64 65 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Nanofocused X-rays offer insights into the cellular life of catalyticmetallodrugs Related publication: Bolitho E. M., Coverdale J. P. C., Bridgewater H. E., Clarkson G. J., Quinn P. D., Sanchez-Cano C., Sadler P. J. Tracking reactions of asymmetric organo-osmium transfer hydrogenation catalysts in cancer cells. Angewandte Chemie International Edition 60 , 6462–6472 (2021). DOI: 10.1002/anie.202016456 Publication keywords: Anticancer catalysts; Bioorganometallic chemistry; X-ray Fluorescence; Organo-osmium complexes; Transfer hydrogenation T ransitionmetal catalysts have potential as therapeutic agents to treat cancer and other diseases. However, there is a need to improve the design of these catalysts tomake themmore efficient, so they can be used in lower doses, and hasten progress towards clinical use. Researchers from the University ofWarwick have designed a series of advanced organo-osmium catalysts that can transformpyruvate (an essential ketone in cell metabolism) to natural L-lactate or unnatural D-lactate inside cancer cells. This can cause selective destruction of cancer cells, but not healthy cells. Using synchrotron X-ray Fluorescence, researchers can track the catalysts in cancer cells and observe how long they remain intact and active. The Hard X-Ray Nanoprobe beamline (I14) allowed the team to study biological samples with a range of X-ray imaging and spectroscopic techniques using a nano-focused photon beam. This allowed direct detection of metals with subcellular resolution, providing valuable insight into the distribution and chemical properties of these metal complexes inside cells. The results obtained suggest a strategy for improving the design of catalytic organo-osmiumanticancer drugs. This work also demonstrated the wider potential of experimental approaches combining simultaneous detection of ligand halogen tags and metals by X-ray based techniques for the fields of chemical biology and medicinal chemistry. This approach can contribute to the design and development of new classes of transitionmetal complexes as therapeutic and biotechnological tools. Transition metal catalysts have potential as therapeutic agents to treat cancer and other diseases. 1 Such catalysts might transform multiple substrate molecules in situ , including exogenous prodrugs, diagnosis agents, and endogenous metabolites, 1,2 whilst requiring only low concentrations to achieve the desired activity. Therefore, therapeutic strategies based on metal catalysts might help to overcome resistance to chemotherapy and reduce unwanted side effects, 1,2 both of which are of current clinical concern. The Sadler group has developed a chiral organo-osmium half-sandwich complex 1 structurally derived from the well-established Noyori Ru(II) industrial catalysts (Fig. 1). 3 Once inside cells, and in the presence of the non-toxic hydride donor formate, this complex catalyses the enantioselective reduction of pyruvate (an essential precursor in cell metabolism) to natural L-lactate or unnatural D-lactate (depending on the chirality of the catalyst). 2 This ability to cause metabolic perturbations in cells requires the presence of an intact catalyst, and gives rise to promising antiproliferative activity in vitro against a variety of human cancer cells, but not healthy cells. 2 Yet, intracellular activity of 1 is marked by low catalytic efficiency, suggesting degradation of the complex inside cells. 2 This seems to be a common problem for most synthetic transition metal catalysts, which are normally designed to work under well-defined chemical conditions, such as in inert atmospheres or pure organic solvents. 4,5 Therefore, it is important to understand the cellular fate of synthetic catalysts to optimise their in cellulo catalytic and therapeutic efficiency. The coordination sphere of metal complexes canbe studied inbiological environments by halogenation of selected ligands, which permits application of a range of techniques, including: 19 F NMR or MRI for fluorinated compounds, ICP-MS and X-ray Fluorescence (XRF) using bromine or iodine tags, or PET/SPECT imaging after radiolabelling with e.g. 131 I. As such, it is possible to probe the stability of catalyst 1 in cells by labelling its chiral chelating ligand with a bromine tag to generate the analogue complex 2 (Fig. 1). This allows comparison of the relative cellular accumulation and localisation of Os centres and Br-tagged ligands, by detecting 189 Os and 79 Br ions using ICP-MS or following XRF emissions from Os (L 3 -M 5 = 8.9 keV) and Br (K-L 3 = 11.9 keV). Such halogen substitution does not affect significantly the chemical, structural and catalytic properties of the new catalyst compared to 1 . Complex 2 shows similar biological properties to the parent complex, having the same mechanism of action and being capable of performing in-cell transfer hydrogenation. Thus, 2 is a good model to study the stability of 1 in cellular environments. Initial ICP-MS studies show that cells accumulate more Br than Os (Fig. 1), but the cellular Br-to-Os ratio decreases when active transport is inhibited. Besides, excretion of the Os- and Br-carrying fragments seems to follow different cellular trafficking and efflux mechanisms. Acquisition of Os and Br XRF elemental maps at the Hard X-ray Nanoprobe beamline (I14) provides a more in-depth picture of the distribution of the different fragments of 2 in cancer cells with sub-cellular spatial resolution (Fig. 2). For this, lung cancer cells were grown on silicon nitride membranes and treated with 2 , before being cryo-fixed and freeze-dried for subsequent analysis under ambient conditions using a nanofocussed X-ray beam (100 x 100 nm 2 ). The XRF maps confirm that cells treated with the catalyst accumulate more Br than Os (2-7x more) after 24 h, confirmed by complementary ICP-MS experiments. Most of the intracellular Br and Os was found in regions of the cytosol (50-70% and 65-85%, respectively), although a significant amount of Br (30-50%), but not Os, also reached the nuclei of the cells. This implies that intracellular degradation of the complex is occurring, in agreement with the ICP-MS data. Nevertheless, within the cytosol, both Os and Br co- localise moderately (Pearson’s Coefficient R=0.17-0.39) in small circular compartments (0.65-0.78 µm 2 ). These organelles are likely to be lysosomes or endosomes, since they are known to be similar in size, and the uptake of 2 occurs through active transport mechanisms such as endocytosis. Remarkably, the Br-to-Os ratio is lower in those vesicular areas than in the rest of the cell, suggesting the presence of higher concentrations of intact complex within endosomes and lysosomes, and supporting transfer hydrogenation catalytic activity in the cytosol of cancer cells. It is also possible that catalysts might be degraded due to processes linked to their active intracellular transport. Remarkably, catalysts 1 and 2 are very stable in acidic environments, but they are degraded, and release their chiral chelated ligands, in presence of biologically relevant concentrations of thiol-containing biomolecules such as glutathione, commonly found in lysosomes. Moreover, reducing cellular levels of glutathione with low doses of L-buthionine sulfoximine, or inhibiting the activity of lysosomes in lung cancer cells with chloroquine diphosphate, leads to reduced degradation of the complex, and hence increased concentrations of intact catalyst inside cells and a significant increase in the anticancer potency of both 1 and 2 . Combining nanoscale synchrotron XRF mapping with ICP-MS, cellular uptake and mechanistic studies has provided new insights into the behaviour of asymmetric transfer hydrogenation catalyst 1 in cancer cells (Fig. 3). These experiments show that the catalysts are internalised by cells via a combination of both passive and active transport, reaching lysosomes, the cytosol and some other organelles. Once inside, in the presence of a hydride donor, intact catalysts facilitate the catalytic reduction of pyruvate to lactate in the cytosol, altering the metabolism of cancer cells and inhibiting their proliferation. Complex 1 also interacts with intracellular thiols. This leads to the release of Os- containing fragments and chelated ligands. The fragments generated during this degradation exhibit different cellular behaviour. Osmium-containing fragments are rapidly excreted from cells, while chelated ligands show much longer in-cell lifetimes. They are highly accumulated by cells, reaching even the cell nuclei before they are excreted. Such reactions help to explain the low intracellular turnover number (TON) observed for these catalysts. Overall, this work demonstrates how the use of halogen tags as probes for MS and X-ray based techniques can elucidate reactions of organometallic anticancer catalysts in cells. Moreover, the reactions observed between catalyst 1 and cellular thiols provide a basis for developing new generations of therapeutic catalysts designed to have enhanced stability towards biological thiols and cause metabolic perturbations with increased efficiency in target cancer cells. References: 1. Soldevila-Barreda J. J. et al . Intracellular catalysis with selected metal complexes and metallic nanoparticles: advances toward the development of catalytic metallodrugs. Chemical Reviews 119 , 829–869 (2019). DOI: 10.1021/acs.chemrev.8b00493 2. Coverdale J. P. C. et al . Asymmetric transfer hydrogenation by synthetic catalysts in cancer cells. Nature Chemistry 10 , 347–354 (2018). DOI: 10.1038/nchem.2918 3. Coverdale J. P. C. et al . Easy to synthesize, robust organo-osmium asymmetric transfer hydrogenation catalysts. Chemistry - A European Journal 21 , 8043–8046 (2015). DOI: 10.1002/chem.201500534 4. Bai Y. et al . Designed transition metal catalysts for intracellular organic synthesis. Chemical Society Reviews 47, 1811–1821 (2018). DOI: 10.1039/ C7CS00447H 5. Martínez-Calvo M. et al . Organometallic catalysis in biological media and living settings. Coordination Chemistry Reviews 359 , 57–79 (2018). DOI: 10.1016/j.ccr.2018.01.011 Funding acknowledgement: The authors thank the Engineering and Physical Sciences Research Council (EPSRC grant no. EP/P030572/1), andWarwick Collaborative Postgraduate Research Scholarship and Diamond Light Source (DLS, Oxford) for a studentship for Elizabeth M. Bolitho. Carlos Sanchez-Cano thanks the Spanish State Research Agency (grant PID2020-118176RJ-I00) and the Gipuzkoa Foru Aldundia (Gipuzkoa Fellows program; grant number 2019- FELL-000018-01/62/2019) for financial support. This work was performed under the Maria de Maeztu (Grant No. MDM-2017-0720) and Severo Ochoa (Grant No. CEX2018-000867-S) Centres of Excellence Programme run by the Spanish State Research Agency. All synchrotron work was performed at the I14 Beamline (DLS, Oxford) under experiment number sp20552-1] Corresponding authors: Prof. Peter J. Sadler, University of Warwick,
[email protected] Dr Carlos Sanchez-Cano, Donostia International Physics Center and Ikerbasque Basque Foundation for Science
[email protected] Imaging andMicroscopy Group Beamline I14 Figure 1: (a) Structures of the organo-osmium catalysts 1 and its brominated analogue 2 . (b) Accumulation of Os and Br (ng / million cells) in cancer cells treated with 2 for different times. Reprinted with permission from DOI: 10.1002/anie.202016456 under a CC-BY License. Figure 2: XRF elemental maps of Os and Br acquired from cryopreserved and freeze-dried lung cancer cells treated with (a) 30, (b) 90 or (c) 150 µM of 2 for 24 h, showing Os-Br co-localisation in small vesicle-like areas highlighted by red circles. Maps were collected using 100 nm step size and 0.1 s dwell time. The calibration bar is in pg mm -2 . Reprinted with permission from DOI: 10.1002/ anie.202016456 under a CC-BY License. Figure 3: Possible mechanisms proposed for the uptake, degradation and efflux of catalysts 1 and 2 in cancer cells. Reprinted with permission from DOI: 10.1002/anie.202016456 under a CC-BY License.