CN115590982A - Deacetylase self-sacrifice system and application thereof - Google Patents
Deacetylase self-sacrifice system and application thereof Download PDFInfo
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Abstract
The invention discloses a deacetylase self-sacrifice system and application thereof, wherein the deacetylase self-sacrifice system comprises a phenol ester connector, an HDAC substrate and a fluorophore; the phenolic ester connector is one of phenolic ester connectors I to VII; the HDAC substrate is 6-acetamido hexanoic acid or 2,2-dimethyl-6-acetamido hexanoic acid; the fluorophore is a carboxylic acid-bearing fluorophore BIX-CA or a hydroxymethyl group-bearing fluorophore BIX-HM. The invention obtains a self-sacrifice system by regulating the reactivity and the steric hindrance of the phenol ester connector, shows the potential of detecting the HDAC activity in tumor cells, and further creates a novel HDAC activated prodrug system to provide effective treatment for tumor mice with high expression of HDAC.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a deacetylase self-sacrifice system and application thereof.
Background
The nucleosomes assembled by histones and DNA are essential components of eukaryotic chromatin. Dynamic modification of the amino terminus of histones plays a crucial regulatory role in controlling the structural organization of chromatin and its transcriptional state. Histone Deacetylases (HDACs) are a class of key enzymes that regulate a variety of cellular processes. Specifically, HDACs catalyze the removal of acetyl groups on histone lysine residues, tightly bind to negatively charged DNA, and chromatin is densely coiled to inhibit transcriptional expression of genes. They also remove acetyl groups from proteins other than histones, such as tubulin, cortical actin, and transcription factors, to alter cell mobility, migration, and proliferation. Aberrant levels of HDAC activity have been implicated in various diseases, such as cancer, making them targets for epigenetic treatment of cancer. The discovery of HDAC inhibitors is a long sought after and a number of inhibitors have been approved for clinical or clinical trials. Therefore, molecular tools for HDAC targeted imaging and therapy play a crucial role for their functional annotation, cancer diagnosis and drug development.
Several examples of reported activated HDAC probes are designed by intramolecular nucleophilic addition and nucleophilic substitution reactions, in which an amino group generated by deacetylation of a substrate is catalyzed by HDAC to undergo intramolecular nucleophilic reaction with an electrophilic moiety (such as dimethyl carbonate, nitrobenzoxadiazole, and aldehyde group), thereby illuminating a fluorescent signal. However, these probes are only suitable for in vitro experiments, limiting their application in biological imaging.
To this end, a highly specific deacetylase self-immolative system is sought that is capable of activating near infrared fluorescence (NIRF) and Photoacoustic (PA) imaging in response to HDACs in vivo and releases the prodrug.
Disclosure of Invention
In order to solve the above technical problems, the present invention discloses a highly specific deacetylase self-immolative system and is used to construct HDAC activated probes and prodrugs, achieve near infrared fluorescence (NIRF) and Photoacoustic (PA) imaging in response to HDAC in vivo, and release prodrugs.
In order to achieve the above object, the present invention provides a deacetylase self-immolative system,
including a phenol ester linker, an HDAC substrate, and a fluorophore;
the phenolic ester connector is one of phenolic ester connectors I to VII,
the phenol ester is connectedThe structural formula of the graft I is:
the structural formula of the phenol ester connector II is as follows:
the structural formula of the phenol ester connector III is as follows:
the structural formula of the phenol ester connector IV is as follows:
the structural formula of the phenol ester connector V is as follows:
the structural formula of the phenol ester connector VI is as follows:
the structural formula of the phenol ester connector VII is as follows:
the above deacetylase is a self-immolative system, and further, the HDAC substrate is 6-acetamidohexanoic acid or 2,2-dimethyl-6-acetamidohexanoic acid.
The deacetylase is a self-immolative system, and further the fluorophore is a carboxylic acid-containing fluorophore BIX-CA or a hydroxymethyl group-containing fluorophore BIX-HM.
The above-mentioned deacetylase self-immolative system, further, the fluorophore as a leaving moiety is BIX-HM-SO 3 - 。
The deacetylase self-immolative system is an HDAC probe having the following structural formula I, II, III, IV:
wherein R is 1 Is H or F, R 2 Is H, F or NO 2 。
Based on a general technical concept, the invention also provides an application of the deacetylase self-sacrifice system in-vivo activation imaging of the deacetylase.
In the above application, further, in the application, the in vivo activation imaging is confocal cell fluorescence imaging, and the specific steps are as follows: cells were incubated with fresh medium containing HDAC probes and imaged with a nikon A1+ confocal microscope with excitation wavelength of 640nm and emission region of 663nm to 738nm.
In the above application, further, in the application, in vivo activation imaging is photoacoustic imaging of cells, and the specific steps are as follows: culturing MDA-MB-231 cells with HDAC probe, collecting cells with trypsin, and collecting cell suspension by microtube centrifugation; tubes with cell particles were inserted into the tube rack and imaged with the MSOT imaging system.
The above application, further, the application specifically is: NIRF/PA imaging of HDAC activity in vivo was performed on a living subject by tail vein injection of HDAC probe.
Based on a general technical concept, the invention also provides the application of the deacetylase self-sacrifice system in prodrug release.
Based on a general technical concept, the invention also provides the application of the deacetylase self-sacrifice system in the evaluation of the efficacy of the HDAC inhibitor.
Compared with the prior art, the invention has the advantages that:
(1) The invention provides a deacetylase self-sacrifice system, which is obtained by regulating and controlling the reactivity and steric hindrance of a phenol ester connector. A novel spiro xanthene dye with ring-opening characteristics is designed, and a molecular basis is provided for high-signal-to-magnification ratio NIRF/PA dual-mode imaging. Research results show that the ortho-position electron-pulling substitution effect of the phenol ester linker is crucial to promoting release kinetics and efficiency, and a feasible strategy is provided for eliminating the interference of carboxylesterase by introducing a geminal dimethyl recognition substrate and increasing the steric hindrance of the ortho-position of an ester bond. In combination with a novel dye and deacetylase self-immolative system, we developed an HDAC activated NIRF/PA probe for in vitro and in vivo detection of HDAC activity.
(2) The invention provides an application of a deacetylase self-sacrifice system in activated imaging of histone deacetylase, and an NIRF/PA probe using a nitro-substituted self-sacrifice linker provides HDAC sensitive imaging with ideal dynamics and high contrast in living cells and living bodies. It shows excellent specificity for HDAC compared to common carboxylesterases such as human carboxylesterase (CES 1, CES 2) and Pig Liver Esterase (PLE), showing the potential to detect HDAC activity in tumor cells.
(3) The invention provides an application of a deacetylase self-sacrifice system in prodrug release, through etherification of a phenolic hydroxyl group of SN-38 with the deacetylase self-sacrifice system, after caging removal, an emission peak of SN-38 is moved from 425nm to 550nm, and the release is monitored through fluorescence spectrum. Fluorescence spectra over time show that SN-38 is gradually activated and reaches a maximum after incubation with HDAC6 for-3 h. The prodrug is specifically released by HDAC6, has ideal resistance to CES1, CES2 and PLE, and provides effective treatment for tumor mice with high expression of HDAC. Our generic deacetylase self-immolative system will open a new paradigm for the development of HDAC activated probes and prodrugs, highlighting its potential for therapeutic applications.
Drawings
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
FIG. 1 shows the structure and principle of the deacetylase self-immolative system of the present invention; (a) the molecular structure of previously reported HDAC probes. (b) Deacetylase, which responds to HDACs, self-sacrifices the design and structure of the system.
FIG. 2 is a schematic diagram showing the mechanism of HDAC-mediated degaussing reaction of the deacetylase self-immolative system of the present invention.
FIG. 3 is a schematic representation of the HDAC-activated NIRF/PA imaging of the deacetylase self-immolative system of the present invention.
FIG. 4 is a schematic representation of HDAC mediated prodrug release from a sacrificial system of a deacetylase according to the present invention.
FIG. 5 shows the structure and physicochemical properties of the fluorophore BIX-HM with hydroxymethyl groups, (a) the main structure of BIX-HM-Me at different pH values. (b) pH dependent absorbance of BIX-HM and BIX-HM-Me.
FIG. 6 shows the results of the reactivity of P1 to P4 to HDAC in examples 1 to 4.
FIG. 7 shows the fluorescence response of P4-P6 to 200nM HDAC6, CES1, CES2 and PLE.
FIG. 8 shows the fluorescent response of P7 to HDAC 6. (a) Fluorescence response of P7 to different concentrations of HDAC6 (0-500 nM), inset: fluorescence intensity and HDAC6 concentration from 0 to 100 nM. (b) UV-visible absorption spectra after response of P7 (20. Mu.M), P7 (20. Mu.M) and HDAC6 (200 nM) in PBS at 37 ℃ in the absence or presence of TSA (10. Mu.M). (c) fluorescent responses of P4 and P7 to different HDAC enzymes. (d) PA spectra and imaging of P7 (20. Mu.M), P7 (20. Mu.M) and HDAC6 (400 nM) in PBS at 37 ℃ in the absence or presence of TSA (10. Mu.M).
FIG. 9 shows the NIRF/PA imaging of HDAC activity in living cells by P7. (a) Confocal fluorescence imaging of MDA-MB-231 cells incubated with P7 (20. Mu.M) without TSA or with TSA (10. Mu.M). (b) MDA-MB-231 cells were analyzed by flow cytometry and incubated with P7 (20. Mu.M) without or with TSA (10. Mu.M). (c) PA spectra and imaging of P7 (20. Mu.M), P7 (20. Mu.M) and MDA-MB-231 cell lysates (5. Mu.L) without or with TSA (10. Mu.M) in PBS at 37 ℃.
FIG. 10 is NIRF/PA imaging of HDAC activity in MDA-MB-231 tumor-bearing mice. (a) NIRF/PA imaging schematic of HDAC activity in vivo. Representative NIRF (b) and PA (c) images of MDA-MB-231 tumor-bearing mice injected intratumorally with TSA or without TSA, P7 (1.85 mg/kg) tail vein injection at various time points, with saline as a control. White-point circles indicate tumor regions. Time-dependent fluorescence (d) and PA intensity (e) at different time points (n = 5) in MDA-MB-231 tumor-bearing mice receiving different treatments. (f) Fluorescence intensity of major organs and tumor tissue dissected from mice 5h after injection. Scale bar =2mm. * P <0.01, p <0.001, p <0.0001.
FIG. 11 is a NIRF imaging of HDAC activity in MDA-MB-231 tumor-bearing mice. (a) Schematic of HDAC inhibitors and real-time NIRF imaging treatment schedules. (b) the chemical structure of the HDAC inhibitor. (c) Representative NIRF imaging of MDA-MB-231 tumor-bearing mice pretreated with different inhibitors after intravenous injection of P7 (1.85 mg/kg) at different time points (n = 5). White circles indicate tumor regions. (d) quantification of NIRF signal at tumor site (n = 5). Test 1: quantification of NIRF signal after 5h injection of P7 (1.85 mg/kg). And (3) testing 2: HDAC activity in homogeneous tumors treated with different inhibitors was analyzed using a commercial HDAC kit. Data are presented as mean ± standard deviation.
FIG. 12 is a graph of the in vivo therapeutic effect of prodrugs on MDA-MB-231 tumor-bearing mice. (a) Schematic representation of tumor model establishment and cancer treatment schedule. (b) a schematic of the mechanism of HDAC activated prodrug release. (c) The normal saline and SN-38 (4.0 mg kg) were intravenously injected every other day -1 ) Or prodrug (7.5 mg kg) -1 ) Five times later, MDA-MB-231 tumor growth curves. (d) body weight of MDA-MB-231 tumor-bearing mice after different treatments. (e) photograph of dissected tumor on day 30. (f) final tumor weight after different treatments. (g) H of tumor sections of MDA-MB-231 tumor-bearing mice receiving different treatments on day 30&E and TUNEL staining. Green fluorescence is the TUNEL staining signal and blue fluorescence is the nuclear staining signal. Scale =50 μm. In all data sets, all groups n =5 mice. * p is a radical of<0.05，**p<0.01，***p<0.001，***p<0.0001。
Detailed Description
The invention is further described below with reference to specific preferred embodiments, without thereby limiting the scope of protection of the invention.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically indicated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods. The methods in the following examples are conventional in the art unless otherwise specified.
Examples
The materials and equipment used in the following examples are commercially available.
The present invention discloses a novel deacetylase self-immolative system that enables in vivo activation imaging and prodrug release specific to HDAC activity with high efficiency and good kinetics. The former HDAC probe is less reactive with free amine groups under physiological conditions and is only suitable for specific fluorophores (a in fig. 1 is a structure diagram of the former HDAC probe molecule). To address these limitations, we designed a novel deacetylase self-immolative system using the reactivity and steric hindrance of the fine-tuned phenyl ester linker. We envision that an Electron Withdrawing Group (EWG) in the phenyl ester linker can facilitate nucleophilic addition-elimination reactions with free amines, providing an effective deacetylase self-immolative system for HDAC targeted activation imaging and prodrug release. Based on this hypothesis, we designed a deacetylase self-immolative system comprising three modules, a phenyl ester linker, an HDAC substrate, and a fluorophore or drug as leaving moiety.
In FIG. 1 b is the design and structure of the HDAC-responsive deacetylase self-immolative system. The invention designs phenyl ester connectors with adjustable reactivity by introducing different Electron Withdrawing Groups (EWG) at the ortho position. The introduction of EWG has two benefits: one is to increase the electrophilicity of the ester moiety, enhancing the nucleophilic addition kinetics, and the other is to weaken the basicity of the phenolate, which imparts a higher leaving efficiency. To improve specificity towards carboxylesterases, we introduced a-dimethyl group on the alpha carbon of the common HDAC substrate 6-acetamidohexanoic acid and selected a bulky, sterically self-immolative linker with an ortho-substituent.
FIG. 2 is a schematic representation of the mechanism of HDAC mediated activation, as seen in the figure: deacetylation of the substrate produces a free amine group, triggering elimination of intramolecular nucleophilic addition to the phenyl ester linker, followed by self-immolative reaction, releasing the protecting group. This deacetylase self-immolative system is universally applicable to the development of HDAC activated probes and prodrugs.
Based on this deacetylase self-immolative system, we developed an activatable probe using a novel spirocyclic xanthene dye for near infrared fluorescence (NIRF) and Photoacoustic (PA) dual mode imaging of HDACs. Fig. 3 is a schematic of HDAC activatable NIRF/PA imaging, as seen from the figure: in HDAC mediated leaving reactions, this dye shows a transition from a colorless spiro "closed" form to a colored xanthene "open-ring" structure.
The present invention provides HDAC sensitive imaging with ideal kinetics and high contrast in living cells and in vivo using NIRF/PA probes with nitro-substituted self-immolative linkers. It shows excellent specificity for HDAC compared to common carboxylesterases such as human carboxylesterase (CES 1, CES 2) and Pig Liver Esterase (PLE). This bimodal capability expands the potential of our probe for in vivo applications, as PA imaging provides high spatial resolution and tissue penetration depth, while NIRF imaging provides excellent sensitivity.
The present invention further develops a novel prodrug system, and fig. 4 is a schematic representation of HDAC mediated prodrug release, as seen in the figure: the prodrug system is capable of targeting therapy in vivo, such as HDAC overexpressed Triple Negative Breast Cancer (TNBC). Our studies created the first generic deacetylase self-immolative system for HDAC-specific NIRF/PA imaging and prodrug release in vivo, highlighting its therapeutic potential.
To develop an HDAC active NIRF/PA probe that is activated in vivo, we designed a novel spirocyclic xanthene dye (BIX) because the spirocyclic xanthene structure can show an adjustable balance between the xanthene "open-loop" and spirocyclic "closed-loop" forms with different absorption and emission profiles. We initially tested a fluorophore BIX-CA with a carboxylic acid. The fluorophore showed a major NIR absorption peak at 730nm, but due to the weak nucleophilicity of the carboxylic acid, the ring-opened form remained even after acetylation of the phenolic hydroxyl group. In order to modify it into a near infrared dye with a ring opening reaction, we designed a new fluorophore BIX-HM with hydroxymethyl groups.
In fig. 5, a is to examine the main structure of BIX-HM-Me at different pH values, which tends to be in an open-loop state under acidic conditions and tends to be in a closed-loop transition state under alkaline conditions.
B of FIG. 5 is the pH dependent absorbance of BIX-HM and BIX-HM-Me. The fluorophore BIX-HM shows a strong absorption peak at pH 7.4 at 730nm, and exhibits a broad blue-shifted absorption band after etherification at about 650 nm. At the same time, the fluorescence peak after etherification becomes negligible. pK of caged BIX-HM (BIX-HM-Me) cycl The value (pH at which the absorbance decreased to half the maximum absorbance due to spiro ring) was 6.6, indicating that at pH 7.4, the etherified BIX-HM was mainly in the form of a closed spiro ring.
Furthermore, in the pH range of 2.5 to 10, BIX-HM shows a strong absorption band in the NIR region, indicating that it is predominantly an open-loop structure. This spectral change is attributed to the equilibrium between the phenolate and phenolic hydroxyl forms. pKa of BIX-HM is 5.8, which means that the fluorophore is predominantly in the phenol form at pH 7.4. Further, the molar absorption coefficient of BIX-HM was 3.74X 10 5 The yield of fluorescence quantum is 11.4%, and the light stability is good. In summary, these results indicate that BIX-HM can lay the foundation for NIRF/PA monitoring HDAC with high signal-to-noise ratio and sensitivity under physiological conditions
Example 1
A self-immolative HDAC probe P1 of this example, having the formula:
wherein R is 1 ＝H，R 2 ＝H。
The self-immolative HDAC probe P1 of this example comprises three modules, a phenolic ester linker I, HDAC substrate and a fluorophore as a leaving moiety.
Wherein phenyl ester linker I has the formula:
the HDAC substrate is 6-acetamidohexanoic acid.
The fluorophore as leaving moiety is the fluorophore BIX-HM carrying a hydroxymethyl group.
Example 2:
a self-immolative HDAC probe P2 of this example, having the formula:
wherein R is 1 ＝H，R 2 ＝F。
The self-immolative HDAC probe P2 of this example comprises three modules, a phenyl ester linker ii, an HDAC substrate, and a fluorophore as a leaving moiety.
Wherein phenyl ester linker II has the formula:
the HDAC substrate is 6-acetamidohexanoic acid;
the fluorophore as leaving moiety is the fluorophore BIX-HM carrying a hydroxymethyl group. .
Example 3:
a self-immolative HDAC probe P3 of this example, having the formula:
wherein R is 1 ＝F，R 2 ＝F。
The self-immolative HDAC probe P3 of this example comprises three modules, a phenyl ester linker III, an HDAC substrate, and a caged fluorophore as a leaving moiety.
Wherein phenyl ester linker III has the formula:
the HDAC substrate is 6-acetamidohexanoic acid;
the fluorophore as leaving moiety is the fluorophore BIX-HM carrying a hydroxymethyl group.
Example 4:
a self-immolative HDAC probe P4 of this example, having the formula:
wherein R is 1 ＝H，R 2 ＝NO 2 。
The self-immolative HDAC probe P4 of this example comprises three modules, phenyl ester linker IV, HDAC substrate and fluorophore as leaving moiety.
Wherein phenyl ester linker IV has the formula:
the HDAC substrate is 6-acetamidohexanoic acid;
the fluorophore as leaving moiety is the fluorophore BIX-HM carrying a hydroxymethyl group.
Experiment one: examples 1 to 4 were tested for reactivity of P1 to P4 towards HDACs:
HDAC6, an HDAC that is widely involved in many biological and pathological processes, is used. The fluorescence response of P1-P4 (20. Mu.M) after reaction with HDAC6 (200 nM) at 37 ℃ for 3h was examined.
FIG. 6 shows the fluorescence intensity at 745nm of P1-P4 in the absence of HDAC6, as shown in the following: these probes have a low fluorescence background. In the presence of HDAC6, fluorescence was significantly enhanced (15-fold, 60-fold, and 90-fold, respectively) for fluoro-, difluoro-, and nitro-substituted phenyl ester linkers II-IV, while unsubstituted linker I showed only a slight increase in fluorescence. The results of absorption spectroscopy analysis are similar. Fluorescence kinetic analysis showed that the reaction rate of the nitro-substituted probe was the fastest. These results demonstrate that the electron-withdrawing substitution effect of the phenol ester linker is critical to the kinetics and efficiency of the self-immolative reaction that promotes HDAC activation.
Considering that HDACs act at a remote site from the ester bond, we assume that a large steric hindrance group near the ester can inhibit interference of carboxylesterase, while having minimal effect on HDAC reaction, and that the geminal dimethyl effect can shield the ester bond by steric hindrance and improve its stability against hydrolysis. We first designed and synthesized a probe with 2,2-dimethyl-6-acetamidohexanoic acid as the HDAC substrate.
Example 5:
a self-immolative HDAC probe P5 of this example, having the formula:
The self-immolative HDAC probe P5 of this example comprises three modules, a phenyl ester linker, an HDAC substrate, and a fluorophore or drug as a leaving moiety.
Wherein the phenyl ester linkage has the formula:
the HDAC substrate is 2,2-dimethyl-6-acetamidohexanoic acid;
the fluorophore as leaving moiety is the fluorophore BIX-HM carrying a hydroxymethyl group.
By changing the near infrared fluorophore from para to ortho, we obtained another probe P6 with a largely sterically hindered self-immolative linker.
Example 6:
a self-immolative HDAC probe P6 of this example, having the formula:
The self-immolative HDAC probe P1 of this example comprises three modules, a phenyl ester linker, an HDAC substrate, and a caged fluorophore or drug as a leaving moiety.
Wherein the phenyl ester linkage has the formula:
the HDAC substrate is 2,2-dimethyl-6-acetamidohexanoic acid;
the fluorophore as leaving moiety is the fluorophore BIX-HM carrying a hydroxymethyl group.
Experiment two: probes P4 to P6 were investigated for their specificity for three carboxylesterases, such as CES1, CES2 and PLE.
Probes P4 to P6 (20. Mu.M) were incubated with CES1, CES2 and PLE (100 nM) for 3h, respectively, and the fluorescence spectra were measured.
FIG. 7 shows the results of the fluorescence response of P4 to P6 to 200nM HDAC6, CES1, CES2 and PLE.
As seen from the figure: probe P4 reacted well with these carboxylesterases probe P5 showed a good fluorescence-enhanced response (30 fold) to HDAC6 and a better resistance (2 fold) to CES1, indicating that 2,2-dimethyl-6-acetamidohexanoic acid is a suitable substrate for HDAC 6. However, it is still susceptible to non-specific interference from CES2 (15 fold) and PLE (18 fold).
By changing the near infrared fluorophore from para to ortho, we obtained another probe P6 with a largely sterically hindered self-immolative linker. Interestingly, the fluorescence enhancement of probe P6 was negligible in the presence of CES1, CES2 and PLE, but showed significant fluorescence activation (30-fold) under the effect of HDAC 6.
This result indicates that the introduction of a large steric hindrance group near the ester bond provides a strategy for eliminating carboxylesterase interference. In addition, probe P6 was found to be ideally resistant to other intracellular small molecules, reactive oxygen species and enzymes. The probe is specific for HDAC activity, as its fluorescence intensity decreases significantly when incubated with the HDAC inhibitor archaea a (TSA). Further testing with HeLa cell lysates under TSA treatment showed negligible fluorescence enhancement. Taken together, these results indicate that we successfully developed a highly efficient deacetylase self-immolative system for the construction of HDAC probes.
To improve the solubility of the probe, a copper (I) -catalyzed azido alkyne cycloaddition was used to modify a sulfonate group onto probe P6, yielding another probe P7.
Example 7:
a self-immolative HDAC probe P7 of this example, having the formula:
The self-immolative HDAC probe P7 of this example comprises three modules, a phenolic ester linker, an HDAC substrate, and a fluorophore as a leaving moiety.
Wherein the structural formula of the phenol ester connector is as follows:
the HDAC substrate is 2,2-dimethyl-6-acetamidohexanoic acid;
the fluorophore as leaving moiety is BIX-HM-SO 3 - 。
Experiment three: examine the fluorescent response of P7 to different concentrations of HDAC 6:
after incubation of P7 (20 μ M) with different concentrations of HDAC6 for 3h, the fluorescence spectra were tested.
FIG. 8 shows the fluorescent response of P7 to HDAC 6. In the figure, a is the fluorescence response of P7 to different concentrations of HDAC6 (0-500 nM). Illustration is shown: linear fit curve of fluorescence intensity and HDAC6 concentration from 0 to 100 nM. As seen from the figure: the probe showed a gradually increasing fluorescence peak at 745nM with increasing HDAC6 concentrations in the range of 7.0nM to 500 nM.
In the figure, b is the UV-visible absorption spectrum after the reaction of P7 (20. Mu.M), P7 (20. Mu.M) and HDAC6 (200 nM). Similarly, the probe has an approximately 20-fold increase in the absorbance peak at 730nM when HDAC6 (400 nM) is present. In contrast, probe P7 showed negligible changes in the absorption spectrum in the mixture of HDAC6 and inhibitor TSA or in the presence of TSA, thus verifying its specific response to HDAC activity.
Further selectivity studies using different deacetylases (including HDAC1, HDAC2, HDAC3, HDAC10, HDAC11, sirt1 and Sirt 2) showed that c in the figure indicates that probe P7 is highly selective for HDAC6, while P4 has a different fluorescent response to different HDACs. This result indicates that 2,2-dimethyl-6-acetamidohexanoic acid can be a selective substrate for HDAC 6.
In the figure d the possibility of PA detection of HDAC activity by probe P7 was investigated. PA spectra and imaging after reaction at 37 ℃ when TSA (10. Mu.M) was added to or to a mixture of P7 (20. Mu.M), P7 (20. Mu.M) and HDAC6 (400 nM). The probe emits very weak PA signal in the 680-850nm range. In contrast, in the presence of HDAC6, it showed a strong PA signal with a signal to fold ratio at 730nm of about 8. Analysis of HDAC6 in the presence of TSA also demonstrated that PA signaling is specific for HDAC activity.
We demonstrate that NIRF/PA probes using nitro substituted self-immolative linkers provide HDAC sensitive imaging with ideal kinetics and high contrast in living cells and in vivo. It shows excellent specificity for HDAC compared to common carboxylesterases such as human carboxylesterase (CES 1, CES 2) and Pig Liver Esterase (PLE). This bimodal capability expands the potential of our probe for in vivo applications, as PA imaging provides high spatial resolution and tissue penetration depth, while NIRF imaging provides excellent sensitivity.
Based on this deacetylase self-immolative system, we developed an activatable probe using a novel spirocyclic xanthene dye for near infrared fluorescence (NIRF) and Photoacoustic (PA) dual mode imaging of HDACs.
Example 8:
the application of a deacetylase self-sacrifice system in vivo activation imaging of histone deacetylase is to perform NIRF/PA imaging on HDAC activity in living cells by using a designed probe P7. TNBC cell line MDA-MB-231 cells with upregulated HDAC activity were selected as a model.
Confocal cell fluorescence imaging experiments were performed as follows: all cells were incubated with 1mL of fresh medium containing probe P7 (20. Mu.M) for 3h at 37 ℃. Cells were washed three times with cold PBS prior to imaging. For inhibition studies, cells were pretreated with TSA (10 μ M) for 1 hour prior to incubation with probe (20 μ M). The fluorescence image is obtained by a Nikon A1+ confocal microscope, the excitation wavelength is 640nm, and the emission region is 663nm to 738nm.
Photoacoustic imaging of cells operates as follows: MDA-MB-231 cells (at 75 cm) 2 About 7X 10 in the cell culture flask of (2) 6 Individual cells) were incubated with P7 (20. Mu.M) at 37 ℃ for 3 hours. Cells were washed three times with 10mL PBS. Cells were harvested with 0.25% trypsin and counted using TC20 TM Automatic cell counter (BIO-RAD, USA). The cell suspension was collected by centrifugation using a microtube (200. Mu.L). Tubes with cell particles were inserted into the tube rack and imaged with the MSOT imaging system. For inhibition studies, MDA-MB-231 cells were pre-treated with TSA (10. Mu.M) for 1 hour prior to incubation with P7 (20. Mu.M).
FIG. 9 shows the NIRF/PA imaging of HDAC activity in living cells by P7.
Panel a is confocal fluorescence imaging of MDA-MB-231 cells incubated with P7 (20 μ M) without TSA or with TSA (10 μ M). From the figure, we observed that P7 showed bright fluorescence in MDA-MB-231 cells (FIG. 3 a). In contrast, cells pretreated with the HDAC inhibitor TSA showed negligible fluorescent signal.
Panel b is a flow cytometry analysis of MDA-MB-231 cells in response to P7 (20 μ M) without or with TSA (10 μ M). As seen from the figure: flow cytometry analysis showed a significant increase in fluorescence of this probe in cell populations with high HDAC activity compared to the inhibited cell population.
Panel c is PA response spectra and imaging of P7 (20 μ M), P7 (5 μ M) and MDA-MB-231 cell lysates (5 μ L) in 37 ℃ PBS with no or no TSA (10 μ M) added. As seen from the figure: further analysis of cell lysates using a commercial kit demonstrated high HDAC activity in MDA-MB-231 cells, whereas HDAC activity was not detected in TSA-pretreated cells. These results indicate that P7 is capable of specific fluorescence detection of HDAC activity in living cells. Furthermore, we used this probe to PA image HDAC activity in living cells by a multispectral photoacoustic tomography (MSOT) system. As expected, MDA-MB-231 cells showed a strong PA signal at 730nm, but a very weak PA response after TSA inhibition. Taken together, these results demonstrate that we have succeeded in developing an activatable HDAC active probe for live cell assays. To our knowledge, this is the first activatable probe for dual mode imaging of HDAC activity NIRF/PA.
Example 10: a self-immolative NIRF/PA probe for in vivo HDAC imaging.
The ability of probe P7 to image HDAC activity in MDA-MB-231 tumor-bearing mice was tested by tail vein injection. FIG. 10 is NIRF/PA imaging of HDAC activity in MDA-MB-231 tumor-bearing mice. As can be seen from the figure: systemic delivery of probes produces delayed NIRF and PA responses compared to intratumoral injection.
Panels b and c are representative NIRF and PA imaging of MDA-MB-231 tumor-bearing mice injected with P7 (1.85 mg/kg), intratumorally injected TSA, or no TSA at different time points, with saline as a control. White circles indicate tumor regions.
In the figure d and e are the time-dependent fluorescence (d) and PA intensity (e) at different time points for MDA-MB-231 tumor-bearing mice receiving different treatments (n = 5). In the figure, f is the fluorescence intensity of the main organs and tumor tissues dissected from the mice 5h after injection. * P <0.01, p <0.001, p <0.0001.
It was found from the figure that significant NIRF and PA responses were observed at the tumor site 3h after injection, with a maximum signal occurring 5h after injection, followed by a gradual decrease until 24h after injection. Taken together, these results indicate that our probe is capable of NIRF/PA dual mode imaging of HDAC activity in vivo.
Example 11: use of a deacetylase self-immolative system for assessing the efficacy of an HDAC inhibitor.
Experimental groups: four HDAC inhibitors, including three approved drugs vorinostat (SAHA), HDAC inhibitor (Quisinostat) and Panobinostat (Panobinostat), and abebestat (Abexinostat), a drug undergoing clinical trials, were administered to MDA-MB-231 tumor-bearing mice according to the recommended dosing regimen, followed by systemic delivery of P7 for NIRF imaging after three consecutive (once daily) injections. The ability of HDAC activity in MDA-MB-231 tumor-bearing mice was assessed using probe P7.
Control group: normal saline instead of P7;
FIG. 11 is a NIRF imaging of HDAC activity in MDA-MB-231 tumor-bearing mice. (a) Schematic of HDAC inhibitors and real-time NIRF imaging treatment schedules. (b) the chemical structure of the HDAC inhibitor. (c) Representative NIRF imaging of MDA-MB-231 tumor-bearing mice pretreated with different inhibitors after intravenous injection of P7 (1.85 mg/kg) at different time points (n = 5). White circles indicate tumor regions. (d) quantification of tumor site NIRF signal (n = 5). Test 1: quantification of NIRF signal after 5h injection of P7 (1.85 mg/kg). And (3) testing 2: HDAC activity in homogeneous tumors treated with different inhibitors was analyzed using a commercial HDAC kit. Data are presented as mean ± standard deviation.
As can be seen from the figure: the intensity of the fluorescence image will also vary greatly with the injection of different inhibitors. NIRF signals were reduced in both mice dosed with inhibitor compared to saline-treated control mice (fig. 8 c). The maximal reduction in NIRF signal in mice treated with Quisinostat indicates that it is the most effective inhibitor of HDAC activity in vivo. This finding suggests that our probes have the ability to evaluate HDAC inhibitors in vivo.
Example 12:
the application of a deacetylase self-immolative system for prodrug release, the principle of which is shown in fig. 1e, enables in vivo targeted treatment of HDAC overexpressed Triple Negative Breast Cancer (TNBC). Our studies created the first generic deacetylase self-immolative system for HDAC-specific NIRF/PA imaging and prodrug release in vivo, highlighting its therapeutic potential.
This deacetylase self-immolative system is universally applicable to fluorophores and drugs for the development of HDAC targeted probes and therapies.
Given the high activity of HDACs in cancer cells, especially aggressive tumors, we have an incentive to develop a HDAC specific prodrug release system. We chose the topoisomerase I inhibitor SN-38, an active camptothecin component commonly used in the treatment of various cancers. Blocking its phenolic hydroxyl group is reported to significantly reduce its efficacy and toxicity. HDAC mediated prodrug systems were designed by etherification of the phenolic hydroxyl group of SN-38 with our deacetylase self-immolative system. 1 HNMR、 13 C NMR and ESI-MS confirmed the successful synthesis of the prodrug. After uncapping, the emission peak of SN-38 shifts from 425nm to 550nm, which allows its release to be monitored by fluorescence spectroscopy. Fluorescence spectra over time show that SN-38 is gradually activated and reaches a maximum after incubation with HDAC6 for-3 h. Further time-dependent HPLC analysis also confirmed that the prodrug was capable of releasing in response to HDAC 6. In addition, the prodrug is specifically released by HDAC6, with ideal resistance to CES1, CES2 and PLE. Taken together, these results indicate that our design provides a highly potent, specific prodrug system for HDACs.
In addition, fluorescent staining of prodrug-treated MCF-10A and MDA-MB-231 cell lines with Calcein Acetyl methyl ester (Calcein AM) and Propidium Iodide (PI) demonstrated selective cytotoxicity against TNBC MDA-MB-231 cells. Taken together, these results demonstrate that our prodrug system can be activated with high specificity in HDAC upregulated cell lines, resulting in specific cytotoxicity on TNBC cells.
The efficacy of the prodrug system was further evaluated in MDA-MB-231 tumor-bearing mice, which were randomized into three groups and injected with prodrug via tail vein, compared to uncapped drug SN-38 and saline controls.
FIG. 12 is a graph of the in vivo therapeutic effect of prodrugs on MDA-MB-231 tumor-bearing mice.
a isSchematic representation of tumor model establishment and cancer treatment schedule. b is a schematic representation of the mechanism by which HDAC activates prodrug release. c is the intravenous injection of normal saline, SN-38 (4.0 mg kg) once every other day -1 ) Or prodrug (7.5 mg kg) -1 ) Five times later, MDA-MB-231 tumor growth curves. d is the body weight of MDA-MB-231 tumor-bearing mice after different treatments. e is a photograph of the dissected tumor on day 30. f is the final tumor weight after different treatments. g is H of tumor sections of MDA-MB-231 tumor-bearing mice receiving different treatments on day 30&E and TUNEL staining. Green fluorescence is the TUNEL staining signal and blue fluorescence is the nuclear staining signal. Scale =50 μm. In all data sets, all groups n =5 mice. * p is a radical of<0.05，**p<0.01，***p<0.001，***p<0.0001。
As can be seen from the figure: the tumor volume was significantly reduced in the prodrug group mice compared to the saline group. The tumor inhibition rate was about 70% (panels c and d). Interestingly, mice given SN-38 showed tumor growth inhibition, but to a lesser extent (panels e and f). We observed that tumor bearing mice treated with our prodrugs showed a greater degree of tumor cell apoptosis compared to tumor bearing mice treated with SN-38. In vitro fluorescence imaging of dissected tumors and major organs of mice treated with the prodrug confirmed the release of SN-38 in tumors. Taken together, these results indicate that our prodrug system provides an effective therapeutic paradigm for HDAC upregulated aggressive cancers, allowing tumor-specific activation of the prodrug with negligible side effects.
In conclusion, we have designed a self-immolative system using deacetylase for in vivo HDAC activation imaging and prodrug release. By fine tuning the reactivity and steric hindrance of the phenolic ester linker, a self-immolative system is obtained. A novel spirocyclic xanthene dye with ring-opening characteristics is designed, and a high signal power ratio is provided for NIRF/PA detection. We demonstrate that electron withdrawing substitution of phenyl ester linkages is critical to promote release kinetics and efficiency, and that the large steric hindrance of substrates with geminal dimethyl substitution and ortho-substitutions near the ester bond provides a viable strategy for eliminating carboxylesterases interference. In combination with a novel dye and deacetylase self-immolative system, we developed an activatable NIRF/PA probe with good kinetics, high contrast and good specificity for in vitro and in vivo detection of HDAC activity. NIRF/PA probes show the potential to detect HDAC upregulation in tumor cells. A novel HDAC specific prodrug system was further created to provide effective treatment for mice bearing HDAC upregulated tumors. Our generic deacetylase self-immolative system will open a new paradigm for the development of HDAC-specific probes and prodrugs, highlighting its potential for therapeutic applications.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Claims (10)
1. A deacetylase self-immolative system comprising a phenolic ester linker, an HDAC substrate, and a fluorophore;
the phenolic ester connector is one of phenolic ester connectors I to VII,
the structural formula of the phenol ester connector I is as follows:
the structural formula of the phenol ester connector II is as follows:
the structural formula of the phenol ester connector III is as follows:
the structural formula of the phenol ester connector IV is as follows:
the structural formula of the phenol ester connector V is as follows:
the structural formula of the phenol ester connector VI is as follows:
the structural formula of the phenol ester connector VII is as follows:
2. the deacetylase self-immolative system according to claim 1, wherein the HDAC substrate is 6-acetamidohexanoic acid or 2,2-dimethyl-6-acetamidohexanoic acid and the fluorophore is the carboxylic acid-bearing fluorophore BIX-CA or the hydroxymethyl group-bearing fluorophore BIX-HM.
3. The deacetylase self-immolative system according to claim 1, wherein the fluorophore is BIX-HM-SO 3 - 。
4. The deacetylase self-immolative system according to any one of claims 1 to 3, wherein the deacetylase self-immolative system is an HDAC probe having the following structural formula I, II, III, IV:
wherein R is 1 Is H or F, R 2 Is H, F or NO 2 。
5. Use of a deacetylase self-immolative system according to any one of claims 1 to 4 for imaging of in vivo activation of a deacetylase.
6. The use according to claim 5, wherein the in vivo activation imaging is confocal cell fluorescence imaging, comprising the steps of: cells were incubated with fresh medium containing HDAC probes and imaged with a confocal microscope.
7. The use according to claim 5, wherein the in vivo activation imaging is photoacoustic imaging of cells, comprising the steps of: culturing MDA-MB-231 cells with HDAC probe, collecting cells with trypsin, and collecting cell suspension by microtube centrifugation; tubes with cell particles were inserted into the tube rack and imaged with the MSOT imaging system.
8. The application according to claim 5, characterized in that it is specifically: NIRF/PA imaging of HDAC activity in vivo was performed on a living subject by tail vein injection of HDAC probe.
9. Use of a deacetylase according to any one of claims 1 to 4 from a sacrificial system for prodrug release.
10. Use of a deacetylase self-immolative system according to any one of claims 1 to 4 for assessing the efficacy of an HDAC inhibitor.
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| CN104120164A (en) * | 2013-04-26 | 2014-10-29 | 中国科学院大连化学物理研究所 | Specific fluorescence probe substrates of human carboxylesterase 2 and application thereof |
| CN108218743A (en) * | 2018-04-02 | 2018-06-29 | 湖南大学 | A kind of histon deacetylase (HDAC) fluorescence probe and its preparation method and application |
| CN114621754A (en) * | 2022-04-18 | 2022-06-14 | 武汉轻工大学 | Preparation of fluorescent probe and application of fluorescent probe in histone deacetylase |
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| CN104120164A (en) * | 2013-04-26 | 2014-10-29 | 中国科学院大连化学物理研究所 | Specific fluorescence probe substrates of human carboxylesterase 2 and application thereof |
| CN108218743A (en) * | 2018-04-02 | 2018-06-29 | 湖南大学 | A kind of histon deacetylase (HDAC) fluorescence probe and its preparation method and application |
| CN114621754A (en) * | 2022-04-18 | 2022-06-14 | 武汉轻工大学 | Preparation of fluorescent probe and application of fluorescent probe in histone deacetylase |
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| VIOLET ENG YEE LEE等: "Strategy for Traceless Codrug Delivery with Platinum(IV) Prodrug Complexes Using Self-Immolative Linkers", 《INORG. CHEM.》, vol. 60, pages 1823 * |
| 李娇: "基于组蛋白去乙酰化酶（HDAC）的近红外荧光探针的设计与合成", 《华东师范大学硕士论文》 * |
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