Clinical relevance and therapeutic predictive ability of hypoxia biomarkers in head and neck cancer tumour models.

Tumour hypoxia promotes poor patient outcomes, with particularly strong evidence for head and neck squamous cell carcinoma (HNSCC). To effectively target hypoxia, therapies require selection biomarkers and preclinical models that can accurately model tumour hypoxia. We established 20 patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of HNSCC that we characterised for their fidelity to represent clinical HNSCC in gene expression, hypoxia status and proliferation and that were evaluated for their sensitivity to hypoxia-activated prodrugs (HAPs). PDX models showed greater fidelity in gene expression to clinical HNSCC than cell lines, as did CDX models relative to their paired cell lines. PDX models were significantly more hypoxic than CDX models, as assessed by hypoxia gene signatures and pimonidazole immunohistochemistry, and showed similar hypoxia gene expression to clinical HNSCC tumours. Hypoxia or proliferation status alone could not determine HAP sensitivity across our 20 HNSCC and two non-HNSCC tumour models by either tumour growth inhibition or killing of hypoxia cells in an ex vivo clonogenic assay. In summary, our tumour models provide clinically relevant HNSCC models that are suitable for evaluating hypoxia-targeting therapies; however, additional biomarkers to hypoxia are required to accurately predict drug sensitivity.

In vitro and in vivo accumulation of the anticancer Ru complexes [RuII(cym)(HQ)Cl] and [RuII(cym)(PCA)Cl]Cl.

The cellular accumulation and the underlying mechanisms for the two ruthenium-based anticancer complexes [RuII(cym)(HQ)Cl] 1 (cym = η6-p-cymene, HQ = 8-hydroxyquinoline) and [RuII(cym)(PCA)Cl]Cl 2 (PCA = N-fluorophenyl-2-pyridinecarbothioamide) were investigated in HCT116 human colorectal carcinoma cells. The results showed that the cellular accumulation of both complexes increased over time and with higher concentrations, and that 2 accumulates in greater quantities in cells than 1. Inhibition studies of selected cellular accumulation mechanisms indicated that both 1 and 2 may be transported into the cells by both passive diffusion and active transporters, similar to cisplatin. Efflux experiments indicated that 1 and 2 are subjected to efflux through a mechanism that does not involve p-glycoprotein, as addition of verapamil did not make any difference. Exploring the influence of the Cu transporter by addition of CuCl2 resulted in a higher accumulation of 1 and 2 whilst the amount of Pt detected was slightly reduced when cells were treated with cisplatin. Complexes 1 and 2 were further explored in zebrafish where accumulation and distribution were determined with ICP-MS and LA-ICP-MS. The results correlated with the in vitro observations and zebrafish treated with 2 showed higher Ru contents than those treated with 1. The distribution studies suggested that both complexes mainly accumulated in the intestines of the zebrafish.

Clonal dynamics limits detection of selection in tumour xenograft CRISPR/Cas9 screens.

Transplantable in vivo CRISPR/Cas9 knockout screens, in which cells are edited in vitro and inoculated into mice to form tumours, allow evaluation of gene function in a cancer model that incorporates the multicellular interactions of the tumour microenvironment. To improve our understanding of the key parameters for success with this method, we investigated the choice of cell line, mouse host, tumour harvesting timepoint and guide RNA (gRNA) library size. We found that high gRNA (80-95%) representation was maintained in a HCT116 subline transduced with the GeCKOv2 whole-genome gRNA library and transplanted into NSG mice when tumours were harvested at early (14 d) but not late time points (38-43 d). The decreased representation in older tumours was accompanied by large increases in variance in gRNA read counts, with notable expansion of a small number of random clones in each sample. The variable clonal dynamics resulted in a high level of 'noise' that limited the detection of gRNA-based selection. Using simulated datasets derived from our experimental data, we show that considerable reductions in count variance would be achieved with smaller library sizes. Based on our findings, we suggest a pathway to rationally design adequately powered in vivo CRISPR screens for successful evaluation of gene function.

Fragment-based and structure-guided discovery of perforin inhibitors.

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands.

On-Resin Conjugation of the Ruthenium Anticancer Agent Plecstatin-1 to Peptide Vectors.

Ruthenium piano-stool complexes have been explored for their anticancer activity and some promising compounds have been reported. Herein, we conjugated a derivative of plecstatin-1 to peptides in order to increase their cancer cell targeting ability. For this purpose, plecstatin-1 was modified at the arene ligand to introduce a functional amine handle (3), which resulted in a compound that showed similar activity in an in vitro anticancer activity assay. The cell-penetrating peptide TAT48-60, tumor-targeting neurotensin8-13, and plectin-targeting peptide were functionalized with succinyl or ß-Ala-succinyl linkers under standard solid-phase peptide synthesis (SPPS) conditions to spatially separate the peptide backbones from the bioactive metal complexes. These modifications allowed for conjugating precursor 3 to the peptides on resin yielding the desired metal-peptide conjugates (MPCs), as confirmed by high-performance liquid chromatography (HPLC), NMR spectroscopy, and mass spectrometry (MS). The MPCs were studied for their behavior in aqueous solution and under acidic conditions and resembled that of the parent compound plecstatin-1. In in vitro anticancer activity studies in a small panel of cancer cell lines, the TAT-based MPCs showed the highest activity, while the other MPCs were virtually inactive. However, the MPCs were significantly less active than the small molecules plecstatin-1 and 3, which can be explained by the reduced cell uptake as determined by inductively coupled plasma MS (ICP-MS). Although the MPCs did not display potent anticancer activities, the developed conjugation strategy can be extended toward other metal complexes, which may be able to utilize the targeting properties of peptides.

Critical evaluation of cell lysis methods for metallodrug studies in cancer cells.

Intracellular accumulation studies are a key step in metallodrug development but often variable results are obtained. Therefore, we aimed here to investigate different protocols for efficient and reproducible lysis of cancer cells in terms of protein content in lysates and in cell uptake studies of the Ru anticancer complex [chlorido(8-oxyquinolinato)(η6-p-cymene)ruthenium(II)] ([Ru(cym)(HQ)Cl]). The physical lysis methods osmosis and sonication were chosen for comparison with chemical lysis with the radioimmunoprecipitation assay (RIPA) buffer. Based on the protein content and the total Ru accumulated in the lysates, the latter determined using inductively coupled plasma-mass spectrometry, RIPA buffer was the most efficient lysis method. Measurements of plastic adsorption blanks revealed that the higher Ru content determined in the RIPA buffer lysis samples may be due a higher amount of Ru extracted from the plastic incubation plates compared with osmosis and sonication. Overall, we found that the choice of lysis method needs to be matched to the information sought and we suggest the least disruptive osmosis method might be the best choice for labile drug-biomolecule adducts. Minimal differences were found for experiments aimed at measuring the overall cell uptake of the Ru complex.

Design, Synthesis and Anticancer Evaluation of Nitroimidazole Radiosensitisers.

The role of hypoxic tumour cells in resistance to radiotherapy, and in suppression of immune response, continues to endorse tumour hypoxia as a bona fide, yet largely untapped, drug target. Radiotherapy innovations such as stereotactic body radiotherapy herald new opportunities for classical oxygen-mimetic radiosensitisers. Only nimorazole is used clinically as a radiosensitiser, and there is a dearth of new radiosensitisers in development. In this report, we augment previous work to present new nitroimidazole alkylsulfonamides and we document their cytotoxicity and ability to radiosensitise anoxic tumour cells in vitro. We compare radiosensitisation with etanidazole and earlier nitroimidazole sulfonamide analogues and we identify 2-nitroimidazole and 5-nitroimidazole analogues with marked tumour radiosensitisation in ex vivo assays of surviving clonogens and with in vivo tumour growth inhibition.

Targeting growth hormone in cancer: future perspectives.

Decades of published research support a role for growth hormone (GH) in cancer. Accordingly, there is increasing interest in targeting GH in oncology, with GH antagonists exhibiting efficacy in xenograft studies as single agents and in combination with anticancer therapy or radiation. Here we discuss challenges associated with using growth hormone receptor (GHR) antagonists in preclinical models and considerations for translation, such as the identification of predictive biomarkers for selecting patients and for monitoring drug efficacy. Ongoing research will determine whether suppressing GH signalling pharmacologically will also reduce the risk of developing cancer. An increase in GH-targeted drugs in preclinical development will ultimately provide new tools to test anticancer efficacy of blocking the GH signalling pathway.

Anticancer Ru and Os complexes of N-(4-chlorophenyl)pyridine-2-carbothioamide: Substitution of the labile chlorido ligand with phosphines.

Half-sandwich MII(cym)Cl (cym = Î·6-p-cymene; M = Ru, Os) complexes of pyridinecarbothioamide (PCA) ligands have demonstrated potential as orally active anticancer agents. In order to investigate the impact of the substitution of the labile chlorido ligand with phosphorous donor ligands on the antiproliferative properties, the triphenylphosphine (PPh3) and 1,3,5-triaza-7-phophaadamantane (pta) analogues were prepared and characterized by spectroscopic techniques and the molecular structures of several complexes were determined by X-diffraction analysis. Interestingly, the molecular structures contained the PCA ligand deprotonated, presumably driven by the reduction in overall charge of the complex. Density Functional Theory (DFT) calculations suggested minor energy differences between the protonated and deprotonated forms. The aqueous stability and the reactivity with the amino acids l-histidine and l-cysteine were investigated by 1H NMR spectroscopy of representative examples. The most potent anticancer agents featured Ru or Os centers and a PPh3 ligand and showed IC50 values in the submicromolar range against four cancer cell lines. This suggests that the antiproliferative activity was mainly dependent on the lipophilic properties of the phosphine ligand with PPh3 having a significantly higher clog P value than pta.

Platinum(terpyridine) complexes with N-heterocyclic carbene co-ligands: high antiproliferative activity and low toxicity in vivo.

Pt(terpyridine) complexes are well-known DNA intercalators. The introduction of an NHC co-ligand rendered such a complex highly antiproliferative in cancer cells compared to its chlorido derivative. Despite the high potency, zebrafish embryos tolerated the compound well, especially compared to cisplatin. DNA interaction studies support a mode of action related to intercalation.

Hydrazone- and imine-containing [PdPtL4]4+ cages: a comparative study of the stability and host-guest chemistry.

A new [PdPtL4]4+ heterobimetallic cage containing hydrazone linkages has been synthesised using the sub-component self-assembly approach. 1H and DOSY nuclear magnetic resonance (NMR) spectroscopy and electrospray ionisation mass spectrometry (ESIMS) data were consistent with the formation of the [PdPtL4]4+ architecture. The cage was stimulus-responsive and could be partially disassembled and reassembled by the addition of dimethylaminopyridine (DMAP) and p-tolenesulfonic acid (TsOH), respectively. Additionally, the stability of the hydrazone cage against hydrolysis in the presence of water and nucleophilic decomposition in the presence of guest molecules was compared to a previously synthesised imine-containing [PdPtL4]4+ cage. It was established that the hydrazone linkage was more resistant to hydrolysis. Furthermore, the host-guest (HG) chemistry with a series of drug and drug-like molecules was examined. The hydrazone cage was shown to interact with cisplatin while the smaller imine cage was shown to interact with 5-fluorouracil and oxaliplatin in CD3CN. No HG interactions were observed in the more polar d6-DMSO. In vitro antiproliferative activity studies demonstrated both cages were active against the cancer cell lines tested and displayed half-maximal inhibitory (IC50) values in the range of 25-35 µM. Most [PdPtL4]4+-drug mixtures tested had higher IC50 values than the hosts. However, the [PdPtL4]4+ cages, and [PdPtL4]4+:drug mixtures were less cytotoxic than the well established anticancer drugs cisplatin, oxaliplatin and 5-fluorouracil.

Impact of Coordination Mode and Ferrocene Functionalization on the Anticancer Activity of N-Heterocyclic Carbene Half-Sandwich Complexes.

The substitution of phenyl rings in established drugs with ferrocenyl moieties has been reported to yield compounds with improved biological activity and alternative modes of action, often involving the formation of reactive oxygen species (ROS). Translating this concept to N-heterocyclic carbene (NHC) complexes, we report here organometallics with a piano-stool structure that feature di- or tridentate ligand systems. The ligands impacted the cytotoxic activity of the NHC complexes, but the coordination modes seemed to have a limited influence, which may be related to the propensity of forming the same species in solution. In general, the stability of the complexes in an aqueous environment and their reactivity to selected biomolecules were largely dominated by the nature of the metal center. While the complexes promoted the formation of ROS, the levels did not correlate with their cytotoxic activity. However, the introduction of ferrocenyl moieties had a significant impact on the antiproliferative potency of the complexes and, in particular, some of the ferrocenyl-functionalized compounds yielded IC50 values in the low µM range.

Preclinical Activity and Pharmacokinetic/Pharmacodynamic Relationship for a Series of Novel Benzenesulfonamide Perforin Inhibitors.

Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis in vitro and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells. All compounds showed >99% binding to plasma proteins and demonstrated perforin inhibitory activity in vitro and in vivo. A lead compound, compound 1, that showed significant increases in allogeneic bone marrow preservation was evaluated for its plasma pharmacokinetics and in vivo efficacy at multiple dosing regimens to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship. The strongest PK/PD correlation was observed between perforin inhibition in vivo and time that total plasma concentrations remained above 900 µM, which correlates to unbound concentrations similar to 3× the unbound in vitro IC90 of compound 1. This PK/PD relationship will inform future dosing strategies of BZS perforin inhibitors to maintain concentrations above 3× the unbound IC90 for as long as possible to maximize efficacy and enhance progression toward clinical evaluation.

Calcium Enabled Remote Loading of a Weak Acid Into pH-sensitive Liposomes and Augmented Cytosolic Delivery to Cancer Cells via the Proton Sponge Effect.

While delivery of chemotherapeutics to cancer cells by nanomedicines can improve therapeutic outcomes, many fail due to the low drug loading (DL), poor cellular uptake and endosomal entrapment. This study investigated the potential to overcome these limitations using pH-sensitive liposomes (PSL) empowered by the use of calcium acetate. An acidic dinitrobenzamide mustard prodrug SN25860 was used as a model drug, with non pH-sensitive liposomes (NPSL) as a reference. Calcium acetate as a remote loading agent allowed to engineer PSL- and NPSL-SN25860 with DL of > 31.1% (w/w). The IC50 of PSL-SN25860 was 21- and 141-fold lower than NPSL and free drug, respectively. At 48 h following injection of PSL-SN25860, NPSL-SN25860 and the free drug, drug concentrations in EMT6-nfsB murine breast tumors were 56.3 µg/g, 6.76 µg/g and undetectable (< 0.015 µg/g), respectively (n = 3). Meanwhile, the ex vivo tumor clonogenic assay showed 9.1%, 19.4% and 42.7% cell survival in the respective tumors. Live-cell imaging and co-localization analysis suggested endosomal escape was accomplished by destabilization of PSL followed by release of Ca2+ in endosomes allowing induction of a proton sponge effect. Subsequent endosomal rupture was observed approximately 30 min following endocytosis of PSL containing Ca2+. Additionally, calcium in liposomes promoted internalization of both PSL and NPSL. Taken together, this study demonstrated multifaceted functions of calcium acetate in promoting drug loading into liposomes, cellular uptake, and endosomal escape of PSL for efficient cytoplasmic drug delivery. The results shed light on designing nano-platforms for cytoplasmic delivery of various therapeutics.

A Solid Support-Based Synthetic Strategy for the Site-Selective Functionalization of Peptides with Organometallic Half-Sandwich Moieties.

The number of donor atoms available on peptides that can competitively coordinate to metal centers renders the site-selective generation of advanced metal-peptide conjugates in high purity a challenging venture. Herein, we present a transmetalation-based synthetic approach on solid support in which an imidazolium pro-ligand can be used to selectively anchor a range of transition metal half-sandwich complexes onto peptides in the presence of multiple coordinative motifs. Amenable to solid support, a range of N-terminus and/or lysine conjugated metal-peptide conjugates were obtained in high purity after cleavage from the resin. The metalated peptides were evaluated for their anticancer properties against human cancer cell lines. While no cytotoxic activity was observed, this platform has the potential to i) provide a pathway to site-selective peptide labelling, ii) be explored as a biorthogonal handle and/or iii) generate a new strategy for ligand design in transition metal catalysts.

Synthetic Strategy Towards Heterodimetallic Half-Sandwich Complexes Based on a Symmetric Ditopic Ligand.

Multimetallic complexes have been shown in several examples to possess greater anticancer activity than their monometallic counterparts. The increased activity has been attributed to altered modes of action. We herein report the synthesis of a series of heterodimetallic compounds based on a ditopic ligand featuring 2-pyridylimine chelating motifs and organometallic half-sandwich moieties. The complexes were characterized by a combination of 1H NMR spectroscopy, electrospray ionization mass spectrometry, elemental analysis and single crystal X-ray diffraction. Investigations into the stability of representative complexes in DMSO-d 6 and 10% DMSO-d 6 /D2O revealed the occurrence of solvent-chlorido ligand exchange. Proliferation assays in four human cancer cell lines showed that the Os-Rh complex possessed minimal activity, while all other complexes were inactive.

Incorporation of ß-Alanine in Cu(II) ATCUN Peptide Complexes Increases ROS Levels, DNA Cleavage and Antiproliferative Activity.

Redox-active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore leads to low ROS generation. In this work, we demonstrate that an exchange of the α-amino acid Gly with the ß-amino acid ß-Ala at position 2 (Gly2→ß-Ala2) of the ATCUN motif reinstates ROS production (• OH and H2 O2 ). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these ß-Ala2-containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for ß-Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where ß-Ala2 peptide complexes had lower IC50 values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP-MS measurements.

Anthracenyl Functionalization of Half-Sandwich Carbene Complexes: In Vitro Anticancer Activity and Reactions with Biomolecules.

N-Heterocyclic carbene (NHC) ligands are widely investigated in medicinal inorganic chemistry. Here, we report the preparation and characterization of a series of half-sandwich [M(L)(NHC)Cl2] (M = Ru, Os, Rh, Ir; L = cym/Cp*) complexes with a N-flanking anthracenyl moiety attached to imidazole- and benzimidazole-derived NHC ligands. The anticancer activity of the complexes was investigated in cell culture studies where, in comparison to a Rh derivative with an all-carbon-donor-atom-based ligand (5a), they were found to be cytotoxic with IC50 values in the low micromolar range. The Ru derivative 1a was chosen as a representative for stability studies as well as for biomolecule interaction experiments. It underwent partial chlorido/aqua ligand exchange in DMSO-d6/D2O to rapidly form an equilibrium in aqueous media. The reactions of 1a with biomolecules proceeded quickly and resulted in the formation of adducts with amino acids, DNA, and protein. Hen egg white lysozyme crystals were soaked with 1a, and the crystallographic analysis revealed an interaction with an l-aspartic acid residue (Asp119), resulting in the cleavage of the p-cymene ligand but the retention of the NHC moiety. Cell morphology studies for the Rh analog 3a suggested that the cytotoxicity is exerted via mechanisms different from that of cisplatin.

Triazolyl-Functionalized N-Heterocyclic Carbene Half-Sandwich Compounds: Coordination Mode, Reactivity and in†vitro Anticancer Activity.

We report investigations on the anticancer activity of organometallic [MII/III (η6 -p-cymene/η5 -pentamethylcyclopentadienyl)] (M=Ru, Os, Rh, and Ir) complexes of N-heterocyclic carbenes (NHCs) substituted with a triazolyl moiety. Depending on the precursors, the NHC ligands displayed either mono- or bidentate coordination via the NHC carbon atom or as N,C-donors. The metal complexes were investigated for their stability in aqueous solution, with the interpretation supported by density functional theory calculations, and reactivity to biomolecules. In vitro cytotoxicity studies suggested that the nature of both the metal center and the lipophilicity of the ligand determine the biological properties of this class of compounds. The IrIII complex 5 d bearing a benzimidazole-derived ligand was the most cytotoxic with an IC50 value of 10 µM against NCI-H460 non-small cell lung carcinoma cells. Cell uptake and distribution studies using X-ray fluorescence microscopy revealed localization of 5 d in the cytoplasm of cancer cells.