Anticancer Properties of Hexosamine Analogs Designed to Attenuate Metabolic Flux through the Hexosamine Biosynthetic Pathway.

Altered cellular metabolism is a hallmark of cancer pathogenesis and progression; for example, a near-universal feature of cancer is increased metabolic flux through the hexosamine biosynthetic pathway (HBP). This pathway produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a potent oncometabolite that drives multiple facets of cancer progression. In this study, we synthesized and evaluated peracetylated hexosamine analogs designed to reduce flux through the HBP. By screening a panel of analogs in pancreatic cancer and glioblastoma multiform (GBM) cells, we identified Ac4Glc2Bz─a benzyl-modified GlcNAc mimetic─as an antiproliferative cancer drug candidate that down-regulated oncogenic metabolites and reduced GBM cell motility at concentrations non-toxic to non-neoplastic cells. More specifically, the growth inhibitory effects of Ac4Glc2Bz were linked to reduced levels of UDP-GlcNAc and concomitant decreases in protein O-GlcNAc modification in both pancreatic cancer and GBM cells. Targeted metabolomics analysis in GBM cells showed that Ac4Glc2Bz disturbed glucose metabolism, amino acid pools, and nucleotide precursor biosynthesis, consistent with reduced proliferation and other anti-oncogenic properties of this analog. Furthermore, Ac4Glc2Bz reduced the invasion, migration, and stemness of GBM cells. Importantly, normal metabolic functions mediated by UDP-GlcNAc were not disrupted in non-neoplastic cells, including maintenance of endogenous levels of O-GlcNAcylation with no global disruption of N-glycan production. Finally, a pilot in vivo study showed that a potential therapeutic window exists where animals tolerated 5- to 10-fold higher levels of Ac4Glc2Bz than projected for in vivo efficacy. Together, these results establish GlcNAc analogs targeting the HBP through salvage mechanisms as a new therapeutic approach to safely normalize an important facet of aberrant glucose metabolism associated with cancer.

Glycoengineering Human Neural and Adipose Stem Cells with Novel Thiol-Modified N-Acetylmannosamine (ManNAc) Analogs.

This report describes novel thiol-modified N-acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of Ac5ManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that Ac5ManNTGc elicited non-canonical activation of Wnt signaling in human embryoid body derived (hEBD) cells but only in the presence of a high affinity, chemically compatible scaffold. Our new analogs Ac5ManNTProp and Ac5ManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, N-acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols. These new analogs showed increased potency in human neural stem cells (hNSCs) and human adipose stem cells (hASCs). In the hNSCs, Ac5ManNTProp upregulated biochemical endpoints consistent with Wnt signaling in the absence of a thiol-reactive scaffold. In the hASCs, both Ac5ManNTProp and Ac5ManNTBut suppressed adipogenic differentiation, with Ac5ManNTBut providing a more potent response, and they did not interfere with differentiation to a glial lineage (Schwann cells). These results expand the horizon for using MGE in regenerative medicine by providing new tools (Ac5ManNTProp and Ac5ManNTBut) for manipulating human stem cells.

Improving the Pharmacodynamics and In Vivo Activity of ENPP1-Fc Through Protein and Glycosylation Engineering.

Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu3 ManNAc. These steps sequentially increased the half-life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N-glycan, to ~ 96 hours with optimized pH-dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6-overexpressing cells with 1,3,4-O-Bu3 ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1-deficient mice when the optimized biologic was administered at a 10-fold lower mass dose less frequently than the parent compound-once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.

Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression.

Sialylation, a post-translational modification that impacts the structure, activity, and longevity of glycoproteins has been thought to be controlled primarily by the expression of sialyltransferases (STs). In this report we explore the complementary impact of metabolic flux on sialylation using a glycoengineering approach. Specifically, we treated three human breast cell lines (MCF10A, T-47D, and MDA-MB-231) with 1,3,4-O-Bu3ManNAc, a "high flux" metabolic precursor for the sialic acid biosynthetic pathway. We then analyzed N-glycan sialylation using solid phase extraction of glycopeptides (SPEG) mass spectrometry-based proteomics under conditions that selectively captured sialic acid-containing glycopeptides, referred to as "sialoglycosites." Gene ontology (GO) analysis showed that flux-based changes to sialylation were broadly distributed across classes of proteins in 1,3,4-O-Bu3ManNAc-treated cells. Only three categories of proteins, however, were "highly responsive" to flux (defined as two or more sialylation changes of 10-fold or greater). Two of these categories were cell signaling and cell adhesion, which reflect well-known roles of sialic acid in oncogenesis. A third category-protein folding chaperones-was unexpected because little precedent exists for the role of glycosylation in the activity of these proteins. The highly flux-responsive proteins were all linked to cancer but sometimes as tumor suppressors, other times as proto-oncogenes, or sometimes both depending on sialylation status. A notable aspect of our analysis of metabolically glycoengineered breast cells was decreased sialylation of a subset of glycosites, which was unexpected because of the increased intracellular levels of sialometabolite "building blocks" in the 1,3,4-O-Bu3ManNAc-treated cells. Sites of decreased sialylation were minor in the MCF10A (<25% of all glycosites) and T-47D (<15%) cells but dominated in the MDA-MB-231 line (~60%) suggesting that excess sialic acid could be detrimental in advanced cancer and cancer cells can evolve mechanisms to guard against hypersialylation. In summary, flux-driven changes to sialylation offer an intriguing and novel mechanism to switch between context-dependent pro- or anti-cancer activities of the several oncoproteins identified in this study. These findings illustrate how metabolic glycoengineering can uncover novel roles of sialic acid in oncogenesis.

Exploiting metabolic glycoengineering to advance healthcare.

Metabolic glycoengineering (MGE) is a technique for manipulating cellular metabolism to modulate glycosylation. MGE is used to increase the levels of natural glycans and, more importantly, to install non-natural monosaccharides into glycoconjugates. In this Review, we summarize the chemistry underlying MGE that has been developed over the past three decades and highlight several recent advances that have set the stage for clinical translation. In anticipation of near-term application to human healthcare, we describe emerging efforts to deploy MGE in diverse applications, ranging from the glycoengineering of biotherapeutic proteins and the diagnosis and treatment of complex diseases such as cancer to the development of new immunotherapies.

Combining Butyrated ManNAc with Glycoengineered CHO Cells Improves EPO Glycan Quality and Production.

Sodium butyrate (NaBu) is not only well-known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4-O-Bu3 ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4-O-Bu3 ManNAc on EPO glycan quality, while evident in wild-type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of ß-1,4- and ß-1,6-N-acetylglucosaminyltransferases (GnTIV and GnTV) and α-2,6-sialyltransferase (ST6) enzymes responsible for N-glycan antennarity and sialylation. Supplementation of 1,3,4-O-Bu3 ManNAc achieves approximately 30% sialylation enhancement on EPO protein in wild-type CHO cells. Overexpression of GnTIV/GnTV/ST6 in CHO cells increases EPO sialylation about 40%. Combining 1,3,4-O-Bu3 ManNAc treatment in glyocengineered CHO cells promotes EPO sialylation about 75% relative to EPO from wild-type CHO cells. Moreover, a detailed mass spectrometric ESI-LC-MS/MS characterization of glycans at each of the three N-glycosylation sites of EPO showed that the 1st N-site is highly sialylated and either the negative impact of NaBu or the beneficial effect 1,3,4-O-Bu3 ManNAc treatments mainly affects the 2nd and 3rd N-glycan sites of EPO protein. In summary, these results demonstrate 1,3,4-O-Bu3 ManNAc can compensate for the negative effect of NaBu on EPO glycan quality while simultaneously enhancing recombinant protein yields. In this way, a platform that integrates glycoengineering with metabolic supplementation can result in synergistic improvements in both production and glycosylation in CHO cells.

Improving Immunotherapy Through Glycodesign.

Immunotherapy is revolutionizing health care, with the majority of high impact "drugs" approved in the past decade falling into this category of therapy. Despite considerable success, glycosylation-a key design parameter that ensures safety, optimizes biological response, and influences the pharmacokinetic properties of an immunotherapeutic-has slowed the development of this class of drugs in the past and remains challenging at present. This article describes how optimizing glycosylation through a variety of glycoengineering strategies provides enticing opportunities to not only avoid past pitfalls, but also to substantially improve immunotherapies including antibodies and recombinant proteins, and cell-based therapies. We cover design principles important for early stage pre-clinical development and also discuss how various glycoengineering strategies can augment the biomanufacturing process to ensure the overall effectiveness of immunotherapeutics.

Integration of genetic and metabolic features related to sialic acid metabolism distinguishes human breast cell subtypes.

In this report we use 'high-flux' tributanoyl-modified N-acetylmannosamine (ManNAc) analogs with natural N-acetyl as well as non-natural azido- and alkyne N-acyl groups (specifically, 1,3,4-O-Bu3ManNAc, 1,3,4-O-Bu3ManNAz, and 1,3,4-O-Bu3ManNAl respectively) to probe intracellular sialic acid metabolism in the near-normal MCF10A human breast cell line in comparison with earlier stage T-47D and more advanced stage MDA-MB-231 breast cancer lines. An integrated view of sialic acid metabolism was gained by measuring intracellular sialic acid production in tandem with transcriptional profiling of genes linked to sialic acid metabolism. The transcriptional profiling showed several differences between the three lines in the absence of ManNAc analog supplementation that helps explain the different sialoglycan profiles naturally associated with cancer. Only minor changes in mRNA transcript levels occurred upon exposure to the compounds confirming that metabolic flux alone can be a key determinant of sialoglycoconjugate display in breast cancer cells; this result complements the well-established role of genetic control (e.g., the transcription of STs) of sialylation abnormalities ubiquitously associated with cancer. A notable result was that the different cell lines produced significantly different levels of sialic acid upon exogenous ManNAc supplementation, indicating that feedback inhibition of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE)-generally regarded as the 'gatekeeper' enzyme for titering flux into sialic acid biosynthesis-is not the only regulatory mechanism that limits production of this sugar. A notable aspect of our metabolic glycoengineering approach is its ability to discriminate cell subtype based on intracellular metabolism by illuminating otherwise hidden cell type-specific features. We believe that this strategy combined with multi-dimensional analysis of sialic acid metabolism will ultimately provide novel insights into breast cancer subtypes and provide a foundation for new methods of diagnosis.

Pharmacological, Physiochemical, and Drug-Relevant Biological Properties of Short Chain Fatty Acid Hexosamine Analogues Used in Metabolic Glycoengineering.

In this study, we catalog structure activity relationships (SAR) of several short chain fatty acid (SCFA)-modified hexosamine analogues used in metabolic glycoengineering (MGE) by comparing in silico and experimental measurements of physiochemical properties important in drug design. We then describe the impact of these compounds on selected biological parameters that influence the pharmacological properties and safety of drug candidates by monitoring P-glycoprotein (Pgp) efflux, inhibition of cytochrome P450 3A4 (CYP3A4), hERG channel inhibition, and cardiomyocyte cytotoxicity. These parameters are influenced by length of the SCFAs (e.g., acetate vs n-butyrate), which are added to MGE analogues to increase the efficiency of cellular uptake, the regioisomeric arrangement of the SCFAs on the core sugar, the structure of the core sugar itself, and by the type of N-acyl modification (e.g., N-acetyl vs N-azido). By cataloging the influence of these SAR on pharmacological properties of MGE analogues, this study outlines design considerations for tuning the pharmacological, physiochemical, and the toxicological parameters of this emerging class of small molecule drug candidates.

Determination of absolute expression profiles using multiplexed miRNA analysis.

Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR's ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes.

Glycoengineering of Esterase Activity through Metabolic Flux-Based Modulation of Sialic Acid.

This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activity. By contrast, hexosamine analogues not targeted to sialic acid biosynthesis (e.g., butanoylated GlcNAc or GalNAc) had minimal impact. Measurement of mRNA and protein confirmed that esterase activity was controlled through glycosylation and not through transcription or translation. Azide-modified ManNAc analogues widely used in MGE also enhanced esterase activity and provided a way to enrich targeted glycoengineered proteins (such as CES2), thereby providing unambiguous evidence that the compounds were converted to sialosides and installed into the glycan structures of esterases as intended. Overall, this study provides a pioneering example of the modulation of intracellular enzyme activity through MGE, which expands the value of this technology from its current status as a labeling strategy and modulator of cell surface biological events.

Harnessing cancer cell metabolism for theranostic applications using metabolic glycoengineering of sialic acid in breast cancer as a pioneering example.

Abnormal cell surface display of sialic acids - a family of unusual 9-carbon sugars - is widely recognized as distinguishing feature of many types of cancer. Sialoglycans, however, typically cannot be identified with sufficiently high reproducibility and sensitivity to serve as clinically accepted biomarkers and similarly, almost all efforts to exploit cancer-specific differences in sialylation signatures for therapy remain in early stage development. In this report we provide an overview of important facets of glycosylation that contribute to cancer in general with a focus on breast cancer as an example of malignant disease characterized by aberrant sialylation. We then describe how cancer cells experience nutrient deprivation during oncogenesis and discuss how the resulting metabolic reprogramming, which endows breast cancer cells with the ability to obtain nutrients during scarcity, constitutes an "Achilles' heel" that we believe can be exploited by metabolic glycoengineering (MGE) strategies to develop new diagnostic methods and therapeutic approaches. In particular, we hypothesize that adaptations made by breast cancer cells that allow them to efficiently scavenge sialic acid during times of nutrient deprivation renders them vulnerable to MGE, which refers to the use of exogenously-supplied, non-natural monosaccharide analogues to modulate targeted aspects of glycosylation in living cells and animals. In specific, once non-natural sialosides are incorporated into the cancer "sialome" they can be exploited as epitopes for immunotherapy or as chemical tags for targeted delivery of imaging or therapeutic agents selectively to tumors.

Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells.

In prior work we reported that advanced stage, drug-resistant pancreatic cancer cells (the SW1990 line) can be sensitized to the EGFR-targeting tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib by treatment with 1,3,4-O-Bu3ManNAc (Bioorg. Med. Chem. Lett. (2015) 25(6):1223-7). Here we provide mechanistic insights into how this compound inhibits EGFR activity and provides synergy with TKI drugs. First, we showed that the sialylation of the EGFR receptor was at most only modestly enhanced (by ~20 to 30%) compared to overall ~2-fold increase in cell surface levels of this sugar. Second, flux-driven sialylation did not alter EGFR dimerization as has been reported for cancer cell lines that experience increased sialylation due to spontaneous mutations. Instead, we present evidence that 1,3,4-O-Bu3ManNAc treatment weakens the galectin lattice, increases the internalization of EGFR, and shifts endosomal trafficking towards non-clathrin mediated (NCM) endocytosis. Finally, by evaluating downstream targets of EGFR signaling, we linked synergy between 1,3,4-O-Bu3ManNAc and existing TKI drugs to a shift from clathrin-coated endocytosis (which allows EGFR signaling to continue after internalization) towards NCM endocytosis, which targets internalized moieties for degradation and thereby rapidly diminishes signaling.

Cell Surface and Membrane Engineering: Emerging Technologies and Applications.

Membranes constitute the interface between the basic unit of life-a single cell-and the outside environment and thus in many ways comprise the ultimate "functional biomaterial". To perform the many and often conflicting functions required in this role, for example to partition intracellular contents from the outside environment while maintaining rapid intake of nutrients and efflux of waste products, biological membranes have evolved tremendous complexity and versatility. This article describes how membranes, mainly in the context of living cells, are increasingly being manipulated for practical purposes with drug discovery, biofuels, and biosensors providing specific, illustrative examples. Attention is also given to biology-inspired, but completely synthetic, membrane-based technologies that are being enabled by emerging methods such as bio-3D printers. The diverse set of applications covered in this article are intended to illustrate how these versatile technologies-as they rapidly mature-hold tremendous promise to benefit human health in numerous ways ranging from the development of new medicines to sensitive and cost-effective environmental monitoring for pathogens and pollutants to replacing hydrocarbon-based fossil fuels.

Metabolic glycoengineering bacteria for therapeutic, recombinant protein, and metabolite production applications.

Metabolic glycoengineering is a specialization of metabolic engineering that focuses on using small molecule metabolites to manipulate biosynthetic pathways responsible for oligosaccharide and glycoconjugate production. As outlined in this article, this technique has blossomed in mammalian systems over the past three decades but has made only modest progress in prokaryotes. Nevertheless, a sufficient foundation now exists to support several important applications of metabolic glycoengineering in bacteria based on methods to preferentially direct metabolic intermediates into pathways involved in lipopolysaccharide, peptidoglycan, teichoic acid, or capsule polysaccharide production. An overview of current applications and future prospects for this technology are provided in this report.

Metabolic glycoengineering sensitizes drug-resistant pancreatic cancer cells to tyrosine kinase inhibitors erlotinib and gefitinib.

Metastatic human pancreatic cancer cells (the SW1990 line) that are resistant to the EGFR-targeting tyrosine kinase inhibitor drugs (TKI) erlotinib and gefitinib were treated with 1,3,4-O-Bu3ManNAc, a 'metabolic glycoengineering' drug candidate that increased sialylation by ∼2-fold. Consistent with genetic methods previously used to increase EGFR sialylation, this small molecule reduced EGF binding, EGFR transphosphorylation, and downstream STAT activation. Significantly, co-treatment with both the sugar pharmacophore and the existing TKI drugs resulted in strong synergy, in essence re-sensitizing the SW1990 cells to these drugs. Finally, 1,3,4-O-Bu3ManNAz, which is the azido-modified counterpart to 1,3,4-O-Bu3ManNAc, provided a similar benefit thereby establishing a broad-based foundation to extend a 'metabolic glycoengineering' approach to clinical applications.

Novel sulfamoyl benzamides as selective CB(2) agonists with improved in vitro metabolic stability.

A lead optimization campaign in our previously reported sulfamoyl benzamide class of CB(2) agonists was conducted to improve the in vitro metabolic stability profile in this series while retaining high potency and selectivity for the CB(2) receptor. From this study, compound 14, N-(3,4-dimethyl-5-(morpholinosulfonyl)phenyl)-2,2-dimethylbutanamide, was identified as a potent and selective CB(2) agonist exhibiting moderate in vitro metabolic stability and oral bioavailability. Compound 14 demonstrated in vivo efficacy in a rat model of post-surgical pain.

Novel pyridine derivatives as potent and selective CB2 cannabinoid receptor agonists.

Replacement of the phenyl ring in our previous (morpholinomethyl)aniline carboxamide cannabinoid receptor ligands with a pyridine ring led to the discovery of a novel chemical series of CB2 ligands. Compound 3, that is, 2,2-dimethyl-N-(5-methyl-4-(morpholinomethyl)pyridin-2-yl)butanamide was identified as a potent and selective CB2 agonist exhibiting in vivo efficacy after oral administration in a rat model of neuropathic pain.

Discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB2 agonists.

Recently sulfamoyl benzamides were identified as a novel series of cannabinoid receptor ligands. Replacing the sulfonamide functionality and reversing the original carboxamide bond led to the discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB(2) agonists. Selective CB(2) agonist 31 (K(i)=2.7; CB(1)/CB(2)=190) displayed robust activity in a rodent model of postoperative pain.

Sulfamoyl benzamides as novel CB2 cannabinoid receptor ligands.

Sulfamoyl benzamides were identified as a novel series of cannabinoid receptor ligands. Starting from a screening hit 8 that had modest affinity for the cannabinoid CB(2) receptor, a parallel synthesis approach and initial SAR are described, leading to compound 27 with 120-fold functional selectivity for the CB(2) receptor. This compound produced robust antiallodynic activity in rodent models of postoperative pain and neuropathic pain without traditional cannabinergic side effects.