@article {3640, title = {Nanobody derived using a peptide epitope from the spike protein receptor-binding motif inhibits entry of SARS-CoV-2 variants.}, journal = {J Biol Chem}, volume = {299}, year = {2022}, month = {2022 Nov 22}, pages = {102732}, abstract = {

The emergence of new escape mutants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS-CoV variants and other viruses in a short period becomes essential. Blocking SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-angiotensin-converting enzyme 2 interaction has already been exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a nine-amino acid peptide from the receptor-binding motif of the spike protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus-containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, using mCherry fluorescence-based reporter assay, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the N1.2 nanobody. In summary, our study presents a rapid and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. We propose that this approach can also be widely extended to target other viruses and pathogens in the future.

}, issn = {1083-351X}, doi = {10.1016/j.jbc.2022.102732}, author = {Mendon, Nivya and Ganie, Rayees A and Kesarwani, Shubham and Dileep, Drisya and Sasi, Sarika and Lama, Prakash and Chandra, Anchal and Sirajuddin, Minhajuddin} } @article {2290, title = {Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors.}, journal = {PLoS Pathog}, volume = {17}, year = {2021}, month = {2021 07}, pages = {e1009706}, abstract = {

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

}, keywords = {Ammonium Chloride, Angiotensin-Converting Enzyme 2, Animals, Antiviral Agents, Cell Line, Chlorocebus aethiops, Chloroquine, Clathrin, COVID-19, Drug Synergism, Endocytosis, Endosomes, Humans, Hydrogen-Ion Concentration, Hydroxychloroquine, Macrolides, Niclosamide, Protein Binding, Protein Domains, SARS-CoV-2, Spike Glycoprotein, Coronavirus, Vero Cells, Virus Internalization}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1009706}, author = {Prabhakara, Chaitra and Godbole, Rashmi and Sil, Parijat and Jahnavi, Sowmya and Gulzar, Shah-E-Jahan and van Zanten, Thomas S and Sheth, Dhruv and Subhash, Neeraja and Chandra, Anchal and Shivaraj, Akshatha and Panikulam, Patricia and U, Ibrahim and Nuthakki, Vijay Kumar and Puthiyapurayil, Theja Parassini and Ahmed, Riyaz and Najar, Ashaq Hussain and Lingamallu, Sai Manoz and Das, Snigdhadev and Mahajan, Bhagyashri and Vemula, Praveen and Bharate, Sandip B and Singh, Parvinder Pal and Vishwakarma, Ram and Guha, Arjun and Sundaramurthy, Varadharajan and Mayor, Satyajit} } @article {2148, title = {Genetically encoded live-cell sensor for tyrosinated microtubules.}, journal = {J Cell Biol}, volume = {219}, year = {2020}, month = {2020 Oct 05}, abstract = {

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.

}, issn = {1540-8140}, doi = {10.1083/jcb.201912107}, author = {Kesarwani, Shubham and Lama, Prakash and Chandra, Anchal and Reddy, P Purushotam and Jijumon, A S and Bodakuntla, Satish and Rao, Balaji M and Janke, Carsten and Das, Ranabir and Sirajuddin, Minhajuddin} }