Exploring novel antimicrobials targeting bacterial pyruvate formate lyase through pharmacophore based virtual screening

Antibiotic resistance has caused a serious impediment in clinical treatment of bacterial infections. This resistance is developed by the micro-organisms over a specific time interval during which the microbes develop or modify metabolic machineries that cause immunity towards the antimicrobial agents, like the development of antibodies in eukaryotes. Thus, development of new antimicrobials, targeting bacterial metabolic pathways not being touched before, is the only solution to fight against drug resistance bacteria. The current project has attempted to explore novel anti-bacterial that act against Pyruvate formate lyase (PFL), an essential enzyme for bacterial survival, using computational methods. PFL couples glycolytic pathway to Krebs’s cycle by converting Pyruvate to Acetyl CoA so that the latter is utilized for ATPs production. Inhibition of PFL leads to energy shutdown in a bacterial cell. To find novel inhibitors of PFL, a structure based pharmacophore consisting of seven chemical features was used to screen a number of databases. Phytochemical (8856 compounds) and PubChem (61967 compounds) databases showed good scoring. A total of 183 drug candidates were selected and further processed by molecular docking and rescoring programs in AutoDock Vina. The 17 binding energy based shortlisted drug candidates were predicted for their toxicity and drug likeness in AdmetSAR tool. The docked poses were visually analyzed for bonding interactions between the ligand-receptor pair in LIGPLOT. The docked poses, one from Phytochemical and five from PubChem databases, showed a good pharmacophore fit score of range of 88.4 to 85.81 and a high binding energy efficiency of less than 6.0 kJ/mol. All the six ligands also passed the drug toxicity and likeness criteria set by ADMET tool. LIGPLOT analysis of the compound further confirmed good interaction with the binding pocket residues Lys600, Asp 661, Gln 428 and Gln 430

Identification of a Potent Inhibitor Targeting the Spike Protein of Pandemic Human Coronavirus, SARS-CoV-2 by Computational Methods

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is an emerging new viral pathogen that causes severe respiratory disease. SARS-CoV-2 is responsible for an outbreak of COVID-19 pandemic worldwide. As there are no confirmed antiviral drugs or vaccines currently available for the treatment of COVID-19, discovering potent inhibitors or vaccines are urgently required for the benefit of humanity. The glycosylated Spike protein (S-protein) directly interacts with human angiotensin-converting enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD) of S-protein. As the S-protein is exposed to the surface and is essential for entry into the host, the S-protein can be considered as a first-line therapeutic target for antiviral therapy and vaccine development. In-silico screening, docking and molecular dynamics simulation studies were performed to identify repurposing drugs using DrugBank and PubChem library against the RBD of S-protein. The study identified a laxative drug, Bisoxatin (DB09219), which is used for the treatment of constipation and preparation of the colon for surgical procedures. It binds nicely at the S-protein – ACE2 interface by making substantial pi-pi interactions with Tyr505 in the ‘Site 1’ hook region of RBD and hydrophilic interactions with Glu406, Ser494 and Thr500. Bisoxatin consistently binds to the protein throughout the 100 ns simulation. Taken together, we propose that the discovered molecule, Bisoxatin may be a potent repurpose drug to develop new chemical libraries for inhibiting SARS-CoV-2 entry into the host.</p

Rational discovery of a SOD1 tryptophan oxidation inhibitor with therapeutic potential for amyotrophic lateral sclerosis.

Formation of Cu, Zn superoxide dismutase 1 (SOD1) protein inclusions within motor neurons is one of the principal characteristics of SOD1-related amyotrophic lateral sclerosis (ALS). A hypothesis as to the nature of SOD1 aggregation implicates oxidative damage to a solvent-exposed tryptophan as causative. Here, we chart the discovery of a phenanthridinone based compound (Lig9) from the NCI Diversity Set III by rational methods by in silico screening and crystallographic validation. The crystal structure of the complex with SOD1, refined to 2.5 Å, revealed that Lig9 binds the SOD1 β-barrel in the β-strand 2 and 3 region which is known to scaffold SOD1 fibrillation. The phenanthridinone moiety makes a substantial π-π interaction with Trp32 of SOD1. The compound possesses a significant binding affinity for SOD1 and inhibits oxidation of Trp32; a critical residue for SOD1 aggregation. Thus, Lig9 is a good candidate from which to develop a new library of SOD1 aggregation inhibitors through protection of Trp32 oxidation. Communicated by Ramaswamy H. Sarma

Aggregation studies of de-metallated and reduced SOD1^WT and SOD1^A4V on treatment with PB.

Analytical SEC of metallated SOD1WT (a), de-metallated SOD1WT (b), metallated SOD1A4V (c) and de-metallated SOD1A4V (d) in the absence of PB were analyzed under reducing conditions before and after 24 hrs and 48 hrs incubation at 37ºC. The monomer (M), dimer (D), trimer (T), and large aggregate (L) species are shown as green, purple, red, and black dotted lines, respectively. All the experiments were performed for n = 3 biological replicates. (DOCX)</p

Detailed analysis of the four binding sites of PB on SOD1.

The polder density map and the intermolecular interactions of PB1 (shown as orange sticks) on the dimer interface of chains C (shown as a pink cartoon) and D (shown as a blue cartoon) are shown in (a); right and left panels, respectively. The polder map for the other poses on the lateral region of chains B (shown as a golden yellow cartoon), C, and I (shown as a wheat brown cartoon) are shown in (b) and (c) (left panels). The intermolecular interactions of PB2 (purple sticks), PB3 (pink sticks), and PB4 (green sticks) are shown in (b) and (c) (right panels). The Polder maps are contoured at the 3σ level for PB1 and PB2, 3.3σ level for PB3, and 3.5σ level for PB4. The maps are shown as green mesh. The binding site residues are shown as yellow sticks.</p

MST interaction analysis of fluorescently labeled SOD1^WT against PB.

The binding of PB to fluorescently Cys-labeled SOD1WT is quantified in PBS buffer. The concentration of PB varied from 30.5 nM to 250 μM, while the protein SOD1WT was kept constant at 25 nM. MST experiments were conducted at 20% LED excitation and medium MST power at 25 °C. The resulting dose-response curves were fitted, and a binding affinity of Kd = 3.7 ± 1.2 μM (orange circles) was calculated. Error bars indicate the standard error of the mean, SEM (n = 3).</p

List of interactions between PB molecules and SOD1 residues as seen in the crystal structure.

List of interactions between PB molecules and SOD1 residues as seen in the crystal structure.</p

Crystal structure of human SOD1 complex with an unknown ligand.

The surface/cartoon model of the homodimer assembly of SOD1-UNK shows binding from an unknown linear molecule, UNK (shown in the pink sticks), at the SOD1 dimer interface. The molecule lies very close to the intra-disulfide bond formed by the residues C57 and C146. The 2mFo–DFc map is contoured at 1σ level and shown as blue mesh. (DOCX)</p

Aggregation studies of de-metallated and metallated SOD1^A4V on treatment with 60x PB.

Analytical SEC of metallated SOD1A4V (a) and de-metallated SOD1A4V (b) in the absence of PB were analyzed under reducing conditions before and after 24 hrs and 48 hrs incubation at 37ºC. The monomer (M), dimer (D), trimer (T), and large aggregate (L) species are shown as green, purple, red, and black dotted lines, respectively. All the experiments were performed for n = 3 biological replicates. (DOCX)</p