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.

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.

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.

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.

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.

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.