Jonathan A. Winger

The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

BackgroundSoluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases.ResultsWe have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 Å resolution, and show that it is a dimeric molecule.ConclusionComparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.

The structure of the leukemia drug imatinib bound to human quinone reductase 2 (NQO2).

BackgroundImatinib represents the first in a class of drugs targeted against chronic myelogenous leukemia to enter the clinic, showing excellent efficacy and specificity for Abl, Kit, and PDGFR kinases. Recent screens carried out to find off-target proteins that bind to imatinib identified the oxidoreductase NQO2, a flavoprotein that is phosphorylated in a chronic myelogenous leukemia cell line.ResultsWe examined the inhibition of NQO2 activity by the Abl kinase inhibitors imatinib, nilotinib, and dasatinib, and obtained IC50 values of 80 nM, 380 nM, and >100 μM, respectively. Using electronic absorption spectroscopy, we show that imatinib binding results in a perturbation of the protein environment around the flavin prosthetic group in NQO2. We have determined the crystal structure of the complex of imatinib with human NQO2 at 1.75 Å resolution, which reveals that imatinib binds in the enzyme active site, adjacent to the flavin isoalloxazine ring. We find that phosphorylation of NQO2 has little effect on enzyme activity and is therefore likely to regulate other aspects of NQO2 function.ConclusionThe structure of the imatinib-NQO2 complex demonstrates that imatinib inhibits NQO2 activity by competing with substrate for the active site. The overall conformation of imatinib when bound to NQO2 resembles the folded conformation observed in some kinase complexes. Interactions made by imatinib with residues at the rim of the active site provide an explanation for the binding selectivity of NQO2 for imatinib, nilotinib, and dasatinib. These interactions also provide a rationale for the lack of inhibition of the related oxidoreductase NQO1 by these compounds. Taken together, these studies provide insight into the mechanism of NQO2 inhibition by imatinib, with potential implications for drug design and treatment of chronic myelogenous leukemia in patients.

Dissociation of Nitric Oxide from Soluble Guanylate Cyclase and Heme-Nitric Oxide/Oxygen Binding Domain Constructs

Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length α1β1 sGC, and the constructs β1(1–194), β1(1–385), and β2(1–217), at 37 and 10 °C with and without the substrate analogue guanosine-5′-[(α,β-methylene]triphosphate (GMPCPP) or the activator 3-(5′-hydroxymethyl-3′-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and β2(1–217) at 37 °C and dramatically accelerated NO dissociation from sGC at 10 °C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 °C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity “closed” form, which releases NO slowly, and a higher activity “open” form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states.

Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput Screen.

ZAP-70 is a critical molecule in the transduction of T cell antigen receptor signaling and the activation of T cells. Upon activation of the T cell antigen receptor, ZAP-70 is recruited to the intracellular ζ-chains of the T cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T cell antigen receptor. The screen is based on a fluorescence polarization assay for peptide binding to ZAP-70.

A New Paradigm in Protecting Ischemic Brain: Preserving the Neurovascular Unit Before Reperfusion

Of the ~795,000 strokes that occur each year in the USA, ~695,000 are ischemic strokes (IS) where a clot occludes a major cerebral artery. About half of these IS patients present with so-called penumbra, defined as a hypoperfused tissue immediately surrounding the ischemic core that is severely deprived of oxygen and at risk for deterioration. Collateral vessels can provide sufficient oxygen and nutrients to temporarily maintain neuronal structure in the penumbra but not enough to support function. Thus, the at-risk tissue has the potential for functional recovery if blood flow is restored, but will irreversibly infarct if recanalization is not achieved, resulting in neurological deterioration. Additionally, though collateral circulation can transiently maintain penumbra viability, injury mechanisms such as excitotoxicity and ATP depletion will have already been initiated. Thus, it is imperative to administer therapies that can alleviate ischemia-induced cell death, restore energy metabolism, and halt pathogenic cascades as soon as possible after occlusion in order to protect the at-risk tissue until reperfusion therapies can be employed. Excitingly, the recent breakthroughs in acute IS reperfusion therapy have opened new opportunities for such adjunct neuroprotective treatments. This chapter provides a description of the penumbra tissue, followed by a brief overview of the emerging standard of care for acute IS based on the recent positive clinical trials using IV tPA and mechanical thrombectomy devices. We will then describe the promising use of adjunctive therapies to enhance the benefits of recanalization therapies. In particular, we will discuss the concept of oxygen therapy and oxygen carriers as a valid approach for “combination therapy” to protect the penumbra until reperfusion. Finally, we will discuss the future challenges of clinical trials in acute IS patients and highlight the need for new trial designs to test the potential benefit of combination therapies.

Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic

The heart exhibits the highest basal oxygen (O2) consumption per tissue mass of any organ in the body and is uniquely dependent on aerobic metabolism to sustain contractile function. During acute hypoxic states, the body responds with a compensatory increase in cardiac output that further increases myocardial O2 demand, predisposing the heart to ischemic stress and myocardial dysfunction. Here, we test the utility of a novel engineered protein derived from the heme-based nitric oxide (NO)/oxygen (H-NOX) family of bacterial proteins as an O2 delivery biotherapeutic (Omniox-cardiovascular [OMX-CV]) for the hypoxic myocardium. Because of their unique binding characteristics, H-NOX–based variants effectively deliver O2 to hypoxic tissues, but not those at physiologic O2 tension. Additionally, H-NOX–based variants exhibit tunable binding that is specific for O2 with subphysiologic reactivity towards NO, circumventing a significant toxicity exhibited by hemoglobin (Hb)-based O2 carriers (HBOCs). Juvenile lambs were sedated, mechanically ventilated, and instrumented to measure cardiovascular parameters. Biventricular admittance catheters were inserted to perform pressure-volume (PV) analyses. Systemic hypoxia was induced by ventilation with 10% O2. Following 15 minutes of hypoxia, the lambs were treated with OMX-CV (200 mg/kg IV) or vehicle. Acute hypoxia induced significant increases in heart rate (HR), pulmonary blood flow (PBF), and pulmonary vascular resistance (PVR) (p < 0.05). At 1 hour, vehicle-treated lambs exhibited severe hypoxia and a significant decrease in biventricular contractile function. However, in OMX-CV–treated animals, myocardial oxygenation was improved without negatively impacting systemic or PVR, and both right ventricle (RV) and left ventricle (LV) contractile function were maintained at pre-hypoxic baseline levels. These data suggest that OMX-CV is a promising and safe O2 delivery biotherapeutic for the preservation of myocardial contractility in the setting of acute hypoxia.

Abstract 1744: Reversal of advanced colitis-associated colon cancer by OMX, a novel oxygen carrier that immunosensitizes the hypoxic tumor microenvironment

Chronic inflammation of the colon increases cancer development risk. Ulcerative colitis, characterized by excessive inflammation initiated by innate immune cells and exacerbated by a dysregulation in adaptive immunity, can give rise to colitis-associated colon cancer (CAC). Whereas overactivity of effector T cells and loss of immunosuppressive cells are hallmarks of ulcerative colitis, the opposite is true for CAC, with CAC tumors exhibiting a lack of effector T cell infiltration and a preponderence of immunosuppressive Treg cells and myeloid-derived suppressor cells (MDSCs). Recently, hypoxia has been identified as a potential driver in the pathogenesis of ulcerative colitis, with hypoxia persisting upon progression to CAC tumor formation. We have previously demonstrated that (i) hypoxia generates an immunosuppressive tumor microenvironment that limits effector T cell infiltration and activation, (ii) OMX, a first-in-class anti-cancer therapy designed to reverse tumor hypoxia to enhance immunotherapeutic efficacy, accumulates in preclinical rodent and spontaneous canine tumors and reduces tumor hypoxia, and (iii) OMX promotes effector T cell infiltration, reduces Treg cells, and enhances checkpoint inhibitor efficacy, resulting in greater tumor control. Given that CAC tumors are hypoxic and immunosuppressed, we hypothesized that hypoxia drives CAC tumor immunosuppression, and accordingly, that reversal of hypoxia with OMX may restore immunosensitivity and elicit an anti-tumor response. Here, using a chemically induced mouse model of CAC generated by administering azoxymethane (AOM) followed by repeated cycles of dextran sulfate sodium (DSS) exposure, we show that OMX treatment exhibits anti-tumor efficacy in advanced CAC tumors. We characterized CAC tumor progression from 8 to 12 weeks post-tumor induction, and confirmed previous reports that advanced CAC tumors are indeed hypoxic, and that immunosuppressive Treg cells and MDSCs are more abundant in CAC tumors relative to adjacent normal mucosa or control non-AOM/DSS-treated colons. Moreover, we observed a negative correlation between hypoxia and CD8+ T cell infiltration into CAC tumors. OMX single agent treatment reduced both CAC tumor number and total CAC tumor burden. Of note, OMX treatment reversed colon length shortening that was characteristic of tumor-bearing mice, indicative of a restoration of colon crypt regeneration and hence normal colon biology. Investigations into the immunological mechanism(s) responsible for OMX anti-tumor efficacy are currently underway. Taken together, our data suggest that OMX, by delivering oxygen to hypoxic CAC tumor regions, may be sufficient to induce an immunological change in the CAC tumor microenvironment from an immunosuppressive to an immunopermissive state, leading to tumor responses and a restoration of normal physiology. Citation Format: Kevin G. Leong, Yuqiong Pan, Changan Guo, Padmini Narayanan, Jonathan A. Winger, Stephen P. Cary, Natacha Le Moan, Ana Krtolica. Reversal of advanced colitis-associated colon cancer by OMX, a novel oxygen carrier that immunosensitizes the hypoxic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1744.

Abstract 1627: Enhancement of anti-cancer immunity by OMX, a novel oxygen carrier immunotherapeutic that ameliorates the hypoxic tumor microenvironment

Hypoxia is a hallmark of cancer and a driver of tumor progression and poor patient outcomes. By generating an immunosuppressive tumor microenvironment that limits cytotoxic T lymphocyte (CTL) infiltration and activation, hypoxia limits the effectiveness of cancer immunotherapy and thus promotes tumor cell evasion of the host immune response. Omniox has developed a first-in-class anti-cancer immunotherapeutic, OMX, specifically designed to reverse tumor hypoxia to enhance cancer immunotherapy efficacy. In preclinical models, we have demonstrated that OMX accumulates in rodent subcutaneous and orthotopic tumors, as well as spontaneous canine melanomas and brain tumors, resulting in significant tumor hypoxia reduction.Here, using multiple subcutaneous syngeneic mouse tumor models (MC38, CT26, 4T1), we assessed OMX effects on intratumoral CTLs and immunosuppressive regulatory T cells (Treg), as well as the anti-tumor potential of OMX as a single agent and in combination with established immunotherapies. Using quantitative immunohistochemistry, we confirmed reports that hypoxic tumor areas are devoid of CTLs. Accordingly, by flow cytometry we observed a negative correlation between tumor hypoxia and CTL infiltration. While OMX single agent treatment did not affect the overall CD45-positive leukocyte population, Treg cells were selectively depleted and the CTL:Treg ratio was substantially increased, suggesting that OMX induced a shift towards immunosensitization. Consistent with this finding, we observed OMX single agent anti-tumor efficacy in MC38 colon tumors. Impressively, anti-tumor effects of OMX single agent were equivalent to that of a single treatment of the checkpoint inhibitor anti-CTLA4. We next assessed whether OMX would enhance the efficacy of checkpoint inhibitors when used in combination. In CT26 colon tumors, OMX exhibited combination anti-tumor activity with anti-CTLA4, giving rise to faster cures and a greater number of complete and durable responders compared to anti-CTLA4 alone. Of note, this enhanced response was observed for both early-stage and late-stage CT26 tumors. In 4T1 breast tumors, known to be insensitive to checkpoint inhibitors, treatment of early-stage (~60mm3) tumors with combination OMX and anti-PD1 resulted in a 27% response rate, compared to a 0% response rate to anti-PD1 alone. Taken together, our data suggest that OMX, by delivering oxygen to hypoxic tumor areas, induces a microenvironmental change from an immunosuppressive to an immunopermissive state. Given that OMX is well-tolerated in both small and large animals, and that its mechanism of action is upstream of numerous major immunosuppressive pathways, OMX holds great clinical potential to synergize with multiple immunotherapeutic agents to enhance tumor control by restoring anti-cancer immune responses in cancer patients. Citation Format: Kevin G. Leong, Yuqiong Pan, Jonathan A. Winger, Stephen P. Cary, Natacha Le Moan, Ana Krtolica. Enhancement of anti-cancer immunity by OMX, a novel oxygen carrier immunotherapeutic that ameliorates the hypoxic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1627. doi:10.1158/1538-7445.AM2017-1627

Abstract 3003: OMX-4.80P, a novel H-NOX oxygen carrier that oxygenates hypoxic tumors in multiple tumor models and canine cancer patients, downregulates HIF-1 pathway and increases response to radiation therapy leading to cures

BACKGROUND: Omniox has engineered OMX-4.80P, a PEGylated H-NOX oxygen carrier, as a long-acting therapeutic candidate to enhance radiotherapy (RT) in the treatment of glioblastoma and other solid tumors. Here, we describe the pre-clinical profile of OMX-4.80P, demonstrating it is well tolerated, long-lasting in circulation and tumors, and it penetrates deep into tumor tissue reducing hypoxia and altering hypoxic phenotype by downregulating HIF-1 pathway. Furthermore, it dramatically enhances RT leading to tumor cures. METHODS: We assessed the ability of OMX-4.80P to penetrate tumor tissue and reduce hypoxia in multiple orthotopic and immunocompetent mouse and rat models of glioblastoma and other tumors as well as in spontaneous canine brain tumors in veterinary patients. We measured the efficacy of OMX-4.80P in NSCLC tumors (H460 and Calu 6), and its activity in intracranial glioblastoma models in nude mice (U251), immunocompetent rats (F98) and in spontaneous canine brain tumors. We assessed exogenous hypoxia markers (pimonidazole and CCI-103F) and hypoxia inducible transcriptional factor HIF-1 by IHC and ELISA, and HIF-1 downstream targets by IHC and qRT PCR. We also conducted toxicology and pharmacokinetic studies in mice, rats and in naive and oncology patient dogs. RESULTS: In xenograft studies of large, hypoxic, radioresistant tumors, single doses of OMX-4.80P in combination with RT result in apparent tumor cures in ∼30-50% of tumors compared to 0% cures in RT-only groups. We observed good penetration into mouse and rat intracranial and subcutaneous tumors (∼1 cm3), and into spontaneous canine brain tumors, that resulted in hypoxia reduction, as assessed by OxyLite pO2 probe and pimonidazole and CCI-103F, leading to downregulation of the HIF-1 pathway. Observed dramatic drop in HIF-1α, VEGF, GLUT-1 and PDL-1 levels suggests OMX-4.80P has profound effects on tumor cell phenotype beyond radiosensitization. Pharmacokinetic and toxicology studies using single or multiple supratherapeutic and therapeutic doses of OMX-4.80P in rodents and dogs demonstrated that it has a circulation half-life of ∼20h in rats and ∼30-40h in dogs, and that it is well tolerated. Finally, OMX-4.80P has no detectable immunogenic response. CONCLUSIONS: The preclinical data demonstrating hypoxia reduction, HIF-1 pathway downregulation and radiation enhancement, and promising PK and toxicology profile of OMX-4.80P support its clinical development as a radiosensitizer for multiple types of hypoxic tumors. Furthermore, its ability to alter key downstream effectors of the HIF-1 pathway suggest it may have potential to alter tumor biology and enhance patient responses to variety of targeted and chemo therapies by affecting tumor drug resistance, immune responsiveness, angiogenesis, metabolism and invasion. Citation Format: Ana Krtolica, Natacha Le Moan, Jen Getz, Tina Davis, Sarah Ng, Catherine Bedard, Andrew Davis, Philberta Leung, Laura Serwer, Kevin Tanaka, Tim Keating, Feng Yan, Teri Guerrero, Michael Kent, Peter Dickinson, Jonathan Winger, Stephen P. L. Cary. OMX-4.80P, a novel H-NOX oxygen carrier that oxygenates hypoxic tumors in multiple tumor models and canine cancer patients, downregulates HIF-1 pathway and increases response to radiation therapy leading to cures. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3003. doi:10.1158/1538-7445.AM2015-3003

Molecular steps in sGC activation

In higher animals, soluble guanylate cyclase (sGC) functions as a selective sensor for NO. sGC belongs to a larger family of proteins termed the H-NOX family (Heme Nitric oxide/OXygen binding proteins) that includes prokaryotic counterparts from aerobic and anaerobic organisms [15]. A molecular basis for the ligand discrimination against O2 in NO-regulated sGCs has been proposed [4,5] and further results support the general aspects of the hypothesis that involve a H-bonding residue in those H-NOXs that bind O2 (Fig. 1).