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

Hot-spot analysis for drug discovery targeting protein-protein interactions

Introduction: Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.This work has been funded by grants BIO2016-79930-R and SEV-2015-0493 from the Spanish Ministry of Economy, Industry and Competitiveness, and grant EFA086/15 from EU Interreg V POCTEFA. M Rosell is supported by an FPI fellowship from the Severo Ochoa program. The authors are grateful for the support of the the Joint BSC-CRG-IRB Programme in Computational Biology.Peer ReviewedPostprint (author's final draft

Fibrogenic Disorders in Human Diseases: From Inflammation to Organ Dysfunction.

Fibrosis is an inadequate response to tissue stress with very few therapeutic options to prevent its progression to organ dysfunction. There is an urgent need to identify drugs with a therapeutic potential for fibrosis, either by designing and developing new compounds or by repurposing drugs already in clinical use which were developed for other indications. In this Perspective, we summarize some pathways and biological targets involved in fibrosis development and maintenance, focusing on common mechanisms between organs and diseases. We review the therapeutic agents under experimental development, clinical trials, or in clinical use for the treatment of fibrotic disorders, evaluating the reasons for the discrepancies observed between preclinical and clinical results. We also discuss the improvement that we envision in the development of therapeutic molecules able to achieve improved potential for treatment, including indirect modulators, targeting approaches, or drug combinations

MicroRNA applications for prostate, ovarian and breast cancer in the era of precision medicine.

The high degree of conservation in microRNA from Caenorhabditiselegans to humans has enabled relatively rapid implementation of findings in model systems to the clinic. The convergence of the capacity for genomic screening being implemented in the prevailing precision medicine initiative and the capabilities of microRNA to address these changes holds significant promise. However, prostate, ovarian and breast cancers are heterogeneous and face issues of evolving therapeutic resistance. The transforming growth factor-beta (TGFβ) signaling axis plays an important role in the progression of these cancers by regulating microRNAs. Reciprocally, microRNAs regulate TGFβ actions during cancer progression. One must consider the expression of miRNA in the tumor microenvironment a source of biomarkers of disease progression and a viable target for therapeutic targeting. The differential expression pattern of microRNAs in health and disease, therapeutic response and resistance has resulted in its application as robust biomarkers. With two microRNA mimetics in ongoing restorative clinical trials, the paradigm for future clinical studies rests on the current observational trials to validate microRNA markers of disease progression. Some of today's biomarkers can be translated to the next generation of microRNA-based therapies

Sphingosine 1-phosphate receptors: do they have a therapeutic potential in cardiac fibrosis?

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is characterized by a peculiar mechanism of action. In fact, S1P, which is produced inside the cell, can act as an intracellular mediator, whereas after its export outside the cell, it can act as ligand of specific G-protein coupled receptors, which were initially named endothelial differentiation gene (Edg) and eventually renamed sphingosine 1-phosphate receptors (S1PRs). Among the five S1PR subtypes, S1PR1, S1PR2 and S1PR3 isoforms show broad tissue gene expression, while S1PR4 is primarily expressed in immune system cells, and S1PR5 is expressed in the central nervous system. There is accumulating evidence for the important role of S1P as a mediator of many processes, such as angiogenesis, carcinogenesis and immunity, and, ultimately, fibrosis. After a tissue injury, the imbalance between the production of extracellular matrix (ECM) and its degradation, which occurs due to chronic inflammatory conditions, leads to an accumulation of ECM and, consequential, organ dysfunction. In these pathological conditions, many factors have been described to act as pro- and anti-fibrotic agents, including S1P. This bioactive lipid exhibits both pro- and anti-fibrotic effects, depending on its site of action. In this review, after a brief description of sphingolipid metabolism and signaling, we emphasize the involvement of the S1P/S1PR axis and the downstream signaling pathways in the development of fibrosis. The current knowledge of the therapeutic potential of S1PR subtype modulators in the treatment of the cardiac functions and fibrinogenesis are also examined

Characterisation of targeted imaging and theranostic agents for cancer and fibrosis

Positron emission tomography (PET) imaging is a non-invasive modality that permits the characterisation of biological processes at the molecular level. Integrin αvβ6 and the chemokine receptor CXCR4 are important receptors in the pathogenesis of various cancers as well other diseases such as idiopathic pulmonary fibrosis (IPF) that may be exploited for the diagnosis, prognosis and assessment of therapeutic response. The gold-standard method for diagnosis of various cancers, such as breast, is immunohistochemistry (IHC), which suffers from a lack of a well-defined and reproducible cut-off value for tumour-positive or benign classification.The integrin αvβ6 is involved in various signalling pathways and it’s expression is generally very low in healthy tissues, and is greatly upregulated in various pathologies such as cancer and IPF, being linked to worse prognosis in pancreatic, head and neck, breast and ovarian cancers. Upregulation of αvβ6 during fibrosis or in tumours makes it an attractive target for a non-invasive imaging agent for the early detection of αvβ6 in cancer or IPF, monitoring of disease progression and the assessment of therapeutic response to existing or novel therapies. This thesis has evaluated αvβ6-targeted peptides for use as non-invasive PET imaging agents. [⁶⁸Ga]Ga-DO3A-JD2-RGD1 was selected as the lead peptide and demonstrated high affinity binding and high specificity for αvβ6 in competitive enzyme-linked immunosorbent assays, surface plasmon resonance and cellular binding assays in vitro as well as imaging and biodistribution studies in vivo using αvβ6+ cancer models, indicating its suitability for the detection of αvβ6 in cancer. Preliminary studies of [natLu]Lu-DO3A-JD2-RGD1 showed that chelation of lutetium-177 did not affect peptide binding to αvβ6, making it a promising potential peptide-receptor radionuclide therapy (PRRT) agent for αvβ6+ cancer.The chemokine receptor CXCR4 and its cognate ligand CXCL12 play a pivotal role in normal physiological processes. CXCR4 overexpression has been associated with over twenty types of human cancer with aggressive, invasive phenotypes, presenting a requirement for a non-invasive imaging agent for the detection of malignant sites including metastatic lesions. This thesis has evaluated the CXCR4-binding characteristics of novel tetraazamacrocycles [natCu]CuCB-bicyclam and [natCu]Cu2CB-bicyclam, synthesised by Archibald and colleagues, by surface plasmon resonance in a comparison study with the clinically used AMD3100. Both novel inhibitors were able to bind to CXCR4 with greater affinity than AMD3100, indicating their suitability for further validation as both PET imaging agents in CXCR4+ cancer detection and therapeutic agents in cancer and IPF

Strategies for anti-fibrotic therapies.

The fibrotic diseases encompass a wide spectrum of entities including such multisystemic diseases as systemic sclerosis, nephrogenic systemic fibrosis and sclerodermatous graft versus host disease, as well as organ-specific disorders such as pulmonary, liver, and kidney fibrosis. Collectively, given the wide variety of affected organs, the chronic nature of the fibrotic processes, and the large number of individuals suffering their devastating effects, these diseases pose one of the most serious health problems in current medicine and a serious economic burden to society. Despite these considerations there is currently no accepted effective treatment. However, remarkable progress has been achieved in the elucidation of their pathogenesis including the identification of the critical role of myofibroblasts and the determination of molecular mechanisms that result in the transcriptional activation of the genes responsible for the fibrotic process. Here we review the origin of the myofibroblast and discuss the crucial regulatory pathways involving multiple growth factors and cytokines that participate in the pathogenesis of the fibrotic process. Potentially effective therapeutic strategies based upon this new information are considered in detail and the major challenges that remain and their possible solutions are presented. It is expected that translational efforts devoted to convert this new knowledge into novel and effective anti-fibrotic drugs will be forthcoming in the near future. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease