WO2018156581A1

WO2018156581A1 - Fluorogenic reporters for phospholipase c isozymes and methods of making and using the same

Info

Publication number: WO2018156581A1
Application number: PCT/US2018/018955
Authority: WO
Inventors: Qisheng Zhang; Xiaoyang Wang; Weigang Huang; John Sondek
Original assignee: University of North Carolina at Chapel Hill
Current assignee: University of North Carolina at Chapel Hill
Priority date: 2017-02-21
Filing date: 2018-02-21
Publication date: 2018-08-30
Anticipated expiration: 2019-08-21

Abstract

The present invention provides fluorogenic compounds, methods for making the same, and methods for using the same including methods of detecting and/or analyzing phospholipase C (PLC) activity. Compounds of the present invention include fluorogenic compounds, such as, for example, a compound of Formula I. Methods of the present invention include a method of identifying a test substance that inhibits phospholipase C activity, a method of detecting phospholipase C activity in a cell, and a method of detecting aberrant phospholipase C activity in a cell.

Description

FLUOROGENIC REPORTERS FOR PHOSPHOLIPASE C ISOZYMES AND METHODS OF MAKING AND USING THE SAME

Related Application Information

This application claims the benefit of and priority to U.S. Provisional Application

Serial No. 62/461,608, filed February 21, 2017, the disclosure of which is hereby incorporated by reference herein in its entirety.

Statement of Government Support

This invention was made with government support under grant numbers GM098984 and GM057391, awarded by the National Institutes of Health. The government has certain rights in this invention.

Statement Regarding Electronic Filing of A Sequence Listing

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821 , entitled

5470-806WO_ST25.txt, 1,256 bytes in size, generated on February 21, 2018 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures. Field of the Invention

This application relates to fluorogenic compounds, methods for making the same, and methods for using the same including methods of detecting and/or analyzing phospholipase C (PLC) activity. Background of the Invention

Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) into two second messengers, inositol 1,4,5- trisphosphate (IP₃) and diacylglycerol (DAG) (Scheme 1). IP₃ mobilizes intracellular stores of Ca²⁺ while DAG activates protein kinase C. Depletion of PIP₂ alters the membrane association and/or activity of many proteins that harbor phosphoinositide binding domains. Consequently, PLC isozymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. The basal activity of PLC isozymes is typically low due to autoinhibition, which is released upon responses to extracellular stimuli such as growth factors, hormones and neurotransmitters. Aberrant regulations of PLC activity have been associated with a wide range of diseases including cancer, rheumatoid arthritis and atherosclerosis. Although extensive studies have been carried out to understand PLC regulation, two limitations remain: 1) it is difficult to monitor PLC activity in living cells; and 2) selective small molecule PLC inhibitors are lacking.

Scheme 1. Cleavage of PIP₂ to IP₃ and DAG by PLC.

R¹ - n-Ci₇H35 R^{2 :}

Summary of the Invention

One aspect of the present invention is directed to fluorogenic compounds, such as, for example, a compound of Formula I. An additional aspect of the present invention is directed to a compound of Formula IA:

wherein:

Pv¹ is each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, -P=0(OH)₂, a cellular localization signal, a quencher, and a cage group (e.g., a photocage group), optionally wherein two adjacent R¹ are taken together to form a cage group (e.g., a photocage group), and optionally wherein R¹ is substituted;

LA and LB are each independently present or absent, and when present are each independently selected from the group consisting of -C(0)NH- -C(O)-, -NH-, -0-, -C(=NR^U)-, -C(=NOH)-, -C(=0)0-, -OC(=0)-, and -NHC(=0)-, wherein R¹¹ is H, alkyl, alkenyl, or alkynyl;

X is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, aliphatic oxide, and -R²-Z-R³-, wherein R² and R³ are each independently selected from the group consisting of a C_\-C_\o unsubstituted or substituted alkyl and a C2-C₁₀ unsubstituted or substituted alkenyl, and Z is oxygen or sulfur;

Y is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aliphatic oxide, aryl, arylalkyl, and -R⁴-CH(D-R⁵)-R⁶-D-R⁷-, wherein R⁴, R⁶, and R⁷ are each independently a Ci-C₁₀ unsubstituted or substituted alkyl or C₂-C₁₀ unsubstituted or substituted alkenyl; R⁵ is H, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, alkylthio, amino, aminoalkyl, alkylamino, cycloalkyl, heteroaryl, heteroalkyl, aryl, arylalkyl, aliphatic oxide, -C(=0)R¹⁰, -C(=0)OR¹⁰, -(CH₂)_mN(R¹⁰)₂, -C(=O)N(R¹⁰)₂, and - (CH₂)_mCOOR¹⁰, where m is 1-20 and R¹⁰ is H, alkyl, alkenyl, or alkynyl; and D is each independently selected from oxygen and sulfur;

Fp is a first fluorophore; and

Q is a quencher or a second fluorophore.

Another aspect of the present invention is directed to a compound Formula II:

wherein:

R¹ is each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, -P=0(OH)₂, a cellular localization signal, a quencher, and a cage group (e.g., a photocage group), optionally wherein two adjacent R¹ are taken together to form a cage group (e.g., a photocage group), and optionally wherein R¹ is substituted;

X is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, aliphatic oxide, and -R²-Z-R³-, wherein R² and R³ are each independently selected from the group consisting of a Ci-Cu) unsubstituted or substituted alkyl and a C₂-C₁₀ unsubstituted or substituted alkenyl, and Z is oxygen or sulfur;

Fp is a first fluorophore; and

Q is a quencher or a second fluorophore.

A further aspect of the present invention is directed to a compound of Formula IIA,

IIB, IIC, III, IV, V, VI, and/or VII.

Another aspect of the present invention is directed to a method of identifying a test substance that inhibits phospholipase C activity, comprising: contacting a compound of the present invention with phospholipase C in the presence and absence of the test substance; detecting the amount of fluorescence in the presence and absence of the test substance; and comparing the amount of fluorescence detected in the presence and absence of the test substance, whereby a decrease in the amount of fluorescence detected in the presence of the test substance identifies that the test substance inhibits phospholipase C activity.

A further aspect of the present invention is directed to a method of detecting phospholipase C activity in a cell, comprising: contacting a compound of the present invention with a cell; and detecting fluorescence in the cell, thereby detecting phospholipase C activity in the cell.

Another aspect of the present invention is directed to a method of detecting aberrant phospholipase C activity in a cell, comprising: contacting a compound of the present invention with a cell; detecting an amount or pattern of fluorescence in the cell; and comparing the amount or pattern of fluorescence detected in the cell with the amount or pattern of fluorescence in a control cell that has been contacted with the compound, whereby an alteration in the amount or pattern of fluorescence in the cell as compared with the control cell detects aberrant phospholipase C activity in the cell.

The foregoing and other aspects of the present invention will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Brief Description of the Drawings

Fig. 1 A is a schematic showing the chemical structure of XY-69 and, after cleavage by PLC, the structures of the dissociated fluorophore portion and quencher portion. The fluorescein derivative of XY-69 is efficiently quenched by close proximity to a DABCYL moiety attached to the 6-position of the inositol. Cleavage of XY-69 by PLCs dissociates the fluorophore/quencher pair so that the fluorescein portion becomes highly fluorescent _ex/em = 485/520 nm). The long alkyl chain (C₁₅H₃₁) serves to increase the affinity of XY-69 for membranes.

Fig. IB is excitation and emission spectra of XY-69 after hydrolysis by wild type (WT) PLC-δΙ and catalytically inactive PLC-δΙ (E341A).

Fig. 1C is a graph showing effects of concentration of free Ca²⁺ on hydrolysis of XY- 69. Hydrolysis of XY-69 (5 μΜ) by PLC-δΙ (0.023 μΜ) was measured in the presence of the indicated concentrations of free calcium. The reaction progression was monitored by fluorescence k_ex/ _em = 485/520 nm). Plots were representatives of three independent experiments. Each experiment was run in triplicates with error bars as standard deviations.

Fig. 2 is a schematic of a chemical synthesis of XY-69 according to embodiments of the present invention.

Fig. 3A is a graph of real-time fluorescence generated by the hydrolysis of XY-69 in detergent micelles upon incubation with purified PLC isozymes. Individual PLCs, including - 51 (25 ng), -δΐ (E341A) (25 ng), -γΐ (100 pg), and -β3 (50 ng), were added to XY-69 (5 μΜ) in 0.5% (w/v) cholate, and reactions were monitored by fluorescence ( _ex/em = 485/520 nm). Plots were representatives of three independent experiments. Each experiment was run in triplicates. For clarity, the error bars are not shown.

Fig. 3B is a schematic showing the chemical structure of XY-23, a fluorescent PtdIns(4,5)P2 derivative.

Fig. 3C illustrates images of thin layer chromatography gels. XY-23 or -69 were incubated with the indicated lipid metabolizing enzymes prior to thin layer chromatography and visualization of fluorescence. Origins of compound additions marked by arrowheads. Uncleaved XY-69 is not fluorescent.

Fig. 4 is a graph of relative fluorescence versus time for XY-69 in cell lysates. HEK293 cells were transfected with plasmids encoding the indicated forms of PLC-P3 or the corresponding parent vector and lysed after 24 h. The resulting lysates were normalized for total protein before addition of XY-69 and monitored for fluorescence ( _ex/e_m ⁼ 485/520 nm). Expression of PLC-P3 constructs verified by western blot (inset). Plots were representatives of two independent experiments. Each experiment was run in triplicates with error bars as standard deviations. Fig. 5 A illustrates images of liposomes incubated with XY-69 and WH-15 prior to and after centrifugation. Liposomes were loaded with 30% sucrose and incubated with either XY-69 or WH-15 prior to a 1 :1 dilution with 25% sucrose and centrifugation. Mixtures were visualized by transillumination at 312 nm.

Fig. 5B is a graph of reporter percentage for the quantification of XY-69 and WH-15 in the liposome layer or aqueous layer after centrifugation. Layers were separated and adjusted to the same volume prior to the addition of PLC-δΙ to hydrolyze the substrates for quantification by fluorescence. Data represented three independent experiments. Each data point is an average of three replicates in one independent experiment.

Fig. 6A is a graph of relative fluorescence versus time for XY-69 incorporated into lipid vesicles. Indicated PLCs, including -51 (11.4 nM), -δΐ (E341A) (18.9 nM), -γΐ (67.3 nM), and -β3 (66.6 nM), were added to XY-69 (5 μΜ) incorporated into lipid vesicles and fluorescence monitored (λ_εχ = 485/520 nm). Plots were representatives of three independent experiments. Each experiment was run in triplicates with error bars as standard deviations.

Fig. 6B is a graph of relative fluorescence versus time for lipid vesicles containing

XY-69 with PLC-P3 activated by Gaq. As indicated, half the vesicles were incubated with purified G_aq (160 nM) activated with aluminum fluoride before addition of either wild- type PLC- 3 (1.7 nM) or the equivalent amount of a form (PH-C2) that does not respond to G_aq. Samples were subsequently monitored by fluorescence. Plots were representatives of three independent experiments. Each experiment was run in triplicates with error bars as standard deviations.

Fig. 6C is a graph of normalized PLC activity. Hydrolysis of [³H]PIP₂ by PLC- 3 was measured with reconstituted phospholipid vesicles for purified PLC-P3 (1.7 nM) or PLC- β3 (1.7 nM) + G_aq (160 nM). [³H]Inositol phosphates were then isolated and quantified. Data represented three independent experiments. Each data point is an average of two replicates in one independent experiment. Figs. 7A, 7B and 7C are each graphs that show the measurement of endogenous PLC activity with XY-69. HEK293 cells were grown in 6-well culture plates at the density of 150,000 cells/well and transfected with either pcHA-GNAQ (Q209L) (30 ng) + pcHA-lic (270 ng) or the control pcHA-lic vector (300 ng) alone. After 48 h, the cell pellets were collected and used for hydrolysis of XY-69 (5 μΜ). Fig. 7 A is a graph of relative fluorescence versus time. The reaction progression was monitored by fluorescence k_ex/X_em = 485/520 nm). Plots were representatives of three independent experiments. Each experiment was run in triplicates with error bars as standard deviations. Fig. 7B is a graph of relative fluorescence increases. The fluorescence increase in Fig. 7A was calculated and plotted. Fig. 7C is a graph of [³H]inositol phosphates produced by PLCs. After 24 h, the medium was replaced with inositol free DMEM and [ H]myo-inositol (1 μθΐ) was subsequently added. The cells were cultured for another 12 h, treated with LiCl (10 mM) for 1 h, and [³H] inositol phosphates were then isolated and quantified. Data represented three independent experiments. Each data point is an average of two replicates in one independent experiment.

Detailed Description of the Invention

The present invention will now be described more fully hereinafter. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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 present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

As used herein, the transitional phrase "consisting essentially of (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. Thus, the term "consisting essentially of as used herein should not be interpreted as equivalent to "comprising."

The term "about," as used herein when referring to a measurable value, such as an amount or concentration and the like, is meant to refer to variations of up to ± 20% of the specified value, such as, but not limited to, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value, as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and/or individual value therein.

"Moiety" and "group" are used interchangeably herein to refer to a portion of a molecule, typically having a particular functional or structural feature, e.g., a linking group (a portion of a molecule connecting two other portions of the molecule).

"Substituted" as used herein to describe chemical structures, groups, or moieties, refers to the structure, group, or moiety comprising one or more substituents. As used herein, in cases in which a first group is "substituted with" a second group, the second group is attached to the first group whereby a moiety of the first group (typically a hydrogen) is replaced by the second group. The substituted group may contain one or more substituents that may be the same or different.

"Substituent" as used herein references a group that replaces another group in a chemical structure. Typical substituents include nonhydrogen atoms (e.g., halogens), functional groups (such as, but not limited to amino, sulfhydryl, carbonyl, hydroxyl, alkoxy, carboxyl, silyl, silyloxy, phosphate, oxygen and the like), hydrocarbyl groups, and hydrocarbyl groups substituted with one or more heteroatoms. Example substituents include, but are not limited to, alkyl, lower alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclo, heterocycloalkyl, aryl, arylalkyl, lower alkoxy, thioalkyl, hydroxyl, thio, oxo, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, boronyl, and modified lower alkyl.

"Alkyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 30 carbon atoms. In some embodiments, the alkyl group may contain 1, 2, or 3 up to 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-diniethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. "Lower alkyl" as used herein, is a subset of alkyl and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like. In some embodiments, alkyl or loweralkyl may be substituted with one or more groups, optionally one or more groups selected from polyalkylene oxides (such as PEG), halo (e.g., haloalkyl), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(0)_m, haloalkyl-S(0)_m, alkenyl-S(0)_m, alkynyl-S(0)_m, cycloalkyl-S(0)_m, cycloalkylalkyl-S(0)_m, aryl-S(0)_m, arylalkyl-S(0)_m, heterocyclo-S(0)_m, heterocycloalkyl- S(0)_m, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m= 0, 1, 2 or 3.

"Alkenyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 2 to 30 carbon atoms (or in loweralkenyl 2 to 4 carbon atoms) which include 1 to 10 double bonds in the hydrocarbon chain. In some embodiments, the alkenyl group may contain 2 or 3 up to 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. Representative examples of alkenyl include, but are not limited to, methylene (=CH₂), vinyl, 2-propenyl, 3- butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like. In some embodiments, alkenyl or loweralkenyl may be substituted with one or more groups, optionally one or more groups such as those described in connection with alkyl and loweralkyl above. "Alkynyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 2 to 30 carbon atoms (or in loweralkynyl 2 to 4 carbon atoms) which include at least one triple bond in the hydrocarbon chain. In some embodiments, the alkynyl group may contain 2, or 3 up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4- pentynyl, 3-pentynyl, and the like. In some embodiments, alkynyl or loweralkynyl may be substituted with one or more groups, optionally one or more groups as set forth in connection with alkyl and loweralkyl above.

"Aliphatic group" as used herein alone or as part of another group refers to a straight- chain, branched- chain, or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated aliphatic groups, such as an alkyl group, an alkenyl group, and an alkynyl group, as defined herein. The aliphatic group may be unsubstituted or substituted with one or more substituents, which may be the same or different. When substituted at both ends, or utilized as part of a chain or "backbone," such groups may also be known as alkylene, alkenylene, and alkynylene groups. In some embodiments, the aliphatic group may contain 1, 2, or 3 up to 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms.

"Aliphatic oxide group" as used herein refers to an aliphatic group as defined herein substituted with one or more oxygen atoms. The aliphatic oxide group may be unsubstituted or substituted with one or more substituents, which may be the same or different. In some embodiments, the aliphatic oxide group may contain 2 or 3 up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon and oxygen atoms. In some embodiments the aliphatic oxide group may be utilized as part of a chain or "backbone," which may comprise, consist of, or consist essentially of an aliphatic group containing from 1 to 10 carbon atoms, then an oxygen atom, followed by another aliphatic group containing from 1 to 10 carbon atoms, wherein the oxygen atom and aliphatic group may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times and the aliphatic groups may be the same or different.

"Cycloalkyl" as used herein alone or as part of another group, refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in a heterocyclic group as discussed below). Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may be optionally substituted with additional substituents as described herein such as halo or loweralkyl.

"Aromatic group" as used herein alone or as part of another group, refers to aromatic hydrocarbons (i.e., "aryl") and heteroaromatic rings (i.e., "heteroaryl"). The aromatic group may be unsubstituted or substituted with one or more substituents, which may be the same or different. If substituted with one or more substitutents the substituents may be at any location on the aromatic group. For instance, in some embodiments the substitutents are in a 1,2 (ortho) and/or 1,4 (para) configuration. Example aromatic hydrocarbons include, but are not limited to, phenyl, as well as bicyclic (e.g., naphthalene), tricyclic (e.g., phenanthrene, anthracene) or higher aromatic hydrocarbons. Example heteroaromatic rings include, but are not limited to, 2,4-imidazole, -thiazole, and -oxazole and 2,5-pyrrole, -furan, and -thiophene. Also included are fused counterparts, i.e., polycyclic aromatic groups containing a 5- membered heteroaromatic ring.

"Aryl" as used herein alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system or higher having one or more aromatic rings. Representative examples of aryl include, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. In some embodiments, aryl may be substituted with one or more groups, optionally one or more groups such as those described in connection with alkyl and loweralkyl above.

"Arylalkyl" as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2- phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.

"Arylalkenyl" as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkenyl group, as defined herein.

"Arylalkynyl" as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkynyl group, as defined herein.

"Heterocyclic group" or "heterocyclo" as used herein alone or as part of another group, refers to an aliphatic (e.g., fully or partially saturated heterocyclo) or aromatic (e.g., heteroaryl) monocyclic- or bicyclic-ring system. Monocyclic ring systems are exemplified by any 5 or 6 membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. The 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic ring system as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3- benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like. These rings include quaternized derivatives thereof and may be optionally substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(0)_m, haloalkyl-S(0)_m, alkenyl- S(0)_m, alkynyl-S(0)_m, cycloalkyl-S(0)_m, cycloalkylalkyl- S(0)_m, aryl-S(0)_m, arylalkyl- S(0)_m, heterocyclo-S(0)_m, heterocycloalkyl-S(0)_m, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m = 0, 1 , 2 or 3.

"Heteroaryl" as used herein is as described in connection with heterocyclo and aryl above.

"Heteroalkyl" as used herein by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical (e.g., "heterocycloalkyl" or "heteroarylalkyl"), or combinations thereof, comprising an alkyl group, as defined herein, and at least one heteroatom selected from the group consisting of O, , and S, and wherein the nitrogen, carbon and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the alkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive, such as, for example, -CH₂-NH-OCH₃. Examples of heterocycloalkyl include, but are not limited to, l-(l,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

"Alkoxy" as used herein alone or as part of another group, refers to an alkyl or loweralkyl group, as defined herein (and thus includes substituted versions such as polyalkoxy), and is appended to the parent molecular moiety through an oxy group, -0-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.

" Aryloxy" as used herein alone or as part of another group refers to an aryl group, as defined herein (and thus includes substituted versions), and is appended to the parent molecular moiety through an oxy group, -0-.

"Hydroxyalkyl" as used herein alone or as part of another group refers to a hydroxyl group, as defined herein, appended to the parent molecular moiety through an alkyl group as defined herein (and thus includes substituted versions). Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, hydroxyethyl, hydroxypropyl and the like.

"Halo" as used herein refers to any suitable halogen, including F, CI, Br and I.

"Mercapto" as used herein refers to an -SH group.

"Azido" as used herein refers to an -N₃ group.

"Cyano" as used herein refers to a -CN group.

"Formyl" as used herein refers to a -C(0)H group.

"Carboxylic acid" or "carboxy" as used herein alone or as part of another group, refers to a -C(0)OH group.

"Hydroxy" as used herein alone or as part of another group, refers to an -OH group. "Nitro" as used herein refers to an -N0₂ group.

"Oxo" as used herein, refers to a =0 moiety.

"Oxy," as used herein refers to a -O- moiety.

"Thio," as used herein refers to a -S- moiety. "Acyl" as used herein alone or as part of another group refers to a -C(0)R radical, where R is any suitable substituent such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl or other suitable substituent as described herein.

"Alkylthio" as used herein alone or as part of another group, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a thio moiety, as defined herein. Representative examples of alkylthio include, but are not limited, methylthio, ethylthio, tert-butylthio, hexylthio, and the like.

"Amino" as used herein refers to the radical -NH₂.

"Alkylamino" as used herein alone or as part of another group refers to the radical - NHR, where R is an alkyl group.

"Aminoalkyl," as used herein refers to an alkyl group which is further substituted with one or more amino groups.

Unless indicated otherwise, nomenclature used to describe chemical groups or moieties as used herein follow the convention where, reading the name from left to right, the point of attachment to the rest of the molecule is at the right-hand side of the name. For example, the group "(C^ alkoxy)C_1-3 alkyl," is attached to the rest of the molecule at the alkyl end. Further examples include methoxyethyl, where the point of attachment is at the ethyl end, and methylamino, where the point of attachment is at the amine end.

Unless indicated otherwise, where a mono or bivalent group is described by its chemical formula, including one or two terminal bond moieties indicated by it will be understood that the attachment is read from left to right.

Unless otherwise stated, structures depicted herein are meant to include all enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C -enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays. The present invention is directed to fluorogenic reporters that detect phospholipase C (PLC) activity upon cleavage by PLC. The fluorogenic reporters comprise a substrate for PLC covalently coupled to at least one fluorophore and optionally a quencher. In some embodiments, a fluorogenic reporter of the present invention may comprise a fluorophore and a quencher. In some embodiments, a fluorogenic reporter of the present invention may comprise at least two fluorophores that are a fluorescent energy transfer (FRET) pair. FRET is a nonradiative process in which an excited dye donor transfers energy to a dye acceptor in the ground state through long-range dipole-dipole interactions. The incorporation of a pair of FRET dyes into an enzymatic substrate may allow for the simultaneous monitoring of both substrate consumption and product generation and/or may provide tracking of both the substrate and product in cells.

In some embodiments, a fluorogenic reporter of the present invention is a membrane- associated fluorogenic reporter. The fluorogenic reporter may selectively report PLC activity. A fluorogenic reporter may partition into liposomes and/or may be stable in cellular lysates that lack active PLCs. In some embodiments, the fluorogenic reporter may recapture the direct activation of PLC activity by Gaq. In some embodiments, the fluorogenic reporter may simultaneously monitor PIP2, IP3, and/or DAG. In some embodiments, a fluorogenic reporter of the present invention may replace PIP2 (e.g., radiolabeled PIP2) in a PLC assay and be used in the PLC assay. A fluorogenic reporter of the present invention may be used to monitor cellular PLC activity with spatiotemporal resolution and/or in a high-throughput screen, such as, e.g., to identify allosteric inhibitors of PLC isozymes in a high-throughput screen.

A substrate for phospholipase C isozymes, as used herein, refers to a chemical molecule or biological molecule (e.g., a phospholipid or glycolipid) that can be cleaved by phospholipase C. The substrate may be natural, synthetic, or an analog or derivative thereof. An analog or derivative of a natural or synthetic substrate may be a natural or synthetic substrate of phospholipase C with one or more modifications (e.g., one or more atoms, substituents, and/or substructures may be replaced with one or more different atoms, substituents, and/or substructures). Substrates for phospholipase C include, but are not limited to, inositol phosphate, phosphatidylinositol, glycosylphosphatidylinositol, or analogs or derivatives thereof. Specific example substrates include, but are not limited to, inositol 1,4,5-triphosphate, D-myo-inositol 1,4-diphosphate, D-myo-inositol 1,2-cyclic phosphate, inositol phosphate analogues, and glycosylphosphatidylinositol analogues. In some embodiments, the substrate is a mammalian PLC isozyme. In some embodiments, a fluorogenic reporter of the present invention comprises at least one linker group. A linker group may be between the substrate for PLC and a fluorophore and/or between the substrate for PLC and a quencher. In some embodiments, the linker group is designed to minimize the potential perturbation of the PLC active site by a fluorophore and/or quencher. In some embodiments, the linker group may minimize interaction of the PLC active site with a fluorophore and/or quencher.

The linker group may comprise a substituent that provides or enhances the hydrophobic or hydrophilic character of the fluorogenic reporter. In some embodiments, the linker group comprises a substituent, as defined herein, that retains and/or provides the hydrophobic character of the fluorogenic reporter, while still allowing for the fluorogenic reporter to be water soluble. Example substituents that retain the hydrophobic character of the fluorogenic reporter, while still allowing for the fluorogenic reporter to be water soluble include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, or arylalkyl.

In some embodiments, a fluorogenic reporter of the present invention may comprise a long alkyl chain (e.g., C₁₅H₃₁), which, upon cleavage by a PLC isozyme, may allow the cleaved fluorophore to stay at the same location as the fluorogenic reporter to report cellular activity of a PLC isozyme with spatial resolution.

Fluorophores of the present invention are chemical compounds, which when excited by exposure to a particular wavelength of light, emit light (fluoresce), for example, at a different wavelength of light. Also encompassed by the term "fluorophore" are luminescent molecules, which are chemical compounds that do not require exposure to a particular wavelength of light to fluoresce; luminescent compounds naturally fluoresce. In some embodiments, the fluorophore is covalently coupled to the substrate or the linker group. Numerous fluorophores are known in the art and may be utilized in the present invention. Example fluorophores include, but are not limited to, fluoresceins, such as TET (Tetramethyl fluorescein), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxyfluorescein (HEX) and 5-carboxyfluorescein (5-FAM); phycoerythrins; resorufin dyes; coumarin dyes; rhodamine dyes, such as 6-carboxy-X-rhodamine (ROX); cyanine dyes; BODIPY dyes; quinolines; pyrenes; N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); acridine; stilbene; Texas Red; as well as derivatives thereof. In some embodiments, the fluorogenic reporter comprises fluorescein or a derivative thereof. In some embodiments, the fluorogenic reporter comprises TAMRA or a derivative thereof.

Example FRET pairs include, but are not limited to, TAMRA/Cy5, Cy3/Cy5, BODIPY FL/BODIPY FL, TAMRA/QSY7, FAM/TAMRA, TexRed/QSY7, and/or fluorescein/TAMRA. In some embodiments, FRET efficiency may be dependent on the FRET dyes themselves and the distance between dyes within the reporter.

"Quencher" as used herein refers to a chemical or biological compound that can absorb energy emitted by a fluorophore so as to reduce the amount of fluorescence emitted (i.e., quench the emission of the fluorescent label). Different fluorophores are quenched by different quenching agents. Quenchers are either non-fluorescent or fluorescent. Generally, non-fluorescent quenchers are capable of quenching the fluorescence of a wide variety of fluorophores, by absorbing energy from the fluorophore and releasing the energy as heat. Examples of non-fluorescent quenchers include, but are not limited to DABCYL, QSY-7, and QSY-33. Fluorescent quenchers are specific to fluorophores that emit at a specific wavelength range. In general, the spectral properties of a particular fluorophore/quenching agent pair are such that one or more absorption wavelengths of the quencher overlaps one or more of the emission wavelengths of the fluorophore. A preferred fluorophore/quencher pair can be selected by one of skill in the art by comparison of emission and excitation wavelengths. In some embodiments, the quencher, when present, may be covalently coupled to any portion of the fluorogenic reporter, such that it quenches the fluorescence emission of the fluorophore. As one of skill in the art would recognize, various moieties may be utilized to conjugate or couple the quencher to the fluorogenic reporter (e.g., a linker group). This may allow the fluorescent group to stay where the reporter is localized, which is desirable for imaging PLC activity in real-time under both normal cellular environments and external stimulation. In some embodiments, the quencher is separated from the fluorogenic reporter upon PLC cleavage.

Example fluorogenic reporters of the present invention include a compound of Formula I:

wherein:

Ps is a substrate for a phospholipase C isozyme;

A is a first linker, wherein the first linker is optionally substituted with one or more functional groups selected from the group consisting of alkyl, alkenyl, alkynyl, hydroxy, alkoxy, alkylthio, amino, aminoalkyl, alkylamino, heteroaryl, heteroalkyl, aryl, arylalkyl, aliphatic oxide, -C(=0)R^1U, -C(=0)OR^lu, -C(=0)N(R^1U)₂, -(CH₂)_mN(R^lu)₂, -C(=0)N(R^1U)₂, and -(CH₂)_mCOOR¹⁰, where m is 1-20 and R¹⁰ is H, alkyl, alkenyl, or alkynyl;

B is a second linker, wherein the second linker is optionally substituted with one or more functional groups selected from the group consisting of alkyl, alkenyl, alkynyl, hydroxy, alkoxy, alkylthio, amino, aminoalkyl, alkylamino, heteroaryl, heteroalkyl, aryl, arylalkyl, aliphatic oxide, -C(=0)R¹⁰, -C(=0)OR¹⁰, -C(=O)N(R¹⁰)₂, -(CH₂)_mN(R¹⁰)₂, -C(=O)N(R¹⁰)₂, and -(CH₂)_mCOOR¹⁰, where m is 1-20 and R¹⁰ is H, alkyl, alkenyl, or alkynyl;

Fp is a first fluorophore; and

Q is a quencher or a second fluorophore.

In some embodiments, the first and/or second linker may comprise an aliphatic oxide. In some embodiments, the first and/or second linker (e.g., an aliphatic oxide) may comprise a -NH-C(O)- moiety to attach the first and/or second linker to the first fluorophore and/or to the quencher or second fluorophore, respectively. In some embodiments, the second linker comprises -R²-Z-R³-NH-C(0)-, wherein R² and R³ are each independently a Ci-Cio unsubstituted or substituted alkyl or C₂-C₁₀ unsubstituted or substituted alkenyl; and Z is oxygen or sulfur. In some embodiments, the first linker comprises -R⁴-CH(D-R⁵)-R⁶-D-R⁷- NH-C(O)-, wherein R⁴, R⁶, and R⁷ are each independently a Q-Cio unsubstituted or substituted alkyl or C₂-C₁₀ unsubstituted or substituted alkenyl; R⁵ is H, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, alkylthio, amino, aminoalkyl, alkylamino, cycloalkyl, heteroaryl, heteroalkyl, aryl, arylalkyl, aliphatic oxide, -C(=0)R¹⁰, -C(=0)OR¹⁰, -C(=O)N(R¹⁰)₂, -(CH₂)_mN(R¹⁰)₂, -C(=O)N(R¹⁰)₂, and -(CH₂)_mCOOR¹⁰, where m is 1-20 and R¹⁰ is H, alkyl, alkenyl, or alkynyl; and D is each independently selected from oxygen and sulfur.

In some embodiments, a fluorogenic reporter of the present invention (e.g., a compound of Formula I) may further comprise a cellular localization signal and/or a cage group (e.g., a photocage group).

"Cellular localization signal" as used herein refers to a signal that is used to direct the fluorogenic reporter to various distinguishable parts, components or organelles of a cell, including without limitation, the nucleus, cytoplasm, plasma membrane, endoplasmic reticulum, Golgi apparatus, filaments such as actin and tubulin filaments, endosomes, peroxisomes and mitochondria. Various cellular localization signals are known in the art and are commercially available. The cellular localization signal may be an amino acid sequence that can be of any size and composition, for example 3 to 100 amino acids in length, e.g., 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids in length. Cellular localization signals can be made by, for example, recombinant techniques or peptide synthesis. Example cellular localization signals include, but are not limited to, cytosol localization signals, nuclear localization signals, including but not limited to, SV40 virus T- antigen NLS and NLS sequence domains derived from viral Tat proteins, such as HIV Tat, and lysosomal localization signals, including but not limited to, lysosome associated membrane protein 1 (LAMP-1) tail sequence: RKRSHAGYQTI (SEQ ID NO: l); lysosomal, acid phosphatase (LAP): RLKRMQAQPPGYRHVADGEDHAV (SEQ ID NO:2), and lysosomal integral membrane protein 2 (LIMP-2): RGQGSTDEGTADERAPLIRT (SEQ ID NO:3).

The cellular localization signal, when present, may be covalently coupled to any portion of the fluorogenic reporter, while still allowing for the PLC to act upon the substrate and for the fluorescent action of the fluorophore(s). There are different pools of PLCs in the cells that function differently. The cellular localization signal will dictate the location of the fluorogenic reporter and thus reports PLC activity at designated sites and/or organelles. As one of skill in the art would recognize, various moieties may be utilized to conjugate or couple the cellular localization signal to the fluorogenic reporter (e.g., a linker group). In some embodiments the cellular localization signal is covalently coupled to the substrate for PLC (Ps) and/or to the fluorophore.

"Cage group" as used herein refers to a group that prevents the fluorogenic reporter from functioning as a PLC substrate. In some embodiments, the cage group may be a photocage group. The cage group may allow for the fluorogenic reporter to become activated at a certain point in time upon removal or modification of the cage group. In some embodiments, the cage group allows for the fluorogenic reporter to be localized to a certain area of the cell. In some embodiments, the cage group allows for PLC activity to be monitored at set time points. For example, in cancer cells or other diseased cells or tissues where PLC activity is abnormally high, delivering PLC reporters into the cells may lead to the quick metabolism of the reporter. Incorporating one or more cage group(s) into the reporter may prevent it from functioning as a PLC substrate unless and until it is decaged (e.g., photo-decaged) even though the reporter is delivered into the cells. The cage group, when present, may be covalently coupled to any portion of the fluorogenic reporter, such that it prevents the fluorogenic reporter from acting as a substrate for PLC. As one of skill in the art would recognize, various moieties may be utilized to conjugate or couple the cage group to the fluorogenic reporter (e.g., a linker group). In some embodiments the cage group may be covalently coupled to the substrate for PLC (Ps).

In some embodiments, a cage group (e.g., photocage group) may be used to mask one or more negatively charged groups (e.g., phosphates) present in the fluorogenic reporter. The cage group may mask one or more phosphates until all or a portion of the cage group is removed, such as, for example, by light. Then, the remainder of the fluorogenic reporter may be used to monitor cellular activity of PLC isozymes, optionally with spatiotemporal resolution. In some embodiments, one or more phosphates of a fluorogenic reporter of the present invention may be caged as esters. In some embodiments, a cage group (e.g., a photocage group) may be present at 2-OH and/or 4,5-bisphosphates of a corresponding PIP2 structure in a fluorogenic reporter of the present invention. Example photocage groups include, but are not limited to photoactivatable 7-diethylamino coumarin-yl-4-methyl groups and derivatives thereof; coumarin derivatives; and/or ester moieties (e.g., acetoxymethyl (AM) esters). In some embodiments, the photocage may comprise and/or is -CH₂-0-C(0)-CH₃ or -CH₂CH₂CN.

In some embodiments, example fluorogenic reporters include a compound of Formula IA:

wherein:

LA and LB are each independently present or absent, and when present are each independently selected from the group consisting of -C(0)NH-, -C(O)-, -NH-, -0-, -C(=NRⁿ)-, -C(=NOH)-, -C(=0)0-, -OC(=0)-, and -NHC(=0)-, wherein R¹¹ is H, alkyl, alkenyl, or alkynyl; X is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, aliphatic oxide, and -R²-Z-R³-, wherein R² and R³ are each independently selected from the group consisting of a C^C^ unsubstituted or substituted alkyl and a C₂-Ci₀ unsubstituted or substituted alkenyl, and Z is oxygen or sulfur;

Y is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aliphatic oxide, aryl, arylalkyl, and -R⁴-CH(D-R⁵)-R⁶-D-R⁷-, wherein R⁴, R⁶, and R⁷ are each independently a C_\-C_\ unsubstituted or substituted alkyl or C₂-Ci₀ unsubstituted or substituted alkenyl; R⁵ is H, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, alkylthio, amino, aminoalkyl, alkylamino, cycloalkyl, heteroaryl, heteroalkyl, aryl, arylalkyl, aliphatic oxide, -C(=0)R¹⁰, -C(=0)OR¹⁰, -(CH₂)_mN(R¹⁰)₂, -C(=O)N(R¹⁰)₂, and - (CH₂)_mCOOR¹⁰, where m is 1-20 and R¹⁰ is H, alkyl, alkenyl, or alkynyl; and D is each independently selected from oxygen and sulfur;

Fp is a first fluorophore; and

Q is a quencher or a second fluorophore.

The quencher or second fluorophore in a compound of Formula IA may be attached to the cyclohexane ring at any suitable location. In some embodiments, the quencher may be attached at the 6-position of the cyclohexane. In some embodiments, X and/or Y may be substituted with a cellular localization signal. In some embodiments, R¹ (optionally R¹ at the 5-position of the inositol) may be a quencher or R¹ may comprise a quencher (e.g., may be an alkyl, alkenyl, alkynyl, aryl, or arylalkyl substituted with a quencher).

In some embodiments, R¹, X, and/or Y may be substituted with one or more substituents selected from alkyl, lower alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclo, heterocycloalkyl, aryl, arylalkyl, lower alkoxy, thioalkyl, hydroxyl, thio, oxo, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, boronyl, and/or modified lower alkyl.

In some embodiments, X is -R²-Z-R³- with R² and R³ each independently selected from a Ci-Cio unsubstituted or substituted alkyl and a C₂-C₁₀ unsubstituted or substituted alkenyl, and Z is oxygen. In some embodiments, R² and R³ are each independently selected from a C₁-C5 unsubstituted alkyl, and Z is oxygen.

In some embodiments, Y is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aliphatic oxide, and -R⁴-CH(D-R⁵)-R⁶-D-R⁷-. In some embodiments, Y is -R⁴-CH(D-R⁵)-R⁶-D-R⁷- with R⁴, R⁶, and R⁷ each independently selected from a Q-C₁₀ unsubstituted or substituted alkyl or C₂-C₁₀ unsubstituted or substituted alkenyl; R⁵ is a Cio-Qo alkyl, Cio-C^ alkenyl, C₁₀-C₃₀ alkynyl, -C(=0)R^1U, -C(=0)OR^lu, -C(=0)N(R^1U)₂, -(CH₂)_mN(R^lu)₂, -C(=0)N(R^1U)₂, or -(CH₂)_mCOOR¹⁰, where m is 1-20 and R¹⁰ is a Ci₀-C₃₀ alkyl, C₁₀-C₃₀ alkenyl, or C₁₀-C₃₀ alkynyl; and each D is oxygen. In some embodiments, R⁴, R⁶, and R⁷ are each independently selected from a C_\-C& unsubstituted or substituted alkyl, R⁵ is -C(=0)R¹⁰, R¹⁰ is a C₁₀-C3₀ alkyl, and each D is oxygen. In some embodiments, R⁷ is a Cj-C₈ substituted alkyl, optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclo, heterocycloalkyl, aryl, arylalkyl, alkoxy, thioalkyl, hydroxyl, thio, oxo, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl. In some embodiments, R⁷ is -C(0)-(CH₂)_1-7.

Additional example fluorogenic reporters include a compound of Formula II:

wherein:

X is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, aliphatic oxide, and -R²-Z-R³-, wherein R² and R³ are each independently selected from the group consisting of a Ct-Cio unsubstituted or substituted alkyl and a C₂-C₁₀ unsubstituted or substituted alkenyl, and Z is oxygen or sulfur;

Y is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aliphatic oxide, aryl, arylalkyl, and -R⁴-CH(D-R⁵)-R⁶-D-R⁷-, wherein R⁴, R⁶, and R⁷ are each independently a C_\-C_\ unsubstituted or substituted alkyl or C₂-C₁₀ unsubstituted or substituted alkenyl; R⁵ is H, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, alkylthio, amino, aminoalkyl, alkylamino, cycloalkyl, heteroaryl, heteroalkyl, aryl, arylalkyl, aliphatic oxide, -C(=0)R^1U, -C(=0)OR^lu, -C(=0)N(R^IU)₂, -(CH₂)_mN(R^iU)₂, -C(=0)N(R^,u)₂, and -(CH₂)_mCOOR¹⁰, where m is 1-20 and R¹⁰ is H, alkyl, alkenyl, or alkynyl; and D is each independently selected from oxygen and sulfur;

Fp is a first fluorophore; and

Q is a quencher or a second fluorophore.

Accordingly, in a compound of Formula II, the quencher or second fluorophore may be attached to the cyclohexane ring at any suitable location. In some embodiments, the quencher may be attached at the 6-position of the cyclohexane. In some embodiments, the carbonyl (-C(O)-) attaching the linker to the first fluorophore may be part of the first fluorophore and/or the carbonyl (-C(O)-) attaching the linker to the quencher or second fluorophore may be part of the second fluorophore or quencher. In some embodiments, X and/or Y may be substituted with a cellular localization signal. In some embodiments, R¹ (optionally R¹ at the 5-position of the inositol) may be a quencher or R¹ may comprise a quencher (e.g., may be an alkyl, alkenyl, alkynyl, aryl, or arylalkyl substituted with a quencher).

In some embodiments, X is -R²-Z-R³- with R² and R³ each independently selected from a Ci-C₁₀ unsubstituted or substituted alkyl and a C₂-Ci₀ unsubstituted or substituted alkenyl, and Z is oxygen. In some embodiments, R² and R³ are each independently selected from a Q-C5 unsubstituted alkyl, and Z is oxygen.

In some embodiments, Y is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aliphatic oxide, and -R⁴-CH(D-R⁵)-R⁶-D-R⁷-. In some embodiments, Y is -R⁴-CH(D-R⁵)-R⁶-D-R⁷- with R⁴, R⁶, and R⁷ each independently selected from a Ci-Ci₀ unsubstituted or substituted alkyl or C₂-Cio unsubstituted or substituted alkenyl; R⁵ is a C₁₀-C₃o alkyl, C₁₀-C₃₀ alkenyl, C₁₀-C₃₀ alkynyl, -C(=0)R¹⁰, -C(=0)OR¹⁰, -C(=O)N(R¹⁰)₂, -(CH₂)_mN(R¹⁰)₂, -C(=O)N(R¹⁰)₂, or -(CH₂)_mCOOR¹⁰, where m is 1-20 and R¹⁰ is a C₁₀-C₃₀ alkyl, C₁₀-C₃₀ alkenyl, or C₁₀-C₃₀ alkynyl; and each D is oxygen. In some embodiments, R⁴, R⁶, and R⁷ are each independently selected from a Cj-C₈ unsubstituted or substituted alkyl, R⁵ is -C(=0)R¹⁰, R¹⁰ is a C₁₀-C₃₀ alkyl, and each D is oxygen. In some embodiments, R⁷ is a Q-Cg substituted alkyl, optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclo, heterocycloalkyl, aryl, arylalkyl, alkoxy, thioalkyl, hydroxyl, thio, oxo, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl. In some embodiments, R⁷ is -C(0)-(CH₂)_1-7.

In some embodiments, a fluorogenic reporter of the present invention has a structure represented b Formula IIA:

In some embodiments, a fluorogenic reporter of the present invention has a structure represented by Formula IIB:

In some embodiments, a fluorogenic reporter of the present invention has a structure represented by Formula IIC:

wherein: G is a cage group (e.g., a photocage group), optionally wherein two adjacent G together form the cage group.

Other example fluorogenic reporters include, but are not limited to, a compound of Formula III:

wherein:

R > 1 , R> 3 , R , R , R°, R , Z, D, Q, and Fp are each as defined above.

In some embodiments, one or more R¹ in a compound of Formula III may be as provided in one of Formulas IIA, IIB, or IIC.

Further example fluorogenic reporters include, but are not limited to, a compound of Formula IV:

wherein R¹, Q, and Fp are each as defined above.

In some embodiments, one or more R¹ in a compound of Formula IV may be as provided in one of Formulas IIA, IIB, or IIC.

In some embodiments, a fluorogenic reporter of the present invention is a compound of Formula V, which has a structure represented by:

The compound of Formula V and/or Formula V may not be fluorescent when excited at 480 nm and monitored at 520 nm, but once cleaved by PLC isozymes, the quencher may be released and result in fluorescence at 520 nm, which may reflect lipase activity (Fig.

I)-

In some embodiments, a fluorogenic reporter of the present invention is a compound of Formula VI, which has a structure represented by:

wherein AM is an acetoxymethyl group (i.e., -CH₂-0-C(0)-CH₃)

The compound of Formula VI may be taken into cells, but inert until irradiated with light. Once uncaged, the compound of Formula VI may remain non-fluorescent until it is hydrolyzed by a PLC. Upon hydrolysis, the TAMRA-containing diacylglycerol derivative may be liberated from the QSY7-modified inositol ring and the TAMRA may no longer be quenched by close proximity to QSY7. Fluorescence (λβχ/em = 546/579 nm) from the TAMRA derivative may accurately and/or robustly report the activation of PLCs with high spatiotemporal resolution. In some embodiments, a fluorogenic reporter of the present invention is a compound of Formula VII, which has a structure represented by:

The compound of Formula VII may have excellent FRET efficiency at long wavelengths of light. When illuminated at 546 nm, TAMRA may be excited and energy transfered to the acceptor Cy5 to result in emission at 666 nm. Once cleaved, FRET may be disrupted and emission of TAMRA at 579 nm may be detected when excited at 546 nm. When excited at 649 nm, the Cy5 dye may absorb photons with emission at 666 nm.

In some embodiments, a fluorogenic reporter of the present invention when in contact with PLC will be cleaved by PLC to generate in tandem reaction products of the cleavage, such as, for example, a quencher portion (i.e., a portion of the fluorogenic reporter comprising the quencher) and a fluorophore portion (i.e., a portion of the fluorogenic reporter comprising the fluorophore). The reaction products of the PLC cleavage may comprise the substrate for PLC, the fluorophore (first and/or second fluorophore), and/or quencher. In some embodiments, another reaction product may be a cellular localization signal. For example, PLC may cleave a compound of Formula V as shown in Fig. 1A to provide a quencher portion and a fluorophore portion. In some embodiments, a fluorogenic reporter of the present invention does not include a sensor described in U.S. Patent No. 8,703,437.

A fluorogenic reporter of the present invention may be used to identify an inhibitor and/or activator of PLC. According to some embodiments of the present invention provided is a method for identifying a test substance that inhibits phospholipase C activity, the method comprising: contacting a fluorogenic reporter of the present invention (e.g., a compound of any of Formulas I- VII) with phospholipase C in the presence and absence of a test substance; detecting the amount of fluorescence in the presence and absence of the test substance; and comparing the amount of fluorescence detected in the presence and absence of the test substance, whereby a decrease in the amount of fluorescence detected in the presence of the test substance identifies that the test substance inhibits phospholipase C activity. In some embodiments, the method comprises (a) contacting a fluorogenic reporter of the present invention with phospholipase C in the presence of a test substance, under conditions whereby fluorescence resulting from reaction of the fluorogenic reporter and phospholipase C can be detected, and detecting the amount of fluorescence; (b) contacting the fluorogenic reporter of step (a) with the phospholipase C of step (a) in the absence of the test substance, under conditions whereby fluorescence resulting from reaction of the fluorogenic reporter and phospholipase C can be detected, and detecting the amount of fluorescence; (c) comparing the amount of fluorescence detected in step (a) with the amount of fluorescence detected in step (b), whereby a decrease in the amount of fluorescence detected in step (a) identifies that the test substance inhibits phospholipase C activity.

In some embodiments of the present invention, provided is a method for identifying a test substance that activates phospholipase C activity, the method comprising: contacting a fluorogenic reporter of the present invention (e.g., a compound of any of Formulas I- VII) with phospholipase C in the presence and absence of a test substance; detecting the amount of fluorescence in the presence and absence of the test substance; and comparing the amount of fluorescence detected in the presence and absence of the test substance, whereby an increase in the amount of fluorescence detected in the presence of the test substance identifies that the test substance activates phospholipase C activity. In some embodiments, the method comprises (a) contacting a fluorogenic reporter of the present invention with phospholipase C in the presence of a test substance, under conditions whereby fluorescence resulting from reaction of the fluorogenic reporter and phospholipase C can be detected, and detecting the amount of fluorescence; (b) contacting the fluorogenic reporter of step (a) with the phospholipase C of step (a) in the absence of the test substance, under conditions whereby fluorescence resulting from reaction of the fluorogenic reporter and phospholipase C can be detected, and detecting the amount of fluorescence; (c) comparing the amount of fluorescence detected in step (a) with the amount of fluorescence detected in step (b), whereby an increase in the amount of fluorescence detected in step (a) identifies that the test substance activates phospholipase C activity.

The test substance may be any chemical or biological compound. The test substance may be natural or synthetic. The test substance can vary in size from small organic molecules to peptides or large proteins. In some embodiments, the test compound is a small molecule. Protocols for the production, selection and testing of small molecules for their inhibitory and/or activating effects are routine and well known in the art and can be readily adapted to the methods of this invention by one of skill in the art. The present invention further provides a method of screening small molecule libraries to identify a small molecule that inhibits and/or activates PLC activity and/or function. Small molecule libraries can be obtained from various commercial entities, for example, SPECS and BioSPEC B.V. (Rijswijk, the Netherlands), Chembridge Corporation (San Diego, CA), Comgenex USA Inc., (Princeton, NJ), Maybridge Chemical Ltd. (Cornwall, UK), and Asinex (Moscow, Russia).

One representative example is known as DIVERSet™, available from ChemBridge

Corporation, 16981 Via Tazon, Suite G, San Diego, Calif. 92127. DIVERSet™ contains between 10,000 and 50,000 drug-like, hand-synthesized small molecules. The compounds are pre-selected to form a "universal" library that covers the maximum pharmacophore diversity with the minimum number of compounds and is suitable for either high throughput or lower throughput screening. For descriptions of additional libraries, see, for example, Tan et al. "Stereoselective Synthesis of Over Two Million Compounds Having Structural Features Both Reminiscent of Natural Products and Compatible with Miniaturized Cell-Based Assays" J. Am. Chem Soc. 120, 8565-8566, 1998; Floyd et al. Prog Med Chem 36:91-168, 1999. Numerous libraries are commercially available, e.g., from AnalytiCon USA Inc., P.O. Box 5926, Kingwood, Tex. 77325; 3-Dimensional Pharmaceuticals, Inc., 665 Stockton Drive, Suite 104, Exton, Pa. 19341-1151; Tripos, Inc., 1699 Hanley Rd., St. Louis, Mo., 63144- 2913, etc. In certain embodiments of the invention the screening methods are performed in a high-throughput format using techniques that are well known in the art, e.g., in multiwell plates, using robotics for sample preparation and dispensing, etc. Representative examples of various screening methods may be found, for example, in U.S. Pat. Nos. 5,985,829, 5,726,025, 5,972,621, and 6,015,692. The skilled practitioner will readily be able to modify and adapt these methods as appropriate. In some embodiments the small molecule has a molecular weight of more than about 10 Daltons and less than about 5,000 Daltons, of more than about 40 Daltons and less than about 3,000 Daltons, or of more than about 100 Daltons and less than about 2,500 Daltons. Example small molecules include, but are not limited to, peptides, peptoids, proteins, nucleotides, oligonucleotides, oligosaccharides, pharmaceuticals, sugars, fatty acids, steroids, derivatives, structural analogs, or combinations thereof.

In some embodiments, a fluorogenic reporter of the present invention may be used to detect PLC activity in a cell. In some embodiments, a method of detecting phospholipase C activity in a cell may comprise: (a) contacting a fluorogenic reporter of the present invention {e.g., a compound of any of Formulas I- VII) with a cell; and (b) detecting fluorescence in the cell, thereby detecting phospholipase C activity in the cell. Contacting the fluorogenic reporter with the cell may be carried out under conditions whereby fluorescence resulting from reaction of the fluorogenic reporter and phospholipase C can be detected.

The cell may be any cell in which the absence, presence, or amount of PLC activity is desired to be determined. Such cells can be from any source, such as, but not limited to, mammalian and bacterial sources. Generally, the cells are from mammalian subjects, such as but not limited to, human subjects, dogs, cats, horses, cows, sheep, monkeys, and chimpanzees.

According to some embodiments, a method of detecting aberrant PLC activity in a cell may be provided. A method of detecting aberrant phospholipase C activity in a cell may comprise: (a) contacting a fluorogenic reporter of the present invention {e.g., a compound of any of Formulas I- VII) with a cell; (b) detecting an amount or pattern of fluorescence in the cell; and (c) comparing the amount or pattern of fluorescence detected in step (b) with the amount or pattern of fluorescence in a control {e.g., normal) cell that has been contacted with the fluorogenic reporter of step (a), whereby an alteration in the amount or pattern of fluorescence in the cell as compared with the control cell detects aberrant phospholipase C activity in the cell. Contacting the fluorogenic reporter with the cell may be carried out under conditions whereby fluorescence resulting from reaction of the fluorogenic reporter and phospholipase C can be detected.

The cell may be any cell as described above. In some embodiments, the cell is a diseased cell or a cell from a subject known to have or suspected of having a disease. In some embodiments, the cell is from a subject at risk of having a disease. In some embodiments, the disease is a disease in which aberrant regulation of PLCs has been implicated. The disease may be caused by aberrant regulation of PLCs, correlated with aberrant regulation of PLCs, associated with aberrant regulation of PLCs, or linked to aberrant regulation of PLCs. In some embodiments, aberrant regulation of PLCs may be suspected to be involved with the disease or to contribute to the disease. Example diseases include, but are not limited to, cancer such as, but not limited to, leukemia, prostate cancer, colorectal cancer, and breast cancer; neurodegenerative disease such as, but not limited to, Alzheimer's disease, Pick's disease, progressive supranuclear palsy, and diffuse Lewy body disease; ischemia; neuropathic pain; Down Syndrome; cardiovascular diseases such as, but not limited to, Tangier disease; and bone diseases. Example cells include, but are not limited to, tumor cells, brain cells, nerve cells, glial cells, endothelial cells, myocardial cells, osteoblasts, and/or stem cells (including, but not limited to, embryonic and/or adult stem cells).

When detecting aberrant PLC activity in a cell (e.g., a diseased cell or a cell from a subject suspected or at risk of having or known to have a disease), the alteration in the amount or pattern of fluorescence may be an increase in the amount of fluorescence in the cell as compared with the control cell. Alternatively, the alteration in the amount or pattern of fluorescence may be a decrease in the amount of fluorescence in the cell as compared to the control cell.

In some embodiments, a fluorogenic reporter of the present invention may be used as a diagnostic tool for various PLC-related diseases, such as, but not limited to, cancer. As discussed above, in some embodiments, the disease is a disease in which aberrant regulation of PLCs has been implicated. The disease may be caused by aberrant regulation of PLCs, correlated with aberrant regulation of PLCs, associated with aberrant regulation of PLCs, and/or linked to aberrant regulation of PLCs. In some embodiments, aberrant regulation of PLCs may be suspected to be involved with the disease or to contribute to the disease. Additionally, the fluorogenic reporter may be used to detect a PLC-related disease, to monitor the treatment of a PLC-related disease, to monitor the progression of a PLC-related disease, and/or to diagnose a PLC-related disease. In some embodiments, the fluorogenic reporter may also be used to determine if a subject has an increased or decreased risk of having a PLC- related disease.

As one of skill in the art would recognize, if the fluorogenic reporter comprises a cage group (e.g., a photocage group), then any method utilizing the fluorogenic reporter may further comprise releasing or modifying the cage group to allow for the fluorogenic reporter to function as a PLC substrate. The cage group may be released or modified by any method known in the art, such as but not limited to, cleavage. Example types of cleavage include, but are not limited to enzymatic cleavage by enzymes such as peptidases, proteases, nucleases, lipases, or sequence specific restriction enzymes; chemical cleavage by a chemical agent that may cause the cage group to dissociate, hydrolyze, or cleave when contacted with the chemical agent; cleavage by environmental cues, such as, for example, changes in temperature, pH, salt concentration, when there is such a change in environment following endocytosis, or by being exposed to energy, such as light, microwave, ultrasound, and radiofrequency.

A method of the present invention may further comprise activating phospholipase C in the cell with a phospholipase C activator. Any chemical or biological agent or compound that activates PLC may be used. .Many different chemical and biological agents or compounds activate PLC activity, but many induce different physiological responses. Accordingly, a specific PLC activator may be used depending on the method or the physiological response of interest. Example PLC activators include, but are not limited to, neurotransmitters, growth hormones, drugs that activate membrane receptors, and growth factors. In some embodiments, the cells are stimulated with a neurotransmitter, a growth hormone, a growth factor, a membrane receptor agonist, or any combination thereof.

A fluorogenic reporter of the present invention may be directly contacted with PLC or vice versa. The fluorogenic reporters may be delivered to cells either in vivo or in vitro by any method known in the art. Example methods for delivering the fluorogenic reporters to cells include, but are not limited to, microinjection, carrier protein histones, and/or protection of the phosphoric acids as their esters.

In some embodiments, the fluorescence reaction of a fluorogenic reporter upon PLC cleavage may have a signal-to-background (S/B) ratio of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and/or a Z' factor of at least about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.

In some embodiments, the fluorogenic reporter may be non-fluorescent until cleaved by PLC to yield a fluorescent product. The fluorophore of the fluorogenic reporter may be quenched prior to PLC cleavage. Quenching of the fluorophore may be by the quencher and/or another compound that is present in the reaction mixture (e.g., water) and later removed or modified to allow for the fluorophore to fluoresce. In some embodiments, the quencher may be released or modified by any method known in the art, such as but not limited to cleavage, as described above.

In some embodiments, prior to PLC cleavage, the fluorophore of the fluorogenic reporter may have an emission maximum that is different from the emission maximum of the fluorophore after PLC cleavage. Thus, the unconjugated fluorophore may experience a shift in wavelength compared to the conjugated fluorophore. This shift may be a red shift or blue shift. The shift in wavelength may be at least about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more nanometers. The present invention is explained in greater detail in the following non-limiting examples.

Examples

Example 1:

Design and synthesis of XY-69

We developed XY-69 to robustly monitor PLC activity at membranes (Fig. 1A). The design of XY-69 relies on the fact that the 6-hydroxyl position of PIP₂ remains solvent exposed within the active site of PLCs such that modifications at this site minimally compromise the capacity of PIP₂ derivatives to function as PLC substrates. Therefore, to create XY-69, fluorescein was introduced at the sn-l position of PIP₂ while 4- (dimethylaminoazo)benzene-4-carboxylic acid (DABCYL) was introduced at the 6-hydroxyl position. While not wishing to be bound to any particular theory, this arrangement may effectively quench the intrinsic fluorescence of fluorescein by the DABCYL moiety. However, once XY-69 is cleaved by PLCs, the fluorophore/quencher pair will be separated resulting in a dramatic increase in the quantum yield of the fluorescein group. XY-69 was also designed to retain a long alkyl chain (C₁₅H₃i) at the sn-2 position in order to favor the partitioning of XY-69 into lipid membranes. Therefore, much like diacylglycerol, the fluorescent product of XY-69 hydrolysis is expected to remain in lipid membranes.

The synthesis of XY-69 (Fig. 2) started with the inositol intermediate 3 that we previously developed. Olefin metathesis of 3 with the terminal alkene N-fert-butoxycarbonyl- 2-allyloxyethanolamine 4 in the presence of Hoveyda-Grubbs catalyst followed by hydrogenation produced 5. The 4- and 5-hydroxyl groups in 5 were phosphorylated through reactions with dibenzyl diisopropylphosphoramidite followed by oxidation with meta- chloroperoxybenzoic acid (mCPBA). The resulting phosphate ester was treated with tetrabutylammonium fluoride (TBAF) to remove the terf-butyldiphenylsilyl (TBDPS) protective group. Phosphorylation of 6 with compound 7, which was prepared according to literature protocols, followed by oxidation by t-BuOOH, generated the coupled intermediate 8 in 85% yield. Removal of the benzyloxycarbonyl (Cbz) protective group through hydro genolysis provided the corresponding primary amine, which was subsequently acylated with the fluorescein derivative 9. The resulting compound was treated with trimethylsilyl bromide (TMSBr) to remove the methoxymethyl (MOM) and benzyl (Bn) protective groups. The tert-butoxycarbonyl (Boc) protective group was also removed during this process to produce 10, which was subsequently reacted with 11 to generate XY-69 in 80% yield.

XY-69 is a PLC-selective reporter

XY-69 was initially incubated with purified PLC-δΙ at 37°C for 1 hour and the fluorescence excitation and emission spectra of the reaction mixture were recorded (Fig. IB). Excitation and emission maxima were 490nm and 518nm, respectively. These maxima are similar to the equivalent numbers for isolated fluorescein (490nm and 525nm). Liquid chromatography-mass spectrometry (LC-MS) analysis of the assay mixture was also carried out and confirmed the formation of the expected product 1. In subsequent experiments, cleavage of XY-69 was detected at 520nm after excitation at 485nm.

PLCs require a Ca cofactor to stabilize the transition state during the hydrolysis of PIP₂. We thus investigated the effect of Ca²⁺ concentration on XY-69 hydrolysis (Fig. 1C). Increasing the concentration of free Ca²⁺ led to increased rates of hydrolysis of XY-69 by PLC-δΙ, and this behavior mirrored earlier work with WH-15, Conversely, without free Ca , there was essentially no hydrolysis of XY-69. Consequently, amounts of free Ca were tightly controlled throughout this work to ensure efficient enzymatic reactions. To demonstrate that XY-69 can be used to monitor PLC activity, the real-time fluorescence of detergent-solubilized XY-69 incubated with purified PLC-δΙ was recorded (Fig. 3A).i Purified PLC-δΙ harboring a single mutation (E341A) within its active site has undetectable lipase activity and was used in a parallel reaction. XY-69 generated approximately a 20-fold increase in fluorescence with PLC-δΙ relative to an identically treated sample containing PLC-δΙ (E341A). While not wishing to be bound to any particular theory, these results suggest that the fluorescence increase arises from the enzymatic action of PLC-δΙ on XY-69.

To further test whether XY-69 functions as a substrate for other PLC isoforms, the reporter was incubated with either purified PLC-P3 or -γΐ in reactions analogous to that described for PLC-δΙ . As shown in Fig. 3 A, XY-69 is hydro lyzed with similar kinetics by each of the PLC isozymes. The catalytic domains of PLC isozymes are highly conserved and consequently, XY-69 is likely a general PLC substrate.

We also tested whether other lipid-metabolizing enzymes that utilize PIP₂ as a substrate could generate fluorescent signals from XY-69. For these tests, we also produced XY-23 (Fig. 3B), which contains a fluorescein moiety but not the DABCYL quencher of XY- 69. Consequently, XY-23 is intrinsically fluorescent and enzymatic transformations of XY-23 should invariably generate new fluorescent products. In this respect, XY-23 is highly similar to PIP₂ and is expected to be a substrate for isozymes of phospho lipase D (PLD), phospholipase A (PLA) and phosphoinositide 3-kinase (PI3K), as well as PLCs. Both XY-23 and XY-69 were subjected to enzymatic reactions with PLCs (-51, -γΐ and -β3), PLD1, PLA2 and PBK . The reaction mixtures were then separated by thin layer chromatography (TLC) and visualized by fluorescence. As shown in Fig. 3C, for XY-23, each lipid-metabolizing enzyme produced a new fluorescent product. In contrast, only the PLCs were capable of hydrolyzing XY-69, attesting to the PLC-selective nature of this fluorogenic reporter.

To assess potential non-specific cleavage of XY-69 by the entire repertoire of cellular phosphodiesterases, the stability of XY-69 was evaluated in lysates derived from HEK293 cells (Fig. 4). Cells transfected with plasmid encoding either wild-type PLC-P3, the catalytically inactive mutant PLC^3 (H323A), constitutively active PLC-p3 (ΔΧΥ) lacking the autoinhibitory XY linker, or the parent vector were lysed 24 h after transfection; the resulting lysates were normalized for total protein and tested for capacity to hydrolyze XY-69 (Fig. 4).

Importantly, lysates from cells transfected with either the parent vector or vector encoding catalytically inactive PLC-P3 (H323A) exhibited minimal capacity to hydrolyze XY-69 as evidenced by minimal increases in fluorescence. In contrast, XY-69 was efficiently hydrolyzed by lysate containing wild-type PLC-P3, and this rate increased further for lysate containing constitutively active PLC-P3 (ΔΧΥ). These results confirm: (i) the expected low basal activities of PLCs prior to upstream stimulation; (ii) the lack of nonspecific hydrolysis of XY-69 by the cellular milieu; and (iii) the capacity of XY-69 to monitor the intrinsic regulation of PLCs. Taken together, these results unequivocally demonstrate that XY-69 is a sensitive and selective reporter of the lipase activity of PLCs in complex cell lysates. The fact that the fluorescence window is as large as 20-fold highlights the potential application of XY- 69 in quantifying PLC activity in different cell lines.

Membrane-association and Gaq activation of XY-69

PIP₂ is a membrane-associated phospholipid and PLCs catalyze the hydrolysis of PIP₂ at the lipid-aqueous interface. Therefore, XY-69 was assessed for capacity to incorporate into membranes used to assay PLC activity. We originally attempted to use a variety of polybasic carriers including histones and polyamines and liposomes to deliver XY-69 or XY-23 into cells with the expectation that these PIP₂ analogs would partition into cellular membranes. However, results were inconsistent with low efficiencies of delivery and high sequestration into endosomal compartments. Instead, we used an in vitro membrane association assay originally developed to measure protein-lipid interactions. In this format, liposomes incubated with either XY-69 or WH-15 were recovered after centrifugation in a sucrose gradient (Fig. 5A). Quantification of the top (lipid) and bottom sucrose layers (Fig. 5B) indicated that approximately 90% of XY-69 incorporated into the lipid fraction. In contrast, WH-15 preferentially (-60%) partitioned into the aqueous fraction. Therefore, XY-69 efficiently partitions into lipid vesicles that mimic cellular membranes.

XY-69 was subsequently reconstituted into lipid vesicles and used to monitor the phospholipase activities of purified PLCs (Fig. 6A). Under these conditions, XY-69 was hydrolyzed with similar kinetics by PLC-δΙ, -β3, and -γΐ. In contrast, catalytically inactive PLC-δΙ (E341A) was unable to hydrolyze XY-69.

We previously showed that the soluble, fluorogenic substrate WH-15 cannot be used to monitor the activation of PLC-β isozymes by membrane-resident G_aq. In contrast, XY-69 reconstituted into lipid vesicles with G_aq readily reports this activation (Fig. 6B). G_aq increased the initial rate of XY-69 hydrolysis by PLC-P3 approximately 12-fold, consistent with measurements when [³H]PIP₂ was used as the substrate (Fig. 6C). For comparison, PLC- 3 (PH-C2), which is not responsive to G_aq in conventional assays, is also unresponsive to G_aq in this assay.

Finally, to demonstrate that XY-69 has the capacity to report activity of endogenous PLCs, HEK293 cells transfected with plasmid encoding G_aq (Q209L) or the parent vector were lysed 48 h after transfection with assay buffer containing XY-69. As expected, cells transfected with the parent vector showed essentially no increase in fluorescence associated with the hydrolysis of XY-69 (Figs. 7A and 7B). In contrast, cells transfected with G_aq (Q209L) showed an approximate 10-fold increase in fluorescence related to the robust hydrolysis of XY-69 (Figs. 7 A and 7B). These results are consistent with the equivalent comparison using conventional labeling of cells with [³H]inositol followed by radioactivity counting of [³H]inositol phosphates produced by PLCs (Fig. 7C). Taken together, these results demonstrate that XY-69 successfully mimics PIP₂ to report the regulation of PLCs operating at membranes. Consequently, XY-69 is able to replace [³H]PIP₂ used in conventional assays used to measure the phospholipase activity of PLCs.

In summary, we have developed a membrane-bound, fluorogenic reporter, XY-69, that monitors the lipase activity of PLC isozymes in real-time with high sensitivity. The reporter works with both detergent micelles and lipid vesicles and can be used with either purified PLCs or cellular lysates. Furthermore, XY-69 captures the activation of PLC activity through intrinsic regulation or activation by G_aq, which requires the presence of membranes. Consequently, XY-69 is suitable to replace radioactive PIP₂ that is used in the canonical enzymatic assay of PLC activity, with the advantages of continuous monitoring, high- throughput and avoidance of using radioactive materials. These features enable XY-69 to be used for development of isozyme-selective inhibitors of PLCs. Given the general significance of PLC isozymes, isozyme-selective inhibitors will likely find applications as novel chemical probes or therapeutics for PLC-associated diseases. Finally, other derivatives of phosphoinsositides such as PIP₃ and PI3P have been delivered into cells through caging negatively charged phosphates to produce the corresponding charge-neutral esters. Accordingly, when a suitable delivery method is available, XY-69 could be used to monitor PLC activity in live cells with high spatiotemporal resolution.

Experimental procedures

Synthetic protocols of XY-69

The second generation Hoveyda-Grubbs catalyst (57 mg, 0.091 mmol) was added to a solution of 3 of Fig. 2 (500 mg, 0.91 mmol) and N-tert-Butoxycarbonyl-2- allyloxyethanolamine (1.1 g, 5.5 mol) in CH₂C1₂ (46 mL), and the reaction mixture was stirred at room temperature. After 2 h, a second portion of the catalyst (57 mg, 0.091 mmol) was added. The solvents were removed after 24 h since the reaction was initiated, and the residue was purified by column chromatography (hexane: acetone = 7:3) over silica. The product thus obtained was contaminated by a brown color and further purified by stirring with activated carbon in MeOH for 12 h. The carbon was removed by filtration and the solvents were removed under vacuum to yield coupling product (394 mg, 0.55 mmol, 60%) as colorless oil. Ή NMR (400 MHz, CDC1₃) δ 7.78-7.68 (m, 4H), 7.42-7.30 (m, 6H), 5.78- 5.62 (m, 2H), 4.91 (br. s, 1H), 4.60 (d, J = 6.5 Hz, 1H), 4.56 (d, J = 6.5 Hz, 1H), 4.48-4.42 (m, 2H), 4.35-4.28 (m, 2H), 3.97 (d, J = 4.3 Hz, 2H), 3.80-3.73 (m, 2H), 3.71 (t, J = 9.0 Hz, 1H), 3.48 (t, J = 5.1 Hz, 1H), 3.36-3.25 (m, 7H), 3.23 (s, 3H), 3.09 (dd, J = 9.9, 2.2 Hz, 1H), 2.78 (br. s, 1H), 2.70 (br. s, 1H), 1.44 (s, 9H), 1.11 (s, 9H). ¹³C NMR (101 MHz, CDC1₃) δ 156.0, 136.2, 136.0, 134.1, 133.0, 130.2, 130.1 , 129.8, 128.9, 127.9, 127.6, 97.5, 95.2, 80.8, 79.3, 76.2, 76.0, 75.0, 74.1, 73.4, 72.2, 71.0, 69.2, 55.6, 55.5, 40.5, 28.4, 27.2, 19.2. MALDI- MS for C₃₇H₅₇NO_! iSiNa: calcd 742.36; found 742.31.

The resulted coupling product (394 mg, 0.55 mmol) as described above and 10% Pd- C (22 mg) were dissolved in MeOH (10 mL) and was stirred under H₂ (1 atm) at room temperature for 12 h. The catalyst was removed by filtration, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (hexane: acetone = 7:3) to afford 5 (394 mg, 0.55 mmol, 99%) as colorless oil. 1H NM (400 MHz, CDC1₃) δ 7.78-7.68 (m, 4H), 7.42-7.30 (m, 6H), 5.21 (br. s, 1H), 4.59 (d, J = 6.2 Hz, 1H), 4.55 (d, J = 6.2 Hz, 1H), 4.48 (d, J = 6.8 Hz, 1H), 4.30 (d, J = 6.8 Hz, 1H), 3.94-3.88 (m, 1H), 3.82-3.74 (m, 3H), 3.63 (t, J = 4.3 Hz, 1H), 3.52-3.31 (m, 4H), 3.28-3.23 (m, 8H), 3.22 (s, 3H), 3.10 (dd, J = 9.7, 1.8 Hz, 1H), 2.77 (br. s, 1H), 1.72-1.50 (m, 4H), 1.44 (s, 9H), 1.11 (s, 9H). ¹³C NMR (101 MHz, CDC1₃) δ 156.1, 136.2, 136.0, 134.2, 133.1, 130.0, 129.7, 127.9, 127.6, 81.4, 79.2, 76.0, 75.1, 74.1, 73.2, 72.3, 71.4, 70.0, 55.5, 55.4, 40.4, 28.4, 27.25, 27.16, 25.9, 19.2. MALDI-MS for C₃7H₅₉NOiiSi a: calcd 744.38; found 744.38.

Tetrazole (3% in CH3CN, 8.55 mL, 2.89 mmol) and 5 (200 mg, 0.28 mmol) were dried under vacuum for 2 h and dissolved in anhydrous CH₂C1₂ (5 mL). Dibenzyl N,N- diisopropylphosphoramidite (368 μΐ,, 1.12 mmol) was then added under argon, and the resulting mixture was stirred at room temperature for 12 h before mCPBA (77%, 480 mg, 2.1 mmol) was added at -40 °C. The reaction solution was slowly warmed to room temperature in 2 h. The solvent was removed, and the residue was re-dissolved in CHC1₃. The organic layer was washed with H₂0 (slightly basic), dried over Na₂S0₄, and concentrated. The residue was purified by column chromatography (hexane: acetone = 3:1) over silica to yield coupling compound (335 mg, 0.27 mmol, 96 %) as white wax. 1H NMR (400 MHz, CDC1₃) δ 7.78- 7.68 (m, 4H), 7.44-7.24 (m, 26H), 5.18-4.91 (m, 8H), 4.83 (q, J = 9.3 Hz, 1H), 4.58 (d, J =

6.3 Hz, 1H), 4.54 (d, J= 6.3 Hz, 1H), 4.30-4.22 (m, 2H), 4.07 (d, J = 7.0 Hz, 1H), 3.83-3.75 (m, 4H), 3.33-3.28 (m, 6H), 3.24-3.13 (m, 5H), 2.96 (s, 3H), 1.61-1.49 (m, 2H), 1.43 (s, 9H), 1.35-1.25 (m, 2H), 1.10 (s, 9H). C NMR (101 MHz, CDC1₃) δ 155.9, 136.23, 136.21, 136.12, 135.9, 134.0, 132.6, 130.1, 129.8, 128.4, 128.4, 128.31, 128.30, 128.19, 128.16, 127.98, 127.95, 127.85, 127.80, 127.65, 97.3, 96.0, 79.8, 79.1, 78.1, 75.7, 74.31, 74.26, 73.6, 70.9, 69.5, 69.4, 69.0, 55.6, 55.5, 40.4, 28.4, 27.2, 26.2, 25.8, 19.2. ³¹P NMR (162 MHz, CDC1₃) δ -1.53 (s, IP), -1.67 (s, IP). MALDI-MS for C₆₅H₈₄N0₁₇P₂Si: calcd 1240.50; found 1239.10.

A solution of above resulted compound (202 mg, 0.16 mmol) in TBAF (0.3 M in THF, 12 mL) was stirred at 0 °C for 12 h and diluted with EtOAc (50 mL). The resulted mixture was washed with H₂0, and the organic layer was dried over Na₂S0₄. Solvent evaporation followed by column chromatography over silica (hexane: acetone = 1 :1) provided 6 (161 mg, 0.16 mmol, 96%) as white wax. 1H NMR (400 MHz, CDC1₃) δ 7.28- 7.23 (m, 20H), 5.19-4.95 (m, 8H), 4.93-4.84 (m, 2H), 4.77 (d, J = 6.7 Hz, 1H), 4.59 (d, J = 7.1 Hz, 1H), 4.50 (d, J= 7.1 Hz, 1H), 4.41 (q, J= 9.1 Hz, 1H), 4.10 (t, J= 2.1 Hz, 1H), 3.77- 3.72 (m, 2H), 3.62 (t, J = 9.1 Hz, 1H), 3.58-3.50 (m, 2H), 3.44 (s, 3H), 3.40-3.30 (m, 3H), 3.28-3.25 (m, 6H), 1.63-1.44 (m, 13H). ¹³C NMR (101 MHz, CDC1₃) δ 156.0, 136.1, 136.08, 136.06, 135.99, 128.40, 128.35, 128.27, 128.17, 127.99, 127.93, 127.87, 127.77, 98.0, 97.2, 80.1, 79.3, 79.1, 78.0, 77.9, 75.7, 72.4, 71.3, 71.2, 69.9, 69.4, 69.2, 55.8, 40.4, 28.4, 27.0, 25.7. ³¹P NMR (162 MHz, CDC1₃) δ -1.50 (s, IP), -1.69 (s, IP). MALDI-MS for C₄₉H₆₆NOi₇P₂: calcd 1002.38; found 1002.38.

Vacuum dried 6 (45 mg, 0.045 mmol) and tetrazole (10.6 mg, 0.136 mmol) were dissolved in anhydrous CH₂C1₂ (2 mL). A solution of 7 of Fig. 2 (104 mg, 0.128 mmol) in CH₃CN (1 mL) was then added under argon and the reaction mixture was stirred overnight. Next, t-BuOOH (410 ί, 0.27 mmol) was added at -40 °C, and the resulting mixture was warmed to room temperature. The solvents were removed under vacuum, and the residue was purified by column chromatography (hexane: acetone = 2: 1) over silica to give 8 (84 mg, 0.051 mmol, 95%) as a 1 :1.2 mixture of two conformational isomers. 1H NMR (400 MHz, CDC1₃) δ 7.35-7.22 (m, 30H), 5.19-4.80 (m, 17H), 4.78-4.71 (m, 2H), 4.54-4.48 (m, 1H), 4.45-4.41 (m, 1H), 4.39-4.34 (m, 1H), 4.32-4.34 (m, 2H), 4.18-4.04 (m, 4H), 3.79-3.74 (m, 1H), 3.70-3.48 (m, 3H), 3.37-3.29 (m, 4H), 3.23-3.11 (m, 7H), 2.26-2.21 (m, 4H), 2.03 (br, s, 2H), 1.59-1.43 (m, 8H), 1.43-1.36 (m, 9H), 1.35-1.16 (m, 26H), 0.85 (t, J= 3.0 Hz, 3H). ¹³C NMR (101 MHz, CDC1₃) δ 172.84, 172.79, 172.74, 156.42, 155.95, 136.71, 136.68, 136.66, 136.07, 136.00, 135.90, 135.49, 135.44, 135.42, 135.38, 135.37, 128.78, 128.71, 128.68, 128.45, 128.42, 128.40, 128.36, 128.35, 128.31, 128.20, 128.05, 128.01, 127.93, 127.89, 127.73, 97.43, 96.98, 79.09, 78.79, 78.10, 77.56, 77.22, 74.73, 74.54, 74.40, 73.38, 73.23, 70.77, 69.78, 69.63, 69.59, 69.51, 69.45, 69.23, 69.18, 69.13, 66.51, 65.40, 61.53, 55.88, 55.86, 55.76, 40.81, 40.42, 34.05, 34.02, 33.93, 33.70, 31.90, 29.68, 29.66, 29.63, 29.48, 29.33, 29.28, 29.08, 28.41, 26.36, 26.31, 26.14, 25.92, 25.86, 24.77, 24.35, 22.67, 22.53, 14.10. ³¹P NMR (162 MHz, CDC1₃) δ -1.37 (s, 1.0P), -1.45 (s, 0.37P), -1.51 (s, 0.16P), -1.71 (s, 0.45P), -1.76 (s, 0.54P), -1.85 (s, 0.43P). ESI-MS for C8₉H₁₂₇N₂0₂6P₃Na: calcd 1755.78; found 1755.62.

Compound 8 (21 mg, 0.013 mmol) was dissolved in MeOH (1.5 mL) and 10% Pd-C (5 mg, 0.004 mmol) were added for hydrogenation under H₂ (1 atm) for 6 h. The catalyst was removed via filtration, and the solvent was evaporated under reduced pressure. The residue was dissolved in TEAB buffer (0.8 M, 1 mL) and then 6-Carboxyfluorescein N- hydroxysuccinimide ester 9 (8 mg, 0.017 mmol) in DMF (1 mL) was added. The reaction mixture was stirred at room temperature for 48 h in the dark. Next, the mixture was thoroughly dried under vacuum and CH₂C1₂ (2 mL) was added. After the solution was cooled to 0 °C, redistilled TMSBr (3 mL, 22.7 mmol) was added in a dropwise manner. The reaction mixture was stirred at room temperature for 1 h, and volatile materials were removed under reduced pressure. The residue was re-dissolved in MeOH (1.5 mL) and stirred at room temperature for 1 h. Then the solution was purified though HPLC to afford 10 (6 mg, 44 μηιοΐ, 34%) as orange solid. 1H NMR (CD₃OD, 400 MHz) δ 8.09-7.99 (m, 2H), 7.64 (s, 1H), 6.68-6.86 (m, 4H), 6.60 (dd, J = 8.8, 1.8 Hz, 2H), 5.18-5.11 (m, 1H), 4.19-4.46 (m, 5H), 3.86-4.18 (m, 5H), 3.40-3.82 (m, 6H), 3.33 (t, J= 6.9 Hz, 2H), 3.07- 3.01 (m, 2H), 2.25-2.40 (m, 4H), 1.50-1.80 (m, 10H), 1.18-1.40 (m, 26H), 0.87 (t, J = 6.9 Hz, 3H); ¹³C NMR (CD₃OD, 125 MHz) δ 174.74, 174.66, 171.33, 168.25, 163.14, 162.86, 155.91, 131.09, 130.01, 127.94, 126.06, 119.42, 117.08, 112.68, 103.82, 80.73, 80.39, 78.86, 77.34, 73.96, 72.92, 72.12, 71.83, 66.89, 64,79, 63.74, 40.99, 40.60, 35.08, 34.80, 34.70, 33.07, 30.79, 30.63, 30.47, 30.36, 30.17, 29.93, 27.71, 27.49, 26.88, 26.01, 25.57, 23.74, 14.46; ³¹P NMR (CD₃OD, 162 MHz) δ 3.54 (IP), -0.65 (IP), -1.12 (IP); MALDI-MS for C₅₈H₈₆N₂0₂₆P₃: calcd 1319.47; found 1319.47.

Compound 10 (4 mg, 0.003 mmol) was dissolved in a mixture of TEAB buffer (1.0 M, 500 μΤ) and then 4-[4-(dimethylamino)phenylazo]benzoic acid N-succinimidyl ester (2 mg, 0.006 mmol) in DMF (2 mL) was added. The reaction mixture was stirred at room temperature for 48 h in the dark. Volatile material was removed under vacuum, and the resulting residue was purified with C8 reverse phase column to afford XY-69 (3.0 mg, 1.8 μηιοΐ, 60%) as red solid. 1H NMR (CD₃OD, 400 MHz) δ 8.12-8.07 (m, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.80-7.96 (m, 4H), 7.63 (s, TH), 6.81 (d, J = 9.1 Hz, 2H), 6.69-6.73 (m, 2H), 6.62 (d, J = 8.8 Hz, 2H), 6.55 (d, J = 8.7 Hz, 2H), 5.18-5.08 (m, 1H), 4.28-4.44 (m, 3H), 3.88-4.26 (m, 10H), 3.67-3.82 (m, 4H), 3.42-3.64 (m, 3H), 3.09 (s, 6H), 2.18-2.32 (m, 4H), 1.15-1.75 (m, 36H), 0.88 (t, J = 7.1 Hz, 3H); ³¹P NMR (CD₃OD, 162 MHz) δ 3.62 (IP), -0.04 (IP), -0.90 (IP); ESI-HRMS for C₇₃H₉₉N₅0₂₇P₃ (M + H)+: calcd 1570.5741 ; found 1570.5747.

PLC assay in detergent micelles

In a PerkinElmer ProxiPlateTM-384 Plus F black plate, XY-69 was added to a final concentration of 5 μΜ in assay buffer containing HEPES (50 mM, pH 7.4), KC1 (70 mM), CaCl₂ (3 mM), EGTA (3 mM), DTT (2 mM), cholate (0.5%), and fatty acid-free BSA (0.2 mg/mL). The free Ca²⁺ concentration in this buffer is calculated as 18.9 μΜ according to the Ca-EGTA Calculator vl .3 program using constants from Theo Schoenmakers' Chelator. The enzyme PLC-δΙ (25 ng), PLC-δΙ (E341A) (25 ng), PLC-γΙ (100 pg) or PLC- 3 (50 ng) in the same buffer was then added to initiate enzymatic reaction at 37 °C. The final volume of the assay was 10 \L. The progression of the assay was monitored continuously by fluorescence intensity of the reaction mixture on a PerkinElmer Wallac En Vision 2103 multilabel reader with an excitation wavelength of 485 nm (bandwidth of 10 nm) and an emission wavelength of 520 nm (bandwidth of 10 nm).

When cell lysates were used in this assay, HEK293 cells were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (10%), penicillin (100 units/ml), and streptomycin (100 μg ml) at 37 °C. Cells were plated in a 12- well dish at a density of 60,000 cells/well in DMEM and 24 hours after plating were transfected with indicated DNA at 300 ng/well using Continuum (Gemini Bio-Sciences). Forty-eight hours post-transfection, cells were washed with PBS, and lysed with RIPA buffer containing NaCl (150 mM), NP-40 (1%), SDS (0.1%), Tris-HCl (50 mM, pH 8), and sodium deoxycholate (12 mM). Western blotting was performed to confirm the expression of PLC-P3 using anti-PLC β3 (Santa Cruz). Lysates were normalized for total protein concentration prior to use in the reporter assay as described above.

PLC assay in lipid vesicles

Lipid vesicles containing liver phosphatidylethanolamine (PE, 330 μΜ), brain PIP₂ (22.5 μΜ), and XY-69 (7.5 μΜ) were generated by mixing the lipids, drying the mixture under a stream of nitrogen, and re-suspending the dried lipid mixture in HEPES (20 mM, pH 7.4) using a probe sonicator. In parallel, PLC isozymes were diluted to the desired concentration with a buffer containing HEPES (20 mM, pH 7.4), NaCl (50 mM), dithiothreitol (DTT, 2 mM), and fatty acid-free BSA (1 mg/mL). To a PerkinElmer ProxiPlateTM-384 Plus F black plate was added 6X buffer (2 μΐ,) containing HEPES (80 mM, pH 7.4), KC1 (420 roM), DTT (10 mM), EGTA (18 mM) and CaCl₂ (14.1 mM). Lipid vesicle (8 μΕ) was then added, followed by addition of PLC protein (2 μί) at concentrations of 11.4, 18.9, 67.3, and 66.6 nM for PLC-δΙ, -δΐ (E341A), -γΐ, and -β3, respectively, to initiate the assay. The assay was incubated at 37 °C and fluorescence was recorded as described above.

Gaq activation of ΡΣΟ-β3

Lipid vesicles containing liver PE (500 μΜ), brain PIP₂ (50 μΜ), and XY-69 (10 μΜ) were generated by drying the lipids under a stream of nitrogen and re-suspending the resulting mixture in HEPES (20 mM, pH 7.4) using a probe sonicator. Wild type PLC-P3 and PLC-P3 (PH-C2) were diluted with 2X assay buffer containing HEPES (20 mM, pH 7.4), KC1 (40 mM), NaCl (60 mM), EGTA (6 mM), A1C1₃ (60 μΜ), MgCl₂ (5 mM), DTT (4 mM), CaCl₂ (3 mM), NaF (20 mM), and 0.3 mg/mL fatty acid-free BSA to the final concentrations of 10 nM and 20 nM, respectively. Gaq was then diluted with the same 2X assay buffer to the final concentration of 480 nM. To a PerkinElmer ProxiPlateTM-384 Plus F black plate was added 2 iL of PLC solution and 4 iL of Gaq solution, followed by the addition of 6 μΕ of lipids to initiate the reaction. The assay was incubated at room temperature and fluorescence was recorded as described above.

Measurement of activity of endogenous PLCs

HEK293 cells were plated in 6-well tissue-culture plates at the density of 150,000 cells/well in DMEM supplemented with 10% fetal bovine serum and incubated overnight in a humidified, 37 °C, 5% C0₂ incubator. Cells were transfected with 300 ng of pcHA-lic control vector or 30 ng of pcHA GNAQ Q209L DNA plus 270 ng of pcHA-lic control using Continuum transfection reagent (Gemini Bio Products). After 48 h, the medium was removed and replaced with 400 μΐ inositol free DMEM. The cells were cultured for another 24 h and then washed with ice-cold PBS before collected with PBS. The suspension was briefly spun to pellet, the supernatant was removed, and the resulting pellets were snap- frozen with liquid N₂ and stored at -80 °C for subsequent uses.

The cell pellets from two wells were combined in a microcentrifuge tube on ice by resuspending cells with 80 μΕ of lipid vesicles containing liver phosphatidyl ethanolamine (PE, 330 μΜ), brain PIP₂ (22.5 μΜ), and XY-69 (7.5 μΜ) in HEPES (20 mM, pH 7.4) and 80 μΐ, of 2X assay buffer containing HEPES (80 mM, pH 7.4), KC1 (420 mM), DTT (10 mM), EGTA (18 mM) and CaCl₂ (14.1 mM), The cells were lysed by passing through a syringe with a 27G hypodermic needle 10 times. The resulting mixture was then incubated at 37 °C. At an indicated time point, 30 μΐ, of mixture was taken out and centrifuged for 40 seconds, and 10 μΐ, of supernatant was transferred to a PerkinElmer ProxiPlateTM-384 Plus F black plate for fluorescence measurement as described above. The activity of endogenous PLCs is calculated from the fluorescence increase as the slope.

For comparison, the endogenous PLC activity was also measured by labeling cells with 1 μθϊ of H-wyo-Inositol (Perkin Elmer) and quantifying [ HJinositol phosphates as previously described. TLC analysis of assay mixtures

XY-69 or XY-23 was added to various assay buffers (30 μί) to reach a final concentration of 67 μΜ. The components for assay buffers included: 1) HEPES (50 mM, pH 7.4), KC1 (70 mM), CaCl₂ (3 mM), EGTA (3 mM), DTT (2 mM), cholate (0.5%), and fatty acid-free BSA (0.2 mg/mL) for PLCs; 2) Tris-HCl (12.5 mM, pH 8.0), SDS (6.2 mM), and EtOH (0.1%) for PLD1 ; 3) Tris-HCl (50 mM, pH 7.5), KC1 (150 mM), and CaCl₂ (10 mM) for PLA2; and 4) MOPS (50 mM, pH 6.5), NaCl^'(100 mM), sodium cholate (0.5 mM), DTT (1 mM), MgCl₂ (10 mM), and ATP (2 mM) for PI3K . The enzyme PLC-δΙ (25 ng), PLC-δΙ (E341A) (25 ng), PLC-γΙ (100 pg), PLC-P3 (50 ng), PLD1 (40 units), PLA2 (2 units), or PI3K (15 ng) was then added to initial enzymatic reactions. The assay mixtures were incubated at 37 °C for 1 h, and samples (1 μΕ) were taken out and spotted on TLC plates (Merck, Silica Gel-60). The solvents used for TLC were CHC1₃: MeOH: H₂0 (100: 20: 1) for reactions with PLCs and PLD and CHC1₃: acetone: MeOH: HOAc: H₂0 (80: 30: 26: 24: 14) for PLA2 and PBKcc, respectively. The products were detected by fluorescence with a Typhoon 9400 Variable Mode Imager (X_ex/em = 488/520 nm). Membrane association assay

The liposome mixture was prepared from stock solutions of lipids in CHC1₃ for a final composition of 45% phosphatidylcholine (PC), 25% PE, 15% cholesterol, 10% phosphatidylinositol, and 5%> phosphatidylserine (PS). The solvent was blown off under a stream of nitrogen followed by drying under vacuum for at least 1 h. The lipid film was then suspended in buffer composed of MOPS (50 mM, pH 6.7), NaCl (100 mM), DTT (1 mM), and MgCl₂ (10 mM) to a concentration of 2 mM. Liposomes were extruded through a 0.03 μΜ pore size polycarbonate filter membrane at least 1 1 times back and forth. XY-69 (10 μΜ) or WH-15 (10 μΜ) and liposomes (1 mM) were incubated in buffer at room temperature for 5 min in a total volume of 150 μΐ,. The suspension was adjusted to 30% sucrose by the addition of 100 μΤ of 75% w/v sucrose in buffer followed by mixing. Buffer (200 μΐ,) containing 25% w/v sucrose was then overlaid on the high-sucrose suspension followed by 50 μΤ of buffer containing no sucrose. The sample was centrifuged at 55,000 rpm in a Beckman swinging- bucket rotor (TLS 55) for 1 h at 4 °C. The bottom 350 μΐ, and top 150 μΐ, were manually collected using a syringe and adjusted to the same volume (350 μΐ,). Each fraction was then diluted with IX PLC buffer containing HEPES (50 mM, pH 7.4), KC1 (70 mM), CaCl₂ (3 mM), EGTA (3 mM), DTT (2 mM), and cholate (0.5%). Fractions containing XY-69 were diluted 1 :50, while WH-15 fractions were diluted 1 :3. PLC-δΙ was diluted to 10 ng/μί in IX PLC buffer with 1 mg/mL fatty acid-free BSA. In a PerkinElmer ProxiPlateTM-384 Plus F black plate, 5 μΕ of above reporter solution was mixed with 5 μΕ PLC-δΙ or PLC dilution buffer. After incubation at room temperature for 6 h, fluorescence intensity was measured on a PerkinElmer Wallac En Vision 2103 multilabel reader as described before. The fluorescence intensity of the samples treated with PLC were subtracted with that of the samples treated with PLC dilution buffer. The fluorescence intensity differences were used to calculate the amount of XY-69 or WH-15, which was then used to calculate the reporter distribution.

Example 2;

Photocaged XY-69

A caged version of XY-69 with photoactivatable 7-diethylamino coumarin-yl-4- methyl group at the 2-OH (Scheme 2) will be synthesized. Among different photoactivatable groups, 7-dialkylamino coumarin-yl-4-methyl derivatives have distinct advantages, including: large extinction coefficients and high photolysis efficiency at longer wavelengths (up to 440 nm); fast photolysis kinetics; and improved stability in the dark. If desired, coumarin derivatives are also excellent for two-photon microscopy since they have large two- photon excitation cross-sections and thus can absorb and combine the energies of two long wavelength photons. Indeed, long wavelength IR light (λ = 720-830 nm) has been used to uncage gamma-aminobutyric acid (GABA) or cyclic nucleotides that are conjugated with coumarin derivatives. Furthermore, we will replace the original fluorescein (λεχ/em = 480/520 nm) in XY-69 with TAMRA (λεχ/em = 546/579 nm) in the caged version. This switch will minimize spectral overlap between the coumarin cage and the TAMRA fluorophore (Scheme 2). The TAMRA fluorophore has the added advantages of being highly photostable and producing orange-red fluorescence with high quantum yield that is pH- insensitive. It is also zwitterionic with a net neutral charge that will facilitate cellular entry.

For similar reasons, the negatively charged phosphates on the inositol ring will be caged and neutralized by the corresponding acetoxymethyl (AM) esters (Scheme 2). These esters are readily hydrolyzed by intracellular esterases and are primarily needed to facilitate cellular permeability. Finally, we will incorporate QSY7 (Scheme 2) into the new, caged version of XY-69 since it efficiently quenches TAMRA52-54. Neither the TAMRA nor the QSY7 moieties are expected to interfere with the efficient uncaging of the coumarin derivative using one-photon photolysis (λ = 405 nm). If desired, two-photon photolysis (λ = 720-830 nm) will be utilized. As a control, the caged fluorescent PIP2 (Scheme 2) without QSY7 will also be generated.

The photocaged version of XY-69 is expected to be readily taken into cells yet inert until irradiated with light. Once uncaged, the newest version of XY-69 remains non- fluorescent until it is hydrolyzed by PLCs. At this point the TAMRA-containing diacylglycerol derivative is liberated from the QS Y7-modified inositol ring and the TAMRA is no longer quenched by close proximity to QSY7. Fluorescence (λεχ/em = 546/579 nm) from the TAMRA derivative should now accurately and robustly report the activation of PLCs with high spatiotemporal resolution. Synthesis:

The synthesis of the reporter is provided in Scheme 2.

Scheme 2: Synthesis for caged XY-69.

Compound 20 has been previously prepared and will be reacted via olefin metathesis with terminal amine 21 followed by hydrogenation to generate 22. The hydroxyl groups in 22 will be phosphorylated in a two-step sequence: first reacted with bis(2- cyanoethyl)diisopropyl phosphoramidite in the presence of tetrazole and then oxidized by 3- chloroperoxybenzoic acid (wCPBA)56. The t-butyldiphenylsilyl (TBDPS) protection will be removed by tetrabutylammonium fluoride (TBAF) to form alcohol 23, which will be coupled to the diacylglycerol side chain 24 to form 25 through phosphorylation followed by oxidation with t-butylperoxide (t-BuOOH). The tert-butyloxycarbonyl (Boc) group will then be removed and the resulting terminal amine coupled to 26. Subsequent removal of the methoxylmethyl (MOM) protective groups with trimethylsilyl bromide (TMSBr) in CH2C1₂ followed by methanolysis will produce 27. The diol in 27 will then be protected with the 7- diethylamino coumarin aldehyde 28 to form the corresponding 29. Both phosphates and carbobenzyloxy (Cbz) groups are removed by aqueous ammonium (NH3), and the free phosphoric acids react with bromomethyl acetate while the terminal amine is coupled with activated TAMRA 30 to generate caged XY-69. The additional compound, caged PIP2 will be synthesized similarly and used in initial experiments designed to optimize cellular loading and uncaging. All proposed chemical steps rely on well-established arid efficient synthetic chemistry. The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. All publications, patent applications, patents, patent publications, sequences identified by GenBank and/or SNP accession numbers, and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

Claims

THAT WHICH IS CLAIMED IS: ompound of Formula IA or Formula II:

wherein:

LA and LB, are each independently present or absent, and when present are each independently selected from the group consisting of -C(0)NH-, -C(O)-, -NH-, -0-, - C(=NRⁿ)-, -C(=NOH)-, -C(=0)0- -OC(=0)-, and -NHC(=0)-, wherein R¹¹ is H, alkyl, alkenyl, or alkynyl;

X is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, aliphatic oxide, and -R²-Z-R³-, wherein R² and R³ are each independently selected from the group consisting of a Cr o unsubstituted or substituted alkyl and a C₂-Cio unsubstituted or substituted alkenyl, and Z is oxygen or sulfur;

Y is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aliphatic oxide, aryl, arylalkyl, and -R⁴-CH(D-R⁵)-R⁶-D-R⁷-, wherein R⁴, R⁶, and R⁷ are each independently a Cj-Cio unsubstituted or substituted alkyl or C2-C₁₀ unsubstituted or substituted alkenyl; R⁵ is H, alkyl, alkenyl, alkynyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, alkylthio, amino, aminoalkyl, alkylamino, cycloalkyl, heteroaryl, heteroalkyl, aryl, arylalkyl, aliphatic oxide, -C(=0)R¹⁰, -C(=0)OR¹⁰, -C(=O)N(R¹⁰)₂, -(CH₂)_mN(R¹⁰)₂, -C(=O)N(R^l0)₂, and -(CH₂)_mCOOR¹⁰, where m is 1-20 and R¹⁰ is H, alkyl, alkenyl, or alkynyl; and D is each independently selected from oxygen and sulfur;

Fp is a first fluorophore; and

Q is a quencher or a second fluorophore.

2. The compound of claim 1, wherein X is -R -Z-R -.

3. The compound of claim 1 or 2, wherein R² and R³ are each independently selected from a Q-Cio unsubstituted or substituted alkyl and a C₂-C₁₀ unsubstituted or substituted alkenyl, and Z is oxygen, optionally wherein R² and R³ are each independently selected from a Ci-C₅ unsubstituted alkyl, and Z is oxygen.

4. The compound of any one of claims 1 -3, wherein Y is selected from the group consisting of unsubstituted or substituted alkyl, alkenyl, alkynyl, aliphatic oxide, and -R⁴- CH(D-R⁵)-R⁶-D-R⁷-.

5. The compound of any one of claims 1-4, wherein Y is -R⁴-CH(D-R⁵)-R⁶-D-R⁷-, optionally wherein R⁴, R⁶, and R⁷ are each independently selected from a C\-C\o unsubstituted or substituted alkyl or C₂-C₁₀ unsubstituted or substituted alkenyl; R⁵ is a C₁₀- C₃₀ alkyl, C₁₀-C₃₀ alkenyl, C₁₀-C₃₀ alkynyl, -C(=0)R¹⁰, -C(=0)OR¹⁰, -C(=O)N(R¹⁰)₂, -(CH₂)_mN(R¹⁰)₂, -C(=O)N(R¹⁰)₂, or -(CH₂)_mCOOR¹⁰, where m is 1 -20 and R¹⁰ is a Ci₀-C₃₀ alkyl, C^-Cso alkenyl, or C₁₀-C₃o alkynyl; and each D is oxygen.

6. The compound of any one of claims 1 -5, wherein R⁴, R⁶, and R⁷ are each independently selected from a -Cg unsubstituted or substituted alkyl, R⁵ is -C(=0)R¹⁰, R¹⁰ is a C₁₀-C₃o alkyl, and each D is oxygen.

7. The compound of any one of claims 1-6 wherein R⁷ is a Ci-C& substituted alkyl, optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclo, heterocycloalkyl, aryl, arylalkyl, alkoxy, thioalkyl, hydroxyl, thio, oxo, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl, optionally wherein, R⁷ is -C(0)-(CH₂)i-₇.

8. The compound of any one of claims 1-7, wherein Q is the quencher.

9. The compound of any one of claims 1-8, wherein Q is the second fluorophore, optionally wherein the first fluorophore and the second fluorophore are a fluorescent energy transfer (FRET) pair.

10. The compound of any one of claims 1-9, wherein the compound has a structure a structure re resented by Formula IIA:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, Z, D, Q, and Fp are each as defined above.

11. The compound of any one of claims 1-10, wherein the compound has a structure a structure represented by Formula IIB:

12. The compound of any one of claims 1-11, wherein the compound has a structure a structure represented by Formula IIC:

(IIC), wherein:

G is a cage group (e.g., a photocage group), optionally wherein two adjacent G together form the cage group; and

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, Z, D, Q, and Fp are each as defined above.

13. The compound of any one of claims 1-12, wherein the compound has a structure represented by a compound of Formula III:

14. The compound of any one of claims 1-13, wherein the compound has a structure represented by a compound of Formula IV:

wherein R¹, Q, and Fp are each as defined above.

15. The compound of any one of claims 1-14, wherein at least one of R¹ comprises the cellular localization signal.

16. The compound of any one of claims 1-15, wherein at least one of R¹ comprises the cage group.

17. The compound of any one of claims 1-16, wherein the compound has a structure

(V), or

55

19. The compound of any one of claims 1-16, wherein the compound has a structure represented by a compound of Formula VII:

20. A method of identifying a test substance that inhibits phospholipase C activity, comprising:

contacting the compound of any of claims 1-19 with phospholipase C in the presence and absence of the test substance;

detecting the amount of fluorescence in the presence and absence of the test substance; comparing the amount of fluorescence detected in the presence and absence of the test substance, whereby a decrease in the amount of fluorescence detected in the presence of the test substance identifies that the test substance inhibits phospholipase C activity.

21. The method of claim 20, wherein the test substance is a small molecule.

22. The method of any of claims 20 or 21, further comprising activating phospholipase C with a phospholipase C activator.

23. A method of detecting phospholipase C activity in a cell, comprising:

contacting the compound of any of claims 1-19 with a cell; and

detecting fluorescence in the cell, thereby detecting phospholipase C activity in the cell.

24. The method of claim 23, further comprising activating phospholipase C with a phospholipase C activator.

25. A method of detecting aberrant phospholipase C activity in a cell, comprising: contacting the compound of any of claims 1-19 with a cell;

detecting an amount or pattern of fluorescence in the cell; and

comparing the amount or pattern of fluorescence detected in the cell with the amount or pattern of fluorescence in a control cell that has been contacted with the compound, whereby an alteration in the amount or pattern of fluorescence in the cell as compared with the control cell detects aberrant phospholipase C activity in the cell.

26. The method of claim 25, wherein the cell is a diseased cell.

27. The method of any of claims 25 or 26, wherein the alteration is an increase in the amount of fluorescence in the cell as compared with the control cell.

28. The method of any of claims 25-27, wherein the alteration is a decrease in the amount of fluorescence in the cell as compared to the control cell.

29. The method of any of claims 25-28, further comprising activating phospholipase C with a phospholipase C activator.

30. The method of any of claims 25-29, wherein the cell is from a subject at risk of having a disease.

31. The method of any of claims 25-30, wherein the cell is from a subject suspected of having a disease.

32. The method of any one of claims 20-31, wherein the contacting is carried out under conditions whereby fluorescence resulting from reaction of the compound and phospholipase C can be detected.