Molecules targeting a novel homotrimer cavity of Spike protein attenuate replication of SARS-CoV-2.

The SARS-CoV-2 Spike glycoprotein (S) utilizes a unique trimeric conformation to interact with the ACE2 receptor on host cells, making it a prime target for inhibitors that block viral entry. We have previously identified a novel proteinaceous cavity within the Spike protein homotrimer that could serve as a binding site for small molecules. However, it is not known whether these molecules would inhibit, stimulate, or have no effect on viral replication. To address this, we employed structural-based screening to identify small molecules that dock into the trimer cavity and assessed their impact on viral replication. Our findings show that a cohort of identified small molecules binding to the Spike trimer cavity effectively reduces the replication of various SARS-CoV-2 variants. These molecules exhibited inhibitory effects on B.1 (European original, D614G, EDB2) and B.1.617.2 (δ) variants, while showing moderate activity against the B.1.1.7 (α) variant. We further categorized these molecules into distinct groups based on their structural similarities. Our experiments demonstrated a dose-dependent viral replication inhibitory activity of these compounds, with some, like BCC0040453 exhibiting no adverse effects on cell viability even at high concentrations. Further investigation revealed that pre-incubating virions with compounds like BCC0031216 at different temperatures significantly inhibited viral replication, suggesting their specificity towards the S protein. Overall, our study highlights the inhibitory impact of a diverse set of chemical molecules on the biological activity of the Spike protein. These findings provide valuable insights into the role of the trimer cavity in the viral replication cycle and aid drug discovery programs aimed at targeting the coronavirus family.

Single Ferritin Nanocages Expressing SARS-CoV-2 Spike Variants to Receptor and Antibodies.

SARS-CoV-2 virus variants of concern (VOCs) have rapidly changed their transmissibility and pathogenicity primarily through mutations in the structural proteins. Herein, we present molecular details with dynamics of the ferritin nanocages stitched with synthetic chimeras displaying the Spike receptor binding domains (RBDs). Our findings demonstrated the potential usage of ferritin-based vaccines that may effectively inhibit viral entry by blocking the Spike-ACE2 network and may induce cross-protective antibody responses. Taking the nanocage constructs into consideration, we evaluated the effects of variants on the docked interface of the SARS-CoV-2 Spike RBD with the ACE2 (angiotensin-converting enzyme 2) host cell receptor and neutralizing antibodies (Abs). Investigating the VOCs revealed that most of the mutations reported a possibly reduced structural stability within the Spike RBD domain. Point mutations have moderate or no effect for VVH-72, CR3022, and S309 Abs when bound with the Spike RBD, whereas a significant effect was observed for B38, CB6, and m396 over the surface of the H-ferritin nanocage. In addition to providing useful therapeutic approaches against COVID-19 (coronavirus disease 2019), these structural details can also be used to fight future coronavirus outbreaks.

In Vitro Cross-Linking MS Reveals SMG1-UPF2-SMG7 Assembly as Molecular Partners within the NMD Surveillance.

mRNAs containing premature stop codons are responsible for various genetic diseases as well as cancers. The truncated proteins synthesized from these aberrant mRNAs are seldom detected due to the nonsense-mediated mRNA decay (NMD) pathway. Such a surveillance mechanism detects most of these aberrant mRNAs and rapidly destroys them from the pool of mRNAs. Here, we implemented chemical cross-linking mass spectrometry (CLMS) techniques to trace novel biology consisting of protein-protein interactions (PPIs) within the NMD machinery. A set of novel complex networks between UPF2 (Regulator of nonsense transcripts 2), SMG1 (Serine/threonine-protein kinase SMG1), and SMG7 from the NMD pathway were identified, among which UPF2 was found as a connection bridge between SMG1 and SMG7. The UPF2 N-terminal formed most interactions with SMG7, and a set of residues emerged from the MIF4G-I, II, and III domains docked with SMG1 or SMG7. SMG1 mediated interactions with initial residues of UPF2, whereas SMG7 formed very few interactions in this region. Modelled structures highlighted that PPIs for UPF2 and SMG1 emerged from the well-defined secondary structures, whereas SMG7 appeared from the connecting loops. Comparing the influence of cancer-derived mutations over different CLMS sites revealed that variants in the PPIs for UPF2 or SMG1 have significant structural stability effects. Our data highlights the protein-protein interface of the SMG1, UPF2, and SMG7 genes that can be used for potential therapeutic approaches. Blocking the NMD pathway could enhance the production of neoantigens or internal cancer vaccines, which could provide a platform to design potential peptide-based vaccines.

Modified Peptide Molecules As Potential Modulators of Shelterin Protein Functions; TRF1.

In this work, we present studies on relatively new and still not well-explored potential anticancer targets which are shelterin proteins, in particular the TRF1 protein can be blocked by in silico designed "peptidomimetic" molecules. TRF1 interacts directly with the TIN2 protein, and this protein-protein interaction is crucial for the proper functioning of telomere, which could be blocked by our novel modified peptide molecules. Our chemotherapeutic approach is based on assumption that modulation of TRF1-TIN2 interaction may be more harmful for cancer cells as cancer telomeres are more fragile than in normal cells. We have shown in vitro within SPR experiments that our modified peptide PEP1 molecule interacts with TRF1, presumably at the site originally occupied by the TIN2 protein. Disturbance of the shelterin complex by studied molecule may not in short term lead to cytotoxic effects, however blocking TRF1-TIN2 resulted in cellular senescence in cellular breast cancer lines used as a cancer model. Thus, our compounds appeared useful as starting model compounds for precise blockage of TRF proteins.

Next-generation sequencing of a combinatorial peptide phage library screened against ubiquitin identifies peptide aptamers that can inhibit the in vitro ubiquitin transfer cascade.

Defining dynamic protein-protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction.

Identification of novel interferon responsive protein partners of human leukocyte antigen A (HLA-A) using cross-linking mass spectrometry (CLMS) approach.

The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein-protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment.

Organic solvents aggregating and shaping structural folding of protein, a case study of the protease enzyme.

Low solubility of reactants or products in aqueous solutions can result in the enzymatic catalytic reactions that can occur in non-aqueous solutions. In current study we investigated aqueous solutions containing different organic solvents / deep eutectic solvents (DESs) that can influence the protease enzyme's activity, structural, and thermal stabilities. Retroviral aspartic protease enzyme is responsible for the cleavage of the polypeptide precursors into mature viral components, a very crucial step for virus life cycle. In molecular dynamic simulations (MDS), the complex of the protease enzyme with Darunavirwas found highly stable in urea aqueous solution compared to when with the ethylene glycol (EG) or glycerol solvents. Particularly, in different organic solvents the presence of Darunavir induced protein-protein interactions within the protease homodimer. For the systems with EG or glycerol solvents, the flap domains of the enzyme formed an "open" conformation which lead to a weak binding affinity with the drug. Conserved D25 and G27 residues among this family of the aspartic protease enzymes made a stable binding with Darunavir in the urea systems. Unfolding of the protease dimer was initiated due to self-aggregation for the EG or glycerol organic solvents, which formed an "open" conformation for the flap domains. On the contrary lack of such clustering in urea solvent, the protease showed conventional structural folding in the presence or absence of the drug molecule. These novel findings may help to better understand the protease enzymes, which could be controlled by deep eutectic solvents.

The Binding Specificity of PAB1 with Poly(A) mRNA, Regulated by Its Structural Folding.

The poly(A)-binding protein cytoplasmic 1 (PAB1 or PABPC1) protein is associated with the long poly(A) mRNA tails, inducing stability. Herein, we investigated the dynamics of the PABPC1 protein, along with tracing its mRNA binding specificity. During molecular dynamics simulations (MDS), the R176-Y408 amino acids (RRM3-4 domains; RNA recognition motifs) initiated a folded structure that resulted in the formation of different conformations. The RRM4 domain formed high-frequency intramolecular interactions, despite such induced flexibility. Residues D45, Y54, Y56, N58, Q88, and N100 formed long-lasting interactions, and specifically, aromatic residues (Y14, Y54, Y56, W86, and Y140) gained a unique binding pattern with the poly(A) mRNA. In addition, the poly(A) mRNA motif assembled a PABPC1-specific conformation, by inducing movement of the center three nucleotides to face towards RRM1-2 domains. The majority of the high-frequency cancer mutations in PAB1 reside within the RRM4 domain and amino acids engaging in high-frequency interactions with poly(A) mRNA were found to be preserved in different cancer types. Except for the G123C variant, other studied cancer-derived mutants hindered the stability of the protein. Molecular details from this study will provide a detailed understanding of the PABPC1 structure, which can be used to modulate the activity of this gene, resulting in production of mutant peptide or neoantigens in cancer.

The Elephant Evolved p53 Isoforms that Escape MDM2-Mediated Repression and Cancer.

The p53 tumor suppressor is a transcription factor with roles in cell development, apoptosis, oncogenesis, aging, and homeostasis in response to stresses and infections. p53 is tightly regulated by the MDM2 E3 ubiquitin ligase. The p53-MDM2 pathway has coevolved, with MDM2 remaining largely conserved, whereas the TP53 gene morphed into various isoforms. Studies on prevertebrate ancestral homologs revealed the transition from an environmentally induced mechanism activating p53 to a tightly regulated system involving cell signaling. The evolution of this mechanism depends on structural changes in the interacting protein motifs. Elephants such as Loxodonta africana constitute ideal models to investigate this coevolution as they are large and long-living as well as having 20 copies of TP53 isoformic sequences expressing a variety of BOX-I MDM2-binding motifs. Collectively, these isoforms would enhance sensitivity to cellular stresses, such as DNA damage, presumably accounting for strong cancer defenses and other adaptations favoring healthy aging. Here we investigate the molecular evolution of the p53-MDM2 system by combining in silico modeling and in vitro assays to explore structural and functional aspects of p53 isoforms retaining the MDM2 interaction, whereas forming distinct pools of cell signaling. The methodology used demonstrates, for the first time that in silico docking simulations can be used to explore functional aspects of elephant p53 isoforms. Our observations elucidate structural and mechanistic aspects of p53 regulation, facilitate understanding of complex cell signaling, and suggest testable hypotheses of p53 evolution referencing Peto's Paradox.

Self-derived peptides from the SARS-CoV-2 spike glycoprotein disrupting shaping and stability of the homotrimer unit.

The structural spike (S) protein from the SARS-CoV-2 ß-coronavirus is shown to make different pre- and post-fusion conformations within its homotrimer unit. To support the ongoing novel vaccine design and development strategies, we report the structure-based design approach to develop self-derived S peptides. A dataset of crucial regions from the S protein were transformed into linear motifs that could act as the blockers or stabilizers for the S protein homotrimer unit. Among these distinct S peptides, the pep02 (537-QQFGRDIAD-545) and pep07 (821-RDLICAQKFNGLTVLPPLLTDE-842) were found making stable folded binding with the S protein (550-750 and 950-1050 regions). Upon inserting SARS-CoV-2 S variants in the peptide destabilized the complexed S protein structure, resulting an allosteric effect in different functional regions of the protein. Particularly, the molecular dynamics revealed that A544D mutation in the pep02 peptide induced instability for the complexed S protein, whereas the N943K variant from pep09 exhibited an opposite behavior. An increased protein-peptide binding affinity and the stable structural folding were observed in mutated systems, compared to that of the wild type systems. The presence of mutation has induced an "up" active conformation of the spike (RBD) domain, responsible for interacting the host cell receptor. Among the lower affinity peptide datasets (e.g., pep01), the S1 and S2 subunit in the protein formed an "open" conformation, whereas with higher affinity peptides (e.g., pep07) these domains gained a "closed" conformation. These findings propose that our designed self-derived S peptides could replace a single S protein monomer, blocking the homotrimer formation or inducing stability.

Drug repositioning against COVID-19: a first line treatment.

COVID-19 disease caused by the SARS-CoV-2 virus has shaken our health and wealth foundations. Although COVID-19 vaccines will become available allowing for attenuation of disease progression rates, distribution of vaccines can create other challenges and delays. Hence repurposed drugs against SARS-CoV-2 can be an attractive parallel strategy that can be integrated into routine clinical practice even in poorly-resourced countries. The present study was designed using knowledge of viral pathogenesis and pharmacodynamics of broad-spectrum antiviral agents (BSAAs). We carried out the virtual screening of BSAAs against the SARS-CoV-2 spike glycoprotein, RNA dependent RNA polymerase (RdRp), the main protease (Mpro) and the helicase enzyme of SARS-CoV-2. Imatinib (a tyrosine kinase inhibitor), Suramin (an anti-parasitic), Glycyrrhizin (an anti-inflammatory) and Bromocriptine (a dopamine agonist) showed higher binding affinity to multiple targets. Further through molecular dynamics simulation, critical conformational changes in the target protein molecules were revealed upon drug binding which illustrates the favorable binding conformations of antiviral drugs against SARS-CoV-2 target proteins. The resulting drugs from the present study in combination and in cocktails from the arsenal of existing drugs could reduce the translational distance and could offer substantial clinical benefit to decrease the burden of COVID-19 illness. This also creates a roadmap for subsequent viral diseases that emerge.Communicated by Ramaswamy H. Sarma.

Molecular Determinants and Specificity of mRNA with Alternatively-Spliced UPF1 Isoforms, Influenced by an Insertion in the 'Regulatory Loop'.

The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the 'regulatory loop (involved in mRNA binding)'. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: 'open (isoform_2; without insertion)' and 'closed (isoform_1; with insertion)'. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the 'open conformation'. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA-UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein-mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.

Structural determinants of peptide-dependent TAP1-TAP2 transit passage targeted by viral proteins and altered by cancer-associated mutations.

The TAP1-TAP2 complex transports antigenic peptide substrates into the endoplasmic reticulum (ER). In ER, the peptides are further processed and loaded on the major histocompatibility class (MHC) I molecules by the peptide loading complex (PLC). The TAP transporters are linked with the PLC; a target for cancers and viral immune evasion. But the mechanisms whereby the cancer-derived mutations in TAP1-TAP2 or viral factors targeting the PLC, interfere peptide transport are only emerging. This study describes that transit of peptides through TAP can take place via two different channels (4 or 8 helices) depending on peptide length and sequence. Molecular dynamics and binding affinity predictions of peptide-transporters demonstrated that smaller peptides (8-10 mers; e.g. AAGIGILTV, SIINFEKL) can transport quickly through the transport tunnel compared to longer peptides (15-mer; e.g. ENPVVHFFKNIVTPR). In line with a regulated and selective peptide transport by TAPs, the immunopeptidome upon IFN-γ treatment in melanoma cells induced the shorter length (9-mer) peptide presentation over MHC-I that exhibit a relatively weak binding affinity with TAP. A conserved distance between N and C terminus residues of the studied peptides in the transport tunnel were reported. Furthermore, by adversely interacting with the TAP transport passage or affecting TAPNBD domains tilt movement, the viral proteins and cancer-derived mutations in TAP1-TAP2 may induce allosteric effects in TAP that block conformation of the tunnel (closed towards ER lumen). Interestingly, some cancer-associated mutations (e.g. TAP1R372Q and TAP2R373H) can specifically interfere with selective transport channels (i.e. for longer-peptides). These results provide a model for how viruses and cancer-associated mutations targeting TAP interfaces can affect MHC-I antigen presentation, and how the IFN-γ pathway alters MHC-I antigen presentation via the kinetics of peptide transport.

The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Interfaces with Structure Dynamics of the Workhorses from Cells Revealed through Cross-Linking Mass Spectrometry (CLMS).

The fundamentals of how protein-protein/RNA/DNA interactions influence the structures and functions of the workhorses from the cells have been well documented in the 20th century. A diverse set of methods exist to determine such interactions between different components, particularly, the mass spectrometry (MS) methods, with its advanced instrumentation, has become a significant approach to analyze a diverse range of biomolecules, as well as bring insights to their biomolecular processes. This review highlights the principal role of chemistry in MS-based structural proteomics approaches, with a particular focus on the chemical cross-linking of protein-protein/DNA/RNA complexes. In addition, we discuss different methods to prepare the cross-linked samples for MS analysis and tools to identify cross-linked peptides. Cross-linking mass spectrometry (CLMS) holds promise to identify interaction sites in larger and more complex biological systems. The typical CLMS workflow allows for the measurement of the proximity in three-dimensional space of amino acids, identifying proteins in direct contact with DNA or RNA, and it provides information on the folds of proteins as well as their topology in the complexes. Principal CLMS applications, its notable successes, as well as common pipelines that bridge proteomics, molecular biology, structural systems biology, and interactomics are outlined.

Functional Interfaces, Biological Pathways, and Regulations of Interferon-Related DNA Damage Resistance Signature (IRDS) Genes.

Interferon (IFN)-related DNA damage resistant signature (IRDS) genes are a subgroup of interferon-stimulated genes (ISGs) found upregulated in different cancer types, which promotes resistance to DNA damaging chemotherapy and radiotherapy. Along with briefly discussing IFNs and signalling in this review, we highlighted how different IRDS genes are affected by viruses. On the contrary, different strategies adopted to suppress a set of IRDS genes (STAT1, IRF7, OAS family, and BST2) to induce (chemo- and radiotherapy) sensitivity were deliberated. Significant biological pathways that comprise these genes were classified, along with their frequently associated genes (IFIT1/3, IFITM1, IRF7, ISG15, MX1/2 and OAS1/3/L). Major upstream regulators from the IRDS genes were identified, and different IFN types regulating these genes were outlined. Functional interfaces of IRDS proteins with DNA/RNA/ATP/GTP/NADP biomolecules featured a well-defined pharmacophore model for STAT1/IRF7-dsDNA and OAS1/OAS3/IFIH1-dsRNA complexes, as well as for the genes binding to GDP or NADP+. The Lys amino acid was found commonly interacting with the ATP phosphate group from OAS1/EIF2AK2/IFIH1 genes. Considering the premise that targeting IRDS genes mediated resistance offers an efficient strategy to resensitize tumour cells and enhances the outcome of anti-cancer treatment, this review can add some novel insights to the field.

Multivalent Display of SARS-CoV-2 Spike (RBD Domain) of COVID-19 to Nanomaterial, Protein Ferritin Nanocages.

SARS-CoV-2, or COVID-19, has a devastating effect on our society, both in terms of quality of life and death rates; hence, there is an urgent need for developing safe and effective therapeutics against SARS-CoV-2. The most promising strategy to fight against this deadly virus is to develop an effective vaccine. Internalization of SARS-CoV-2 into the human host cell mainly occurs through the binding of the coronavirus spike protein (a trimeric surface glycoprotein) to the human angiotensin-converting enzyme 2 (ACE2) receptor. The spike-ACE2 protein-protein interaction is mediated through the receptor-binding domain (RBD) of the spike protein. Mutations in the spike RBD can significantly alter interactions with the ACE2 host receptor. Due to its important role in virus transmission, the spike RBD is considered to be one of the key molecular targets for vaccine development. In this study, a spike RBD-based subunit vaccine was designed by utilizing a ferritin protein nanocage as a scaffold. Several fusion protein constructs were designed in silico by connecting the spike RBD via a synthetic linker (different sizes) to different ferritin subunits (H-ferritin and L-ferritin). The stability and the dynamics of the engineered nanocage constructs were tested by extensive molecular dynamics simulation (MDS). Based on our MDS analysis, a five amino acid-based short linker (S-Linker) was the most effective for displaying the spike RBD over the surface of ferritin. The behavior of the spike RBD binding regions from the designed chimeric nanocages with the ACE2 receptor was highlighted. These data propose an effective multivalent synthetic nanocage, which might form the basis for new vaccine therapeutics designed against viruses such as SARS-CoV-2.

Viruses, cancer and non-self recognition.

Virus-host interactions form an essential part of every aspect of life, and this review is aimed at looking at the balance between the host and persistent viruses with a focus on the immune system. The virus-host interaction is like a cat-and-mouse game and viruses have developed ingenious mechanisms to manipulate cellular pathways, most notably the major histocompatibility (MHC) class I pathway, to reside within infected cell while evading detection and destruction by the immune system. However, some of the signals sensing and responding to viral infection are derived from viruses and the fact that certain viruses can prevent the infection of others, highlights a more complex coexistence between the host and the viral microbiota. Viral immune evasion strategies also illustrate that processes whereby cells detect and present non-self genetic material to the immune system are interlinked with other cellular pathways. Immune evasion is a target also for cancer cells and a more detailed look at the interfaces between viral factors and components of the MHC class I peptide-loading complex indicates that these interfaces are also targets for cancer mutations. In terms of the immune checkpoint, however, viral and cancer strategies appear different.

Hydrogen deuterium exchange mass spectrometry identifies the dominant paratope in CD20 antigen binding to the NCD1.2 monoclonal antibody.

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.

Highly Conserved Homotrimer Cavity Formed by the SARS-CoV-2 Spike Glycoprotein: A Novel Binding Site.

An important stage in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) life cycle is the binding of the spike (S) protein to the angiotensin converting enzyme-2 (ACE2) host cell receptor. Therefore, to explore conserved features in spike protein dynamics and to identify potentially novel regions for drugging, we measured spike protein variability derived from 791 viral genomes and studied its properties by molecular dynamics (MD) simulation. The findings indicated that S2 subunit (heptad-repeat 1 (HR1), central helix (CH), and connector domain (CD) domains) showed low variability, low fluctuations in MD, and displayed a trimer cavity. By contrast, the receptor binding domain (RBD) domain, which is typically targeted in drug discovery programs, exhibits more sequence variability and flexibility. Interpretations from MD simulations suggest that the monomer form of spike protein is in constant motion showing transitions between an "up" and "down" state. In addition, the trimer cavity may function as a "bouncing spring" that may facilitate the homotrimer spike protein interactions with the ACE2 receptor. The feasibility of the trimer cavity as a potential drug target was examined by structure based virtual screening. Several hits were identified that have already been validated or suggested to inhibit the SARS-CoV-2 virus in published cell models. In particular, the data suggest an action mechanism for molecules including Chitosan and macrolides such as the mTOR (mammalian target of Rapamycin) pathway inhibitor Rapamycin. These findings identify a novel small molecule binding-site formed by the spike protein oligomer, that might assist in future drug discovery programs aimed at targeting the coronavirus (CoV) family of viruses.