A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins avß3, avß5, avß6, avß8, a5ß1, aIIbß3, using homogenous ELISA-like solid phase binding assay.Postprint (published version

Functionalization of Ruthenium(II) terpyridine complexes with cyclic RGD peptides to target integrin receptors in cancer cells

The lack of selectivity for cancer cells and the resulting negative impact on healthy tissue is a severe drawback of actual cancer chemotherapy. Tethering of cytotoxic drugs to targeting vectors such as peptides, which recognize receptors overexpressed on the surface of tumor cells, is one possible strategy to overcome such a problem. The pentapeptide cyc(RGDfK) targets the integrin receptor αvβ3, important for tumor growth and metastasis formation. In this work, two terpyridine based Ru(II) complexes were prepared and for the first time conjugated to cyc(RGDfK) via amide bond formation resulting in a monomeric and a dimeric bioconjugate. Both Ru(II) complexes bind strongly and selectively to integrin αvβ3, with the dimeric molecule displaying a 20-fold higher affinity to the receptor than the monomeric one. However, the cytotoxicity of the complexes and related bioconjugates against human A549 and SKOV-3 cell lines is still not sufficient for application as anticancer agents. Nevertheless, considering the high selectivity for integrin receptor αvβ3, the synthesis of Ru-based bioconjugates with cyc(RGDfK) paves a promising way towards the design of effective targeted anticancer agents

Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly

Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3′ splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Guideline for Care of Patients with the Diagnoses of Craniosynostosis: Working Group on Craniosynostosis

This guideline for care of children with craniosynostosis was developed by a national working group with representatives of 11 matrix societies of specialties and the national patients' society. All medical aspects of care for nonsyndromic and syndromic craniosynostosis are included, as well as the social and psychologic impact for the patient and their parents. Managerial aspects are incorporated as well, such as organizing a timely referral to the craniofacial center, requirements for a dedicated craniofacial center, and centralization of this specialized care. The conclusions and recommendations within this document are founded on the available literature, with a grading of the level of evidence, thereby highlighting the areas of care that are in need of high-quality research. The development of this guideline was made possible by an educational grant of the Dutch Order of Medical Specialists. The development of this guideline was supported by an educational grant of the Dutch Order of Medical Specialists

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

N-Methylation of isoDGR Peptides: Discovery of a Selective ?5?1-Integrin Ligand as a Potent Tumor Imaging Agent.

Specific targeting of the integrin subtype ?5?1 possesses high potential in cancer diagnosis and therapy. Through sequential N-methylation, we successfully converted the biselective ?5?1/?v?6 peptide c(phg- isoDGR-k) into a potent peptidic RGD binding ?5?1 subtype selective ligand c(phg- isoDGR-( NMe)k). Nuclear magnetic resonance spectroscopy and molecular modeling clarified the molecular basis of its improved selectivity profile. To demonstrate its potential in vivo, c(phg- isoDGR-( NMe)k) was trimerized with the chelator TRAP and used as a positron-emission tomography tracer for monitoring ?5?1 integrin expression in a M21 mouse xenograft

A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins ?v?3, ?v?5, ?v?6, ?v?8, ?5?1, ?IIb?3, using homogenous ELISA-like solid phase binding assay

Synthesis and evaluation of bioactive ligands for integrins and the CXCR4 receptor

Literaturbekannte Integrinliganden wurden erstmalig in einem einheitlichen in-vitro-Testsystem auf deren Bindungsaffinität gegenüber sechs RGD-bindenden Integrinen evaluiert. Ein peptidomimetischer Integrinligand wurde funktionalisiert und ein cyclisches Pentapeptid durch einen N-Methylscan optimiert und in der molekularen Bildgebung eingesetzt. Durch die Modifizierung der Guanidiniumgruppe von Arginin in Integrinliganden konnte die Subtypselektivität stark beeinflusst werden. Des weiteren wurde eineSynthesemethode zur Funktionalisierung der Guanidiniumgruppe bei der Peptidfestphasensynthese entwickelt, damit konnten CXCR4-Liganden nahezu ohne Affinitätsverluste funktionalisiert werden. Außerdem wurde das Adhäsionsverhalten von humanen mesenchymalen Stammzeller auf einer mit CXCR4-Liganden beschichteten Titanoberfläche untersucht.Several well known integrin ligands were, for the first time, homogenously evaluated in an in-vitro testing system for their binding affinity to six different RGD-binding integrin subtyptes. A peptidomimetic integrin ligand was optimized and functionalized, a cyclic pentapeptide ligand optimized through a N-methylscan and used for molecular imaging. Different modifications of the guanidine group of arginine in integrin ligands strongly influenced their subtype selectivity profiles. Furthermore, a method for the functionalization via the guanidine group was developed to be used during solid phase peptide synthesis. Using this protocol, CXCR4 ligands could be functionalized almost without influencing the binding affinity. Moreover, the adhesion of human mesenchumal stem cells on titan surfaces coated with CXCR4 ligands was investigated