Design,Synthesis And Evaluation Of Environment-Sensitive Fluorescent Inhibitors For Histone Deacetylases

Histone deacetylases(HDACs)are proteases that can catalyze the deacetylation of histones so as to inhibit gene transcription.HDACs also deacetylate non-histones and interact with other proteins to regulate various biological processes.Abnormal levels of HDAC are thought to be associated with a variety of diseases,including cancer,metabolic syndrome,and neurodegenerative diseases.Considering that mutations and/or aberrant expression of various HDACs are frequently involved in human diseases,particularly cancer,HDACs are important therapeutic targets for many human cancers.HDACs induces tumors not only by overexpression,but also by truncation or passivation mutations.Moreover,HDACs are associated with tumorigenesis through a variety of mechanisms,and more than one mechanism of HDAC has been determined in carcinogenesis so far.There are currently five HDAC inhibitors on the market,and more than 20 HDAC inhibitors are available in the phase II/III clinical trials.In addition,preclinical studies have shown that HDAC inhibitors can act synergistically with a variety of anticancer drugs against several tumors with different mechanisms of action.Overall,these evidences are sufficient to demonstrate that HDAC inhibitors have great potential for anti-malignant tumors.Because cancer and other diseases are highly demanding HDAC inhibitors with high potency and excellent selectivity,it is imperative to develop new HDAC probes.Currently,probes for HDAC are mainly divided into activity-based probes,PET probes and small molecule fluorescent probes.Small molecule fluorescent probes not only are sensitive,fast,high-throughput and easy to automate,but also examine timeand/or cell-dependent HDAC activity,and as a result,small molecule fluorescent probes are used to detect HDAC activity in vitro and in cellulo.However,there are still relatively few studies on HDAC small molecule fluorescent probes.This thesis designed a class of environmentally sensitive fluorescent inhibitors with switching mechanism,and selected SAHA,the most commonly used hydroxamic acid inhibitor of HDAC,as the recognition motif.Considering that small volume fluorophores can minimally affect the binding affinity of the parent ligand,a relatively small fluorophore SBD is designed to incorporate into the structure of SAHA.In classical design strategies,the fluorophore does not participate in the binding of the target protein,so that the binding activity of the parent ligand is more or less reduced.In our design strategy,based on the most commonly used hydroxamic acid HDAC inhibitor SAHA,a series of fluorescent HDAC inhibitors were designed by substituting the aromatic ring moiety of the inhibitor structure with the SBD fluorophore.The fluorophores in the structure of such fluorescent inhibitors are themselves pharmacophores,thus avoiding the effect of fluorophores on the binding of inhibitors and enzymes in conventional fluorescent probes.We hope to find HDAC fluorescent probes that have strong inhibitory activity against HDAC,good physicochemical properties,and can be used both for disease diagnosis and for disease treatment.Compound Z4 was selected after a series of bioactivity evaluations,in which its in vitro inhibitory activity was comparable to that of the positive control SAHA.The subtype selectivity was similar to that of SAHA,where both of them had high selectivity for HDAC1 and HDAC3 in the Class I family.The selectivity to HDAC2 and HDAC8 is poor,the selectivity to HDAC6 in the Class Ⅱa family is best,and the selectivity to HDAC7 in the Class Ⅱb family is poor.However,in vitro anti-tumor activity proliferation assay and apoptosis assay demonstrated less cytotoxicity than the positive control drug SAHA.Overall,Z4 has good optical properties,suitable cytotoxicity and high affinity with HDAC,and reasonable cell imaging results.In cell imaging study,MOLT4 cells and PC-3 cells with high expression of HDAC were stained,while HEK293 cells with low expression of HDAC were hardly stained,indicating that compound Z4 selectively labeled cells with high expression of HDAC.Co-localization imaging experiments also revealed that the main staining site of compound Z4 is cytoplasm,which is consistent with the reported results.However,compound Z4 has a high co-dyeing rate of 62.1%with membrane dye and almost no co-staining with nuclear dyes,which may be caused by the low membrane permeability of compound Z4.In the future work,the lipophilic portion of the compound should be increased to improve the membrane penetration.In the follow-up study,we will explore the application of this probe in the determination of HDAC enzyme activity and HDAC inhibitor screening,and try to use the more active HDAC inhibitor as the parent structure to improve the affinity of the compound with HDAC.In the future,in the process of designing compounds,it is considered to introduce a lipophilic group to increase the membrane permeability of the compound.