Imaging Sites of Inhibition of Proteolysis in Pathomimetic Human Breast Cancer Cultures by Light-Activated Ruthenium Compound

Ramalho, Suelem D.; Sharma, Rajgopal; White, Jessica K.; Aggarwal, Neha; Chalasani, Anita; Sameni, Mansoureh; Moin, Kamiar; Vieira, Paulo C.; Turró, Claudia; Kodanko, Jeremy J.; Sloane, Bonnie F.

doi:10.1371/journal.pone.0142527

Cited by 23 publications

(41 citation statements)

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“…While there was some level of inhibition by 5 in the dark, which was not statistically significant, DQ collagen-I degradation was reduced by over 50% under light conditions. Regarding inhibition in the dark, recent studies with 5 and other nitrile-based inhibitors caged with ruthenium complexes have shown that the caged complexes are less stable in cell growth media than in simple aqueous buffers or DMSO, where half-lives of > 1800 days have been observed, suggesting that some level of inhibitor release may occur under prolonged exposure to cell culture conditions (Respondek et al, 2014; Ramalho et al, 2015). The method used herein represented a significant challenge for our caged complexes, which need to be stable over the course of the assay (48 h) to provide high levels of light control.…”

Section: Discussionmentioning

confidence: 99%

Photoactivated inhibition of cathepsin K in a 3D tumor model

Herroon

Sharma

Rajagurubandara

et al. 2016

Biological Chemistry

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Collagenolytic activity of cathepsin K is important for many physiological and pathological processes including osteoclast-mediated bone degradation, macrophage function and fibroblast-mediated matrix remodeling. Here, we report application of a light-activated inhibitor for controlling activity of cathepsin K in a 3D functional imaging assay. Using prostate carcinoma cell line engineered to overexpress cathepsin K, we demonstrate the utility of the proteolytic assay in living tumor spheroids for the evaluation and quantification of the inhibitor effects on cathepsin K-mediated collagen I degradation. Importantly, we also show that utilizing the ruthenium-caged version of a potent nitrile cathepsin K inhibitor (4), cis-[Ru(bpy)2(4)2](BF4)2 (5), offers significant advantage in terms of effective concentration of the inhibitor and especially its light-activated control in the 3D assay. Our results suggest that light activation provides a suitable, attractive approach for spatial and temporal control of proteolytic activity, which remains a critical, unmet need in treatment of human diseases, especially cancer.

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Section: Discussionmentioning

confidence: 99%

Photoactivated inhibition of cathepsin K in a 3D tumor model

Herroon

Sharma

Rajagurubandara

et al. 2016

Biological Chemistry

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“…Data shown are from three independent experiments (48 fields); * ≤0.05; mean ± SD. Adapted from Ramalho et al [10]…”

Section: Figmentioning

confidence: 99%

Live-Cell Imaging of Protease Activity: Assays to Screen Therapeutic Approaches

Chalasani

Sameni

et al. 2017

Methods in Molecular Biology

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Methodologies to image and quantify the activity of proteolytic enzymes have been developed in an effort to identify protease-related druggable pathways that are involved in malignant progression of cancer. Our laboratory has pioneered techniques for functional live-cell imaging of protease activity in pathomimetic avatars for breast cancer. We analyze proteolysis in the context of proliferation and formation of structures by tumor cells in 3-D cultures over time (4D). In order to recapitulate the cellular composition and architecture of tumors in the pathomimetic avatars, we include other tumor-associated cells (e.g., fibroblasts, myoepithelial cells, microvascular endothelial cells). We also model noncellular aspects of the tumor micro-environment such as acidic pericellular pH. Use of pathomimetic avatars in concert with various types of imaging probes has allowed us to image, quantify, and follow the dynamics of proteolysis in the tumor microenvironment and to test interventions that impact directly or indirectly on proteolytic pathways. To facilitate use of the pathomimetic avatars for screening of therapeutic modalities, we have designed and fabricated custom 3D culture chambers with multiple wells that are either individual or connected by a channel to allow cells to migrate between wells. Optical glass microscope slides underneath an acrylic plate allow the cultures to be imaged with an inverted microscope. Fluid ports in the acrylic plate are at a level above the 3D cultures to allow introduction of culture media and test agents such as drugs into the wells and the harvesting of media conditioned by the cultures for immunochemical and biochemical analyses. We are using the pathomimetic avatars to identify druggable pathways, screen drug and natural product libraries and accelerate entry of validated drugs or natural products into clinical trials.

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“…12,33,49 The Ru(bpy) 2 fragment has also been used extensively to cage enzyme inhibitors and small-molecule drugs in live-cell assays. 21,28,34,36,50,51 Apart from Ru(bpy) 2 , Ru(II)-based photocaging groups are frequently composed of bi- or tridentate ancillary ligands, including 2,2′:6′,2″-terpyridine (tpy) and 1,10-phenanthroline (phen). 8,21,28,35,39,52,53 These imine-based photocages have been effectively applied to release bioactive molecules bearing nitriles, thioethers, and aromatic heterocycles, which generally cannot be protected by traditional organic photocages.…”

Section: Introductionmentioning

confidence: 99%

Ru(II) Polypyridyl Complexes Derived from Tetradentate Ancillary Ligands for Effective Photocaging

Turró

Kodanko

2018

Acc. Chem. Res.

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CONSPECTUS Metal complexes have many proven applications in the caging and photochemical release of biologically active compounds. Photocaging groups derived from Ru(II) traditionally have been composed of ancillary ligands that are planar and bi- or tridentate, such as 2,2′-bipyridine (bpy), 2,2′:6′,2″-terpyridine (tpy), and 1,10-phenanthroline (phen). Complexes bearing ancillary ligands with denticities higher than three represent a new class of Ru(II)-based photocaging groups that are grossly underdeveloped. Because high-denticity ancillary ligands provide the ability to increase the structural rigidity and control the stereochemistry, our groups initiated a research program to explore the applications of such ligands in Ru(II)-based photocaging. Ru(TPA), bearing the tetradentate ancillary ligand tris(2-pyridylmethyl)amine (TPA), has been successfully utilized to effectively cage nitriles and aromatic heterocycles. Nitriles and aromatic heterocycles caged by the Ru(TPA) group show excellent stability in aqueous solutions in the dark, and the complexes can selectively release the caged molecules upon irradiation with light. Ru(TPA) is applicable as a photochemical agent to offer precise spatiotemporal control over biological activity without undesired toxicity. In addition, Ru(II) polypyridyl complexes with desired photochemical properties can be synthesized and identified by solid-phase synthesis, and the resulting complexes show properties to similar to those of complexes obtained by solution-phase synthesis. Density functional theory (DFT) calculations reveal that orbital mixing between the π* orbitals of the ancillary ligand and the Ru−N dσ* orbital is essential for ligand photodissociation in these complexes. Furthermore, the introduction of steric bulk enhances the photoliability of the caged molecules, validating that steric effects can largely influence the quantum efficiency of photoinduced ligand exchange in Ru(II) polypyridyl complexes. Recently, two new photocaging groups, Ru(cyTPA) and Ru(1-isocyTPQA), have been designed and synthesized for caging of nitriles and aromatic heterocycles, and these complexes exhibit unique photochemical properties distinct from those derived from Ru(TPA). Notably, the unusually greater quantum efficiency for the ligand exchange in [Ru(1-isocyTPQA)(MeCN)2](PF6)2, Φ400 = 0.033(3), uncovers a trans-type effect in the triplet metal-to-ligand charge transfer (3MLCT) state that enhances photoinduced ligand exchange in a new manner. DFT calculations and ultrafast transient spectroscopy reveal that the lowest-energy triplet state in [Ru(1-isocyTPQA)(MeCN)2](PF6)2 is a highly mixed 3MLCT/3ππ* excited state rather than a triplet metal-centered ligand-field (3LF) excited state; the latter is generally accepted for ligand photodissociation. In addition, Mulliken spin density calculations indicate that a majority of the spin density in [Ru(1-isocyTPQA)(MeCN)2](PF6)2 is localized on the isoquinoline arm, which is opposite to the cis MeCN, rather than on the ruthenium center. This ...

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Imaging Sites of Inhibition of Proteolysis in Pathomimetic Human Breast Cancer Cultures by Light-Activated Ruthenium Compound

Cited by 23 publications

References 50 publications

Photoactivated inhibition of cathepsin K in a 3D tumor model

Photoactivated inhibition of cathepsin K in a 3D tumor model

Live-Cell Imaging of Protease Activity: Assays to Screen Therapeutic Approaches

Ru(II) Polypyridyl Complexes Derived from Tetradentate Ancillary Ligands for Effective Photocaging

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