Developing Covalent Protein Drugs via Proximity-Enabled Reactive Therapeutics

Cell. 2020 Jul 9;182(1):85-97.e16. doi: 10.1016/j.cell.2020.05.028. Epub 2020 Jun 23.


Small molecule covalent drugs provide desirable therapeutic properties over noncovalent ones for treating challenging diseases. The potential of covalent protein drugs, however, remains unexplored due to protein's inability to bind targets covalently. We report a proximity-enabled reactive therapeutics (PERx) approach to generate covalent protein drugs. Through genetic code expansion, a latent bioreactive amino acid fluorosulfate-L-tyrosine (FSY) was incorporated into human programmed cell death protein-1 (PD-1). Only when PD-1 interacts with PD-L1 did the FSY react with a proximal histidine of PD-L1 selectively, enabling irreversible binding of PD-1 to only PD-L1 in vitro and in vivo. When administrated in immune-humanized mice, the covalent PD-1(FSY) exhibited strikingly more potent antitumor effect over the noncovalent wild-type PD-1, attaining therapeutic efficacy equivalent or superior to anti-PD-L1 antibody. PERx should provide a general platform technology for converting various interacting proteins into covalent binders, achieving specific covalent protein targeting for biological studies and therapeutic capability unattainable with conventional noncovalent protein drugs.

Keywords: Genetic code expansion; PERx; SuFEx; bioreactive amino acid; cancer immunotherapy; click chemistry; covalent protein drug; immuno-checkpoint; proximity-enabled reactive therapeutics; proximity-enabled reactivity; sulfur fluoride exchange; unnatural amino acid.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antineoplastic Agents / metabolism
  • B7-H1 Antigen / chemistry
  • B7-H1 Antigen / metabolism
  • Cell Membrane / metabolism
  • Cell Proliferation
  • Dendritic Cells / metabolism
  • Humans
  • Kinetics
  • Ligands
  • Lymphocyte Activation / immunology
  • Mice
  • Monocytes / metabolism
  • Pharmaceutical Preparations / metabolism*
  • Phenotype
  • Proteins / chemistry
  • Proteins / therapeutic use*
  • Receptors, Chimeric Antigen / metabolism
  • T-Lymphocytes / cytology
  • T-Lymphocytes / immunology
  • Xenograft Model Antitumor Assays


  • Antineoplastic Agents
  • B7-H1 Antigen
  • Ligands
  • Pharmaceutical Preparations
  • Proteins
  • Receptors, Chimeric Antigen