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John L Magnani - One of the best experts on this subject based on the ideXlab platform.
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administration of the dual e selectin cxcr4 antagonist gmi 1359 results in a unique profile of tumor mobilization from the bone marrow and facilitation of chemotherapy in a murine model of flt3 itd aml
Blood, 2016Co-Authors: William E. Fogler, Helen M Thackray, Henry Flanner, Curt Wolfgang, Jeffrey Allen Smith, John L MagnaniAbstract:FLT3 mutated AML confers poor prognosis despite treatment with standard chemotherapy, attributable to bone marrow (BM) induced tumor cell resistance in the microenvironment via molecules such as CXCR4 and E-selectin. We recently reported that simultaneous targeting of CXCR4 and E-selectin with GMI-1359, a novel small molecule dual antagonist, mobilized leukemic cells into the circulation; further, combination with AraC and DNR resulted in prolonged survival in a FLT3-ITD AML murine xenograft model. Here we report a novel profile of leukemic cell mobilization induced by GMI-1359, its impact on survival when combined with chemotherapy, and toxicity profile supporting entry to the clinic. A comparison of tumor mobilization by GMI-1359 to E-selectin or CXCR4 antagonists was determined in NCR mice with established BM infiltrative MV4-11 FLT3 ITD human AML tumor (modified with mCherry and luciferase). Mice (n=18/group) were injected with GMI-1359 (40 mg/kg), a potent E-selectin antagonist (40 mg/kg), or plerixafor (5 mg/kg) and blood samples were assessed by flow cytometry at multiple times for human AML cells and murine WBC. Appearance of human AML cells in peripheral blood increased ~16-fold compared to untreated mice 8 h post treatment with GMI-1359, and remained elevated for the duration of the study (72 h). Tumor cell mobilization following treatment with the E-selectin antagonist was gradual, reaching a 9.7-fold increase 48 h post injection and trending down at 72 h. Administration of plerixafor induced a rapid 11-fold mobilization of tumor cells by 1 h returning to baseline at 24 h. Murine WBC count did not change in any group at the time points assessed. These data establish that the profile of GMI-1359 induced tumor cell mobilization is distinct from both plerixafor (rapid mobilization of short duration) and an E-selectin antagonist (gradual mobilization of long duration). As E-selectin but not CXCR4 has been reported to be critical for tumor cell entry into the BM, the sustained presence of tumor cells in circulation after GMI-1359 treatment could be the result of blocking not only CXCR4 but also E-selectin, thereby inhibiting their re-entry into the BM. The therapeutic impact of tumor mobilization by GMI-1359 was evaluated for therapeutic consequence in combination with AraC and DNR in MV4.11 tumor bearing mice. Mice (n=10/group) were treated with saline; GMI-1359 (40 mg/kg) alone; chemotherapy alone; or GMI-1359 and AraC/DNR. GMI-1359 was initiated at onset of chemotherapy and given for 1, 3 or 14 days. Treatment of mice with GMI-1359 alone or together with AraC/DNR was well-tolerated. Median survival times (MST) of mice treated with saline, GMI-1359 or AraC/DNR was 47, 50 and 50 days, respectively; all mice succumbed to progressive disease by study conclusion (Day 71). In contrast, the MST of tumor bearing mice treated with GMI-1359 qdx3 or qdx14 and AraC/DNR was 63.5 days (p GMI-1359 was tested for off-target interactions against 87 potential targets, with no significant activity observed. Toxicology studies showed a safety profile consistent with that of plerixafor. The pharmacokinetic (PK) profile was characterized in mice, rats, dogs, and monkeys, and was linear with t 1/2 In summary, GMI-1359, an innovative dual E-selectin and CXCR4 antagonist, more rapidly mobilized tumor cells and maintained them in circulation for a longer time when compared to either an antagonist of E-selectin or CXCR4 alone in a murine model of FLT3 ITD AML. This novel kinetic signature of mobilization by dual inhibition of two important adhesion molecules and accompanying improvement in survival over standard chemotherapy support the potential for combination with current treatment to improve outcomes for patients with FLT3-mutated AML. First-in-human trials are being initiated. Disclosures Fogler: Glycomimetics, Inc.: Employment. Flanner: Glycomimetics, Inc.: Employment. Wolfgang: Glycomimetics, Inc.: Employment. Smith: Glycomimetics, Inc.: Employment. Thackray: Glycomimetics: Employment, Equity Ownership. Magnani: Glycomimetics: Employment, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees.
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e selectin ligand expression increases with progression of myeloma and induces drug resistance in a murine transplant model which is overcome by the Glycomimetic e selectin antagonist gmi 1271
Blood, 2015Co-Authors: Alessandro Natoni, Theodore Smith, Isabela Oliva, William E. Fogler, Niamh Keane, Silvia Locatellihoops, John L Magnani, Michael OdwyerAbstract:There is increasing evidence that E-selectin and its ligands play an important role in the progression of multiple myeloma (MM) and drug resistance. We reported that the sialyltransferase ST3GAL6 influences homing and survival in MM, and postulated that it may function in the synthesis of E-selectin ligands (Glavey et al Blood, 2014). We also found that a small subpopulation of cells (~ 5%) from MM cell lines express functional E-selectin ligands, which could be expanded under hypoxic conditions typical of the bone marrow (BM) microenvironment. These cells were identified by reactivity with an antibody (HECA452), which binds the same carbohydrate epitope required for binding to E-selectin. Rolling of MM1S cells on E-selectin was blocked by a small molecule Glycomimetic antagonist to E-selectin (GMI-1271). Moreover, GMI-1271 significantly enhanced the anti-myeloma activity of bortezomib (BTZ) in an in vivo murine transplant model (Natoni et al Blood, 2014). We now extend these observations to obtain a more complete understanding of the role E-selectin plays in MM biology and chemotherapy resistance for its potential clinical relevance. The parental, heterogeneous MM cell lines MM1S and RPMI8226 (MM1S par , RPMI8226 par , respectively) were sequentially sorted to obtain cell lines highly enriched (>85% and 80%) for the expression of cell surface carbohydrates bound by HECA452, and designated MM1S HECA452 and RPMI8226 HECA452 . The cell lines could be passaged in vitro and were stable for enriched E-selectin ligand expression. In contrast to parental cells, both MM1S HECA452 and RPMI8226 HECA452 showed strong binding to E-selectin in static adhesion assays. Both MM1S HECA452 and RPMI8226 HECA452 exhibited strong rolling on E-selectin under shear stress. MM1S par or RPMI8226 par failed to roll well on E-selectin. The addition of GMI-1271 during culture conditions led to a marked reduction in adhesion of MM1S HECA452 and profoundly inhibited rolling on E-selectin of both HECA452 enriched MM cell lines. The significance of these in vitro findings was studied in vivo. MM1S par or MM1S HECA452 cells were injected i.v. into SCID beige mice. Beginning 5 days post tumor injection, the survival impact of treatment with saline control, GMI-1271, BTZ or a combination of both was determined. Mice transplanted with MM1S HECA452 had more aggressive disease with significantly shorter survival compared to those transplanted with MM1S par . In contrast to MM1S par cells, mice engrafted with MM1S HECA452 demonstrated a marked resistance to BTZ treatment. Whereas GMI-1271 treatment alone had no impact on survival, the combination of GMI-1271 and BTZ led to a highly significant improvement in survival of MM1S par engrafted mice (P=0.0363), and more importantly broke the resistance and restored the anti-myeloma activity of BTZ in MM1S HECA452 engrafted mice (P=0.0123) (figure 1). The number of peripheral blood (PB) human CD138+ cells was increased in MM1S HECA452 -engrafted mice within 60 min following a single injection of GMI-1271, and persisted for at least 24 hours (2.37% v. 0.03%, p HECA-452 cells from the BM niche. Given these findings we wished to see if samples from patients with MM express E-selectin ligands and whether higher levels are seen with disease progression. BM and/or PB were obtained following informed consent from patients with MM and plasma cells (CD38+/CD138+) were analyzed for E-selectin ligand expression by flow cytometry using the HECA452 antibody. To date all primary MM samples (n=25) contained HECA452-reactive cell populations (median 22%). A consistently higher proportion of circulating MM cells express HECA452 when compared with paired BM samples (n=14), with a median difference of 33% (Wilcoxon signed rank test, p=0.02). HECA452 expression of MM in PB was significantly higher (on average 40% higher) in samples taken at relapse vs. diagnosis, (unpaired t test, p = 0.0008) These data provide compelling evidence that E-selectin ligand bearing cells play an important role in disease progression and drug resistance in MM, and a strong rationale for clinical strategies incorporating GMI-1271 to improve patient outcome. Disclosures Smith: Glycomimetics, Inc.: Employment. Locatelli-Hoops: Glycomimetics, Inc.: Employment. Oliva: Glycomimetics, Inc.: Employment. Fogler: Glycomimetics, Inc.: Employment. Magnani: Glycomimetics: Employment, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees. O9Dwyer: Celgene: Honoraria, Research Funding.
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abstract 4503 a small molecule Glycomimetic antagonist of e selectin gmi 1271 prevents pancreatic tumor metastasis and offers a novel treatment for improved efficacy of chemotherapy
Cancer Research, 2014Co-Authors: Maria M Steele, John L Magnani, Prakash Radhakrishnan, Michael A HollingsworthAbstract:The processes of intra- and extravasation of tumor cells into and out of the blood and lymphatic systems are crucial steps during metastasis to distant organ sites. These processes are tightly regulated by the initial binding of sialyl Lewis A and sialyl Lewis X (sialyl Le A/X) carbohydrate moeities found on tumor cells to the adhesion protein E-selectin expressed on the activated endothelium. GMI-1271 is a small molecule Glycomimetic rationally designed based on the bioactive conformation of sialyl Lea/x and is a potent and specific antagonist of E-selectin. In vitro treatment of human lymphatic endothelial cells with GMI-1271 resulted in a decrease in the number of sialyl Lewis A-expressing pancreatic cancer cells (S2.013 and BxPC-3) binding to the endothelium in a dose-dependent manner. GMI-1271 treatment also inhibited the transendothelial migration of S2.013 and BxPC-3 cells through a lymphatic cell monolayer. We evaluated the in vivo efficacy of GMI-1271 following orthotopic implantation of pancreatic tumor cell line S2.013, which expresses high levels of sialyl Lewis A (CA19-9), into nude mice. Following 2 weeks of tumor growth, mice were treated by intraperitoneal injections for 4 weeks with either PBS once daily, once daily with 40mg/kg GMI-1271 (low dose), twice daily with 40mg/kg GMI-1271 (high dose), twice a week with 60mg/kg gemcitabine injections, combination low dose GMI-1271 and gemcitabine injections, or combination high dose GMI-1271 and gemcitabine injections. Co-treatment of either low or high dose GMI-1271 with gemcitabine resulted in a significant decrease in the number of metastasis to the lymph nodes (p=0.02 low dose; p=0.04 high dose). In addition, compared with gemcitabine alone, low dose GMI-1271 plus gemcitabine was found to be effective at reducing the number of metastatic lesions (per histological section) in the liver (p=0.001), lung (p=0.026) and diaphragm (p=0.01). Based on the significant effects of combination therapy on tumor metastasis, the small molecule Glycomimetic antagonist to E-selectin, GMI-1271, offers great promise in preventing pancreatic tumor cell entry into the blood and lymphatic systems and offers a novel treatment for the improved efficacy of standard chemotherapy. Citation Format: Maria M. Steele, Prakash Radhakrishnan, John L. Magnani, Michael A. Hollingsworth. A small molecule Glycomimetic antagonist of E-selectin (GMI-1271) prevents pancreatic tumor metastasis and offers a novel treatment for improved efficacy of chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4503. doi:10.1158/1538-7445.AM2014-4503
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Glycomimetic drugs source of novel therapeutics
2014Co-Authors: John L MagnaniAbstract:Complex carbohydrates contain dense structural information that is decoded in recognition processes among cells, their pathogens and products, as well as many other functional proteins and represent a new source of novel therapeutics. Native carbohydrate ligands, however, lack classic drug-like properties and in general suffer from poor pharmacokinetics, bioavailability, stability, and low affinity. One solution is to rationally design Glycomimetic drugs based on the bioactive conformation of the native carbohydrate ligand obtained from empirical data using techniques such as STD-NMR and X-ray crystallography. Modifications to improve binding affinity include stabilizing the bioactive core structure thereby reducing entropy costs of the interaction as well as adding second site interactions to enhance enthalpic contributions. Improvements in bioavailability and pharmacokinetics can be realized by lowering the molecular weight, reducing polar surface area and hydrogen bond donors and acceptors, as well as increasing log P. Examples of approved Glycomimetic drugs are discussed and highlight the great potential of using this technology to uncover an entire class of innovative drugs to address current unmet medical needs. J.L. Magnani (*) Department of Biochemistry and Drug Discovery, Glycomimetics, Gaithersburg, MD, USA e-mail: jmagnani@Glycomimetics.com # Springer Japan 2015 N. Taniguchi et al. (eds.), Glycoscience: Biology and Medicine, DOI 10.1007/978-4-431-54841-6_201 1517
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GMI 1070: Reduction In Time To Resolution Of Vaso-Occlusive Crisis and Decreased Opioid Use In a Prospective, Randomized, Multi-Center Double Blind, Adaptive Phase 2 Study In Sickle Cell Disease
Blood, 2013Co-Authors: Marilyn J Telen, Lakshmanan Krishnamurti, Lewis L Hsu, Seungshin Rhee, Theodore Wun, Timothy L. Mccavit, Laura M. De Castro, Sophie Lanzkron, Wally R. Smith, John L MagnaniAbstract:Background Treatment for vaso-occlusive crisis (VOC) in sickle cell disease (SCD) remains supportive, focusing largely on symptom relief with opioids. Animal models have suggested a role for both red cell and leukocyte selectin-mediated adhesion in VOC, and the pan-selectin inhibitor GMI 1070 restored blood flow in a mouse model of VOC. We therefore explored safety and possible efficacy of GMI 1070 in patients with VOC. Methods This prospective, randomized multi-center double blind, adaptive Phase 2 study enrolled patients ages 12-60 yrs with HbSS or HbSβ thalassemia presenting with VOC. Patients were randomized 1:1, stratified by site. A loading dose of GMI 1070 to achieve steady state, followed by ≤14 subsequent doses, were given q 12 h. Other treatment was per institutional standard of care. The primary efficacy endpoint was resolution of VOC, defined as: 1.5 cm sustained decrease in visual analog scale (VAS) pain score from baseline and transition to oral analgesia; or documented readiness for discharge; or order for discharge. Pre-planned population PK was examined after 11 subjects, and dose was subsequently escalated, per protocol. Secondary endpoints included cumulative opioid use (morphine equivalent units [MEU]/kg) and adverse events. Differences by treatment were analyzed by ANCOVA (means, adjusted for age and sex) and Kaplan-Meier (KM, medians), stratified by age and dose. Results Seventy-six subjects were enrolled (ages 12-51; 45 F, 31 M). Drug dose was doubled to reach target nadir levels after interim PK analysis. All 76 patients reached the primary endpoint: 58 subjects continued study drug until criteria for resolution of VOC were met or the maximum number of doses were given; 18 discontinued drug for AEs, no improvement at day 5, or other reasons. In the primary efficacy analysis, reduction in mean time to VOC resolution was consistently observed in all treatment groups (pooled high and low dose GMI 1070, 103.6 h ± 20.9 (SE), n=43, vs placebo, 144.6 h ± 23.5, n=33, p=0.192). KM analysis showed median times to resolution of 69.6 h (CI 44.3, 115.5) for the GMI 1070 group vs 132.9 h (CI 67.0, 164.2) for the placebo group (p=0.187, Log-rank, [Fig 1a][1]). Thus, GMI 1070 resulted in differences in time to resolution of 41 h (mean) and 63 h (median). Similar differences were seen when analysis was stratified by age. Proportionally more subjects receiving GMI 1070 than placebo achieved resolution of VOC at 48, 72, 96 and 120 h. Analyses by how endpoint was reached were consistent with composite findings. The time to transition to oral pain medications was substantially decreased in all treatment groups: this reduction was 76 h in the GMI 1070 group (KM p=0.089) and reached statistical significance in the pediatric population (87.8 h, KM p=0.037). Time to discharge was reduced from a median of 156.1 h (CI 75.4, 185.8) for placebo to 72.2 h (CI 59.9, 121.0) for GMI 1070 (p=0.092). Stratified analysis by age and dose showed similar reductions in time to discharge. Although 24 subjects (31.6%) did not achieve a sustained reduction in VAS while hospitalized, active treatment was associated with a decrease in time to first sustained reduction in VAS. Median times to VAS reduction were: GMI 1070, 72.0 h (CI 34.5, 115.7); placebo, 125.3 h (CI 19.7, 180.2). Time to agreement about readiness for discharge was documented in 88% and 85% of the treatment and placebo groups, with medians of 72.5 h (CI 60.9, 139.1) and 137.4 h (CI 83.2, 165.7), respectively (p=0.151). An 83% reduction in mean cumulative IV opioid analgesic use (mg/kg MEU) was seen across all treatment groups (p=0.010, [Fig 1b][1]). The effect on opioid use was seen within 24 h. Total adverse event (AE) rates, serious AEs, and ‘treatment related’ AEs were comparable across all groups and ages (high dose, low dose, and pooled GMI 1070, or placebo; pediatric vs. adult). No subjects discontinued the study due to AEs. ![Figure][2] Conclusion Treatment with GMI 1070 was associated with substantial reductions in mean and median time to resolution of VOC, analyzed either as a composite or component endpoints, that were near statistical significance. The magnitude of reduction was clinically meaningful. GMI 1070 was also associated with reduction in both hourly and daily opioid use. There was no evidence for heterogeneity of effect among patient groups. These promising results support a role for selectins in the pathophysiology of VOC and justify a Phase 3 study of GMI 1070 for SCD patients with VOC. Disclosures: Telen: Dilaforette, NA: Research Funding; Pfizer, Inc.: Consultancy; Glycomimetics, Inc.: Research Funding. Wun: Emmaus, Inc.: Clinical Adjudication Committee Other; Pfizer, Inc.: Consultancy; Glycomimetics: Research Funding. McCavit: Glycomimetics, Inc.: Research Funding; Pfizer, Inc.: Consultancy. De Castro: Glycomimetics, Inc.: Research Funding; Novella Clinical: Consultancy. Krishnamurti: Glycomimetics, Inc.: Research Funding. Lanzkron: Glycomimetics, Inc.: Research Funding. Hsu: Glycomimetics, Inc.: Research Funding. Smith: Glycomimetics, Inc.: Research Funding. Rhee: Rho, Inc.: Employment; Glycomimetics, Inc.: Research Funding. Magnani: Glycomimetics, Inc.: Employment, Equity Ownership. Thackray: Glycomimetics, Inc.: Employment, Equity Ownership. [1]: #F1 [2]: pending:yes
Ayman M Mahmoud - One of the best experts on this subject based on the ideXlab platform.
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small molecule Glycomimetics inhibit vascular calcification via c met notch3 hes1 signalling
Cellular Physiology and Biochemistry, 2019Co-Authors: Ayman M Mahmoud, Alan M Jones, Gp Sidgwick, Ayman M Arafat, Yvonne Alexander, Fiona L WilkinsonAbstract:© Copyright by the Author(s). Published by Cell Physiol Biochem Press. BACKGROUND/AIMS: Vascular calcification represents a huge clinical problem contributing to adverse cardiovascular events, with no effective treatment currently available. Upregulation of hepatocyte growth factor has been linked with vascular calcification, and thus, represent a potential target in the development of a novel therapeutic strategy. Glycomimetics have been shown to interrupt HGF-receptor signalling, therefore this study investigated the effect of novel Glycomimetics on osteogenic signalling and vascular calcification in vitro. METHODS: Primary human vascular smooth muscle cells (HVSMCs) were induced by β-glycerophosphate (β-GP) and treated with 4 Glycomimetic compounds (C1-C4). The effect of β-GP and C1-C4 on alkaline phosphatase (ALP), osteogenic markers and c-Met/Notch3/HES1 signalling was determined using colorimetric assays, qRT-PCR and western blotting respectively. RESULTS: C1-C4 significantly attenuated β-GP-induced calcification, as shown by Alizarin Red S staining and calcium content by day 14. In addition, C1-C4 reduced ALP activity and prevented upregulation of the osteogenic markers, BMP-2, Runx2, Msx2 and OPN. Furthermore, β-GP increased c-Met phosphorylation at day 21, an effect ameliorated by C2 and C4 and the c-Met inhibitor, crizotinib. We next interrogated the effects of the Notch inhibitor DAPT and confirmed an inhibition of β-GP up-regulated Notch3 protein by C2, DAPT and crizotinib compared to controls. Hes-1 protein upregulation by β-GP, was also significantly downregulated by C2 and DAPT. GOLD docking analysis identified a potential binding interaction of C1-C4 to HGF which will be investigated further. CONCLUSION: These findings demonstrate that Glycomimetics have potent anti-calcification properties acting via HGF/c-Met and Notch signalling.
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a novel role for small molecule Glycomimetics in the protection against lipid induced endothelial dysfunction involvement of akt enos and nrf2 are signaling
Biochimica et Biophysica Acta, 2017Co-Authors: Ayman M Mahmoud, Fiona L Wilkinson, Alan M Jones, James A Wilkinson, Miguel Romero, Juan Duarte, Yvonne M AlexanderAbstract:Background: Glycomimetics are a diverse array of saccharide-inspired compounds, designed to mimic the bioactive functions of glycosaminoglycans. Therefore, Glycomimetics represent a unique source of novel therapies to target aberrant signaling and protein interactions in a wide range of diseases. We investigated the protective effects of four newly synthesized small molecule Glycomimetics against lipid-induced endothelial dysfunction, with an emphasis on nitric oxide (NO) and oxidative stress. Methods: Four aromatic sugar mimetics were synthesized by the stepwise transformation of 2,5- dihydroxybenzoic acid to derivatives (C1–C4) incorporating sulfate groups tomimic the structure of heparan sulfate. Results: Glycomimetic-treated human umbilical vein endothelial cells (HUVECs)were exposed to palmitic acid to model lipid-induced oxidative stress. Palmitate-induced impairment of NO production was restored by the Glycomimetics, through activation of Akt/eNOS signaling. Furthermore, C1-C4 significantly inhibited palmitateinduced reactive oxygen species (ROS) production, lipid peroxidation, and activity and expression of NADPH oxidase. These effectswere attributed to activation of the Nrf2/ARE pathway and downstreamactivation of cellular antioxidant and cytoprotective proteins. In ex vivo vascular reactivity studies, the Glycomimetics (C1–C4) also demonstrated a significant improvement in endothelium-dependent relaxation and decreased ROS production and NADPH oxidase activity in isolated mouse thoracic aortic rings exposed to palmitate. Conclusions: The small molecule Glycomimetics, C1–C4, protect against lipid-induced endothelial dysfunction through up-regulation of Akt/eNOS and Nrf2/ARE signaling pathways. Thus, carbohydrate-derived therapeutics are a new class of Glycomimetic drugs targeting endothelial dysfunction, regarded as the first line of defense against vascular complications in cardiovascular disease.
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a new class of Glycomimetic drugs to prevent free fatty acid induced endothelial dysfunction
Cardiovascular Research, 2016Co-Authors: Fiona L Wilkinson, Ayman M Mahmoud, Alan M Jones, James A Wilkinson, Miguel Romero, Juan Duarte, My AlexanderAbstract:Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of Glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule Glycomimetics against free fatty acidinduced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four Glycomimetics were synthesized by the stepwise transformation of 2,5dihydroxybenzoic acid to a range of 2,5substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholineinduced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without Glycomimetics (1µM). DAF2 and H2DCFDA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RTPCR and western blotting were utilized to measure Akt, eNOS, Nrf2, NQO1 and HO1 expression. Results: Ex vivo endotheliumdependent relaxation was significantly improved by the Glycomimetics under palmitateinduced oxidative stress. In vitro studies showed that the Glycomimetics protected HUVECs against the palmitateinduced oxidative stress and enhanced NO production. We demonstrate that the protective effects of preincubation with Glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROSinduced lipid peroxidation. Conclusion: We have developed a novel set of small molecule Glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease.
Eike-christian Wamhoff - One of the best experts on this subject based on the ideXlab platform.
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a specific Glycomimetic langerin ligand for human langerhans cell targeting
ACS central science, 2019Co-Authors: Felix F Fuchsberger, Jessica Schulze, Lydia Bellmann, Mareike Rentzsch, Gunnar Bachem, Eike-christian Wamhoff, Juliane RademacherAbstract:Langerhans cells are a subset of dendritic cells residing in the epidermis of the human skin. As such, they are key mediators of immune regulation and have emerged as prime targets for novel transcutaneous cancer vaccines. Importantly, the induction of protective T cell immunity by these vaccines requires the efficient and specific delivery of both tumor-associated antigens and adjuvants. Langerhans cells uniquely express Langerin (CD207), an endocytic C-type lectin receptor. Here, we report the discovery of a specific, Glycomimetic Langerin ligand employing a heparin-inspired design strategy and structural characterization by NMR spectroscopy and molecular docking. The conjugation of this Glycomimetic to liposomes enabled the specific and efficient targeting of Langerhans cells in the human skin. We further demonstrate the doxorubicin-mediated killing of a Langerin+ monocyte cell line, highlighting its therapeutic and diagnostic potential in Langerhans cell histiocytosis, caused by the abnormal proliferation of Langerin+ myeloid progenitor cells. Overall, our delivery platform provides superior versatility over antibody-based approaches and novel modalities to overcome current limitations of dendritic cell-targeted immuno- and chemotherapy.
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a specific Glycomimetic langerin ligand for human langerhans cell targeting
ACS central science, 2019Co-Authors: Felix F Fuchsberger, Jessica Schulze, Lydia Bellmann, Mareike Rentzsch, Gunnar Bachem, Eike-christian Wamhoff, Juliane RademacherAbstract:Langerhans cells are a subset of dendritic cells residing in the epidermis of the human skin. As such, they are key mediators of immune regulation and have emerged as prime targets for novel transcutaneous cancer vaccines. Importantly, the induction of protective T cell immunity by these vaccines requires the efficient and specific delivery of both tumor-associated antigens and adjuvants. Langerhans cells uniquely express Langerin (CD207), an endocytic C-type lectin receptor. Here, we report the discovery of a specific, Glycomimetic Langerin ligand employing a heparin-inspired design strategy and structural characterization by NMR spectroscopy and molecular docking. The conjugation of this Glycomimetic to liposomes enabled the specific and efficient targeting of Langerhans cells in the human skin. We further demonstrate the doxorubicin-mediated killing of a Langerin+ monocyte cell line, highlighting its therapeutic and diagnostic potential in Langerhans cell histiocytosis, caused by the abnormal prolifera...
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A Specific, Glycomimetic Langerin Ligand for Human Langerhans Cell Targeting
2019Co-Authors: Eike-christian Wamhoff, Felix F Fuchsberger, Jessica Schulze, Lydia Bellmann, Mareike Rentzsch, Gunnar Bachem, Juliane Rademacher, Martin Hermann, Barbara Del Frari, Rob Van DalenAbstract:Langerhans cells are a subset of dendritic cells residing in the epidermis of the human skin. As such, they are key mediators of immune regulation and have emerged as prime targets for novel transcutaneous cancer vaccines. Importantly, the induction of protective T cell immunity by these vaccines requires the efficient and specific delivery of both tumor-associated antigens and adjuvants. Langerhans cells uniquely express Langerin (CD207), an endocytic C-type lectin receptor. Here, we report the discovery of a specific, Glycomimetic Langerin ligand employing a heparin-inspired design strategy and structural characterization by NMR spectroscopy and molecular docking. The conjugation of this Glycomimetic to liposomes enabled the specific and efficient targeting of Langerhans cells in the human skin. We further demonstrate the doxorubicin-mediated killing of a Langerin+ monocyte cell line, highlighting its therapeutic and diagnostic potential in Langerhans cell histiocytosis, caused by the abnormal proliferation of Langerin+ myeloid progenitor cells. Overall, our delivery platform provides superior versatility over antibody-based approaches and novel modalities to overcome current limitations of dendritic cell-targeted immuno- and chemotherapy
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19f nmr guided design of Glycomimetic langerin ligands
ACS Chemical Biology, 2016Co-Authors: Eike-christian Wamhoff, Jonas Aretz, Jonas Hanske, Lennart Schnirch, Maurice Grube, Daniel Varon Silva, Christoph RademacherAbstract:C-type lectin receptors (CLRs) play a pivotal role in pathogen defense and immune homeostasis. Langerin, a CLR predominantly expressed on Langerhans cells, represents a potential target receptor for the development of anti-infectives or immunomodulatory therapies. As mammalian carbohydrate binding sites typically display high solvent exposure and hydrophilicity, the recognition of natural monosaccharide ligands is characterized by low affinities. Consequently, Glycomimetic ligand design poses challenges that extend to the development of suitable assays. Here, we report the first application of 19F R2-filtered NMR to address these challenges for a CLR, i.e., Langerin. The homogeneous, monovalent assay was essential to evaluating the in silico design of 2-deoxy-2-carboxamido-α-mannoside analogs and enabled the implementation of a fragment screening against the carbohydrate binding site. With the identification of both potent monosaccharide analogs and fragment hits, this study represents an important advanc...
Michel Thepaut - One of the best experts on this subject based on the ideXlab platform.
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Development of C-type lectin-oriented surfaces for high avidity glycoconjugates: towards mimicking multivalent interactions on the cell surface
Organic and Biomolecular Chemistry, 2020Co-Authors: Vanessa Porkolab, Michel Thepaut, Corinne Vives, Ieva Sutkeviciute, Anna Bernardi, Carlo Pifferi, Stefania Ordanini, Marwa Taouai, Mohammed Benazza, Olivier RenaudetAbstract:Multivalent interactions between complex carbohydrates and oligomeric C-type lectins govern a wide range of immune responses. Up to date, standard SPR (surface plasmon resonance) competitive assays have largely been to evaluate binding properties from monosaccharide units (low affinity, mM) to multivalent elemental antagonists (moderate affinity, μM). Herein, we report typical case-studies of SPR competitive assays showing that they underestimate the potency of glycoclusters to inhibit the interaction between DC-SIGN and immobilized glycoconjugates. This paper describes the design and implementation of a SPR direct interaction over DC-SIGN oriented surfaces, extendable to other C-type lectin surfaces as such Langerin. This setup provides an overview of intrinsic avidity generation emanating simultaneously from multivalent glycoclusters and from DC-SIGN tetramers organized in nanoclusters at the cell membrane. To do so, covalent biospecific capture of DC-SIGN via StreptagII/StrepTactin interaction preserves tetrameric DC-SIGN, accessibility and topology of its active sites, that would have been dissociated using standard EDC-NHS procedure under acidic conditions. From the tested glycoclusters libraries, we demonstrated that the scaffold architecture, the valency and the Glycomimetic-based ligand are crucial to reach nanomolar affinities for DC-SIGN. The glycocluster 3·D illustrates the tightest binding partner in this set for a DC-SIGN surface (KD = 18 nM). Moreover, the selectivity at monovalent scale of Glycomimetic D can be easily analyzed at multivalent scale comparing its binding over different C-type lectin immobilized surfaces. This approach may give rise to novel insights into the multivalent binding mechanisms responsible for avidity and make a major contribution to the full characterization of the binding potency of promising specific and multivalent immodulators.
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enhancing potency and selectivity of a dc sign Glycomimetic ligand by fragment based design structural basis
Chemistry: A European Journal, 2019Co-Authors: Laura Medve, Silvia Achilli, Michel Thepaut, Corinne Vives, Sara Sattin, L. Senaldi, C. Ebel, Joan Guzmancaldentey, Aline Le Roy, Sonsoles MartinsantamariaAbstract:Chemical modification of pseudo-dimannoside ligands guided by fragment-based design allowed for the exploitation of an ammonium-binding region in the vicinity of the mannose-binding site of DC-SIGN, leading to the synthesis of a Glycomimetic antagonist (compound 16) of unprecedented affinity and selectivity against the related lectin langerin. Here, the computational design of pseudo-dimannoside derivatives as DC-SIGN ligands, their synthesis, their evaluation as DC-SIGN selective antagonists, the biophysical characterization of the DC-SIGN/16 complex, and the structural basis for the ligand activity are presented. On the way to the characterization of this ligand, an unusual bridging interaction within the crystals shed light on the plasticity and potential secondary binding sites within the DC-SIGN carbohydrate recognition domain.
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development of c type lectin oriented surfaces for high avidity glycoconjugates towards mimicking multivalent interactions on the cell surface
bioRxiv, 2019Co-Authors: Vanessa Porkolab, Michel Thepaut, Corinne Vives, Ieva Sutkeviciute, Anna Bernardi, Carlo Pifferi, Stefania Ordanini, Marwa Taouai, Mohammed Benazza, Olivier RenaudetAbstract:ABSTRACT Multivalent interactions between complex carbohydrates and oligomeric C-type lectins govern a wide range of immune responses. Up to date, standard SPR (surface plasmon resonance) competitive assays have largely been to evaluate binding properties from monosaccharide units (low affinity, mM) to multivalent elemental antagonists (moderate affinity, µM). Herein, we report typical case-studies of SPR competitive assays showing that they underestimate the potency of glycoclusters to inhibit the interaction between DC-SIGN and immobilized glycoconjugates. This paper describes the design and implementation of a SPR direct interaction over DC-SIGN oriented surfaces, extendable to other C-type lectin surfaces as such Langerin. This setup provides a microscopic overview of intrinsic avidity generation emanating simultaneously from multivalent glycoclusters and from DC-SIGN tetramers that are organized in nanoclusters on the cell membrane. For this purpose, covalent biospecific capture of DC-SIGN via StreptagII /StrepTactin interaction offers the preservation of tetrameric DC-SIGN and the accessibility/functionality of all active sites. From the tested glycoclusters libraries, we demonstrated that the scaffold architecture, the valency and the Glycomimetic-based ligand are crucial to reach nanomolar affinities for DC-SIGN. The glycocluster 3.D illustrates the tightest binding partner in this set for a DC-SIGN surface (Kd= 18 nM). Moreover, the selectivity at monovalent scale of Glycomimetic D can be easily analyzed at multivalent scale comparing its binding over different C-type lectin immobilized surfaces. This approach may give rise to novel insights into the multivalent binding mechanisms responsible to avidity and make a major contribution to the full characterization of the binding potency of promising specific and multivalent immunomodulators.
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on chip screening of a Glycomimetic library with c type lectins reveals structural features responsible for preferential binding of dectin 2 over dc sign r and langerin
Chemistry: A European Journal, 2018Co-Authors: Laura Medve, Silvia Achilli, Sonia Serna, Fabio Zuccotto, Norbert Varga, Michel Thepaut, Monica Civera, Corinne Vives, Franck Fieschi, Niels ReichardtAbstract:A library of mannose- and fucose-based Glycomimetics was synthesized and screened in a microarray format against a set of C-type lectin receptors (CLRs) that included DC-SIGN, DC-SIGNR, langerin, and dectin-2. Glycomimetic ligands able to interact with dectin-2 were identified for the first time. Comparative analysis of binding profiles allowed their selectivity against other CLRs to be probed.
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On-Chip Screening of a Glycomimetic Library with C-Type Lectins Reveals Structural Features Responsible for Preferential Binding of Dectin-2 over DC-SIGN/R and Langerin
'Wiley', 2018Co-Authors: Laura Medve, Silvia Achilli, Sonia Serna, Fabio Zuccotto, Norbert Varga, Michel Thepaut, Monica Civera, Corinne Vives, Franck Fieschi, Niels ReichardtAbstract:A library of mannose- and fucose-based Glycomimetics was synthesized and screened in a microarray format against a set of C-type lectin receptors (CLRs) that included DC-SIGN, DC-SIGNR, langerin and dectin-2. Glycomimetic ligands able to interact with dectin-2 were identified for the first time. Comparative analysis of binding profiles allowed to probe their selectivity against other CLRs
Fiona L Wilkinson - One of the best experts on this subject based on the ideXlab platform.
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small molecule Glycomimetics inhibit vascular calcification via c met notch3 hes1 signalling
Cellular Physiology and Biochemistry, 2019Co-Authors: Ayman M Mahmoud, Alan M Jones, Gp Sidgwick, Ayman M Arafat, Yvonne Alexander, Fiona L WilkinsonAbstract:© Copyright by the Author(s). Published by Cell Physiol Biochem Press. BACKGROUND/AIMS: Vascular calcification represents a huge clinical problem contributing to adverse cardiovascular events, with no effective treatment currently available. Upregulation of hepatocyte growth factor has been linked with vascular calcification, and thus, represent a potential target in the development of a novel therapeutic strategy. Glycomimetics have been shown to interrupt HGF-receptor signalling, therefore this study investigated the effect of novel Glycomimetics on osteogenic signalling and vascular calcification in vitro. METHODS: Primary human vascular smooth muscle cells (HVSMCs) were induced by β-glycerophosphate (β-GP) and treated with 4 Glycomimetic compounds (C1-C4). The effect of β-GP and C1-C4 on alkaline phosphatase (ALP), osteogenic markers and c-Met/Notch3/HES1 signalling was determined using colorimetric assays, qRT-PCR and western blotting respectively. RESULTS: C1-C4 significantly attenuated β-GP-induced calcification, as shown by Alizarin Red S staining and calcium content by day 14. In addition, C1-C4 reduced ALP activity and prevented upregulation of the osteogenic markers, BMP-2, Runx2, Msx2 and OPN. Furthermore, β-GP increased c-Met phosphorylation at day 21, an effect ameliorated by C2 and C4 and the c-Met inhibitor, crizotinib. We next interrogated the effects of the Notch inhibitor DAPT and confirmed an inhibition of β-GP up-regulated Notch3 protein by C2, DAPT and crizotinib compared to controls. Hes-1 protein upregulation by β-GP, was also significantly downregulated by C2 and DAPT. GOLD docking analysis identified a potential binding interaction of C1-C4 to HGF which will be investigated further. CONCLUSION: These findings demonstrate that Glycomimetics have potent anti-calcification properties acting via HGF/c-Met and Notch signalling.
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Diabetic endothelial colony forming cells have the potential for restoration with Glycomimetics
Nature Publishing Group, 2019Co-Authors: Alexander W. W. Langford-smith, Fiona L Wilkinson, Alan M Jones, Ahmad Hasan, Ria Weston, Nicola Edwards, Andrew J. M. Boulton, Frank L. Bowling, Tawqeer S. Rashid, Yvonne M AlexanderAbstract:Abstract Endothelial colony forming progenitor cell (ECFC) function is compromised in diabetes, leading to poor vascular endothelial repair, which contributes to impaired diabetic foot ulcer healing. We have generated novel Glycomimetic drugs with protective effects against endothelial dysfunction. We investigated the effect of Glycomimetic C3 on the functional capacity of diabetic ECFCs. ECFCs were isolated from healthy controls and patients with diabetes with neuroischaemic (NI) or neuropathic (NP) foot ulcers. Functionally, diabetic ECFCs demonstrated delayed colony formation (p
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a novel role for small molecule Glycomimetics in the protection against lipid induced endothelial dysfunction involvement of akt enos and nrf2 are signaling
Biochimica et Biophysica Acta, 2017Co-Authors: Ayman M Mahmoud, Fiona L Wilkinson, Alan M Jones, James A Wilkinson, Miguel Romero, Juan Duarte, Yvonne M AlexanderAbstract:Background: Glycomimetics are a diverse array of saccharide-inspired compounds, designed to mimic the bioactive functions of glycosaminoglycans. Therefore, Glycomimetics represent a unique source of novel therapies to target aberrant signaling and protein interactions in a wide range of diseases. We investigated the protective effects of four newly synthesized small molecule Glycomimetics against lipid-induced endothelial dysfunction, with an emphasis on nitric oxide (NO) and oxidative stress. Methods: Four aromatic sugar mimetics were synthesized by the stepwise transformation of 2,5- dihydroxybenzoic acid to derivatives (C1–C4) incorporating sulfate groups tomimic the structure of heparan sulfate. Results: Glycomimetic-treated human umbilical vein endothelial cells (HUVECs)were exposed to palmitic acid to model lipid-induced oxidative stress. Palmitate-induced impairment of NO production was restored by the Glycomimetics, through activation of Akt/eNOS signaling. Furthermore, C1-C4 significantly inhibited palmitateinduced reactive oxygen species (ROS) production, lipid peroxidation, and activity and expression of NADPH oxidase. These effectswere attributed to activation of the Nrf2/ARE pathway and downstreamactivation of cellular antioxidant and cytoprotective proteins. In ex vivo vascular reactivity studies, the Glycomimetics (C1–C4) also demonstrated a significant improvement in endothelium-dependent relaxation and decreased ROS production and NADPH oxidase activity in isolated mouse thoracic aortic rings exposed to palmitate. Conclusions: The small molecule Glycomimetics, C1–C4, protect against lipid-induced endothelial dysfunction through up-regulation of Akt/eNOS and Nrf2/ARE signaling pathways. Thus, carbohydrate-derived therapeutics are a new class of Glycomimetic drugs targeting endothelial dysfunction, regarded as the first line of defense against vascular complications in cardiovascular disease.
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a new class of Glycomimetic drugs to prevent free fatty acid induced endothelial dysfunction
Cardiovascular Research, 2016Co-Authors: Fiona L Wilkinson, Ayman M Mahmoud, Alan M Jones, James A Wilkinson, Miguel Romero, Juan Duarte, My AlexanderAbstract:Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of Glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule Glycomimetics against free fatty acidinduced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four Glycomimetics were synthesized by the stepwise transformation of 2,5dihydroxybenzoic acid to a range of 2,5substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholineinduced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without Glycomimetics (1µM). DAF2 and H2DCFDA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RTPCR and western blotting were utilized to measure Akt, eNOS, Nrf2, NQO1 and HO1 expression. Results: Ex vivo endotheliumdependent relaxation was significantly improved by the Glycomimetics under palmitateinduced oxidative stress. In vitro studies showed that the Glycomimetics protected HUVECs against the palmitateinduced oxidative stress and enhanced NO production. We demonstrate that the protective effects of preincubation with Glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROSinduced lipid peroxidation. Conclusion: We have developed a novel set of small molecule Glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease.