CHEMICAL BIOLOGY APPROACHES TO STUDY ANTIBODY-DRUG CONJUGATE SYNTHESIS, GENETIC CODE EXPANSION AND MAIN PROTEASE OF SARS-COV-2
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2021-08-18
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Abstract
Over the last two decades, the genetic code expansion technique became an important toolkit to study and bring new functionalities to proteins and peptides. Incorporating non-canonical amino acids that are native structures of post-translational modifications (PTMs) or PTM mimics enables researchers to study its function. One of the main limitations of this technique is the low incorporation rates of non-canonical amino acids into proteins. A way to circumvent this problem is by developing efficient aminoacyl-tRNA synthetase (aaRS) non-canonical amino acid pairs. In chapter 3, studies on introducing mutations to Methanosarcina mazei pyrrolysyl-tRNA synthetase (Mm PylRS) enzyme and studying the mutant enzymes` substrate preference is presented. Our investigation started with testing L-homoarginine (HAr) incorporation using a previously reported mutant PylRS and its cognate tRNAPyl. To better understand the PylRS enzymatic activity, the mutations were introduced step-wise and the effects of these mutations on PylRS substrate preference were analyzed. During these studies, efficient incorporation of ortho-chloro-L-phenylalanine (o-ClF) into a mutant PylRS (o-ClFRS) is serendipitously discovered. O-ClFRS contains three mutations from the PylRS mutant that incorporates HAr. O-ClF incorporation was further studied by crystal structure of o-ClFRS with o-ClF. Next, extending the HAr incorporation system to incorporate methylated derivatives of HAr was aimed. However, the incorporation of HAr derivatives was inefficient due to an uncharacterized modification taking place in vivo.
In chapter 2, a kinetics study on ligand-accelerated copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and its application to synthesize antibody-drug conjugates (ADCs) is presented. ADCs found widespread use due to offering lower side toxicity in cancer treatment. The current bioconjugation approaches have drawbacks such as plasma instability and conjugation efficiency. Researchers look for new conjugation methods, to prepare stable conjugates in a homogenous form. We started studying CuAAC reaction with a kinetic investigation by using fluorogenic dyes. Next, we adopted the CuAAC reaction to synthesize ADCs by using the Pertuzumab antibody. We tested the effectivity of the conjugated ADCs against cancer cell line MDA-MB-453. The synthesized ADCs performed similar to widely used cysteine-maleimide conjugation and showed lower side toxicity. Then, to improve the conjugation process, we developed a methodology to synthesize ADCs on-resin by immobilizing antibodies on Protein A Sepharose resin using copper-chelating azide modified drug molecule.
In chapters 4 and 5, studies on the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are described. The turmoil created by SARS-CoV-2 prompted the study of the main protease (Mpro), one of the key enzymes in life cycle of SARS-CoV-2. The studies began by docking selected FDA/EMA (Food Drug Administration/European Medical Agency) drugs to the active site of Mpro. Next, the promising drugs were purchased and their IC50 values were determined. Then, three of the most promising drugs were tested in live-virus microneutralization assay. Bepridil (an antiginal medicine) showed complete eradication of SARS-CoV-2 replication in low-micromolar concentrations showing promise for animal studies. Next, guided by the known SARS-CoV Mpro inhibitors, 9 aldehyde-based peptide structures were synthesized and their inhibiton against Mpro was studied. Inhibition constants (Ki) at low-nanomolar level were obtained. Compound MPI8 eradicated SARS-CoV-2 replication in A549/ACE2 cells at 0.3 µM concentration. Next, attention was turned to engineering the Mpro enzyme to improve its catalytic activity. An azide containing ncAAwas incorporated into Mpro to achieve this goal. Then, two Mpro units were cross-linked using a di-cyclooctyne linker and locked the enzyme in its active dimer form. This process yielded a more active Mpro derivative that allows measuring inhibition constants at lower enzyme concentrations.
In the final chapter, the findings of the studies presented in this thesis are summarized and the future directions for these studies are discussed.
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copper-catalyzed ligand-assisted azide-alkyne cycloaddition, click chemistry, antibody-drug conjugates, non-canonical amino acid incorporation, SARS-CoV-2 main protease