-Activated protein kinase 15 polypeptide corresponding to UniProt identifier Q8TD08; the calcium / calmodulin dependent serine protein kinase polypeptide corresponding to UniProt identifier O14936; and the calcium / calmodulin- dependent protein kinase polypeptide corresponding to UniProt identifier Q96RR4.

35. A kit for measuring the binding activity of a test compound to human constitutive androstane receptor (hCAR) and / or a kinase comprising: at least one compound of any of claims 1-19, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; and optionally, one or more of: a) instructions for carrying out an in vivo fluorescence assay using at least one compound of any of claims 1-19; b) instructions for carrying out an in vitro fluorescence assay using at least one compound of any of claims 1-19; c) at least one polypeptide that can bind the at least one compound of any of claims 1-19, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; d) at least one compound that binds to hCAR and / or a kinase; e) one or more test compounds; and / or f) at least one buffer and / or salt.ATTORNEY DOCKET NO.331904-2010 36. The kit of claim 35, wherein the in vivo fluorescence assay is a homogeneous time resolved fluorescence (HTRF) assay.

37. The kit of claim 35, wherein the in vivo fluorescence assay is a fluorescence polarization assay.

38. The kit of claim 35, wherein the in vitro fluorescence assay is a homogeneous time resolved fluorescence (HTRF) assay.

39. The kit of claim 35, wherein the in vitro fluorescence assay is a fluorescence polarization assay.

40. The kit of any one of claims 35-39, wherein the kit further comprises an hCAR co- activator.

41. The kit of claim 40, wherein the hCAR co-activator comprises a Cy5-PGC1α peptide, a Cy5-SRC peptide, a PGC1α peptide, or a combination thereof.

42. The kit of any one of claims 35-39, wherein the kit comprises instructions for carrying out an in vitro fluorescence assay; and further comprises the at least one polypeptide that binds a moiety of the least one compound of any of claims 1-19 having a structure given by the formula: .

43. The kit of claim 42,the in vitro fluorescence assay is a time-resolved fluorescence energy transfer (TR-FRET) assay.

44. The kit of claim 42 or claim 43, wherein the kit further comprises a fluorescent donor molecule comprising a fluorescence donor molecule bound to one or more moieties capable of binding the at least one polypeptide.

45. The kit of claim 44, wherein the fluorescence donor molecule is a chelated terbium.

46. The kit of claim 45, wherein the chelated terbium comprises at least one diethylenetriaminepentaacetic acid residue and Tb+3.

47. The kit of any one of claims 44-46, wherein the one or more moieties capable of binding the at least one polypeptide comprises an antibody.

48. The kit of claim 47, wherein the antibody recognizes an epitope on the at least one polypeptide.ATTORNEY DOCKET NO.331904-2010 49. The kit of claim 48, wherein the epitope on the at least one polypeptide comprises one or more affinity tags or epitope tags.

50. The kit of claim 49, wherein the affinity tag is selected from histidine tag, maltose binding protein tag, chitin binding protein tag, and glutathione-S-transferase tag.

51. The kit of claim 49, wherein the epitope tag is selected from T7 tag, FLAG tag, HA tag, VSV-G tag, V5 tag, and c-myc tag.

52. The kit of claim 48, wherein the at least one polypeptide epitope is a native sequence of the at least one polypeptide.

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