Caloxin-derived peptides for the inhibition of plasma membrane calcium ATPases.

Plasma membrane calcium ATPases (PMCAs) are a family of transmembrane proteins responsible for the extrusion of cytosolic Ca2+ to the extracellular milieu. They are important players of the calcium homeostasis possibly implicated in some important diseases. The reference inhibitors of PMCA extruding activity are on one hand ortho-vanadate (IC50 in the 30 mM range), and on the other a series of 12- to 20-mer peptides named caloxins (IC50 in the 100 µM scale). As for all integral membrane proteins, biochemistry and pharmacology are difficult to study on isolated and/or purified proteins. Using a series of reference blockers, we assessed a pharmacological window with which we could study the functionality of PMCAs in living cells. Using this system, we screened for alternative versions of caloxins, aiming at shortening the peptide backbone, introducing non-natural amino acids, and overall trying to get a glimpse at the structure-activity relationship between those new peptides and the protein in a cellular context. We describe a short series of equipotent 5-residue long analogues with IC50 in the low µM range.

MCH-R1 Antagonist GPS18169, a Pseudopeptide, Is a Peripheral Anti-Obesity Agent in Mice.

Melanin-concentrating hormone (MCH) is a 19 amino acid long peptide found in the brain of animals, including fishes, batrachians, and mammals. MCH is implicated in appetite and/or energy homeostasis. Antagonists at its receptor (MCH-R1) could be major tools (or ultimately drugs) to understand the mechanism of MCH action and to fight the obesity syndrome that is a worldwide societal health problem. Ever since the deorphanisation of the MCH receptor, we cloned, expressed, and characterized the receptor MCH-R1 and started a vast medicinal chemistry program aiming at the discovery of such usable compounds. In the present final work, we describe GPS18169, a pseudopeptide antagonist at the MCH-R1 receptor with an affinity in the nanomolar range and a Ki for its antagonistic effect in the 20 picomolar range. Its metabolic stability is rather ameliorated compared to its initial parent compound, the antagonist S38151. We tested it in an in vivo experiment using high diet mice. GPS18169 was found to be active in limiting the accumulation of adipose tissues and, correlatively, we observed a normalization of the insulin level in the treated animals, while no change in food or water consumption was observed.

VHH characterization. Comparison of recombinant with chemically synthesized anti-HER2 VHH.

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody-like protein, using an anti-HER2 VHH as a model. The total chemical synthesis of the 125 amino-acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its "antigen." Using this combination of orthogonal techniques, it was possible to show that the three VHHs-whether synthetic or recombinant ones-were properly and similarly folded and recognized the "antigen" HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH-drug conjugates.

Dual Alleviation of Acute and Neuropathic Pain by Fused Opioid Agonist-Neurokinin 1 Antagonist Peptidomimetics.

Herein, the synthesis and biological evaluation of dual opioid agonists-neurokinin 1 receptor (NK1R) antagonists is described. In these multitarget ligands, the two pharmacophores do not overlap, and this allowed maintaining high NK1R affinity and antagonist potency in compounds 12 and 13. Although the fusion of the two ligands resulted in slightly diminished opioid agonism at the µ- and δ-opioid receptors (MOR and DOR, respectively), as compared to the opioid parent peptide, balanced MOR/DOR activities were obtained. Compared to morphine, compounds 12 and 13 produced more potent antinociceptive effects in both acute (tail-flick) and neuropathic pain models (von Frey and cold plate). Similarly to morphine, analgesic tolerance developed after repetitive administration of these compounds. To our delight, compound 12 did not produce cross-tolerance with morphine and high antihyperalgesic and antiallodynic effects could be reinstated after chronic administration of each of the two compounds.

Stabilisation of a short α-helical VIP fragment by side chain to side chain cyclisation: a comparison of common cyclisation motifs by circular dichroism.

A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several conventional cyclisation methods on the α-helical conformation in short peptide fragments. Three of the classical macrocyclisation techniques (i.e. lactamisation, ring-closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity of the triazole-tethered peptide after azide-alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short peptide fragment can differ significantly depending on the chemical strategy used to covalently cross-link side chain residues in a 'helical' fragment.