d-Glucose- and d-mannose-based antimetabolites. Part 4: Facile synthesis of mono- and di-acetates of 2-deoxy-d-glucose prodrugs as potentially useful antimetabolites.

2-Deoxy-d-glucose (2-DG), a compound known to interfere with d-glucose and d-mannose metabolism, has been tested as a potential anticancer and antiviral agent. Preclinical and clinical studies focused on 2-DG have highlighted several limitations related to 2-DG drug-like properties, such as poor pharmacokinetic properties. To overcome this problem, we proposed design and synthesis of novel 2-DG prodrugs that subsequently could be tested using a variety of biochemical and molecular methods. We narrowed here our focus to esters of 2-DG as potential prodrugs based on the hypothesis that ubiquitous esterases will regenerate 2-DG, leading to increased circulation time of drug and adequate organ and tumor penetration. Testing this hypothesis in vitro and, especially, in vivo requires significant amounts of respective pure mono- and previously unknown di-acetylated water-soluble derivatives of 2-DG. Development of their efficient and practical method of synthesis was imperative. We describe novel facile and scalable syntheses of seven selectively acetylated water-soluble derivatives of 2-DG and present a detailed 1H and 13C NMR analysis of all final products. X-ray diffraction analysis has been performed for compound WP1122 that was selected for detailed preclinical and subsequent clinical evaluation as potential anticancer or antiviral agent.

The Antiviral Effects of 2-Deoxy-D-glucose (2-DG), a Dual D-Glucose and D-Mannose Mimetic, against SARS-CoV-2 and Other Highly Pathogenic Viruses.

Viral infection almost invariably causes metabolic changes in the infected cell and several types of host cells that respond to the infection. Among metabolic changes, the most prominent is the upregulated glycolysis process as the main pathway of glucose utilization. Glycolysis activation is a common mechanism of cell adaptation to several viral infections, including noroviruses, rhinoviruses, influenza virus, Zika virus, cytomegalovirus, coronaviruses and others. Such metabolic changes provide potential targets for therapeutic approaches that could reduce the impact of infection. Glycolysis inhibitors, especially 2-deoxy-D-glucose (2-DG), have been intensively studied as antiviral agents. However, 2-DG's poor pharmacokinetic properties limit its wide clinical application. Herein, we discuss the potential of 2-DG and its novel analogs as potent promising antiviral drugs with special emphasis on targeted intracellular processes.

X-ray wavefunction refinement and comprehensive structural studies on bromo-substituted analogues of 2-deoxy-d-glucose in solid state and solution.

The structural studies on two bromo-substituted derivatives of 2-deoxy-d-glucose (2-DG), namely 2-deoxy-2-bromo-d-glucose (2-BG) and 2-deoxy-2-bromo-d-mannose (2-BM) are described. 2-DG itself is an inhibitor of hexokinase, the first enzyme in the glycolysis process, playing a vital role in both cancer cell metabolism and viral replication in host cells. Because of that, 2-DG derivatives are considered as potential anti-cancer and anti-viral drugs. An X-ray quantum crystallography approach allowed us to obtain more accurate positions of hydrogen atoms by applying Hirshfeld atom refinement, providing a better description of hydrogen bonding even in the case of data from routine X-ray experiments. Obtained structures showed that the introduction of bromine at the C2 position in the pyranose ring has a minor influence on its conformation but still, it has a noticeable effect on the crystal structure. Bromine imposes the formation of a layered supramolecular landscape containing hydrogen bonds, which involves the bromine atom. Periodic DFT calculations of cohesive and interaction energies (at the B3LYP level of theory) have supported these findings and highlighted energetic changes upon bromine substitution. Based on molecular wavefunction from the refinement, we calculated the electrostatic potential, Laplacian, and ELI-D, and applied them to charge-density studies, which confirmed the geometry of hydrogen bonding and involvement of the bromine atom with these intermolecular interactions. NMR studies in the solution show that both compounds do not display significant differences in their anomeric equilibria compared to 2-DG, and the pyranose ring puckering is similar in both aqueous and solid state.

Correction: X-ray wavefunction refinement and comprehensive structural studies on bromo-substituted analogues of 2-deoxy-d-glucose in solid state and solution.

[This corrects the article DOI: 10.1039/D1RA08312K.].

Synergistic Anticancer Effect of Glycolysis and Histone Deacetylases Inhibitors in a Glioblastoma Model.

Over the last decade, we have seen tremendous progress in research on 2-deoxy-D-glucose (2-DG) and its analogs. Clinical trials of 2-DG have demonstrated the challenges of using 2-DG as a monotherapy, due to its poor drug-like characteristics, leading researchers to focus on improving its bioavailability to tissue and organs. Novel 2-DG analogs such as WP1122 and others have revived the old concept of glycolysis inhibition as an effective anticancer strategy. Combined with other potent cytotoxic agents, inhibitors of glycolysis could synergistically eliminate cancer cells. We focused our efforts on the development of new combinations of anticancer agents coupled with 2-DG and its derivatives, targeting glioblastoma, which is in desperate need of novel approaches and therapeutic options and is particularly suited to glycolysis inhibition, due to its reliance on aerobic glycolysis. Herein, we present evidence that a combined treatment of 2-DG analogs and modulation of histone deacetylases (HDAC) activity via HDAC inhibitors (sodium butyrate and sodium valproate) exerts synergistic cytotoxic effects in glioblastoma U-87 and U-251 cells and represents a promising therapeutic strategy.

Experimental and Computational Studies on Structure and Energetic Properties of Halogen Derivatives of 2-Deoxy-D-Glucose.

The results of structural studies on a series of halogen-substituted derivatives of 2-deoxy-D-glucose (2-DG) are reported. 2-DG is an inhibitor of glycolysis, a metabolic pathway crucial for cancer cell proliferation and viral replication in host cells, and interferes with D-glucose and D-mannose metabolism. Thus, 2-DG and its derivatives are considered as potential anticancer and antiviral drugs. X-ray crystallography shows that a halogen atom present at the C2 position in the pyranose ring does not significantly affect its conformation. However, it has a noticeable effect on the crystal structure. Fluorine derivatives exist as a dense 3D framework isostructural with the parent compound, while Cl- and I-derivatives form layered structures. Analysis of the Hirshfeld surface shows formation of hydrogen bonds involving the halogen, yet no indication for the existence of halogen bonds. Density functional theory (DFT) periodic calculations of cohesive and interaction energies (at the B3LYP level of theory) have supported these findings. NMR studies in the solution show that most of the compounds do not display significant differences in their anomeric equilibria, and that pyranose ring puckering is similar to the crystalline state. For 2-deoxy-2-fluoro-D-glucose (2-FG), electrostatic interaction energies between the ligand and protein for several existing structures of pyranose 2-oxidase were also computed. These interactions mostly involve acidic residues of the protein; single amino-acid substitutions have only a minor impact on binding. These studies provide a better understanding of the structural chemistry of halogen-substituted carbohydrates as well as their intermolecular interactions with proteins determining their distinct biological activity.

Drug Conjugates for Targeting Eph Receptors in Glioblastoma.

Glioblastoma (GBM) is a complex and heterogeneous tumor that warrants a comprehensive therapeutic approach for treatment. Tumor-associated antigens offer an opportunity to selectively target various components of the GBM microenvironment while sparing the normal cells within the central nervous system. In this study, we conjugated a multivalent vector protein, QUAD 3.0, that can target four receptors: EphA3, EphA2, EphB2, and also IL-13RA2, spanning virtually 100% of the GBM microenvironment, to doxorubicin derivatives. The conjugates effectively bound to all four receptors, although to varying degrees, and delivered cytotoxic loads to both established and patient-derived GBM cell lines, with IC50 values in the low nM range. The conjugates were also non-toxic to animals. We anticipate that the QUAD 3.0 Dox conjugates will be further used in preclinical models and possibly clinics in the foreseeable future.

Novel Molecular Multilevel Targeted Antitumor Agents.

A multifunctional fusion protein, IL-13.E13K-D2-NLS, effectively recognizes glioblastoma (GBM) cells and delivers its portion to the cell nucleus. IL-13.E13K-D2-NLS is composed of a cancer cell targeting ligand (IL-13.E13K), specialized cytosol translocation bacterial toxin domain 2 of Pseudomonas exotoxin A (D2) and SV40 T antigen nuclear localization signal (NLS). We have now tested whether we can produce proteins that would serve as a delivery vehicle to lysosomes and mitochondria as well. Moreover, we examined whether IL-13.E13K-D2-NLS can deliver anti-cancer drugs like doxorubicin to their nuclear site of action in cancer cells. We have thus constructed two novel proteins: IL-13.E13K-D2-LLS which incorporates lysosomal localization signal (LLS) of a human lysosomal associated membrane protein (LAMP-1) for targeting to lysosomes and IL-13-D2-KK2, which incorporates a pro-apoptotic peptide (KLAKLAK)2 (KK2) exerting its action in mitochondria. Furthermore, we have produced IL-13.E13K-D2-NLS and IL-13.E13K-D2-LLS versions containing a cysteine for site-specific conjugation with a modified doxorubicin, WP936. We found that single-chain recombinant proteins IL-13.E13K-D2-LLS and IL-13-D2-KK2 are internalized and localized mostly to the lysosomal and mitochondrial compartments, respectively, without major trafficking to cells' nuclei. We also determined that IL-13.E13K-D2-NLS-cys[WP936], IL-13.E13K-D2-LAMP-cys[WP936] and IL-13-D2-KK2 were cytotoxic to GBM cells overexpressing IL-13RA2, while much less cytotoxic to GBM cell lines expressing low levels of the receptor. IL-13.E13K-D2-NLS-cys[WP936] was the most potent of the tested anti-tumor agents including free WP936. We believe that our receptor-directed intracellular organelle-targeted proteins can be employed for numerous specific and safer treatment applications when drugs have specific intracellular sites of their action.

Bis-anthracycline WP760 abrogates melanoma cell growth by transcription inhibition, p53 activation and IGF1R downregulation.

Anthracycline chemotherapeutics, e.g. doxorubicin and daunorubicin, are active against a broad spectrum of cancers. Their cytotoxicity is mainly attributed to DNA intercalation, interference with topoisomerase activity, and induction of double-stranded DNA breaks. Since modification of anthracyclines can profoundly affect their pharmacological properties we attempted to elucidate the mechanism of action, and identify possible molecular targets, of bis-anthracycline WP760 which previously demonstrated anti-melanoma activity at low nanomolar concentrations. We studied the effect of WP760 on several human melanoma cell lines derived from tumors in various development stages and having different genetic backgrounds. WP760 inhibited cell proliferation (IC50 = 1-99 nM), impaired clonogenic cell survival (100 nM), and inhibited spheroid growth (≥300 nM). WP760 did not induce double-stranded DNA breaks but strongly inhibited global transcription. Moreover, WP760 caused nucleolar stress and led to activation of the p53 pathway. PCR array analysis showed that WP760 suppressed transcription of ten genes (ABCC1, MTOR, IGF1R, EGFR, GRB2, PRKCA, PRKCE, HDAC4, TXNRD1, AKT1) associated with, inter alia, cytoprotective mechanisms initiated in cancer cells during chemotherapy. Furthermore, WP760 downregulated IGF1R and upregulated PLK2 expression in most of the tested melanoma cell lines. These results suggest that WP760 exerts anti-melanoma activity by targeting global transcription and activation of the p53 pathway and could become suitable as an effective therapeutic agent.

IGF-1R and mTOR Blockade: Novel Resistance Mechanisms and Synergistic Drug Combinations for Ewing Sarcoma.

BACKGROUND: Therapies cotargeting insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) have demonstrated remarkable, albeit short-lived, clinical responses in a subset of Ewing sarcoma (ES) patients. However, the mechanisms of resistance and applicable strategies for overcoming drug resistance to the IGF-1R/mTOR blockade are still undefined. METHODS: To elucidate predominant mechanism(s) of acquired drug resistance while identifying synergistic drug combinations that improve clinical efficacy, we generated more than 18 ES cell lines resistant to IGF-1R- or mTOR-targeted therapy. Two small-molecule inhibitors of IGF-1R were chosen, NVP-ADW-742 (IGF-1R-selective) and OSI-906 (a dual IGF-1R/insulin receptor alpha [IR-α] inhibitor). Reverse-phase protein lysate arrays (RPPAs) revealed proteomic changes linked to IGF-1R/mTOR resistance, and selected proteins were validated in cell-based assays, xenografts, and within human clinical samples. All statistical tests were two-sided. RESULTS: Novel mechanisms of resistance (MOR) emerged after dalotuzumab-, NVP-ADW-742-, and OSI-906-based targeting of IGF-1R. MOR to dalotuzumab included upregulation of IRS1, PI3K, and STAT3, as well as p38 MAPK, which was also induced by OSI-906. pEIF4E(Ser209), a key regulator of Cap-dependent translation, was induced in ridaforolimus-resistant ES cell lines. Unique drug combinations targeting IGF-1R and PI3K-alpha or Mnk and mTOR were synergistic in vivo and vitro (P < .001) as assessed respectively by Mantel-Cox and isobologram testing. CONCLUSIONS: We discovered new druggable targets expressed by chemoresistant ES cells, xenografts, and relapsed human tumors. Joint suppression of these newfound targets, in concert with IGF-1R or mTOR blockade, should improve clinical outcomes.

Development of novel molecular probes of the Rio1 atypical protein kinase.

The RIO kinases are essential protein factors required for the synthesis of new ribosomes in eukaryotes. Conserved in archaeal organisms as well, RIO kinases are among the most ancient of protein kinases. Their exact molecular mechanisms are under investigation and progress of this research would be significantly improved with the availability of suitable molecular probes that selectively block RIO kinases. RIO kinases contain a canonical eukaryotic protein kinase fold, but also display several unusual structural features that potentially create opportunity for the design of selective inhibitors. In an attempt to identify structural leads to target the RIO kinases, a series of pyridine caffeic acid benzyl amides (CABA) were tested for their ability to inhibit the autophosphorylation activity of Archeaoglobus fulgidus Rio1 (AfRio1). Screening of a small library of CABA molecules resulted in the identification of four compounds that measurably inhibited AfRio1 activity. Additional biochemical characterization of binding and inhibition activity of these compounds demonstrated an ATP competitive inhibition mode, and allowed identification of the functional groups that result in the highest binding affinity. In addition, docking of the compound to the structure of Rio1 and determination of the X-ray crystal structure of a model compound (WP1086) containing the desired functional groups allowed detailed analysis of the interactions between these compounds and the enzyme. Furthermore, the X-ray crystal structure demonstrated that these compounds stabilize an inactive form of the enzyme. Taken together, these results provide an important step in identification of a scaffold for the design of selective molecular probes to study molecular mechanisms of Rio1 kinases in vitro and in vivo. In addition, it provides a rationale for the future design of potent inhibitors with drug-like properties targeting an inactive form of the enzyme. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

D-Glucose and D-mannose-based metabolic probes. Part 3: Synthesis of specifically deuterated D-glucose, D-mannose, and 2-deoxy-D-glucose.

Altered carbohydrate metabolism in cancer cells was first noted by Otto Warburg more than 80 years ago. Upregulation of genes controlling the glycolytic pathway under normoxia, known as the Warburg effect, clearly differentiates malignant from non-malignant cells. The resurgence of interest in cancer metabolism aims at a better understanding of the metabolic differences between malignant and non-malignant cells and the creation of novel therapeutic and diagnostic agents exploiting these differences. Modified d-glucose and d-mannose analogs were shown to interfere with the metabolism of their respective monosaccharide parent molecules and are potentially clinically useful anticancer and diagnostic agents. One such agent, 2-deoxy-d-glucose (2-DG), has been extensively studied in vitro and in vivo and also clinically evaluated. Studies clearly indicate that 2-DG has a pleiotropic mechanism of action. In addition to effectively inhibiting glycolysis, 2-DG has also been shown to affect protein glycosylation. In order to better understand its molecular mechanism of action, we have designed and synthesized deuterated molecular probes to study 2-DG interference with d-glucose and d-mannose metabolism using mass spectrometry. We present here the synthesis of all desired probes: 2-deutero-d-glucose, 2-deutero-d-mannose, 6-deutero-d-glucose, 6-deutero-d-mannose, and 2-deutero-2-deoxy-d-glucose as well as their complete chemical characterization.

Novel small molecular inhibitors disrupt the JAK/STAT3 and FAK signaling pathways and exhibit a potent antitumor activity in glioma cells.

JAK (Janus kinase)/STAT (signal transducers and activators of transcription) signaling is involved in the regulation of cell growth, differentiation and apoptosis. Constitutive activation of STATs, in particular STAT3, is observed in a large number of human tumors, including gliomas and may contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis, thus it emerges as a promising target for anti-cancer therapy. To investigate the therapeutic potential of blocking STAT3 in glioma cells a set of small synthetic molecules - caffeic acid derivatives, structurally related to AG490 was screened for its ability to inhibit STAT3. Inhibitor 2 (E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide was the most effective in inhibition of JAK/STAT3 signaling and at doses ≥ 25 µM significantly reduced the level of phosphorylated JAK1, JAK2 and STAT3 (at Tyr705) and downregulated the expression of known STAT3 targets. In treated cells we observed rapid detachment and rounding of cells associated with reduction of focal adhesion kinase phosphorylation and activity, followed by upregulation of phosphorylated p38, JNK and ERK1/2 levels. Accumulation of cells with fragmented DNA, increases of the cleaved caspase 3 and fragmented PARP levels were detected 24 h after the treatment suggesting ongoing apoptotic cell death. Three human malignant glioblastoma cell lines defective in tumor suppressors TP53 and/or PTEN were susceptible to inhibitor 2 that induced the programmed cell death. Global gene expression profiling revealed modulation of numerous genes in cells treated with inhibitor 2 revealing novel, potential JAK/STAT targets. Our study demonstrates that suitably modified caffeic acid molecules exhibit significant cytotoxic potential toward glioma cells.

Induction of cell-cycle arrest and apoptosis in glioblastoma stem-like cells by WP1193, a novel small molecule inhibitor of the JAK2/STAT3 pathway.

Glioma stem-like cells (GSCs) may be the initiating cells in glioblastoma (GBM) and contribute to the resistance of these tumors to conventional therapies. Development of novel chemotherapeutic agents and treatment approaches against GBM, especially those specifically targeting GSCs are thus necessary. In the present study, we found that a novel Janus kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway inhibitor (WP1193) significantly decreased the proliferation of established glioma cell lines in vitro and inhibit the growth of glioma in vivo. To test the efficacy of WP1193 against GSCs, we then administrated WP1193 to GSCs isolated and expanded from multiple human GBM tumors. We revealed that WP1193 suppressed phosphorylation of JAK2 and STAT3 with high potency and demonstrated a dose-dependent inhibition of proliferation and neurosphere formation of GSCs. These effects were at least due in part to G1 arrest associated with down-regulation of cyclin D1 and up-regulation of p21( Cip1/Waf-1 ). Furthermore, WP1193 exposure decreased expression of stem cell markers including CD133 and c-myc, and induced cell death in GSCs through apoptosis. Taken together, our data indicate that WP1193 is a potent small molecule inhibitor of the JAK2/STAT3 pathway that shows promise as a therapeutic agent against GBM by targeting GSCs.

Drug-tolerant cancer cells show reduced tumor-initiating capacity: depletion of CD44 cells and evidence for epigenetic mechanisms.

Cancer stem cells (CSCs) possess high tumor-initiating capacity and have been reported to be resistant to therapeutics. Vice versa, therapy-resistant cancer cells seem to manifest CSC phenotypes and properties. It has been generally assumed that drug-resistant cancer cells may all be CSCs although the generality of this assumption is unknown. Here, we chronically treated Du145 prostate cancer cells with etoposide, paclitaxel and some experimental drugs (i.e., staurosporine and 2 paclitaxel analogs), which led to populations of drug-tolerant cells (DTCs). Surprisingly, these DTCs, when implanted either subcutaneously or orthotopically into NOD/SCID mice, exhibited much reduced tumorigenicity or were even non-tumorigenic. Drug-tolerant DLD1 colon cancer cells selected by a similar chronic selection protocol also displayed reduced tumorigenicity whereas drug-tolerant UC14 bladder cancer cells demonstrated either increased or decreased tumor-regenerating capacity. Drug-tolerant Du145 cells demonstrated low proliferative and clonogenic potential and were virtually devoid of CD44(+) cells. Prospective knockdown of CD44 in Du145 cells inhibited cell proliferation and tumor regeneration, whereas restoration of CD44 expression in drug-tolerant Du145 cells increased cell proliferation and partially increased tumorigenicity. Interestingly, drug-tolerant Du145 cells showed both increases and decreases in many "stemness" genes. Finally, evidence was provided that chronic drug exposure generated DTCs via epigenetic mechanisms involving molecules such as CD44 and KDM5A. Our results thus reveal that 1) not all DTCs are necessarily CSCs; 2) conventional chemotherapeutic drugs such as taxol and etoposide may directly target CD44(+) tumor-initiating cells; and 3) DTCs generated via chronic drug selection involve epigenetic mechanisms.

Glucose, not glutamine, is the dominant energy source required for proliferation and survival of head and neck squamous carcinoma cells.

BACKGROUND: Tumor metabolism is an essential contributor to disease progression and response to treatment. An understanding of the metabolic phenotype of head and neck squamous cell carcinoma (HNSCC) will allow the development of appropriate antimetabolic strategies for this tumor type. METHODS: A panel of 15 HNSCC cell lines was assayed for glucose and glutamine dependence and sensitivity to metabolic inhibitors. In addition, broad-spectrum metabolomic analysis using mass spectrometry/liquid chromatography was combined with individual measurements of reducing potential, adenosine triphosphate, and lactate production to characterize cellular metabolic phenotypes. RESULTS: HNSCC energy and reducing potential levels closely mirrored extracellular glucose concentrations. Glucose starvation induced cell death despite the activation of secondary energetic pathways. Conversely, glutamine was not required for HNSCC survival and did not serve as a significant source of energy. 2-deoxyglucose (2-DG) and its fluorinated derivative decreased glycolytic and Krebs cycle activity, cellular energy, and reducing potential and inhibited HNSCC cell proliferation. 2-DG effects were potentiated by the addition of metformin, but not by inhibitors of the pentose phosphate pathway or glutaminolysis. Despite dependence on glucose catabolism, the authors identified a subset of cell lines with relative resistance to starvation. Exploration of 1 such cell line (HN30) suggested that the presence of wild-type p53 can partially protect tumor cells from glucose starvation. CONCLUSIONS: HNSCC tumor cells are dependent on glucose, not glutamine, for energy production and survival, providing a rationale for treatment strategies that target glucose catabolism. However, antimetabolic strategies may need to be tailored to the tumor background, more specifically, p53 status.

A genistein derivative, ITB-301, induces microtubule depolymerization and mitotic arrest in multidrug-resistant ovarian cancer.

PURPOSE: To investigate the mechanistic basis of the anti-tumor effect of the compound ITB-301. METHODS: Chemical modifications of genistein have been introduced to improve its solubility and efficacy. The anti-tumor effects were tested in ovarian cancer cells using proliferation assays, cell cycle analysis, immunofluorescence, and microscopy. RESULTS: In this work, we show that a unique glycoside of genistein, ITB-301, inhibits the proliferation of SKOv3 ovarian cancer cells. We found that the 50% growth inhibitory concentration of ITB-301 in SKOv3 cells was 0.5 µM. Similar results were obtained in breast cancer, ovarian cancer, and acute myelogenous leukemia cell lines. ITB-301 induced significant time- and dose-dependent microtubule depolymerization. This depolymerization resulted in mitotic arrest and inhibited proliferation in all ovarian cancer cell lines examined including SKOv3, ES2, HeyA8, and HeyA8-MDR cells. The cytotoxic effect of ITB-301 was dependent on its induction of mitotic arrest as siRNA-mediated depletion of BUBR1 significantly reduced the cytotoxic effects of ITB-301, even at a concentration of 10 µM. Importantly, efflux-mediated drug resistance did not alter the cytotoxic effect of ITB-301 in two independent cancer cell models of drug resistance. CONCLUSION: These results identify ITB-301 as a novel anti-tubulin agent that could be used in cancers that are multidrug resistant. We propose a structural model for the binding of ITB-301 to α- and ß-tubulin dimers on the basis of molecular docking simulations. This model provides a rationale for future work aimed at designing of more potent analogs.

Inhibition of p-STAT3 enhances IFN-alpha efficacy against metastatic melanoma in a murine model.

PURPOSE: Melanoma is a common and deadly tumor that upon metastasis to the central nervous system has a median survival duration of <6 months. Activation of the signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma malignancy, including immune suppression in melanoma patients. We hypothesized that WP1193, a novel inhibitor of STAT3 signaling, would enhance the antitumor activity of IFN-alpha against metastatic melanoma. EXPERIMENTAL DESIGN: Combinational therapy of STAT3 blockade agents with IFN-alpha was investigated in a metastatic and an established syngeneic intracerebral murine tumor model of melanoma. The immunologic in vivo mechanisms of efficacy were investigated by T-cell and natural killer (NK) cell cytotoxic assays. RESULTS: IFN-alpha immunotherapy was synergistic with WP1193 showing marked in vivo efficacy against metastatic and established intracerebral melanoma. At autopsy, it was noted that there was a decreased trend in mice with melanoma developing leptomeningeal disease treated with combinational therapy. The combinational approach enhanced both NK-mediated and T-cell-mediated antitumor cytotoxicity. CONCLUSIONS: The immune modulatory effects of STAT3 blockade can enhance the therapeutic efficacy of IFN-alpha immunotherapy by enhancing both innate and adaptive cytotoxic T-cell activities. This combination therapy has the potential in the treatment of metastatic melanoma that is typically refractory to this type of immune therapeutic approach.

D-glucose- and D-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-D-glucoses and -D-mannoses.

Modified D-glucose and D-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of D-glucose and D-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-D-glucal.

A novel phosphorylated STAT3 inhibitor enhances T cell cytotoxicity against melanoma through inhibition of regulatory T cells.

The activation of signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma malignancy, including immune suppression in melanoma patients. Increasing evidence also suggests that regulatory T cells (Tregs) are important in suppressing anti-tumor immunity and play a dominant role in negating efficacious immunotherapy approaches. We hypothesized that WP1066, a novel inhibitor of STAT3 signaling, reverses immune suppression through the inhibition of Tregs and that this contributes to the antitumor activity of this agent against melanoma brain metastases. We found that the mean percentage of peripheral blood mononuclear cells expressing phosphorylated STAT3 (p-STAT3) was significantly elevated in samples from patients with melanoma brain metastases compared to healthy donors, 16.13 +/- 2.48% versus 4.17 +/- 1.79%. The p-STAT3 inhibitor WP1066 enhanced CD3+ (which contained Tregs) but not CD8+ T cell cytotoxicity against human A375 melanoma cells, indicating that this p-STAT3 blockade agent did not directly activate CD8+ T cells. Furthermore, the p-STAT3 inhibitor did not enhance the cytotoxicity of CD3+CD25- T cells (from which Tregs were excluded), indicating that the enhanced cytotoxicity of WP1066 is secondary to its inhibition of Tregs. This was confirmed by demonstrating that WP1066 inhibited FoxP3+ Treg induction in a dose-dependent manner. Moreover, CD3+ T cells exhibited markedly enhanced levels of phosphorylated ZAP-70, a critical proximal signal in T cell activation, after exposure to WP1066. Similar effects were not observed in Treg-depleted CD3+CD25- T cell populations, confirming that the T cell activation by WP compounds is secondary to their inhibition of the Tregs. These results suggest that WP1066 enhances T cell cytotoxicity against melanoma through inhibition of Tregs.