Crystallography-based exploration of non-covalent interactions for the design and synthesis of coumarin for stronger protein binding.

Protein molecules are a good target for the inhibition or promotion of biological processes. Different methods like QSAR and molecular docking have been developed to accurately design small binder molecules for target proteins. An alternative model has been developed wherein a statistical method is used to find the propensity of different non-covalent interactions between small molecules and amino acid residues of the protein. The results give hints as to the choice of substituents required at the SM to strongly bind to a protein. In this case, 75 different types of proteins bound with coumarin derivatives have been investigated and the non-covalent interactions observed between the basic coumarin moiety and amino acids have been analyzed. Density functional theory (DFT) calculations were used to identify the electronic features of coumarin to understand the feasibility of the observed non-covalent interactions and to find appropriate groups that can modulate these interactions. The binding affinity towards a protein (ß-lactoglobulin (BLG)) and the stability of the protein complex have been investigated through docking and molecular dynamics of 100 ns, respectively. The modeled compounds were synthesized and investigated with regards to their interactions with the model carrier protein. The thermodynamics of the interactions were also investigated and the binding is governed by the Le Chatelier principle.

A crystallography-based investigation of weak interactions for drug design against COVID-19.

Interactions between proteins and small molecules play important roles in the inhibition of protein function. However, a lack of proper knowledge about non-covalent interactions can act as a barrier towards gaining a complete understanding of the factors that control these associations. To find effective molecules for COVID-19 inhibition, we have quantitatively investigated 143 X-ray crystal structures of the SARS-CoV-2 Mpro protein of coronavirus with covalently or non-covalently bound small molecules (SMs). Our present study is able to explain ordinary and perceptive aspects relating to protein inhibition. The active site of the protein consists of 21 amino acid residues, but only nine are actively involved in the ligand binding process. The H41, M49, and C145 residues have highest priority with respect to interactions with small molecules through hydrogen bond, CH-π, and van der Waals interactions. At the active site, this ranking of amino acids is clear, based on different spatial orientations of ligands, and consistent with the electronic properties. SMs with aromatic moieties that bind to the active site of the protein play a distinct role in the determination of the following order of interaction frequency with the amino acids: CH-π > H-bonding > polar interactions. This present study revealed that the G143 and C145 residues play crucial roles in the recognition of the carbonyl functionality of SMs through hydrogen bonding. With this knowledge in mind, an effective inhibitor small-molecule for SARS-CoV-2 Mpro was designed: docking studies showed that the designed molecule has strong binding affinity towards the protein. The non-covalent interactions in the protein-ligand complex are in good agreement with the results obtained from X-ray crystallography. Moreover, the present study focused on weak forces and their influence on protein inhibition, henceforth shedding much light on the essential requirements for moieties that should be present in a good inhibitor and their orientations at the ligand binding site.

Participatory role of zinc in structural and functional characterization of bioremediase: a unique thermostable microbial silica leaching protein.

A unique protein, bioremediase (UniProt Knowledgebase Accession No.: P86277), isolated from a hot spring bacterium BKH1 (GenBank Accession No.: FJ177512), has shown to exhibit silica leaching activity when incorporated to prepare bio-concrete material. Matrix-assisted laser desorption ionization mass spectrometry analysis suggests that bioremediase is 78% homologous to bovine carbonic anhydrase II though it does not exhibit carbonic anhydrase-like activity. Bioinformatics study is performed for understanding the various physical and chemical parameters of the protein which predicts the involvement of zinc encircled by three histidine residues (His94, His96 and His119) at the active site of the protein. Isothermal titration calorimetric-based thermodynamic study on diethyl pyrocarbonate-modified protein recognizes the presence of Zn(2+) in the enzyme moiety. Exothermic to endothermic transition as observed during titration of the protein with Zn(2+) discloses that there are at least two binding sites for zinc within the protein moiety. Addition of Zn(2+) regains the activity of EDTA chelated bioremediase confirming the presence of extra binding site of Zn(2+) in the protein moiety. Revival of folding pattern of completely unfolded urea-treated protein by Zn(2+) explains the participatory role of zinc in structural stability of the protein. Restoration of the λ max in intrinsic fluorescence emission study of the urea-treated protein by Zn(2+) similarly confirms the involvement of Zn in the refolding of the protein. The utility of bioremediase for silica nanoparticles preparation is observed by field emission scanning electron microscopy.

Phosphorylation drives an apoptotic protein to activate antiapoptotic genes: paradigm of influenza A matrix 1 protein function.

During infection, viral proteins target cellular pathways that regulate cellular innate immune responses and cell death. We demonstrate that influenza A virus matrix 1 protein (M1), an established proapoptotic protein, activates nuclear factor-κB member RelB-mediated survival genes (cIAP1, cIAP2, and cFLIP), a function that is linked with its nuclear translocation during early infection. Death domain-associated protein 6 (Daxx) is a transcription co-repressor of the RelB-responsive gene promoters. During influenza virus infection M1 binds to and stabilizes Daxx protein by preventing its ubiquitination and proteasomal degradation. Binding of M1 with Daxx through its Daxx binding motif prevents binding of RelB and Daxx, resulting in up-regulation of survival genes. This interaction also prevents promoter recruitment of DNA methyltransferases (Dnmt1 and Dnmt3a) and lowers CpG methylation of the survival gene promoters, leading to the activation of these genes. Thus, M1 prevents repressional function of Daxx during infection, thereby exerting a survival role. In addition to its nuclear localization signal, translocation of M1 to the nucleus depends on cellular kinase-mediated phosphorylation as the protein kinase C inhibitor calphostin C effectively down-regulates virus replication. The study reconciles the ambiguity of dual antagonistic function of viral protein and potentiates a possible target to limit virus infection.

Rotavirus-encoded nonstructural protein 1 modulates cellular apoptotic machinery by targeting tumor suppressor protein p53.

p53, a member of the innate immune system, is triggered under stress to induce cell growth arrest and apoptosis. Thus, p53 is an important target for viruses, as efficient infection depends on modulation of the host apoptotic machinery. This study focuses on how rotaviruses manipulate intricate p53 signaling for their advantage. Analysis of p53 expression revealed degradation of p53 during initial stages of rotavirus infection. However, in nonstructural protein-1 (NSP1) mutant strain A5-16, p53 degradation was not observed, suggesting a role of NSP1 in this process. This function of NSP1 was independent of its interferon or phosphatidylinositol 3-kinase (PI3K)/AKT modulation activity since p53 degradation was observed in Vero cells as well as in the presence of PI3K inhibitor. p53 transcript levels remained the same in SA11-infected cells (at 2 to 14 h postinfection), but p53 protein was stabilized only in the presence of MG132, suggesting a posttranslational process. NSP1 interacted with the DNA binding domain of p53, resulting in ubiquitination and proteasomal degradation of p53. Degradation of p53 during initial stages of infection inhibited apoptosis, as the proapoptotic genes PUMA and Bax were downregulated. During late viral infection, when progeny dissemination is the main objective, the NSP1-p53 interaction was diminished, resulting in restoration of the p53 level, with initiation of proapoptotic signaling ensuing. Overall results highlight the multiple strategies evolved by NSP1 to combat the host immune response.

Rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of apoptosis during infection.

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca(2+) homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61-83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.

Active participation of cellular chaperone Hsp90 in regulating the function of rotavirus nonstructural protein 3 (NSP3).

Heat shock protein 90 (Hsp90) has been reported to positively regulate rotavirus replication by modulating virus induced PI3K/Akt and NFκB activation. Here, we report the active association of Hsp90 in the folding and stabilization of rotavirus nonstructural protein 3 (NSP3). In pCD-NSP3-transfected cells, treatment with Hsp90 inhibitor (17-N,N-dimethylethylenediamine-geldanamycin (17DMAG)) resulted in the proteasomal degradation of NSP3. Sequence analysis and deletion mutations revealed that the region spanning amino acids 225-258 within the C-terminal eIF4G-binding domain of NSP3 is a putative Hsp90 binding region. Co-immunoprecipitation and mammalian two-hybrid experiments revealed direct interaction of the C-terminal 12-kDa domain of Hsp90 (C90) with residues 225-258 of NSP3. NSP3-Hsp90 interaction is important for the formation of functionally active mature NSP3, because full-length NSP3 in the presence of the Hsp90 inhibitor or NSP3 lacking the amino acid 225-258 region did not show NSP3 dimers following in vitro coupled transcription-translation followed by chase. Disruption of residues 225-258 within NSP3 also resulted in poor RNA binding and eIF4G binding activity. In addition, inhibition of Hsp90 by 17DMAG resulted in reduced nuclear translocation of poly(A)-binding protein and translation of viral proteins. These results highlight the crucial role of Hsp90 chaperone in the regulation of assembly and functionality of a viral protein during the virus replication and propagation in host cells.

Coumarin derivatives inhibit the aggregation of ß-lactoglobulin.

The binding of a small molecule to a protein through non-covalent interactions mainly depends on its size and electronic environment. Such binding can change the stability of the three dimensional protein structure which sometimes may destabilize it to accelerate or to inhibit protein aggregation. Coumarin is a widely used fluorescent dye with several biological applications. Different substituents (electron-donating and electron-withdrawing) at different positions of the coumarin moiety can influence its molecular volume, physical and chemical properties. Here we investigate the effect of such substituents of coumarin on the aggregation of a model protein, beta-lactoglobulin (ß-lg) through a multi spectroscopic approach. It was observed that coumarin methyl ester with an 8-hydroxyl group can inhibit the ß-lg aggregation. This compound can bind the hydrophobic site of beta-lactoglobulin and stabilize a particular protein conformation through the formation of hydrogen bond and hydrophobic interactions. Thus a properly designed compound can inhibit protein-protein interactions through protein-small molecule interactions. Other coumarinoid compounds also are effective in the prevention of thermal aggregation of ß-lg.

Rotavirus nonstructural protein 1 suppresses virus-induced cellular apoptosis to facilitate viral growth by activating the cell survival pathways during early stages of infection.

Following virus infection, one of the cellular responses to limit the virus spread is induction of apoptosis. In the present study, we report role of rotavirus nonstructural protein 1 (NSP1) in regulating apoptosis by activating prosurvival pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and NF-kappaB (nuclear factor kappaB) during early hours of infections (2 to 8 hpi). The NSP1 mutant strain A5-16 induces weak and transient activation of Akt (protein kinase B) and p65 NF-kappaB compared to the isogenic wild-type strain A5-13 in MA104 or HT29 cells. The weak NF-kappaB promoter activity or Akt phosphorylation after A5-16 infection could be complemented in cells transfected with plasmid expressing NSP1 after infection with the rotavirus A5-16 strain. In cells either infected with A5-13 or transfected with pcD-NSP1, coimmunoprecipitation of NSP1 with phosphoinositide 3-kinase (PI3K) was observed, indicating that strong activation of PI3K/Akt could be due to its interaction with NSP1. In addition, after infection with same multiplicity of infection, A5-16 showed reduced number of viral particles compared to the A5-13 strain at the end of the replication cycle. A lower growth rate could be due to weak induction of PI3K/Akt and NF-kappaB, since the A5-13 strain also showed reduced growth in the presence of PI3K or NF-kappaB inhibitors. This effect was interferon independent; however, it was partly due to significantly higher caspase-3 activity, poly-ADP ribose polymerase (PARP) cleavage, and apoptosis during earlier stages of infection with the NSP1 mutant. Thus, our data suggest that NSP1 positively supports rotavirus growth by suppression of premature apoptosis for improved virus growth after infection.

Modulation of amyloid fibrillation of bovine ß-lactoglobulin by selective methionine oxidation.

Deposition of oxidation-modified proteins during normal aging and oxidative stress are directly associated with systemic amyloidoses. Methionine (Met) is believed to be one of the most readily oxidisable amino acid residues of protein. Bovine beta-lactoglobulin (ß-lg), a model globular whey protein, has been presented as a subsequent paradigm for studies on protein aggregation and amyloid formation. Herein, we investigated the effect of t-butyl hydroperoxide (tBHP)-induced oxidation on structure, compactness and fibrillation propensity of ß-lg at physiological pH. Notably, whey protein modification, specifically Met residues, plays an important role in the dairy industry during milk processing and lowering nutritional value and ultimately affecting their technological properties. Several bio-physical studies revealed enhanced structural flexibility and aggregation propensity of oxidised ß-lg in a temperature dependent manner. A molecular docking study is used to predict possible interactions with tBHP and infers selective oxidation of methionine residues at 7, 24 and 107 positions. From our studies, it can be corroborated that specific orientations of Met residues directs the formation of a partially unfolded state susceptible to fibrillation with possible different cytotoxic effects. Our studies have greater implications in deciphering the underlying mechanism of different whey proteins encountering oxidative stress. Our findings are also important to elucidate the understanding of oxidation induced amyloid fibrillation of protein which may constitute a new route to pave the way for a modulatory role of oxidatively stressed proteins in neurological disorders.

In silico fight against novel coronavirus by finding chromone derivatives as inhibitor of coronavirus main proteases enzyme.

Novel coronavirus, 2019-nCoV is a danger to the world and is spreading rapidly. Very little structural information about 2019-nCoV make this situation more difficult for drug designing. Benzylidenechromanones, naturally occurring oxygen heterocyclic compounds, having capability to inhibit various protein and receptors, have been designed here to block mutant variety of coronavirus main protease enzyme (SARC-CoV-2 Mpro) isolated from 2019-nCoV with the assistance of molecular docking, bioinformatics and molecular electrostatic potential. (Z)-3-(4'-chlorobenzylidene)-thiochroman-4-one showed highest binding affinity to the protein. Binding of a compound to this protein actually inhibits the replication and transcription of the virus and, ultimately, stop the virus multiplication. Incorporation of any functional groups to the basic benzylidenechromanones enhances their binding ability. Chloro and bromo substitutions amplify the binding affinity. ADME studies of all these compounds indicate they are lipophilic, high gastro intestine absorbable and blood-brain barrier permeable. The outcome reveals that the investigated benzylidenechromanones can be examined in the case of 2019-nCoV as potent inhibitory drug of SARC-CoV-2 Mpro, for their strong inhibition ability, high reactivity and effective pharmacological properties.

Silver nanoparticle modulates the aggregation of beta-lactoglobulin and induces to form rod-like aggregates.

Silver nanoparticles (SNPs) have been increasingly used in medicines and biomaterials as a drug carriers and diagnostic or therapeutic material due to their smaller size, large surface area and cell penetration ability. Here we report the preparation of SNPs of diameter 10 ±â€¯3 nm by using silver nitrate and sodium borohydride and the interaction of synthesized SNPs with our model protein ß-lactoglobulin (ß-lg) in 10 mM phosphate buffer at pH 7.5 after thermal exposure at 75 °C. Heat exposed ß-lg forms amyloidal fibrillar aggregates whereas this protein aggregates adopt rod-like shape instead of fibrillar structure in presence of SNP under the same conditions. Size of the synthesized SNPs is confirmed by UV-Visible spectroscopy, SEM and TEM. Interactions and subsequent formation of molecular assembly of heat stressed ß-lg with SNP were investigated using Th-T assay and ANS binding assay, DLS, RLS, CD, FT-IR, SEM, TEM. Docking study parallely also support the experimental findings.

Bone marrow stem cells to destroy circulating HIV: a hypothetical therapeutic strategy.

Human immunodeficiency virus (HIV) still poses enigmatic threats to human life. This virus has mastered in bypassing anti retroviral therapy leading to patients' death. Circulating viruses are phenomenal for the disease outcome. This hypothesis proposes a therapeutic strategy utilizing receptor-integrated hematopoietic, erythroid and red blood cells. Here, HIV specific receptors trap circulating viruses that enter erythrocyte cytoplasm and form inactive integration complex. This model depicts easy, effective removal of circulating HIV without any adverse effect.

LC-MS/MS assay for quantitation of enalapril and enalaprilat in plasma for bioequivalence study in Indian subjects.

BACKGROUND: Enalapril (EPL) is an angiotensin-converting enzyme inhibitor for the treatment of hypertension and chronic heart failure. Enalaprilat (EPLT) is an active metabolite that contributes to the overall activity of EPL. AIM: To quantitate EPL along with its metabolite EPLT using LC-MS/MS, a bioanalytical method was developed and validated with tolbutamide in human plasma using a protein precipitation technique. RESULTS: The sensitive and selective method has an LLOQ of 1 ng/ml with a linearity range of 1-500 ng/ml for both EPL and EPLT using 300 µl of plasma without any matrix effect. CONCLUSION: Linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples after oral administration of 20 mg of EPL maleate in healthy volunteers demonstrate applicability to bioavailability/bioequivalence studies.

Insight into the co-solvent induced conformational changes and aggregation of bovine ß-lactoglobulin.

Many proteins form ordered irreversible aggregates called amyloid fibrils which are responsible for several neurodegenerative diseases. ß-lactoglobulin (ß-lg), an important globular milk protein, self-assembles to form amyloid-like fibrils on heating at low pH. The present study investigated the effects of two commonly used organic solvents acetonitrile (MeCN) and antimicrobial preservative benzyl alcohol (BA) on the conformation and self-assembly of ß-lg at ambient condition. Both MeCN and BA induced a concentration-dependent conformational change showing exposure of hydrophobic patches, loss of tertiary structure and higher α-helical structure at moderate concentrations. In the presence of 50-80% (v/v) MeCN and 1.5-3% (v/v) BA further structural transitions from α-helical to non-native ß-sheet structure were observed with a molten globule-like intermediate at 70% MeCN. These non-native ß-sheet structures have high tendency to form aggregates. The formation of ß-lg self-assembly was confirmed by Thioflavin T studies, Congo red assay, Rayleigh scattering and dynamic light scattering analysis. Transmission electron microscopy studies showed amyloid fibril formation in both MeCN and BA. Our results showed that BA enhances the unfolding and self-assembly of ß-lg at much lower concentration than MeCN. Thus solvent composition forces the protein to achieve the non-native structures which are responsible for protein aggregation.

Amyloid fibril formation by ß-lactoglobulin is inhibited by gold nanoparticles.

The endogenous deposition of protein fibrillar aggregates in the tissues is the cause of several neurodisorders. In the present study the native ß-lactoglobulin (ß-lg) has been driven towards amyloid fibrillar aggregates when it was exposed to heat at 75°C for 1h at pH 7.5. The citrate stabilized gold nanoparticle (AuNPs) of 20nm diameter is shown to inhibit the thermal aggregation of ß-lg. The stability of the ß-lg against heat stress was assessed by studying its aggregation at 75°C, either in presence or in absence of AuNPs. AuNPs stabilizes the monomeric and dimeric forms of the ß-lg inhibiting the nucleation and elongation for the formation of higher aggregates. Incubation of ß-lg with AuNPs at 75°C results in the formation of smaller ragged aggregates. Results show that AuNPs possess the capability of inhibiting the formation of amyloid fibrillar aggregates of ß-lg in a concentration-dependent manner and may thus facilitate the refolding into native like structure. AuNPs thus serve as nano-chaperon to inhibit the protein aggregation. Thus chaperon like activity of AuNP may be exploited in the design of rational therapeutics for the neurodegenerative diseases.