Developing covalent protein therapeutics using unnatural amino acids

covalent protein therapeutics

Date: 29th June 2020

Covalent drugs have made a huge impact on human health and within the pharmaceutical industry.  However, as they block protein function by forming a specific bond between a ligand and the target protein there has been anxiety concerning their potential for off-target activity and toxicity.  As such they are often overlooked, and remain largely unexplored.   Now scientists report a proximity-enabled reactive therapeutics (PERx) approach to generate covalent protein drugs through genetic code expansion, reducing the potential for off-target effects.

There are currently only around 40 covalent drugs approved by the US Food and Drug Administration (FDA). They include vital drugs such as penicillin and aspirin and, in the main, most were identified by chance and not by design, with their covalent action only unearthed after their clinical use had been well established.

However, whilst scientists have been quick to dismiss them in drug development programs they offer advantages over non-covalent drugs.  They have a long duration of action, have increased biochemical efficiency and potency, can completely inactivate their targets, and provide an opportunity to inhibit intractable targets.

Now a team of scientists from the University of California-San Francisco, US, the Southern Medical University, the Sun Yat-sen University and the Chinese Academy of Sciences, China, have developed a proximity-enabled reactive therapeutics (PERx) strategy to develop covalent protein drugs, by introducing a latent bioreactive unnatural amino acid (Uaa) into the protein, which has latent chemical reactivity and remains inert inside the protein and in vivo.

The team started by genetically encoding the latent bioreactive Uaa, fluorosulfate-L-tyrosine (FSY), into the ectodomain of the human programmed cell death protein-1 (PD-1). PD-1 is a checkpoint protein on T cells and acts as an “off switch” keeping the T cells from attacking other cells in the body. It interacts with its ligand PD-L1, which is often found on cancerous cells, and together they inhibit T-lymphocyte proliferation, release of cytokines, and cytotoxicity.

The blockage of the PD-1/PD-L1 interaction has been a target for treating cancer, and several monoclonal antibodies have been developed. The theory here is that upon binding of PD-1(FSY) to its target PD-L1, the Uaa would be brought into close proximity to a natural residue of PD-L1, this would promote Uaa to react with the natural residue selectively due to proximity-enabled reactivity. By generating a covalent linkage between PD-1 and PD-L1 the team hoped to create an irreversible antagonist.

  • The team showed that PD-1(FSY) covalently bound to its natural ligand PD-L1 in vitro, on the surface of cancer cells, and in tumours in vivo.
  • PD-1(FSY) was able to enhance T cell activation in vitro by blocking PD-1/PD-L1 interaction.
  • Tumour growth in a xenograft mouse model was inhibited by PD-1(FSY).
  • CAR-T cell activation was enhanced by PD-1(FSY) in vitro.
  • PD-1(FSY) enhanced the anti-tumour activity of CAR-T cells in vivo, showing increased cell infiltration and cytokine activity in tumours. Conversely, T cell exhaustion-associated genes were downregulated.
  • PD-1(FSY) inhibited tumour growth in xenografts in a humanised mouse model which had a sustained human immune system. The wild type version of PD-1 did not affect tumour growth.
  • To demonstrate the broad application of PERx, the team converted an affibody (an alternative to immunoglobulin-based binding proteins) to a covalent protein using FSY. The affibody (FSY) covalently bound selectively to its target in vitro and to the surface of cancer cells.

Conclusions and future applications:

The team have shown here that PD-1(FSY) can efficiently and covalently bind to its target ligand and inhibit tumour growth effectively.  Whilst most protein drugs need to be modified to extend their half-life, the irreversible binding affinity of PERx drugs circumvents this requirement, as the covalent binding decouples drug efficacy from pharmacokinetics.

The safety of new drugs is always paramount, PERx minimises off-target effects through its unique mechanism. As both drug-target binding and Uaa-natural residue pairing, has to occur for covalent linking, this affords the system enhanced specificity and target selectivity.

PERx should offer a highly useful, general platform to convert a variety of proteins into covalent binders.  It is hoped that this technology will accelerate therapeutic applications impacting protein therapeutics.  As the PERx can be applied to existing protein drugs and binders such as antibodies, nanobodies, and affibodies it may lead to the development of a new class of protein therapies.


Li, Q., Q. Chen, P. C. Klauser, M. Li, F. Zheng, N. Wang, X. Li, Q. Zhang, X. Fu, Q. Wang, Y. Xu and L. Wang “Developing Covalent Protein Drugs via Proximity-Enabled Reactive Therapeutics.” Cell.