Increased neutralization of SARS-CoV-2 Delta variant by nanobody (Nb22) and the structural basis

Delta variant, also known as B.1.617.2, has become a predominant circulating variant in many countries since it first emerged in India in December 2020. Delta variant is less sensitive to serum neutralization from COVID-19 convalescent individuals or vaccine recipients, relative to Alpha strains. It was also resistant to neutralization by some anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies in clinics. Previously, we reported the discovery of nanobodies isolated from an alpaca immunized with spike protein, exhibiting ultrahigh potency against SARS-CoV-2 and its mutated variants, where a novel inhalable bispecific Nb15 protected SARS-CoV-2 infection in hACE2 mice. Here, we found that Nb22-Fc, among our previously reported nanobodies, exhibited 8.4-fold increased neutralization potency against Delta variant with an IC50 value of 0.41 ng/ml (5.13 pM) relative to Alpha variant. Furthermore, our crystal structural analysis reveals that the binding of Nb22 on SARS-CoV-2 RBD effectively blocks the binding of RBD to ACE2 during virus infection. Furthermore, the L452R mutation in RBD of Delta variant forms an additional hydrogen bond with the hydroxy group of T30 of Nb22, leading to the increased neutralization potency of Nb22 against Delta variant. Thus, Nb22 is a potential therapeutic agent against SARS-CoV-2, especially the highly contagious Delta variant.

Alteration in H-bond strength affects the stability of codon-anticodon interaction at in-frame UAG stop codon during in vitro translation

We have studied the decoding ability of a non-standard nucleobase modified tRNA for non-natural amino acid mutagenesis. The insertion of 2, 6-diaminopurine (D) base at the 3rd position of a tRNA anticodon enabled us to evaluate the effect of an additional hydrogen bond during translation. The presence of D at the tRNA anticodon led to stabilization of the codon-anticodon interaction due to an additional H-bond between the N2-exocyclic amine of D and the C2 carbonyl group of uracil during protein translation. While decoding UAG codons using stop codon suppression methodology, the enhanced codon-anticodon interaction improved codon readthrough and synthesis of modified protein with a non-natural amino acid at multiple sites. Our findings imply that the number of hydrogen bonds at the tRNA-mRNA duplex interface is an important criterion during mRNA decoding and improves protein translation at multiple UAG stop sites. This work provides valuable inputs towards improved non-natural amino acid mutagenesis for creating functional proteins.

Alteration in H-bond strength affects the stability of codon-anticodon interaction at in-frame UAG stop codon during in vitro translation

We have studied the decoding ability of a non-standard nucleobase modified tRNA for non-natural amino acid mutagenesis. The insertion of 2, 6-diaminopurine (D) base at the 3rd position of a tRNA anticodon enabled us to evaluate the effect of an additional hydrogen bond during translation. The presence of D at the tRNA anticodon led to stabilization of the codon-anticodon interaction due to an additional H-bond between the N2-exocyclic amine of D and the C2 carbonyl group of uracil during protein translation. While decoding UAG codons using stop codon suppression methodology, the enhanced codon-anticodon interaction improved codon readthrough and synthesis of modified protein with a non-natural amino acid at multiple sites. Our findings imply that the number of hydrogen bonds at the tRNA-mRNA duplex interface is an important criterion during mRNA decoding and improves protein translation at multiple UAG stop sites. This work provides valuable inputs towards improved non-natural amino acid mutagenesis for creating functional proteins.

Structural and calorimetric studies reveal specific determinants for the binding of a high-affinity NLS to mammalian importin-alpha

The classical nuclear import pathway is mediated by importin (Impα and Impβ), which recognizes the cargo protein by its Nuclear Localization Sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far N-terminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3 the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs.

Mutation Effect Investigation on Cytochrome C552 Protein Instability and Electron Transfer Improvement in the Acidithiobacillus Ferrooxidans Bacteria Respiratory Chain

Cytochrome c552 (Cyc1) is a protein in the electron transport chain of the Acidithiobacillus ferrooxidans (Af) bacteria which obtain their energy from oxidation Fe2+ to Fe+3. The electrons are directed through Cyc2, RCY (rusticyanin), Cytochrome c552, and Cox aa3 proteins to O2. Cytochrome c552 protein consists of two chains, A and B. In the present study, a new mutation (E121D) in the A chain of cytochrome c552 protein was selected due to electron receiving from Histidine 143 of RCY. Then, the changes performed in the E121D mutant were evaluated by MD simulations analyzes. Cytochrome c552 and RCY proteins were docked by a Patchdock server. By E121D mutation, the connection between the two chains in Cytochrome c552 was enhanced by an additional hydrogen bond between Zn1388 and aspartate 121. Asp 121 in chain A gets farther from Zn 1388 in chain B. Therefore, the aspartate gets closer to Cu 1156 of the RCY leading to the higher stability of the RCY/Cytochrome c552 complex. Further, an acidic residue (Glu121) becomes a more acidic residue (Asp121) and improving the electron transfer to Cytochrome c552 protein. The results of RMSF analysis showed further ligand flexibility in mutation. This leads to fluctuation of the active site and increases redox potential at the mutation point and the speed of electron transfer. This study also predicts that in all respiratory chain proteins, electrons probably enter the first active site via glutamate and exit through the second active site of each respiratory chain protein and through histidine.

The basis of a more contagious 501Y.V1 variant of SARS-COV-2

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing a world-wide pandemic. A variant of SARS-COV-2 (20I/501Y.V1) recently discovered in the United Kingdom has a single mutation from N501 to Y501 within the receptor binding domain (Y501-RBD), of the Spike protein of the virus. This variant is much more contagious than the original version (N501-RBD). We found that this mutated version of RBD binds to human Angiotensin Converting Enzyme 2 (ACE2) a ~10 times more tightly than the native version (N501-RBD). Modeling analysis showed that the N501Y mutation would allow a potential aromatic ring-ring interaction and an additional hydrogen bond between the RBD and ACE2. However, sera from individuals immunized with the Pfizer-BioNTech vaccine still efficiently block the binding of Y501-RBD to ACE2 though with a slight compromised manner by comparison with their ability to inhibit binding to ACE2 of N501-RBD. This may raise the concern whether therapeutic anti-RBD antibodies used to treat COVID-19 patients are still efficacious. Nevertheless, a therapeutic antibody, Bamlanivimab, still binds to the Y501-RBD as efficiently as its binds to N501-RBD.

Biological Evaluation and Docking Studies of New Carbamate, Thiocarbamate, and Hydrazide Analogues of Acyl Homoserine Lactones as Vibrio fischeri-Quorum Sensing Modulators

A series of carbamate, thiocarbamate, and hydrazide analogues of acylhomoserine lactones (AHLs) were synthesized and their ability to modulate Vibrio fischeri-quorum sensing was evaluated. The compounds in the series exhibit variable side chain length and the possible presence of a diversely substituted phenyl substituent. Biological evaluation on the Vibrio fischeri quorum sensing system revealed that the ethyl substituted carbamate (1) display a weak agonistic activity whereas compounds with longer chain length or benzyl substituents display significant antagonistic activity. The most active compounds in the series were the 4-nitrobenzyl carbamate and thiocarbamate 7 and 11 which exhibited an IC50 value of about 20 µM. These activities are in the range of other reported of AHL-structurally related quorum sensing (QS) inhibitors. Docking experiments conducted on the LuxR model showed that, compared to the natural ligand OHHL, the additional heteroatom of the carbamate group induces a new hydrogen bond with Tyr70 leading to a different global hydrogen-bond network. Tyr70 is an important residue in the binding site and is strictly conserved in the LuxR family. For the 4-nitrobenzyl carbamate and thiocarbamate analogues, the docking results highlight an additional hydrogen bond between the nitro group and Lys178. For hydrazide analogues, which are deprived of any activity, docking shows that the orientation of the carbonyl group is opposite as compared with the natural ligand, leading to the absence of a H-bond between the C=O with Tyr62. This suggests that, either this later interaction, or the influence of the C=O orientation on the overall ligand conformation, are essential for the biological activity.

Synthesis and Biological Evaluation of NH2-Sulfonyl Oseltamivir Analogues as Influenza Neuraminidase Inhibitors

A series of NH2-sulfonyl oseltamivir analogues were designed, synthesized, and their inhibitory activities against neuraminidase from H5N1 subtype evaluated. The results indicated that the IC50 value of compound 4a, an oseltamivir analogue via methyl sulfonylation of C5-NH2, was 3.50 μM. Molecular docking simulations suggested that 4a retained most of the interactions formed by oseltamivir carboxylate moieties and formed an additional hydrogen bond with the methylsulfonyl group. Meanwhile, 4a showed high stability towards human liver microsomes. More importantly, 4a without basic moieties is not a zwitterion as reported on the general structure of neuraminidase inhibitors. This research will provide valuable reference for the research of new types of neuraminidase inhibitors.

An investigation to elucidate the factors dictating the crystal structure of seven ammonium carboxylate molecular salts

The crystal structures of seven ammonium carboxylate salts are reported, namely (RS)-1-phenylethan-1-aminium isonicotinate, C8H12N+·C6H4N1O2 −, (I), (RS)-1-phenylethan-1-aminium flurbiprofenate [or 2-(3-fluoro-4-phenylphenyl)propanoate], C8H12N+·C15H12FO2 −, (II), (RS)-1-phenylethan-1-aminium 2-chloro-4-nitrobenzoate, C8H12N+·C7H3ClNO4 −, (III), (RS)-1-phenylethan-1-aminium 4-iodobenzoate, C8H12N+·C7H4IO2 −, (IV), (S)-1-cyclohexylethan-1-aminium 2-chloro-4-nitrobenzoate, C8H18N+·C7H3ClNO4 −, (V), 2-(cyclohex-1-en-1-yl)ethan-1-aminium 4-bromobenzoate, C8H16N+·C7H4BrO2 −, (VI), and (S)-1-cyclohexylethan-1-aminium 4-bromobenzoate, C8H18N+·C7H4BrO2 −, (VII). Salts (II) to (VII) feature three N+—H...O− hydrogen bonds, which form one-dimensional hydrogen-bonded ladders. Salts (II), (III), (IV), (V) and (VII) have a type II ladder system despite the presence of halogen bonding and other intermolecular interactions, whereas (VI) has a type III ladder system. Salt (I) has a unique hydrogen-bonded system of ladders, featuring both N+—H...O− and N+—H...N hydrogen bonds owing to the presence of the pyridine functional group. The presence of an additional hydrogen-bond acceptor on the carboxylate cation disrupts the formation of the ubiquitous type II and III ladder found predominately in ammonium carboxylate salts. Halogen bonding, however, has no influence on their formation.

(E)-4-Methoxy-N′-(2,3,4-trimethoxybenzylidene)benzohydrazide monohydrate

The asymmetric unit of the title compound, C18H20N2O5·H2O, consists of a benzohydrazide molecule which exists in anEconformation with respect to the C=N imine bond and a water molecule. The dihedral angle between the aromatic rings is 41.67 (9)°. The methoxy substituent of the 4-methoxyphenyl group is twisted at an angle of 6.8 (3)° out of the plane of the attached benzene ring. In the 2,4,5-trimethoxyphenyl unit, thepara-methoxy group is coplanar with the ring [C—C—O—C = −1.5 (3)°], whereas theortho- andmeta-methoxy groups are twisted out of the plane of the ring [C—C—O—C = 75.4 (2) and −67.1 (2)°, respectively]. Two molecules are connected by two water moleculesviaO—H...O hydrogen bonds, generating anR22(8) ring motif. One of the water H atoms forms an additional hydrogen bond to an N atom. The water molecules act as an acceptor for an N—H...O hydrogen bond. As a result, a three-dimensional network is formed.