Development and rational design of enzymes for enhanced performance

Abstract

L-arginine (L-Arg) depletion for the treatment of cancer has attracted great attention in the world. Although three types of arginine depleting enzymes, PEGylated arginine deiminase (ADI-PEG20) and PEGylated human arginase I (BCT-100 and Co-hArgI-KPEG-5K) are undergoing clinical trials, these kinds of random site PEGylation technology fail to produce a homogenous drug. In addition, low enzyme kinetic affinity of BCT-100 resulted in poor drug efficacy for treating various kinds of cancer while high enzyme kinetic affinity of Co-hArgI-KPEG-5K displayed a strong side effect on body weight loss resulting in mouse mortality. In order to synthesize a more defined, potent and less toxic arginine depleting drugs, the molecular cloning, expression, purification, chemical modification, drug characterization and anti-cancer properties of human arginase I and thermostable Bacillus caldovelox (B. caldovelox) arginase were explored in this thesis. Human arginase I mutant (Cys168 and 303 → Ser168 and 303: HAI) and B. caldovelox arginase mutant (Ser161 → Cys161: BCA-M) without the 6xHis-tag were highly expressed in E. coli by adding 0.06% lactose and purified by heat treatment and ion-exchange chromatography. Also, divalent metal ion screening (co-factor of arginase enzymes) was applied on both HAI and BCA-M so as to produce a better drug formulation. Our results suggested that the trends of catalytic efficiency (kcat/KM) and the drug potency (IC50) of HAI and BCA-M were Co2+>Ni2+>>Mn2+ and Ni2+>Co2+>>Mn2+, respectively. Even though the catalytic efficiency and IC50 value of Ni2+ enriched BCA-M were improved about 7-fold and 11-fold, respectively compared to wild-type BCA, the anti-cancer ability of it was far from satisfactory. Further protein structural engineering of wild-type BCA was conducted. Our results demonstrated that double mutations of wild-type BCA (Val20 → Pro20 and Ser161 →Cys161: V20P) combined with Ni2+ ion substitution could significantly improve the catalytic efficiency and IC50 value by approximately 19-fold and 146-fold, respectively in comparison with wild-type BCA. Furthermore, we have successfully produced a site-specific PEGylated HAI (HAI-PEG20L/20B/40B) and PEGylated BCA-M (BCA-M-PEG20L) by covalently conjugating the Cys45 and Cys161 residues, respectively to the 20L (linear), 20B (branched) and 40B (branched) kDa PEG-maleimide (polyethylene glycol with maleimide functional group). PEGylation on the specific residue was beneficial for minimizing the decrease in activity and producing a homogeneous product. Additionally, the results of enzyme kinetic studies and circular dichroism (CD) spectrometry demonstrated that the 20 or 40 kDa PEG attached on the HAI surface did not affect the enzyme activity and the protein secondary structure. Moreover, the new drugs of Co-HAI-PEG20L/20B/40B were successfully lyophilized into powder forms which could retain the enzyme catalytic activity up to at least 10 months when stored at 4 °C. When tested in mice, the combination of site-specific PEGylation and divalent metal ion substitution, Co-HAI-PEG20L and Ni-HAI-PEG20L, could sustain long-lasting arginine depletion for approximately 7 days after one injection and the body weights of mice returned to normal levels within five days after injection, suggesting that these drugs were very well tolerated. Therefore, both the new arginine-depleting drugs Ni-HAI-PEG20L and Co-HAI-PEG20L may act as promising candidates for cancer therapy.