Fluorescence probing and molecular docking analysis of the binding interaction of bovine serum albumin (BSA) with the polarity probe AICCN.

In this work, the fluorescent probe 2-amino-4-(1H-indol-3-yl)-4H-chromene-3-carbonitrile (AICCN) has been used to evaluate its potential as a prospective polarity probe. From detailed fluorescence studies of the probe, it could be shown that AICCN can indeed function as an effective polarity probe. The calculated dipole moments of AICCN in both the ground state and excited state in various solvents lend support to the steady state fluorescence results. It was also shown that AICCN can be used to probe the micropolarity of micelles and can be used successfully for the determination of CMC of the surfactants. The binding process of the probe AICCN to BSA has been followed by plotting the binding isotherms and Scatchard Plots. The time-resolved fluorescence data indicate that the preferred binding site of AICCN in BSA lies close to the buried Trp residue Trp-213 in Domain II. This contention is further supported by the molecular docking studies. The interaction study of the probe AICCN with proteins is relevant for future use of AICCN as a hydrophobic drug. Information was also obtained about the effect of probe binding on the serum albumin structure, which may be correlated to its physiological activity. Thus, the probe AICCN can serve not only as a good reporter of polarity of the microenvironment in biological systems but also as an efficient fluorophore to monitor conformational changes in proteins in future.

A drug repurposing endeavor to discover a multi-targeting ligand against RhlR and LasR proteins from opportunistic human pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen. It synthesizes the poison called Hydrogen Cyanide (HCN). The synthesis of HCN is mediated by the enzyme HCN synthase which is obtained from the hcnABC operon and the transcription of the hcnABC operon is mediated by three proteins LasR, RhlR, and ANR. In our previous works, we analyzed the activation process of RhlR and LasR proteins by their cognate auto-inducer ligands (N-butanoyl-L-homoserine lactone and N-(3-oxododecanoyl)-homoserine lactone respectively). In this work, we attempted to identify some multi-targeting ligands which would be able to destroy the structural integrity of both the RhlR and LasR proteins using steered MD simulations. We used the virtual screening of ligand libraries, and for that purpose, we used the NCI drug database. We selected the top 4 ligands from our virtual screening experiments. We then tried to check their relative binding affinities with the LasR and RhlR proteins in comparison to their native auto-inducer ligands. Through this work, we were able to identify 4 such ligands which were capable of binding to both the RhlR and LasR proteins in a better way than their native auto-inducer ligands. The efficacies of these ligands to actually perturb the structural integrity of RhlR and LasR proteins could be tested in wet lab. The work is the first work in the field of structure-based drug design to come up with possible multi-targeting drug-like structures against the RhlR and LasR proteins from Pseudomonas aeruginosa.

Anticancer, Antibacterial, Antioxidant, and DNA-Binding Study of Metal-Phenalenyl Complexes.

Phenalenyl (PLY)-based metal complexes are a new addition to the metal complex family. Various applications of metal-based phenalenyl complexes (metal-PLY) have been reported, such as catalyst, quantum spin simulators, spin electronic devices, and molecular conductors, but the biological significance of metal-PLY (metal = Co(II), Mn(III), Ni(II), Fe(III), and Al(III)) systems has yet to be explored. In this study, the anticancer properties of such complexes were investigated in ovarian cancer cells (SKOV3 and HEY A8), and the cytotoxicity was comparable to that of other platinum-based drugs. Antibacterial activity of the metal-PLY complexes against both gram-negative (E. coli) and gram-positive (S. aureus) bacteria was studied using a disk diffusion test and minimum inhibitory concentration (MIC) methods. All five metal-PLY complexes showed significant antibacterial activity against both bacterial strains. The antioxidant properties of metal-PLY complexes were evaluated following the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging method and were acceptable. The DNA-binding properties of these metal-PLY complexes were investigated using absorption spectroscopy, fluorescence spectroscopy, viscosity measurements, and thermal denaturation methods. Experimental evidence revealed that the complexes bind to DNA through intercalation, and the molecular docking study supported this conclusion.

A novel plant lectin, NTL-125, interferes with SARS-CoV-2 interaction with hACE2.

COVID-19 caused by SARS-CoV-2 virus has had profound impact on the world in the past two years. Intense research is going on to find effective drugs to combat the disease. Over the past year several vaccines were approved for immunization. But SARS-CoV-2 being an RNA virus is continuously mutating to generate new variants, some of which develop features of immune escape. This raised serious doubts over the long-term efficacy of the vaccines. We have identified a unique mannose binding plant lectin from Narcissus tazetta bulb, NTL-125, which effectively inhibits SARS-CoV-2 replication in Vero-E6 cell line. In silico docking studies revealed that NTL-125 has strong affinity to viral Spike RBD protein, preventing it from attaching to hACE2 receptor, the gateway to cellular entry. Binding analyses revealed that all the mutant variants of Spike protein also have stronger affinity for NTL-125 than hACE2. The unique α-helical tail of NTL-125 plays most important role in binding to RBD of Spike. NTL-125 also interacts effectively with some glycan moieties of S-protein in addition to amino acid residues adding to the binding strength. Thus, NTL-125 is a highly potential antiviral compound of natural origin against SARS-CoV-2 and may serve as an important therapeutic for management of COVID-19.

Omeprazole prevents stress induced gastric ulcer by direct inhibition of MMP-2/TIMP-3 interactions.

The healing of damaged tissues in gastric tract starts with the extracellular matrix (ECM) remodeling by the action of matrix metalloproteinases (MMPs). Particularly, MMP-2 (gelatinase-A) maintains ECM structure and function by degrading type IV collagen, the major component of basement membranes and by clearing denatured collagen. The proteolytic activities of MMPs are critically balanced by endogenous tissue inhibitors of metalloproteinases (TIMPs) and disruption of this balance results in several diseases. The well-known drug omeprazole is a proton pump inhibitor used for curing gastric ulcer. However, the action of omeprazole in ECM remodeling on gastroprotection has never been explored. Herein, using rat model of gastric ulcer, we report that restraint cold stress caused increase apoptosis to surface epithelia of gastric tissues along with TIMP-3 upregulation and inhibition of MMP-2 activity thereon. In contrast, omeprazole treatment suppressed TIMP-3 while increasing MMP-2 activity and thereby, restoring MMP-2/TIMP-3 balance. Additionally, nanomolar binding constant (Kd = 318 nM) of omeprazole with purified MMP-2 indicates a direct effect of omeprazole in restoring MMP-2 activity. Further in silico simulations revealed a plausible mechanism of action of omeprazole for TIMP-3 deactivation. Altogether, omeprazole restores MMP-2 activity and reduces apoptosis while preventing acute stress-induced gastric ulcer that occurs via suppression of nuclear factor kappa B (NF-κB) activity and peroxisome proliferator-activated receptor gamma activity (PPAR-γ). This represents an unprecedented correlation between physical docking of drug molecule to a protease and the severity of organ injury and provides a novel therapeutic approach to prevent stress induced tissue damage.

Identification of the binding interactions of some novel antiviral compounds against Nsp1 protein from SARS-CoV-2 (COVID-19) through high throughput screening.

The SARS-CoV-2 pandemic has become a global threat. It has become very difficult to control the spreading of the virus. The virus is a RNA virus and the virulence of the virus is mediated by three virulence causing proteins, viz., Nsp1, Nsp3c and ORF7. So far the drug designing endeavors against the virus have been being targeted towards the spike protein which is responsible for the entry of the virus inside human host as well as the RNA dependent RNA polymerase. However, no effective treatment against the virus has so far been developed. In the present situation, an attempt has been made to target the virulence protein factor Nsp1 which binds to the 40S ribosomal subunit of the human host. We tried to target the Nsp1 by in-silico virtual screening of ligand libraries. We built the three dimensional structure of Nsp1 and used the structure to screen the ChEMBL drug library. We used molecular docking simulations of the top6 screened ligands with Nsp1 and subjected the liagnd-Nsp1 complexes to molecular dynamics simulations to analyze the behaviors of the ligands in a virtual cell. From our analysis we could predict that the ligands bearing the ChEMBL identifiers, CHEMBL1096281, CHEMBL2022920, CHEMBL175656, had the best binding affinity values with Nsp1. Therefore, these ligand molecules may be tested in wet-lab for further analysis. This is the first report to target the virulence factor Nsp1 from SARS-CoV-2. Communicated by Ramaswamy H. Sarma.

Engineered Bio-inspired Multifunctional Peptide- and Protein-based Therapeutic Biomolecules for Better Wound Care.

Developing non-immunogenic therapeutic biomolecules for facilitating blood clotting followed by wound healing via therapeutic angiogenesis, still remains a formidable challenge. Excessive blood loss of accident victims and battalions cause a huge number of deaths worldwide. Patients with inherited bleeding disorders face acute complications during injury and post-surgery. Biologically-inspired peptide-based hemostat can act as a potential therapeutic for handling coagulopathy. Additionally, non-healing wounds for patients having ischemic diseases can cause severe clinical complications. Advancement in stabilized growth-factor-based proangiogenic therapy may offer effective possibilities for the treatment of ischemic pathology. This review will discuss nature-inspired biocompatible stabilized peptide- and protein-based molecular medicines to serve unmet medical challenges for handling traumatic coagulopathy and impaired wound healing.

Structural Characterization of the Hidden Peptide SHPRH-146aa Encoded by Non-Coding circ-SHPRH to Act as Tumor Suppressor.

Circular RNAs belong to the class of non-coding RNA molecules, though surprisingly some of them have protein-coding potentials. However, the circular RNA circ-SHPRH is known to code for an unusual protein known as SHPRH-146aa. However, the molecular level details of the protein are not yet identified. It was proposed that the protein has its role in glioblastoma. Therefore, in this work, an attempt was made to decipher the various structural features of SHPRH-146aa. The binding interactions of the protein SHPRH-146aa with its partner protein DTL were also analyzed. The main aim of the work was to decipher the characteristics features of this unusual protein and the region on SHPRH-146aa that would form different types of non-covalent binding interactions both among itself as well as with its binding partner. In this work, we tried to elucidate the various structural and physico-chemical features of the protein as well as its mode of interactions with its binding partner. The study would therefore pave the pathway to design future wet lab experiments to delineate the appropriate structural features of the protein as well as its association with glioblastoma and neuro-degenerative diseases.

Elucidation of the hetero-dimeric binding activity of LasR and RhlR proteins with the promoter DNA and the role of a specific Phe residue during the biosynthesis of HCN synthase from opportunistic pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen. It causes secondary infections in patients suffering from cancer and other immunological disorders. The pathogenicity of the organism is dependent on the ability of the organism to code for hydrogen cyanide (HCN), the synthesis of which is mediated by HCN synthase enzyme. HCN synthase is encoded by hcnABC operon. The transcription of the operon is controlled by a complex interplay between the proteins LasR and RhlR. Till date, there is no report that deals with the binding interactions of the RhlR-LasR heterodimer with the promoter DNA region of the hcnABC operon. We, for the first time, tried to analyse the binding modes of the RhlR-LasR heterodimer with the promoter DNA regions. From our work, we could predict the importance of a specific amino acid residue Phe214 from RhlR which might be considered to have the desired specificity to bind to the promoter DNA. Therefore, the amino acid Phe214 may be targeted to develop suitable ligands to eradicate the spread of secondary infections by Pseudomonas aeruginosa.

Repurposed drugs and nutraceuticals targeting envelope protein: A possible therapeutic strategy against COVID-19.

COVID-19 pandemic caused by SARS-CoV-2 has already claimed millions of lives worldwide due to the absence of a suitable anti-viral therapy. The CoV envelope (E) protein, which has not received much attention so far, is a 75 amino acid long integral membrane protein involved in assembly and release of the virus inside the host. Here we have used artificial intelligence (AI) and pattern recognition techniques for initial screening of FDA approved pharmaceuticals and nutraceuticals to target this E protein. Subsequently, molecular docking simulations have been performed between the ligands and target protein to screen a set of 9 ligand molecules. Finally, we have provided detailed insight into their mechanisms of action related to the varied symptoms of infected patients.

Design, synthesis and biological evaluation of vortioxetine derivatives as new COX-1/2 inhibitors in human monocytes.

In order to identify a suitable alternative to non-steroidal anti-inflammatory drugs (NSAIDs) we aimed to develop derivatives of vortioxetine, a multimodal anti-depressive drug that has been shownpreviously to be endowed withanti-inflammatory activity in human monocytes/macrophages. Vortioxetine (1) was synthesized in good yield and different alkyl and aryl derivatives were prepared based on their structural diversity and easy availability. The compounds were tested on human monocytes isolated from healthy donors for theireffect on superoxide anion production and cytokine gene expression, and for COX-1/2 gene expression and activity modulation. Moreover, a docking study was performed to predict the interactions between the synthesized compounds and COX-1 and COX-2. Correlating experimental biological data to the molecular modelling studies, it emerged that among the novel compounds, 6 was endowed of antioxidant and anti-COX-1 activity, vortioxetine and 3 were good antioxidants and mild anti-COX-1/2 inhibitors, while 7 was a good anti-COX-1/2 inhibitor and 11 was more specific versus COX-2.

Removal of an atypical region from a staphylococcal cyclophilin affects its structure, function, stability, and shape.

SaCyp, a cyclophilin having 197 amino acid residues, acts both as a protein-folding catalyst and a virulence factor in Staphylococcus aureus. Interestingly, a region, homologous to the SaCyp region carrying 121-148 amino acid residues, is present in many putative cyclophilins but absent in well-studied cyclophilins. To determine the exact roles of this unusual region in SaCyp and related proteins, we have investigated a deletion mutant (rCypΔ) of a recombinant SaCyp (rCyp) using various probes. The data reveal that rCypΔ has significantly less catalytic activity and possesses altered structure and hydrophobic surface compared to rCyp. Conversely, the deletion substantially increased inhibitor binding affinity and altered the shape of rCyp. However, both proteins were unfolded by a non-two-state mechanism in the presence of urea. Additionally, the stability of rCyp was significantly reduced due to the deletion of the residues 121-148. Our MD simulation study also indicated the considerable alteration in structure, shape, and fluctuations of SaCyp due to the removal of the region carrying 121-148 residues. Hence, the atypical region located in SaCyp might be vital for maintaining its unique structure, function, stability, and shape.

Structure based virtual screening of natural products to disrupt the structural integrity of TRAF6 C-terminal domain homotrimer.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ligase which takes part in different cellular pathways. TRAF6 is seen to be highly expressed in various cancers and most importantly is known to drive cancer metastasis. This makes TRAF6 a potential therapeutic target. In our previous studies, we observed that the C-terminal domain of TRAF6 forms a mushroom shaped trimer structure. Lys340 and Glu345 were identified to be the most critical residues in the trimer interface. In this current work, we screened for more than 14000 small molecules derived from various natural sources and they were screened against TRAF6 C-terminal trimer interaction interface to prevent the formation of the interface. All the obtained molecules were tested for their drug-likeliness properties. The ligands which qualified the filter were considered for protein-ligand docking or structure based virtual screening in GOLD 5.2. Pose selection was carried out on the basis of GoldScore and ChemScore function of GOLD 5.2. Top 20 molecules binding the TRAF6 trimeric interface were tested for their ADME properties. From the top 20 molecules, top 3 ligands were chosen based on their abilities to pass the maximum numbers of ADME filters.

Identification and analyses of natural compounds as potential inhibitors of TRAF6-Basigin interactions in melanoma using structure-based virtual screening and molecular dynamics simulations.

The interaction of the proteins, tumor necrosis factor receptor-associated factor6 (TRAF6) and Basigin (CD147), is known to be associated with the over-expression of matrix metalloproteinases (MMPs) in melanoma cells. MMPs are known to be responsible for melanoma metastasis. Hence, the TRAF6-Basigin complex can act as a potential therapeutic target. In previous studies, amino acid residues Lys340, Lys 384, Glu417 and Glu511 of TRAF6 were identified as the most vital residues on the basis of their contributions to interaction energy, relative solvent accessibility and electrostatic interactions in the TRAF6-Basigin protein-protein interaction (PPI) scheme. In our current work, we performed structure-based virtual screenings of some natural compounds obtained from ZINC database (n = 14509) to search for molecules which can act as inhibitors against the formation of TRAF6-Basigin complex. Three potential inhibitors were identified which were observed to make intermolecular interactions with Lys384 and Glu511 of TRAF6. Molecular dynamics simulation results suggested the substantial pharmacological importance of the ligand molecules as it was observed that there was total destabilization of TRAF6-Basigin complex upon binding of the molecule ZINC02578057. From our studies, we could conclude that the ligands termed as ZINC49048033, ZINC02578057 and ZINC72320240 could have great potentials to act as inhibitors to prevent melanoma metastasis.

Identification of ligand binding activity and DNA recognition by RhlR protein from opportunistic pathogen Pseudomonas aeruginosa-a molecular dynamic simulation approach.

RhlR protein from opportunistic pathogen Pseudomonas aeruginosa is involved in the transcription of virulence genes of the organism. The RhlR protein functions as a dimer and binds to the cognate promoter DNA with the help of an autoinducer ligand BHL to initiate the transcription of the virulence genes. Till date, there are no reports that detail the mechanism of virulence gene expression by RhlR protein in P. aeruginosa. In this work, we tried to analyse the molecular aspects of the various binding interactions of the RhlR protein while formimg the dimmer as well as with the promoter DNA. We analysed the mode of dimerisation of the RhlR protein and its binding interactions with the autoinducer BHL ligand. From our analyses, we could identify the potential amino acid residues which are involved in the binding interactions. We also predicted how the autoinducer BHL would help in making contacts with the DNA as well as with itself. Thus, the autoinducer BHL would serve as an important mediator of molecular interactions involved in binding the RhlR protein to itself as well as with the promoter DNA. Therefore, any other molecule which would be able to compete with the autoinducer ligand BHL to bind to RhlR protein but would not let the RhlR protein bind the promoter DNA would be an ideal drug candidate to prevent the transcription process of the virulence genes in P. aeruginosa. Our future aim is to predict suitable ligands which would compete with BHL to thwart the transcription process.

Structural Insight into the Gene Expression Profiling of the hcn Operon in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common opportunistic human pathogen. It generally attacks immunosuppressed patients like AIDS, cancer, cystic fibrosis, etc. The virulence of P. aeruginosa is mediated by various virulence factors. One of such potential virulence factors is HCN synthesized by HCN synthase enzyme, which is encoded by the hcnABC operon. The expressions of the genes of this operon are regulated by three transcriptional regulators, viz., LasR, ANR, and RhlR. In our previous work, we analyzed the molecular details of the functionalities of LasR. In this work, we focused on ANR. ANR is a regulatory protein which belongs to the FNR family and works in anaerobic condition. ANR binds to the promoter DNA, named ANR box, as a dimer. The dimerization of this ANR protein is regulated by Fe4S4, an iron-sulfur cluster. This dimer of ANR (ANR-Fe4S4/ANR-Fe4S4) recognizes and binds the promoter DNA sequence and regulates the transcription of this hcnABC operon. Till date, the biomolecular details of the interactions of ANR dimer with the promoter DNA are not fully understood. Thus, we built the molecular model of ANR-Fe4S4/ANR-Fe4S4. We docked the complex with the corresponding promoter DNA region. We analyzed the mode of interactions with the promoter DNA under different conditions. Thus, we tried to analyze the functionality of the ANR protein during the expressions of the genes of the hcnABC operon. So far, this is the first report to detail the molecular mechanism of the gene expression in P. aeruginosa.

Molecular insight into the activity of LasR protein from Pseudomonas aeruginosa in the regulation of virulence gene expression by this organism.

Pseudomonas aeruginosa is an opportunistic human pathogen. This organism attacks human patients suffering from diseases like AIDS, cancer, cystic fibrosis, etc. One of the important virulent factors produced by this organism is Hydrogen Cyanide. This is expressed from the genes encoded by the hcnABC operon. The expressions of the genes encoded by hcnABC operon are mediated mainly by the interactions of LasR protein with the corresponding promoter region of the hcnABC operon. The LasR protein acts as a dimer and binds to the promoter DNA with the help of an autoinducer ligand. However, till date the detailed molecular mechanism of how the LasR protein interacts with the promoter DNA is not clearly known. Therefore, in this work, an attempt has been made to analyze the mode of interactions of the LasR protein with the promoter DNA region of the hcnABC operon. We analyzed the three dimensional structure of the LasR protein from Pseudomonas aeruginosa and docked the protein with the autoinducer ligand. We then docked the ligand-bound-LasR-protein as well the LasR-protein-without-the-autoinducer-ligand on to the promoter DNA region of hcnABC operon. We analyzed the details of the interaction profiles of LasR protein with the autoinducer ligand. We also deciphered the details of the LasR promoter-DNA interactions. We compared the modes of DNA bindings by the LasR protein in presence and absence of the autoinducer ligand and tried to analyze the molecular details of the binding of LasR protein with the promoter DNA region of hcnABC operon during hcnABC gene expression. This study may therefore pave the pathway for future experiments to determine the relative effects of the amino acid residues of LasR protein in DNA binding during the transcription of hcnABC operon.