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

Oxopyrido[2,3‑<i>d</i>]pyrimidines as Covalent L858R/T790M Mutant Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors

In nonsmall cell lung cancer (NSCLC), the threonine⁷⁹⁰–methionine⁷⁹⁰ (T790M) point mutation of EGFR kinase is one of the leading causes of acquired resistance to the first generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Herein, we describe the optimization of a series of 7-oxopyrido­[2,3-<i>d</i>]­pyrimidinyl-derived irreversible inhibitors of EGFR kinase. This led to the discovery of compound <b>24</b> which potently inhibits gefitinib-resistant EGFR^L858R,T790M with 100-fold selectivity over wild-type EGFR. Compound <b>24</b> displays strong antiproliferative activity against the H1975 nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and promising antitumor activity in an EGFR^L858R,T790M driven H1975 xenograft model sparing the side effects associated with the inhibition of wild-type EGFR

Structure-Based Design of a Novel Series of Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3-Kinases

A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor <b>5</b>, a structure-based approach was used to improve potency and selectivity, resulting in the identification of <b>54</b> as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound <b>54</b> demonstrated a robust PD–PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway