Smoothened Agonist

Highly sensitive detection of Smoothened based on the drug binding and rolling cycle amplification

Metastases are the leading causes of death in cancer patients. Due to intimate connection with metastasis, Smoothened (Smo) has become a therapeutic target for antimetastatic drugs and can provide early warning of metastasis in breast cancer. Thus, we have developed an electrochemical method in Smo analysis based on small-molecule drugs. Smo on the metastatic cell surface can be internalized after combination with the small-molecule drug. The surplus small-molecule drug and rolling circle amplification (RCA) primer are competitively binding with capture probe on the electrode surface through the click chemical reaction. After RCA reaction, methylene blue is used to label the RCA product. In this process, the more Smo on the metastatic cell surface, the more RCA primer is bound with peptide on the electrode. Therefore, the obtained signal response is positively correlated to Smo on the cancer cells. Moreover, the RCA provides sufficiently high sensitivity, enabling the limit of detection of Smo to be calculated as 0.1 pM (S/N = 3). Owing to its desirable sensitivity, excellent reproducibility, and high selectivity, the proposed method may hold great potential in clinical practice in the future.

Breast cancer has the highest incidence in female cancers threating women’s health seriously, and most of breast cancer deaths are attributed to metastases [1–4]. It is found that me- tastasis originates from the metastatic transformation of a few tumor cells in the primary tumor sites [5, 6]. So, the detection Electronic supplementary material The online version of this article ( contains supplementary material, which is available to authorized users of metastatic activity of breast cancer cells is crucial for in- creasing the survival rate [7].Significantly, Smoothened (Smo), a G protein–coupled re- ceptor, could be frequently upregulated in tumor cells and can provide early warning of metastasis in primary tumors [8–12]. Thus, it is needed to develop a specific and sensitive assay for Smo. Currently, the detection of Smo is commonly achieved by the traditional immunoassay, which mainly involves anti- bodies [13, 14]. However, antibodies have some disadvan- tages in the field of protein analysis, such as high cost, easy degradation, and complicated operation [15–18].In recent years, Smo has become a therapeutic target for antimetastatic drugs, due to its intimate connection with me- tastasis. While many Smo receptor inhibitors have been re- ported, the relationship between structure and activity of these small drug molecules is clear [19–22]. Moreover, these chem- ical synthesized drug molecules possess uniform and stable properties. Thus, we consider utilizing these drug molecules as probes in the design of a new protein-sensing scheme in- stead of antibodies.

Herein, we report a sensitive and specific assay for Smo based on SAG (Smo-agonist) derivatives, which has special groups and a derived non-core group (see Electronic Supplementary Material (ESM) Fig. S1) [22–26]. After the special group of SAG binds with Smo specifically, both of them can be internalized into the cytosol [5]. The Smo can be quantified by testing the unbound SAG derivatives in solu- tion. In order to achieve the above design strategy, a compet- itive detection method is designed for the fabrication of the biosensor. The unbound SAG derivatives and rolling circle amplification (RCA) primer can bind to the peptide modified on the electrode surface competitively by click chemical ligation. After RCA reaction, methylene blue is labeled to RCA product. As a result, we can obtain the signal positively related with the abundance of Smo on the tumor cells. The results demonstrate that the new method can detect metastatic activity of the tumor cells in complex biological samples effectively, and there is a certain correlation between the concentration of Smo and the malignancy degree of tumor cells.

Human recombinant Smo protein was purchased from Abnova. T4 DNA ligase and phi29 DNA polymerase were purchased from New England Biolabs Corporation. 9- Mercapto nonanol (MN), human epidermal growth factor re- ceptor 2 (HER2), and CelLytic™ MEM Protein Extraction Kit were purchased from Sigma-Aldrich Corporation. SAG deriv- atives and peptides were synthesized by Shanghai Science Peptide Biological Technology Co., Ltd. (purity > 95%, ly- ophilized powder). Other chemical reagents were of analytical grade. The ultrapure water employed for the dilution of all the solutions was produced by a water purification system (Milli- Q), the purity of which was warranted by purification until the gold standard resistance of 18 MΩ cm.The oligonucleotides used in the experiment were synthe- sized by TaKaRa (Dalian, China) and the sequences are given as follows: primer DNA: N3-5′-AGGTGAACGTGTTTTTTGCG CTATCTTCA-3′, linear template DNA: 5′-TTATAGGGTTAGGGTTAGGG TTAGGGTAT-3′.T47D and MCF-7, from Type Collection of Chinese Academy of Sciences, were cultured in Dulbecco’s modified Eagle’s medium (DMEM, from Gibco Co.) containing 10% fetal cattle serum (FCS, from Hyclone Co.) and maintained in a humidified atmosphere with 5% CO2 at 37 °C.Gold electrode was treated according to the literature previ- ously reported [27–29]. Then, the electrode was immersed into the 10 mM PBS (pH 7.4) containing 5-μM peptides at 4 °C for 16 h and further rinsed with PBS (10 mM, pH 7.4) and dried again with nitrogen, followed by being dipped in 1 μM 9-mercapto nonanol for 3 h. The peptide can be modi- fied on the gold electrode through the Au-S, the efficiency of immobilization is around 10% [5]. The electrode was thor- oughly washed to remove non-specific adsorption with PBS (10 mM, pH 7.4).

The solution containing standard Smo or cell sample, SAG derivatives (20 nM), and RCA primer (1 nM) were incubated at 37 °C for 60 min. Then, the electrode modified with pep- tides was immersed into the above solution at 37 °C for 60 min and washed with PBS (10 mM, pH 7.4). The above electrode was incubated in 0.1-mL template DNA (1 μM) at 37 °C for 30 min. Later, T4 DNA ligase (6 unit/mL) was dropped on the electrode at 37 °C for 60 min; the template DNA could be converted into a circular DNA, which would serve as a template for rolling circle amplification reaction simultaneously. The rolling circle amplification reaction was carried out in phi29 DNA polymerase reaction buffer contain- ing phi29 DNA polymerase (50 unit/mL) and dNTP at 37 °C for 60 min. Finally, 5% detergent was mixed with the reaction solution to deactivate phi29 DNA polymerase. The above electrode was incubated in methylene blue solution (100 mM) for 40 min and washed with PBS (10 mM, pH 7.4). The modified electrode was finally ready for measurement.Cells were cultured in a 6-well plate and total protein was extracted using CelLytic™ MEM Protein Extraction Kit. The protein concentration was determined with the bicinchoninic acid assay. Proteins (10 μg) were separated on a 10% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes followed by blocking with 5% non-fat milk at 25 °C for 1 h. The membranes were incubated overnight with primary mouse monoclonal antibodies against Smo or GAPDH. Membranes were then washed and incubated with HRP-conjugated secondary antibody for 1 h at room temper- ature. Protein was determined using an enhanced chemilumi- nescence kit.A three-electrode system consisting of the modified gold disk electrode, platinum pole counter electrode, and saturated cal- omel reference electrode was used for all the electrochemical measurements. Electrochemical impedance spectra (EIS) were tested in 5 mM of [Fe(CN)6]3−/4− solution. Square wave voltammetry (SWV) was recorded in 10 mM Tris-HCl (pH 7.4). The detailed experimental parameters were as fol- lows: EIS: initial potential, 0.224 V; amplitude, 5 mV; fre- quency range, 0.1 Hz–10 kHz. SWV: the scan range, − 0.6 to 0.0 V; square wave amplitude, 25 mV; the data was obtain- ed at least three independent repetitions of the experiment. Error bars were shown in the figures.

Results and discussion
The principle of the proposed method for the detection of Smo is illustrated in Scheme 1. A mixed solution including cell sample, SAG derivatives, and RCA primer is incubated to- gether. SAG derivatives can bind with the Smo on the meta- static cell surface that induce internalization. The RCA primer and unbound SAG probe are separated from the solution through low-speed centrifugation and incubated with the modified electrode. They can bind to peptides modified on the electrode surface competitively through the click chemical ligation between the DBCO group at the termini of peptide and the azide group at the termini of the RCA primer or SAG probe. After RCA reaction, methylene blue is labeled to the RCA product. In this process, the more Smo are on the met- astatic cell surface, the more SAG derivatives are internalized into the cytoplasm. So more RCA primer can be bind with peptides modified on the electrode surface, resulting in a
The electrode modification process has been confirmed by EIS. As shown in Fig. 1, the bare gold electrode almost shows a straight line (curve a), implying a very low electron transfer resistance. However, the peptides are modified on the elec- trode surface, and the electron transfer resistance increases owing to the poor conductivity of peptides (curve b). After the unbound SAG derivatives and RCA primer react with the peptides modified on the electrode surface, resulting in a sig- nificant electron transfer resistance (curve c), the reason is that the RCA primer forms a sequential charged interface on the electrode surface, excluding [Fe(CN)6]3−/4− with negative charge in the solution. After the amplification by rolling circle replication, electron transfer resistance becomes larger be- cause more negatively charged DNA is modified in the gold electrode (curve d). The above results indicate the successful fabrication of the biosensor.

The affinity between SAG derivatives and Smo can be exam- ined by investigating the thermodynamic aspects of the SAG derivatives-Smo interactions. For each interaction, a charac- teristic thermo-titration plot can be obtained. The essential thermodynamic parameters can be deduced from the plot (Table 1). As shown in Fig. 2, there is a strong specific inter- action between SAG derivatives and Smo, which is sufficient for the binding in the actual biological reactions.To achieve the optimal performance of the assay, various ex- perimental conditions are then optimized, including the con- centrations of SAG derivatives and RCA primer, the incubation time between SAG derivatives and Smo, as well as the reaction time of the RCA. As shown in Fig. 2, the affinity is about 25 nM between SAG derivatives and Smo, and the concentration of the SAG derivatives cannot be substantially higher than its affinity. So, the concentration of SAG deriva- tives is selected to be about 20 nM. In that case, the low concentration of Smo can also be detected by signal amplifi- cation. As shown in Fig. S2 (see ESM), 1 nM of RCA primer is chosen for the following experiment, not only the absolute value of peak current is big but also the signal-to-noise ratio is satisfactory. As is shown in Fig. S3 (see ESM), the peak cur- rent increases along with the incubation time, and then it re- mains nearly unchanged. The results indicate 60 min is enough for the interaction between SAG derivatives and Smo. So, 60 min is chosen as the optimal reaction time. It can be observed in Fig. S4 (see ESM) that the peak current is positively related to the reaction time of the RCA, and it reaches a plateau at 60 min. Therefore, 60 min is selected in the subsequent experiments.

Under the optimal conditions, the assay method is then employed to detect Smo. It can be observed in Fig. 3 that the peak current increases along with the concentration of Smo. Because a large number of Smo can bind with SAG derivatives, the level of remaining SAG derivatives becomes lower and the competitiveness of which becomes weak. So, more RCA primer can be bound with peptides modified on the electrode surface, resulting in a positive signal associated with metastatic activity of the cancer cells. A plot of the peak cur- rents vs the Smo concentrations reveals a positive correlation in the range from 0.1 to 3200 pM. The average coefficient of variation shows a desirable reproducibility of our data, which is below 5%, indicating a good accuracy and repeatability of the testing system.In order to verify the specificity of the assay method for detect- ing Smo, we have challenged the biosensor with other interfere proteins, including denatured Smo, HER-2, and MUC-1. As shown in Fig. 4, there is a significant peak current of 3200 pM Smo compared with denatured Smo protein, MUC- 1, HER2, and blank control solution. The peak currents of all control proteins are nearly the same as those in the blank control. This result is reasonable because SAG derivatives can- not bind to other non-specific proteins and only a few RCA primers bind to the peptides modified on the electrode [30]. Therefore, our method has a good specificity for the determi- nation of Smo.

To further investigate the possibilities and reliability of the assay method in clinical practice application, we have detected the Smo on cancer cell surface with this assay method. As shown in Figs. 5 and 6, the peak currents increase along with the con- centration of T47D cells and MCF-7 cells respectively. In addi- tion, we find the level of Smo on the MCF cells is lower than that on T47D cells, which is in accordance with the result of Western blot and previous literature reports (Fig. 7). These results indicate the expression difference of Smo among different cell lines, the higher degree of malignancy, and the higher expression of Smo.

In summary, we have proposed a new electrochemical method for detecting Smo on metastatic cells based on SAG deriva- tives, which can bind with Smo on the cell surface specifically. Quantitative detection of Smo on metastatic cells is achieved with high sensitivity and specificity by utilizing competitive detection mechanisms and RCA (Table 2). The experimental results obtained in this work suggest the prospective applica- tion of the proposed method in combating metastatic cancer in the future. We believe the proposed method in this study can be developed as a universal approach for the assay of other important Smoothened Agonist cell membrane proteins.