Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARλ 3 full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARλ 3, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARλ 3 provides a rationale for the different activation of the ligand towards PPARα and PPARλ 3, suggesting a novel basis for ligand design

PHA-680626 Is an Effective Inhibitor of the Interaction between Aurora-A and N-Myc.

Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcription factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation by directly binding to a highly conserved N-Myc region. As a result, elevated levels of N-Myc are observed. During recent years, it has been demonstrated that some ATP competitive inhibitors of AURKA also cause essential conformational changes in the structure of the activation loop of the kinase that prevents N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complexes with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results identify PHA-680626 as an amphosteric inhibitor both in vitro and in MYCN overexpressing cell lines, thus expanding the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex and confirming that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells

Affinity-based separation methods for the study of biological interactions: The case of peroxisome proliferator-activated receptors in drug discovery

Affinity-based methods using immobilized proteins are important approaches for understanding the interactions between small molecules and biological targets. This review is intended to provide an overview of different affinity based separation methods that have been applied to the study of peroxisome proliferator activated receptors (PPARs). The screening of compound to increase screening rates for synthetic and natural ligands of PPAR are reported. Pros and cons of the approaches in ligand discovery initiatives are discusse

Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex

Cancer cells reprogramme one­carbon metabolism (OCM) to support enhanced growth and proliferation, in this contest Serine hydroxymethyltransferase (SHMT) is a pivotal enzyme. SHMT mainly exists in three isoforms; two localized in the cytosol (SHMT1/SHMT2 α) and one (SHMT2) in the mitochondria. SHMT1 undergoes to a nuclear localization during the S­phase of the cell cycle to sustain de novo dTMP synthesis1. The de novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), the latter two are targets of widely used chemotherapeutics such as antifolates and 5­fluorouracil. It had been suggested that these three proteins assemble in the nucleus into the thymidylate synthesis complex (dTMP­SC)1. We have recently understood the intracellular dynamics of dTMP synthesis complex in lung cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result strongly indicates that the role of the dTMP­SC assembly may go beyond dTMP synthesis. We have also successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR by using a different array of techniques. Moreover, we have demonstrated that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionary selected in eukaryotes to optimize protein­protein interactions. Lastly, we have set­up an activity assay of the complete thymidylate cycle in vitro, which may provide a useful tool to develop drugs targeting the entire complex instead of the individual components. [1] Anderson, D. and Stover, P. (2009). SHMT1 and SHMT2 Are Functionally Redundant in Nuclear De novo Thymidylate Biosynthesis. PLoS ONE, 4(6), p.e5839

Screening of saponins and sapogenins from Medicago species as potential PPARγ agonists and X-ray structure of the complex PPARγ/caulophyllogenin

A series of saponins and sapogenins from Medicago species were tested for their ability to bind and activate the nuclear receptor PPARγ by SPR experiments and transactivation assay, respectively. The SPR analysis proved to be a very powerful and fast technique for screening a large number of compounds for their affinity to PPARγ and selecting the better candidates for further studies. Based on the obtained results, the sapogenin caulophyllogenin was proved to be a partial agonist towards PPARγ and the X-ray structure of its complex with PPARγ was also solved, in order to investigate the binding mode in the ligand binding domain of the nuclear receptor. This is the first known crystal structure of a sapogenin directly interacting with PPARγ. Another compound of the series, the echinocistic acid, showed antagonist activity towards PPARγ, a property that could be useful to inhibit the adipocyte differentiation which is a typical adverse effect of PPARγ agonists. This study confirms the interest on saponins and sapogenins as a valuable natural resource exploitable in the medical and food industry for ameliorating the metabolic syndrome

Selective MMP inhibitors for the treatment of skeletal malignancy.

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