Echinomycin, a member of the quinoxaline family of antibiotics, is known to be a small-molecule inhibitor of hypoxia inducible factor–1 (HIF-1) DNA binding activity. Recently, it has been shown to suppress mammalian target of rapamycin (mTOR) signaling and growth in leukemia cell lines. In this study, we investigated whether echinomycin interacts with the FKBP12 protein. Molecular docking was used, and the predicted binding energy was −10.61 kcal/mol. Moreover, surface plasmon resonance imaging and fluorescence quenching techniques were used to validate this interaction. Echinomycin binds to FKBP12 with a strong binding affinity comparable with rapamycin. Furthermore, the echinomycin-FKBP12 complex has been shown to affect calcineurin activity when tested in a calcineurin phosphatase inhibition assay. All of these studies have shown that echinomycin may have a double impact on HIF signaling by direct inhibition and through mTOR.
The label-free and sensitive detection of small molecule drugs on SPRi is still a challenging task, mainly due to the limited surface immobilization capacity of the sensor. In this research, a dextran hydrogel-coated gold sensor chip for SPRi was successfully fabricated via photo-cross-linking for enhanced surface immobilization capacity. The density of the dextran hydrogel was optimized for protein immobilization and sensitive small molecule detection. The protein immobilization capacity of the hydrogel was 10 times greater than a bare gold surface, and 20 times greater than an 11-mercaptoundecanoic acid (MUA) surface. Such a drastic improvement in immobilization capacity allowed the SPRi sensor to detect adequate response signals when probing small molecule binding events. The binding signal of 4 nM liquid-phase biotin to streptavidin immobilized on the dextran surface reached 435 RU, while no response was observed on bare gold or MUA surfaces. The dextran hydrogel-coated SPRi sensor was also applied in a kinetic study of the binding between an immunosuppressive drug (FK506) and its target protein (FKBP12) in a high-throughput microarray format. The measured binding affinity was shown to be consistent with reported literature values, and a detection limit of 0.5 nM was achieved.
Systematic and rapid profiling of the interactions between small molecule drugs (or candidates) and target proteins is vital in studying drug promiscuity and drug discovery. However, a label-free and high-throughput method for the detection of small molecules binding on protein microarray is still lacking. In this research, we introduced surface plasmon resonance imaging (SPRi) technology combined with protein microarray on 3D-dextran hydrogel chip surface to realize the detection of small molecule drugs. With the development and optimization of this method, we obtained the ideal binding signals of small molecular drugs versus their target proteins, with good quality and uniformity (CV = 10.3%, n = 48). Then we demonstrated three applications, including in situ mimicking and detecting the formation of FK506-mediated trimer complex (FKBP12-FK506-calcineurin), rapid validation of FK506-FKBP12 recognition via FKBP12 mutant microarray (1 vs n mode), and 3 × 4 orthogonal studying the drug binding specificity on protein microarray (m vs n mode). Fortunately, these trials enable the label-free and kinetic study of drug-protein interactions on SPRi in a high-throughput manner, which could be a promising solution in screening/forecasting side effect of drugs (or candidates), drug repositioning or repurposing, target identification of natural products and personalized medicine, etc.
危险品化学品经营许可证（带存储）