Drug Targets In Cells Research Articles

Identification of PDCD2 as a Candidate Target of Andrographolide That Arrests the Tumor Cell Cycle by Human Proteome-Scale Screening.

Andrographolide (andro) and its derivatives have been reported to have antitumor activity by arresting the cell cycle. However, the more precise mechanism has been controversial. Here, a proteome chip was used to screen drug targets in cells, and we discovered that andro can bind to PDCD2 (PD2), which has been shown to be associated with the cell cycle and mRNA nuclear export. Then, RNA-binding protein immunoprecipitation for PD2 was used to detect the quantity of cell cycle-related mRNAs, and the nuclear distribution difference analyses of these mRNAs in tumor cells after andro intervention, followed by systematic experiments, were performed to assess the downstream effects of this event in vivo and in vitro. Thus, the target spectrum of andro was revealed at the level of the human proteome chip for the first time, and this work demonstrated that andro, through targeting PD2, blocks the nuclear output of CDK mRNAs in the nucleus of tumor cells, further reduces the expression of cell CDK proteins, and finally causes tumor cell cycle arrest in phenotype and tumor tissue growth arrest in vivo.

Abstract 2093: Imaging single cell drug target engagement in vivo reveals heterogeneous fractional occupancy

Abstract To produce effective therapeutic treatment, drugs need to reach and engage the cognate target in the cells of interest. Incomplete target occupancy may lead to cell survival and disease progression. Therefore, for maximum efficacy, it is critical to achieve high target occupancy within each cell of a tumor. However, the tumor microenvironment likely creates a dynamic, heterogenous distribution of drug that limits target occupancy in some cells. Unfortunately, we lack the tools to determine target engagement at the cellular level, which prevents studies on heterogeneous target occupancy and contributes to the poor understanding of drug failure mechanisms. Here, we developed an approach to measure target engagement of small molecule inhibitors within single cells in mouse xenograft in vivo tumor models. Our method uses fluorescently labeled, target specific companion imaging drugs, fluorescence polarization imaging, and intravital microscopy, to measure target occupancy of unlabeled, clinical drugs in single cells. We calculated the equivalent in vitro exposure of ibrutinib, a covalent BTK inhibitor, in vivo, but found increased cellular heterogeneity, highlighting differences between in vivo activity and in vitro measurements. Additionally, following systemic delivery of the reversible PARP inhibitor olaparib, we found cells with substantial uninhibited target when average measurements indicated all target was occupied, illustrating drug activity heterogeneity that may lead to tumor survival. These results demonstrate that our technique, which is the first to measure unlabeled drug engagement with target in single cells, can quantitate cellular fractional occupancy heterogeneity during drug treatment. We expect this approach to serve as a valuable tool when measuring cellular pharmacology in vivo, providing insight on dosing, drug activity in the tumor and sources of heterogeneous drug distribution. Citation Format: Matt Dubach, Ralph Weissleder. Imaging single cell drug target engagement in vivo reveals heterogeneous fractional occupancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2093.

The Heme Oxygenase-1-Targeting Compound PEG-ZnPP Inhibits Growth of Imatinib-Resistant BCR/ABL-Transformed Cells.

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the BCR/ABL oncogene and an increased survival of leukemic cells. The BCR-ABL tyrosine kinase inhibitor imatinib has successfully been introduced as a treatment of CML. However, resistance after an intitial response is common in patients with advanced disease, and it is not yet clear if responses in early disease phases will be durable. Therefore, current studies focus on novel potential drug-targets in CML cells. We have recently identified heme oxygenase-1 (HO-1) as a novel BCR/ABL-dependent survival-molecule in primary CML cells. In this study, we analyzed signal transduction pathways underlying BCR/ABL-induced expression of HO-1 and evaluated the role of HO-1 as a potential new target of drug therapy. We found that the PI3-kinase inhibitor LY294002 and MEK inhibitor PD98059 downregulate expression of HO-1 in CML cells. In addition, constitutively active Ras- and Akt -mutants were found to promote expression of HO-1 in Ba/F3 cells, further supporting the involvement of the PI3-kinase/Akt as well as the MAPK pathway in regulating HO-1 expression. To establish a role for HO-1 in survival of CML cells, expression of HO-1 was silenced by siRNAs which resulted in apoptosis of K562 cells. Next, HO-1 was targeted in CML cells by pegylated zinc protoporphyrin (PEG-ZnPP), a competitive inhibitor of HO-1. Exposure to PEG-ZnPP resulted in growth inhibition and induction of apoptosis in primary CML cells as well as in the CML-derived cell lines K562 and KU812 with IC50 values ranging between 1–10 μM. The growth-inhibitory effects of PEG-ZnPP were not only observed in CML cells responsive to imatinib, but also in imatinib-resistant K562 cells and Ba/F3 cells expressing various imatinib-resistant mutants of BCR/ABL (T315I, E255K, M351T, Y253F, Q252H, H396P). Moreover, imatinib and PEG-ZnPP were found to exert synergistic growth inhibitory effects on imatinib-resistant leukemic cells. Together, these data suggest that HO-1 represents a novel drug target in cells expressing BCR/ABL, including those with resistance to imatinib.