Investigating single amino acid substitutions in PIM1 kinase: A structural genomics approach.

PIM1, is a serine/threonine proto-oncogene kinase, involved in many biological functions, including cell survival, proliferation, and differentiation, thus play a key role in oncogenesis. It plays a crucial role in the onset and progression of various hematopoietic and non-hematopoietic malignancies, including acute myeloid leukemia and prostate cancer. Mutations in PIM1, especially in its kinase domain, can induce abnormal structural changes and thus alter functionalities that can lead to disease progression and other complexities. Herein, we have performed an extensive analysis of the PIM1 mutations at sequence and structure level while utilizing state-of-the-art computational approaches. Based on the impact on PIM1, numerous pathogenic and destabilizing mutations were identified and subsequently analyzed in detail. Finally, two amino acid substitutions (W109C and F147C) in the kinase domain of PIM1 were selected to explore their impact on the PIM1 structure in a time evolution manner using all-atom molecular dynamics (MD) simulations for 200 ns. MD results indicate significant conformational altercations in the structure of PIM1, especially upon F147C mutation. This study provides a significant insight into the PIM1 dysfunction upon single amino acid substitutions, which can be utilized to get insights into the molecular basis of PIM1-associated disease progression.

Exploring insights of syntaxin superfamily proteins from Entamoeba histolytica: a prospective simulation, protein-protein interaction, and docking study.

Entamoeba histolytica (Eh), a parasitic protozoan and the causative agent of invasive Amoebiasis, invade the host tissue through an effective secretory pathway. There are several lines of evidence suggesting that amoebic trophozoite pore-forming complex amoebapore and a large class of proteases enzymes including rhomboid proteases, cysteine proteases, and metalloproteases are implicated in host tissue invasion. For successful delivery of these molecules/cargos, trophozoites heavily rely on sorting machinery from the endoplasmic reticulum, Golgi to plasma membrane. Although, sole secretion machinery in E. histolytica is not characterized yet. Therefore, here our aim is to understand the properties of key molecules N-ethylmaleimide-sensitive fusion protein attached to protein receptors (SNAREs) in E. histolytica. SNAREs proteins are an important component of the membrane-trafficking machinery and have been associated in a range of processes including vesicle tethering, fusion as well as specificity of vesicular transport in all eukaryotic cells. SNARE proteins are architecturally simple, categorized by the presence of one copy of a homologous coiled-coil forming motif. However, the structural information and protein-protein interaction study of Eh-associated syntaxin proteins are still not known. Here, we characterize the syntaxin 1 like molecule and VAMP from Eh through physiochemical profiling, modeling, atomistic simulation, protein-protein interaction, and docking approaches on the proteins containing SNARE and synaptobrevin domain. The modeled structures and the critical residues recognized through protein interaction and docking study may provide better structural and functional insights into these proteins and may aid in the development of newer diagnostic assays.

Impact of amino acid substitution in the kinase domain of Bruton tyrosine kinase and its association with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a rare disease that affects the immune system, characterized by a serial development of bacterial infection from the onset of infantile age. Bruton tyrosine kinase (BTK) is a non-receptor cytoplasmic kinase that plays a crucial role in the B-lymphocyte maturation. The altered expression, mutation and/or structural variations of BTK are responsible for causing XLA. Here, we have performed extensive sequence and structure analyses of BTK to find deleterious variations and their pathogenic association with XLA. First, we screened the pathogenic variations in the BTK from a pool of publicly available resources, and their pathogenicity/tolerance and stability predictions were carried out. Finally, two pathogenic variations (E589G and M630K) were studied in detail and subjected to all-atom molecular dynamics simulation for 200 ns. Intramolecular hydrogen bonds (H-bonds), secondary structure, and principal component analysis revealed significant conformational changes in variants that support the structural basis of BTK dysfunction in XLA. The free energy landscape analysis revealed the presence of multiple energy minima, suggests that E589G brings a large destabilization and consequently unfolding behavior compared to M630K. Overall, our study suggests that amino acid substitutions, E589G, and M630K, significantly alter the structural conformation and stability of BTK.

Identification of High-Affinity Inhibitors of Cyclin-Dependent Kinase 2 Towards Anticancer Therapy.

Cyclin-dependent kinase 2 (CDK2) is an essential protein kinase involved in the cell cycle regulation. The abnormal activity of CDK2 is associated with cancer progression and metastasis. Here, we have performed structure-based virtual screening of the PubChem database to identify potent CDK2 inhibitors. First, we retrieved all compounds from the PubChem database having at least 90% structural similarity with the known CDK2 inhibitors. The selected compounds were subjected to structure-based molecular docking studies to investigate their pattern of interaction and estimate their binding affinities with CDK2. Selected compounds were further filtered out based on their physicochemical and ADMET properties. Detailed interaction analysis revealed that selected compounds interact with the functionally important residues of the active site pocket of CDK2. All-atom molecular dynamics simulation was performed to evaluate conformational changes, stability and the interaction mechanism of CDK2 in-complex with the selected compound. We found that binding of 6-N,6-N-dimethyl-9-(2-phenylethyl)purine-2,6-diamine stabilizes the structure of CDK2 and causes minimal conformational change. Finally, we suggest that the compound (PubChem ID 101874157) would be a promising scaffold to be further exploited as a potential inhibitor of CDK2 for therapeutic management of cancer after required validation.

Identification and evaluation of quercetin as a potential inhibitor of naphthoate synthase from Enterococcus faecalis.

Enterococcus faecalis is a gram-positive, rod-shape bacteria responsible for around 65% to 80% of all enterococcal nosocomial infections. It is multidrug resistant (MDR) bacterium resistant to most of the first-line antibiotics. Due to the emergence of MDR strains, there is an urgent need to find novel targets to develop new antibacterial drugs against E. faecalis. In this regard, we have identified naphthoate synthase (1,4-dihydroxy-2-naphthoyl-CoA synthase, EC: 4.1.3.36; DHNS) as an anti-E. faecalis target, as it is an essential enzyme for menaquinone (vitamin K2 ) synthetic pathway in the bacterium. Thus, inhibiting naphtholate synthase may consequently inhibit the bacteria's growth. In this regard, we report here cloning, expression, purification, and preliminary structural studies of naphthoate synthase along with in silico modeling, molecular dynamic simulation of the model and docking studies of naphthoate synthase with quercetin, a plant alkaloid. Biochemical studies have indicated quercetin, a plant flavonoid as the potential lead compound to inhibit catalytic activity of EfDHNS. Quercetin binding has also been validated by spectrofluorimetric studies in order to confirm the bindings of the ligand compound with EfDHNS at ultralow concentrations. Reported studies may provide a base for structure-based drug development of antimicrobial compounds against E. faecalis.