What you need to know about peptide modifications - Fatty Acid Conjugation

Fatty acids are a family of carboxylic acids with long aliphatic chains, which are conjugated to the N-terminus, or to the side-chain of a lysine in peptides. Meanwhile, cysteine residues can also be modified by fatty acids to produce the corresponding thioester derivatives. Fatty acids modified peptides can use for a number of different applications, such as increasing antimicrobial activity or prolonging half-life. The commonly used fatty acids are Caprylic acid (C8, also called Octanoic acid), Capric acid (C10, as known as Decanoic acid), Lauric acid (C12), Myristic acid (C14), Palmitic acid (C16) or Stearic acid (C18) etc.

The structure of commonly used fatty acids

Applications of fatty acid conjugation peptides

l  Increasing antimicrobial activity

Antimicrobial peptides (AMPs), also known as host defense peptides, have the ability to kill microbial pathogens directly or indirectly. AMPs becomes the promising antimicrobial alternatives for infection treatment. In spite of large number of AMPs going through clinical development, some bottlenecks still impede the further development of AMPs, for instance, low antimicrobial activity, instability under physiological conditions. Inducing fatty acids into AMPs significantly increases the antimicrobial activity of AMPs.

Chu-Kung et al. found that fatty acid conjugation may improve the usefulness of peptides as antimicrobial agents by enhancing their ability to form secondary structures upon interacting with the bacterial membranes. Two peptides, YGAA [KKAAKAA] 2 (AKK) and KLFKRHLKWKII (SC4) were conjugated with lauric acid and showed increased antimicrobial activity relative to unconjugated peptides. The antimicrobial activities of the analogues increased in a fatty acid chain length dependent manner but decreased when the minimal active concentration was above the critical micelle concentration (CMC) of the peptide.

l  Prolonging the half-life of therapeutic peptides

Many studies have shown that fatty acid conjugation is used to delay the absorption rate, extend the stability of therapeutic peptides and protect against proteolysis. Liraglutide is a GLP-1 analog with a 97% homology to endogenous human GLP-1. It has two modifications, a substitution of lysine to arginine at position 34 and attachment of a C16 fatty acid (palmitic acid) at position 26. The attached fatty acid moiety fosters binding to serum albumin, delaying renal excretion and extending the half-life approximately 13 h.

l  Improving peptide-mediated nucleic acid delivery

Cell-penetrating peptides (CPPs) are short amino acid sequences characterized by crossing the cell membrane and transporting a wide range of molecules, including DNA/RNA, peptides, nanoparticles and even large active proteins. In 2001, Futaki and colleagues firstly successfully introduced fatty acids into CPPs. Modifying CPPs with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. Introducing stearic acid at the N-terminus of CPP increased transfection efficiency by approximately 100 times, while lauryl and cholesteryl groups showed modest increase in COS-7 cells.

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Reference

1. Chu-Kung, Alexander F., et al. "Promotion of peptide antimicrobial activity by fatty acid conjugation." Bioconjugate chemistry 15.3 (2004): 530-535.

2. Li, Ying, et al. "Variant fatty acid-like molecules Conjugation, novel approaches for extending the stability of therapeutic peptides." Scientific reports 5.1 (2015): 1-9.

3. Reinhardt, Andre, and Ines Neundorf. "Design and application of antimicrobial peptide conjugates." International journal of molecular sciences 17.5 (2016): 701.

4. Zhong, Chao, et al. "Antimicrobial peptides conjugated with fatty acids on the side chain of D-amino acid promises antimicrobial potency against multidrug-resistant bacteria." European Journal of Pharmaceutical Sciences 141 (2020): 105123.

5. Aronson, J. K. "A worldwide yearly survey of new data in adverse drug reactions. Vol. 33." (2011): 669-690.

6. Lear, Sam, Zaid Amso, and Weijun Shen. "Engineering PEG-fatty acid stapled, long-acting peptide agonists for G protein-coupled receptors." Methods in enzymology 622 (2019): 183-200.

7. Shukla, Arun K. Chemical and Synthetic Biology Approaches to Understand Cellular Functions. Academic Press, 2019.

8. Knudsen, Lotte Bjerre, and Jesper Lau. "The discovery and development of liraglutide and semaglutide." Frontiers in Endocrinology 10 (2019).

9. Lehto, Tõnis, et al. "Saturated fatty acid analogues of cell-penetrating peptide PepFect14: role of fatty acid modification in complexation and delivery of splice-correcting oligonucleotides." Bioconjugate chemistry 28.3 (2017): 782-792.

10. Futaki, Shiroh, et al. "Stearylated arginine-rich peptides: a new class of transfection systems." Bioconjugate chemistry 12.6 (2001): 1005-1011.

11. Lehto, Tõnis. Characterization of nucleic acid delivery with fatty acid modified cell-penetrating peptide nanoparticle formulations. Diss. Department of Biochemistry and Biophysics, Stockholm University, 2018.