Discovery of Melanocortin Ligands via a Double Simultaneous Substitution Strategy Based on the Ac-His‑dPhe-Arg-Trp-NH₂ Template

The melanocortin system regulates an array of diverse physiological functions including pigmentation, feeding behavior, energy homeostasis, cardiovascular regulation, sexual function, and steroidogenesis. Endogenous melanocortin agonist ligands all possess the minimal messaging tetrapeptide sequence His-Phe-Arg-Trp. Based on this endogenous sequence, the Ac-His¹-dPhe²-Arg³-Trp⁴-NH₂ tetrapeptide has previously been shown to be a useful scaffold when utilizing traditional positional scanning approaches to modify activity at the various melanocortin receptors (MC1–5R). The study reported herein was undertaken to evaluate a double simultaneous substitution strategy as an approach to further diversify the Ac-His¹-dPhe²-Arg³-Trp⁴-NH₂ tetrapeptide with concurrent introduction of natural and unnatural amino acids at positions 1, 2, or 4, as well as an octanoyl residue at the N-terminus. The designed library includes the following combinations: (A) double simultaneous substitution at capping group position (Ac) together with position 1, 2, or 4, (B) double simultaneous substitution at positions 1 and 2, (C) double simultaneous substitution at positions 1 and 4, and (D) double simultaneous substitution at positions 2 and 4. Several lead ligands with unique pharmacologies were discovered in the current study including antagonists targeting the neuronal mMC3R with minimal agonist activity and ligands with selective profiles for the various melanocortin subtypes. The results suggest that the double simultaneous substitution strategy is a suitable approach in altering melanocortin receptor potency or selectivity or converting agonists into antagonists and vice versa

Engineering Antibody Reactivity for Efficient Derivatization to Generate Na_V1.7 Inhibitory GpTx‑1 Peptide–Antibody Conjugates

The voltage-gated sodium channel Na_V1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional Na_V1.7 targeting antibodies are not known. In this report, we describe Na_V1.7 inhibitory peptide–antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified Na_V1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the Na_V1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked Na_V1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to <i>in vivo</i> half-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not Na_V1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit Na_V1.7 in engineered and neuronal cells