Structure elucidation of a human melanocortin-4 receptor specific orthosteric nanobody agonist.

The melanocortin receptor 4 (MC4R) belongs to the melanocortin receptor family of G-protein coupled receptors and is a key switch in the leptin-melanocortin molecular axis that controls hunger and satiety. Brain-produced hormones such as α-melanocyte-stimulating hormone (agonist) and agouti-related peptide (inverse agonist) regulate the molecular communication of the MC4R axis but are promiscuous for melanocortin receptor subtypes and induce a wide array of biological effects. Here, we use a chimeric construct of conformation-selective, nanobody-based binding domain (a ConfoBody Cb80) and active state-stabilized MC4R-ß2AR hybrid for efficient de novo discovery of a sequence diverse panel of MC4R-specific, potent and full agonistic nanobodies. We solve the active state MC4R structure in complex with the full agonistic nanobody pN162 at 3.4 Å resolution. The structure shows a distinct interaction with pN162 binding deeply in the orthosteric pocket. MC4R peptide agonists, such as the marketed setmelanotide, lack receptor selectivity and show off-target effects. In contrast, the agonistic nanobody is highly specific and hence can be a more suitable agent for anti-obesity therapeutic intervention via MC4R.

TLR ligands and cytokines induce CXCR3 ligands in endothelial cells: enhanced CXCL9 in autoimmune arthritis.

CXC chemokines are potent attractants of neutrophil granulocytes, T cells or natural killer cells. Toll-like receptors (TLR) recognize microbial components and are also activated by endogenous molecules possibly implicated in autoimmune arthritis. In contrast to CXC chemokine ligand 8 (CXCL8), no CXC chemokine receptor 3 (CXCR3) ligand (ie CXCL9, CXCL10 and CXCL11) was induced by bacterial TLR ligands in human microvascular endothelial cells (HMVEC). However, peptidoglycan (PGN), double-stranded (ds) RNA or lipopolysaccharide (LPS) (TLR2, TLR3 or TLR4 ligands, respectively) synergized with interferon-gamma (IFN-gamma) at inducing CXCL9 and CXCL10. In contrast, enhanced CXCL11 secretion was only obtained when IFN-gamma was combined with TLR3 ligand. Furthermore, flagellin, loxoribine and unmethylated CpG oligonucleotide (TLR5, TLR7 and TLR9 ligands, respectively) did not enhance IFN-gamma-dependent CXCR3 ligand production in HMVEC. In analogy with TLR ligands, tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), in combination with IFN-gamma, synergistically induced CXCL9 and CXCL11 in HMVEC and human fibroblasts, two fundamental cell types delineating the joint cavity. Etanercept, a humanized soluble recombinant p75 TNF-receptor/IgG(1)Fc fusionprotein, neutralized synergistic CXCL9 production induced by TNF-alpha plus IFN-gamma, but not synergy between IFN-gamma and the TLR ligands PGN or LPS. Synovial chemokine concentrations exemplify the physiopathological relevance of the observed in vitro chemokine production patterns. In synovial fluids of patients with spondylarthropathies (ie ankylosing spondylitis or psoriatic arthritis) or rheumatoid arthritis, significantly enhanced CXCL9, but not CXCL11 levels, were detected compared to concentrations in synovial fluids of patients with metabolic crystal-induced arthritis. Thus, CXCL9 is an important chemokine in autoimmune arthritis.