An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif.

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.

Optimization of co-agonism at GLP-1 and glucagon receptors to safely maximize weight reduction in DIO-rodents.

The ratio of GLP-1/glucagon receptor (GLP1R/GCGR) co-agonism that achieves maximal weight loss without evidence of hyperglycemia was determined in diet-induced obese (DIO) mice chronically treated with GLP1R/GCGR co-agonist peptides differing in their relative receptor agonism. Using glucagon-based peptides, a spectrum of receptor selectivity was achieved by a combination of selective incorporation of GLP-1 sequences, C-terminal modification, backbone lactam stapling to stabilize helical structure, and unnatural amino acid substitutions at the N-terminal dipeptide. In addition to α-amino-isobutyric acid (Aib) substitution at position two, we show that α,α'-dimethyl imidazole acetic acid (Dmia) can serve as a potent replacement for the highly conserved histidine at position one. Selective site-specific pegylation was used to further minimize enzymatic degradation and provide uniform, extended in vivo duration of action. Maximal weight loss devoid of any sign of hyperglycemia was achieved with a co-agonist comparably balanced for in vitro potency at murine GLP1R and GCGR. This peptide exhibited superior weight loss and glucose lowering compared to a structurally matched pure GLP1R agonist, and to co-agonists of relatively reduced GCGR tone. Any further enhancement of the relative GCGR agonist potency yielded increased weight loss but at the expense of elevated blood glucose. We conclude that GCGR agonism concomitant with GLP1R agonism constitutes a promising approach to treatment of the metabolic syndrome. However, the relative ratio of GLP1R/GCGR co-agonism needs to be carefully chosen for each species to maximize weight loss efficacy and minimize hyperglycemia.

Functional association of the N-terminal residues with the central region in glucagon-related peptides.

GLP-1 is an incretin peptide involved in the regulation of glucose metabolism and the glucose-dependent stimulation of insulin secretion. Ex-4 is a paralog of GLP-1 that has comparable GLP-1R potency but extended physiological action. GLP-1 and Ex-4 are helical peptides that share ∼50% sequence homology but differ at several residues, notably the second amino acid which controls susceptibility to DPP-IV cleavage. This single amino acid difference yields divergent receptor potency when studied in the context of the two hormone sequences. Ex-4 uniquely tolerates Gly2 through select amino acid differences in the middle region of the peptide that are absent in GLP-1. We report that substitution of Ex-4 amino acids Glu16, Leu21, and Glu24 to the GLP-1 sequence enabled Gly2 tolerance. The coordination of the N-terminus with these central residues shows an interaction of substantial importance not only to DPP-IV stability but also to receptor activation. Extension of this observation to glucagon-based co-agonist peptides showed different structural requirements for effective communication between the N-terminus and the mid-section of these peptides in achieving high potency agonism at the GLP-1 and GCGRs.

A new glucagon and GLP-1 co-agonist eliminates obesity in rodents.

We report the efficacy of a new peptide with agonism at the glucagon and GLP-1 receptors that has potent, sustained satiation-inducing and lipolytic effects. Selective chemical modification to glucagon resulted in a loss of specificity, with minimal change to inherent activity. The structural basis for the co-agonism appears to be a combination of local positional interactions and a change in secondary structure. Two co-agonist peptides differing from each other only in their level of glucagon receptor agonism were studied in rodent obesity models. Administration of PEGylated peptides once per week normalized adiposity and glucose tolerance in diet-induced obese mice. Reduction of body weight was achieved by a loss of body fat resulting from decreased food intake and increased energy expenditure. These preclinical studies indicate that when full GLP-1 agonism is augmented with an appropriate degree of glucagon receptor activation, body fat reduction can be substantially enhanced without any overt adverse effects.