Cilengitide: The First Anti-Angiogenic Small Molecule Drug Candidate. Design, Synthesis and Clinical Evaluation

Cilengitide, a cyclic RGD pentapeptide, is currently in clinical phase III for treatment of glioblastomas and in phase II for several other tumors. This drug is the first anti-angiogenic small molecule targeting the integrins αvβ3, αvβ5 and α5β1. It was developed by us in the early 90s by a novel procedure, the spatial screening. This strategy resulted in c(RGDfV), the first superactive αvβ3 inhibitor (100 to 1000 times increased activity over the linear reference peptides), which in addition exhibited high selectivity against the platelet receptor αIIbβ3. This cyclic peptide was later modified by N-methylation of one peptide bond to yield an even greater antagonistic activity in c(RGDf(NMe)V). This peptide was then dubbed Cilengitide and is currently developed as drug by the company Merck-Serono (Germany)

A molecular toolkit for the functionalization of titanium-based biomaterials that selectively control integrin-mediated cell adhesion

We present a click chemistry- based molecular toolkit for the bio- functionalization of materials to selec- tively control integrin-mediated cell ad- hesion. To this end, a 5 b 1-selective RGD peptidomimetics were covalently immobilized on Ti-based materials, and the capacity to promote the selective binding of a 5 b 1 was evaluated using a solid-phase integrin binding assay. This functionalization strategy yielded surfa- ces with a nine-fold increased affinity for a 5 b 1, in comparison to control samples, and total selectivity against the binding of the closely related integ- rin a v b 3. Moreover, our methodology allowed the screening of several phos- phonic acid containing anchoring units to find the best spacer–anchor moiety required for establishing an efficient binding to titanium and to promote se- lective integrin binding. The integrin subtype specificity of these biofunc- tionalized surfaces was further exam- ined in vitro by inducing selective ad- hesion of genetically modified fibro- blasts, which express exclusively the a 5 b 1 integrin. The versatility of our molecular toolkit was proven by shift- ing the cellular specificity of the mate- rials from a 5 b 1- to a v b 3-expressing fi- broblasts by using an a v b 3-selective peptidomimetic as coating molecule. The results shown here represent the first functionalization of Ti-based mate- rials with a 5 b 1- or a v b 3-selective pep- tidomimetics that allow an unprece- dented control to discriminate between a 5 b 1- and a v b 3-mediated adhesions. The role of these two integrins in dif- ferent biological events is still a matter of debate and is frequently discussed in literature. Thus, such bioactive titanium surfaces will be of great relevance for the study of integrin-mediated cell ad- hesion and the development of new bi- omaterials targeting specific cell typesPeer ReviewedPostprint (published version

Interface Immobilization Chemistry of cRGD-based Peptides Regulates Integrin Mediated Cell Adhesion

The interaction of specifi c surface receptors of the integrin family with different extracellular matrix-based ligands is of utmost importance for the cellular adhesion process. A ligand consists of an integrin-binding group, here cyclic RGDfX, a spacer molecule that lifts the integrin-binding group from the surface and a surface anchoring group. c(-RGDfX-) peptides are bound to gold nanoparticle structured surfaces via polyproline, polyethylene glycol or aminohexanoic acid containing spacers of different lengths. Although keeping the integrin-binding c(-RGDfX-) peptides constant for all compounds, changes of the ligand´s spacer chemistry and length reveal signifi cant differences in cell adhesion activation and focal adhesion formation. Polyproline-based peptides demonstrate improved cell adhesion kinetics and focal adhesion formation compared with common aminohexanoic acid or polyethylene glycol spacers. Binding activity can additionally be improved by applying ligands with two head groups, inducing a multimeric effect. This study gives insights into spacer-based differences in integrin-driven cell adhesion processes and remarkably highlights the polyproline-based spacers as suitable ligand-presenting templates for surface functionalization.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

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

Substrate engagement of integrins α5 β1 and αv β3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5 β1 and αv β3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5 β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αv β3 or α5 β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration.Instituto de Investigaciones Fisicoquímicas Teóricas y AplicadasConsejo Nacional de Investigaciones Científicas y Técnica

Substrate engagement of integrins α5 β1 and αv β3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5 β1 and αv β3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5 β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αv β3 or α5 β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration.Instituto de Investigaciones Fisicoquímicas Teóricas y AplicadasConsejo Nacional de Investigaciones Científicas y Técnica

Novel peptide-based strategies to install osteointegrative properties on metallic biomaterials

Postprint (published version

Functionalizing avß3- or a5ß1-selective integrin antagonists for surface coating: A new tool to discriminate integrin subtypes in vitro

Thumbnail image of graphical abstract Stuck with the right choice: avß3- or a5ß1-selective integrin ligands were functionalized for surface coating without losing activity and selectivity. The coating of nanostructured gold surfaces with these compounds stimulated subtype-selective cell adhesion of genetically modified avß3- or a5ß1-expressing fibroblasts in vitro.Postprint (published version

Clinical Efficacy of a Novel Therapeutic Principle, Anakoinosis

Classic tumor therapy, consisting of cytotoxic agents and/or targeted therapy, has not overcome therapeutic limitations like poor risk genetic parameters, genetic heterogeneity at different metastatic sites or the problem of undruggable targets. Here we summarize data and trials principally following a completely different treatment concept tackling systems biologic processes: the principle of communicative reprogramming of tumor tissues, i.e., anakoinosis(ancient greek for communication), aims at establishing novel communicative behavior of tumor tissue, the hosting organ and organism via re-modeling gene expression, thus recovering differentiation, and apoptosis competence leading to cancer control – in contrast to an immediate, “poisoning” with maximal tolerable doses of targeted or cytotoxic therapies. Therefore, we introduce the term “Master modulators” for drugs or drug combinations promoting evolutionary processes or regulating homeostatic pathways. These “master modulators” comprise a broad diversity of drugs, characterized by the capacity for reprogramming tumor tissues, i.e., transcriptional modulators, metronomic low-dose chemotherapy, epigenetically modifying agents, protein binding pro-anakoinotic drugs, such as COX-2 inhibitors, IMiDs etc., or for example differentiation inducing therapies. Data on 97 anakoinosis inducing schedules indicate a favorable toxicity profile: The combined administration of master modulators, frequently (with poor or no monoactivity) may even induce continuous complete remission in refractory metastatic neoplasia, irrespectively of the tumor type. That means recessive components of the tumor, successively developing during tumor ontogenesis, are accessible by regulatory active drug combinations in a therapeutically meaningful way. Drug selection is now dependent on situative systems characteristics, to less extent histology dependent. To sum up, anakoinosis represents a new substantive therapy principle besides novel targeted therapies

Low-density lipoprotein balances T cell metabolism and enhances response to anti-PD-1 blockade in a HCT116 spheroid model

IntroductionThe discovery of immune checkpoints and the development of their specific inhibitors was acclaimed as a major breakthrough in cancer therapy. However, only a limited patient cohort shows sufficient response to therapy. Hence, there is a need for identifying new checkpoints and predictive biomarkers with the objective of overcoming immune escape and resistance to treatment. Having been associated with both, treatment response and failure, LDL seems to be a double-edged sword in anti-PD1 immunotherapy. Being embedded into complex metabolic conditions, the impact of LDL on distinct immune cells has not been sufficiently addressed. Revealing the effects of LDL on T cell performance in tumor immunity may enable individual treatment adjustments in order to enhance the response to routinely administered immunotherapies in different patient populations. The object of this work was to investigate the effect of LDL on T cell activation and tumor immunity in-vitro. MethodsExperiments were performed with different LDL dosages (LDLlow = 50 μg/ml and LDLhigh = 200 μg/ml) referring to medium control. T cell phenotype, cytokines and metabolism were analyzed. The functional relevance of our findings was studied in a HCT116 spheroid model in the context of anti-PD-1 blockade.ResultsThe key points of our findings showed that LDLhigh skewed the CD4+ T cell subset into a central memory-like phenotype, enhanced the expression of the co-stimulatory marker CD154 (CD40L) and significantly reduced secretion of IL-10. The exhaustion markers PD-1 and LAG-3 were downregulated on both T cell subsets and phenotypical changes were associated with a balanced T cell metabolism, in particular with a significant decrease of reactive oxygen species (ROS). T cell transfer into a HCT116 spheroid model resulted in a significant reduction of the spheroid viability in presence of an anti-PD-1 antibody combined with LDLhigh.DiscussionFurther research needs to be conducted to fully understand the impact of LDL on T cells in tumor immunity and moreover, to also unravel LDL effects on other lymphocytes and myeloid cells for improving anti-PD-1 immunotherapy. The reason for improved response might be a resilient, less exhausted phenotype with balanced ROS levels