Targeting cancer lactate metabolism with synergistic combinations of synthetic catalysts and monocarboxylate transporter inhibitors.

Synthetic anticancer catalysts offer potential for low-dose therapy and the targeting of biochemical pathways in novel ways. Chiral organo-osmium complexes, for example, can catalyse the asymmetric transfer hydrogenation of pyruvate, a key substrate for energy generation, in cells. However, small-molecule synthetic catalysts are readily poisoned and there is a need to optimise their activity before this occurs, or to avoid this occurring. We show that the activity of the synthetic organometallic redox catalyst [Os(p-cymene)(TsDPEN)] (1), which can reduce pyruvate to un-natural D-lactate in MCF7 breast cancer cells using formate as a hydride source, is significantly increased in combination with the monocarboxylate transporter (MCT) inhibitor AZD3965. AZD3965, a drug currently in clinical trials, also significantly lowers the intracellular level of glutathione and increases mitochondrial metabolism. These synergistic mechanisms of reductive stress induced by 1, blockade of lactate efflux, and oxidative stress induced by AZD3965 provide a strategy for low-dose combination therapy with novel mechanisms of action.

Tracking Reactions of Asymmetric Organo-Osmium Transfer Hydrogenation Catalysts in Cancer Cells.

Most metallodrugs are prodrugs that can undergo ligand exchange and redox reactions in biological media. Here we have investigated the cellular stability of the anticancer complex [OsII [(η6 -p-cymene)(RR/SS-MePh-DPEN)] [1] (MePh-DPEN=tosyl-diphenylethylenediamine) which catalyses the enantioselective reduction of pyruvate to lactate in cells. The introduction of a bromide tag at an unreactive site on a phenyl substituent of Ph-DPEN allowed us to probe the fate of this ligand and Os in human cancer cells by a combination of X-ray fluorescence (XRF) elemental mapping and inductively coupled plasma-mass spectrometry (ICP-MS). The BrPh-DPEN ligand is readily displaced by reaction with endogenous thiols and translocated to the nucleus, whereas the Os fragment is exported from the cells. These data explain why the efficiency of catalysis is low, and suggests that it could be optimised by developing thiol resistant analogues. Moreover, this work also provides a new way for the delivery of ligands which are inactive when administered on their own.

Ligand-Controlled Reactivity and Cytotoxicity of Cyclometalated Rhodium(III) Complexes.

We report the synthesis, characterisation and cytotoxicity of six cyclometalated rhodium(III) complexes [CpXRh(C^N)Z]0/+, in which CpX = Cp*, Cpph, or Cpbiph, C^N = benzo[h]quinoline, and Z = chloride or pyridine. Three x-ray crystal structures showing the expected "piano-stool" configurations have been determined. The chlorido complexes hydrolysed faster in aqueous solution, also reacted preferentially with 9-ethyl guanine or glutathione compared to their pyridine analogues. The 1-biphenyl-2,3,4,5,-tetramethylcyclopentadienyl complex [CpbiphRh(benzo[h]quinoline)Cl] (3a) was the most efficient catalyst in coenzyme reduced nicotinamide adenine dinucleotide (NADH) oxidation to NAD+ and induced an elevated level of reactive oxygen species (ROS) in A549 human lung cancer cells. The pyridine complex [CpbiphRh(benzo[h]quinoline)py]+ (3b) was the most potent against A549 lung and A2780 ovarian cancer cell lines, being 5-fold more active than cisplatin towards A549 cells, and acted as a ROS scavenger. This work highlights a ligand-controlled strategy to modulate the reactivity and cytotoxicity of cyclometalated rhodium anticancer complexes.

Glycoconjugated Metallohelices have Improved Nuclear Delivery and Suppress Tumour Growth In Vivo.

Monosaccharides are added to the hydrophilic face of a self-assembled asymmetric FeII metallohelix, using CuAAC chemistry. The sixteen resulting architectures are water-stable and optically pure, and exhibit improved antiproliferative selectivity against colon cancer cells (HCT116 p53+/+ ) with respect to the non-cancerous ARPE-19 cell line. While the most selective compound is a glucose-appended enantiomer, its cellular entry is not mainly glucose transporter-mediated. Glucose conjugation nevertheless increases nuclear delivery ca 2.5-fold, and a non-destructive interaction with DNA is indicated. Addition of the glucose units affects the binding orientation of the metallohelix to naked DNA, but does not substantially alter the overall affinity. In a mouse model, the glucose conjugated compound was far better tolerated, and tumour growth delays for the parent compound (2.6 d) were improved to 4.3 d; performance as good as cisplatin but with the advantage of no weight loss in the subjects.

Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia.

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE2) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4+/CD44+/CD62L+ and CD4+/CD44+/CD62L-/CD27+ T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation. This secondary immune activation drives local tissue-resident T cell development while limiting tissue injury.

Unlocking Dendritic Cell-Based Vaccine Efficacy through Genetic Modulation-How Soon Is Now?

The dendritic cell (DC) vaccine anti-cancer strategy involves tumour-associated antigen loading and maturation of autologous ex vivo cultured DCs, followed by infusion into the cancer patient. This strategy stemmed from the idea that to induce a robust anti-tumour immune response, it was necessary to bypass the fundamental immunosuppressive mechanisms of the tumour microenvironment that dampen down endogenous innate immune cell activation and enable tumours to evade immune attack. Even though the feasibility and safety of DC vaccines have long been confirmed, clinical response rates remain disappointing. Hence, the full potential of DC vaccines has yet to be reached. Whether this cellular-based vaccination approach will fully realise its position in the immunotherapy arsenal is yet to be determined. Attempts to increase DC vaccine immunogenicity will depend on increasing our understanding of DC biology and the signalling pathways involved in antigen uptake, maturation, migration, and T lymphocyte priming to identify amenable molecular targets to improve DC vaccine performance. This review evaluates various genetic engineering strategies that have been employed to optimise and boost the efficacy of DC vaccines.

Albumin-mediated extracellular zinc speciation drives cellular zinc uptake.

The role of the extracellular medium in influencing metal uptake into cells has not been described quantitatively. In a chemically-defined model system containing albumin, zinc influx into endothelial cells correlates with the extracellular free zinc concentration. Allosteric inhibition of zinc-binding to albumin by free fatty acids increased zinc flux.

Osmium-arene complexes with high potency towards Mycobacterium tuberculosis.

The treatment of tuberculosis (TB) poses a major challenge as frontline therapeutic agents become increasingly ineffective with the emergence and spread of drug-resistant strains of Mycobacterium tuberculosis (Mtb). To combat this global health problem, new antitubercular agents with novel modes of action are needed. We have screened a close family of 17 organometallic half-sandwich Os(II) complexes [(arene)Os(phenyl-azo/imino-pyridine)(Cl/I)]+Y- containing various arenes (p-cymene, biphenyl, or terphenyl), and NMe2, F, Cl, or Br phenyl or pyridyl substituents, for activity towards Mtb in comparison with normal human lung cells (MRC5). In general, complexes with a monodentate iodido ligand were more potent than chlorido complexes, and the five most potent iodido complexes (MIC 1.25-2.5 µM) have an electron-donating Me2N or OH substituent on the phenyl ring. As expected, the counter anion Y (PF6-, Cl-, I-) had little effect on the activity. The pattern of potency of the complexes towards Mtb is similar to that towards human cells, perhaps because in both cases intracellular thiols are likely to be involved in their activation and their redox mechanism of action. The most active complex against Mtb is the p-cymene Os(II) NMe2-phenyl-azopyridine iodido complex (2), a relatively inert complex that also exhibits potent activity towards cancer cells. The uptake of Os from complex 2 by Mtb is rapid and peaks after 6 h, with temperature-dependence studies suggesting a major role for active transport. Significance to Metallomics Antimicrobial resistance is a global health problem. New advances are urgently needed in the discovery of new antibiotics with novel mechanisms of action. Half-sandwich organometallic complexes offer a versatile platform for drug design. We show that with an appropriate choice of the arene, an N,N-chelated ligand, and monodentate ligand, half-sandwich organo-osmium(II) complexes can exhibit potent activity towards Mycobacterium tuberculosis (Mtb), the leading cause of death from a single infectious agent. The patterns of activity of the 17 azo- and imino-pyridine complexes studied here towards Mtb and normal lung cells suggest a common redox mechanism of action involving intracellular thiols.

Dose- and time-dependent tolerability and efficacy of organo-osmium complex FY26 and its tissue pharmacokinetics in hepatocarcinoma-bearing mice.

The organo-osmium complex [OsII(ɳ6-p-cym)(PhAzPy-NMe2)I]+ (FY26) exhibits promising in vitro antitumour activity against mouse hepatocarcinoma Hepa1-6 and other mouse or human cancer cell lines. Here, we drastically enhance water solubility of FY26 through the replacement of the PF6- counter-anion with chloride using a novel synthesis method. FY26⋅PF6 and FY26⋅Cl displayed similar in vitro cytotoxicity in two cancer cell models. We then show the moderate and late anticancer efficacy of FY26⋅PF6 and FY26⋅Cl in a subcutaneous murine hepatocarcinoma mouse model. Both efficacy and tolerability varied according to FY26 circadian dosing time in hepatocarcinoma tumour-bearing mice. Tumour and liver uptake of the drug were determined over 48 h following FY26⋅Cl administration at Zeitgeber time 6 (ZT6), when the drug is least toxic (in the middle of the light span when mice are resting). Our studies suggest the need to administer protracted low doses of FY26 at ZT6 in order to optimize its delivery schedule, for example through the use of chrono-releasing nanoparticles.

The Epstein-Barr Virus-Encoded EBNA1 Protein Activates the Bone Morphogenic Protein (BMP) Signalling Pathway to Promote Carcinoma Cell Migration.

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) protein is expressed in all virus-associated malignancies, where it performs an essential role in the maintenance, replication and transcription of the EBV genome. In recent years, it has become apparent that EBNA1 can also influence cellular gene transcription. Here, we demonstrate that EBNA1 is able to stimulate the expression of the Transforming growth factor-beta (TGFß) superfamily member, bone morphogenic protein 2 (BMP2), with consequential activation of the BMP signalling pathway in carcinoma cell lines. We show that BMP pathway activation is associated with an increase in the migratory capacity of carcinoma cells, an effect that can be ablated by the BMP antagonist, Noggin. Gene expression profiling of authentic EBV-positive nasopharyngeal carcinoma (NPC) tumours revealed the consistent presence of BMP ligands, established BMP pathway effectors and putative target genes, constituting a prominent BMP "signature" in this virus-associated cancer. Our findings show that EBNA1 is the major viral-encoded protein responsible for activating the BMP signalling pathway in carcinoma cells and supports a role for this pathway in promoting cell migration and possibly, metastatic spread.

Structure-activity relationships for osmium(II) arene phenylazopyridine anticancer complexes functionalised with alkoxy and glycolic substituents.

Twenty-four novel organometallic osmium(II) phenylazopyridine (AZPY) complexes have been synthesised and characterised; [Os(η6-arene)(5-RO-AZPY)X]Y, where arene = p-cym or bip, AZPY is functionalized with an alkoxyl (O-R, R = Me, Et, nPr, iPr, nBu) or glycolic (O-{CH2CH2O}nR*, n = 1-4, R* = H, Me, or Et) substituent on the pyridyl ring para to the azo-bond, X is a monodentate halido ligand (Cl, Br or I), and Y is a counter-anion (PF6-, CF3SO3- or IO3-). X-ray crystal structures of two complexes confirmed their 'half-sandwich' structures. Aqueous solubility depended on X, the AZPY substituents, arene, and Y. Iodido complexes are highly stable in water (X = I â‹™ Br > Cl), and exhibit the highest antiproliferative activity against A2780 (ovarian), MCF-7 (breast), SUNE1 (nasopharyngeal), and OE19 (oesophageal) cancer cells, some attaining nanomolar potency and good cancer-cell selectivity. Their activity and distinctive mechanism of action is discussed in relation to hydrophobicity (RP-HPLC capacity factor and Log Po/w), cellular accumulation, electrochemical reduction (activation of azo bond), cell cycle analysis, apoptosis and induction of reactive oxygen species (ROS). Two complexes show ca. 4× higher activity than cisplatin in the National Cancer Institute (NCI) 60-cell line five-dose screen. The COMPARE algorithm of their datasets reveals a strong correlation with one another, as well as anticancer agents olivomycin, phyllanthoside, bouvardin and gamitrinib, but only a weak correlation with cisplatin, indicative of a different mechanism of action.

Induction of immunogenic cell death in cancer cells by a photoactivated platinum(IV) prodrug.

The platinum(IV) prodrug trans,trans,trans-[Pt(N3)2(OH)2(py)2] (1) is stable and non-toxic in the dark, but potently cytotoxic to cancer cells when irradiated by visible light, including cisplatin-resistant cells. On irradiation with visible light, it generates reactive Pt(II) species which can attack DNA, and produces reactive oxygen species (ROS) and reactive nitrogen species (RNS) which exert unusual effects on biochemical pathways. We now show that its novel mechanism of action includes induction of immunogenic cell death (ICD). Treatment of cancer cells with 1 followed by photoirradiation with visible light induces calreticulin (CRT) expression at the surface of dying cancer cells. This is accompanied by release of high mobility group protein-1B (HMGB1) and the secretion of ATP. Autophagy appears to play a key role in this chemotherapeutically-stimulated ICD. The observed uneven distribution of ecto-CRT promotes phagocytosis, confirmed by the observation of engulfment of photoirradiated CT26 colorectal cancer cells treated with 1 by J774.A1 macrophages. The photoactivatable prodrug 1 has a unique mechanism of action which distinguishes it from other platinum drugs due to its immunomodulating properties, which may enhance its anticancer efficacy.

Ligand-centred redox activation of inert organoiridium anticancer catalysts.

Organometallic complexes with novel activation mechanisms are attractive anticancer drug candidates. Here, we show that half-sandwich iodido cyclopentadienyl iridium(iii) azopyridine complexes exhibit potent antiproliferative activity towards cancer cells, in most cases more potent than cisplatin. Despite their inertness towards aquation, these iodido complexes can undergo redox activation by attack of the abundant intracellular tripeptide glutathione (GSH) on the chelated azopyridine ligand to generate paramagnetic intermediates, and hydroxyl radicals, together with thiolate-bridged dinuclear iridium complexes, and liberate reduced hydrazopyridine ligand. DFT calculations provided insight into the mechanism of this activation. GS- attack on the azo bond facilitates the substitution of iodide by GS-, and leads to formation of GSSG and superoxide if O2 is present as an electron-acceptor, in a largely exergonic pathway. Reactions of these iodido complexes with GSH generate Ir-SG complexes, which are catalysts for GSH oxidation. The complexes promoted elevated levels of reactive oxygen species (ROS) in human lung cancer cells. This remarkable ligand-centred activation mechanism coupled to redox reactions adds a new dimension to the design of organoiridium anticancer prodrugs.

Metallodrugs are unique: opportunities and challenges of discovery and development.

Metals play vital roles in nutrients and medicines and provide chemical functionalities that are not accessible to purely organic compounds. At least 10 metals are essential for human life and about 46 other non-essential metals (including radionuclides) are also used in drug therapies and diagnostic agents. These include platinum drugs (in 50% of cancer chemotherapies), lithium (bipolar disorders), silver (antimicrobials), and bismuth (broad-spectrum antibiotics). While the quest for novel and better drugs is now as urgent as ever, drug discovery and development pipelines established for organic drugs and based on target identification and high-throughput screening of compound libraries are less effective when applied to metallodrugs. Metallodrugs are often prodrugs which undergo activation by ligand substitution or redox reactions, and are multi-targeting, all of which need to be considered when establishing structure-activity relationships. We focus on early-stage in vitro drug discovery, highlighting the challenges of evaluating anticancer, antimicrobial and antiviral metallo-pharmacophores in cultured cells, and identifying their targets. We highlight advances in the application of metal-specific techniques that can assist the preclinical development, including synchrotron X-ray spectro(micro)scopy, luminescence, and mass spectrometry-based methods, combined with proteomic and genomic (metallomic) approaches. A deeper understanding of the behavior of metals and metallodrugs in biological systems is not only key to the design of novel agents with unique mechanisms of action, but also to new understanding of clinically-established drugs.

In Vivo Selectivity and Localization of Reactive Oxygen Species (ROS) Induction by Osmium Anticancer Complexes That Circumvent Platinum Resistance.

Platinum drugs are widely used for cancer treatment. Other precious metals are promising, but their clinical progress depends on achieving different mechanisms of action to overcome Pt-resistance. Here, we evaluate 13 organo-Os complexes: 16-electron sulfonyl-diamine catalysts [(η6-arene)Os( N, N')], and 18-electron phenylazopyridine complexes [(η6-arene)Os( N, N')Cl/I]+ (arene = p-cymene, biphenyl, or terphenyl). Their antiproliferative activity does not depend on p21 or p53 status, unlike cisplatin, and their selective potency toward cancer cells involves the generation of reactive oxygen species. Evidence of such a mechanism of action has been found both in vitro and in vivo. This work appears to provide the first study of osmium complexes in the zebrafish model, which has been shown to closely model toxicity in humans. A fluorescent osmium complex, derived from a lead compound, was employed to confirm internalization of the complex, visualize in vivo distribution, and confirm colocalization with reactive oxygen species generated in zebrafish.