Most downloaded biology preprints, all time
in category biochemistry
4,451 results found. For more information, click each entry to expand.
24,747 downloads bioRxiv biochemistry
Zhenming Jin, Xiaoyu Du, Yechun Xu, Yongqiang Deng, Meiqin Liu, Yao Zhao, Bing Zhang, Xiaofeng Li, Leike Zhang, Chao Peng, Yinkai Duan, Jing Yu, Lin Wang, Kailin Yang, Fengjiang Liu, Rendi Jiang, Xinglou Yang, Tian You, Xiaoce Liu, Xiuna Yang, Fang Bai, Hong Liu, Xiang Liu, Luke W. Guddat, Wenqing Xu, Gengfu Xiao, Chengfeng Qin, Zhengli Shi, Ruotian Jiang, Zihe Rao, Haitao Yang
A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan–. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus,. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 μM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available. : #ref-1 : #ref-4 : #ref-5 : #ref-6
19,249 downloads bioRxiv biochemistry
Michael Schoof, Bryan Faust, Reuben A. Saunders, Smriti Sangwan, Veronica V Rezelj, Nicholas Hoppe, Morgane Boone, Christian Billesbolle, Cristina Puchades, Caleigh M. Azumaya, Huong T. Kratochvil, Marcell Zimanyi, Ishan Deshpande, Jiahao Liang, Sasha Dickinson, Henry C. Nguyen, Cynthia M Chio, Gregory E. Merz, Michael C Thompson, Devan Diwanji, Kaitlin Schaefer, Aditya A. Anand, Niv Dobzinski, Beth Shoshana Zha, Camille R. Simoneau, Kristoffer E Leon, Kris M White, Un Seng Chio, Meghna Gupta, Mingliang Jin, Fei Li, Yanxin Liu, Kaihua Zhang, David Bulkley, Ming Sun, Amber M Smith, Alexandrea N. Rizo, Frank R. Moss, Axel F. Brilot, Sergei Pourmal, Raphael Trenker, Thomas Pospiech, Sayan Gupta, Benjamin Barsi-Rhyne, Vladislav Belyy, Andrew W. Barile-Hill, Silke Nock, Yuwei Liu, Nevan J. Krogan, Corie Y. Ralston, Danielle L Swaney, Adolfo Garcia-Sastre, Melanie Ott, Marco Vignuzzi, QCRG Structural Biology Consortium, Peter Walter, Aashish Manglik
Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century. ### Competing Interest Statement M.Schoof, B.Faust, R.Saunders, N.Hoppe, P.Walter, and A.Manglik are inventors on a provisional patent describing anti-Spike nanobodies described in this manuscript.
15,682 downloads bioRxiv biochemistry
SARS-CoV-2 is continually evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2 resulted in the more contagious mutations, S477N, E484K, and N501Y, to be among the first selected, explaining the convergent evolution of the European (20E-EU1), British (501.V1), South African (501.V2), and Brazilian variants (501.V3). Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R epistatic to N501Y. Nevertheless, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9A Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.
14,857 downloads bioRxiv biochemistry
The recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
13,731 downloads bioRxiv biochemistry
Angiotensin-converting enzyme 2 (ACE2) is the surface receptor for SARS coronavirus (SARS-CoV) through interaction with its spike glycoprotein (S protein). ACE2 is also suggested to be the receptor for the new coronavirus (2019-nCoV), which is causing a serious epidemic in China manifested with severe respiratory syndrome. BAT1 (SLC6A19) is a neutral amino acid transporter whose surface expression in intestinal cells requires ACE2. Here we present the 2.9 Å resolution cryo-EM structure of full-length human ACE2 in complex with BAT1. The complex, assembled as a dimer of ACE2-BAT1 heterodimers, exhibits open and closed conformations due to the shifts of the peptidase domains of ACE2. A newly resolved Collectrin-like domain (CLD) on ACE2 mediates homo-dimerization. The extended TM7 in each BAT1 clamps CLD of ACE2. Structural analysis suggests that the ACE2-BAT1 complex can bind two S proteins simultaneously, providing important clues to the molecular basis for coronavirus recognition and infection.
13,542 downloads bioRxiv biochemistry
In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed in Wuhan, China. Due to international travel and human-to-human transmission, the virus spread rapidly inside and outside of China. Currently, there is no effective antiviral treatment for coronavirus disease 2019 (COVID-19); therefore, research efforts are focused on the rapid development of vaccines and antiviral drugs. The SARS-CoV-2 main protease constitutes one of the most attractive antiviral drug targets. To address this emerging problem, we have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases, using natural and a large panel of unnatural amino acids. On the basis of these findings, we designed and synthesized an inhibitor and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. Using this approach we visualized SARS-CoV-2 active Mpro within nasopharyngeal epithelial cells of a patient with active COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents or diagnostic tests. ### Competing Interest Statement Wroclaw University of Science and Technology has filed a patent application covering compounds: Ac-Abu-Tle-Leu-Gln-VS, Biotin-PEG(4)-Abu-Tle-Leu-Gln-VS and Cy5-PEG(4)-Abu-Tle-Leu-Gln-VS as well as related compounds with W.R. and M.D. as inventors.
11,868 downloads bioRxiv biochemistry
Many pathogens take advantage of the dependence of the host on the interaction of hundreds of extracellular proteins with the glycosaminoglycans heparan sulphate to regulate homeostasis and use heparan sulphate as a means to adhere and gain access to cells. Moreover, mucosal epithelia such as that of the respiratory tract are protected by a layer of mucin polysaccharides, which are usually sulphated. Consequently, the polydisperse, natural products of heparan sulphate and the allied polysaccharide, heparin have been found to be involved and prevent infection by a range of viruses including S-associated coronavirus strain HSR1. Here we use surface plasmon resonance and circular dichroism to measure the interaction between the SARS-CoV- 2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) and heparin. The data demonstrate an interaction between the recombinant surface receptor binding domain and the polysaccharide. This has implications for the rapid development of a first-line therapeutic by repurposing heparin and for next-generation, tailor-made, GAG-based antivirals. ### Competing Interest Statement The authors have declared no competing interest.
10,471 downloads bioRxiv biochemistry
Yan Gao, Liming Yan, Yucen Huang, Fengjiang Liu, Yao Zhao, Lin Cao, Tao Wang, Qianqian Sun, Zhenhua Ming, Lianqi Zhang, Ji Ge, Litao Zheng, Ying Zhang, Haofeng Wang, Yan Zhu, Chen Zhu, Tianyu Hu, Tian Hua, Bing Zhang, Xiuna Yang, Jun Li, Haitao Yang, Zhijie Liu, Wenqing Xu, Luke W. Guddat, Quan Wang, Zhiyong Lou, Zihe Rao
A novel coronavirus (2019-nCoV) outbreak has caused a global pandemic resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp, also named nsp12), which catalyzes the synthesis of viral RNA, is a key component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. Here we report the cryo-EM structure of 2019-nCoV full-length nsp12 in complex with cofactors nsp7 and nsp8 at a resolution of 2.9-Å. Additional to the conserved architecture of the polymerase core of the viral polymerase family and a nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain featured in coronaviral RdRp, nsp12 possesses a newly identified β-hairpin domain at its N-terminal. Key residues for viral replication and transcription are observed. A comparative analysis to show how remdesivir binds to this polymerase is also provided. This structure provides insight into the central component of coronaviral replication/transcription machinery and sheds light on the design of new antiviral therapeutics targeting viral RdRp. One Sentence Summary Structure of 2019-nCov RNA polymerase. ### Competing Interest Statement
10,362 downloads bioRxiv biochemistry
The rapid and escalating spread of SARS coronavirus 2 (SARS-CoV-2) poses an immediate public health emergency. The viral spike protein S binds ACE2 on host cells to initiate molecular events that release the viral genome intracellularly. Soluble ACE2 inhibits entry of both SARS and SARS-2 coronaviruses by acting as a decoy for S binding sites, and is a candidate for therapeutic, prophylactic and diagnostic development. Using deep mutagenesis, variants of ACE2 are identified with increased binding to the receptor binding domain of S. Mutations are found across the interface, in the N90-glycosylation motif, and at buried sites where they are predicted to enhance local folding and presentation of the interaction epitope. When single substitutions are combined, large increases in binding can be achieved. The mutational landscape offers a blueprint for engineering high affinity proteins and peptides that block receptor binding sites on S to meet this unprecedented challenge. ### Competing Interest Statement E.P. is the inventor on a provisional patent filing by the University of Illinois claiming mutations in ACE2 described here that enhance binding to S. E.P. is a cofounder of Orthogonal Biologics Inc, which has a license from the University of Illinois.
9,920 downloads bioRxiv biochemistry
In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning. ### Competing Interest Statement F.E.O. declares competing financial interests as co-founders and shareholder of UbiQ Bio BV. M.B. is an employee and shareholder of Arvinas, Inc. The remaining authors declare no competing interests.
8,586 downloads bioRxiv biochemistry
Angiotensin-converting enzyme 2 (ACE2) has been suggested to be the cellular receptor for the new coronavirus (2019-nCoV) that is causing the coronavirus disease 2019 (COVID-19). Like other coronaviruses such as the SARS-CoV, the 2019-nCoV uses the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) to engage ACE2. We most recently determined the structure of the full-length human ACE2 in complex with a neutral amino acid transporter BAT1. Here we report the cryo-EM structure of the full-length human ACE2 bound to the RBD of the 2019-nCoV at an overall resolution of 2.9 Å in the presence of BAT1. The local resolution at the ACE2-RBD interface is 3.5 Å, allowing analysis of the detailed interactions between the RBD and the receptor. Similar to that for the SARS-CoV, the RBD of the 2019-nCoV is recognized by the extracellular peptidase domain (PD) of ACE2 mainly through polar residues. Pairwise comparison reveals a number of variations that may determine the different affinities between ACE2 and the RBDs from these two related viruses.
8,576 downloads bioRxiv biochemistry
Courtney J. Mycroft-West, Dunhao Su, Isabel Pagani, Timothy R. Rudd, Stefano Elli, Scott Guimond, Gavin Miller, Maria C. Z. Meneghetti, Helena B. Nader, Yong Li, Quentin Nunes, Patricia Procter, Nicasio Mancini, Massimo Clementi, Antonella Bisio, Nicholas R. Forsyth, Jeremy E. Turnbull, Marco Guerrini, David Fernig, Elisa Vicenzi, Edwin A Yates, Marcelo Lima, Mark Andrew Skidmore
The dependence of the host on the interaction of hundreds of extracellular proteins with the cell surface glycosaminoglycan heparan sulphate (HS) for the regulation of homeostasis is exploited by many microbial pathogens as a means of adherence and invasion. The closely related polysaccharide heparin, the widely used anticoagulant drug, which is structurally similar to HS and is a common experimental proxy, can be expected to mimic the properties of HS. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Heparin prevents infection by a range of viruses if add-ed exogenously, including S-associated coronavirus strain HSR1. Here, we show that the addition of heparin to Vero cells between 6.25 - 200 μg/mL, which spans the concentration of heparin in therapeutic use, and inhibits invasion by SARS-CoV-2 at between 44 and 80%. We also demonstrate that heparin binds to the Spike (S1) protein receptor binding domain and induces a conformational change, illustrated by surface plasmon resonance and circular dichroism spectroscopy studies. The structural features of heparin on which this interaction depends were investigated using a library of heparin derivatives and size-defined fragments. Binding is more strongly dependent on the presence of 2-O or 6-O sulphation, and the con-sequent conformational consequences in the heparin structure, than on N-sulphation. A hexasaccharide is required for conformational changes to be induced in the secondary structure that are comparable to those that arise from heparin binding. Enoxaparin, a low molecular weight clinical anticoagulant, also binds the S1 RBD protein and induces conformational change. These findings have implications for the rapid development of a first-line therapeutic by repurposing heparin as well as for next-generation, tailor-made, GAG-based antiviral agents against SARS-CoV-2 and other members of the Coronaviridae. ### Competing Interest Statement The authors have declared no competing interest.
8,380 downloads bioRxiv biochemistry
Homology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.
8,186 downloads bioRxiv biochemistry
Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.
7,854 downloads bioRxiv biochemistry
Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of Nov 15, 2020 has claimed 1,319,946 lives worldwide. Vaccine development focuses on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralisation. Immunogen integrity is therefore important for glycoprotein-based vaccine candidates. Here we show how site-specific glycosylation differs between virus-derived spikes and spike proteins derived from a viral vectored SARS-CoV-2 vaccine candidate. We show that their cellular secretion pathways are unique, resulting in different protein glycosylation and secretion, which may have implications for the resulting immune response and future vaccine design. ### Competing Interest Statement W.B.S. is a shareholder and consultant to Refeyn Ltd. All other authors declare no conflict of interest.
7,700 downloads bioRxiv biochemistry
A recent outbreak of novel coronavirus (SARS-CoV-2), the causative agent of COVID-19, has spread rapidly all over the world. Human immunodeficiency virus (HIV) is another deadly virus and causes acquired immunodeficiency syndrome (AIDS). Rapid and early detection of these viruses will facilitate early intervention and reduce disease transmission risk. Here, we present an All-In-One Dual CRISPR-Cas12a (termed "AIOD-CRISPR") assay method for simple, rapid, ultrasensitive, one-pot, and visual detection of coronavirus SARS-CoV-2 and HIV virus. In our AIOD CRISPR assay, a pair of crRNAs was introduced to initiate dual CRISPR-Cas12a detection and improve detection sensitivity. The AIOD-CRISPR assay system was successfully utilized to detect nucleic acids (DNA and RNA) of SARS-CoV-2 and HIV with a sensitivity of few copies. Also, it was evaluated by detecting HIV-1 RNA extracted from human plasma samples, achieving a comparable sensitivity with real-time RT-PCR method. Thus, our method has a great potential for developing next-generation point-of-care molecular diagnostics.
7,337 downloads bioRxiv biochemistry
SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Currently, there are no antiviral drugs or vaccines with proven efficacy, and development of these treatments are hampered by our limited understanding of the molecular and structural biology of the virus. Like many other RNA viruses, RNA structures in coronaviruses regulate gene expression and are crucial for viral replication. Although genome and transcriptome data were recently reported, there is to date little experimental data on predicted RNA structures in SARS-CoV-2 and most putative regulatory sequences are uncharacterized. Here we report the secondary structure of the entire SARS-CoV-2 genome in infected cells at single nucleotide resolution using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq). Our results reveal previously undescribed structures within critical regulatory elements such as the genomic transcription-regulating sequences (TRSs). Contrary to previous studies, our in-cell data show that the structure of the frameshift element, which is a major drug target, is drastically different from prevailing in vitro models. The genomic structure detailed here lays the groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics. ### Competing Interest Statement The authors have declared no competing interest.
7,261 downloads bioRxiv biochemistry
Three anti-HIV drugs, ritonavir, lopinavir and darunavir, might have therapeutic effect on coronavirus disease 2019 (COVID-19). In this study, the structure models of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteases, coronavirus endopeptidase C30 (CEP\_C30) and papain like viral protease (PLVP), were built by homology modeling. Ritonavir, lopinavir and darunavir were then docked to the models, respectively, followed by energy minimization of the protease-drug complexes. In the simulations, ritonavir can bind to CEP\_C30 most suitably, and induce significant conformation changes of CEP\_C30; lopinavir can also bind to CEP\_C30 suitably, and induce significant conformation changes of CEP\_C30; darunavir can bind to PLVP suitably with slight conformation changes of PLVP. It is suggested that the therapeutic effect of ritonavir and lopinavir on COVID-19 may be mainly due to their inhibitory effect on CEP\_C30, while ritonavir may have stronger efficacy; the inhibitory effect of darunavir on SARS-CoV-2 and its potential therapeutic effect may be mainly due to its inhibitory effect on PLVP.
6,762 downloads bioRxiv biochemistry
Justin D Walter, Cedric A.J. Hutter, Iwan Zimmermann, Marianne Wyss, Pascal Egloff, Michèle Sorgenfrei, Lea M. Hürlimann, Imre Gonda, Gianmarco Meier, Sille Remm, Sujani Thavarasah, Philippe Plattet, Markus A Seeger
The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has resulted in a global health and economic crisis of unprecedented scale. The high transmissibility of SARS-CoV-2, combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2. In an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. We obtained six strongly enriched sybody pools against the isolated RBD and identified 63 unique anti-RBD sybodies which also interact in the context of the full-length SARS-CoV-2 spike ectodomain. Among the selected sybodies, six were found to bind to the viral spike with double-digit nanomolar affinity, and five of these also showed substantial inhibition of RBD interaction with human angiotensin-converting enzyme 2 (ACE2). Additionally, we identified a pair of anti-RBD sybodies that can simultaneously bind to the RBD. It is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against COVID-19. Moreover, generation of polyvalent antivirals, via fusion of anti-RBD sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. We present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource. ### Competing Interest Statement Iwan Zimmermann, Pascal Egloff and Markus A. Seeger are founders and shareholders of Linkster Therapeutics AG.
6,569 downloads bioRxiv biochemistry
Using mRNA-Seq and de novo transcriptome assembly, we identified, cloned and characterized nine previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from avGFP. Among these FPs are the brightest GFP homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including two that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.
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