阅读说明：本技术 化合物及其作为脂肪酸合酶活性探针上的应用 (Compounds and their use as probes for fatty acid synthase activity ) 是由 周怡青 石悦 徐悦 邓张双 许静远 李文龙 于 2019-10-17 设计创作，主要内容包括：本发明公开了一种化合物及其作为脂肪酸合酶活性探针上的应用。并公开了一种评估潜在的脂肪酸合酶抑制剂在哺乳动物中的效力的方法,其步骤包括：A、将待评估的脂肪酸合酶抑制剂施用于哺乳动物中；B、将权利要求1-3任一项所述的化合物施用于该哺乳动物或从该哺乳动物分离的细胞中；C、测量所述化合物的报告物部分的活性,将报告物部分的活性与标准进行比较,得到脂肪酸合酶抑制剂在哺乳动物中的效力。本发明提供一种针对脂肪酸合酶(FAS)的高选择性探针,能够选择性地标记FAS并提供在活细胞中直接测量FAS活性及抑制剂的靶结合效力。(The invention discloses a compound and application thereof as a fatty acid synthase activity probe. And a method of assessing the efficacy of a potential fatty acid synthase inhibitor in a mammal comprising the steps of: A. administering a fatty acid synthase inhibitor to be evaluated into a mammal; B. administering a compound of any one of claims 1-3 to the mammal or to a cell isolated from the mammal; C. measuring the activity of the reporter moiety of the compound and comparing the activity of the reporter moiety to a standard to obtain the efficacy of the fatty acid synthase inhibitor in the mammal. The present invention provides a highly selective probe for Fatty Acid Synthase (FAS), capable of selectively labeling FAS and providing direct measurement of FAS activity and the target binding potency of inhibitors in living cells.)

1. The use of a compound as a probe for the activity of fatty acid synthase, the compound having the general formula shown in formula (I):

wherein:

R₁is an optional substituent of a phenyl group selected from the group consisting of a label, a dye, a photocrosslinker, a cytotoxic compound, a drug, an active label, a photoactive label, a reactive compound, an antibody or antibody fragment, a biological material, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, an alkynyl group, an azido group, a radioactive moiety, a novel functional group, a group that interacts covalently or non-covalently with other molecules, a photocaged moiety, an actinic radiation-labile moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analog, a heavy atom-incorporating moiety, a chemically cleavable group, an redox agent, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron dense group, a magnetic group, an intercalating group, a chromophore, an energy transfer agent, a bioactive agent, A detectable label or a combination thereof;

R₂is an optional substituent of phenyl, selected from halogen, NH₂-、CN-，OH-，HS-，-NO₂，-CF₃，-COOH，CH₃CO-，(C_1-10Alkyl) -COO-, -O-C_1-10Alkyl radical, C_1-10Alkyl-, C_2-10Alkenyl-, C_2-10Alkynyl-;

the linking site is selected from a bond, an optionally substituted alkyl moiety, an optionally substituted heterocyclic moiety, an optionally substituted amide moiety, a ketone moiety, an optionally substituted carbamate moiety, an ester moiety, or a combination thereof.

2. Use according to claim 1, characterized in that: the structural formula of the compound is:

3. use of a pharmaceutically acceptable salt of a compound of claim 1 as a probe for fatty acid synthase activity.

4. Use of a pharmaceutically acceptable salt of a compound of claim 2 as a probe for fatty acid synthase activity.

5. A method of assessing the efficacy of a potential fatty acid synthase inhibitor in a mammal, comprising the steps of:

A. administering a fatty acid synthase inhibitor to be evaluated into a mammal;

B. administering a compound of any one of claims 1-3 to the mammal or to a cell isolated from the mammal;

C. measuring the activity of the reporter moiety of the compound and comparing the activity of the reporter moiety to a standard to obtain the efficacy of the fatty acid synthase inhibitor in the mammal.

Technical Field

The invention belongs to the field of medicine detection, and particularly relates to a compound capable of being used as a fatty acid synthase activity probe and application thereof.

Background

3-Fatty Acid Synthase (FAS) is a key enzyme catalyzing acetyl CoA and malonyl CoA to synthesize endogenous long-chain Fatty acids, and is normally expressed in tissues such as brain, lung, liver, fat, and mammary gland during lactation.

Fatty Acid Synthase (FAS) plays an important role in energy metabolism. Inhibition of FAS, the most critical enzyme in fat synthesis, can significantly reduce fat anabolism, and inhibition of FAS is one of the effective strategies for treating obesity. At the same time, FAS inhibitors have their remarkable anticancer effects, which can destroy cancer cells by controlling fatty acids produced by the proliferation of cancer cells. Therefore, the FAS inhibitor may provide a new approach for treating obesity and cancer, and the research on the FAS inhibitor is expected to provide a new target for treating obesity and cancer.

In the last 20 years, a large number of FAS inhibitors have been designed, synthesized and reported. However, all methods for evaluating the efficacy and activity of FAS inhibitors are limited to in vitro biochemical experiments, and no method for directly detecting the activity of FAS and evaluating the kinetics of FAS inhibitors in vivo has been reported.

Active probes generally comprise three components: a recognition group, a reactive group and a reporter group (Annu. Rev. biochem.2008,77, 383-414.). The recognition group introduces the probe into the binding pocket of the targeted protein and facilitates the formation of a covalent bond between the reactive group and the biomolecule. The reporter group provides a convenient means of identifying the probe-bound proteins within a complex proteome.

Orlistat (Orlistat) is currently the only marketed FAS inhibitor approved by the FDA. Yao et al developed Orlistat-based molecular probes for FAS labeling and imaging. However, due to the large off-target effect of orlistat, the probe covalently labels several tens of proteins at the same time, and thus its selectivity limits further applications of the probe (j.am. chem. soc.2010,132, 656-666).

The fluorophosphate probe was used for serine hydrolase labeling. The probe is found to be capable of labeling FAS protein in vitro. However, since this probe mainly labels serine hydrolase, its selectivity is also problematic. Moreover, the probe is unstable in vivo, and no in vivo labeling and imaging are reported (Proc. Natl. Acad. Sci. USA 1999,96, 14694-14699).

The structure of the PF-yne probe is similar to that of the compounds of the present application, and it has been reported that tyrosine 111 residues of Schistosoma japonicum SjGST proteins are covalently modified, and thus can be used for labeling and immobilization of GST fusion proteins, but the binding of the probe in mammalian proteomes is not clear (chem. Commun.2018,54, 4661-4664).

Target engagement (Target engagement) refers to the expected occupation of a biological Target by a drug molecule (nat. rev. drug discov.2006,5, 730-. This information is crucial for in vivo pharmacokinetic studies, particularly for establishing correlations at the molecular level between phenotypic observations and inhibitor-biomolecule interactions. At the same time, molecular probes play an important role in determining the appropriate drug dose for a patient in clinical trials.

The general protocol for an assay to check for target binding of a drug molecule is as follows: by sequentially adding the inhibitor and probe to the biological sample (cells, tissue, etc.), the intensity of the probe-labeled bands will give a direct readout of those biological targets not occupied by the inhibitor. As the concentration of the inhibitor increases, a decrease in the intensity of the band indicates that a portion of the biological target is bound by the inhibitor (Annu. Rev. biochem.2008,77, 383-414).

Disclosure of Invention

1. The invention aims to provide a novel method.

The present invention provides a highly selective probe for Fatty Acid Synthase (FAS).

2. The technical scheme adopted by the invention is disclosed.

The use of a compound as a probe for the activity of fatty acid synthase, which compound has the general formula shown in formula (I):

wherein:

R₁(ii) is a phenyl group as a reporter group, an optional substituent selected from the group consisting of a label, a dye, a photocrosslinker, a cytotoxic compound, a drug, an active label, a photoactive label, a reactive compound, an antibody or antibody fragment, a biological material, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, an alkynyl group, an azido group, a radioactive moiety, a novel functional group, a group that interacts covalently or non-covalently with other molecules, a photocaged moiety, an actinic radiation-labile moiety, a ligand, a photoisomerizable moiety, biotin, a biotin analog, a heavy atom-incorporating moiety, a chemically cleavable group, a photolyzable group, an redox agent, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminescent group, an electron dense group, a magnetic group, an intercalating group, a chromophore, an energy transfer agent, A bioactive agent, a detectable label, or a combination thereof;

the fluorophosphate in the formula (1) is a reactive group;

the moiety other than the reactive group and the reporter group is an identifying group, wherein:

R₂is an optional substituent of phenyl, selected from halogen, NH₂-、CN-，OH-，HS-，-NO₂，-CF₃，-COOH，CH₃CO-，(C_1-10Alkyl) -COO-, -O-C_1-10Alkyl radical, C_1-10Alkyl-, C_2-10Alkenyl-, C_2-10Alkynyl-;

the linking site is selected from a bond, an optionally substituted alkyl moiety, an optionally substituted heterocyclic moiety, an optionally substituted amide moiety, a ketone moiety, an optionally substituted carbamate moiety, an ester moiety, or a combination thereof.

Preferably, the structural formula is:

also includes the application of the above compound in the pharmaceutically acceptable salt as a fatty acid synthase activity probe.

The present invention also discloses a method of assessing the efficacy of a potential fatty acid synthase inhibitor in a mammal, comprising the steps of:

A. administering a fatty acid synthase inhibitor to be evaluated into a mammal;

B. administering the compound described above to the mammal or to cells isolated from the mammal;

C. measuring the activity of the reporter moiety of the compound and comparing the activity of the reporter moiety to a standard to obtain the efficacy of the fatty acid synthase inhibitor in the mammal.

3. The technical effect produced by the invention.

The present invention provides a highly selective probe for Fatty Acid Synthase (FAS), capable of selectively labeling FAS and providing direct measurement of FAS activity and Target-binding (Target engagement) efficacy of an inhibitor in living cells.

Drawings

FIG. 1: probe 1(Probe 1) marks a 250-300 kDa high molecular weight protein band in a HeLa cell lysate in a concentration-dependent manner. Left panel: fluorescence scanning; right panel: coomassie brilliant blue staining.

FIG. 2: enrichment and identification of the labeled band of Probe 1(Probe 1) in HeLa cell lysate. (A) Detecting the enriched protein of the probe 1 in the HeLa cell lysate by silver staining; (B) mass spectrometry analysis; (C) the probe 1-enriched protein was verified using the FAS antibody.

FIG. 3: probe 1(Probe 1) labeled FAS protein in HeLa cells in a concentration-dependent manner. (A) Fluorescence scanning and coomassie brilliant blue staining; (B) the concentration interval of probe 1 selective marker FAS was determined based on fluorescence quantification.

FIG. 4: probe 1(Probe 1) time-dependently labeled FAS protein in HeLa cells. (A) Fluorescence scanning and coomassie brilliant blue staining; (B) and (4) quantitatively analyzing a fluorescence band.

FIG. 5: probe 1 selectively labels the FAS protein in various cells. Left panel: fluorescence scanning; right panel: coomassie brilliant blue staining.

FIG. 6: probe 1 was used to evaluate the intracellular kinetic processes of the FDA-approved marketed FAS inhibitor drug orlistat.

Detailed Description

The structural formula of the probe 1 is as follows:

synthetic routes are described in literature (chem. Commun.2018,54, 4661-4664):

and (3) biological experiments:

HeLa cell lysate label based on gel electrophoresis fluorescence scanning

In reaction buffer (50mM HEPES, pH 7.4,150mM NaCl,5mM MgCl)₂) HeLa cell lysate was diluted to a final concentration of 5. mu.g/. mu.L and incubated with 0.01-10. mu.M probe 1 at room temperature for 1 hour. The "click" reaction was performed after adding a final concentration of 1% SDS to the protein sample: for each reaction, 19.2. mu.L of protein sample was added to 0.2. mu.L each of TAMRA-N₃(10mM stock solution in DMSO, Lumiprobe), CuSO₄(100mM stock in water), THPTA (10mM stock in water, Sigma) and sodium ascorbate (100mM stock in water). The samples were incubated at room temperature in the dark for 1 hour. The reaction was then quenched by the addition of 5 μ L of 4 XSDS loading buffer (Invitrogen) and boiling for 15 minutes at 95 ℃. Samples were applied to NUPAGE 12% Bis-tris denaturing gel (Invitrogen) and in-gel fluorescence scans were performed with a 4600SF imaging system (shanghai energy). As shown in FIG. 1, probe 1 can label a large molecular weight protein of 250-300 kDa in a HeLa cell lysate in a concentration-dependent manner.

Enrichment and identification of Probe 1-labeled high molecular weight proteins

In reaction buffer (50mM HEPES, pH 7.4,150mM NaCl,5mM MgCl)₂) HeLa cell lysate was diluted to a final concentration of 5. mu.g/. mu.L and incubated with DMSO or 1. mu.M probe 1, respectively, for 1 hour at room temperature. The "click" reaction was performed after adding a final concentration of 1% SDS to the protein sample: for each reaction, 300. mu.L of protein sample was added to 3. mu.L each of Biotin-N₃(100mM in DMSO)Stock solution, Biomatrix), CuSO₄(100mM stock in water), THPTA (10mM stock in water, Sigma) and sodium ascorbate (100mM stock in water). The samples were incubated at room temperature for 1 hour in the dark. 0.6mL of methanol, 0.15mL of chloroform and 0.4mL of water were sequentially added to precipitate the protein, the mixture was mixed well and centrifuged at 14000g for 5 minutes, and the precipitate was collected, washed with 0.5mL of methanol and air-dried. The protein was reconstituted by addition of 200. mu.L PBS, 1% SDS, and 200. mu.L PBS containing 50. mu.L of LStreptavidin Sepharose resin was added and incubated at room temperature for 2 hours. The bound proteins on the resin were eluted by sequentially washing with 0.5mL PBS (3 times), PBS +0.5M NaCl (3 times), PBS (1 times), deionized water (1 time), and then by adding 30 μ L of 4 x SDS loading buffer (Invitrogen) and boiling for 15 minutes at 95 ℃. Samples were applied to NUPAGE 4-12% Bis-tris denaturing gel (Invitrogen) and developed with silver staining. As shown in the figure, probe 1 was successfully enriched in the large molecular weight protein labeled in the previous experiment. This band was cleaved and then subjected to an intracollagen digestion, and identified as human Fatty Acid Synthase (FAS) by mass spectrometry. Meanwhile, the eluted sample was separated by NUPAGE 4-12% Bis-tris denaturing gel (Invitrogen), transferred to a PVDF membrane, and the mass spectrometry result was verified by using FAS antibody. FIG. 2 shows that the high molecular weight protein enriched by probe 1 is indeed FAS.

HeLa live cell marker (probe concentration gradient) based on gel electrophoresis fluorescence scanning

HeLa cells were cultured in serum-free DMEM medium. Add probe 1 to a final concentration of 0.1-10. mu.M and incubate at 37 ℃ for 1 hour. Lysis buffer (50mM HEPES, pH 7.4,150mM NaCl, 0.1% Triton X-100,5mM MgCl) was added₂) K562 cells were lysed and the lysate solution diluted to a final concentration of 5 μ g/μ L. The "click" reaction was performed after adding a final concentration of 1% SDS to the protein sample: for each reaction, 19.2. mu.L of protein sample was added to 0.2. mu.L each of TAMRA-N₃(10mM stock solution in DMSO, Lumiprobe), CuSO₄(100mM stock in water), THPTA (10mM stock in water, Sigma) and sodium ascorbate (100mM stock in water). The samples were incubated at room temperature in the dark for 1 hour. Then added by 5. mu.L of 4 XSDS loading buffer (Invitrogen) and boiled at 95 ℃ for 15The reaction was terminated in minutes. Samples were applied to NUPAGE 12% Bis-tris denaturing gel (Invitrogen) and in-gel fluorescence scans were performed with a 4600SF imaging system (shanghai energy). As shown in FIG. 3, probe 1 selectively labeled FAS in HeLa cells at a concentration dependent manner, and probe 1 selectively labeled a single FAS protein band in the range of 100nM to 1. mu.M.

HeLa live cell marker (incubation time gradient) based on gel electrophoresis fluorescence scanning

HeLa cells were cultured in serum-free RPMI medium. Add probe 1 to a final concentration of 0.1. mu.M and incubate at 37 ℃ for 5 minutes to 2 hours. Lysis buffer (50mM HEPES, pH 7.4,150mM NaCl, 0.1% Triton X-100,5mM MgCl) was added₂) HeLa cells were lysed and the lysate liquid was diluted to a final concentration of 5. mu.g/. mu.L. The "click" reaction was performed after adding a final concentration of 1% SDS to the protein sample: for each reaction, 19.2. mu.L of protein sample was added to 0.2. mu.L each of TAMRA-N₃(10mM stock solution in DMSO, Lumiprobe), CuSO₄(100mM stock in water), THPTA (10mM stock in water, Sigma) and sodium ascorbate (100mM stock in water). The samples were incubated at room temperature in the dark for 1 hour. The reaction was then quenched by the addition of 5 μ L of 4 XSDS loading buffer (Invitrogen) and boiling for 15 minutes at 95 ℃. Samples were applied to NUPAGE 12% Bis-tris denaturing gel (Invitrogen) and in-gel fluorescence scans were performed with a 4600SF imaging system (shanghai energy). As shown in FIG. 4, 100nM probe 1 was able to time-dependently selectively label FAS protein in HeLa cells, and saturation labeling was achieved in 30 min.

Various cell markers based on gel electrophoresis fluorescence scanning

HeLa, MCF-7, HCT116, HepG2, Hep3B, K562, BJAB, Jurkat, HEK293T cells were cultured in serum-free DMEM/RPMI medium. Add probe 1 to a final concentration of 100nM and incubate at 37 ℃ for 1 hour. Lysis buffer (50mM HEPES, pH 7.4,150mM NaCl, 0.1% Triton X-100,5mM MgCl) was added₂) HeLa cells were lysed and the lysate liquid was diluted to a final concentration of 5. mu.g/. mu.L. The "click" reaction was performed after adding a final concentration of 1% SDS to the protein sample: for each reaction, 19.2. mu.L of protein sample was added to each0.2. mu.L of TAMRA-N₃(10mM stock solution in DMSO, Lumiprobe), CuSO₄(100mM stock in water), THPTA (10mM stock in water, Sigma) and sodium ascorbate (100mM stock in water). The samples were incubated at room temperature in the dark for 1 hour. The reaction was then quenched by the addition of 5 μ L of 4 XSDS loading buffer (Invitrogen) and boiling for 15 minutes at 95 ℃. Samples were applied to NUPAGE 12% Bis-tris denaturing gel (Invitrogen) and in-gel fluorescence scans were performed using a 4600SF imaging system (shanghai energy). As shown in FIG. 5, 100nM probe 1 selectively labels the FAS protein in the cells tested.

FAS inhibitor Orlistat (Orlistat) target binding assay based on gel electrophoresis fluorescence scanning

Examination of whether probe 1 can measure the extent of occupancy of FAS by the inhibitor in living cells. The marketed FAS drug orlistat was examined. HeLa cells were cultured in serum-free DMEM medium. Cells were first incubated with orlistat at various concentrations for 60 minutes at 37 ℃ followed by 100nM probe 1 for 30 minutes. Lysis buffer (50mM HEPES, pH 7.4,150mM NaCl, 0.1% Triton X-100,5mM MgCl) was added₂) HeLa cells were lysed and the lysate liquid was diluted to a final concentration of 5. mu.g/. mu.L. The "click" reaction was performed after adding a final concentration of 1% SDS to the protein sample: for each reaction, 19.2. mu.L of protein sample was added to 0.2. mu.L each of TAMRA-N₃(10mM stock solution in DMSO, Lumiprobe), CuSO₄(100mM stock in water), THPTA (10mM stock in water, Sigma) and sodium ascorbate (100mM stock in water). The samples were incubated at room temperature in the dark for 1 hour. The reaction was then quenched by the addition of 5 μ L of 4 XSDS loading buffer (Invitrogen) and boiling for 15 minutes at 95 ℃. Samples were applied to NUPAGE 12% Bis-tris denaturing gel (Invitrogen) and in-gel fluorescence scans were performed with a 4600SF imaging system (shanghai energy). As shown in fig. 6A, orlistat effectively blocked the labeling of FAS by probe 1 at 50 μ M.

Similarly, it was examined whether probe 1 can measure the extent of occupancy of FAS by the inhibitor in living cells. The marketed FAS drug orlistat was examined. HeLa cells were cultured in serum-free DMEM medium. Cell headThe orlistat was incubated at 37 ℃ for various times with 50. mu.M followed by 30 minutes with 100nM probe 1. Lysis buffer (50mM HEPES, pH 7.4,150mM NaCl, 0.1% Triton X-100,5mM MgCl) was added₂) HeLa cells were lysed and the lysate liquid was diluted to a final concentration of 5. mu.g/. mu.L. The "click" reaction was performed after adding a final concentration of 1% SDS to the protein sample: for each reaction, 19.2. mu.L of protein sample was added to 0.2. mu.L each of TAMRA-N₃(10mM stock solution in DMSO, Lumiprobe), CuSO₄(100mM stock in water), THPTA (10mM stock in water, Sigma) and sodium ascorbate (100mM stock in water). The samples were incubated at room temperature in the dark for 1 hour. The reaction was then quenched by the addition of 5 μ L of 4 XSDS loading buffer (Invitrogen) and boiling for 15 minutes at 95 ℃. Samples were applied to NUPAGE 12% Bis-tris denaturing gel (Invitrogen) and in-gel fluorescence scans were performed with a 4600SF imaging system (shanghai energy). As shown in fig. 6C, 50 μ M orlistat effectively blocked the labeling of FAS by probe 1 after 60 minutes.

Orlistat target binding assay based on fluorescence confocal microscope live cell in-situ labeling

HeLa cells were cultured in serum-free DMEM medium. Cells were first incubated with orlistat at various concentrations for 1 hour at 37 ℃ followed by 100nM probe 1 and 0.5 hour incubation in a 37 ℃ incubator. PBS was washed three times, fixed for 10 minutes on ice with PBS containing 0.4% paraformaldehyde, washed three times with PBS, and penetrated with cell membrane by adding PBS containing 0.5% Triton X-100. After three washes with PBS, 0.2. mu.L each of TAMRA-N was added₃(10mM stock solution in DMSO, Lumiprobe), CuSO₄1mL PBS of (100mM stock in water), THPTA (10mM stock in water, Sigma) and sodium ascorbate (100mM stock in water) were incubated for 1 hour in the dark. After 5 washes with PBS, DAPI dye was added and incubated for 30 minutes, followed by 3 washes with PBS and imaging with Leica TSC SP8 STED 3X ultra high resolution laser scanning confocal microscope. As shown in the results of fig. 6B, orlistat effectively blocked the labeling of FAS by probe 1 at 50 μ M under 60 min incubation conditions. As shown in the results of FIG. 6D, 50. mu.M orlistat effectively blocked the FAS labeling by probe 1 after 60 minutes.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.