| Author: | Tam, Suet Ying |
| Title: | The utilization of amino acid-depleting enzymes |
| Advisors: | Leung Y. C. Thomas (ABCT) |
| Degree: | Ph.D. |
| Year: | 2023 |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | 156 pages : color illustrations |
| Language: | English |
| Abstract: | Throughout the past few decades, amino acid-depleting enzymes have drawn lots of attention in various applications. Herein, we further bioengineered arginine deiminase (ADI) as a biosensor to rapidly detect a wide range of arginine (L-Arg) concentration levels and genetically engineered Erwinia chrysanthemi asparaginase to fuse with an albumin-binding domain (ABD) to unleash its potential for the treatment of a solid tumor. ADI was rationally mutated to become a "turn-off" fluorescent biosensor that can rapidly determine the concentration of L-Arg. Two-point mutations (Cys251→Ser251 and Thr265→Cys265) were introduced in the recombinant ADI, resulting in only one cysteine residue located on the mutated ADI for the site-directed attachment of a fluorophore (fluorescein-5-maleimide). The fluorescein-labeled form of ADI (265Cf) displayed similar catalytic efficiency when compared to the native form of ADI and the mutated form of ADI (265C). When binding to L-Arg, 265Cf showed significant structural conformational changes that induced the fluorescent moiety to move closer to Trp 264, leading to a significant quenching of the fluorescence intensity. The duration of fluorescence quenching was linearly proportional to L-Arg concentration levels, ranging from 12 to 100 µM with R² = 0.9988. Also, the assay time was less than 4 min. The limit of detection of 265Cf was 4 µM. 256Cf was able to detect the concentration of L-Arg in fetal bovine serum by using the standard addition method without the need for sample pre-treatment. The results were in alignment with the mass spectrometry. Therefore, our 256Cf biosensor has great potential to achieve rapid detection of L-Arg in biological and clinical samples. Asparaginase is highly effective in treating acute lymphoblastic leukemia (ALL) by depleting an essential amino acid, asparagine, from leukemia cells. Most leukemia cells are auxotrophic to asparagine due to the lack of or low protein expression of asparagine synthetase (ASNS), rendering them sensitive to asparagine depletion. The deprivation of asparagine in leukemia cells can significantly inhibit their cell growth and eventually lead to cell death. However, almost all solid tumors are resistant to asparagine depletion due to their expression of ASNS. Unlike asparagine depletion, glutamine depletion demonstrated significant anti-cancer effects on multiple solid tumors. Meanwhile, it has been reported that asparagine could promote cancer cell growth as well as protect cancer cells from death under glutamine deficiency. Therefore, we utilized dual enzymatic activities of Erwinia chrysanthemi asparaginase, which has been approved by the US Food and Drug Administration as a second-line treatment for ALL, for further optimization to achieve double amino acid depletions for treating solid tumors. We rationally fused this asparaginase with an albumin-binding domain (ABD) via a soft linker (GGGGS)5 to become ABD-ErS5. It demonstrated anti-cancer effects by inducing cell death in BGC-823 and MKN-45 gastric cancer cell lines through multiple pathways, including S-phase arrest, autophagy and apoptosis. The ABD allowed ABD-ErS5 to bind with serum albumin, and to simultaneously deplete asparagine for ~15 days and glutamine for ~11 days. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed the tumor growth in an ASNS- and glutamine synthetase (GS-) positive MKN-45 gastric cancer xenograft model, demonstrating a novel approach for the treatment of gastric cancer by double amino acid depletions. More importantly, ABD-ErS5 could remain active for a period even anti-ABD-ErS5 antibodies were present in a multiple administration analysis, suggesting the ABD part could bind to serum albumin and thus protect ABD-ErS5 from the attack of antibodies. Moreover, multiple administrations of ABD-ErS5 imposed no noticeable histopathological abnormalities on key organs, indicating the absence of acute toxicity to the animals. It is suggested that our ABD-ErS5 is a promising drug candidate for the treatment of gastric cancer. |
| Rights: | All rights reserved |
| Access: | open access |
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