Structure-based identification of potential natural compound inhibitors targeting bacterial cytoskeleton protein FtsZ from Acinetobacter baumannii by computational studies.

Acinetobacter baumannii is one of the multi-drug-resistant pathogens responsible for hospital-acquired infections reported worldwide. Clinically it is challenging to treat these pathogens as they have developed resistance against the existing class of antibiotics. Hence, there is an urgent need to develop a new class of antibiotics against these pathogens to prevent the spread of infections and mortality. In Acinetobacter baumannii, the filamentous temperature-sensitive mutant Z protein polymerizes at the imminent division site to form a Z-ring at the mid-point of the cell and act as a scaffold to recruit other cell division proteins involved in orchestrating septum synthesis in bacteria. Perturbation in the assembly of FtsZ affects bacterial cell dynamics and survival. Hence, FtsZ has emerged as a new drug target in antibiotic discovery to identify compounds that inhibit bacterial cell division. In this study, we have performed a virtual screening of 30,000 compounds from the ZINC Biogenic natural compound library targeting the nucleotide-binding site of FtsZ from Acinetobacter baumannii. We have identified 8 new natural compounds with binding energy in the range of -8.66 to -6.953 kcal/mol and analyzed them by 200 ns molecular dynamics simulations. Out of these eight compounds, ZINC14708526 showed the best binding with relatively optimal drug-likeness and medicinal chemistry as a potent inhibitor of abFtsZ. Thus, the identified FtsZ inhibitor ZINC14708526 is a promising lead compound to develop potent antimicrobial agents against Acinetobacter baumannii infections.Communicated by Ramaswamy H. Sarma.

Structural Modelling of Platelet Activating Factor Acetyl Hydrolase in Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei: Implications on Therapeutics for Leishmaniasis, Chagas Disease, and Sleeping Sickness.

Purpose: Leishmaniasis, Chagas disease, and sleeping sickness are caused by protozoa Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei, respectively. Platelet activating factor acetyl hydrolase (PAF-AH) is an inflammatory protein implicated in pathogenesis of these three infections, thereby making them attractive drug targets. Methods: PAF-AH sequences were retrieved from UniProt and aligned using Clustal Omega. Homologous models of parasitic proteins were built based on crystal structure of human PAF-AH and validated using PROCHECK server. Volumes of substrate-binding channel were calculated using the ProteinsPlus program. High throughput virtual screening using Glide program in Schrodinger was done with ZINC drug library against parasitic PAF-AH enzymes. Complexes with best hits were energy-minimized and subjected to 100 ns molecular dynamic simulation and analyzed. Results: PAF-AH enzyme sequences from protozoa Leishmania donovani, Trypanosoma cruzi, Trypanosoma brucei, and human have a minimum of 34% sequence similarity with each other. Corresponding structures show a globular conformation consisting of twisted ß-pleated sheets, flanked by α-helices on either side. Catalytic triad of serine-histidine-aspartate is conserved. Substrate-binding channel residues are conserved to an extent, with a lower channel volume in human as compared to target enzymes. Drug screening resulted in identification of three molecules that had better affinities than the substrate to the target enzymes. These molecules fulfill Lipinski's rules for drug likeness and also bind with less affinity to the human counterpart, thereby establishing a high selective index. Conclusion: Structures of PAF-AH from protozoan parasites and humans belong to the same family of enzymes and have a similar three-dimensional fold. However, they show subtle variations in residue composition, secondary structure composition, substrate-binding channel volume, and conformational stability. These differences result in certain specific molecules being potent inhibitors of the target enzymes while simultaneously having weaker binding to human homologue.

Association of Hsp90 with p53 and Fizzy related homolog (Fzr) synchronizing Anaphase Promoting Complex (APC/C): An unexplored ally towards oncogenic pathway.

The intricate molecular interactions leading to the oncogenic pathway are the consequence of cell cycle modification controlled by a bunch of cell cycle regulatory proteins. The tumor suppressor and cell cycle regulatory proteins work in coordination to maintain a healthy cellular environment. The integrity of this cellular protein pool is perpetuated by heat shock proteins/chaperones, which assist in proper protein folding during normal and cellular stress conditions. Among these versatile groups of chaperone proteins, Hsp90 is one of the significant ATP-dependent chaperones that aid in stabilizing many tumor suppressors and cell cycle regulator protein targets. Recently, studies have revealed that in cancerous cell lines, Hsp90 stabilizes mutant p53, 'the guardian of the genome.' Hsp90 also has a significant impact on Fzr, an essential regulator of the cell cycle having an important role in the developmental process of various organisms, including Drosophila, yeast, Caenorhabditis elegans, and plants. During cell cycle progression, p53 and Fzr coordinately regulate the Anaphase Promoting Complex (APC/C) from metaphase to anaphase transition up to cell cycle exit. APC/C mediates proper centrosome function in the dividing cell. The centrosome acts as the microtubule organizing center for the correct segregation of the sister chromatids to ensure perfect cell division. This review examines the structure of Hsp90 and its co-chaperones, which work in synergy to stabilize proteins such as p53 and Fizzy-related homolog (Fzr) to synchronize the Anaphase Promoting Complex (APC/C). Dysfunction of this process activates the oncogenic pathway leading to the development of cancer. Additionally, an overview of current drugs targeting Hsp90 at various phases of clinical trials has been included.

Screening of plant-based natural compounds as an inhibitor of FtsZ from Salmonella Typhi using the computational, biochemical and in vitro cell-based studies.

Salmonella Typhi is emerging as a drug-resistant pathogen, particularly in developing countries. Hence, the progressive development of new antibiotics against novel drug targets is essential to prevent the spread of infections and mortality. The cell division protein FtsZ is an ideal drug target as the cell wall synthesis in bacteria is driven by the dynamic treadmilling nature of the FtsZ. The polymerization of the FtsZ provides the essential mechanical constricting force and flexibility to modulate the cell wall synthesis. Any alteration in FtsZ polymerization leads to the bactericidal or bacteriostatic effect. In this study, we have evaluated the secondary metabolites of natural compounds berberine chloride, cinnamaldehyde, scopoletin, quercetin and eugenol as potential inhibitors of FtsZ from Salmonella Typhi (stFtsZ) using computational, biochemical, and in vivo cell-based assays. Out of these five compounds, berberine chloride and cinnamaldehyde exhibited the best binding affinity of Kd = 7 µM and 10 µM, respectively and inhibit stFtsZ GTPase activity and polymerization by 70 %. The compound berberine chloride showed the best MIC of 500 µg/mL and 175 µg/mL against gram-negative and gram-positive bacterial strains. The findings support that these natural compounds can be used as a backbone structure to develop a broad spectrum of antibacterial agents.

Comparative Structural Analysis of Human ACE2 Receptor with Spike Protein of SARS-CoV-2 Variants: Implications to Understand Infectivity of the Virus.

Purpose: Spike protein on SARS-CoV-2 virus plays an integral part during infection as cell entry depends on binding of this protein to human ACE2 receptor. Understanding of infectivity by these variants necessitates a comparative structural analysis of complexes of spike protein-receptor binding domain (RBD) of these variants to receptor. Methodology: Wild type SARS-CoV-2 spike protein sequence was retrieved from the UniProt database, and mutations of five variants at receptor binding domain were manually incorporated and aligned using Clustal Omega. Crystal structure complexes of human ACE2 receptor with spike protein RBD domain of SARS-CoV-2 variants of wild type, α, ß, and δ were extracted from the RCSB database. Wild type SARS-CoV-2 complex with receptor was used as template to generate model complexes of receptor with spike protein RBD of γ and omicron variants through WinCoot program. These were energy minimized and validated and molecular dynamic simulation was performed using Desmond simulation program. Results: Mutations are distributed across the entire length of RBD, but the maximum number of mutations are seen at 11 positions within binding interface motifs of six variant sequences. Interface of spike protein RBDs with human ACE2-receptor shows different mix of hydrogen bonded and ionic interactions. Alpha and ß variants have few interactions, while γ and δ variants have higher number of interactions compared to wild type variant. Omicron variant, with 10 polar interactions including two ionic bonds, has the highest binding energy. Conclusion: Different mutations on RBD of spike protein results in varying quantity and quality of interactions, thereby affecting potency of each variant. Variations in binding are due to interactions of mutant residues and induced conformational changes on loops of RBDs. Variants α and ß have a low potency, while, γ, δ, and omicron have a higher potency. These results correlate with viral infectivity and place clinical observations in the right perspective.

Deciphering the mechanism of p73 recognition of p53 response elements using the crystal structure of p73-DNA complexes and computational studies.

p73 belongs to p53 family transcription factor activating more than 50% of cell fate p53 target genes involved in cell cycle, apoptosis, DNA damage response alongside neuronal system development and differentiation by binding to 20-bp response elements (REs) having sequence motif (PPPC-A/T-T/A-GYYY) where P-purines and Y-pyrimidines with each 10-bp separated by minimum 0 to 13-bp spacer. The promiscuous nature of recognizing both cell fate and development genes and the underlying RE selectivity mechanism by p73 is not well understood. Here, we report the molecular details of p73 recognizing the REs using the crystal structure of p73 DNA binding domain (DBD) in complex with 12 base pair DNA sequence 5'-cAGGCATGCCTg-3' and molecular dynamics simulations with six different p53 natural promoter sequences. Each 20-base pair natural promoter forms a different major/minor groove due to the presence of nucleotides A/T, A/C, G/G, T/T and G/T at positions 3, 8, 13, 18 uniquely recognized by p73 key residues Lys138 and Arg268. The loops L1 and L3 bearing these residues influence inter-and intra-dimer interfaces interactions and hence p73 forms a unique tetramer with each natural promoter sequence. Structural features of the DNA and the spacing between half-sites influence p73 tetramerization and its transactivation function.

Structural modeling of Omicron spike protein and its complex with human ACE-2 receptor: Molecular basis for high transmissibility of the virus.

Omicron is a new variant of SARS-CoV-2, which is currently infecting people around the world. Spike glycoprotein, an important molecule in pathogenesis of infection has been modeled and the interaction of its Receptor Binding Domain with human ACE-receptor has been analysed by simulation studies. Structural analysis of Omicron spike glycoprotein shows the 30 mutations to be distributed over all domains of the trimeric protein, wherein the mutant residues are seen to be participating in higher number of intra-molecular interactions including two salt bridges emanating from mutant residues thereby stabilizing their conformation, as compared to wild type. Complex of Receptor Binding Domain (RBD) with human ACE-2 receptor shows seven mutations at interacting interface comprising of two ionic interactions, eight hydrogen bonds and seven Van der Waals interactions. The number and quality of these interactions along with other binding biophysical parameters suggests more potency of RBD domain to the receptor as compared to the wild type counterpart. Results of this study explains the high transmissibility of Omicron variant of SARS-CoV-2 that is currently observed across the world.

Structural analysis of COVID-19 spike protein in recognizing the ACE2 receptor of different mammalian species and its susceptibility to viral infection.

The pandemic COVID-19 was caused by a novel Coronavirus-2 (SARS-CoV-2) that infects humans through the binding of glycosylated SARS-CoV-2 spike 2 protein to the glycosylated ACE2 receptor. The spike 2 protein recognizes the N-terminal helices of the glycosylated metalloprotease domain in the human ACE2 receptor. To understand the susceptibility of animals for infection and transmission, we did sequence and structure-based molecular interaction analysis of 16 ACE2 receptors from different mammalian species with SARS-CoV-2 spike 2 receptor binding domain. Our comprehensive structure analysis revealed that the natural substitution of amino acid residues Gln24, His34, Phe40, Leu79 and Met82 in the N-terminal α1 and α2 helices of the ACE2 receptor results in loss of crucial network of hydrogen-bonded and hydrophobic interactions with receptor binding domain of SARS-CoV-2 spike protein. Another striking observation is the absence of N-glycosylation site Asn103 in all mammals and many species, lack more than one N-linked glycosylation site in the ACE2 receptor. Based on the loss of crucial interactions and the absence of N-linked glycosylation sites we categorized Felis catus, Equus caballus, Panthera tigris altaica, as highly susceptible while Oryctolagus cuniculus, Bos Tauras, Ovis aries and Capra hircus as moderately susceptible species for infection. Similarly, the E. asinus, Bubalus bubalis, Canis lupus familiaris, Ailuropoda melaleuca and Camelus dromedarius are categorized as low susceptible with Loxodonta Africana, Mus musculus, Sus scrofa and Rattus rattus as least susceptible species for SARS-CoV-2 infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02599-2.