Antiviral peptides inhibiting the main protease of SARS-CoV-2 investigated by computational screening and in vitro protease assay.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in viral replication and transcription and received great attention as a vital target for drug/peptide development. Therapeutic agents such as small-molecule drugs or peptides that interact with the Cys-His present in the catalytic site of Mpro are an efficient way to inhibit the protease. Although several emergency-approved vaccines showed good efficacy and drastically dropped the infection rate, evolving variants are still infecting and killing millions of people globally. While a small-molecule drug (Paxlovid) received emergency approval, small-molecule drugs have low target specificity and higher toxicity. Besides small-molecule drugs, peptide therapeutics are thus gaining increasing popularity as they are easy to synthesize and highly selective and have limited side effects. In this study, we investigated the therapeutic value of 67 peptides targeting Mpro using molecular docking. Subsequently, molecular dynamics (MD) simulations were implemented on eight protein-peptide complexes to obtain molecular-level information on the interaction between these peptides and the Mpro active site, which revealed that temporin L, indolicidin, and lymphocytic choriomeningitis virus (LCMV) GP1 are the best candidates in terms of stability, interaction, and structural compactness. These peptides were synthesized using the solid-phase peptide synthesis protocol, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and authenticated by mass spectrometry (MS). The in vitro fluorometric Mpro activity assay was used to validate the computational results, where temporin L and indolicidin were observed to be very active against SARS-CoV-2 Mpro with IC50 values of 38.80 and 87.23 µM, respectively. A liquid chromatography-MS (LC-MS) assay was developed, and the IC50 value of temporin L was measured at 23.8 µM. The solution-state nuclear magnetic resonance (NMR) structure of temporin L was determined in the absence of sodium dodecyl sulfate (SDS) micelles and was compared to previous temporin structures. This combined investigation provides critical insights and assists us to further develop peptide inhibitors of SARS-CoV-2 Mpro through structural guided investigation.

Structural dynamics and functional analysis of Saprolegnia parasitica chitin synthases 5 in a phospholipid bilayer.

Saprolegnia parasitica is an oomycete responsible for a fish disease called saprolegniosis, which poses an economic and environmental burden on aquaculture production. In Saprolegnia, CHS5 of S. parasitica (SpCHS5) contains an N-terminal domain, a catalytic domain of the glycosyltransferase -2 family containing a GT-A fold, and a C-terminal transmembrane domain. No three-dimensional structure of SpCHS5 is reported yet disclosing the structural details of this protein. We have developed a structural model of full-length SpCHS5 and validated it by molecular dynamics simulation technique. From the 1 microsecond simulations, we retrieved the stable RoseTTAFold model SpCHS5 protein to explain characteristics and structural features. Furthermore, from the analysis of the movement of chitin in the protein cavity, we assumed that ARG 482, GLN 527, PHE 529, PHE 530, LEU 540, SER 541, TYR 544, ASN 634, THR 641, TYR 645, THR 641, ASN 772 residues as a main cavity lining site. In SMD analysis, we investigated the opening of the transmembrane cavity required for chitin translocation. The pulling of chitin from the internal cavity to the extracellular region was observed through steered molecular dynamics simulations. A comparison of the initial and final structures of chitin complex showed that there's a transmembrane cavity opening in the simulations. Overall, this present work will help us understand the structural and functional basis of CHS5 and design inhibitors against SpCHS5.Communicated by Ramaswamy H. Sarma.

Therapeutic Potential of Antiviral Peptides against the NS2B/NS3 Protease of Zika Virus.

The NS2B/NS3 protease is highly conserved among various proteases of the Zika virus, making it an important therapeutic target for developing broad-spectrum antiviral drugs. The NS2B/NS3 protease is a crucial enzyme in the replication cycle of Zika virus and plays a significant role in viral maturation and assembly. Inhibiting the activity of this protease can potentially prevent viral replication, making it an attractive target for developing therapies against Zika virus infection. This work screens 429 antiviral peptides in comparison with substrate peptide against the NS2B/NS3 of Zika virus using molecular docking and molecular dynamics (MD) simulation. Based on the docking screening, MD simulation conducted for the best four peptides including AVP0239, AVP0642, AVP0660, and AVP2044, could be effective against NS2B/NS3. These results were compared with the control substrate peptide. Further analysis indicates that AVP0642 and AVP2044 are the most promising candidates. The interaction analysis showed that the catalytic site residues including His51, Asp75, Ser135 and other non-catalytic residues such as Asp129, Asp83, and Asp79 contribute substantial interactions. Hydrogen bonds (41%) and hydrophobic interactions (33%) are observed as the prominent non-covalent interaction prompting the peptide-protein complex formation. Furthermore, the structure-activity relationship (SAR) illustrates that positively charged (Lys, Arg) residues in the peptides dominate the interactions. This study provides the basis for developing novel peptide-based protease inhibitors for Zika virus.

Elucidating the Structure, Dynamics, and Interaction of a Choline Chloride and Citric Acid Based Eutectic System by Spectroscopic and Molecular Modeling Investigations.

Eutectic solvent systems are versatile solvents that have found widespread use in numerous applications. Traditional solvents are homogeneous, having only one component, and their chemistry is relatively simple, with some exceptions. On the other hand, deep eutectic solvents (DESs) comprise binary components, generally a donor and an acceptor in hydrogen bonding with varying ratios. The interaction chemistry among the donor and acceptor involved in hydrogen bonding in DESs is complicated. Although numerous research is focused on the synthesis and application of DESs, few studies are reported to elucidate the complex structure and dynamic and interaction behavior of DESs. In this study, we employed calorimetry, vibrational spectroscopy techniques including FTIR and Raman, and nuclear magnetic resonance to derive insight into the structural feature and noncovalent contact of choline chloride (ChCl) and citric acid (CA) while they formed DESs. The 1:1 ChCl/CA eutectic system showed phase transitions and melting peaks with the most pronounced peak at 156.22 °C, suggesting the DESs melting at a lower temperature than the melting temperatures of ChCl and CA. In addition to IR and Raman findings, 1H NMR investigations demonstrate hydrogen bonding intermolecular interactions between ChCl and CA, supporting the formation of 1:1 ChCl/CA DESs based on the deshielded chemical shifts of the proton for Ch. The interaction of the chloride anion with the methyl protons (H4) and methylene protons (H3) of ChCl as well as the strong hydrogen bonding interactions between the hydroxyl hydrogen (H1) of ChCl with one of CA's carbonyl oxygens both supported the formation of conformer E. In addition, molecular dynamics followed by the density functional theory (DFT) was employed to visualize the structure and interaction of DESs using the ωB97XD theory and 6-311++G (d,p) basis set. Both experimental and theoretical IR, Raman, and structural analyses provided evidence of the formation of DESs by possessing hydrogen bonds. These multifaceted experimental and computational investigations provide details of structural and intermolecular interactions of ChCl/CA DESs.

Unraveling the impact of ORF3a Q57H mutation on SARS-CoV-2: insights from molecular dynamics.

ORF3a is a conserved accessory protein of SARS-CoV-2, linked to viral infection and pathogenesis, with acquired mutations at various locations. Previous studies have shown that the occurrence of the Q57H mutation is higher in comparison to other positions in ORF3a. This mutation is known to induce conformational changes, yet the extent of structural alteration and its role in the viral adaptation process remain unknown. Here we performed molecular dynamics (MD) simulations of wt-ORF3a, Q57H, and Q57A mutants to analyze structural changes caused by mutations compared to the native protein. The MD analysis revealed that Q57H and Q57A mutants show significant structural changes in the dimer conformation than the wt-ORF3a. This dimer conformer narrows down the ion channel cavity, which reduces Na + or K + permeability leading to decrease the antigenic response that can help the virus to escape the host immune system. Non-bonding interaction analysis shows the Q57H mutant has more interacting residues, resulting in more stability within dimer conformation than the wt-ORF3a and Q57A. Moreover, both mutant dimers (Q57H and Q57A) form a novel salt-bridge interaction at the same position between A:Asp142 and B:Lys61, whereas such an interaction is absent in the wt-ORF3a dimer. We have also noticed that the TM3 domain's flexibility in Q57H is increased because of strong inter-domain interactions of TM1 and TM2 within the dimer conformation. These unusual interactions and flexibility of Q57H mutant can have significant impacts on the SARS-CoV-2 adaptations, virulence, transmission, and immune system evasion. Our findings are consistent with the previous experimental data and provided details information on the structural perturbation in ORF3a caused by mutations, which can help better understand the structural change at the molecular level as well as the reason for the high virulence properties of this variant.Communicated by Ramaswamy H. Sarma.

Structure and dynamics of whole-sequence homology model of ORF3a protein of SARS-CoV-2: An insight from microsecond molecular dynamics simulations.

The ORF3a is a large accessory protein in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which plays an important role in virulence and viral replication; especially in inflammasome activation and apoptosis. However,, the existing cryo-EM structure of SARS-CoV-2 ORF3a is incomplete, . making it challenging to understand its structural and functional features. The aim of this study is to investigate the dynamic behaviors of the full-sequence homology model of ORF3a and compare it with the cryo-EM structure using microsecond molecular dynamics simulations. The previous studies indicated that the unresolved residues of the cryo-EM structure are not only involved in the pathogenesis of the SARS-CoV-2 but also exhibit a significant antigenicity. The dynamics scenario of homology model revealed higher RMSD, Rg, and SASA values with stable pattern when compared to the cryo-EM structure. Moreover, the RMSF analysis demonstrated higher fluctuations at specific positions (1-43, 97-110, 172-180, 219-243) in the model structure, whereas the cryo-EM structure displayed lower overall drift (except 1-43) in comparison to the model structure.Secondary structural features indicated that a significant unfolding in the transmembrane domains and ß-strand at positions 166 to 172, affecting the stability and compactness of the cryo-EM structure , whereas the model exhibited noticeable unfolding in transmembrane domains and small-coiled regions in the N-terminal. , The results from molecular docking and steered molecular dynamics investigations showed the model structure had a greater number of non-bonding interactions, leading to enhanced stability when compared to the cryo-EM structure. Consequently, higher forces were necessary for unbinding of the baricitinib and ruxolitinib inhibitors from the model structure.. Our findings can help better understanding of the significance of unresolved residues at the molecular level. Additionally, this information can guide researchers for experimental endeavors aimed at completing the full-sequence structure of the ORF3a.Communicated by Ramaswamy H. Sarma.

Structural and functional effects of the L84S mutant in the SARS-COV-2 ORF8 dimer based on microsecond molecular dynamics study.

The L84S mutation has been observed frequently in the ORF8 protein of SARS-CoV-2, which is an accessory protein involved in various important functions such as virus propagation, pathogenesis, and evading the immune response. However, the specific effects of this mutation on the dimeric structure of ORF8 and its impacts on interactions with host components and immune responses are not well understood. In this study, we performed one microsecond molecular dynamics (MD) simulation and analyzed the dimeric behavior of the L84S and L84A mutants in comparison to the native protein. The MD simulations revealed that both mutations caused changes in the conformation of the ORF8 dimer, influenced protein folding mechanisms, and affected the overall structural stability. In particular, the 73YIDI76 motif has found to be significantly affected by the L84S mutation, leading to structural flexibility in the region connecting the C-terminal ß4 and ß5 strands. This flexibility might be responsible for virus immune modulation.  The free energy landscape (FEL) and principle component analysis (PCA) have also supported our investigation. Overall, the L84S and L84A mutations affect the ORF8 dimeric interfaces by reducing the frequency of protein-protein interacting residues (Arg52, Lys53, Arg98, Ile104, Arg115, Val117, Asp119, Phe120, and Ile121) in the ORF8 dimer.  Our findings provide detail insights for further research in designing structure-based therapeutics against the SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

In silico peptide-based therapeutics against human colorectal cancer by the activation of TLR5 signaling pathways.

OBJECTIVE: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in both men and women. Toll-like receptor 5 (TLR5), an autoimmune signaling receptor that plays a role in cancer, can be exploited for the suppression of human colon cancer. Salmonella flagellin protein, a novel agonist of TLR5 activating downstream signaling, could be a basis for designing anticancer peptides. METHODS: The three-dimensional crystal structure of TLR5 (PDB ID: 3J0A, Resolution = 26.0 Å) was optimized using the AMBER force field in the YASARA suit. In silico enzymatic digestion tool, PeptideCutter, was used to identify peptides from Salmonella flagellin, an agonist against human TLR5. The 3D structure of the peptides was generated using PEP-FOLD3. These peptides were screened against human TLR5 using shape complementarity principles based on the binding affinity and interactions with the active residue of TLR5 monomer, and the selected peptides were further validated by molecular dynamic (MD) simulation. RESULTS: In this study, we generated 42 peptides from Salmonella flagellin protein by in silico protein digestion. Then, based on a new hidden Markov model sub-optimal conformation sampling approach as well as the size of the fragments, we select 38 effective peptides from these 42 cleavages. These peptides were screened against the monomeric Xray structure of human TLR5 using shape complementarity principles. Based on the binding affinity and interactions with the active residue of TLR5 monomer (residues 294 and 366 of TLR5), nine top-scored peptides were selected for the initial molecular dynamic (MD) simulation. Among these peptides, Clv10, Clv17, and Clv28 showed high stability and less flexibility during MD simulation. A 1 µs MD simulation was performed on TLR5-Clv10, TLR-Clv17, and TLR5-Clv28 complexes to further analyze the stability, conformational changes, and binding mode (Clv10, Clv17, and Clv28). During this MD study, the peptides showed high salt bridges and ionic interactions with residue ASP294 and residue ASP366 throughout the simulation and remained in the concave of the human TLR5 monomer. The RMSD and Rg values showed that the peptide-protein complexes become stable after 200 ns of contraction and extraction. CONCLUSION: These findings can facilitate the rational design of selected peptides as an agonist of TLR5, which have antitumor activity, suppress colorectal cancer tumors, and can be used as promising candidates and novel agonists of TLR5.

Potential antiviral peptides against the nucleoprotein of SARS-CoV-2.

Nucleoprotein is a conserved structural protein of SARS-CoV-2, which is involved in several functions, including replication, packaging, and transcription. In this research, 21 antiviral peptides that are known to have inhibitory function against nucleoprotein in several other viruses, were screened computationally against the nucleoprotein of SARS-CoV-2. The complexes of five best performing peptides (AVP1142, AVP1145, AVP1148, AVP1150, AVP1155) with nucleoprotein were selected for subsequent screening via 5 ns molecular dynamics (MD) simulation. Two peptides, namely AVP1145 and AVP1155, came out as promising candidates and hence were selected for 200 ns MD simulation for further validation, incorporating a DMPC-based membrane environment. In the long MD simulation, both AVP1155 and AVP1145 utilized multiple residues-mainly aromatic, acidic, and nonpolar residues-as interacting points to remain in contact with the nucleoprotein and formed predominantly hydrogen bonds along with hydrophobic and electrostatic interactions. However, AVP1155 proved to be superior to AVP1145 when its complex with nucleoprotein was analyzed in terms of root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessible surface area and free energy landscape. In a nutshell, the findings of this research may guide future studies in the development of selective peptide inhibitors of SARS-CoV-2 nucleoprotein. Supplementary Information: The online version contains supplementary material available at 10.1007/s11696-022-02514-4.

Large scale peptide screening against main protease of SARS CoV-2.

The COVID-19 pandemic has been a public health emergency, with deadly forms constantly emerging around the world, highlighting the dire need for highly effective antiviral therapeutics. Peptide therapeutics show significant potential for this viral disease due to their efficiency, safety, and specificity. Here, two thousand seven hundred eight antibacterial peptides were screened computationally targeting the Main protease (Mpro) of SARS CoV-2. Six top-ranked peptides according to their binding scores, binding pose were investigated by molecular dynamics to explore the interaction and binding behavior of peptide-Mpro complexes. The structural and energetic characteristics of Mpro-DRAMP01760 and Mpro-DRAMP01808 complexes fluctuated less during a 250 ns MD simulation. In addition, three peptides (DRAMP01760, DRAMP01808, and DRAMP01342) bind strongly to Mpro protein, according to the free energy landscape and principal component analysis. Peptide helicity and secondary structure analysis are in agreement with our findings. Interaction analysis of protein-peptide complexes demonstrated that Mpro's residue CYS145, HIS41, PRO168, GLU166, GLN189, ASN142, MET49, and THR26 play significant contributions in peptide-protein attachment. Binding free energy analysis (MM-PBSA) demonstrated the energy profile of interacting residues of Mpro in peptide-Mpro complexes. To summarize, the peptides DRAMP01808 and DRAMP01760 may be highly Mpro specific, resulting disruption in a viral replication and transcription. The results of this research are expected to assist future research toward the development of antiviral peptide-based therapeutics for Covid-19 treatment.