Target Identification Using Nanoprobe And Dual-target Anticancer Mechanism Of Thiazolidinone Compounds

Small organic molecules can affect cellular processes and phenotypes by modulating macromolecules function in the cells. It has made small molecules valuable as drugs in medicine and as probes for biological studies. As many drugs are developed by phenotype screening, the targets and mechanisms of these drugs need to be further investigated. Elucidating the targets and mechanisms is useful for showing the therapeutic and side effects of the drugs and developing second-generation drugs with a better profile.Generally, the strategies of target identification are divided into direct identification strategies and indirect identification strategies. In the direct identification strategies, the target proteins are separated from a cell or organ lysate and then identified. The indirect identification strategies is based on technology-driven functional genetic or bioinformatic platforms. The information of target proteins is obtained by the changes of gene or protein level in the cells treated with the test molecules. Compared with the strategies, the direct identification strategies facilitate to observe the target protein which binding with the molecule directly. The direct identification strategies are based on affinity purification, which requires that the test molecules are covalently attached to solid supports or radiolabel/imaging probes. The strategies can employ affinity chromatograph, biotinylated probes, radiolabeled or fluorescent probes and photoaffinity probes. Among them, the most simple and classic one is the target identification approches employing affinity chromatograph, which involves the linkage of modified compounds to solid supports, followed by incubation with cell or organ lysates. This approach separates the bound proteins from all other proteins by simple filtration, washing and elution. The advantages of this approach are easy operating and wide range of application. However, one of the limitations of this approach is that the biological activity of the solid support attached with compounds cannot be validated.We designed a generic approach for target identification using gold nanoprobes. The nanoprboes were constructed by attaching modified test compounds to gold nanoparticles. Using the nanoprobes, the binding proteins of test compounds were pulled down from the cellular proteome. The nanoprobes can enter live cells to confirm the desired biological activity and targeting specificity of the test compounds, which made up the drawback of the strategy employing affinity chromatograph. To prove this idea, the target proteins of thiazolidinone compound1which showed high efficacy of killing H460cancer cells were identified using gold nanoprobes. Before the target identification, it was necessary to test whether the gold nanoprobe could still kill cancer cells. After they were proven to be active in live cells, the nanoprobe attached with compound1(GNP-2) and another nanoprobe attached inactive thiazolidinone compound3(GNP-4) were incubated with H460cell lysates to identify the target proteins. According to the comparison with negative control GNP-4, two proteins were selected as the specific target proteins of compound1. Using MALDI-TOF MS and the Mascot search engine, the target proteins of compound1were identified to be a-tubulin and HSP90. Then, the target tubulin was validated by microtubule polymerization assay in vitro, microtubule immunofluorescence and JNK phosphorylation that a hallmark event for microtubule damage. The other target HSP90was validated by the change of the level of its client proteins, including CRAF-1, ERBB2and p-AKT. Our findings demonstrated the power of nanotechnology in drug discovery and chemical biology research. Target identification for therapeutic compounds has been a severely under developed area in drug discovery, and the validation of uncertain targets is tedious and expensive. Nanoprobes will possibly play a pivotal role in this area. In addition, this study indicates that the thiazolidinone compounds have the ability of tubulin interfering and dual-targeting.Microtubules are a very suitable and popular target for chemotherapeutic drugs against cancer cells that divide rapidly. The processes of microtubule damage to cell death are regulated by some signaling molecules, which are involved survival or pro-apoptotic signaling pathways. Among these signaling molecules, kinases are a kind of critical signals activated by microtubule damage. The activation of some kinases is related to survival signaling pathway, inducing cell survival; other kinases play a crucial role in the cytotoxicity after microtubule damage. However, more kinases related to microtubule damage-induced cell death need to be investigated. A better understanding of the interaction between microtubule damage and kinases will guide to design more efficient cancer chemotherapy. As our thiazolidinone compounds possibly have the ability of tubulin interfering and dual-targeting, we employed a pair of small molecule probes (probes A and B) from the thiazolidinone compound library synthesized previously by our lab, which both inhibit tubulin polymerization, to map the signaling events between microtubule damage and kinases. Although probes A and B had similar tubulin inhibition abilities in vitro, they induced different cellular phenotypes. Then, using kinase target identification, the second target of probe A that Dyrk1B kinase was identified. Probe A inhibited Dyrk1B activity in vitro, but not probe B. Then, Dyrk1B activity in RD cells was further investigated by an immune-complex kinase assay. The results showed that both probes A and B increased Dyrk1B activity after damaging microtubule, indicating that microtubule damage triggered a signaling pathway that led to the activation of Dyrk1B kinase. However, the increase of the Dyrk1B activity in the RD cells treated with probe A was less than the increase after probe B treatment, which further confirmed the DyrklB kinase inhibition of probe A. On the basis of the reported pro-survival function of Dyrk1B kinase and our previous results, the differences in cell phenotypes induced by A and B can be explained by the strong inhibition of probe A on the DyrklB-related survival pathway. It indicated that Dyrk1B kinase is a key survival factor that is activated by microtubule damage. In addition, we found that Dyrk1B triggered cell survival involves at least two paths:MAP4overexpression to promote microtubule stabilization and cell cycle recovery from G2/M arrest and p21 mitochondrial translocation to avoid apoptosis by inhibiting caspase-3activity. Besides, this work reports the first dual-targeting compound damaging a cancer target and its self-repair pathway simultaneously. A10-fold increase in cell viability inhibitory potency is realized by the double attacks. This finding opens a new revenue for future drug discovery strategic considerations.