The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform, Microfluidic Image Cytometry (MIC), capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000-2,800 cells. Using cultured cell lines, we demonstrate simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt and phospho-S6) within the oncogenic PI3K/Akt/mTOR signaling pathway. To demonstrate the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking inter- and intra-tumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters which predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine.
We
report the synthesis and photochemical and biological characterization
of the first selective and potent metal-based inhibitors of cytochrome
P450 3A4 (CYP3A4), the major human drug metabolizing enzyme. Five
Ru(II)-based derivatives were prepared from two analogs of the CYP3A4
inhibitor ritonavir, 4 and 6: [Ru(tpy)(L)(6)]Cl2 (tpy = 2,2′:6′,2″-terpyridine)
with L = 6,6′-dimethyl-2,2′-bipyridine (Me2bpy; 8), dimethylbenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine
(Me2dppn; 10) and 3,6-dimethyl-10,15-diphenylbenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (Me2Ph2dppn; 11), [Ru(tpy)(Me2bpy)(4)]Cl2 (7) and [Ru(tpy)(Me2dppn)(4)]Cl2 (9). Photochemical release of 4 or 6 from 7–11 was demonstrated, and the spectrophotometric evaluation of 7 showed that it behaves similarly to free 4 (type
II heme ligation) after irradiation with visible light but not in
the dark. Unexpectedly, the intact Ru(II) complexes 7 and 8 were found to inhibit CYP3A4 potently and specifically
through direct binding to the active site without heme ligation. Caged
inhibitors 9–11 showed dual action
properties by combining photoactivated dissociation of 4 or 6 with efficient 1O2 production.
In prostate adenocarcinoma DU-145 cells, compound 9 had
the best synergistic effect with vinblastine, the anticancer drug
primarily metabolized by CYP3A4 in vivo. Thus, our
study establishes a new paradigm in CYP inhibition using metalated
complexes and suggests possible utilization of photoactive CYP3A4
inhibitory compounds in clinical applications, such as enhancement
of therapeutic efficacy of anticancer drugs.
Highlights d We identified an allosteric inhibitory site for an activating enzyme d The lead compound inhibits the ATP-dependent step of SUMO E1 catalysis d The compound has specificity to 1 out of 18 non-disulfide bonded Cys residues d The compound increased miR-34b and reduced c-Myc in cellular and xenograft models
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.