Design,Synthesis And Biological Evaluation Of HDAC8 Selective Fluorescent Probe Based On Inhibitors

Histone deacetylase(HDAC)is involved in the proliferation of a variety of cancer cells and has been found to be abnormally high expressed in a variety of tumors.At present,the biological process of HDAC enzyme and its inhibitor has been studied comprehensively,especially the specific subtype enzyme has been widely studied.Among them,HDAC8 is a class Ⅰ subtype enzyme in the large HDAC family,which has been widely concerned due to its unique structural characteristics.The expression of HDAC8 is associated with a variety of cancers,but the function and biological process of HDAC8 protease still need to be further explored.The design and discovery of more effective selective HDAC8 inhibitors is also an important and challenging task in the field of anticancer therapy,especially for the design and development of subtype proteases.Therefore,HDAC8 was selected as a biomarker for cancer in this paper,with the hope of using fluorescence imaging technology to develop a selective fluorescent probe of HDAC8 to evaluate the relative expression of HDAC 8 in cells and tissues,predict the effect of inhibitors,and promote the research and development of HDAC8-targeted drugs.In this paper,two fluorescent probes(NP-C6-PCI and AM-C6-PCI)targeting HDAC8 protein were designed and synthesized by coupling PCI-34051,the most widely used specific inhibitor of HDAC8,with the fluorescent dye naphthalimide.The structure and spectral properties of the two probe compounds were determined.The binding force between the probe and HDAC8 was determined by biofilm interference(BLI)technique,and the HDAC enzyme inhibitory activity of the probe compound and the proliferation inhibitory activity of SH-SY5Y cells and MDA-MB-231 cells were evaluated.The laser confocal microscope(LSCM)was used to study the uptake conditions of the probe compound and the evaluation of cell fluorescence imaging,and the tissue slice imaging experiment was used to further verify the target binding effect of the probe and HDAC8 and the tissue imaging effect.The results showed that the synthesized probe had a selective binding ability and inhibitory activity against HDAC8.In tumor cells with high expression of HDAC8,the probe had a specific binding ability against HDAC8 protein and showed a good cell uptake process.The probe NP-C6-PCI could target and retain HDAC8 in tumor tissues with high HDAC8 expression,which was conducive to the evaluation of the subcellular location and relative expression of HDAC8 in surgically excised tissues or biopsy specimens,and is conducive to the prediction of patients’ response to drugs,thus promoting the research and development of HDAC8-related drugs.Specific research contents are as follows:Ⅰ.Design,synthesis and spectroscopic characterization of HDAC8 fluorescent probeIn this paper,the most widely used HDAC8 specific inhibitor PCI-34051 was selected as the targeted binding part,and the easily modified naphthalimide dye was used as the fluorescent signal part;The aromatic naphthalimide part and N-imide site of the dye were respectively coupled by flexible link chain to design a selective HDAC8 fluorescent probe.The molecular docking virtual screening was used to design the structure of the probe compounds,and the effects of the partial substituents of the fluorophor and the length of the link chain on the binding of the protein were investigated.Finally,the selective HDAC8 fluorescent probes NP-C6-PCI and AM-C6-PCI were designed and determined.The compounds were confirmed by HRMS-ESI,1H NMR and 13C NMR.Then,the spectroscopic properties of the probe at different concentrations and different excitation wavelengths were investigated.The ultraviolet absorption spectra of the probe compound were detected under the multi-functional enzyme plate analyzer.Finally,the excitation wavelength and emission wavelength of probe compounds NP-C6-PCI and AM-C6-PCI were 450nm and 550nm,respectively.Moreover,the fluorescence intensity of probe compounds NP-C6-PCI and AM-C6-PCI was linear within 80 μM,which could be used in subsequent biological evaluation experiments.Ⅱ.Bioactivity evaluation of HDAC8 fluorescent probe in vitro Biofilm interference(BLI)technique was used to detect the affinity of probe compounds with HDAC6 and HDAC8 proteins using Octet intermolecular interaction instrument.The results showed that NP-C6-PCI and AM-C6-PCI had a fast binding and disassociative process on HDAC8 protein.The Kd values of the binding force between NP-C6-PCI and AM-C6-PCI were lower than those of PCI-34051,which were 8.06×10-6 M and 7.42×10-6 M,respectively,and the binding effect was better than that of PCI-3405 1.The concentration dependence of NP-C6-PCI on HDAC8 protein was good,while the dependence trend of AM-C6-PCI on HDAC8 protein was slightly worse,but NP-C6-PCI and AM-C6-PCI did not bind to HDAC6.These results indicate that the two probe compounds obtained by fluorophore coupling based on PCI-34051 have similar binding ability to HDAC8 as PCI-34051,and the binding effect of NP-C6-PCI to HDAC8 is better.In this study,BPS fluorescence kit was used to further verify the enzyme inhibitory activity and selectivity relationship of probe compounds against HDAC1,HDAC6 and HDAC8,and PCI-34051 was selected to determine the IC50 concentration of HDAC8(10 nM).The results showed that the probes had similar inhibitory activity to the inhibitor PCI-34051,with half inhibitory effect on HDAC8,while the probes increased to 5 μM had no inhibitory activity on HDAC1 and HDAC6.These results proved that NP-C6-PCI and AM-C6-PCI had selective binding effect on HDAC8 protein and certain inhibitory effect on HDAC8 enzyme.This article further used QRT-PCR technology to verify the cell model SH-SY5Y cells and MDA-MB-231 cells with high expression of HDAC8.,cell proliferation activity determined by the MTT method,two probes of cell proliferation inhibition effect and the effect of inhibitors,illustrates the fluorophore stitching on inhibitors modify the two probes do not influence HDAC8 binding force;The probe NP-C6-PCI had a more similar effect to the inhibitor,while the probe AM-C6-PCI had a weaker effect.The results of in vitro enzyme inhibition and cell proliferation inhibition indicated that the probe NP-C6-PCI could be used as a potential fluorescent inhibitor of HDAC8,and the probe NP-C6-PCI was selected for imaging experiments in vitro and in vivo.Ⅲ.In vitro and in vivo imaging of HDAC8 fluorescent probeIn this chapter,the probe NP-C6-PCI with good enzyme binding ability and cell activity was selected to carry out the next cell imaging experiment.Firstly,the cytotoxicity test of the probe compound NP-C6-PCI was carried out on SH-SY5Y cells and MDA-MB-231 cells to verify the low toxicity of the probe to cells.The time and concentration of cell uptake were investigated,and the probe NP-C6-PCI was selected to incubate 5 μM for 30 min to investigate the inhibition of cell uptake competition.The inhibition effect of SH-SY5Y cells was stronger than that of MDA-MB-231 cells,possibly due to the expression of HDAC8 in SH-SY5Y cells and the stronger inhibitory activity of the inhibitor on SH-SY5Y cells.In order to further verify the targeted binding and relative quantitative effect of probe NP-C6-PCI on the target protein HDAC8 at the tumor tissue level,this paper established a mouse model of SH-SY5Y tumor cells and made tissue sections to conduct tissue fluorescence imaging experiments.In the staining test of the probe compound NP-C6-PCI tissue sections,the fluorescence intensity of tumor tissue sections was inhibited when co-incubated with the inhibitor PCI-34051,indicating that the retention of NP-C6-PCI in cells was mediated by HDAC8;Through the establishing a staining method for the probe NP-C6-PCI tissue section,and comparing the staining conditions of the probe between the tumor and normal tissue,it was found that under the same conditions,the normal tissue almost had no fluorescence,while the tumor tissue still had a certain amount of fluorescence,which proved that the probe NP-C6-PCI could be retained in the tumor tissue by targeting HDAC8.It can be used to evaluate the relative expression of HDAC8 in tumor tissue sections,predict the effect of inhibitors,and promote the development of HDAC8 targeting drugs.In a word,NP-C6-PCI is affordable and rapid for imaging in living cells and tissue slices mediated by HDAC8,which is expected for applications in target drug screening as well as in pathologic diagnosis.