Rapid Development of Piperidine Carboxamides as Potent and Selective Anaplastic Lymphoma Kinase Inhibitors

Piperidine carboxamide <b>1</b> was identified as a novel inhibitor of anaplastic lymphoma kinase (ALK enzyme assay IC₅₀ = 0.174 μM) during high throughput screening, with selectivity over the related kinase insulin-like growth factor-1 (IGF1R). The X-ray cocrystal structure of <b>1</b> with the ALK kinase domain revealed an unusual DFG-shifted conformation, allowing access to an extended hydrophobic pocket. Structure–activity relationship (SAR) studies were focused on the rapid parallel optimization of both the right- and left-hand side of the molecule, culminating in molecules with improved potency and selectivity over IGF1R

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