Researchers Discovered Water-Soluble Zolinium Selectively Modifying Lysine Residues of Peptides and Proteins

Lysine has a high abundance (5.9%) in the human proteome and is involved in important physiological functions. Site-selective modification of lysine residues of target proteins under physiological conditions has broad applications in the field of pharmacy. Usually, the ε-amino group of lysine is easily protonated under physiological conditions, so it is very challenging to covalently modify lysine residues of target proteins under physiological conditions. Generally, the reported lysine covalent modifiers have poor water solubility and high reactivity, leading to some possible problems such as selectivity and toxicity. So far, no rationally designed drug covalently targeting lysine residues has been marketed. Therefore, there are actual requirements to develop lysine modifiers with good selectivity, water solubility and low toxicity to meet the needs of various biomedical applications.
In a study published in J. Med. Chem. on August 23, 2022, LI Bo and ZHU Weiliang from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, together with Professor GE Guangbo from Shanghai University of Traditional Chinese Medicine, designed and developed a new type of lysine modifiers, called "Zolinium", based on their 10-year optimization results from the natural product Berberine. 
The new water-soluble "Zolinium" could undergo the selective modification of lysine residues of peptides and proteins in a physiological environment without adding any catalyst. The mechanism of this chemo-selective modification was explored by quantum chemistry calculation. Further applications to mapping the lysine in living cells were implemented.
This study showed that the new water-soluble zolinium was capable of selectively modifying the lysine residues of the peptidic drugs such as pramorelin derivatives, octreotide and semaglutide, as well as bovine serum albumin K350 and two highly conserved lysine residues (K5 and K61) of SARS-CoV-2 3CLpro, and the SARS-CoV-2 3CLpro was inactivated by the covalent modification of lysine. At the cellular level, the zolinium can also be used to map lysine-modified proteins in living cells, and fluorescent molecules with zolinium warheads can image subcellular organelles such as lysosomes. Protein pooling experiments suggested that site-selective modifications occur primarily in hydrophobic or catalytically active pockets and may be enhanced by the neighboring acidic amino acid residues. 
In addition, modification experiments with mixtures of 8 different amino acids and different peptides with potentially reactive residues showed that the novel water-soluble zolinium exhibited good selectivity for the lysine quinolinylation of peptides and proteins, and was highly selective and well tolerated for various functional groups. 
Quantum chemistry calculation results showed that, compared with other reactive groups on various amino acid residues, the reaction of ε-amino group of lysine with zolinium had the lowest free energy barrier. Since the quinoline ring of zolinium can be further modified to obtain various derivatives, it is expected to play an important role in the research of peptides and proteins for specific purposes and protein-lysine modification at living cells.
This new type of water-soluble zolinium developed in this study can chemically and site-selectively modify lysine in peptides, proteins and living cell proteins, which should have potential application value in chemical biology and innovative drug research.

Water-soluble "zolinium" modifies lysine residues of peptides and proteins and its applications at the cellular level (Image by the authors of the JMC paper)

DIAO Wentong
Shanghai Institute of Materia Medica