Possible Link between Higher Transmissibility of Alpha,Kappa and Delta Variants of SARS-CoV-2 and Increased Structural Stability of Its Spike Protein and hACE2 Affinity

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has created alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests. It has also increased the chances of reinfection and immune escape. Recently various lineages namely, B.1.1.7 (Alpha), B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.617.3 have caused rapid infection around the globe. To understand the biophysical perspective, we have performed molecular dynamic simulations of four different spikes (receptor binding domain)-hACE2 complexes, namely wildtype (WT), Alpha variant (N501Y spike mutant), Kappa (L452R, E484Q) and Delta (L452R, T478K), and compared their dynamics, binding energy and molecular interactions. Our results show that mutation has caused significant increase in the binding energy between the spike and hACE2 in Alpha and Kappa variants. In the case of Kappa and Delta variants, the mutations at L452R, T478K and E484Q increased the stability and intra-chain interactions in the spike protein, which may change the interaction ability of neutralizing antibodies to these spike variants. Further, we found that the Alpha variant had increased hydrogen interaction with Lys353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.     

Preparation,characterization and X-ray structure of {[(3,2,3-tet)Ni(μ-CO3)Ni(3,2,3-tet)][(3,2,3-tet)Cu]}(ClO4)4: a heteronuclear trimetallic complex containing hydrogen bonds

A heteronuclear trimetallic complex {[(3,2,3-tet)Ni(μ-CO₃)Ni(3,2,3-tet)][(3,2,3-tet)Cu]}⁴⁺ (3,2,3-tet=N,N′-bis(3-aminopropyl)ethylenediamine) has been prepared by the reaction of Ni(II), 3,2,3-tet, Na₂CO₃ and Cu(3,2,3-tet)(ClO₄)₂ in situ. The complex is composed of two units, [(3,2,3-tet)Ni(μ-CO₃)Ni(3,2,3-tet)]²⁺ and [Cu(3,2,3-tet)]²⁺, which are linked by hydrogen bonding. The carbonate ion in the former unit acts as a bis-μ-bidentate ligand and adopts cis arrangement, and the resulting dinuclear complex has cis-β configuration for both Ni(II) ions.     

The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

The DNA binding mechanism of box B in HMG1, a member of thesequence non-specific DNA binding HMG1/2-box family of proteins,has been examined by both mutation analyses and molecular modelingtechniques. Substitution of the residue 102F, which is characteristicallyexposed to solvent, with a small hydrophobic amino acid affectedits DNA binding activity. However, no additional effect wasobserved by the further mutation of flanking 101F. Moleculardynamics simulation and modeling studies revealed that 102Fintercalates into DNA base-pairs, being supported by the flanking101F. The mutants with a small hydrophobic residue at position102 tolerated the substitution for 101F because the side chainat position 102 is too short to intercalate. Thus the intercalationof 102F and the positive effect of the flanking 101F residueare important for the sequence non-specific DNA binding of theHMG1/2-box. The conserved basic residues of 95K, 96R and 109Rwere also examined for their roles in DNA binding. These residuesinteracted with DNA mainly by electrostatic interaction andmaintained the location of the box on the DNA, which prescribedthe intercalation of 102F. The DNA intercalation by HMG1 consistsof an ingenious mechanism which brings DNA conformational changesnecessary for biological functions.     

Theoretical study of the human bradykinin-bradykinin B2 receptor complex

The interaction of bradykinin (BK) with the bradykinin B2 receptor (B2R) was analyzed by using molecular modeling (MM) and molecular dynamics (MD) simulations. A homology model for B2R has been generated and the recently determined receptor-bound solid-state NMR spectroscopic structure of BK (Lopez et al., Angew. Chem. 2008, 120, 1692-1695; Angew. Chem. Int. Ed. 2008, 47, 1668-1671) has been modeled into the binding pocket of the receptor to probe the putative ligand-receptor interface. The experimental hormone structure fitted well into the binding pocket of the receptor model and remained stable during the MD simulation. We propose a parallel orientation of the side chains for Arg1 and Arg9 in BK that is bound to B2R. The MD simulation study also allows the conformational changes that lead to the activated form of B2R to be analyzed. The hydrogen bond between N140 (3.35) and W283 (6.48) is the key interaction that keeps the receptor in its inactive form. This hydrogen bond is broken during the MD simulation due to rotation of transmembrane helix 3 (TM3) and is replaced by a new hydrogen bond between W283 (6.48) and N324 (7.45). We propose that this interaction is specific for the activated form of the bradykinin B2 receptor. Additionally, we compared and discussed our putative model in the context of the structural model of the partially activated rhodopsin (Rh*) and with the known biochemical and structural data.     

SARS-CoV-2 Virus−Host Interaction: Currently Available Structures and Implications of Variant Emergence on Infectivity and Immune Response

Coronavirus disease 19, or COVID-19, is an infection associated with an unprecedented worldwide pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which has led to more than 215 million infected people and more than 4.5 million deaths worldwide. SARS-CoV-2 cell infection is initiated by a densely glycosylated spike (S) protein, a fusion protein, binding human angiotensin converting enzyme 2 (hACE2), that acts as the functional receptor through the receptor binding domain (RBD). In this article, the interaction of hACE2 with the RBD and how fusion is initiated after recognition are explored, as well as how mutations influence infectivity and immune response. Thus, we focused on all structures available in the Protein Data Bank for the interaction between SARS-CoV-2 S protein and hACE2. Specifically, the Delta variant carries particular mutations associated with increased viral fitness through decreased antibody binding, increased RBD affinity and altered protein dynamics. Combining both existing mutations and mutagenesis studies, new potential SARS-CoV-2 variants, harboring advantageous S protein mutations, may be predicted. These include mutations S13I and W152C, decreasing antibody binding, N460K, increasing RDB affinity, or Q498R, positively affecting both properties.     

Dihydroimidazophenanthridinium (DIP)-based DNA binding agents with tuneable structures and biological activity

We have synthesised a library of dihydroimidazophenanthridinium cations (DIPs) with large structural diversity (1-29) using a "one-pot" approach. The DNA binding constants of DIPs range from 2x10(4) to 1.3x10(5) M(-1), and the free energies for binding range from -5.9 to -6.40 kcal mol(-1). Viscosity measurements demonstrated that the binding of the compounds caused DNA lengthening, thus signifying binding by intercalation. The cytotoxicities of the compounds were determined by tetrazolium dye-based microtitration assays and showed a large range of values (0.09-11.7 microM). Preliminary molecular modelling studies of the DNA-DIP interactions suggested that the DIP moieties can interact with DNA by intercalation, and some R groups might facilitate binding by minor-groove binding. The results provide insight into how to design biologically active DNA binding agents that can interact in these ways.     

Design and characterisation of an artificial DNA-binding cytochrome

We aim to design novel proteins that link specific biochemical binding events, such as DNA recognition, with electron transfer functionality. We want these proteins to form the basis of new molecules that can be used for templated assembly of conducting cofactors or for thermodynamically linking DNA binding with cofactor chemistry for nanodevice applications. The first examples of our new proteins recruit the DNA-binding basic helix region of the leucine zipper protein GCN4. This basic helix region was attached to the N and C termini of cytochrome b(562) (cyt b(562)) to produce new, monomeric, multifunctional polypeptides. We have fully characterised the DNA and haem-binding properties of these proteins, which is a prerequisite for future application of the new molecules. Attachment of a single basic helix of GCN4 to either the N or C terminus of the cytochrome does not result in specific DNA binding but the presence of DNA-binding domains at both termini converts the cytochrome into a specific DNA-binding protein. Upon binding haem, this chimeric protein attains the spectral characteristics of wild-type cyt b(562). The three forms of the protein, apo, oxidised holo and reduced holo, all bind the designed (ATGAcgATGA) target DNA sequence with a dissociation constant, K(D), of approximately 90 nM. The protein has a lower affinity (K(D) ca. 370 nM) for the wild-type GCN4 recognition sequence (ATGAcTCAT). The presence of only half the consensus DNA sequence (ATGAcgGGCC) shifts the K(D) value to more than 2500 nM and the chimera does not bind specifically to DNA sequences with no target recognition sites. Ultracentrifugation revealed that the holoprotein-DNA complex is formed with a 1:1 stoichiometry, which indicates that a higher-order protein aggregate is not responsible for DNA binding. Mutagenesis of a loop linking helices 2 and 3 of the cytochrome results in a chimera with a haem-dependent DNA binding affinity. This is the first demonstration that binding of a haem group to a designed monomeric protein can allosterically modulate the DNA binding affinity.     

A Spectroscopic Study on PtCl(4) Binding to Rabbit Skeletal Muscle G-Actin

It was found that the binding of PtCl(4) (2-) to G-actin and the consequent conformational changes are different with those for hard acids. It is a two-step process depending on molar ratio PtCl(4) (2-)/actin (R). In the first step, R less than 25, the PtCl(4) (2-) ions are bound to sulfur-containing groups preferentially. These high-affinity sites determined by Scatchard approach are characterized by n(1) = 30 with average binding constant K(1)=1.0x10(7)M(-1). The conformational changes are significant as characterized by N-(1-pyrenyl) maleimide(NPM) labeled fluorescence, intrinsic fluorescence and CD spectra. EPR spectroscopy of maleimide spin labeled(MSL) actin demonstrated that even PtCl(4) (2-)binding is limited to a very small fraction of high-affinity sites(R<1), it can bring about a pronounced change of conformation. In the range of R=25-40, high-affinity sites accessible are saturated. In the second step(R>40) , deep-buried binding sites turn out to be accessible as a result of the accumulated conformational changes. These new binding sites are estimated to be n(2)=26 with average binding constant K(2)=2.1x10(6)M(-1). Although in this step the quenching of intrinsic fluorescence goes on and the NPM-labled thiols moves to more hydrophilic environment, no change in alpha-helix content was found. These results suggested that with increasing in PtCl(4) ((2-)) binding, the G-actin turns to an open and loose structure in a discontinuous mode.     

DNA compaction by divalent cations: structural specificity revealed by the potentiality of designed quaternary diammonium salts

DNA interaction with quaternary diammonium dications, R(CH(3))(2)N(+)(CH(2))(n)N(+)(CH(3))(2)R, having various intercharge distances, lengths, and branching, and the chemical nature of the hydrophobic substituents were investigated by fluorescent microscopy and circular dichroism (CD) spectroscopy to reveal their structural specificity for binding to DNA. The conformational behavior of DNA was found to be highly sensitive to the structure of the dications with separated charges. The distance between two ammonium groups greatly influences the compaction activity of the dications. To explain this situation, we proposed a model that demonstrates that the charge density of the dication and the geometric fit between DNA phosphates and the ammonium groups in the dications play an important role in providing efficient DNA collapse. Elongation of the alkyl substituents (R) in the diammonium salts from ethyl to hexyl did not generate any significant alterations in the compaction activities, whereas the branching of substituents caused a drastic decrease in their compaction ability. Based on the results of CD spectroscopy, it was found that the ability of the dications to provoke a DNA transition from the B-form to A-form was also specific: it depended on their intercharge distances and was independent of the length of alkyl substituents.     

Synthesis, Crystal Structure and Interaction With DNA of N,N'-(Butane-1,4-Diyl)Bis(Guanidinium) Tetrachloroplatinate (II)

The design, synthesis, crystal structure and interaction with DNA of the N,N'-(butane-1,4-diyl)bis(guanidinium) tetrachloroplatinate(ll) are described. Crystal data: a = 8.152(1), b = 8.889(4), c = 10.700(3) A , alpha = 81.59(3), beta = 87.99(5), gamma = 78.48(6) degrees , V = 752(1) A(3), Z = 2 , space group P-1. The structure was refined to R = 0.039 and Rw = 0.046 from 1853 reflections (I > 3sigma(I)). This compound, named PtC(4)Gua, does not exhibit a center of symmetry and the center linker chain C(2) - C(3) - C(4) - C(5) is in gauche conformation. The cation is bisprotonated with the H(+) attached to the imine group of each terminal guanidinium function. The presence of the platinum moiety reinforces the binding of the butane(bis)guanidinium structure with double stranded DNA as judged from thermal denaturation studies and DNA unwinding experiments.     

Mutational analyses of restriction endonuclease--HindIII mutant E86K with higher activity and altered specificity

We have performed mutational analyses of restriction endonucleaseHindIII in order to identify the amino acid residues responsiblefor enzyme activity. Four of the seven HindIII mutants, whichhad His-tag sequences at the N-termini, were expressed in Escherichiacoli, and purified to homogeneity. The His-tag sequence didnot affect enzyme activity, whereas it hindered binding of theDNA probe in gel retardation assays. A mutant E86K in whichLys was substituted for Glu at residue 86 exhibited high endonucleaseactivity. Gel retardation assays showed high affinity of thismutant to the DNA probe. Surprisingly, in the presence of atransition metal, Mo²⁺ or Mn²⁺, the E86K mutant cleaved substrateDNA at a site other than HindIII. Substitution of Glu for Valat residue 106 (V106E), and Asn for Lys at residue 125 (K125N)resulted in a decrease in both endonucleolytic and DNA bindingactivities of the enzyme. Furthermore, substitution of Leu forAsp at residue 108 (D108L) abolished both HindIII endonucleaseand DNA binding activities. CD spectra of the wild type andthe two mutants, E86K and D108L, were similar to each other,suggesting that there was little change in conformation as aresult of the mutations. These results account for the notionthat Asp108 could be directly involved in HindIII catalyticfunction, and that the substitution at residue 86 may bringabout new interactions between DNA and cations.     

Steroid conjugates of dichloro(6-aminomethylnicotinate)platinum(II): effects on DNA, sex hormone binding globulin, the estrogen receptor, and various breast cancer cell lines

Esters of 6-aminomethylnicotinic acid with various steroidal alcohols were treated with K(2)PtCl(4) to give the N,N-chelated dichloroplatinum(II) complex conjugates 4. Their interaction with plasmid DNA was monitored by electrophoretic mobility measurements. Their affinities towards sex hormone binding globulin (SHBG) and towards the nuclear estrogen receptor ER(alpha) were assessed by competitive displacement radioassays. The inhibitory effect of 4 on breast tumour cells MCF-7 ER(+)/ER(-) and MDA-MB-231 was investigated in vitro. Conjugates with 3-O-linked estrogens 4 a,b or 17-O-linked androgens 4 g bound strongly to SHBG. The conjugate complex 4 b, featuring a 3-O-linked estradiol, also bound strongly and agonistically to the estrogen receptor. It also elicited distinct growth retardation of MCF-7 (ER(+)) cells, presumably by a mechanism different from that of cisplatin.     

Structure‐Based Mutational Study of an Archaeal DNA Ligase towards Improvement of Ligation Activity

DNA ligases catalyze the joining of strand breaks in duplex DNA. The DNA ligase of Pyrococcus furiosus (PfuLig), which architecturally resembles the human DNA ligase I (hLigI), comprises an N‐terminal DNA‐binding domain, a middle adenylylation domain, and a C‐terminal oligonucleotide‐binding (OB)‐fold domain. Here we addressed the C‐terminal helix in the OB‐fold domain of PfuLig by mutational analysis. The crystal structure of PfuLig revealed that this helix stabilizes a closed conformation of the enzyme by forming several ionic interactions with the adenylylation domain. The C‐terminal helix is oriented differently in hLigI when DNA is bound; this suggested that disruption of its interaction with the adenylylation domain might facilitate the binding of DNA substrates. We indeed identified one of its residues, Asp540, as being critical for ligation efficiency. The D540R mutation improved the overall ligation activity relative to the wild‐type enzyme, and at lower temperatures; this is relevant to applications such as ligation amplification reactions. Physical and biochemical analyses indicated that the improved ligation activity of the D540R variant arises from effects on the ligase adenylylation step and on substrate DNA binding in particular.     

Synthesis and biological evaluation of 1,3,3,4-tetrasubstituted pyrrolidine CCR5 receptor antagonists. Discovery of a potent and orally bioavailable anti-HIV agent

A series of 1,3,3,4-tetrasubstituted pyrrolidine containing CCR5 receptor antagonists were designed, which were elaborated either by condensation of a lithium salt of 3-(N,N-dibenzyl)aminopropionic acid methyl ester with ethyl benzoformate or by Baylis-Hillman reaction of ethyl acrylate with ethyl benzoformate and subsequent 1,4-addition of benzylamine, in the key steps. These compounds bearing 4-(N,N-disubstituted)amino piperidine units showed low nanomolar potency against the CCR5 receptor, whereas molecules with a 4-phenylpiperidine moiety displayed poor activity. Asymmetric synthesis of the most potent compound 23 a gave rise to the (3R,4S)-enantiomer 30 and the (3S,4R)-enantiomer 31, which showed IC(50) values of 2.9 and 385.9 nM, respectively. These results indicated that (3R,4S)-configuration in the series of compounds is favored for their interaction with the CCR5 receptor. The possible binding mode of these antagonists with the CCR5 receptor was discussed using a computer-modeling method. Compound 30 displayed excellent replication inhibition of seven genetically diverse R5 HIV-1 strains in the PBMC model, in a concentration-dependent manner with EC(50) values ranging from 0.3 nM to 30 nM. This molecule showed oral bioavailabilities of 41.2 % and 21.6 % in rats and dogs, respectively. Thus, compound 30 is a promising candidate for the treatment of HIV-1 infection.     

DNA stability in ionic liquids and deep eutectic solvents

DNA molecules are known as the genetic information carriers. Recently, they have been explored as a new generation of biocatalysts or chiral scaffolds for metal catalysts. There is also growing interest in finding alternative solvents for DNA preservation and stabilization, including two unique types of solvents: ionic liquids (ILs) and deep eutectic solvents (DES). Therefore, it is important to understand how DNA molecules interact with these novel ionic solvent systems (i.e. ILs and DES). It is well known that inorganic divalent and monovalent ions preferentially bind with major and minor grooves of DNA structures. However, in the case of ILs and DES, organic cations may intrude into the DNA minor grooves; more importantly, electrostatic attraction between organic cations and the DNA phosphate backbone becomes a predominant interaction, accompanied by hydrophobic and polar interactions between ILs and DNA major and minor grooves. In addition, anions may form hydrogen bonds with cytosine, adenine and guanine bases. Despite these strong interactions, DNA molecules maintain a double helical structure in most ionic solvent systems, especially in aqueous IL solutions. The exciting advances of G‐quadruplex DNA structures in ILs and DES are also discussed. © 2014 Society of Chemical Industry     

Nanomolar competitive inhibitors of Mycobacterium tuberculosis and Streptomyces coelicolor type II dehydroquinase

Isomeric nitrophenyl and heterocyclic analogues of the known inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid have been synthesized and tested as inhibitors of M. tuberculosis and S. coelicolor type II dehydroquinase, the third enzyme of the shikimic acid pathway. The target compounds were synthesized by a combination of Suzuki and Sonogashira cross-coupling and copper(I)-catalyzed 2,3-dipolar cycloaddition reactions from a common vinyl triflate intermediate. These studies showed that a para-nitrophenyl derivative is almost 20-fold more potent as a competitive inhibitor against the S. coelicolor enzyme than that of M. tuberculosis. The opposite results were obtained with the meta isomer. Five of the bicyclic analogues reported herein proved to be potent competitive inhibitors of S. coelicolor dehydroquinase, with inhibition constants in the low nanomolar range (4-30 nM). These derivatives are also competitive inhibitors of the M. tuberculosis enzyme, but with lower affinities. The most potent inhibitor against the S. coelicolor enzyme, a 6-benzothiophenyl derivative, has a K(i) value of 4 nM-over 2000-fold more potent than the best previously known inhibitor, (1R,4R,5R)-1,5-dihydroxy-4-(2-nitrophenyl)cyclohex-2-en-1-carboxylic acid (8 microM), making it the most potent known inhibitor against any dehydroquinase. The binding modes of the analogues in the active site of the S. coelicolor enzyme (GOLD 3.0.1), suggest a key pi-stacking interaction between the aromatic rings and Tyr 28, a residue that has been identified as essential for enzyme activity.     

XPS investigation of surface oxidation layers on a platinum electrode in alkaline solution

A thick oxidation layer on a platinum electrode has been grown in 1 N NaOH at 3 V vs Ag/AgCl reference electrode. After transferring the Pt electrode into an ultrahigh vacuum chamber the surface layer was analysed by X-ray photoelectron spectroscopy. Pt4f_5/2 amd O1s electron binding energies of 74.3, 77.6 and 530.9 eV respectively, as well as the broad peak shape of the O1s signal and the oxygen-to-platinum intensity ratio of 3.08 point towards a platinum—oxyhydroxide PtO(OH)₂. This formula is in good agreement with cyclic voltammetry curves, measured for the same electrode, that revealed two cathodic reduction peaks for oxygen surface coverages equivalent to more than two hydrogen monolayers. These two peaks were assigned to PtOH and PtO. XPS analysis at elevated temperatures showed that the thick (5 nm) oxidation layer decomposes at 400 K to a mixture of several oxides and hydroxides of Pt⁴⁺ and Pt²⁺ and Pt metal with a ratio of O-to-Pt of 1. This mixture further gradually decomposes to only a monolayer of oxygen at 770–870 K. Sodium cations were found to be present in trace amounts in the adlayer and to strongly shift the O1s binding energy to lower values.     

Hyrtiosal, from the marine sponge Hyrtios erectus, inhibits HIV-1 integrase binding to viral DNA by a new inhibitor binding site

HIV-1 integrase (IN) is composed of three domains, the N-terminal domain (NTD, residues 1-50), the catalytic core domain (CCD, residues 51-212), and the C-terminal domain (CTD, residues 213-288). All the three domains are required for the two known integration reactions. CCD contains the catalytic triad and is believed to bind viral DNA specifically, and CTD binds viral DNA in a nonspecific manner. As no clear evidence has confirmed the involvement of NTD in DNA binding directly, NTD has not been seriously considered and less is known about its function in viral replication. In the current work, using a SPR technology-based assay, the HIV-1 viral DNA was determined to bind directly to NTD with a K(D) value of 8.8 microM, suggesting that the process of preintegrated complex formation for HIV-1 IN might involve the direct interaction of NTD with viral DNA in addition to binding of viral DNA to the catalytic core domain and C-terminal domain. Moreover, such viral DNA/IN binding could be inhibited by the marine product hyrtiosal from the marine sponge Hyrtios erectus with an IC(50) of 9.60+/-0.86 microM. Molecular dynamic analysis correlated with a site-directed mutagenesis approach further revealed that such hyrtiosal-induced viral DNA/IN binding inhibition was caused by the fact that hyrtiosal could bind HIV-1 NTD at Ser17, Trp19, and Lys34. As hyrtiosal was recently discovered by us as a protein tyrosine phosphatase 1B (PTP1B) inhibitor,1 this work might also supply multiple-target information for this marine product, and the verified HIV-NTD/HIV-1 IN interaction model could have further implications for new HIV-1 IN inhibitor design and evaluation.     

Factors mediating activity, selectivity, and substrate specificity for the thiamin diphosphate-dependent enzymes benzaldehyde lyase and benzoylformate decarboxylase

Benzaldehyde lyase from Pseudomonas fluorescens and benzoylformate decarboxylase from Pseudomonas putida are homologous thiamin diphosphate-dependent enzymes that catalyze carboligase and carbolyase reactions. Both enzymes catalyze the formation of chiral 2-hydroxy ketones from aldehydes. However, the reverse reaction has only been observed with benzaldehyde lyase. Whereas benzaldehyde lyase is strictly R specific, the stereoselectivity of benzoylformate decarboxylase from P. putida is dependent on the structure and orientation of the substrate aldehydes. In this study, the binding sites of both enzymes were investigated by using molecular modelling studies to explain the experimentally observed differences in the activity, stereo- and enantioselectivity and substrate specificity of both enzymes. We designed a detailed illustration that describes the shape of the binding site of both enzymes and sufficiently explains the experimental effects observed with the wild-type enzymes and different variants. These findings demonstrate that steric reasons are predominantly responsible for the differences observed in the (R)-benzoin cleavage and in the formation of chiral 2-hydroxy ketones.