| Author: | Zhang, Dong |
| Title: | Development of zein-based bionanoparticles for highly efficient targeted glioblastoma therapy and investigation of conformational dynamics of SARS-CoV-2 variants RBDs and their interactions with ACE2 by mass spectrometry |
| Advisors: | Yao, Zhongping (ABCT) Wang, Yi (ABCT) |
| Degree: | Ph.D. |
| Year: | 2023 |
| Subject: | Gliomas -- Treatment Brain -- Cancer COVID-19 (Disease) Cell receptors Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xxv, 198 pages : color illustrations |
| Language: | English |
| Abstract: | Dactolisib (Dac) is an effective dual PI3K/mTOR inhibitor for cancer treatment. It was the first PI3K inhibitor to enter clinical trials, which, however, were terminated because of the toxicity of Dac to normal tissues. To apply Dac to cancer therapy while avoiding its toxicity, we developed a new brain-targeting drug delivery system self-assembled from zein, a cell membrane-penetrating amphiphilic protein found in corn. Specifically, the amphiphilicity of zein drives its self-assembly into nanoparticles (NPs) that encapsulate Dac with high efficiency. RVG29, a 29-mer brain-targeting peptide, is chemically conjugated to zein that constitutes the NPs to form Dac-encapsulated NPs (zein-RVG-Dac_NPs). Both zein and RVG29 facilitate the resultant NPs to cross the blood-brain tumor barrier (BBTB) and they are taken up by the glioblastoma (GBM) cells, with RVG29 being more efficient than zein in the BBB permeation. Administration of zein-RVG-Dac_NPs through tail veins significantly increased the accumulation of Dac in the orthotopic brain tumor of mice and effectively inhibited tumor growth. Neither toxicity nor adverse effects in the major organs were found due to the excellent biocompatibility of zein and the targeted delivery of Dac into brain tumor cells. These results showed that integrating a brain-targeting peptide (RVG29) to a cell-penetrating natural protein (zein) could form NPs that could effectively penetrate the blood-brain barriers (BBB) and BBTB and then enter brain tumor cells to release Dac, leading to highly effective targeted brain cancer therapy. The use of such NPs can be extended to the development of therapeutics for treating different brain diseases due to their unique combination of biocompatibility as well as brain-targeting, BBB-crossing and cell-penetrating properties. COVID-19 has been posing serious health threat to and significant impact on the social life and economy globally since its first report in December 2019. The rapid evolution of SARS-CoV-2 to multiple variants with enhanced infectivity and transmissibility has made the combat against the pandemic more challenging. It is thus of great importance to understand how SARS-CoV-2 variants-associated mutations mediated the interaction between the SARS-CoV-2 spike protein, particularly its receptor binding domain (RBD), and the receptor in host cells, angiotensin-converting enzyme 2 (ACE2), which is closely related to the transmission of SARS-CoV-2. Until now, knowledge about this interaction is mainly based on the static structures obtained by Cryogenic electron microscopy (Cryo-EM) or X-ray crystallography, and no systematic study on this interaction has been conducted for various variants of SARS-CoV-2. In this study, RBDs of SARS-CoV-2 wild type (WT) and several major variants, including Alpha, Beta, Zeta, Kappa, Delta and Omicron, as well as their ACE2 complexes were investigated using hydrogen/deuterium exchange mass spectrometry (HDX-MS), a powerful technique for exploring in-solution conformational dynamics of proteins, in an effort to reveal the information that cannot be obtained by Cryo-EM or X-ray crystallography and understand how SARS-CoV-2 mutations affect the conformational dynamics of RBD and its interaction with ACE2. HDX-MS of the unbound RBDs revealed the reduced flexibility of several regions in SARS-CoV-2 RBD variants, especially for those with the mutation N501Y in the key interaction loops, which helped to stabilize the loop (Y485-Y501, L1) at the closed conformation of RBD. In addition, the core of RBD became more rigid as SARS-CoV-2 variants developed. The Kappa variant (L452R/E484Q) presented the higher flexibility for several vital binding interfaces, which emphasized the conformational effects of the mutation L452R. Compared with the Kappa variant, the Delta variant (L452R/T478K) showed a more rigid conformation in another interface loop (residues 444-452), suggesting that the dual mutations of E478K and L452R closely correlated with the increased binding affinity. Upon binding to ACE2, reduced deuterium uptake was observed at two binding interface regions covering residues 495-512 and 464-480. Increased HDX and thus higher flexibility of the sheet within the RBD core (residues 405-419) were observed for the Beta variant containing the mutation at N417K, which would break the existing salt bridge with D30 of ACE2. The most dramatically reduced HDX of L1 upon the binding was observed for the Alpha and Delta RBDs, which supported the higher transmission of the two variants. Moreover, the positively charged mutations Q493R and Q498R of Omicron created more favorable connections with the negatively charged E35 and D38 in ACE2, resulting in a more compact binding interface. The conformational dynamics of ACE2 and the changes upon binding to WT and variant RBDs were investigated as well. Among all the studied RBDs, the Alpha RBD (N501Y) was observed to induce the most dramatically decreased HDX uptake for the region covering residues F28-S43 of ACE2, which was associated with a newly formed π-π stacking between Y501 and Y41 of ACE2, resulting in the more compact interfaces of the RBD-ACE2 complex. Allosteric effects on the loops at the edge of ACE2, especially upon binding to the Beta, Delta or Omicron variants, which were the determinants for viral acceptance, were observed. The binding with the Beta variant induced higher flexibility of the edge loop covering S280-T294. Interestingly, the Delta RBD allosterically led to the more flexible regions at the edge of ACE2, while more compact regions were detected in the Omicron RBD-ACE2 complex. These findings might provide evidence to explain the different performances and clinical symptoms for the patients infected with the Delta or Omicron variants. Overall, this study about the conformational dynamics of WT and variant RBDs and their binding to ACE2 provides valuable information for understanding the evolution of SARS-CoV-2 and improving the design of drugs and vaccines against SARS-CoV-2 variants. |
| Rights: | All rights reserved |
| Access: | open access |
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