CD and NMR Conformational Preferences of Intrinsically Disordered Amphiphilic Peptides: A New Class of Potential Targets in Drug Discovery

Owing to the large panel of biological functions of peptides and their high specificity and potency, the development of peptide-based therapeutic and diagnostic tools has received increased interest. Peptide amphiphiles (PAs) are an emerging class of molecules in which a bioactive peptide is covalently conjugate to a hydrophobic moiety. Due to the coexistence in the molecule of a hydrophilic peptide sequence and a hydrophobic group, PAs are able to selfassemble spontaneously into a variety of nanostructures, such as monolayers, bilayers, and vesicles. In this work we have synthesized predicted by MEDOR disordered peptide sequences3 functionalized by alkyl chains, and connected by ethoxylic-based linker. The structural properties in solution of these new PAs were investigated using CD, NMR and DLS. The presence of the alkyl chains induces not only the self-assembly of these new PAs into supramolecular aggregates but also a gain of structure within the disordered peptide. The design of supramolecular systems, generated by joining a disordered peptide and a lipophilic moiety, could drive the disordered peptide to fold into a stable structure. This structural modification could be a promising route to develop a new class of bio-molecules for processes in which a specific conformational rearrangement is required

Metabolomic and oxidative effects of quantum dots-indolicidin on three generations of Daphnia magna

This study evaluated the effect of QDs functionalized with the antimicrobial peptide indolicidin on oxidative stress and metabolomics profiles of Daphnia magna across three generations (F0, F1, and F2). Exposing D. magna to sub-lethal concentrations of the complex QDs-indolicidin, a normal survival of daphnids was observed from F0 to F2, but a delay of first brood, fewer broods per female, a decrease of length of about 50% compared to control. In addition, QDs-indolicidin induced a significantly higher production of reactive oxygen species (ROS) gradually in each generation and an impairment of enzymes response to oxidative stress such as superoxide dismutase (SOD), catalase (CAT) and glutathione transferase (GST). Effects were confirmed by metabolomics profiles that pointed out a gradual decrease of metabolomics content over the three generations and a toxic effect of QDs-indolicidin likely related to the higher accumulation of ROS and decreased antioxidant capacity in F1 and F2 generations. Results highlighted the capability of metabolomics to reveal an early metabolic response to stress induced by environmental QDs-indolicidin complex

Cyclic mimetics of kinase-inhibitory region of Suppressors of Cytokine Signaling 1: Progress toward novel anti-inflammatory therapeutics

Herein we investigated the structural and cellular effects ensuing from the cyclization of a potent inhibitor of JAK2 as mimetic of SOCS1 protein, named PS5. The introduction of un-natural residues and a lactam internal bridge, within SOCS1-KIR motif, produced candidates that showed high affinity toward JAK2 catalytic domain. By combining CD, NMR and computational studies, we obtained valuable models of the interactions of two peptidomimetics of SOCS1 to deepen their functional behaviors. Notably, when assayed for their biological cell responses mimicking SOCS1 activity, the internal cyclic PS5 analogues demonstrated able to inhibit JAK-mediated tyrosine phosphorylation of STAT1 and to reduce cytokine-induced proinflammatory gene expression, oxidative stress generation and cell migration. The present study well inserts in the field of low-molecular-weight proteomimetics with improved longtime cellular effects and adds a new piece to the puzzled way for the conversion of bioactive peptides into drugs

Ad-hoc modifications of cyclic mimetics of SOCS1 protein: Structural and functional insights

Suppressors of cytokine signaling 1 (SOCS1) protein, a negative regulator of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, possesses a small kinase inhibitory region (KIR) involved in the inhibition of JAK kinases. Several studies showed that mimetics of KIR-SOCS1 can be potent therapeutics in several disorders (e.g., neurological, autoimmune or cardiovascular diseases). In this work, starting from a recently identified cyclic peptidomimetic of KIR-SOCS1, icPS5(Nal1), to optimize the peptide structure and improve its biological activity, we designed novel derivatives, containing crucial amino acids substitutions and/or modifications affecting the ring size. By combining microscale thermophoresis (MST), Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR) and computational studies, we showed that the cycle size plays a key role in the interaction with JAK2 and the substitution of native residues with un-natural building blocks is a valid tool to maintain low-micromolar affinity toward JAK2, greatly increasing their serum stability. These findings contribute to increase the structural knowledge required for the recognition of SOCS1/JAK2 and to progress towards their conversion into more drug-like compounds

Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?

Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short β-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains

Peptide targeting chemokine receptor CXCR4: structural behavior and biological binding studies

CXCR4 is a G-protein-coupled receptor involved in a number of physiological processes in the hematopoietic and immune systems. CXCL12/CXCR4 axis plays a central role in diseases, such as HIV, cancer, WHIM syndrome, rheumatoid arthritis, pulmonary fibrosis and lupus and, hence, indicated as putative therapeutic target. Although multiple CXCR4 antagonists have been developed there is only one marketed drug, Plerixafor, indicated for stem cell mobilization in poor mobilizer patients. In this work we have designed and synthesized two peptides, six and seven residues long, using as template the N-terminal region of CXCL12, analyzed their conformations by circular dichroism, NMR, and molecular dynamics simulations, simulated their complexes with CXCR4 by docking methods, and validated these data by in vitro studies. The results showed that two peptides, are rather flexible in aqueous solution lacking ordered secondary structure elements and present a promising affinity for CXCR4. This affinity is not revealed for CXCR7, indicating a specificity for CXCR

A Diruthenium Metallodrug as a Potent Inhibitor of Amyloid-β Aggregation: Synergism of Mechanisms of Action

The physical and chemical properties of paddlewheel diruthenium compounds are highly dependent on the nature of the ligands surrounding the bimetallic core. Herein, we compare the ability of two diruthenium compounds, [Ru2Cl(D-p-FPhF)- (O2CCH3)3]·H2O (1) (D-p-FPhF− = N,N′-bis(4-fluorophenyl)- formamidinate) and K3[Ru2(O2CO)4]·3H2O (2), to act as inhibitors of amyloid aggregation of the Aβ1−42 peptide and its peculiar fragments, Aβ1−16 and Aβ21−40. A wide range of biophysical techniques has been used to determine the inhibition capacity against aggregation and the possible mechanism of action of these compounds (Thioflavin T fluorescence and autofluorescence assays, UV−vis absorption spectroscopy, circular dichroism, nuclear magnetic resonance, mass spectrometry, and electron scanning microscopy). Data show that the most effective inhibitory effect is shown for compound 1. This compound inhibits fiber formation and completely abolishes the cytotoxicity of Aβ1−42. The antiaggregatory capacity of this complex can be explained by a binding mechanism of the dimetallic units to the peptide chain along with π−π interactions between the formamidinate ligand and the aromatic side chains. The results suggest the potential use of paddlewheel diruthenium complexes as neurodrugs and confirm the importance of the steric and charge effects on the properties of diruthenium compoundsUniversidad Complutense de MadridUniversidad Complutense de MadridMinisterio de Ciencia e InnovaciónDepto. de Química InorgánicaFac. de Ciencias QuímicasTRUEpu