Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.

Given the similar chemical properties of zinc and cadmium, zinc finger domains have been often proposed as mediators of the toxic and carcinogenic effects exerted by this xenobiotic metal. The effects of zinc replacement by cadmium in different eukaryotic zinc fingers have been reported. In the present work, to evaluate the effects of such substitution in the prokaryotic zinc finger, we report a detailed study of its functional and structural consequences on the Ros DNA binding domain (Ros87). We show that this protein, which bears important structural differences with respect to the eukaryotic domains, appears to structurally tolerate the zinc to cadmium substitution and the presence of cadmium does not affect the DNA binding activity of the protein. Moreover, we show for the first time how zinc to cadmium replacement can also take place in a cellular context. Our findings both complement and extend previous results obtained for different eukaryotic zinc fingers, suggesting that metal substitution in zinc fingers may be of relevance to the toxicity and/or carcinogenicity mechanisms of this metal.

Background Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A 2 (cPLA 2 ) metabolite glycerophosphoinositol 4-phosphate (GroPIns4 P ), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4 P activates Src remains unknown. Methods Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4 P- interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4 P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. Results We find that Shp1 is the direct cellular target of GroPIns4 P . GroPIns4 P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4 P show significantly enhanced wound healing capability, indicating that GroPIns4 P has a stimulatory role to activate fibroblast migration. GroPIns4 P is produced by cPLA 2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4 P was shown to mediate the EGF-induced cell motility. Conclusions This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA 2 metabolite GroPIns4 P . We show that GroPIns4 P is required for EGF-induced fibroblast migration and that it is part of a cPLA 2 /GroPIns4 P/ Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer. El...

Indoleamine 2,3-dioxygenase 1 (IDO1) is attracting a great deal of interest as drug target in immune-oncology being highly expressed in cancer cells and participating to the tumor immune-editing process. Although several classes of IDO1 inhibitors have been reported in literature and patent applications, only few compounds have proved optimal pharmacological profile in preclinical studies to be advanced in clinical trials. Accordingly, the quest for novel structural classes of IDO1 inhibitors is still open. In this paper, we report a fragment-based screening campaign that combines Water-LOGSY NMR experiments and microscale thermophoresis approach to identify fragments that may be helpful for the development of novel IDO1 inhibitors as therapeutic agents in immune-oncology disorders.