Dess-Martin periodinane-mediated oxidation of the primary alcohol of cytidine into a carboxylic acid.

Herein the first example of conversion of alcohols into carboxylic acids by use of the Dess-Martin Periodinane (DMP), which is otherwise routinely employed for the conversion to aldehydes, is reported. This methodology will have significant potential utility in the synthesis of cytidine analogues and other related biologically important molecules.

Cancer-specific glycosylation of CD13 impacts its detection and activity in preclinical cancer tissues.

Harnessing the differences between cancer and non-cancer tissues presents new opportunities for selective targeting by anti-cancer drugs. CD13, a heavily glycosylated protein, is one example with significant unmet clinical potential in cancer drug discovery. Despite its high expression and activity in cancers, CD13 is also expressed in many normal tissues. Here, we report differential tissue glycosylation of CD13 across tissues and demonstrate for the first time that the nature and pattern of glycosylation of CD13 in preclinical cancer tissues are distinct compared to normal tissues. We identify cancer-specific O-glycosylation of CD13, which selectively blocks its detection in cancer models but not in normal tissues. In addition, the metabolism activity of cancer-expressed CD13 was observed to be critically dependent on its unique glycosylation. Thus, our data demonstrate the existence of discrete cancer-specific CD13 glycoforms and propose cancer-specific CD13 glycoforms as a clinically useful target for effective cancer-targeted therapy.

Chemoselective Solution- and Solid-Phase Synthesis of Disulfide-Linked Glycopeptides.

Glycosylation of peptides and proteins is a widely employed strategy to mimic important post-translational modifications or to modulate the physicochemical properties of peptides to enhance their delivery. Furthermore, glycosylation via a sulfur atom imparts increased chemical and metabolic stability to the resulting glycoconjugates. Herein, we report a simple and chemoselective procedure to prepare disulfide-linked glycopeptides. Acetate-protected glycosylsulfenyl hydrazines are shown to be highly reactive with the thiol group of cysteine residues within peptides, both in solution and as part of conventional solid-phase peptide synthesis protocols. The efficiency of this glycosylation methodology with unprotected carbohydrates is also demonstrated, which avoids the need for deprotection steps and further extends its utility, with disulfide-linked glycopeptides produced in excellent yields. Given the importance of glycosylated peptides in structural glycobiology, pharmacology, and therapeutics, the methodology outlined provides easy access to disulfide-linked glycopeptides as molecules with multiple biological applications.

Targeted delivery of a colchicine analogue provides synergy with ATR inhibition in cancer cells.

Despite significant preclinical promise as anticancer agents, vascular-disrupting agents have yet to fulfil their clinical potential due to systemic toxicities. ICT2588 is a tumour-selective MT1-MMP-targeted prodrug of azademethylcolchicine, ICT2552. We investigate activation of ICT2588 and subsequent release of ICT2552 in tumour cells, and examine its ability to induce G2/M cell cycle arrest. We also explore synergism between ICT2588 and ATR inhibition, since colchicine, in addition to its vascular-disrupting properties, is known to induce G2/M arrest, DNA damage, and trigger apoptosis. Several ATR inhibitors are currently undergoing clinical evaluation. The cellular activation of ICT2588 was observed to correlate with MT1-MMP expression, with selective release of ICT2552 not compromised by cellular uptake and prodrug activation mechanisms. ICT2588 induced G2/M arrest, and triggered apoptosis in MT1-MMP-expressing cells, but not in cells lacking MT1-MMP expression, while ICT2552 itself induced G2/M arrest and triggered apoptosis in both cell lines. Interestingly, we uncovered that the intracellular release and accumulation dynamics of ICT2552 subsequent to prodrug activation provided synergism with an ATR inhibitor in a way not observed with direct administration of ICT2552. These findings have important potential implications for clinical combinations of ICT2588 and DNA repair inhibitors.

Progress towards a clinically-successful ATR inhibitor for cancer therapy.

The DNA damage response (DDR) is now known to play an important role in both cancer development and its treatment. Targeting proteins such as ATR (Ataxia telangiectasia mutated and Rad3-related) kinase, a major regulator of DDR, has demonstrated significant therapeutic potential in cancer treatment, with ATR inhibitors having shown anti-tumour activity not just as monotherapies, but also in potentiating the effects of conventional chemotherapy, radiotherapy, and immunotherapy. This review focuses on the biology of ATR, its functional role in cancer development and treatment, and the rationale behind inhibition of this target as a therapeutic approach, including evaluation of the progress and current status of development of potent and specific ATR inhibitors that have emerged in recent decades. The current applications of these inhibitors both in preclinical and clinical studies either as single agents or in combinations with chemotherapy, radiotherapy and immunotherapy are also extensively discussed. This review concludes with some insights into the various concerns raised or observed with ATR inhibition in both the preclinical and clinical settings, with some suggested solutions.

Is tumour-expressed aminopeptidase N (APN/CD13) structurally and functionally unique?

Aminopeptidase N (APN/CD13) is a multifunctional glycoprotein that acts as a peptidase, receptor, and signalling molecule in a tissue-dependent manner. The activities of APN have been implicated in the progression of many cancers, pointing toward significant therapeutic potential for cancer treatment. However, despite the tumour-specific functions of this protein that have been uncovered, the ubiquitous nature of its expression in normal tissues as generally reported remains a limitation to the potential utility of APN as a target for cancer therapeutics and drug discovery. With this in mind, we have extensively explored the literature, and present a comprehensive review that for the first-time provides evidence to support the suggestion that tumour-expressed APN may in fact be unique in structure, function, substrate specificity and activity, contrary to its nature in normal tissues. The review also focuses on the biology of APN, and its "moonlighting" functional roles in both normal physiology and cancer development. Several APN-targeting approaches that have been explored over recent decades as therapeutic strategies in cancer treatment, including APN-targeting agents reported both in preclinical and clinical studies, are also extensively discussed. This review concludes by posing critical questions about APN that remain unanswered and unexplored, hence providing opportunities for further research.

An assay for quantitative analysis of polysialic acid expression in cancer cells.

Polysialic acid (polySia) is a linear polysaccharide comprised of N-acetylneuraminic acid residues and its over-expression in cancer cells has been correlated with poor clinical prognosis. An assay has been developed for quantitative analysis of cellular polySia expression. This was achieved by extracting and purifying released polySia from glycoproteins by mild acid hydrolysis and optimised organic extraction. The polySia was further hydrolysed into Sia monomers, followed by fluorescent labelling and quantitative analysis. The assay was qualified utilising endoneuraminidase-NF to remove polySia from the surface of C6-ST8SiaII cancer cells (EC50 = 2.13 ng/mL). The result was comparable to that obtained in a polySia-specific cellular ELISA assay. Furthermore, the assay proved suitable for evaluation of changes in polySia expression following treatment with a small molecule inhibitor of polysialylation. Given the importance of polySia in multiple disease states, notably cancer, this is a potentially vital tool with applications in the fields of drug discovery and glycobiology.

Glycosyl disulfides: importance, synthesis and application to chemical and biological systems.

The disulfide bond plays an important role in the formation and stabilisation of higher order structures of peptides and proteins, while in recent years interest in this functional group has been extended to carbohydrate chemistry. Rarely found in nature, glycosyl disulfides have attracted significant attention as glycomimetics, with wide biological applications including lectin binding, as key components of dynamic libraries to study carbohydrate structures, the study of metabolic and enzymatic studies, and even as potential drug molecules. This interest has been accompanied and fuelled by the continuous development of new methods to construct the disulfide bond at the anomeric centre. Glycosyl disulfides have also been exploited as versatile intermediates in carbohydrate synthesis, particularly as glycosyl donors. This review focuses on the importance of the disulfide bond in glycobiology and in chemistry, evaluating the different methods available to synthesise glycosyl disulfides. Furthermore, we review the role of glycosyl disulfides as intermediates and/or glycosyl donors for the synthesis of neoglycoproteins and oligosaccharides, before finally considering examples of how this important class of carbohydrates have made an impact in biological and therapeutic contexts.

An efficient assay for identification and quantitative evaluation of potential polysialyltransferase inhibitors.

The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors.

Theranostic nanoparticles enhance the response of glioblastomas to radiation.

Despite considerable progress with our understanding of glioblastoma multiforme (GBM) and the precise delivery of radiotherapy, the prognosis for GBM patients is still unfavorable with tumor recurrence due to radioresistance being a major concern. We recently developed a cross-linked iron oxide nanoparticle conjugated to azademethylcolchicine (CLIO-ICT) to target and eradicate a subpopulation of quiescent cells, glioblastoma initiating cells (GICs), which could be a reason for radioresistance and tumor relapse. The purpose of our study was to investigate if CLIO-ICT has an additive therapeutic effect to enhance the response of GBMs to ionizing radiation. Methods: NSG™ mice bearing human GBMs and C57BL/6J mice bearing murine GBMs received CLIO-ICT, radiation, or combination treatment. The mice underwent pre- and post-treatment magnetic resonance imaging (MRI) scans, bioluminescence imaging (BLI), and histological analysis. Tumor nanoparticle enhancement, tumor flux, microvessel density, GIC, and apoptosis markers were compared between different groups using a one-way ANOVA and two-tailed Mann-Whitney test. Additional NSG™ mice underwent survival analyses with Kaplan-Meier curves and a log rank (Mantel-Cox) test. Results: At 2 weeks post-treatment, BLI and MRI scans revealed significant reduction in tumor size for CLIO-ICT plus radiation treated tumors compared to monotherapy or vehicle-treated tumors. Combining CLIO-ICT with radiation therapy significantly decreased microvessel density, decreased GICs, increased caspase-3 expression, and prolonged the survival of GBM-bearing mice. CLIO-ICT delivery to GBM could be monitored with MRI. and was not significantly different before and after radiation. There was no significant caspase-3 expression in normal brain at therapeutic doses of CLIO-ICT administered. Conclusion: Our data shows additive anti-tumor effects of CLIO-ICT nanoparticles in combination with radiotherapy. The combination therapy proposed here could potentially be a clinically translatable strategy for treating GBMs.

Recent advances in the analysis of polysialic acid from complex biological systems.

Polysialic acid (polySia) is a unique, well-characterised carbohydrate polymer highly-expressed on the cell surface of neurons in the early stages of mammalian brain development. Post-embryogenesis, it is also re-expressed in a number of tumours of neuroendocrine origin. It plays important roles in modulating cell-cell, and cell-matrix adhesion and migration, tumour invasion and metastasis. Techniques for structural and quantitative characterisation of polySia from tumours and cancer cells are thus essential in exploring the relationship between polySia expression levels and structural and functional changes associated with cancer progression and metastasis. A variety of techniques have been developed to structurally and quantitatively analyse polySia in clinical tissues and other biological samples. In this review, analytical approaches used for the determination of polySia in biological matrices in the past 20 years are discussed, with a particular focus on chemical approaches, and quantitative analysis.

Novel Ran-RCC1 Inhibitory Peptide-Loaded Nanoparticles Have Anti-Cancer Efficacy In Vitro and In Vivo.

The delivery of anticancer agents to their subcellular sites of action is a significant challenge for effective cancer therapy. Peptides, which are integral to several oncogenic pathways, have significant potential to be utilised as cancer therapeutics due to their selectivity, high potency and lack of normal cell toxicity. Novel Ras protein-Regulator of chromosome condensation 1 (Ran-RCC1) inhibitory peptides designed to interact with Ran, a novel therapeutic target in breast cancer, were delivered by entrapment into polyethylene glycol-poly (lactic-co-glycolic acid) PEG-PLGA polymeric nanoparticles (NPs). A modified double emulsion solvent evaporation technique was used to optimise the physicochemical properties of these peptide-loaded biodegradable NPs. The anti-cancer activity of peptide-loaded NPs was studied in vitro using Ran-expressing metastatic breast (MDA-MB-231) and lung cancer (A549) cell lines, and in vivo using Solid Ehrlich Carcinoma-bearing mice. The anti-metastatic activity of peptide-loaded NPs was investigated using migration, invasion and colony formation assays in vitro. A PEG-PLGA-nanoparticle encapsulating N-terminal peptide showed a pronounced antitumor and anti-metastatic action in lung and breast cancer cells in vitro and caused a significant reduction of tumor volume and associated tumor growth inhibition of breast cancer model in vivo. These findings suggest that the novel inhibitory peptides encapsulated into PEGylated PLGA NPs are delivered effectively to interact and deactivate Ran. This novel Ran-targeting peptide construct shows significant potential for therapy of breast cancer and other cancers mediated by Ran overexpression.

A Novel Theranostic Strategy for MMP-14-Expressing Glioblastomas Impacts Survival.

Glioblastoma (GBM) has a dismal prognosis. Evidence from preclinical tumor models and human trials indicates the role of GBM-initiating cells (GIC) in GBM drug resistance. Here, we propose a new treatment option with tumor enzyme-activatable, combined therapeutic and diagnostic (theranostic) nanoparticles, which caused specific toxicity against GBM tumor cells and GICs. The theranostic cross-linked iron oxide nanoparticles (CLIO) were conjugated to a highly potent vascular disrupting agent (ICT) and secured with a matrix-metalloproteinase (MMP-14) cleavable peptide. Treatment with CLIO-ICT disrupted tumor vasculature of MMP-14-expressing GBM, induced GIC apoptosis, and significantly impaired tumor growth. In addition, the iron core of CLIO-ICT enabled in vivo drug tracking with MR imaging. Treatment with CLIO-ICT plus temozolomide achieved tumor remission and significantly increased survival of human GBM-bearing mice by more than 2-fold compared with treatment with temozolomide alone. Thus, we present a novel therapeutic strategy with significant impact on survival and great potential for clinical translation. Mol Cancer Ther; 16(9); 1909-21. ©2017 AACR.

Rationalized Computer-Aided Design of Matrix-Metalloprotease-Selective Prodrugs.

Matrix metalloproteinases (MMPs) are central to cancer development and metastasis. They are highly active in the tumor environment and absent or inactive in normal tissues; therefore they represent viable targets for cancer drug discovery. In this study we evaluated in silico docking to develop MMP-subtype-selective tumor-activated prodrugs. Proof of principle for this therapeutic approach was demonstrated in vitro against an aggressive human glioma model, with involvement of MMPs confirmed using pharmacological inhibition.

Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen.

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

Polysialic acid sustains cancer cell survival and migratory capacity in a hypoxic environment.

Polysialic acid (polySia) is a unique carbohydrate polymer expressed on the surface of NCAM (neuronal cell adhesion molecule) in a number of cancers where it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis and is strongly associated with poor clinical prognosis. We have carried out the first investigation into the effect of polySia expression on the behaviour of cancer cells in hypoxia, a key source of chemoresistance in tumours. The role of polysialylation and associated tumour cell migration and cell adhesion were studied in hypoxia, along with effects on cell survival and the potential role of HIF-1. Our findings provide the first evidence that polySia expression sustains migratory capacity and is associated with tumour cell survival in hypoxia. Initial mechanistic studies indicate a potential role for HIF-1 in sustaining polySia-mediated migratory capacity, but not cell survival. These data add to the growing body of evidence pointing to a crucial role for the polysialyltransferases (polySTs) in neuroendocrine tumour progression and provide the first evidence to suggest that polySia is associated with an aggressive phenotype in tumour hypoxia. These results have significant potential implications for polyST inhibition as an anti-metastatic therapeutic strategy and for targeting hypoxic cancer cells.

An optimised assay for quantitative, high-throughput analysis of polysialyltransferase activity.

The polysialyltransferases are biologically important glycosyltransferase enzymes responsible for the biosynthesis of polysialic acid, a carbohydrate polymer that plays a critical role in the progression of several diseases, notably cancer. Having improved the chemical synthesis and purification of the fluorescently-labelled DMB-DP3 acceptor, we report optimisation and validation of a highly sensitive cell-free high-throughput HPLC-based assay for assessment of human polysialyltransferase activity.

The use of thermographic imaging to evaluate therapeutic response in human tumour xenograft models.

Non-invasive methods to monitor tumour growth are an important goal in cancer drug development. Thermographic imaging systems offer potential in this area, since a change in temperature is known to be induced due to changes within the tumour microenvironment. This study demonstrates that this imaging modality can be applied to a broad range of tumour xenografts and also, for the first time, the methodology's suitability to assess anti-cancer agent efficacy. Mice bearing subcutaneously implanted H460 lung cancer xenografts were treated with a novel vascular disrupting agent, ICT-2552, and the cytotoxin doxorubicin. The effects on tumour temperature were assessed using thermographic imaging over the first 6 hours post-administration and subsequently a further 7 days. For ICT-2552 a significant initial temperature drop was observed, whilst for both agents a significant temperature drop was seen compared to controls over the longer time period. Thus thermographic imaging can detect functional differences (manifesting as temperature reductions) in the tumour response to these anti-cancer agents compared to controls. Importantly, these effects can be detected in the first few hours following treatment and therefore the tumour is observable non-invasively. As discussed, this technique will have considerable 3Rs benefits in terms of reduction and refinement of animal use.

The Effect of Polysialic Acid Expression on Glioma Cell Nano-mechanics.

Polysialic acid (polySia) is an important carbohydrate bio-polymer that is commonly over-expressed on tumours of neuroendocrine origin and plays a key role in tumour progression. polySia exclusively decorates the neural cell adhesion molecule (NCAM) on tumour cell membranes, modulating cell-cell interactions, motility and invasion. In this preliminary study, we examine the nano-mechanical properties of isogenic C6 rat glioma cells-transfected cells engineered to express the enzyme polysialyltransferase ST8SiaII, which synthesises polySia (C6-STX cells) and wild-type cells (C6-WT). We demonstrate that polySia expression leads to reduced elastic and adhesive properties but also more viscoelastic compared to non-expressing wild-type cells. Whilst differences in cell elasticity between healthy and cancer cells are regularly assigned to changes in the cytoskeleton, we show that in this model system, the change in properties at the nano-level is due to the polySia on the transfected cell membrane surface.

Novel strategies for the synthesis of unsymmetrical glycosyl disulfides.

Novel strategies for the efficient synthesis of unsymmetrical glycosyl disulfides are reported. Glycosyl disulfides are increasingly important as glycomimetics and molecular probes in glycobiology. Sialosyl disulfides are synthesised directly from the chlorosialoside Neu5Ac2Cl, proceeding via a thiol-disulfide exchange reaction between the sialosyl thiolate and symmetrical disulfides. This methodology was adapted and found to be successfully applicable to the synthesis of unsymmetrical glucosyl disulfides under mild conditions.