Methods for measuring binding of ligands to target proteins and cell engagement

Abstract

A method for detecting and quantifying the binding properties of a compound to a target macromolecule is disclosed, wherein the target macromolecule is denatured and linked to a labeled peptide, such as a short fragment of an enzyme. The method uses a fluid mixture comprising (i) a chimeric molecule containing the target macromolecule linked to the labeled peptide, wherein the target macromolecule may be a chimeric protein expressed and present in intact living cells, and (ii) a compound whose binding to the target macromolecule is measured, wherein the target macromolecule is denatured. After allowing the compound (e.g., a small molecule inhibitor of the target macromolecule) to bind, a signal from the labeled peptide is detected, for example, by enzyme fragment complementation. This signal indicates the difference between denatured and undenatured target macromolecules, and thus respectively indicates the difference between target macromolecules not bound to the compound and target macromolecules bound to the compound.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Application Serial No. 14 / 830,328, filed August 19, 2015, entitled "Homogenous Thermal Shift Ligand Binding Assay," which is incorporated herein by reference in its entirety.

[0003] Statement regarding government funding

[0004] none.

[0005] References to sequence lists, computer programs, or optical discs

[0006] This application contains a sequence list that has been submitted as an ASCII text file and is incorporated herein by reference in its entirety. This text file was created on December 20, 2015, is named "3817_55_PCT_seq_list.txt", and is 4,096 bytes in size. Background of the Invention

[0007] Invention Field

[0008] This invention generally relates to methods for monitoring the binding of compounds to target macromolecules, wherein the target macromolecules are expressed in vitro or in transfected cells, and the cells are not separated from or isolated from the target macromolecules. This invention also relates to a novel method using enzyme fragment complementation and employing enzyme fragment complementation assays for thermotransformation.

[0009] Related technologies

[0010] The following is background information regarding certain aspects of the invention, as they may relate to technical features mentioned but not necessarily described in detail in the detailed description. That is, the various compositions or methods used in the invention can be described in more detail in the publications and patents discussed below, which may provide further guidance to those skilled in the art in making or using certain aspects of the claimed invention. The following discussion should not be construed as an admission of relevance to the patents or publications mentioned or of prior art effects.

[0011] Many fields of biology and pharmaceuticals rely on and have developed assays to detect the binding of ligands to proteins and / or protein fragments. Binding assays help identify targets that should be safe and druggable. The multi-billion dollar pharmaceutical industry relies on these assays to discover drug compounds that can bind to their target proteins to perform their functions.

[0012] Numerous protein-ligand interaction assays have been developed and discussed, including labeled and unlabeled ligand binding assays, structure-based ligand binding assays, thermodynamic ligand binding assays, and whole-cell ligand binding assays. In addition, fluorescence-based ligand binding assays exist, in which fluorescently labeled ligands bind to target macromolecules. However, these assays are susceptible to various fluorescence interferences, leading to undesirable alterations in ligand binding properties. Radiolabeled binding assays are popular for membrane-based targets; however, they are costly and involve handling high levels of radioactivity, thus limiting the laboratories and personnel performing these assays. NMR-based analyses have also been used to analyze the detailed structure of proteins, thus aiding in structure-based drug design, but they are costly and require lengthy spectroscopic analysis times.

[0013] Previously developed thermal transformation assays (also known as differential scanning fluorescence (DSF)) are a type of thermal denaturation assay that measures the thermal stability of a target protein and the subsequent increase in protein melting temperature after ligand binding. Changes in thermal stability are measured by profiling the protein in the presence of a fluorescent dye such as Sypro Orange. These methods also involve steps of centrifugation and oil partitioning.

[0014] Therefore, there is a need for a binding assay that provides a simple, sensitive, and accurate detection of ligand-protein interactions in a homogeneous assay format.

[0015] Specific patents and publications

[0016] US Patent 6,020,141, "Microplate thermal shiftassay for ligand development and multi-variable protein chemistry optimization," granted to Pantoliano et al. on February 1, 2000, discloses a thermal shift assay that involves contacting a target molecule with one of a variety of different molecules in each of a plurality of containers while heating the containers, and measuring physical changes in each container that are associated with the thermal denaturation of the target molecule.

[0017] Jung et al., “Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1,” J. Biol. Chem., 2014, Feb. 2, 289:9304-9319, disclose a thermal conversion assay in which BRD4BD1 protein is mixed with 5 μl Sypro Orange (Molecular Probe).

[0018] Molina et al., “Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay,” Science, July 5, 2013: Vol. 341, No. 6141, pp. 84-87, disclose a method utilizing the thermal stability shift of proteins induced by drug binding to target proteins. This method was used to monitor drug target binding in cancer cells and mouse liver and kidneys. However, according to the authors, this method is not suitable for highly heterogeneous proteins or proteins in which the unfolding of ligand-binding domains does not promote aggregation. Invention Overview

[0020] The following brief overview is not intended to include all features and aspects of the invention, nor does it mean that the invention must include all features and aspects discussed in this overview.

[0021] This invention includes a method for measuring the binding of a test compound to a target macromolecule. Furthermore, this invention discloses a homogeneous method for measuring the binding of a test compound to a target protein under thermal denaturation of a macromolecule having a defined natural secondary and optionally tertiary structure, wherein denaturation disrupts the secondary and / or tertiary structure of the target protein but does not destroy the labeled peptides attached thereto. Therefore, this method can be performed in a homogeneous manner, eliminating the need for centrifugation or filtration steps between sample addition and result readout. This method induces aggregation and accessibility of the labeled peptides attached to the target macromolecule; the labeled peptides can be conveniently added to the N- or C-terminus of the protein-based macromolecule. As will be described below, multiple short, relatively low-temperature heating pulses are preferred for a single heating step; similarly, complete denaturation of the protein under study is not preferred.

[0022] The method of the present invention can be performed using whole cells engineered to express a fusion protein comprising a target macromolecule and a labeled peptide expressed in the cells from a vector introduced into the cells. Preferably, the cells are eukaryotic or mammalian cells. The target compound is incubated with the whole-cell preparation, and heated together with the whole cells using the heating conditions described below; also as described below, some or all of the cells in the preparation are lysed in the second labeling and labeled peptide reaction step.

[0023] As explained below, the present invention as described above can be combined with various steps and features described below. In some embodiments, the invention includes the step of detecting signals that respectively indicate differences between (i) denatured and (ii) non-denatured target macromolecules, and thereby indicate differences between target macromolecules not bound to the compound and target macromolecules bound to the compound.

[0024] In one embodiment, the method includes preparing a chimeric molecule, said chimeric molecule being a fusion protein comprising a labeled peptide and a protein target macromolecule. In another embodiment, the method includes preparing a fusion protein comprising a nucleotide-binding domain, a labeled peptide, and a target macromolecule. The nucleotide-binding domain may be a DNA-binding domain. The target macromolecule may be a protein, peptide, carbohydrate, or lipid molecule.

[0025] In other embodiments, the present invention is a method for measuring the binding between a compound and a target macromolecule, comprising: (a) preparing a fluid mixture comprising (i) intact living cells expressing a chimeric protein, the chimeric protein comprising a target macromolecule linked to a labeled peptide and (ii) a compound bound to the target macromolecule, wherein the target macromolecule is denatured; (b) incubating the fluid mixture of step (a) under conditions allowing the compound to bind to the target macromolecule; (c) after incubation in step (b) denaturing the target macromolecule in the fluid mixture under conditions producing a combined mixture of (i) denatured chimeric molecules not bound to the compound and (ii) undenatured chimeric molecules bound to the compound; and (d) contacting the combined mixture with a second label, the second label being bound to and reacting with a labeled peptide in the chimeric molecule to form a detectable signal, wherein (e) the detectable signal in step (d) depends on the denaturation of the target macromolecule and represents the binding nature between the compound in the chimeric protein and the target macromolecule.

[0026] In other embodiments, the invention is a method as described above, wherein the partial denaturation comprises heating a fluid mixture as prepared in step (b) in one step, said step being one of (a) a plurality of heating steps and at least one cooling step between heating steps and (b) a single heating step. In other embodiments, the invention is a method as described above, wherein the target macromolecule is a protein. In other embodiments, the invention is a method wherein the protein further comprises an inactive exogenous polypeptide (“IEP”) ​​linked to a protein distal to the terminator of the labeled peptide. The protein will have an IEP-target protein-labeled peptide arrangement. In other embodiments, the invention is a method as described above, wherein the length of the labeled peptide is between 10 and 100 amino acids.

[0027] In another embodiment, the invention includes a method wherein the labeled peptide is an enzyme fragment, and a second label is a complementary enzyme fragment that binds to the labeled peptide to produce an active enzyme (i.e., to produce enzyme complementarity), the method further comprising the step of lysing living cells. In another embodiment, the invention includes a method wherein the labeled peptide is an enzyme donor (“ED”) active in enzyme fragment complementarity to β-galactosidase and is fused to the terminus of a protein that is a target macromolecule. In other embodiments, the invention includes the method as described above, wherein the ED is one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0028] In other embodiments, the invention is the method described above, wherein the labeled peptide is an epitope tag. In other embodiments, the invention is the method described above, wherein the compound is a small molecule. In other embodiments, the invention is the method described above, wherein the small molecule is a small molecule that binds to an active site on a target macromolecule.

[0029] In other embodiments, the invention is a method as described above, wherein the step of processing the fluid mixture includes heating the fluid mixture to one of a temperature between (a) 25°C and 100°C and (b) 30°C and 60°C. In other embodiments, the invention is a method as described above, wherein the heating step includes multiple heating steps lasting for a defined time period between 0.1 and 5 minutes. In other embodiments, the invention is a method as described above, wherein the heating step includes applying heat to the mixture between 40°C and 60°C, and further includes multiple heating steps lasting for a time of 0.1 to 5 minutes. In other embodiments, the invention is a method as described above, wherein the multiple heating steps include respective cooling steps lasting for a duration between 10 seconds and 2 minutes between the respective heating steps. In other embodiments, the invention is a method as described above including three to ten cooling steps. In other embodiments, the invention is a method as described above, wherein the temperature of the mixture is maintained above the temperature in the previous cooling steps during the cooling steps. In other embodiments, the invention is a method as described above, wherein the cooling step includes actively cooling the mixture.

[0030] In other embodiments, the invention is the method described above, wherein the denaturation step includes at least one of (a) heating, (b) hydrostatic pressure, (c) an organic solvent, and (d) radiation, thereby maintaining cell integrity after denaturation. In other embodiments, the invention is the method described above, wherein the organic solvent is an alcohol or chloroform.

[0031] In another embodiment, the invention includes the method having steps (a) to (e) as further described above, wherein steps (a) to (e) are repeated in a mixture containing the compound at different dilutions. In another embodiment, the invention includes the method having steps (a) to (e) as described above, wherein repeated steps (a) to (e) can be used to calculate the binding constant (K) of the compound to the target macromolecule. D ).

[0032] In another embodiment, the present invention includes the method described above, wherein the target macromolecule is a protein selected from bromine domain proteins, protein kinases, hydrolases, or histone methyltransferases.

[0033] In other embodiments, the present invention is a method for measuring the binding properties between a small molecule compound and a target protein, comprising: (a) preparing a fluid mixture containing cells expressing a chimeric protein, the chimeric protein being a fusion of a target protein and a labeled peptide, the labeled peptide being a β-galactosidase fragment of a length between 10 and 100 amino acids, the fluid mixture further containing a small molecule compound whose binding to the target protein is measured; (b) incubating the fluid mixture of step (a) under conditions allowing the small molecule compound to bind to the target protein in the cells; and (c) heating the fluid mixture of step (b) under conditions causing denaturation of proteins not bound to the compound and also causing less denaturation of proteins bound to the compound, thereby enabling the detection of a signal from a protein bound to the compound of step (c) by adding a second label, the second label being a β-galactosidase fragment that reacts with the labeled peptide on a protein not denatured during one or more heating steps, the mixture further containing lysed cells. In other embodiments, the invention is a method as described above, wherein the assay mixture is prepared in a single container via steps (a) to (c). This can be referred to as a homogeneous assay, requiring no separation steps. In other embodiments, the invention is a method as described above, wherein repeat samples containing different concentrations of the compound are prepared. In other embodiments, the invention is a method as described above, wherein the target macromolecule is one of a bromine-domain protein and an enzyme.

[0034] Additionally, the present invention includes a kit. In another embodiment, the present invention includes a kit comprising instructions for measuring the binding of a potential ligand to a target protein comprising components I to III, wherein component I is a target protein fused at the terminal to a first β-galactosidase fragment, component II is a β-galactosidase fragment complementary to the first β-galactosidase fragment, and component III is a β-galactosidase substrate, the instructions comprising instructions for: (a) preparing a fluid reaction mixture comprising cells containing (i) a target protein fused at the terminal to a first β-galactosidase fragment. (a) the target protein of the fragment fusion (component I), and (ii) the potential ligand of the target protein; (b) heating the reaction mixture and cells of step (a) to denature at least a portion of the target protein group; (c) after step (b), by adding to the mixture a β-galactosidase enzyme fragment complementary to the first β-galactosidase enzyme fragment (component II) and a substrate indicating the complementarity of the β-galactosidase and indicating the binding of the potential ligand (component III), measuring in the reaction mixture the amount of the undenatured fusion protein in step (b) as the inverse function of the heating step of step (b).

[0035] In another embodiment, the invention includes a kit in which the target protein is a bromine domain protein and an enzyme, wherein the enzyme may be one of a protein kinase or a histone methyltransferase. In another embodiment, the invention includes a kit in which a first β-galactosidase fragment is substantially identical to one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In another embodiment, the invention includes a kit in which the substrate is chromogenic, fluorescent, or chemiluminescent and generates a signal in the presence of an active β-galactosidase rather than an inactive β-galactosidase. Brief description of the attached diagram

[0037] Figure 1 This is a schematic diagram of the assay, illustrating how labeled macromolecules (e.g., fusion proteins and labeled peptides) are protected from denaturation in their native form (i.e., native secondary and tertiary structures) in the presence of a test compound that binds to the labeled native macromolecule; thus, this protection can be detected by an enzyme complementation assay.

[0038] Figure 2 Is it like this? Figure 1 The schematic diagram of the determination principle is shown in the figure. The exception is that in this case, the test compound does not bind to the labeled natural macromolecule, causing the labeled natural macromolecule to denature and further making the label (labeled peptide) unusable for complementation.

[0039] Figure 3 BRD4(1)-ProLabel in crude cell extract was shown after incubation with the indicator (substrate) and then heating at approximately 45°C for 30 seconds. TM A diagram of the ED fusion protein. Inhibitors binding to BRD4(1) were added at different concentrations between 0 and 100 μM. It can be seen that the dose-response protection of the fusion / BRD4(1) is achieved through the binding of the inhibitor. In all the diagrams illustrating BRD4(1), the construct used is NFκB DNA-binding domain-linker-BRD4(1)-linker-ED. The NFκB DNA-binding domain sequence in the fusion protein does not play an active role in these examples.

[0040] Figure 4 The non-inhibitor DMSO (labeled "2") is combined with JQ1, (6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza, a known selective BET bromine domain inhibitor. A graph comparing 6-acetic acid and 1,1-dimethyl ethyl ester. As can be seen, the enzyme complementarity of BRD4(1) decreased significantly at temperatures between 45 and 55 °C, while the presence of 10 μM JQ1 protected BRD4(1) from denaturation, and the decrease in enzyme complementarity was much smaller.

[0041] Figure 5 The graph shows the protection of BRD4(1) at 45°C, 50°C, and 55°C with or without JQ1 bound to BRD4(1). As indicated by the arrows, the thermal denaturation protection at 45°C is more significant than at 25°C, 50°C, or 55°C. Incubation at 25°C was performed as a control and does not indicate thermal denaturation.

[0042] Figure 6 The graph shows the RLU (Relative Luminescent Units), as indicated by the dense points at 602 and 604, indicating that there is almost no difference whether centrifugation was performed.

[0043] Figure 7 This is a pair of bar graphs showing the dose-response relationship between the potent BRD4(1) inhibitor JQ1 (top) and the known less potent BRD4(1) inhibitor GW334556X (bottom) in a ligand binding assay. BRD4(1)-ProLabel in crude cell extract... TM The ED fusion protein was incubated with the indicator and then heated to the target temperature in a PCR thermal cycler. An asterisk indicates K. D (Dissociation constant) concentration, as obtained from DiscoveRx Corp. The measurement was performed using a technical platform. This indicates that the assay can determine the dose-response relationship. The coefficient of variation observed here is less than 1%. The assay also exhibits very high reproducibility. GW334556X is further described in Chung et al., J.Med.Chem. (2012) 55:576.

[0044] Figures 8A and 8B are a pair of graphs showing an example of a pulsed denaturation protocol. Figure 8A shows a standard denaturation curve for one cycle, with a high temperature (45°C) over a 3-minute time period; Figure 8B shows a pulsed denaturation curve for 25 pulses under a cooling condition of 40°C, where each pulse represents a brief denaturation pulse time of 7 seconds (i.e., a heating period of 7 seconds), and the PCR thermal cycler is reset to a lower temperature (e.g., ambient temperature) after each heating pulse.

[0045] Figure 9 This is a graph showing the mathematical model for calculating the measurement window relative to the number of cycles of deformation. It demonstrates that the measurement window increases exponentially with the number of cycles. Measurement windowThe luminescence signal is defined as the luminescence signal after x cycles in the presence of a known ligand divided by the luminescence signal in the absence of a known ligand. This model assumes that a small fraction of the folded protein denatures in each cycle and cannot refold / reactivate in subsequent cycles. The model also assumes that the known ligand rescues 50% of the protein from denaturation in each cycle. Furthermore, the model assumes that during the temperature transition from the set point to room temperature, compound binding returns to room temperature levels in each cycle. The degree to which compound binding protects the protein from denaturation and the total amount of denaturation / naturalization without the compound control indicate the assay window. The assay window can be estimated from the data presented here by comparing the baseline signal (RLU) with the maximum signal in a given experiment.

[0046] Figure 10 This is a graph comparing the "standard" (constant heat) protocol and the pulsed protocol. The measurement window for pulsed denaturation is shown by black circles. Over time, the pulsed system results in an increased measurement window compared to the standard denaturation, shown by white circles. The thermal melting measurement windows of the "standard" and "pulsed" protocols for measuring the interaction with BRD9-Bromosporine are shown. Bromosporine is N-[(6-3-methanesulfonylamino-4-methylphenyl)-3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl]carbamate, commercially available from Tocris Biosciences.

[0047] Protein samples were exposed to a single standard thermal denaturation step (“Standard”) or repeated 0.5-minute heat pulses. Both protocols used a denaturation temperature of 45°C. “Total denaturation time” is the total amount of time the protein sample was exposed to 45°C; therefore, a single 3-minute step using the Standard protocol (i.e., no pulses, but a single 3-minute heating step at isothermal temperature) is equivalent to six repeated 0.5-minute steps using the pulsed protocol. In other words, six 0.5-minute heating pulses can be compared to one 3-minute heating step.

[0048] Figure 11This is a graph showing the different signals, in RLU (relative luminescent units), of binding to CREBBP (Gene ID1387, NCBI) at different concentrations of SGC-CBP30 (CREBBP / EP300-selective chemical probe) present in pulse denaturation relative to standard denaturation. SGC-CBP30 is commercially available from Tocris Biosciences and is 8-(3-chloro-4-methoxy-phenethyl)-4-(3,5-dimethyl-isoxazol-4-yl)-9-(2-(morpholin-4-yl)-propyl)-7,9-diaza-bicyclo[4.3.0]non-1(6),2,4,7-tetraene. Dose-response curves of CREBBP were measured using SGC-CBP30 at 45°C using the "standard protocol" or at 40°C using the "pulse protocol". Multiple cycles of mild denaturation at 40°C produced the apparent EC. 50 This value is close to the 0.021 μM K value measured by isothermal titration calorimetry. D (See Picaud et al., “RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain,” PNAS, December 3, 2013, Vol. 110, No. 49: 19754-19759, for a description of isothermal titration calorimetry). Mild denaturation is less than the melting point of CREBBP, i.e., 46 °C.

[0049] Figure 12 This is a graph showing the dose-response curves of seven ABL1 protein kinase inhibitors. Dose-response curves were obtained using ED-labeled ABL1 and the inhibitors in a pulse denaturation protocol. For example, it shows that the test compound dasatinib (N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolyl carboxamide monohydrate) binds significantly to the test protein ABL1 at a concentration of 1 nM, while other compounds such as imatinib (4-[(4-methylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]phenyl]benzamide) do not bind significantly at a concentration of 1 nM, as measured by the thermal pulse assay of this invention. Very significant differences between the different inhibitors are readily apparent.

[0050] Figure 13This is a graph showing the dose-response curve of UNC-0638 (2-cyclohexyl-6-methoxy-N-[1-(1-methylethyl)-4-piperidinyl]-7-[3-(1-pyrrolidinyl)propoxy]-4-aminoquinazoline, CAS 1255580-76-7), a G9a protein methyltransferase inhibitor. Dose-response curves were obtained using ED-labeled G9a and the inhibitor in a pulse denaturation protocol.

[0051] Figure 14 This is a schematic diagram of the intracellular implementation scheme for this assay, showing cell 1106 transfected with a protein labeled with the target enzyme fragment; this cell has been modified to overexpress a fusion protein containing a peptide marker (a small PL enzyme fragment of β-galactosidase) and the target macromolecule. The cells were incubated with a potential ligand 1104 that can cross the cell membrane. The binding of the compound to the target protein is being evaluated. The cells and ligand were then subjected to pulse denaturation as described above. In terms of the extent to which labeled macromolecules (e.g., fusion proteins / nucleic acids and labeled peptides) bind to the test compound in cells, they are protected from denaturation, as shown at 1108, and PL can interact with EA (enzyme receptor, i.e., a large fragment of β-galactosidase) to form an active β-galactosidase, which, as shown herein, will produce a luminescent readout (“light”). In cells where the labeled macromolecule does not bind to the test ligand (or if the test compound cannot cross the plasma membrane), protein aggregates form; a reduced signal is generated by the substrate. The binding and non-binding states shown at 1108 and 1110, respectively, exist in an equilibrium state, and binding curves can be constructed using different concentrations of the test compound bound to the labeled macromolecule expressed in recombinant cells. The method of the present invention can also be used to evaluate the ability of the test compound to cross the plasma membrane of engineered cells as described. This provides information on bioavailability and binding properties.

[0052] Figure 15 An exemplary intracellular pulse assay is shown using it in combination with the tested inhibitors dasatinib and VX-680, which bind to ABL1 tyrosine kinase.

[0053] Figure 16 An exemplary intracellular pulse assay using the inhibitor JQ1 on the bromine domain of BRD4(1) is shown.

[0054] Figure 17 An exemplary intracellular pulse assay using the MTH1 hydrolase domain and the inhibitor SCH 51344 is shown, employing two preliminary denaturation protocols indicated in the illustration.

[0055] Figure 18 An exemplary intracellular pulse assay using the G9a methyltransferase catalytic domain and the inhibitor UNC0638 is shown.

[0056] Detailed Explanation

[0057] definition

[0058] Unless otherwise defined, all 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 pertains. While any methods and materials similar to or equivalent to those described herein may be used to practice or test the invention, preferred methods and materials will be described. Generally, terms and techniques related to cell and molecular biology and chemistry are those well-known and commonly used in the art. Certain experimental techniques that are not explicitly defined are typically performed according to conventional methods well-known in the art and methods described in the various general and more specific references cited and discussed throughout this specification. For clarity, the following terms are defined as follows.

[0059] According to the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the scope of the art can be used. Such techniques are well explained in the literature. See, for example, Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: A Practical Approach”, Volumes I and II (edited by DN Glover, 1985); “Oligonucleotide synthesis” (edited by MJ Gait, 1984); “Nucleic Acid Hybridization” (edited by BD Hames & S.J. Higgins, 1985); “Transcription and Translation” (edited by BD Hames & S.J. Higgins, 1984); “Animal Cell Culture” (edited by RI Freshney, 1986); “Immobilized Cells and Enzymes” (IRL Press, 1986); B. Perbal, “A Practical Guide to Molecular Cloning” (1984).

[0060] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple indicators unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a priori basis for the proprietary terms “merely,” “only,” etc., in relation to the statement or “negative” limitation of claim elements.

[0061] scope For the sake of brevity, unless otherwise stated, any range presented is intended to include any subrange of the specified range. As a non-limiting example, a range of 2 to 8 minutes includes 3-4 minutes, 2-7 minutes, etc. A temperature range of 40-45°C includes 41-45°C, 42-43°C, etc. The term " about It has its approximate general meaning and can be determined in context by experimental variability. In case of doubt, the term "about" means plus or minus 5% of the specified value.

[0062] Upon reading this disclosure, it will be apparent to those skilled in the art that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any stated method may be performed in the order of the stated events or in any other logically possible order.

[0063] As used in this article, the term " peptides "" refers to any polymer compound produced by amide formation between the α-carboxyl group of one amino acid and the α-amino group of another group. As used herein, peptides can be labeled peptides that are relatively small in size, have little or no secondary structure (i.e., ring) linked to a macromolecule, and are used for the detection of fusions.

[0064] As used in this article, the term " protein A peptide is a polypeptide with a specific sequence of more than approximately 50 residues. Although all proteins are peptides, the term "peptide" generally refers to a fragment of a protein; the term "fusion protein" is used to refer to fusion proteins and fusions with peptides, such as fusions with labeled peptides like ED. Regarding this assay, it should be understood that the protein studied need not be a full-length protein sequence. The target macromolecule can actually be a protein that has been truncated to isolate the domains under study, modified for ease of processing, etc. Therefore, for simplicity, the protein fragments in this assay are referred to as "proteins".

[0065] As used in this article, the term " Fusion proteinA fusion protein is a protein produced from two or more proteins / peptides through genetic engineering. Typically, this is achieved by generating a "fusion gene" (a nucleic acid encoding a fusion protein). For example, a fusion gene encoding a fusion protein can be prepared by removing a stop codon from a first DNA sequence encoding a first protein and then appending a DNA sequence encoding a second protein within the frame. The resulting fusion gene sequence is then expressed by cells as a single fusion protein. Fusion proteins may include linker (or "spacer") sequences that can promote the appropriate folding and activity of each domain of the fusion protein. Fusion proteins may also include epitope tags (tagged peptides) for identification (e.g., Western blotting, immunofluorescence, etc.) and / or purification. Non-limiting examples of currently used epitope tags include HA, myc, FLAG, and 6-HIS. These known epitope tags are relatively short peptides that can be specifically detected by monoclonal antibodies that bind to a second tag attached to the epitope tag on a target macromolecule.

[0066] If a molecule contains two sequences that are not normally found together in the same polypeptide chain, then the fusion protein will be a " Chimeric Chimeric molecules can also contain fusions of two different polymers, such as a single polypeptide chain comprising a target macromolecule and a labeled peptide. Chimeric molecules can also contain a labeled peptide chemically linked to the target macromolecule. As defined, a chimeric molecule may contain a... Labeled peptides Connection or fusion Target macromolecules Furthermore, the target macromolecule is fused or linked to a third polypeptide, which is used to increase the effective concentration of the target macromolecule during partial denaturation and detection.

[0067] For the purposes of this invention, the proteins used in the assay are typically target human proteins intended as drug targets, and are prepared in active form using recombinant methods and contain known protein binding sites. However, the definition of "protein" herein specifically includes protein fragments that are not full-length proteins, but only fragments containing sufficient structure to have the necessary secondary and tertiary structures and binding sites with the target compound.

[0068] As used in this article, the term " Target macromolecules"Macromolecule" refers to a variety of variable macromolecules. That is, they have secondary or tertiary structures that can be eliminated by heating or other alternative reagents. These are, for example, DNA, RNA, and / or proteins (which include protein fragments such as protein domains). In some cases, a "macromolecule" can have a relatively small molecular weight compared to a full-length protein, provided that it has a strictly defined native three-dimensional structure through cross-linking, hydrogen binding, etc. For example, desmin peptides have a strictly defined tertiary structure that can be measured by this assay. The term "macromolecule" also refers to polynucleotides, polypeptides, or complex carbohydrates with a defined tertiary structure. For example... Glycans, typically existing as glycoproteins or glycolipids, form highly complex structures. In mammals, ten monosaccharides are used to construct glycoconjugates in the form of oligomers (up to about twelve monomers) and polysaccharides. The “macromolecule” used in this paper is a macromolecule that can be denatured by significantly disrupting this three-dimensional structure. The “target” macromolecule is a macromolecule capable of specifically binding to a third molecule, typically a small molecule or other pharmaceutical candidate. The target macromolecule used in this assay may be purified, present as a cell extract, or in other forms. Target macromolecules may inherently possess specific binding partners, and small molecules are designed to target this binding.

[0069] the term" Small molecules "Small organic molecules" is a term recognized in the art and refers to compositions with a molecular weight of less than about 2000 amu, or less than about 1000 amu and even less than about 500 amu. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptide nucleic acids, peptide mimics, carbohydrates, lipids, or other organic (carbon-containing) or inorganic molecules. Many pharmaceutical companies have large libraries of chemical and / or biological mixtures, typically fungal, bacterial, or algal extracts, which can be screened using any of the assays of this invention. The term "small organic molecule" refers to synthetic or purified natural small molecules that are typically identified as organic or pharmaceutical compounds and does not include molecules containing nucleic acids, peptides, or polypeptides.

[0070] As used in this article, the term " Combination "Binding" refers to the binding of a small molecule, protein, or compound to a protein in a cell or solution. The term "binding partner" or "member of a binding pair" refers to a molecule that specifically binds to another molecule to form a binding complex, such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc. In a particularly preferred embodiment, the binding is primarily mediated by non-covalent (e.g., ionic, hydrophobic, etc.) interactions and occurs between the small molecule and its target and / or between two proteins that specifically bind to each other during cellular processes.

[0071] the term" Enzyme fragment complementarity (EFC)As explained below, this involves the use of an enzyme fragment, which may be referred to as a labeled peptide or ED (enzyme donor), that does not possess enzymatic activity until it is combined with another enzyme fragment, called an EA or enzyme acceptor or second labeled enzyme fragment. Refer to β-galactosidase for the use of the terms ED and EA. However, the terms EFC or labeled peptide are not limited to β-galactosidase systems. EFC is a general term describing the combination of enzyme fragments to form an active enzyme, which is then detected by measuring hydrolysis (usually by colorimetry, fluorescence, or chemiluminescence). As part of the detection system, it has the advantage of providing an amplification step due to enzyme conversion.

[0072] As an illustration, EFC assays based on dihydrofolate reductase or β-lactamase have been used to quantify the effects of the drug rapamycin on its targets in live cells (Remy, I. and Michnick, SW, Clonal Selection and InVivo Quantitation of Protein Interactions with Protein Fragment Complementation Assays. Proc Natl Acad Sci USA, 96:5394-5399, 1999; Galarneau, A., Primeau, M., Trudeau, L.-E. and Michnick, SW, A Protein fragment Complementation Assay based on TEM1β-lactamase for detection of protein-protein interactions, Nature Biotech. 20:619-622, 2002) and to study the phosphorylation-dependent interaction between the two domains of the cyclic AMP response element binding to the protein CREB (JM Spotts, RE Dolmetsch, & ME Greenberg, 2002, Time-lapse imaging of a dynamic phosphorylation-dependent protein-protein interaction). inmammalian cells, Proc. Natl. Acad. Sci. USA 99:15142-15147.).

[0073] Another example of an EFC available in this article is the Promega NanoBiT. TMThis technology utilizes NanoLuc, a small (19 kDa) luciferase engineered for structural stability and the generation of strong, stable bioluminescent signals. It is described in detail in Dixon et al., “NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells,” ACS Chem. Biol., a recently accepted manuscript, published online: November 16, 2015. Through design, the NanoBiT subunits (i.e., the 1.3 kDa peptide and the 18 kDa polypeptide) weakly associate, such that their assembly into luminescent complexes is determined by the interaction characteristics of the target proteins to which they are attached.

[0074] the term" Labeled peptides "Labeled peptide" refers to a peptide that essentially lacks secondary structure (i.e., random coil) and is used as a label for detecting proteins or protein fragments (e.g., target peptides) fused with it, without substantially affecting the stability of the target peptide. Labeled peptides are typically less than 100 amino acids in length. They can be used as labels on their own, or they can provide epitopes for antibody recognition. As explained below, labeled peptides are selected so as not to affect the stability of the fusion partner of the labeled peptide.

[0075] As is known in the art, the term " ED "This refers to the enzyme donor fragment used in the β-galactosidase fragment complementation assay. Examples of EDs are given below. Except for up to two amino acid changes, an ED that is "substantially identical" to one of SEQ ID NO: 1-3 will be 100% identical.

[0076] As is known in the art, the term " EA "This refers to the enzyme acceptor fragment used for the assay of β-galactosidase enzyme fragment complementation."

[0077] the term" transsexual "When used in its conventional sense, 'natural' refers to the process in which a protein, nucleic acid, or other macromolecule or macromolecular structure (e.g., ribosome) at least partially loses its quaternary, tertiary, and / or secondary structure. This loss of the native state occurs through the application of external stress or compounds such as strong acids or bases, concentrated inorganic salts, organic solvents (e.g., alcohols or chloroform), radiation, or heating. The term used herein specifically includes partial denaturation, where only a portion of the molecules (e.g., proteins) in a mixture are denatured. The term 'protein melting,' referring to protein denaturation, is also used herein. As is known, the melting temperature (Tm) can be determined from a protein denaturation study. See US 2013 / 0217137 for details."

[0078] the term" Pulse denaturation "Pulsed denaturation" refers to a process in which proteins or nucleic acids are denatured by applying short thermal pulses for more than one cycle, followed by a short period of reequilibration at room temperature or below. A "mild temperature" is considered to be at least about 5°C lower than the melting temperature of the heated macromolecule. The melting point of a protein can be determined by known methods, such as those described in US20140057368 mentioned below. Pulsed denaturation protocols can induce a small amount of denaturation (5%-10%) in a given step. However, denaturation accumulates over multiple cycles and can reach 80% or more denaturation after the application of the pulse. For example, a pulsed denaturation protocol may comprise 10-70 pulses at 37-50°C for 5-10 seconds, separated by cooling pulses of 15-20 seconds. As shown below, pulsed denaturation can be performed under conditions where a portion of the denatured protein in one cycle does not recrystallize upon cooling or during another pulsed denaturation step.

[0079] the term" protein denaturation "" refers to protein denaturation involving the disruption or potential disruption of secondary and tertiary structures.

[0080] Denaturation disrupts the normal α-helices and β-sheets of proteins, unfolding them into arbitrary shapes, thereby causing protein precipitation or coagulation. Protein denaturation leads to the loss of its biological function and / or activity. Denaturation can be caused by external stresses or compounds such as strong acids or bases, concentrated inorganic salts, organic solvents, radiation, heat, or cold. Denaturation can be complete or partial, but is sufficient to cause insolubility for the purposes of this invention. Pressure denaturation of proteins is described, for example, by Johnson et al., “Pressure and Protein Denaturation,” J. Biol. Chem. 1946, 163: 689-698. Frick, “Effect of Ionizing Radiation on Protein Denaturation,” Nature 169, 965-966 (June 7, 1952), describes the use of ionizing radiation to denature protein solutions.

[0081] the term" Inactive exogenous peptides "or" Inactive exogenous polypeptide sequences "" refers to a sequence engineered to be expressed in the chimeric protein used in this method, which is added to provide additional sequence to the fusion protein without participating in or affecting the binding events in the assay. It is used to modulate the denaturation of the chimeric protein. The peptide can have any length, such as 20-1000 amino acids, and is exogenous in the sense that it is not part of the fusion protein unless artificially encoded, and furthermore, it does not participate in the binding of the peptide label to the second label. Additionally, it does not bind to the target compound.

[0082] the term" thermal stability"Refined" refers to the property of macromolecules such as proteins to resist irreversible changes in their chemical or physical structure at high relative temperatures.

[0083] the term" Thermal transformation determination This refers to assays based on the principle that purified proteins denature and unfold at specific temperatures, and that the binding of ligands to the protein makes it thermally stable. Further details on various thermal shift assays can be found in Nordlund, US20140057368, "Methods for determining ligand binding to a target protein using a thermalshift assay." As mentioned above, such assays can be performed using unpurified samples.

[0084] the term" homogeneous "Used in its standard sense, it refers to a form and method of determination that does not require a separation step. This allows for measurement of results through a simple mixing and reading procedure, without the need to process the sample during the determination via separation or washing steps."

[0085] Overview

[0086] It is well known that ligand binding protects proteins from thermal denaturation, and this concept has been used to measure drug-target interactions in cellular and solution environments (“Cellular Thermal Shift Assay”; “CETSA”) (Science (2013), cited above). CETSA is based on the principle that denatured proteins aggregate into complexes that can be removed by centrifugation, and subsequent publications by the same group and others have shown that this concept can be applied to intracellular proteins and intact membrane proteins (see Reinhardt et al., “Thermal proteome profiling monitors ligand interactions with cellular membrane proteins,” Nature Methods 12, 1129–1131 (2015)). This article describes an extended thermal proteome profiling analysis to detect transmembrane protein-small molecule interactions in cultured human cells. When they assessed the effect of detergents on ATP-binding characteristics, the authors observed a shift in the denaturation temperature of ATP-binding transmembrane proteins. Aliquots were heated to different temperatures, digested, labeled with a 10-fold tandem mass spectrometry tag (TMT10), and analyzed by mass spectrometry.

[0087] The method of this invention can be used to measure other binding interactions, such as those described by Huber et al., “Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling,” Nature Methods 12, 1055–1057 (2015). In that article, the authors demonstrate a binding quantitative mass spectrometry method that allows for the systematic investigation of protein binding between cellular metabolites and drugs. The authors dissected the targets of the drugs methotrexate and (S)-crizotinib, as well as the metabolite 2'3'-cGAMP, in intact cells and identified the 2'3'-cGAMP homologous transmembrane receptor STING, which is involved in immune signaling.

[0088] However, it should be noted that this prior art method requires the removal of denatured and aggregated proteins through impractical high-gravity centrifugation steps, and the quantification of soluble proteins protected by ligand binding at high temperatures through cumbersome, relatively imprecise, and low-linear dynamic range assays (such as Western blotting, ELISA, and mass spectrometry). Most reported CETSA data have been collected for naturally expressed proteins present at native levels in a given cell line or tissue; however, in at least one report, membrane proteins expressed as fusions with green fluorescent protein (GFP) have been described [Structure 20, 1293–1299, August 8, 2012]. In this study, the GFP component of the fusion protein was used for protein detection in analytical size exclusion chromatography and not for direct assessment of the protein's structural state.

[0089] This invention relates to the inventor's patent application entitled "Homogeneous Thermal Ligand Binding Assay," which is incorporated herein by reference. It describes a biochemical (in vitro, non-cellular) method for detecting ligand-protected target proteins after thermal denaturation using a recombinant target protein fused to a short "enzyme donor" (ED) peptide derived from β-galactosidase. By simply adding a β-galactosidase enzyme receptor (EA) protein, the ED peptide (also known as ProLabel or PL) can be detected at femtomolar concentrations in a homogeneous assay. This EA protein is complementary to the trans-ED, resulting in stable β-galactosidase activity (enzyme fragment complementation (EFC)). This detection system is not only sensitive but also exhibits a linear dynamic range of 4-5 orders of magnitude. Furthermore, ED-labeled proteins denatured / aggregated due to heat-induced unfolding are significantly insufficient to complement the EA, resulting in lower activity in this form. Therefore, for ED-labeled proteins, the impractical centrifugation steps required in the aforementioned scientific paper are unnecessary, as are immunoassays or mass spectrometry-based readouts. Furthermore, this invention differs from the use of GFP fusions that serve only as detection tags (see the reference above) because the ED tag in our system is both a detection tag and reports the protein's folding state.

[0090] Therefore, the use of recombinant ED-labeled proteins (purified or in crude cell extracts, and possibly in the cellular environment) solves all the procedural bottlenecks mentioned above for current heat-denaturing-based ligand screening methods: 1. No need for pure proteins; 2. Requirement for low target protein concentrations (<10 nM); 3. Simplicity, sensitivity, dynamic range, and accuracy of assays (homogeneous EFC reaction versus immunoassay); 4. High throughput (no centrifugation step or immunoassay required).

[0091] The methods described herein can be applied to target macromolecules, such as proteins, in a cellular environment. That is, the target macromolecule can be expressed within cells (or cells in tissues), preferably mammalian cells, and the compound can then be exposed to the cell in its intact state, i.e., across the intact plasma membrane. The method of the present invention for thermal denaturation in the presence of the test compound can be performed simultaneously with the target molecule and the compound contained within a living cell. The term “in cell” is broadly used to refer to target macromolecules that are entirely within the cell (e.g., cytosol, nucleus, inner membrane, etc.) or macromolecules attached to the cell membrane. In this case, the target molecule may have extracellular domains. (The term is being used in the context of “InCELL Pulse”.) TM The system name of this invention, a commercially developed method, includes heating cells to produce a thermal transition in enzyme complementarity, such as... Figure 14-18As illustrated, the InCELL Pulse system can quantitatively detect the binding of small intracellular ligands to cytoplasmic target protein domains expressed as ED-enzyme fragments (e.g., PL) fusions, and has been demonstrated with various target classes, including kinases, bromodomain proteins, methyltransferases, and hydrolases. This method can also be applied to intact membrane proteins, for which cell penetration of the ligand may or may not be required to achieve target binding.

[0092] Furthermore, Examples 13-16 use a single heating pulse instead of a series of short heating pulses (pulse denaturation), as used in the previous examples herein. Additionally, cell-free biochemical applications of this technique (e.g., Examples 8-11) have been demonstrated using N-terminal fusions of target proteins with the N-terminal NFκB DNA-binding domain (not shown in the examples). In some cases, the addition of additional amino acid sequences (hereinafter referred to as “inactive exogenous peptides”) enhances assay performance. The NFκB moiety is expected to be more critical for cell-free assays (e.g., Examples 8-11) than for cellular assays (e.g., Examples 13-16), but may be beneficial in some InCELL Pulse assays. Inactive exogenous peptides can be attached to the N-terminus of the target macromolecule (protein), and labeled peptides can be attached to the C-terminus of the target macromolecule.

[0093] This method monitors and measures the binding properties between a compound and a target macromolecule. It utilizes the known ability of ligands, such as small molecules, to protect macromolecules like proteins from denaturation and aggregation due to unfolding, and demonstrates that this method can be applied to evaluate ligands within whole cells (e.g., mammalian cells already transfected with chimeric proteins). Existing methods require high-gravity centrifugation steps and insensitive readouts. The method of the present invention, exemplified by the use of β-galactosidase enzyme fragment complementation, utilizes a small peptide (less than 100 amino acids) fused to a macromolecule. The small peptide can be a known enzyme donor, such as ProLabel, commercially available from DiscoveRxCorporation, Fremont, CA. TM (“ED”). Enzyme fragment complementarity provides a sensitive, simple, and universal readout. Furthermore, the assay can be performed in a homogeneous form, meaning that no physical separation reagents are required, thus eliminating the need for filtration, decantation, centrifugation, etc. This invention is broadly applicable to many ligand binding assays.

[0094] The ligand is typically a small molecule whose binding properties with a target molecule are being studied, which would be a large molecule susceptible to thermal denaturation and thus loss of buoyancy or stability in the fluid. Binding properties can be measured in the presence of a competitor or with different target macromolecules. This invention is illustrated by studying BRD4 (bromine-containing domain protein 4), but is not limited thereto. As demonstrated herein, this method provides:

[0095] 1. Universal homogeneous direct ligand binding assay;

[0096] A simple and quick method (no washing, filtering, or centrifugation steps required)

[0097] - No protein purification required

[0098] - No fluorescent tag or antibody

[0099] - Target proteins present in <10 nM (in vitro implementation method)

[0100] -Utilizing DiscoveRx proprietary enzyme fragment complementation

[0101] (EFC) technology

[0102] 2. BRD4(1) Working Example

[0103] - Highly accurate (median %CV (variance coefficient)) );excellent

[0104] Signal-to-noise ratio

[0105] - Correct order of high and low potency inhibitors

[0106] 3. Application

[0107] - High-throughput screening (384-well compatible)

[0108] - Hit confirmation and potency ranking

[0109] 4. Target Category

[0110] - Bromodomain; pharmaceutical target proteins such as kinases, G protein-coupled receptors, methyltransferases, RAS, MAPK and MSK1 signaling molecules, nuclear receptors, ion channels, methyltransferases, nucleoside hydrolases, etc. Preferred target categories are human drug targets.

[0111] This method can also be performed using a series of separate heating pulses followed by cooling. The heat applied to the protein and (potential) ligand mixture described above is used to denature the protein; the resulting activity can be as follows: Figure 1 and Figure 2Measurements are performed as shown. That is, the described method can include pulse denaturation protocols in which a protein sample (+ / - ligands) is subjected to several cycles (e.g., 10-200 cycles) of short heating pulses at a mild temperature, followed by a short reequilibration at room temperature or lower. These “pulse” protocols are readily performed using standard programmable thermal cycling instruments. This method avoids the need for high denaturation temperatures and long denaturation times, thus increasing the sensitivity of methods for detecting inhibitor binding. In light of this teaching, optimal heating time, cooling time, cooling temperature, and heating temperature can be routinely determined. These will be based on the biophysical properties of the target macromolecule under study, including the temperature at which 50% of the molecule has unfolded (this temperature is referred to as the melting temperature or T). m Other factors to consider include the expected affinity of the compound to be tested, the concentration in the study, etc.

[0112] General methods

[0113] Proteins, along with other macromolecules, are among the most studied and targeted macromolecules in the pharmaceutical industry. Numerous methods have been devised and developed to study proteins, their structures, their chemistry, protein-protein interactions, protein-test compound interactions, and the biological pathways involved in proteins, as well as to determine the binding of small molecules to target proteins. However, all these processes and measurements require proteins to be stable and active, as proteins are susceptible to degradation or aggregation due to a variety of factors.

[0114] Macromolecules such as proteins denature when exposed to high temperatures, leading to precipitation and aggregation. The binding of compounds to macromolecules can increase their thermal stability and can therefore be used in binding assays, where the target macromolecule bound to the compound can be measured by measuring its thermal stability. Thus, compounds binding to the active or allosteric sites of macromolecules such as proteins will form complexes and affect their thermal stability. This will result in the compound remaining stable even at higher temperatures. As is known, thermal transition assays measure the thermal stability of a target protein and the increase in the protein's melting temperature after subsequent ligand binding. Numerous assays have been designed to investigate the thermal stability of proteins and to study buffering conditions, ligands, cofactors, drugs, and other compounds that affect this stability to identify and further characterize protein complexes. Another form of thermal transition assay for measuring ligand binding to target proteins has also been disclosed. This assay works for both purified and unpurified protein samples, even at low concentrations. However, this assay requires the removal of denatured, precipitated proteins by centrifugation and quantification of stable proteins by low dynamic range immunoassays (e.g., Western blotting or ELISA).

[0115] Therefore, a simple, sensitive, high-throughput, and accurate readout assay is still lacking to determine ligand binding in extracts, cells, and tissues.

[0116] This invention discloses a homogeneity binding assay to determine how ligand-macromolecule interactions and further interactions lead to the stabilization of macromolecules. More specifically, this invention discloses a homogeneity binding assay to determine how ligand-protein interactions and further interactions lead to the stabilization of proteins under heat stress.

[0117] Now for reference Figure 1 and Figure 2 A macromolecule 101 with a secondary or tertiary structure is chemically linked to a tag 102, which can be an enzyme fragment or a fusion protein containing a protein macromolecule. The test compound 104 is mixed with the labeled native macromolecule in a fluid environment under buffer and physical conditions, which allows the test compound to bind to the native macromolecule with the evaluated specificity. Non-specific binding is minimized by the buffer conditions. The buffer may contain a low concentration of detergent (0.05% Tween 20). Non-specific binding is also minimized because all other proteins in the crude extract hinder non-specific binding. Target binding is typically specific and high-affinity binding. The test compound can bind to a naturally designed specific binding site to accept and bind ligands. During this incubation, as shown at 106, the test compound is bound to a chimeric molecule containing a target macromolecule linked to a labeled peptide. As shown at arrow 108, a thermal denaturation step is applied to the construct in the mixture. Then, as indicated by arrow 110, complementary enzyme fragment 112 is added to the mixture, complementing tag 102 on the macromolecule, where denaturation is minimized by testing the binding of the compound. As indicated by 114, the enzyme substrate or substrate mixture is added to the active enzyme found in the mixture, and the luminescent reaction is read using standard optical methods.

[0118] exist Figure 2 In, it is shown Figure 1 The deduction from the schematic diagram. Here, in relation to... Figure 1 Under similar conditions, test compound 104 does not bind to macromolecule 101. The thermal denaturation treatment in 208 causes macromolecules in the mixture to form aggregates 210. Aggregation increases with the degree of denaturation. Therefore, as shown in step 214, when enzyme fragment 112 is added at 110, no active enzyme is produced or minimally active enzyme is produced. The denaturation of some or all of the macromolecules effectively isolates their tags 102, thereby reducing the signal from the mixture.

[0119] Figure 1 and Figure 2The process may occur simultaneously in the same mixture and reaction. The degree of binding can be measured at different concentrations and / or in the absence or presence of binding inhibitors. In this case, the "test compound" can be a known binding agent, and the evaluation is an evaluation of the test inhibitor.

[0120] macromolecules

[0121] This invention provides an assay system for detecting macromolecule-ligand interactions using EFC or fluorescent protein complementation after heat stress and denaturation. The macromolecules tested are typically large molecules, usually polymerized from smaller subunits (amino acids and nucleotides). Target macromolecules include polynucleotides, proteins, and carbohydrates with defined three-dimensional structures. The macromolecules of this invention are denatured, i.e., lose their quaternary (subunit), tertiary, and / or secondary structures by applying external stress (such as external compounds, radiation, or heat).

[0122] The target macromolecule can be a full-length protein or a target protein domain, which can be complexed and bound, for example, by a small molecule agonist or antagonist of the target macromolecule. The target macromolecule is prepared in a chimeric form using recombinant DNA methods. The chimeric protein may include a peptide tag located at the amino or carboxyl terminus of the target macromolecule. Additionally, the target macromolecule can be further fused to an inactive exogenous polypeptide sequence that has no binding activity in the assay, but provides additional amino acid sequence (mass) to the target macromolecule for partial denaturation. For example, the NF-κB DNA-binding domain described in Table 1 of U.S. Patent 9,110,054 (SEQ ID NO: 5 in that document), entitled “Detectable nucleic acid tag,” can be conveniently fused to the target macromolecule at an end opposite to the terminus containing the labeled peptide. The NF-κB binding amino acid sequence binds to a specific DNA sequence and is not known to bind to any small molecule drug candidate. Other inactive exogenous polypeptide sequences can be cloned into the target polypeptide and used, provided they are known not to interact with the test compound, labeled peptide, or target macromolecule.

[0123] Labeled peptides

[0124] Attaching labeled peptides to target macromolecules facilitates the detection of macromolecular stability, compound-to-macromolecule binding, inhibitors of compound-to-macromolecule binding, and allosteric modulators, among other applications. Many labeled moieties can be used to detect ligand-to-macromolecule binding. Complementary reporter subunits derived from β-lactamases can be constructed and utilized. The activity of complementary β-lactamases can be detected using β-lactamase substrates developed in the art, comprising a fluorescent donor moiety and a quencher, wherein the attached group is hydrolyzed after the substrate enters the cell. The fluorescence energy transfer between the donor and quencher can then be monitored as an indicator of β-lactamase activity, as described in PCT WO 96 / 30540, published October 3, 1996.

[0125] Green fluorescent protein (GFP): This protein is isolated from marine organisms and exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. GFP is labeled with target proteins to create fusion proteins, which can be measured using fluorescence microscopy after binding to compounds that affect the protein's thermal stability.

[0126] Alternatively, chromogenic peptide substrates can be used, where the enzymatic cleavage of the p-nitroaniline amide bond in the chromogenic peptide substrate leads to the release of the chromophore p-nitroaniline. The reaction can be monitored spectrophotometrically.

[0127] Other labeling motifs include those from ras-based recruitment systems (RRS and SOS), fusion protein-based systems such as the yeast two-hybrid system, etc. The labeling motif can be coupled to the target macromolecule using any suitable method. The labeling motif can be directly attached to the target macromolecule or via a linker. Enzymes capable of catalyzing the direct or indirect conversion of substrates into detectable reaction products, such as β-glucuronidase, alkaline phosphatase, peroxidase, luciferase, and β-galactosidase, can also be used as labeling motifs.

[0128] β-galactosidase (β-gal) is encoded by the *E. coli* lacZ gene and can be used as a marker peptide. Enzyme activity can be monitored by various methods, including live-cell flow cytometry and histochemical staining using chromogenic substrates. β-gal enzymes and their fragments (see U.S. Patent No. 4,708,929) need to possess several characteristics. These fragments are essentially inactive individually, as the background (if any) of only one fragment in the presence of the substrate is negligible. Secondly, the fragments have sufficient affinity for each other, i.e., the fragments will bind to provide an active enzyme in the absence of other bindings, such as through an entity fused with the fragment. Small fragments (“ED” or “PL”) can be engineered and will not interfere with the biological activity of the gene or protein with which they are fused. As determined herein, the resulting fusion protein will fold appropriately and retain active sites, including enzyme activity, binding activity with other proteins, translocation capacity, etc. EDs are typically at least about 37 amino acids, typically at least about 40 amino acids, and typically no more than about 110, more typically no more than about 90.

[0129] The β-galactosidase complementation system described herein is a system composed of two or more β-galactosidase fragments or variants thereof. For example, in some embodiments, the complementation system comprises first and second fragments of a β-galactosidase (e.g., α and ω fragments). In other embodiments, the complementation system may comprise more than two β-galactosidase fragments, such as first, second, and third β-galactosidase fragments (e.g., α, β, and ω fragments). In this application, the small fragment of β-gal (which is also the signal-generating peptide) will be referred to as the enzyme donor (ED). The signal-generating peptide is one of a pair of fragments of an enzyme reconstructed when the two fragments, namely the enzyme donor (“ED”) and the enzyme acceptor (“EA”), are complexed together. The ED will be a fragment of an enzyme that is complementary to the other fragment EA to form an active enzyme. Due to the affinity between the fragments, the ED fragment of the fusion protein will complex with the EA fragment.

[0130] In other embodiments, the complement fragment is a high-affinity fragment. The high-affinity component is typically two fragments of the enzyme, wherein the fragments have sufficiently high affinity that they can spontaneously bind together and reform into a fully functional enzyme or enzyme subunit. Typically, when mixed under appropriate conditions in solution, at least 5% of the native enzyme's enzymatic activity is obtained, sometimes about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of the native enzyme's enzymatic activity. This activity can be determined using conventional methods, wherein the concentrations when comparing the complementary enzyme and the parent enzyme are typically, for example, 10... -3 Up to 10 -6The ranges between M are the same. The high-affinity component allows monitoring of the presence or increase of complementary fragments forming complexes with interacting partners. For example, if a complementary fragment is present in a fusion protein (fused with a target macromolecule) and forms a complex with a test compound, incubation with the second fragment after cell lysis will result in detectable enzymatic activity, allowing for the analysis of cell interactions. Therefore, if the amount of the high-affinity component in the assay system increases, the amount of detectable enzymatic activity will increase proportionally. Typically, a significant increase in activity can be detected with a reporter component ratio as high as 1:1. A rational approach for use with an initial high-affinity β-galactosidase complementary reporter system is reviewed, for example, in the experimental section below and in U.S. Patent Application Serial No. 11 / 132,764, filed May 18, 2005.

[0131] In one specific implementation, the α-peptide used is a peptide stably complementary to the ω-peptide in mammalian cells. The minimal α-peptide with high affinity allows for sensitive and precise analysis of cell-cell interaction events between various intracellular entities, where detection requires only minimal interaction. Exemplary α-peptides (enzyme donors) include...

[0132] (Wild-type ED) SEQ ID NO:

[0133] MGVITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQL

[0134] (ProLabel TM Or “PL”ED)SEQ ID NO.2:

[0135] NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR and

[0136] (Modified ED, W34Y), SEQ ID NO.3:

[0137] MGVITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASYRNSEEARTDRPS

[0138] QQL.

[0139] A range of methods are available for measuring the enzymatic activity of β-galactosidase, including live-cell flow cytometry and histochemical staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal). See, for example, Nolan et al., Proc. Natl. Acad. Sci., USA, 85:2603-2607 (1988); and Lojda, Z., Enzyme Histochemistry: A laboratory Manual, Springer, Berlin, (1979). Currently disclosed methods and materials also include important substrates for β-galactosidase that can be used in live cells. For example, the fluorescent substrate, halogenated β-galactosidase bis-aminopropyl polyethylene glycol 1900 (RGPEG), has been described. Minden (1996) BioTechniques 20(1):122-129. This compound can be delivered to cells via microinjection, electroporation, or various bulk-loading techniques. Once inside the cell, the substrate cannot escape through the plasma membrane or gap junctions. Another important substrate that can be used to practice the methods and materials of this disclosure is fluorescein di-β-D-galactopyranoside (FDG), which is particularly suitable for analysis by fluorescence-activated cell sorting (FACS) and flow cytometry. Nolan et al., Proc. Natl. Acad. Sci., USA, 85:2603-2607 (1988) and Rotman et al. (1963) Proc. Natl. Acad. Sci., USA 50:1-6.

[0140] Reconstituted β-galactosidase can also be detected using chemiluminescence assays. For example, cells containing β-galactosidase fusions are lysed (with or without contact with a cross-linking agent) in a buffer mixture containing a Galactolight Plus substrate from the Galactolight Plus Assay Kit (Tropix, Bedford Mass.). Bronstein et al., J. Biolumin. Chemilumin., 4:99-111 (1989). After adding a luminescence promoter solution, luminescence is measured in a luminometer or scintillation counter.

[0141] Fusion protein

[0142] As disclosed in this application, a target macromolecule is fused with a labeled peptide to form a fusion protein. The fusion protein comprises a single, continuous linear polymer of amino acids, which includes all or part of the sequences of two or more macromolecules or two or more different proteins. Methods for constructing fusion proteins are known in the art. The fusion protein gene construct may also comprise a single, continuous linear polymer of nucleotides encoding all or part of the sequences of two or more different proteins within the same unbroken reading frame. Furthermore, the fusion gene construct of the present invention is introduced into cells to determine ligand binding after denaturation. The fusion gene construct can be introduced into cells by any nucleic acid transfer method known in the art. Different fusion gene constructs encoding unique fusion proteins may be present on different nucleic acid molecules or on the same nucleic acid molecule.

[0143] Fusion proteins may also contain a target macromolecule fused to the β-gal fragment as a labeled peptide. Therefore, in some embodiments, such as those used in this assay, the fusion protein comprises a target protein and a labeled peptide, which, upon processing, is contacted with a second label (enzyme fragment, antibody, etc.) to generate a signal.

[0144] Using proteins as target macromolecules (target proteins)

[0145] The protein used as a target macromolecule can be any desired polypeptide or protein that could potentially be a target (e.g., for study or other characterization). Target proteins can include transferases, oxidoreductases, hydrolases, ligases, and isomerases, along with kinases, phosphatases, carboxylases, phosphodiesterases, dehydrogenases, oxidases, peroxidases, proteases, signaling proteins, metalloproteins, cytoplasmic proteins, and nuclear localization proteins. Target proteins can be obtained from any source, such as naturally occurring sources like cells, tissues, biological fluids, tissue biopsies, soil, water, etc.

[0146] The bromodomain protein family is described in the SCOP database: http: / / scop.mrc-lmb.cam.ac.uk / scop-1.75 / data / scop.bbec.bbhtml. Of particular interest are the BET family of bromodomain proteins, such as BRD1-BRD4 and related proteins, described by Filippakopoulos et al., “Selective inhibition of BET bromodomains,” Nature 468:1067-1073 (December 23, 2010).

[0147] Target proteins may also include cell membrane proteins, defined as proteins that interact with biological membranes and encompass both intact and peripheral membrane proteins. Target proteins may also include signaling proteins that control basic cellular activity and coordinate cellular processes.

[0148] engineered cells

[0149] This invention considers the use of transgenic cells engineered to carry a fusion protein that expresses (or overexpresses) a target macromolecule and a labeling peptide as described herein. The labeling peptide may be the PL at the C-terminus of the target protein.

[0150] The host cell used in the embodiments herein is HEK293 (human embryonic kidney cells). This cell line is available from ATCC and other suppliers. Methods for engineering HEK cells to express transgenes are known. For example, see Doering et al., “Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A,” Mol Ther. July 2009; 17(7):1145–1154 and US 20130344537, “Mammalian expression vectors and uses thereof,” published December 26, 2013, for details on the generation of cells that can be used herein.

[0151] Other cell lines can be engineered for use in this method. These include CHO (Chinese hamster ovary cells), BHK (juvenile hamster kidney cells), NSO (mouse myeloma cells), SP2 / 0 (ATCC CRL-1581), etc. Suitable engineered cells with the PL tag are commercially available from DiscoveRx Corporation, Fremont, CA.

[0152] Reagent test kit

[0153] The present invention also provides kits for practicing one or more of the above-described applications. In some embodiments, the kit includes at least cells constitutively or inducibly expressing a fusion protein comprising a target protein and a β-galactosidase fragment as reviewed above. In some embodiments, the kit includes elements for preparing such cells, such as nucleic acids encoding the fusion protein present on a vector and / or nucleic acids encoding the β-galactosidase fragment, to which the target protein can be fused as reviewed above using standard molecular biology techniques. The kit may also include one or more other components for practicing certain embodiments of the invention, including but not limited to enzyme substrates, cell growth media, etc.

[0154] In some embodiments, the kit may include (a) cells containing an expression vector for expressing a fusion protein comprising a target protein (component I) fused to an ED (enzyme donor) fragment of β-galactosidase, (b) an EA (enzyme acceptor) fragment of β-galactosidase for addition to the cells after incubation with a test compound (component II), and (c) a β-galactosidase substrate for detecting the binding of EA to ED in the fusion protein (component III), and optionally a lysis buffer for lysing cells before or during EA addition. Cell lysis facilitates contact between the labeled peptide (ED) and EA. The kit may also include an expression vector containing multiple cloning sites, allowing the user to insert a selected target protein. The kit may also include an expression vector for expressing a fusion protein comprising a foreign sequence fused to the inserted target protein. The foreign sequence may encode any protein known not to interact in the assay, such as, for example, plant or non-mammalian viral proteins.

[0155] The kit may also contain positive and negative controls associated with the target protein.

[0156] In addition to the components mentioned above, the thematic kit may also include instructions for practicing the thematic methods and complete cells for use with the intracellular implementation plan. These instructions may be present in a variety of forms within the thematic kit, one or more of which may be included in the kit. One form of these instructions is as printed information on a suitable medium or substrate, such as one or more sheets of paper with the information printed on them, in the kit packaging, in packaging inserts, etc. Another form would be a computer-readable medium on which the information is recorded, such as a floppy disk, CD, etc. Yet another possible form is as access to the information at a remote location via a website address accessible via the Internet. Any convenient means may be included in the kit. Example

[0157] This work demonstrates that the thermal denaturation of the BRD4(1)-ED fusion can be measured in a simple, sensitive, and accurate manner using enzyme fragment complementation (EFC). Figure 4 and 5 Furthermore, the observed denaturation can be rescued in a dose-dependent manner in the presence of a small molecule BRD4(1) inhibitor. Figure 3-7 This method is homogeneous and requires no washing or centrifugation. Figure 6 Therefore, it is suitable for high-throughput applications; and the obtained data has high precision. Figure 3 and 7 This method can also be used to distinguish molecules with different efficiencies towards BRD4(1). Figure 7 ).

[0158] Example 1: Measurement of binding of BRD4(1)ED fusion protein to a known ligand (JQ1) .

[0159] The purpose of this experiment is to investigate the binding of the ligand to the BRD4(1) fusion protein under heat stress, and whether ligand binding protects the fusion protein from denaturation.

[0160] To perform this determination, BRD4(1) and ProLabel were used. TM The ED fragment was constructed as a fusion protein [BRD4(1)-ED] containing an N-terminal NFκB DNA-binding domain. The fusion protein from crude cell extract was incubated with a known effective inhibitor (JQ1) at concentrations ranging from 0 to 100 μmol. Incubation was carried out at room temperature for 1 hour. The sample was then heated to 45°C for 30 seconds. EA (along with the luminescent substrate of β-galactosidase) was subsequently added to the sample, and complementarity was measured by measuring luminescence. Figure 3 As shown, the binding and interaction of the inhibitor with the BRD4(1) fusion protein can rescue the fusion protein from denaturation in a dose-dependent manner. Precise accuracy is obtained for duplicate samples.

[0161] Example 2: Measurement of thermal denaturation of BRD4(1) with and without JQ1 .

[0162] This experiment was conducted to investigate BRD4(1) denaturation / aggregation in the presence and absence of an inhibitor. The inhibitor used in this experiment was JQ1.

[0163] For the assay, two separate samples were prepared: one containing BRD4(1) (fusion) without the inhibitor, and the other containing both BRD4(1) (fusion) and the inhibitor. The inhibitor used in this experiment was JQ1 at a concentration of 10 μmol. After incubation, the samples were exposed to elevated temperatures (25°C, 45°C, 50°C, and 55°C, respectively). The precipitate was then separated by centrifugation. Figure 4As can be seen, compared with BRD4(1), JQ1 bound to the fusion protects BRD4(1) from denaturation, without the binding of an inhibitor.

[0164] Example 3: Measurement of denaturation of BRD4(1) fusion protein in the presence of JQ1 .

[0165] This experiment was conducted to investigate how binding to JQ1 protects the BRD4(1) fusion protein from temperature-induced denaturation.

[0166] For experimental purposes, a fusion protein containing NFκB-BRD4(1)-ED was constructed. The fusion protein from cell lysates was incubated with JQ1 (10 μM). Samples were then exposed to multiple temperatures (25 °C, 45 °C, 50 °C, and 55 °C). Figure 5 As can be seen, the folded soluble BRD4(1) is rescued to the greatest extent at 45°C, and the protein slowly begins to maintain its denatured state at higher temperatures.

[0167] Example 4: The effect of centrifugation on measurement readout .

[0168] This experiment was conducted to investigate the importance of centrifugation in the separation of denatured fusion proteins from compositions that also contain soluble-bound fusion proteins, and how it affects the final readout.

[0169] To conduct this experiment, a model containing NFκB-BRD4(1)-ProLabel was constructed. TM (ED) fusion protein. The fusion protein was then incubated with the ligand and luminescently read out in PBS in the presence of cell lysates containing detergent. The samples were then stored at room temperature or exposed to 45°C. Each sample was aliquoted, one aliquot was centrifuged and the other was not centrifuged.

[0170] from Figure 6 As can be seen, there is almost no difference in the determination readout whether the sample was centrifuged to separate the precipitate. Therefore, this experiment shows that centrifugation is not required in this determination, and ligand binding can be determined using EFC from crude cell extract without a physical separation step.

[0171] Example 5: Dose-response curves of known BRD4(1) inhibitors after ligand binding assay .

[0172] This experiment was conducted to determine the dose-dependent binding of ligand proteins after the ligand binding assay described in this application.

[0173] For assay purposes, the fusion protein was constructed as described above. The fusion protein was then incubated separately with ligands (JQ1 and GW334556X), as follows: Figure 7As shown. Incubation at the specified concentration was carried out for 1 hour at room temperature with the extract diluted 1:100 (approximately 10 nMBRD4(1) concentration). The sample was then heated to 45°C and held for 3 minutes, followed by the addition of EA and the luminescent β-galactosidase substrate (rapid substrate) to measure complementarity. Error bars represent 3X standard deviation (99.7% confidence interval). When the compound is in ≥3X K D During concentration screening, interactions with high statistical significance were detected.

[0174] * indicates another method Measured inhibitor KD value (align * with concentration values ​​on the x-axis). Developed by DiscoveRx Corporation, Fremont, CA. The platform measures the interaction between the test compound and a set of bromine domains. For more information, please see: http: / / www.discoverx.com / technologies-platforms / competitive-binding-technology / bromoscan-technology-platform#sthash.ZjaxX6mR.dpuf.

[0175] Example 6: Comparison between the standard modification scheme and the pulse modification scheme .

[0176] The methods of Examples 1-5 can be implemented through multiple heating steps. The following examples relate to a scheme in which a macromolecule (e.g., a protein) is heated to a temperature at which significant denaturation (melting) is not induced in a single heating step but is induced in a series of heating steps. Applying these steps results in a series of heating steps that cumulatively induce denaturation (in the absence of ligands) following a heating step that cannot effectively melt the protein. To achieve this, thermoelectric heating and cooling devices can be employed and pre-programmed to perform predetermined heating and cooling (non-heating) steps. Preferred thermoelectric heating and cooling devices are based on Peltier junctions. These devices can perform active heating and cooling; alternatively, the device can be equipped with a large heat sink. Details of Peltier heating and cooling of the substrate and mask can be found in U.S. Patent 3,161,542. The methods described below can include multiple heating steps separated by cooling steps, and a series of one or more heating temperatures. These figures can be determined using the teachings herein. The method can be carried out based on the properties of the macromolecule as a binding target during the heating steps. Once the temperature T and time t are determined, T can be reduced incrementally, preferably below the melting temperature of the macromolecules, and time t can be subdivided into a series of heating steps, the total of which is at least initially the total time. The cooling step can be based on equipment used to bring the reaction mixture back to or near ambient temperature (about 25°C). Possible, non-limiting combinations include 10-70 pulses at 37-50°C for 5-10 seconds, with cooling intervals of 15 seconds to 2 minutes. Other embodiments are given below. The volume of the reaction mixture should also be considered, with larger volumes indicating the use of more and longer pulses.

[0177] As shown in Figure 8A, the standard denaturation curve consists of a single cycle of high-temperature pulses, followed by an extended denaturation time for protein denaturation. Conversely, the pulsed denaturation curve consists of several cycles of heating pulses at a mild temperature, followed by a brief denaturation and reset to a lower temperature (Figure 8B). The temperatures in Figures 8A and 8B represent heat settings. It is known that the temperature in the mixture will follow a curve. According to this embodiment, the cooling step in Figure 8B will cause the mixture to slope upwards over time. That is, the existing temperature of the mixture during the cooling step remains higher than the temperature that might have been present and measured in the mixture during the previous cooling step.

[0178] The temperature for standard denaturation protocols may be 45°C or higher, while the temperature for pulse denaturation may be 40°C or lower. Denaturation of macromolecules occurs over several minutes in standard denaturation protocols, while in pulse denaturation protocols, a brief denaturation step takes place over several seconds, followed by a temperature reduction to room temperature or even lower before the next heating pulse.

[0179] Example 7: Mathematical Modeling Research .

[0180] Figure 9 The mathematical modeling results show the advantages of pulsed denaturation. The Y-axis in the figure represents the measurement window, defined as the emission signal in the presence of a known ligand divided by the emission signal in the absence of a known ligand after X cycles. The X-axis in the figure represents the number of denaturation cycles, where denaturation is performed by pulsed heating.

[0181] The model is based on the assumption that a small fraction of the folded protein denatures during each cycle and cannot refold or reactivate in subsequent cycles, while incubating the protein with a known ligand saves half or more of the protein from denaturation. When the temperature is lowered to room temperature or below, the protein ligand binding level returns to room temperature or below during each cycle.

[0182] The mathematical modeling diagram shows that even if the denaturation temperature is below the melting temperature, multiple cycles of mild denaturation are expected to produce a stable measurement window.

[0183] Example 8: Study of BRD9-bromosporin binding using standard and pulse denaturation protocols .

[0184] Figure 10 The dose-response curves for BRD9-bromosporin binding following standard and pulse denaturation protocols are shown. Cell extracts containing ED-labeled BRD9 were diluted in PBST and incubated at room temperature with 25 μM bromoxysporin or with 0.1% DMSO / 0.9% MEG (49.5 μL cell extract + 0.5 μL 2.5 mM bromoxysporin or 0.5 μL 10% DMSO / 90% MEG) for 1 hour. The samples were then subjected to thermal denaturation at 45 °C using either the pulse method or the standard (one-step) denaturation protocol. In the first case, the samples were repeatedly exposed to 45 °C for a total heating time ranging from 0.5 to 3 minutes (with 1-minute intervals between heating pulses at room temperature) (“Pulse Denaturation”, black circles). In the second case, the samples were heated for the same total time (0.5 to 3 minutes), but in one step (“Standard Denaturation”, white circles).

[0185] Soluble proteins were then quantified by EFC (heat denaturation makes ED less complementary to EA). The EFC reaction setup was as follows: 5 μL of cell extract was incubated for 30 minutes with 10 μL EA, 10 μL EA dilution buffer, 20 μL rapid lysis buffer, 20 μL rapid substrate, and 135 μL PBS. The assay window was calculated by dividing the RLU signal of the protein under bromoxynil conditions by the RLU signal of the protein under DMSO / MEG conditions.

[0186] As can be seen from the curves, the measurement window was improved by pulse denaturation (6 repeated pulses, a total of 3 minutes of heating), providing improved combined measurement results without prolonged heating.

[0187] Example 9: Study of CREBBP / SGC-CBP-30 combination using standard and pulse denaturation protocols .

[0188] Figure 11 The dose-response curves for the CREBBP / SGC-CBP-30 combination following the standard and pulse denaturation protocols are shown. (CREBBP is the CREB-binding protein with the official symbol CREBBP; SGC-CBP-30 is a small molecule that is a selective inhibitor of CREBBP, with the IUPAC name (S)-4-(1-(2-(3-chloro-4-methoxyphenethyl)-5-(3,5-dimethylisoxazol-4-yl)-1H-benzo[d]imidazol-1-yl)prop-2-yl)morpholine. Cell extracts containing ED-labeled CREBBP and also fused to the DNA-binding domain of NFκB were incubated at room temperature with serially diluted SGC-CBP30 (49.5 μL of cell extract diluted in PBST + 0.5 μL of 100X compound in 10% DMSO / 90% MEG) for 1 hour, followed by heat denaturation under the conditions indicated in the figure. Soluble proteins were quantified by EFC using the same protocol as BRD9. Data were fitted with the Hill equation and EC was calculated.) 50 . Figure 11 The results showed that pulse denaturation exhibited higher signal gain at lower inhibitor concentrations and maintained a dose-response profile across the entire range between the minimum and maximum detectable concentrations; therefore, the sensitivity of assays using pulse denaturation was improved compared to assays using standard denaturation methods. The addition of a second peptide (NFκB) increased the assay window for this target protein (CREBBP).

[0189] Example 10: Studying the binding of ABL1 to a series of small molecule inhibitors using pulsed denaturation. .

[0190] Figure 12 The pulse denaturation technique applied to protein kinase (ABL1) was used to test a range of small molecule inhibitors known in the literature for binding to the target with broad affinity. EC50 of seven inhibitors against ED-labeled ABL1 is shown on the graph. 50Dose-response curves (inhibitor name labels and corresponding curves in the same left-to-right order). The entire procedure used was similar to that described in Example 9, using the following pulse denaturation sequence for 30 cycles: 7 seconds at 42°C, followed by 60 seconds at 25°C. The affinity of the inhibitors for ABL1 decreased from left to right on the graph; the leftmost curve identified the inhibitor with the highest affinity, and the rightmost curve identified the inhibitor with the lowest affinity. The inhibitors, ranked from highest to lowest affinity, were dasatinib, ponatinib, imatinib, VX-680, astrococcus, SU-14813, and purvalanol B. The potency ranking of these seven inhibitors was consistent with the published values ​​(Davis et al. Nat Biotechnol. 20 Oct 2011; 29(11):1046-51. doi:10.1038 / nbt.1990.).

[0191] At each concentration, the inhibitors could be compared in terms of their binding to ABL1. For example, at an inhibitor concentration of 1 nM, dasatinib showed the strongest signal, while asteroidin showed the weakest signal. This means that at this concentration, dasatinib protects ABL1 from denaturation to a much greater extent than asteroidin, a result reflecting dasatinib's higher affinity for ABL1.

[0192] Example 11: Study on the binding of methyltransferase G9a / UNC-0638 using standard denaturation protocols .

[0193] Figure 13 The denaturation protocol applied to the protein methyltransferase (G9a) is shown to test the known inhibitor UNC-0638 using dose-response mapping. UNC-0638 was tested against ED-labeled G9a (containing an inactive exogenous polypeptide sequence with NFκB), and the entire procedure used was similar to that described for Example 9. This proof-of-concept G9a study was performed using a single denaturation step at 50°C for 3 minutes. The measured signal ranged from approximately 10... 2 nM begins to increase and continues to increase until it reaches 10. 4 nM, showing that the protective effect against G9a denaturation increases in a dose-dependent manner with increasing inhibitor concentration. Protection derives from inhibitor binding, and its associated EC50. 50 The value can be derived from this curve. This example, along with other examples disclosed herein, demonstrates that this assay can be used to measure the binding of compounds to a variety of macromolecules.

[0194] This embodiment demonstrates that a single heating pulse lasting 3 minutes at 50°C produces a useful assay for the administration of methyltransferase, and indicates the broad applicability of this method. Reference can be made to current "standard" methods or comparisons between single-pulse and multi-pulse protocols, and improvements to the assay window of this invention are expected through pulsed protocols.

[0195] Example 12: Kit for measuring BRD4(1) inhibitors .

[0196] This exemplary kit was prepared as a 96-well plate format kit for the in vitro biochemical evaluation of BRD4(1) inhibitor potency. The assay uses enzyme fragment complementation (EFC) and pulse denaturation techniques to measure the ligand-dependent thermostability of the target protein. This method is capable of measuring quantitative inhibitory epigenetic EC. 50 Value. This kit provides sufficient reagents for four 96-well plate experiments (16x12 point dose-response curves, duplicate copies), and is targeted at the inhibitor EC. 50 The measurement of values ​​is optimized. The simplified and rapid assay protocol eliminates the need for cumbersome sample handling, plate transfer, or centrifugation steps. The assays provided by this kit are not optimized for single-concentration screening of a set of compounds. The kit can be used to validate screening hits identified by correlation or orthogonal methods; monitor compound potency improvements during lead optimization; obtain rapid results (less than 4 hours from start to finish), with a setup time of less than 60 minutes; and measure inhibitor EC over a wide potency range. 50 Values ​​(high picomol to millimole). This kit includes a fusion of the target protein with a β-galactosidase fragment (“PL”); a positive control; a dilution buffer; and two enzyme substrate reagents – a complementary β-galactosidase fragment PL; and a β-galactoside substrate.

[0197] Instructions for use of the exemplary reagent kit Step 1 This was to prepare serially diluted test and control compounds. A series of dilutions were prepared. Then, BRD4-PL reagent was added and the mixture was incubated. Step 2 It is to establish a complex combination reaction. Step 3 This is the instruction manual for the pulse denaturation protocol. The pulse denaturation protocol is summarized as follows: a. Transfer the PCR plate to a thermal cycler and perform 40 pulse denaturation cycles. One denaturation cycle is defined as: 7 seconds at 40°C, followed by 60 seconds at 25°C. b. After completing the cycling program, the plate can be read immediately via EFC, or stored at -80°C if read later. Ideally, the heating cap of the thermal cycler should be set to 40°C, but some instruments have a default setting of ≥95°C, which is also acceptable. (The manual is incomplete.) step 4 The EFC detection scheme for each well of the measuring plate is described. Step 5 Includes instructions for reading samples on a light-emitting plate reader at 1 second / well and data analysis that will display the dose response of one or more compounds being tested.

[0198] The following examples describe the intracellular method (InCELL Pulse) as described above. TMThe use of ). Generally, these examples use a target macromolecule (protein or protein domain or portion) linked to a labeled peptide, referred to herein as ProLabel. TM The β-galactosidase fragment. These fusion proteins still undergo denaturation in the cells expressing them. Vectors for expressing the fusion proteins in cells to be treated (e.g., heated) can be prepared by conventional recombinant methods, as described in Horecka et al., “Analysis of intracellular modifications,” US20060019285, published January 26, 2006, which are also described in the last paragraph here. Chimeric molecules can be prepared in any convenient cells. Preferably, the same cells used in the assay are partially denatured and are mammalian cells. The following examples use the following scheme:

[0199]

[0200] Example 13: Intracellular methods using kinases and their inhibitors: Figure 15

[0201] Measurement indicators (n=3)

[0202] "n=3" means that the experiment is conducted in triplicate. The displayed value is + / - one standard deviation.

[0203] Inhibitor EC 50 (nM) Measurement window

[0204] Dasatinib 2.8 ± 0.5 g, 5.2 ± 0.38 g

[0205] VX-680 570+ / -230 3.6+ / -0.058

[0206] The published cell titer of dasatinib against ABL1-driven cells: IC50 50 =1nM

[0207] Blood. 2006; 108(7): 2332-2338. The assay used the ABL1 kinase domain expressed together with the C-terminal PL tag (SEQ ID NO: 2).

[0208] Measurement scheme

[0209] ABL1 measurement is basically performed as follows:

[0210] 1. Discard the growth medium from a 10cm plate containing HEK293 cells transfected with a plasmid expressing the ABL1-PL fusion, and wash the cells with PBS. 2. Use 1ml 3. Dilute cells with 10 ml of 1X DMEM 1% FBS medium. 4. Count the cells. 5. Dilute the cells to 100,000 cells / ml in 1X DMEM 1% FBS medium using an appropriate volume for cell counting. 6. Dispense 1 μl of the serial dilution of the compound. 7. Add 50 μl of cells. 8. Incubate at 37°C for 5 hours. 9. Perform pulse denaturation using a standard PCR thermal cycler (MJ Research DNA Engine) in a 96-well black PCR plate from Thermo Scientific (product number AB-0800 / K) under the following conditions:

[0211] Pulse temperature (°C) Pulse duration (seconds) Comparison No pulse 48 180 50 180 50 90 52 90

[0212] 10. Add 60 μL of enzyme fragment complement (EFC) mixture (containing EA, EA dilution buffer, lysis buffer and substrate); 11. Incubate at room temperature for 30 minutes and then read the plate.

[0213] The measurement parameters shown in Example 13 are for a pulse temperature of 48°C and a denaturation time of 90 seconds.

[0214] Example 14: Intracellular methods using bromine domain proteins and their inhibitors: Figure 16 Measurement indicators (n=2)

[0215] Inhibitor EC 50 (nM) Measurement window

[0216] JQ1 390+ / -23 3.5+ / -0.14

[0217] Published JQ1 cell titer for reducing c-Myc expression: IC50 50 ≈100 nM, see Ciceri et al., “Dualkinase-bromodomain inhibitors for rationally designed polypharmacology,” Nat Chem Biol. April 2014; 10(4):305-12. doi:10.1038 / nchembio.1471. Electronic version March 2, 2014.

[0218] Measurement scheme

[0219] The BRD4(1) determination was performed as described in Example 13 above.

[0220] Example 15: Intracellular methods using hydrolases and their inhibitors: Figure 17 Determination of SCH51344 binding index

[0221] Temperature / Time EC 50 (nM) Measurement window

[0222] 45℃ / 1.5min. 370 1.9

[0223] 48℃ / 3.0min. 4600 9.7

[0224] The published SCH 51344MTH1 titer: intracellular IC50 50 <20µM;

[0225] In vitro EC 50 =50nM.

[0226] Measurement scheme

[0227] MTH1 determination is basically performed as follows:

[0228] The MTH1 hydrolase catalytic domain, which is expressed together with the C-terminal PL tag and is also fused with the DNA-binding domain of NF-κB at the N-terminus, was incubated with an inhibitor at 37°C for 3 hours, followed by a denaturation step as described in Example 13.

[0229] For details on MTH1 and its inhibitors, see Huber et al., “Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy,” Nature 508, 222–227 (10 April 2014) and Bessman et al., “The MutT Proteins or “Nudix” Hydrolases, a Family of Versatile, Widely Distributed, “Housecleaning” Enzymes,” J. Biol. Chem. 271(41):25059-25062 (1996).

[0230] Example 16: Intracellular methods using G9a methyltransferase and its inhibitors: Figure 18 UNC0538 combined measurement Set targets

[0231] Inhibitor EC 50 (nM) Measurement window

[0232] UNC0538 13 5.0

[0233] The published titer of UNC0538 cells used to reduce H3K9me2: IC50 50 ≈81nM;

[0234] For details on methyltransferases and their inhibitors, see Vedadi et al., “A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells,” Nature Chemical Biology 7, 566–574 (2011).

[0235] Measurement scheme

[0236] The G9a determination was performed essentially as described in the preceding embodiments. The G9a catalytic domain was expressed together with the C-terminal PL tag.

[0237] After incubating with the inhibitor at 37°C for 3 hours, a single 3-minute denaturation step was performed at 48°C.

[0238] in conclusion

[0239] The above detailed description is intended to illustrate and explain the invention, and should not be construed as limiting the scope of the invention, which is defined by the words and equivalents of the appended claims. Any patents or publications mentioned in this specification are intended to convey details of methods and materials used in carrying out certain aspects of the invention, which may not be explicitly stated but will be understood by those skilled in the art. For the purpose of describing and implementing the mentioned methods or materials, such patents or publications are hereby incorporated by reference to the extent that each is specifically and individually incorporated herein by reference. sequence list <110> THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY Treiber, Daniel K Menichelli, Elena <120> Methods for measuring ligand binding to target proteins and cell conjugation. <130> 3817.55 PCT <150> US 14 / 830,328 <151> 2015-08-19 <150> US 62 / 087,671 <151> 2014-12-04 <150> US 62 / 040,294 <151> 2014-08-21 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 52 <212> PRT <213> E. coli <220> <221> peptides <222> (1)..(52) <223> Wild type ED <400> 1 Met Gly Val Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp 1 5 10 15 Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro 20 25 30 Pro Phe Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro 35 40 45 Ser Gln Gln Leu 50 <210> 2 <211> 42 <212> PRT <213> E. coli <220> <221> peptides <222> (1)..(42) <223> ProLabel ED <400> 2 Asn Ser Leu Ala Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly 1 5 10 15 Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Trp 20 25 30 Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg 35 40 <210> 3 <211> 52 <212> PRT <213> Large intestinal bacteria <220> <221> peptide <222> (1)..(52) <223> W34Y ED <400> 3 Met Gly Val Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp 1 5 10 15 Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro 20 25 30 Pro Phe Ala Ser Tyr Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro 35 40 45 Ser Gln Gln Leu 50

Claims

1. A method for measuring the binding between a compound and a target macromolecule, comprising: (a) Preparing a fluid mixture comprising (i) intact living cells expressing a chimeric protein, the chimeric protein being a fusion protein linking a target macromolecule to a labeled peptide, and (ii) a compound measured to bind to and protect the target macromolecule from denaturation, wherein the target macromolecule is subject to denaturation. (b) Incubate the fluid mixture of step (a) under conditions that allow the compound to bind to the target macromolecule; (c) After incubation in step (b), the target macromolecules in the fluid mixture are denatured under conditions that produce a combination mixture of (i) denatured chimeric molecules that are not bound to the compound and (ii) non-denatured chimeric molecules that are bound to the compound; (d) Contact the combined mixture of step (c) with a second label bound to the labeled peptide to form a complex, wherein less of the complex is formed in (i) a denatured chimeric molecule not bound to the compound than in (ii) a non-denatured chimeric molecule bound to the compound, wherein the complex is an active enzyme, wherein the labeled peptide and the second label are enzyme fragments, and wherein the labeled peptide does not possess enzymatic activity until it is complexed with the second label; and (e) Detect the complex in step (d) to generate a signal indicating a measurement of the binding between the compound and the target macromolecule. The target macromolecules mentioned above are proteins, and The labeled peptide is an enzyme donor ED that is active in complementarity with an enzyme fragment of β-galactosidase and is fused to the C-terminus of a protein that is the target macromolecule, wherein the ED is as shown in SEQ ID NO:

2.

2. The method of claim 1, wherein the chimeric protein may further be a fusion protein of an inactive exogenous polypeptide-target macromolecule-labeled peptide, wherein an inactive exogenous polypeptide is linked to the N-terminus of the target macromolecule, and an enzyme donor ED as shown in SEQ ID NO: 2 is linked to the C-terminus of the target macromolecule, wherein the inactive exogenous polypeptide is an NFκB DNA-binding domain.

3. The method of claim 1, wherein the length of the labeled peptide is between 10 and 100 amino acids.

4. The method of claim 3, further comprising the step of lysing the cells.

5. The method of claim 1, 2, 3, or 4, wherein the compound is a small molecule.

6. The method of claim 5, wherein the small molecule is a small molecule that binds to an active site on the target macromolecule.

7. The method of claim 1, 2, 3, or 4, wherein the denaturation step comprises a single step of heating the fluid mixture to a temperature between 25°C and 100°C and (b) between 30°C and 60°C.

8. The method of claim 1, 2, 3, or 4, wherein the heating comprises multiple heating steps and lasts for a defined time period between 0.1 and 5 minutes.

9. The method of claim 8, wherein the heating step comprises applying heat to the mixture between 40°C and 60°C, and further comprises a plurality of steps of continuously heating for 0.1 to 5 minutes.

10. The method of claim 9, wherein the plurality of heating steps include a cooling step lasting from 10 seconds to 2 minutes between the respective heating steps.

11. The method of claim 10, comprising between three and ten cooling steps.

12. The method of claim 11, wherein the temperature of the mixture is maintained at a higher temperature than in the previous cooling step during the cooling step.

13. The method of claim 12, wherein the cooling step comprises actively cooling the mixture.

14. The method of claim 1, wherein steps (a) to (e) are carried out in a mixture containing the compound at different dilutions.

15. The method of claim 14, wherein repeated steps (a) to (e) can be used to calculate the binding constant (K) of the compound to the target macromolecule. D ).

16. The method of claim 1, wherein the target macromolecule is a protein selected from the group consisting of bromine-domain proteins, protein kinases, hydrolases, and histone methyltransferases.

17. A method for measuring the binding properties between a small molecule compound and a target protein, comprising: (a) Prepare a fluid mixture containing cells expressing a target protein and a fusion of a labeled peptide as a β-galactosidase fragment of 10 to 100 amino acids in length, the fluid mixture also containing a small molecule compound that is measured to bind to the target protein and protect the target protein from denaturation. (b) Incubate the fluid mixture of step (a) under conditions that allow the small molecule compound to bind to the target protein in the cell; (c) Heating the fluid mixture from step (b), thereby denaturing the target proteins that are not bound to the compound; (d) Lyse the heated cells from step (c) and add a second label, a β-galactosidase fragment, to the mixture. This second label reacts more readily with the labeled peptide on the undenatured target protein than with the labeled peptide on the denatured target protein. A substrate is then added, which generates a signal indicating the degree of denaturation of the target protein. The detection of the signal indicates the binding between the small molecule and the target protein. The target protein is connected to a labeled peptide at its C-terminus and to an inactive exogenous polypeptide at its N-terminus. The labeled peptide is as shown in SEQ ID NO: 2, and the inactive exogenous polypeptide is an NFκB DNA-binding domain.

18. The method of claim 17, wherein the mixture is prepared in a single container by steps (a) to (d).

19. The method of claim 17, wherein repeatable samples containing different concentrations of the compound are prepared.

20. The method of claim 17, wherein the target macromolecule is selected from the group consisting of proteins with bromine domains and enzymes.

21. Use of the kit in a method for measuring binding between a potential ligand and a target protein, said method comprising: (a) Prepare a fluid reaction mixture containing cells, the cells containing (i) a target protein having a first β-galactosidase fragment fused to the C-terminus of the target protein, and (ii) a potential ligand for the target protein, wherein the potential ligand protects the target protein from denaturation upon binding to the target protein. (b) Heating the reaction mixture and cells from step (a) to denature at least a portion of the target protein group; (c) Following step (b), by adding a β-galactosidase fragment complementary to the first β-galactosidase fragment and a substrate indicating the complementarity of the β-galactosidase and the binding of the potential ligand to the mixture, the amount of the undenatured fusion protein in step (b) is measured in the reaction mixture as the inverse function of the heating step in step (b), wherein the target protein is a protein selected from the group consisting of bromodomain proteins and enzymes, wherein the enzyme may be one of a protein kinase, a hydrolase, or a histone methyltransferase, wherein the enzyme fragment of the first β-galactosidase is as shown in SEQ ID NO: 2; The kit contains: Component I, comprising cells containing an expression vector for expressing a target protein fused with a first β-galactosidase fragment at its C-terminus; Component II, the β-galactosidase fragment complementary to the first β-galactosidase fragment in step c; Component III, substrate of β-galactosidase.

22. Use of the kit as claimed in claim 21, wherein the substrate is chromogenic, fluorescent, or chemiluminescent, and generates a signal in the presence of an active β-galactosidase but not an inactive β-galactosidase.

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