The Application of Non-Canonical Amino Acids in Drug Discovery

Amino acids, the building blocks of life, are known for their critical roles in virtually all biological processes and play important roles in the agrochemical and pharmaceutical industries. Traditionally, 20 amino acids—referred to as canonical amino acids— (Two additional amino acids are in some species, Selenocysteine and pyrrolysine) are recognized for protein synthesis and are vital in physiological processes, from tissue repair to nutrient absorption. However, a less commonly discussed group, known as non-canonical amino acids (ncAAs), is expanding our understanding of biology and its applications in drug discovery.

Non-canonical amino acids, or unnatural amino acids differ from canonical amino acids as they are not found in natural polypeptide chains that make up proteins in living cells. ncAAs can occur naturally in some plants, bacteria, and marine organisms or can be synthesized chemically. Two major groups of ncAAs exist; ncAAs that are derivatives of the 20 canonical amino acids that can be used as substitutes, and ncAAs that differ from the canonical amino acids. Despite what group they coincide, the distinct chemical structures and properties of ncAAs from their differing side chains and backbones make them an attractive strategy in protein engineering and biotechnological applications.

Currently, ncAAs are used in expanding the chemical diversity of proteins, promoting enhanced activity, and enabling the creation of novel protein structures and functions. This expansion in the genetic code has been shown to be effectual in the pharmaceutical industry, as seen in drug discovery, clinical trials, and biopharmaceutical manufacturing.

Non-Canonical Amino Acids: Mechanism and Importance

The incorporation of ncAAs into proteins involves specialized techniques, often utilizing modified tRNA molecules, engineered ribosomes, and specific codon-recoding strategies. Genetic code expansion is one of the most used methods in incorporating ncAAs into proteins, where the ncAA is inserted at specific positions along the protein during translation, where they are not naturally occurring.

One key mechanism in selectively incorporating non-canonical amino acids in tRNA engineering is using orthogonal translation systems that work independently from the cell’s main translation machinery. This consists of creating an orthogonal pair, with an engineered tRNA and an aminoacyl-tRNA synthetase specific to the ncAA. The orthogonal tRNA, a modified tRNA molecule, is designed to specifically recognize the unique codon in the mRNA sequence that is not used by the natural tRNAs.

A selective orthogonal aminoacyl-tRNA synthetase, a specially engineered enzyme, is also designed to recognize ncAAs and attach them to the modified tRNA molecule. It first forms an aminoacyl-tRNA complex, which pairs the ncAA with the anticodon sequence of the orthogonal tRNA. The mRNA sequence being translated contains a specific codon that matches the orthogonal tRNA’s anticodon and is recoded to now specify the incorporation of the ncAA. The incorporation of the ncAA being carried by the aminoacyl-tRNA complex is initiated when the ribosome recognizes this codon in the mRNA and pairs it with the anticodon of the orthogonal tRNA. A peptide bond is formed between the ncAA from the aminoacyl-tRNA complex and the growing protein chain, resulting in the ncAA being incorporated at a specified position in the protein.

Application of ncAAs in Drug Discovery – Biochemical tools, Medicinal Chemistry, Novel therapeutic targets identified + Challenges

The capabilities and importance of ncAAs are progressively being recognized in biological processes and disease development. They have been shown to play roles in cellular regulation, metabolic pathways, and certain disease pathologies, making them exciting targets for drug discovery. Current research trends involve understanding ncAA functions, biosynthesis, and their roles in health and disease. When incorporated into proteins, ncAAs permit an extensive range of novel functions and can be used as biochemical tools in medicinal chemistry and in drug delivery systems.

Genetic code expansion is beneficial in studying protein function in living cells, where the incorporation of ncAAs into a protein can allow monitorization or manipulation of interactions, translocation, and function modification. This approach can aid in the identification of drug targets, highlight molecular mechanisms, and contribute to the development of more effected and targeted therapeutic strategies. With ncAAs being capable of making modifications to protein function, it is possible for researchers investigating drug targets to examine the impact of these modifications on a protein’s role in cellular processes and disease pathways. The molecular mechanisms and interactions underlying diseases can also be investigated through the introduction of ncAAs at specific interaction sites within a protein. By closely tracking protein-protein interactions, and tagging interaction sites via ncAAs, protein complexes used for understanding signaling pathways and drug targets can be identified. The new functionality and tags introduced into proteins by ncAAs may allow researchers to gain further insights into interactions that might be otherwise challenging to study using traditional methods. In addition, inserting ncAAs into disease-related proteins and understanding how these now modified proteins contribute to disease development and progression allow for the study of their roles in complex biological systems and can be a versatile tool in identifying potential intervention points.

Drug delivery systems incorporating ncAAs are an emerging area of research with the potential to revolutionize targeted drug delivery. These systems take advantage of the unique properties of ncAAs to improve the specificity, stability, and effectiveness of drug delivery. An example includes introducing ncAAs to peptide-based delivery systems to enhance their stability against enzymatic degradation. This can also improve how the drug navigates through the body and prolong its circulation time, leading to better delivery and sustained therapeutic effects. ncAAs can also be integrated into drug delivery systems via targeted ligands, which recognize specific cell surface receptors or biomarkers. Engineering ligands with ncAAs can lead to enhanced ligand-receptor interactions which improve the selectivity of drug delivery to target cells or tissues.

The application of ncAAs in drug discovery is a promising but complex endeavor that comes with its own set of challenges and limitations. One primary concern when using ncAAs is ensuring their biocompatibility and safety. The introduction of a non-natural chemical functionality could lead to unexpected interactions with cellular components, potentially causing toxic effects or unwanted immune responses. There may also be difficulties in the genetic code expansion process, where some ncAAs might not be incorporated as efficiently as desired, leading to incomplete modification of the protein of interest. Developing orthogonal aminoacyl-tRNA synthetases and tRNA pairs to specifically bind to ncAAs can be technically complex as well, requiring high specificity and efficiency while avoiding interference with cellular machinery.

Future of ncAA’s in Drug Discovery

With the emergence of ncAA use, it is anticipated that they will be incorporated in more future drug discovery research. Advances in synthetic biology, protein engineering, and bioinformatics are making it easier to explore the potential of ncAAs. Moreover, the development of artificial intelligence and machine learning tools can facilitate the process of investigating ncAA functionality, and their potential use in drug development, instituting a frontier in drug discovery.

In conclusion, ncAAs make up a progressive set of biochemical tools with the potential to go beyond what is possible with the 20 standard amino acids. Although the use of ncAAs present technical challenges, technological advances and the potential for new drug development suggest that their role in drug discovery will continue to expand. As we advance in our understanding of ncAAs, we can anticipate significant contributions to the field of drug discovery and development.

Tiffany Zhou
Tiffany Zhou
Tiffany Zhou is a dedicated scientific explorer in the field of biology at the University of British Columbia. Her scholarly pursuits are imbued with a profound ardor for scientific inquiry, notably directed towards the domain of cell biology and the intricacies of health sciences. Beyond her academic pursuits, Tiffany derives leisure from harmonious melodies, leisurely strolls in nature, and the art of culinary confectionery.

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