CN111378010A

CN111378010A - Synthesis of targeting caspase-8 probe and application thereof in inhibitor screening

Info

Publication number: CN111378010A
Application number: CN201811612128.8A
Authority: CN
Inventors: 张崇敬; 林浩
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2018-12-27
Filing date: 2018-12-27
Publication date: 2020-07-07

Abstract

The invention relates to a novel lightening probe synthesized by combining aggregation induced emission molecules (TPETH) and hydrophilic polypeptide fragments. The probe does not emit light in aqueous solution, and when caspase-8 is acted on, the polypeptide fragment is cut off, the water solubility of the probe is deteriorated, and the fluorescence intensity of the probe is gradually enhanced. The change of fluorescence intensity when the probe reacts with caspase-8 can be used for screening the inhibitor of caspase-8 enzyme. The probe has larger Stokes shift and can avoid the background fluorescence signal of the screened compound, so that the interference is less when the small molecule caspase-8 inhibitor with fluorescence property is screened, the probe has more obvious advantages compared with other types of probes, and the obtained result is more objective.

Description

Synthesis of targeting caspase-8 probe and application thereof in inhibitor screening

Technical Field

The invention relates to a novel caspase-8 enzyme lightening probe with aggregation-induced emission property. The invention relates to chemical synthesis of a probe and application of the probe in screening of caspase-8 enzyme inhibitors, belonging to the field of pharmaceutical chemistry.

Background

Conventional fluorescent molecules have good luminescence properties in a solution state, but generally exhibit a phenomenon of reduced luminescence efficiency or even no luminescence in an aggregation state, which is called aggregation-quenching (ACQ). This is due to intermolecular interactions that result in non-radiative energy transfer or the formation of species that are not conducive to fluorescent emission. In practical application, fluorescent materials are often required to be made into solid or thin film forms, aggregation between fluorescent molecules is inevitable, and fluorescence quenching of traditional materials is also inevitable.

Contrary to the properties of conventional fluorescent molecules, compounds having Aggregation-induced emission (AIE) properties hardly emit light in a dissolved state, but fluorescence is greatly enhanced after Aggregation, thus fundamentally overcoming the problem of fluorescence quenching caused by Aggregation, and arousing extensive research interest. The common point of the molecular structures of the class is that the centers of the peripheral aromatic substituents are connected by a rotatable single bond. In solution, the free rotation of these aromatic substituents around single bonds consumes excited state energy, becoming a non-radiative decay channel, leading to reduced fluorescence; in the aggregated state, however, due to space constraints, such molecular rotation is greatly impeded, the above-mentioned non-radiative pathways are suppressed, and excited-state molecules can only decay back to the ground state by radiation, thereby significantly enhancing fluorescence. Therefore, intramolecular restricted rotation (RIR) is the mechanism by which the AIE phenomenon occurs.

Apoptosis is a process of physiological autonomous cell death, and a group of proteolytic systems of proteases called caspases are central to this process. Fas (APO-1/CD95) is a transmembrane receptor that, when activated, triggers apoptosis. A series of caspases are activated in tandem during Fas-initiated apoptosis, with activation of caspase-8 being its first step response. Activated caspase-8 triggers activation of downstream caspases, including caspase-9, which in turn induces apoptosis. Caspase-8 is involved in pathological changes of many diseases and injuries in human, such as cancer, neurodegenerative diseases, parasitic diseases and wounds. Therefore, caspase-8 is a promising medicinal target, and the screening of caspase-8 inhibitors has important application.

The invention utilizes the combination of AIE molecule TPETH and hydrophilic polypeptide fragment to synthesize a novel lightening probe. The probe has larger Stoke shift and can effectively react with caspase-8 enzyme. The probe does not emit light in aqueous solution, and when the probe reacts with caspase-8, the polypeptide fragment is cut off, the water solubility of the probe is reduced, and the fluorescence intensity of the probe is gradually enhanced. The AIE probe overcomes the problem that the traditional fluorescent dye is easy to cause aggregation to cause fluorescence quenching. By using the change of the fluorescence intensity of the probe, the inhibitor of the caspase-8 enzyme can be screened, in particular the small molecule caspase-8 inhibitor with fluorescence property can be screened.

Disclosure of Invention

The invention relates to a novel lightening probe synthesized by combining a molecule TPETH with aggregation-induced emission property and a hydrophilic polypeptide fragment. The probe related by the invention has been proved to be capable of effectively acting with caspase-8, and can be effectively applied to detecting the activity of caspase-8, such as screening the inhibitor of caspase-8 enzyme, through the change of fluorescence intensity when the probe acts with the caspase-8 enzyme.

The probe comprises a structural general formula shown in a formula (I),

or a pharmaceutically acceptable salt thereof, wherein:

X₁-Cys- (semiphotoacyl),

X₂-Ile-Glu-Thr-Asp- (isoleucyl-glutamyl-threonyl-aspartyl)

X₃-Asp- (aspartyl), -Asp- (aspartyl-aspartyl), -Asp- (aspartyl-)

X₄-Cys- (semiphotoacyl),

X₅-Ile-Glu-Thr-Asp- (isoleucyl-glutamyl-threonyl-aspartyl)

X₆-Asp- (aspartyl), -Asp- (aspartyl-aspartyl), -Asp- (aspartyl-)

R₁Ac (acetyl), CH₃CH₂CO- (propionyl), CH₃CH₂CH₂CO- (butyryl)

R₂Ac (acetyl), CH₃CH₂CO- (propionyl), CH₃CH₂CH₂CO- (butyryl)

The mother-nucleus structure of the probe is TPETH with AIE property, hydrophilic polypeptide fragments IETDC or DIETDC are connected to two ends of the TPETH, and the probe molecules with good activity on caspase-8 are obtained through modification of hydrophilic amino acid.

The probes are prepared by a chemical synthesis way. The synthetic route of the probe is as follows:

TPETH-OMe is used as a starting material, and two-step reaction is carried out to obtain TPETH-2MAL, and the TPETH-2MAL and a hydrophilic polypeptide fragment with specificity to caspase-8 are subjected to thiol-ene reaction to obtain a target probe C8-P1.

The probes described above have good activity against caspase-8 enzyme. Such probes do not emit light in aqueous solution, and when caspase-8 is reacted, the polypeptide fragment is cleaved to release AIE molecule, and the fluorescence intensity of the probe is gradually increased.

The probe related to the invention has been proved to be capable of effectively acting with caspase-8, the fluorescence intensity of the probe is continuously enhanced when the probe and the caspase-8 act, and the intensity is basically saturated after 90 minutes of acting. Through the change of fluorescence intensity when the probe and the caspase-8 enzyme act, the inhibitor of the caspase-8 enzyme can be screened, in particular the micromolecule caspase-8 inhibitor with fluorescence property can be screened.

Drawings

1. FIG. 1 optical Properties of Probe C8-P1 and chemical formula of TPETH-2OMe

2. FIG. 2C 8-P1 shows the change of fluorescence intensity with time upon caspase-8 interaction

3. FIG. 3 Probe C8-P1 was able to detect the inhibitory activity of known inhibitors on caspase-8

4. FIG. 4 screening results of caspase-8 inhibitory Activity of Compounds 1-88

5. FIG. 5 shows the inhibition of caspase-8 by (+) gossypol and (-) gossypol at different concentrations

6. FIG. 6HeLa cells with C8-P1, inhibitors and H₂O₂Confocal imaging when applied.

Detailed Description

Example 1

Synthesis of Compound C8-P1:

synthesis route of Sheme 1 probe C8-P1

① Synthesis of TPETH-2MAL TPETH-2OH (80mg,0.15mmol) was dissolved in 10mL acetonitrile, A (113mg,0.3mmol) and K were added₂CO₃(90mg,0.66 mmol). The reaction solution is stirred for 12 hours at 50 ℃, cooled to room temperature, filtered, and the filtrate is concentrated and then separated by column chromatography to obtain an intermediate. The intermediate was dissolved in 5mL of toluene and heated at reflux for 20 hours. The reaction solution was concentrated and separated by column chromatography to give a red solid (46.9mg, 34.9% yield) of TPETH-2 MAL.

② Synthesis of C8-P1 TPETH-2MAL (5.0mg, 6.27. mu.M) was dissolved in 0.4mL DMSO, and Ac-DIETDC-NH was added₂(11mg, 15. mu.M), triphenylphosphine (4.27mg, 16.3. mu.M), DIEA (1.94mg, 15. mu.M). The reaction was stirred at room temperature for 3 hours and then purified by HPLC to give a red solid (4.08mg, 28.7% yield).¹H NMR(600MHz,DMSO-d₆):δ8.25-8.27(d,1.02H),8.23-8.24(d,2.92H),8.10-8.13(d,1.86H),8.05-8.06(d,1.95H),7.65-7.69(m,4.58H),7.37-7.38(t,1.06H),7.34-7.35(d,1.96H),7.18-7.28(m,5.92H),7.13-7.15(d,1.04H),7.10-7.12(d,2.16H),7.02-7.03(d,1.98H),6.89-6.91(d,2.01H),6.84-6.85(d,2.04H),6.67-6.70(t,4.04H),4.57-4.60(m,3.99H),4.32-4.39(m,4.45H),4.25(m,2.2H),4.16-4.19(t,2.26H),3.95-4.00(m,4.73H),3.89-3.90(m,4.19H),3.51-3.53(m,6.77H),3.14-3.21(m,4.77H),2.95-3.06(m,3.31H),2.83-2.85(m,1.86H),2.73-2.76(m,1.86H),2.73-2.76(m,3.06H),2.62-2.68(m,4.08H),2.22-2.27(m,5.76H),1.83(s,5.99H),1.24-1.27(m,5.09H),1.01-0.85(d,10.82H)ESI,calcd for[M+2H]²⁺1133.8923,found[M+2H]²⁺:1134.4018

Experimental example 1

Optical Properties of Probe C8-P1

1. Experimental Material

Probe C8-P1 was synthesized by the present inventors (institute of drug research, academy of Chinese medical sciences), dissolved in DMSO to prepare a 1mM stock solution, and diluted by HEPES buffer solution in multiple proportions before use to prepare a working solution. Caspase-8 enzyme was purchased from Youning vitamin Co., Ltd,

2. the instrument microplate reader was purchased from Tecan, switzerland under the model Spark.

3. As a result: FIG. 1 shows the absorption spectrum and fluorescence spectrum of a probe in DMSO/HEPES buffer (1:99) solution at a concentration of 10. mu.M. As can be seen from the left panel of FIG. 1, the UV absorption spectrum of probe C8-P1(probe C8-P1) is similar to that of AIE mother nucleus TPETH-2OMe (2- ((4- (2,2-bis (4-methoxyphenyl) -1-phenylvinyl) phenyl) (thiophen-2-yl) methyl) malononitrile); the fluorescence spectrum (right panel of FIG. 1) shows that the fluorescence intensity of the probe in aqueous solution is much lower than that of the AIE mother nucleus TPETH-2OMe, and there is almost no fluorescence in aqueous solution.

Experimental example 2

Fluorescence intensity changes with time when Probe C8-P1 reacts with caspase-8

1. Experimental Material

2. the instrument was a fully automatic enzyme marker available from Tecan, Switzerland under the model Spark.

3. As a result: as shown in FIG. 2, the fluorescence intensity (E) of the probe C8-P1 (10. mu.M) reacted with caspase-8(100pM)_x:420nm；E_m630nm) gradually increased with the passage of time. Experiments prove that after the probe is cut off by caspase-8, the fluorescence intensity is obviously enhanced and is 14.4 times of that of the original probe.

Experimental example 3

Detection of inhibition of commercial caspase-8 inhibitor Z-IETD-FMK on caspase-8 Using Probe C8-P1 1. Experimental materials

Probe C8-P1 was synthesized by the present inventors (institute of drug research, academy of Chinese medical sciences), dissolved in DMSO to prepare a 1mM stock solution, and diluted by HEPES buffer solution in multiple proportions before use to prepare a working solution. Caspase-8 enzyme is available from Youning vitamin Co. caspase-8 inhibitor Z-IETD-FMK was purchased from Sigma-Aldrich.

3. As a result: after caspase-8 and inhibitor Z-IETD-FMK are incubated for 30min in advance, probe C8-P1 is added, and the change of fluorescence intensity along with time is detected. FIG. 3 shows that when caspase-8 and inhibitor Z-IETD-FMK act for 30 minutes and then probe is added, the fluorescence intensity of the probe is kept unchanged all the time, which indicates that the activity of caspase-8 is inhibited and the probe cannot be lightened; while the fluorescence intensity of the control wells without inhibitor increased with time. These experimental results indicate that the probe can effectively screen compounds with caspase-8 inhibitory activity.

FIG. 3 Experimental example 4

Caspase-8 inhibitor screening experiment on natural product by using probe C8-P1

1. Experimental Material

Probe C8-P1 was synthesized by the present inventors (institute of drug research, academy of Chinese medical sciences), dissolved in DMSO to prepare a 1mM stock solution, and diluted by HEPES buffer solution in multiple proportions before use to prepare a working solution. Caspase-8 enzyme is available from Youning vitamin Co. Natural compounds are purchased from shanghai ceramic biochemical technologies, ltd; caspase-8 inhibitor Z-IETD-FMK was purchased from Sigma-Aldrich.

2. Instrument full-automatic enzyme marking instrument the full-automatic enzyme marking instrument is purchased from Tecan corporation of Switzerland, and is the Spark.

3. And (4) screening results: and (3) incubating caspase-8 with the small molecular compound for 30min, adding a probe C8-P1, and detecting the change of fluorescence intensity. As shown in Table 2 and FIG. 4, 1-86 are 86 natural fluorescent products (see Table 1 for structure), compound 87 is a reported caspase-8 inhibitor Z-IETD-FMK, and 88 is a probe plus caspase-8 control. The results of the screening (results for compounds 1-88 from left to right in the figure) are shown in FIG. 4, and compounds 13, 21, 25, 34, 69 have better caspase-8 inhibitory activity. As can be seen from the screening results, the probe can well screen out the small-molecule inhibitor of caspase-8.

TABLE 1.1-86 structures of fluorescent Small molecule Compounds

TABLE 2 screening results for Compounds 1-88

Experimental example 5

The probe C8-P1 is used for inhibiting capsase-8 enzyme IC of natural products (+) Gossypol, (-) Gossypol₅₀Measurement of (2)

1. Experimental Material

Probe C8-P1 was synthesized by the present inventors (institute of drug research, academy of Chinese medical sciences), dissolved in DMSO to prepare a 1mM stock solution, and diluted by HEPES buffer solution in multiple proportions before use to prepare a working solution. Caspase-8 enzyme is available from Youning vitamin Co. Gossypol was purchased from Aladdin reagents Inc.; (+) Gossypol, (-) Gossypol was obtained by resolution by the present inventors (institute of medicine, national academy of medicine).

3. And (4) screening results: the caspase-8 enzyme is incubated with (+) Gossypol and (-) Gossypol for 30min, then the probe C8-P1 is added, and the fluorescence intensity is measured by a microplate reader after 30 min. The results of the experiment showed that the compound (+) Gossypol IC₅₀IC with value of 0.48 μ M, (-) Gossypol₅₀The value was 0.44. mu.M. As can be seen from the screening results in FIG. 5, the probe can well determine the IC of the small molecule inhibitor of caspase-8₅₀。

Experimental example 6

Cell imaging experiments were performed on the natural products (+) Gossypol, (-) Gossypol using probe C8-P1

1. Experimental Material

Probe C8-P1 was synthesized by the present inventors (institute of drug research, academy of Chinese medical sciences), dissolved in DMSO to prepare a 1mM stock solution, and diluted by HEPES buffer solution in multiple proportions before use to prepare a working solution. Caspase-8 enzyme is available from Youning vitamin Co. Gossypol was purchased from Aladdin reagents Inc.; (+) Gossypol, (-) Gossypol was obtained by resolution by the present inventors (institute of medicine, national academy of medicine). Z-IETD-FMK was purchased from Sigma-Aldrich.

2. Laser scanning confocal microscopy: leica TCS SP8X common Microscope.

3. The experimental results are as follows: adding (+) Gossypol and a probe C8-P1 into a 60% HeLa cell culture dish at a hole 1; add (-) Gossypol and Probe C8-P1 into well 2; adding Z-IETD-FMK and probes C8-P1 into the hole 3; add DMSO to well 4 with probes C8-P1. After 2 hours of incubation, H was added₂O₂. Confocal imaging was performed after 1 hour. As can be seen from the experimental results (FIG. 6), the fluorescence intensity of the wells without the inhibitor is strong, and the fluorescence intensity of the wells with the inhibitor is weak or almost no fluorescence exists, indicating that the probe can well screen out the effective caspase-8 small molecule inhibitor at the cellular level.

Claims

1. A compound shown in a formula (I) and pharmaceutically acceptable salts thereof,

wherein:

X₁＝-Cys-；

X₂＝-Ile-Glu-Thr-Asp-；

X₃＝-Asp-,-Asp-Asp-,

or-Asp-Asp-Asp-;

X₄＝-Cys-；

X₅＝-Ile-Glu-Thr-Asp-；X₆＝Asp-,-Asp-Asp-,

or-Asp-Asp-Asp-;

R₁＝Ac,CH₃CH₂CO-, or CH₃CH₂CH₂CO-；

R₂＝Ac,CH₃CH₂CO-, or CH₃CH₂CH₂CO-。

2. The compound of claim 1, and pharmaceutically acceptable salts thereof, wherein the compound is selected from the group consisting of,

3. use of a compound according to any one of claims 1 to 2, and pharmaceutically acceptable salts thereof, for the detection of caspase-8 activity.

4. The use according to claim 3, wherein said detecting caspase-8 activity comprises screening for caspase-8 enzyme inhibitors.