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Related Publications
Journal articles
J. Biomed. Opt.
Extended dynamic range imaging for noise mitigation in fluorescence anisotropy imaging
Feruglio†, P. F.,
Vinegoni#†, C.,
and Weissleder, R.
Significance: Fluorescence polarization (FP) and fluorescence anisotropy (FA) microscopy are powerful imaging techniques that allow to translate the common IT assay capabilities into the in vitro and in vivo cellular domain. As a result, they have found potential for mapping drug- protein or protein-protein interactions. Unfortunately, these imaging modalities are ratiometric in nature and as such they suffer from excessive noise even under regular imaging conditions, preventing accurate image-feature analysis of fluorescent molecules behaviors. Aim: We present a high dynamic range (HDR)-based FA imaging modality for improving image quality in FA microscopy. Approach: The method exploits ad hoc acquisition schemes to extend the dynamic range of individual FP channels, allowing to obtain FA images with increased signal-to-noise ratio. Results: A direct comparison between FA images obtained with our method and the standard, clearly indicates how an HDR-based FA imaging approach allows to obtain high-quality images, with the ability to correctly resolve image features at different values of FA and over a substantially higher range of fluorescence intensities. Conclusion: The method presented is shown to outperform standard FA imaging microscopy narrowing the spread of the propagated error and yielding higher quality images. The method can be effectively and routinely used on any commercial imaging system and could be also translated to other microscopy ratiometric imaging modalities. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License.
32820624
PMC7439791
10.1117/1.Jbo.25.8.086003
Adv. Drug Deliv. Rev.
Fluorescence anisotropy imaging in drug discovery
Vinegoni#, C.,
Feruglio, P. F.,
Gryczynski, I.,
Mazitschek, R.,
and Weissleder, R.
Non-invasive measurement of drug-target engagement can provide critical insights in the molecular pharmacology of small molecule drugs. Fluorescence polarization/fluorescence anisotropy measurements are commonly employed in protein/cell screening assays. However, the expansion of such measurements to the in vivo setting has proven difficult until recently. With the advent of high-resolution fluorescence anisotropy microscopy it is now possible to perform kinetic measurements of intracellular drug distribution and target engagement in commonly used mouse models. In this review we discuss the background, current advances and future perspectives in intravital fluorescence anisotropy measurements to derive pharmacokinetic and pharmacodynamic measurements in single cells and whole organs. (C) 2018 Elsevier B.V. All rights reserved.
29410158
6072632
10.1016/j.addr.2018.01.019
Sci. Rep.
The anti-tumor diterpene oridonin is a direct inhibitor of Nucleolin in cancer cells
Vasaturo, M.,
Cotugno, R.,
Fiengo, L.,
Vinegoni, C.,
Dal Piaz, F.,
and De Tommasi#, N.
The bioactive plant diterpene oridonin displays important pharmacological activities and is widely used in traditional Chinese medicine; however, its molecular mechanism of action is still incompletely described. In vitro and in vivo data have demonstrated anti-tumor activity of oridonin and its ability to interfere with several cell pathways; however, presently only the molecular chaperone HSP70 has been identified as a direct potential target of this compound. Here, using a combination of different proteomic approaches, innovative Cellular Thermal Shift Assay (CETSA) experiments, and classical biochemical methods, we demonstrate that oridonin interacts with Nucleolin, effectively modulating the activity of this multifunctional protein. The ability of oridonin to target Nucleolin and/or HSP70 could account for the bioactivity profile of this plant diterpene. Recently, Nucleolin has attracted attention as a druggable target, as its diverse functions are implicated in pathological processes such as cancer, inflammation, and viral infection. However, up to now, no small molecule as Nucleolin binders has been reported, thus our finding represents the first evidence of Nucleolin modulation by a small inhibitor.
30425290
6233161
10.1038/s41598-018-35088-x
Nat. Chem. Biol.
Quantitating drug-target engagement in single cells in vitro and in vivo
Dubach, J. M.,
Kim, E.,
Yang, K.,
Cuccarese, M.,
Giedt, R. J.,
Meirnetis, L. G.,
Vinegoni#, C.,
and Weissleder#, R.
Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.
27918558
PMC5630128
10.1038/nchembio.2248
Theranostics
Design and Development of Fluorescent Vemurafenib Analogs for In Vivo Imaging
Mikula, H.,
Stapleton, S.,
Kohler, R. H.,
Vinegoni, C.,
and Weissleder#, R.
Herein we describe fluorescent derivatives of vemurafenib to probe therapeutic BRAF inhibition in live cells and in vivo. The compounds were evaluated and compared by determining target binding, inhibition of mutant BRAF melanoma cell lines and live cell imaging. We show that vemurafenib-BODIPY is a superior imaging drug to visualize the targets of vemurafenib in live cells and in vivo in non-resistant and resistant melanoma tumors.
28435463
PMC5399591
10.7150/thno.18238
Nat. Protoc.
Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging
Vinegoni#, C.,
Fumene Feruglio, P.,
Brand, C.,
Lee, S.,
Nibbs, A. E.,
Stapleton, S.,
Shah, S.,
Gryczynski, I.,
Reiner, T.,
Mazitschek, R.,
and Weissleder, R.
The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPEPE microscope, the protocol can be extended to other commercial or custom-built microscopes.
28686582
PMC5928516
10.1038/nprot.2017.043
IEEE J.S.T.Q.E.
Two-Photon Fluorescence Anisotropy Microscopy for Imaging and Direct Measurement of Intracellular Drug Target Engagement
Vinegoni#, C.,
Dubach, J. M.,
Feruglio, P. F.,
and Weissleder, R.
Ieee Journal of Selected Topics in Quantum Electronics 2016
Small molecule therapeutic drugs must reach their intended cellular targets (pharmacokinetics) and engage them to modulate therapeutic effects (pharmacodynamics). These processes are often difficult to measure in vivo due to their complexities and occurrence within single cells. It has been particularly difficult to directly measure cellular drug target binding. Fluorescence polarization is commonly used in pharmacological screening assays to measure drug-protein or protein-protein interactions. We hypothesized that fluorescence polarization imaging could be adapted and used with fluorescently labeled drugs to measure drug target engagement in vivo. Here, we summarize recent results using two photon fluorescence anisotropy microscopy. Our imaging technique offers quantitative pharmacological binding information of diverse molecular interactions at themicroscopic level, differentiating between bound, and unbound states. Used in combination with other recent advances in the development of novel fluorescently labeled drugs, we expect that the described imaging modality will provide a window into the distribution and efficacy of drugs in real time and in vivo at the cellular and subcellular level.
27440991
PMC4946648
10.1109/jstqe.2015.2501384
Drug Discov. Today
Advances in measuring single-cell pharmacology in vivo
Vinegoni#, C.,
Dubach, J. M.,
Thurber, G. M.,
Miller, M. A.,
Mazitschek, R.,
and Weissleder, R.
Measuring key pharmacokinetic and pharmacodynamic parameters in vivo at the single cell level is likely to enhance drug discovery and development. In this review, we summarize recent advances in this field and highlight current and future capabilities.
26024776
PMC4567932
10.1016/j.drudis.2015.05.011
Nat. Commun.
In vivo imaging of specific drug-target binding at subcellular resolution
Dubach†, J. M.,
Vinegoni#†, C.,
Mazitschek, R.,
Fumene Feruglio, P.,
Cameron, L. A.,
and Weissleder, R.
The possibility of measuring binding of small-molecule drugs to desired targets in live cells could provide a better understanding of drug action. However, current approaches mostly yield static data, require lysis or rely on indirect assays and thus often provide an incomplete understanding of drug action. Here, we present a multiphoton fluorescence anisotropy microscopy live cell imaging technique to measure and map drug-target interaction in real time at subcellular resolution. This approach is generally applicable using any fluorescently labelled drug and enables high-resolution spatial and temporal mapping of bound and unbound drug distribution. To illustrate our approach we measure intracellular target engagement of the chemotherapeutic Olaparib, a poly(ADP-ribose) polymerase inhibitor, in live cells and within a tumour in vivo. These results are the first generalizable approach to directly measure drug-target binding in vivo and present a promising tool to enhance understanding of drug activity.
24867710
PMC4362617
10.1038/ncomms4946
Neoplasia
Imaging Therapeutic PARP Inhibition In Vivo through Bioorthogonally Developed Companion Imaging Agents
Reiner, T.,
Lacy, J.,
Keliher, E. J.,
Yang, K. S.,
Ullal, A.,
Kohler, R. H.,
Vinegoni, C.,
and Weissleder#, R.
A number of small-molecule poly (ADP-ribose) polymerase (PARP) inhibitors are currently undergoing advanced clinical trials. Determining the distribution and target inhibitory activity of these drugs in individual subjects, however, has proven problematic. Here, we used a PARP agent for positron emission tomography-computed tomography (PET-CT) imaging (F-18-BO), which we developed based on the Olaparib scaffold using rapid bioorthogonal conjugation chemistries. We show that the bioorthogonal F-18 modification of the parent molecule is simple, highly efficient, and well tolerated, resulting in a half maximal inhibitory concentration (IC50) of 17.9 +/- 1.1 nM. Intravital imaging showed ubiquitous distribution of the drug and uptake into cancer cells, with ultimate localization within the nucleus, all of which were inhibitable. Whole-body PET-CT imaging showed tumoral uptake of the drug, which decreased significantly, after a daily dose of Olaparib. Standard F-18-fludeoxyglucose imaging, however, failed to detect such therapy-induced changes. This research represents a step toward developing a more generic approach for the rapid codevelopment of companion imaging agents based on small-molecule therapeutic inhibitors.