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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

The Nrf2 pathway is an essential defense pathway in a cell. It responds to oxidative and electrophilic stress via derepression of Nrf2 from Keap1-Cul3-mediated degradation, accumulation of Nrf2 in the nucleus and transcriptional activation of a number of detoxifying and cell protective Nrf2 target genes. Here we report that normal and cancer cells also express the N-terminally truncated Nrf2 isoform (ΔN-Nrf2), which originates from an alternative promoter. Co-immunoprecipitation together with molecular dynamics simulation showed that the binding between ΔN-Nrf2 and Keap1 is impaired, resulting in the much higher stability of this form. ΔN-Nrf2 is retained in the cytoplasm in response to electrophilic stress, indicating that it does not regulate transcription under the same stress stimuli as the full-length Nrf2. Altogether this data suggests that Nrf2 has other functions in cells than transcriptional activation of genes, which most probably rely on the protein-protein interactions in the cytoplasm. The regulation between these functions takes place on the level of transcription.Significance StatementThis work signifies the importance of alternative transcription in assigning the function to the produced protein. Nrf2 transcripts produced from the second promoter of the Nrf2 gene give rise to the N-terminally truncated Nrf2 form (ΔN-Nrf2), which is retained in the cytoplasm upon stress, thus it has a different role in cells than transcriptional regulation. ΔN-Nrf2 is resistant to the Keap1-Cul3 degradation pathway and is highly expressed in all tested cell types. This work points to the new, cytoplasmic role of Nrf2 in cells, determined at the level of transcription.

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.

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.

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.

Overexpression of telomerase is one of the hallmarks of human cancer. Telomerase is important for maintaining the integrity of the ends of chromosomes, which are called telomeres. A growing number of human disease syndromes are associated with organ failure caused by mutations in telomerase (hTERT or hTR). Mutations in telomerase lead to telomere shortening by decreasing the stability of the telomerase complex, reducing its accumulation, or directly affecting its enzymatic activity. In this work, potential human telomerase mutations were identified by a systematic computational approach. Moreover, molecular docking methods were used to predict the effects of these mutations on the affinity of certain ligands (C_9i, C_9k, 16A, and NSC749234). The C_9k inhibitor had the best binding affinity for wild-type (WT) telomerase. Moreover, C_9i and C_9k had improved interactions with human telomerase in most of the mutant models. The R631 and Y717 residues of WT telomerase formed interactions with all studied ligands and these interactions were also commonly found in most of the mutant models. Residues forming stable interactions with ligands in molecular dynamics (MD) were traced, and the MD simulations showed that the C_9k ligand formed different conformations with WT telomerase than the C_9i ligand.

International audience; 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.

International audience; 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.

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.

Graphical abstract, 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 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.

Nonsense-mediated messenger RNA (mRNA) decay (NMD) is a surveillance pathway used by cells to control the quality mRNAs and to fine-tune transcript abundance. NMD plays an important role in cell cycle regulation, cell viability, DNA damage response, while also serving as a barrier to virus infection. Disturbance of this control mechanism caused by genetic mutations or dys-regulation of the NMD pathway can lead to pathologies, including neurological disorders, immune diseases and cancers. The role of NMD in cancer development is complex, acting as both a promoter and a barrier to tumour progression. Cancer cells can exploit NMD for the downregulation of key tumour suppressor genes, or tumours adjust NMD activity to adapt to an aggressive immune microenvironment. The latter case might provide an avenue for therapeutic intervention as NMD inhibition has been shown to lead to the production of neoantigens that stimulate an immune system attack on tumours. For this reason, understanding the biology and co-option pathways of NMD is important for the development of novel therapeutic agents. Inhibitors, whose design can make use of the many structures available for NMD study, will play a crucial role in characterizing and providing diverse therapeutic options for this pathway in cancer and other diseases.

EmrE is a bacterial transporter protein that forms an anti-parallel homodimer with four transmembrane helices in each monomer. EmrE transports positively charged aromatic compounds, such as TPP+ and its derivatives. We performed molecular dynamics (MD) simulations of EmrE in complex with TPP+, MeTPP+, and MBTPP+ embedded in a membrane. The detailed molecular properties and interactions were analysed for all EmrE-ligand complexes. Our MD results identified that Lys22, Tyr40, Phe44, Trp45, and Trp63 formed potential π interactions with all three ligands and further confirmed the essential role of Glu14. Moreover, distance analysis and structural changes in the EmrE translocation pathway suggest that ligand recognition and protein conformational changes depend on the structural properties of the substrate. Analysis of the movement of the ligand in the protein binding site and rotation of the ligand's aromatic rings confirm that substrates with aromatic moieties, such as MBTPP+, exhibit relatively stable binding to EmrE. Interestingly, the aromatic rings of Tyr40, Phe44, Trp45, and Trp63 underwent parallel movements with the aromatic rings of TPP+. Based on the MD results, we propose that π interactions, as well as the mutual rotation of the aromatic rings in the protein and ligand, can be regarded as sources of ligand movement, and thus, the whole complex may work as a "molecular propeller".

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.

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.

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.

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.

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.

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 &ldquo, up&rdquo, and &ldquo, down&rdquo, state. In addition, the trimer cavity may function as a &ldquo, bouncing spring&rdquo, 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.

Down regulation of the major histocompatibility class (MHC) I pathway plays an important role in tumour development, and can be achieved by suppression of HLA expression or mutations in the MHC peptide-binding pocket. The peptide-loading complex (PLC) loads peptides on the MHC-I molecule in a dynamic multi-step assembly process. The effects of cancer variants on ERp57 and tapasin components from the MHC-I pathway is less known, and they could have an impact on antigen presentation. Applying computational approaches, we analysed whether the ERp57-tapasin binding might be altered by missense mutations. The variants H408R(ERp57) and P96L, D100A, G183R(tapasin) at the protein&ndash, protein interface improved protein stability (&Delta, &Delta, G) during the initial screen of 14 different variants. The H408R(ERp57) and P96L(tapasin) variants, located close to disulphide bonds, were further studied by molecular dynamics (MD). Identifying intramolecular a-a' domain interactions, MD revealed open and closed conformations of ERp57 in the presence and absence of tapasin. In wild-type and mutant ERp57-tapasin complexes, residues Val97, Ser98, Tyr100, Trp405, Gly407(ERp57) and Asn94, Cys95, Arg97, Asp100(tapasin) formed common H-bond interactions. Moreover, comparing the H-bond networks for P96L and H408R with each other, suggests that P96L(tapasin) improved ERp57-tapasin binding more than the H408R(ERp57) mutant. During MD, the C-terminus domain (that binds MHC-I) in tapasin from the ERp57(H408R)-tapasin complex moved away from the PLC, whereas in the ERp57-tapasin(P96L) system was oppositely displaced. These findings can have implications for the function of PLC and, ultimately, for the presentation of MHC-I peptide complex on the tumour cell surface.

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.

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.

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.

Display Omitted N-phenyl-1H-indol-2-amine was selected as a base for novel FTO protein inhibitors.Designed ligands were studied by molecular docking and dynamic simulation approach.Hydroxyl (OH) and carbonyl (CO) group of ligand played a key role in FTO inhibition. Fat mass and obesity-associated (FTO) protein contributes to non-syndromic human obesity which refers to excessive fat accumulation in human body and results in health risk. FTO protein has become a promising target for anti-obesity medicines as there is an immense need for the rational design of potent inhibitors to treat obesity. In our study, a new scaffold N-phenyl-1H-indol-2-amine was selected as a base for FTO protein inhibitors by applying scaffold hopping approach. Using this novel scaffold, different derivatives were designed by extending scaffold structure with potential functional groups. Molecular docking simulations were carried out by using two different docking algorithm implemented in CDOCKER (flexible docking) and AutoDock programs (rigid docking). Analyzing results of rigid and flexible docking, compound MU06 was selected based on different properties and predicted binding affinities for further analysis. Molecular dynamics simulation of FTO/MU06 complex was performed to characterize structure rationale and binding stability. Certainly, Arg96 and His231 residue of FTO protein showed stable interaction with inhibitor MU06 throughout the production dynamics phase. Three residues of FTO protein (Arg96, Asp233, and His231) were found common in making H-bond interactions with MU06 during molecular dynamics simulation and CDOCKER docking.

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 -R

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.

During previous years, many studies on synthesis, as well as on anti-tumor, anti-inflammatory and anti-bacterial activities of the pyrazole derivatives have been described. Certain pyrazole derivatives exhibit important pharmacological activities and have proved to be useful template in drug research. Considering importance of pyrazole template, in current work the series of novel inhibitors were designed by replacing central ring of acridine with pyrazole ring. These heterocyclic compounds were proposed as a new potential base for telomerase inhibitors. Obtained dibenzopyrrole structure was used as a novel scaffold structure and extension of inhibitors was done by different functional groups. Docking of newly designed compounds in the telomerase active site (telomerase catalytic subunit TERT) was carried out. All dibenzopyrrole derivatives were evaluated by three docking programs: CDOCKER, Ligandfit docking (Scoring Functions) and AutoDock. Compound C_9g, C_9k and C_9l performed best in comparison to all designed inhibitors during the docking in all methods and in interaction analysis. Introduction of pyrazole and extension of dibenzopyrrole in compounds confirm that such compound may act as potential telomerase inhibitors.

Based on the structure of the most potential inhibitor diamidophosphate, various novel groups of inhibitors were developed by knowledge-based design approach with covalent carbon-phosphorus or carbon-phosphorus-carbon bond to improve hydrolytic stability to inhibit the microbial ureases. Designed compounds were evaluated with 10 (LigScore1, LigScore2, PLP1, PLP2, JAIN, PMF, PMF04, LUDI_1, LUDI_2 and LUDI_3) different scoring functions implemented in Discovery Studio and conformation analysis by AutoDock package.

Cathepsin C is a cysteine protease required for activation of various pro-inflammatory serine proteases and, essentially, is of interest as a therapeutic target. Cathepsin C coordinate system was employed as a model to study the interaction of some already available inhibitors of Cathepsin C. Compounds containing Gly-Phe fragment with functional groups at its ends were designed by knowledge based approach. Using AutoDock and Discovery Studio Client 3.1 software packages, binding energy of different conformations and ten scoring functions (LigScore1, LigScore2, PLP1, PLP2, JAIN, PMF, PMF04, LUDI_1, LUDI_2 and LUDI_3) were calculated for newly designed compounds. These docking studies revealed favorable energy scores which also helps to understand interaction of ligands with enzyme.

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., S.S. acknowledges Postdoc Mobility fellowship no. P400PB 180889 from the Swiss National Science Foundation. E.M.M. and E.V.A. acknowledge funding from the NIH (R35 GM122480 and R01 DK110520 to E.M.M.), Welch Foundation (F1515 to E.M.M. and F-0046 to E.V.A.), Army Research Office grant W911NF-12-1-0390 and Erisyon. E.M.M. and E.V.A. are co-founders and shareholders of Erisyon. R.T.K. acknowledges funding from NIGMS (R01 GM138931). P.Y. acknowledges funding from an NIH Director’s New Innovator Award (1DP2OD007292), an NIH Transformative Research Award (1R01EB018659), an NIH Pioneer Award (1DP1GM133052), and the Molecular Robotics Initiative fund at the Wyss Institute for Biologically Inspired Engineering. M.D. acknowledges funding from a Systems Biology Department Fellowship from Harvard Medical School and a Technology Development Fellowship from Wyss Institute for Biologically Inspired Engineering. C.C. acknowledges the Peter and Traudl Engelhorn Foundation. C.D. acknowledges the ERC Advanced Grant Looping DNA (no. 883684) and the NWO programs NanoFront and Basyc., S.O. acknowledges the support of the Azrieli fellowship foundation. N.L.K. acknowledges funding from the Paul G. Allen Frontiers Program (11715), the NIH HuBMAP program (UH3 CA246635) and NIGMS (P41 GM108569). J.P.M. and Z.K. acknowledge internal funding from NIST and are co-inventors on patents relevant to this work. M. Wanunu acknowledges funding from the NIH (HG009186). K.S. and A.A. acknowledge funding from the NSF (PHY-1430124). C.J., C.D. and R.E. acknowledge funding from NWO-I (SMPS). C.J. acknowledges funding from HFSP (RGP0026/2019). A.P. acknowledges Bekker fellowship no. PPN/BEK/2018/1/00296 from the Polish National Agency for Academic Exchange. C.M. and S.H. acknowledge funding from the European Research Council (ERC ‘Enlightened’, GA 616251) and the CEA Transverse Program ‘Instrumentation and Detection’ (PTC-ID VIRIONEMS). Support from the Proteomics French Infrastructure (PROFI) is also gratefully acknowledged. G.D. acknowledges funding from FNR (C17/BM/11642138). M.M. acknowledges funding from the Adolphe Merkle Foundation, the Michael J. Fox Foundation for Parkinson’s Research (grant 17924) and the Swiss National Science Foundation (grant no. 200021-169304). A.M. acknowledges funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 833399-ERC NanoProt-ID and ISF award 3485/19. M.C. acknowledges computational resources from CINECA (NATWE project) and the Swiss National Super-Computing Centre (CSCS), under projects sm11 and s865. E.C. acknowledges funding from I-Site Lille, Région Hauts-de-France, and the European Union’s Horizon 2020 Marie Skłodowska-Curie no. 843052. The study was supported by the project ‘International Centre for Cancer Vaccine Science’ that is carried out within the International Agendas Programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. D.G. thanks Genome Canada and Genome British Columbia for financial support for Genomics Technology Platforms (GTP) funding for operations and technology development (264PRO).