Haibin Shi

Specific Detection of Integrin αvβ3 by Light-Up Bioprobe with Aggregation-Induced Emission Characteristics

Specific bioprobes with fluorescence turn-on response are highly desirable for high contrast biosensing and imaging. In this work, we developed a new generation bioprobe by integrating tetraphenylsilole, a fluorogenic unit with aggregation-induced emission (AIE) characteristic, with cyclic arginine-glycine-aspartic acid tripeptide (cRGD), a targeting ligand to integrin α(v)β(3) receptor. Emission of the AIE probe is switched on upon its specific binding to integrin α(v)β(3), which allows quantitative detection of integrin α(v)β(3) in solution and real-time imaging of the binding process between cRGD and integrin α(v)β(3) on cell membrane. The probe can be used for tracking integrin α(v)β(3) and for identifying integrin α(v)β(3)-positive cancer cells.

Cell-based proteome profiling of potential dasatinib targets by use of affinity-based probes.

Protein kinases (PKs) play an important role in the development and progression of cancer by regulating cell growth, survival, invasion, metastasis, and angiogenesis. Dasatinib (BMS-354825), a dual Src/Abl inhibitor, is a promising therapeutic agent with oral bioavailability. It has been used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). Most kinase inhibitors, including Dasatinib, inhibit multiple cellular targets and do not possess exquisite cellular specificity. Recent efforts in kinase research thus focus on the development of large-scale, proteome-wide chemical profiling methods capable of rapid identification of potential cellular (on- and off-) targets of kinase inhibitors. Most existing approaches, however, are still problematic and in many cases not compatible with live-cell studies. In this work, we have successfully developed a cell-permeable kinase probe (DA-2) capable of proteome-wide profiling of potential cellular targets of Dasatinib. In this way, highly regulated, compartmentalized kinase-drug interactions were maintained. By comparing results obtained from different proteomic setups (live cells, cell lysates, and immobilized affinity matrix), we found DA-2 was able to identify significantly more putative kinase targets. In addition to Abl and Src family tyrosine kinases, a number of previously unknown Dasatinib targets have been identified, including several serine/threonine kinases (PCTK3, STK25, eIF-2A, PIM-3, PKA C-α, and PKN2). They were further validated by pull-down/immunoblotting experiments as well as kinase inhibition assays. Further studies are needed to better understand the exact relevance of Dasatinib and its pharmacological effects in relation to these newly identified cellular targets. The approach developed herein should be amenable to the study of many of the existing reversible drugs/drug candidates.

Fluorescent Light-up Probe with Aggregation-Induced Emission Characteristics for Alkaline Phosphatase Sensing and Activity Study

Fluorogens with aggregation-induced emission (AIE) characteristics have attracted intensified research interest in biosensing applications, and those with specific targeting ability are especially desirable. In this work, we designed and synthesized an AIE fluorescent probe by functionalizing a tetraphenylethylene (TPE) fluorogen with two phosphate groups (TPE-phos) for the detection of alkaline phosphatase (ALP) and its enzymatic activity based on the specific interaction between the probe and ALP. The probe is virtually nonfluorescent in aqueous media due to good water solubility. In the presence of ALP, the phosphate groups are cleaved through enzymatic hydrolysis, yielding a highly fluorescent product as a result of activated AIE process. This light-up probe shows excellent selectivity toward ALP among a group of proteins. The detection limit is found to be 11.4 pM or 0.2 U L(-1) in Tris buffer solution with a linear quantification range of 3-526 U L(-1). The assay is also successfully performed in diluted serum with a linear range up to 175 U L(-1), demonstrating its potential application in clinical analysis of ALP levels in real samples. Furthermore, by conducting kinetic analysis of the enzyme using TPE-phos as the substrate, the kinetic parameter kcat/KM is determined to be 5.1×10(5) M(-1) s(-1), indicating a high efficiency of the substrate.

Light-up Bioprobe with Aggregation-induced Emission Characteristics for Real-time Apoptosis Imaging in Target Cancer Cells

Specific bioprobes that are capable of real-time and targeted monitoring and imaging of cancer cell apoptosis are highly desirable for cancer diagnosis and the evaluation of cancer therapy efficacy. In this work, an asymmetric fluorescent light-up bioprobe with aggregation-induced emission (AIE) characteristics was designed and synthesized by the conjugation of two different hydrophilic peptides, caspase-specific Asp-Glu-Val-Asp (DEVD) and cyclic Arg-Gly-Asp (cRGD), onto a typical AIE luminogen of a tetraphenylsilole (TPS) unit. The asymmetric probe is almost non-emissive in aqueous solution and its fluorescence is significantly switched on in the presence of caspase-3. The fluorescence turn-on is due to the cleavage of the DEVD moiety by caspase-3, and the aggregation of released TPS-cRGD residues, which restricts the intramolecular rotations of TPS phenyl rings and populates the radiative decay channels. Application of the asymmetric light-up probe for real-time targeted imaging of cancer cell apoptosis is successfully demonstrated using integrin αvβ3 receptor overexpressing U87MG human glioblastoma cells as an example. The probe shows specific targeting capability to U87MG cancer cells by virtue of the efficient binding between cRGD and integrin αvβ3 receptors and is able to real-time monitor and image cancer cell apoptosis in a specific and sensitive manner.

Distinct Optical and Kinetic Responses from E/Z Isomers of Caspase Probes with Aggregation-induced Emission Characteristics

A dual-labeled probe for monitoring caspase activity was designed and synthesized based on a tetraphenylethene (TPE) fluorogen with aggregation-induced emission characteristics and a caspase-specific Asp-Glu-Val-Asp (DEVD) peptide. Two stereoisomers were furnished and successfully separated by HPLC. We demonstrated for the first time the effect of isomerization on the reaction kinetics between the probes and caspase. It was revealed that caspase can produce a much higher light-up ratio for the Z-TPE-2DEVD probe, while its kinetics favor E-TPE-2DEVD due to enhanced probability of optimal binding between the two. Understanding the stereoisomers and their biological functions will open new opportunities for bioprobe design with optimized performance.

Peptide microarrays for high-throughput studies of Ser/Thr phosphatases.

Protein phosphorylation and dephosphorylation play an important role in regulation of intracellular signal transduction pathways in the biological system. A key step in the biological characterization of phosphatases and their use as drug targets is the identification of their cellular partners and suitable substrates for potential inhibitor development. Herein we describe a microarray-based protocol to map the substrate specificity of protein Ser/Thr phosphatases. This protocol uses Pro-Q dye to sensitively and quantitatively detect the amount of dephosphorylation that occurs from many putative peptide substrates in parallel, and therefore could be used to generate the so-called peptide substrate fingerprints as well as detailed kinetic information of a target phosphatase. Excluding the synthesis of the peptide substrates, the whole protocol takes a total of 11 h to complete and in future can be readily extended to the study of other classes of phosphatases, i.e., protein tyrosine phosphatases.

Fluorescence turn-on detection of live cell apoptosis using a hyperbranched conjugated polyelectrolyte

A fluorescence “turn-on” assay for live cell apoptosis imaging is developed based on self-assembled complexes between a cationic water-soluble hyperbranched conjugated polyelectrolyte (HCPE) and an Asp-Glu-Val-Asp (DEVD) peptide substrate labelled with a fluorescence quencher (DEVD-dabcyl). The polymer fluorescence can be significantly quenched upon DEVD-dabcyl binding, and the fluorescence is switched on upon treatment of the complexes with caspase-3/7, which are activated in the cell apoptotic process and are able to cleave the DEVD moiety. The light-up nature of the assay allows monitoring of caspase-3/7 activities both in solutions and in living cells with a high signal to noise ratio, which provides a new opportunity to screen caspase inhibitors and detect cell apoptosis.

A Method for Small Molecule Microarray-Based Screening for the Rapid Discovery of Affinity-Based Probes

We describe herein a new method for the high-throughput identification of affinity-based probes (AfBPs) using a small molecule microarray (SMM) approach. A hydroxylethylene-based small molecule library was first generated by solid-phase combinatorial synthesis. The library was tagged with biotin to facilitate immobilization on avidin-coated slides. Preliminary screening with γ-secretase (both the recombinantly purified protein as well as cellular lysates overexpressing the enzyme) was carried out, in order to identify potential small molecule binders, which were subsequently redesigned into AfBPs. Several specific and potent probes for γ-secretase were thus identified through the binding profiles observed on the SMMs. The SMM platform was able to sensitively and conveniently report activity-based binding interactions between aspartic proteases and their small molecule inhibitors. This new approach thus provides a potentially more rapid and efficient method for developing AfBPs using SMMs.

Expedient solid-phase synthesis of both symmetric and asymmetric diol libraries targeting aspartic proteases.

C(2)-symmetric diols have been shown to be highly potent against HIV-1 protease (PR). However, gaining access to these compounds has been hampered by the need of multistep solution-phase reactions which are often tedious and inefficient. In this Letter, we have disclosed a solid-phase strategy for rapid preparation of small molecule-based, symmetric and asymmetric diols as potential HIV-1 protease inhibitors. Upon biological screening, we found one of them, SYM-5, to be a potent and selective inhibitor (K(i)=400 nM) against HIV-1 protease.