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.

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.

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.

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.

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.

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.

Insights into the Effects of Cancer Associated Mutations at the UPF2 and ATP-Binding Sites of NMD Master Regulator: UPF1.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that recognizes post-transcriptionally abnormal transcripts and mediates their degradation. The master regulator of NMD is UPF1, an enzyme with intrinsic ATPase and helicase activities. The cancer genomic sequencing data has identified frequently mutated residues in the CH-domain and ATP-binding site of UPF1. In silico screening of UPF1 stability change as a function over 41 cancer mutations has identified five variants with significant effects: K164R, R253W, T499M, E637K, and E833K. To explore the effects of these mutations on the associated energy landscape of UPF1, molecular dynamics simulations (MDS) were performed. MDS identified stable H-bonds between residues S152, S203, S205, Q230/R703, and UPF2/AMPPNP, and suggest that phosphorylation of Serine residues may control UPF1-UPF2 binding. Moreover, the alleles K164R and R253W in the CH-domain improved UPF1-UPF2 binding. In addition, E637K and E833K alleles exhibited improved UPF1-AMPPNP binding compared to the T499M variant; the lower binding is predicted from hindrance caused by the side-chain of T499M to the docking of the tri-phosphate moiety (AMPPNP) into the substrate site. The dynamics of wild-type/mutant systems highlights the flexible nature of the ATP-binding region in UPF1. These insights can facilitate the development of drug discovery strategies for manipulating NMD signaling in cell systems using chemical tools.

The structurally similar TRFH domain of TRF1 and TRF2 dimers shows distinct behaviour towards TIN2.

The telomere repeat binding-factor 1 and 2 (TRF1 and TRF2) proteins of the shelterin complex bind to duplex telomeric DNA as homodimers, and the homodimerization is mediated by their TRFH (TRF-homology) domains. We performed molecular dynamic (MD) simulations of the dimer forms of TRF1TRFH and TRF2TRFH in the presence/absence of the TIN2TBM (TIN2, TRF-interacting nuclear protein 2, TBM, TRF-binding motif) peptide. The MD results suggest that TIN2TBM is necessary to ensure the stability of TRF1TRFH homodimer but not the TRF2TRFH homodimer. In TRF1-TIN2-TRF2 complex, the peptide enhances the protein-protein interactions to yield a stable heterodimer. Both monomers in TRF1TRFH homodimer interact almost equally with the peptide, whereas in TRF2TRFH homodimer, monomer TRF2TRFH(M1) exhibits more dominant interactions than the TRF2TRFH(M2). The common residues of TRF1/2TRFH(M1) that form interactions with TIN2TBM in all peptide-bound systems originate from the H3 (helix) and L3 (loop) regions. Additionally, in the homodimer systems, residues of TRF1/2TRFH(M2) also interact with the peptide. The residue pair E71-K213 is responsible for different conformations of TRF1TRFH homodimers; specifically, this residue pair enhances the protein-peptide/protein interactions in peptide-bound/unbound systems, respectively. TRF1TRFH and TRF2TRFH proteins have a conserved but different interface responsible for the protein-protein/peptide interactions that exist in the corresponding dimers.

Extracting functional groups of ALLINI to design derivatives of FDA-approved drugs: Inhibition of HIV-1 integrase.

HIV-1 integrase (IN) is crucial for integration of viral DNA into the host genome and a promising target in development of antiretroviral inhibitors. In this work, six new compounds were designed by linking the structures of two different class of HIV-1 IN inhibitors (active site binders and allosteric IN inhibitors (ALLINIs)). Among newly designed compounds, INRAT10b was found most potent HIV-1 IN inhibitor considering different docking results. To further validate protein-ligand interactions obtained from dockings, molecular dynamics simulations were performed for inhibitor raltegravir and INRAT10b placed either at active site or allosteric site of HIV-1 IN (monomer or dimer). Results suggest that both raltegravir and INRAT10b were interacting with residue Gln62, Gly140, Ile141, and Ser147. However, INRAT10b interacts better with high H-bond occupancy, which can explain the strong binding affinity of INRAT10b than raltegravir with the HIV-1 IN protein. Subdomains rearrangements in HIV-1 IN suggest that the C-terminal and catalytic core domains develop their closeness in the presence of ligand. More significantly, the newly designed derivatives represent novel compounds targeting catalytic site and C-terminal (protein-protein interaction) domains simultaneously. And we also propose INRAT10b as a promising lead compound for the development of potent HIV-1 IN inhibitors.

Molecular basis and quantitative assessment of TRF1 and TRF2 protein interactions with TIN2 and Apollo peptides.

Shelterin is a six-protein complex (TRF1, TRF2, POT1, RAP1, TIN2, and TPP1) that also functions in smaller subsets in regulation and protection of human telomeres. Two closely related proteins, TRF1 and TRF2, make high-affinity contact directly with double-stranded telomeric DNA and serve as a molecular platform. Protein TIN2 binds to TRF1 and TRF2 dimer-forming domains, whereas Apollo makes interaction only with TRF2. To elucidate the molecular basis of these interactions, we employed molecular dynamics (MD) simulations of TRF1TRFH-TIN2TBM and TRF2TRFH-TIN2TBM/ApolloTBM complexes and of the isolated proteins. MD enabled a structural and dynamical comparison of protein-peptide complexes including H-bond interactions and interfacial residues that may regulate TRF protein binding to the given peptides, especially focusing on interactions described in crystallographic data. Residues with a selective function in both TRF1TRFH and TRF2TRFH and forming a stable hydrogen bond network with TIN2TBM or ApolloTBM peptides were traced. Our study revealed that TIN2TBM forms a well-defined binding mode with TRF1TRFH as compared to TRF2TRFH, and that the binding pocket of TIN2TBM is deeper for TRF2TRFH protein than ApolloTBM. The MD data provide a basis for the reinterpretation of mutational data obtained in crystallographic work for the TRF proteins. Together, the previously determined X-ray structure and our MD provide a detailed view of the TRF-peptide binding mode and the structure of TRF1/2 binding pockets. Particular TRF-peptide interactions are very specific for the formation of each protein-peptide complex, identifying TRF proteins as potential targets for the design of inhibitors/drugs modulating telomere machinery for anticancer therapy.

Molecular basis and potential activity of HIV-1 reverse transcriptase toward trimethylamine-based compounds.

Reverse transcriptase (RT) inhibitors are currently used to treat human immunodeficiency virus (HIV)-1 infections. In this work, novel triethylamine derivatives were designed and studied by rigid and flexible docking and molecular dynamics (MD) approaches. An apo form of HIV-1 RT was also studied by MD simulation to analyze comparative response of protein in ligand-bound and ligand-unbound forms. Among newly designed HIV-1 RT inhibitors, compound HIV104 was the most potent inhibitor considering different docking results. Molecular docking results were further validated by MD simulations of an HIV-1 RT/HIV104 complex using two independent software (Discovery Studio Client 3.1 and GROMACS) to perform comparative analysis. Results suggest that hydroxyl and carboxyl groups present at -R1 position in compounds favored strong H-bond contacts as well as good interaction energy profile. Our MD results are consistent with the observations that conformational dynamics between the thumb and finger subdomains of HIV-1 RT controls its dynamics on substrate binding and subsequent activity. MD studies of HIV-1 RT/HIV104 provide insight into interrelatedness of residue scale interactions and global conformational change and also hint at the complex nature of allosteric inhibition. Thus, the results obtained from this study facilitate the design of potent HIV-1 RT inhibitors.

Comparative molecular dynamics study of dimeric and monomeric forms of HIV-1 protease in ligand bound and unbound state.

Human immunodeficiency virus type 1 protease is a viral-encoded enzyme and it is essential for replication and assembly of the virus. Inactivation of HIV-1 protease causes production of immature, noninfectious viral particles and thus HIV-1 protease is an attractive target in anti-AIDS drug design. In our current work, we performed molecular dynamics (MD) calculations (500 ns) for two different ligands (COM5 - designed in our previous study, and Darunavir) and made effort to understand dynamics behaviour of our designed compound COM5. An apo form of HIV-1 protease as monomer and dimer form was also studied in order to analyze response of protein to the ligand. MD results suggest that presence of ligand in hinders the stability of HIV-1 protease and one monomer from dimer systems is dominant on other monomer in terms of interaction made with ligands. We were able to trace functional residues as well as continuous motion of opening and closing (clapping) of flap region in HIV-1 protease (apo form) during entire 1000 ns of MD simulation. COM5 showed almost similar behaviour towards HIV-1 protease enzyme as Darunavir and propose as promising lead compound for the development of new inhibitor for HIV-1 protease.

Structural and dynamic changes adopted by EmrE, multidrug transporter protein--Studies by molecular dynamics simulation.

EmrE protein transports positively charged aromatic drugs (xenobiotics) in exchange for two protons and thus provides bacteria resistance to variety of drugs. In order to understand how this protein may recognize ligands, the monomer and asymmetric apo-form of the EmrE dimer embedded in a heterogeneous phospholipid (POPE+POPG) membrane were studied by molecular dynamics simulations. Dimer is regarded as a functional form of the transporter, but to understand molecular aspects of its mode of action, a monomer was also included in our work. We analyzed hydrogen bonds which include inter- and intra-molecular interactions. Analyzing the long-lasting H-bond interactions, we found that water access to the internal transmembrane segments is regulated by residues with aromatic or basic side chains and fluctuating transmembrane helices. Our finding supports that GLU14 in EmrE apo-form is ready to interact or bind with substrate molecule. The analysis of distance center of masses and water entrance area indicate the feasibility of the dimer to undergo induced fit in order to accommodate a ligand. The results indicate that a binding pattern can be formed in the EmrE in such a way that GLU14 binds to the positively charged fragment of a substrate molecule, and other aromatic residues (i.e., TRP63 and TYR40) located in vicinity may accommodate other non-polar parts of substrate molecule. The results of our simulation also allow us to support experimentally testable hypotheses concerning functional inward-outward conformational changes of the protein.

Identification of 1H-indene-(1,3,5,6)-tetrol derivatives as potent pancreatic lipase inhibitors using molecular docking and molecular dynamics approach.

Pancreatic lipase is a potential therapeutic target to treat diet-induced obesity in humans, as obesity-related diseases continue to be a global problem. Despite intensive research on finding potential inhibitors, very few compounds have been introduced to clinical studies. In this work, new chemical scaffold 1H-indene-(1,3,5,6)-tetrol was proposed using knowledge-based approach, and 36 inhibitors were derived by modifying its functional groups at different positions in scaffold. To explore binding affinity and interactions of ligands with protein, CDOCKER and AutoDock programs were used for molecular docking studies. Analyzing results of rigid and flexible docking algorithms, inhibitors C_12, C_24, and C_36 were selected based on different properties and high predicted binding affinities for further analysis. These three inhibitors have different moieties placed at different functional groups in scaffold, and to characterize structural rationales for inhibitory activities of compounds, molecular dynamics simulations were performed (500 nSec). It has been shown through simulations that two structural fragments (indene and indole) in inhibitor can be treated as isosteric structures and their position at binding cleft can be replaced by each other. Taking into account these information, two lines of inhibitors can further be developed, each line based on a different core scaffold, that is, indene/indole.

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.

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.

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.

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.

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.