Cysteine as a Monothiol Reducing Agent to Prevent Copper-Mediated Oxidation of Interferon Beta During PEGylation by CuAAC

Bioconjugation by copper-catalyzed azide–alkyne cycloaddition (CuAAC) provides a powerful means to produce site-specifically modified proteins. However, the use of a copper catalyst brings about the possible generation of reactive oxygen species that could cause degradation of vulnerable amino acid residues. We investigated whether PEGylation by CuAAC caused any modifications to the therapeutic protein interferon beta-1b, which was produced via global amino acid substitution with azidohomo-alanine at the N-terminus and contains no methionine residues. Using previously reported reaction conditions, LC-MS peptide mapping detected +32 Da and +48 Da oxidation modifications of tryptic peptides 28–33 (LEYCLK) and 137–147 (EYSHCAWTIVR) in the protein post-PEGylation. The oxidative degradation increased with reaction time, whereas reducing the copper concentration slowed the PEGylation rate as well as the oxidation rate. Replacing dithiothreitol (DTT) with any of five different monothiol reducing agents in anaerobic conditions allowed efficient PEGylation in 2–4 h and abrogated oxidative degradation. Free cysteine provided reproducible reaction results as a reducing agent in this system and has been successfully applied to other protein conjugations. Monothiol reducing agents, such as cysteine, may be useful tools as protective reducing agents for CuAAC in some bioconjugation systems

Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody–Drug Conjugates Using Click Cycloaddition Chemistry

Antibody–drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain <i>N</i>6-((2-azidoethoxy)­carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide–alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2<i>H</i>)‑ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound <b>18</b>. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound <b>31.</b> Compound <b>31</b> displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound <b>18</b>. Inhibitor <b>31</b> also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2<i>H</i>)‑ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound <b>18</b>. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound <b>31.</b> Compound <b>31</b> displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound <b>18</b>. Inhibitor <b>31</b> also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes

Correction to Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2<i>H</i>)‑ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

Correction to Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2<i>H</i>)‑ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitor