Krishnasamy Gunasekaran

Structural modeling, in vitro antiproliferative activity, and the effect of substituents on the DNA fastening and scission actions of heteroleptic copper(II) complexes with terpyridines and naproxen

A series of heteroleptic copper(II) complexes of the type [Cu(L1–6)(nap)Cl] (1–6) (L1–6 = 4′-(4-substituted)-2,2′:6′,2′′-terpyridines, nap = naproxen) has been synthesized and characterized. The single crystal analyses of complexes 1 and 6 show distorted octahedral geometry around the copper(II) ion. Structural parameters from the crystallographic and DFT studies are in good agreement with each other. HOMO–LUMO energy levels are constructed and the corresponding theoretical frontier energy gaps are calculated to understand the charge transfer occurring in the molecule, and the lowering of the HOMO–LUMO band gap supports the bioactive properties of the molecule. Electrochemical studies show a one-electron irreversible reduction process in the cathodic potential (Epc) region from −0.75 to −0.82 V. The obtained room-temperature magnetic moment values (1.82–1.93 BM), XRD and EPR spectral data support a distorted octahedral geometry for the copper(II) complexes. The binding studies of complexes 1, 5 and 6 with CT-DNA imply a groove mode of binding, and complex 5 exhibits a higher binding affinity than the other complexes. The binding results are further supported by molecular docking studies. The higher binding propensity of complex 5, containing R5, was proved by computationally derived factors such as chemical potential (μ), chemical hardness (η), electrophilicity (ω) and nuclease-independent chemical shift (NICS). All the complexes display pronounced nuclease activity against supercoiled pBR322 DNA. The in vitro antiproliferative activity of complexes 1, 5 and 6 against human breast cancer cells (MCF-7) was assessed by MTT assay, which shows the potency of 1 and 5, with lower IC50 values than cisplatin and values comparable to doxorubicin. The complexes induce mitochondrial-mediated and caspase-dependent apoptosis with an increase in G0–G1 and subsequent arrest in the S phase in cell cycle evaluation.

Phytochemical Analyses and Activity of Herbal Medicinal Plants of North- East India for Anti-Diabetic, Anti-Cancer and Anti-Tuberculosis and their Docking Studies

The traditional knowledge of medicinal plants that are in use by the indigenous Jaintia tribes residing in few isolated pockets of North-East India is documented here. The present study was carried out through the personal discussion with the president of the Jaintia Indigenous Herbal Medicine Association, Dr.H.Carehome Pakyntein from Jowai, Meghalaya. The plants being used generation after generation by his family of herbalists to cure ailments like tuberculosis, cancer and diabetes were selected for the present study. In order to scientifically validate the use of these selected plants for the cure of selected diseases, phytochemical analyses, characterization and molecular docking studies of some of the selected compounds from these plants have been carried out. The compounds 2-hydroxy-4-methoxy- Benzaldehyde from methanolic extract of Strophanthus Wallichii and DL tetrahydropalmatine from Stephania Hernandifolia have been confirmed after determining their molecular structures, justifying the activity of these two plants against TB and cancer, respectively. The present study covers the potentials of some of the medicinal plants of North east India in curing common diseases due to which millions of people suffer and die. The presence of certain compounds in these plants related to the cure of the diseases deserves further studies.

Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: a human Lymphatic Filarial parasite.

Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33 kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.

Identification of new BACE1 inhibitors using Pharmacophore and Molecular dynamics simulations approach

Inhibition of β-Secretase (BACE1) is crucial for the treatment of Alzheimers disease (AD). Availability of BACE1 crystal structures in both apo and complexed forms enables to find structure-based BACE1 inhibitors for controlling AD. There are two catalytic aspartates (ASP32 and ASP228) presents in the active domain of BACE1. In order to understand the binding mechanism and structure-activity relationship of amidine-containing BACE1 inhibitors, molecular docking, and pharmacophore and 3D-QSAR studies have been carried out with 34 amidine derivatives to develop a pharmacophore model. Pharmacophore-based virtual screening (PBVS) has been performed against BACE1 (PDB ID: 2FDP), using three chemical databases (CoCoCo, Enamine, Zinc), which yielded 6000 hit compounds. These compounds were further analyzed using structure-based docking in hierarchical filtering approaches of Glide such as HTVS, SP, and XP precision modes. The docking results show that binding orientations of the inhibitors at Asp dyad active site amino acid residues of β-Secretase. Results from glide XP docking and induced fit docking showed that four leads (Lead1, Lead3, Lead4 and Lead5) have good interactions with the target protein in comparison with cocrystal (amino-ethylene BACE1 inhibitor). Further, molecular dynamics (MD) simulation for these leads bound with BACE1 shows conformational stability and difference in dynamical flap behaviors of the active site with cocrystal inhibitor. Binding free energetic using MM-GB/SA approaches suggest lead 1 and lead 3 has comparably favorable binding to cocrystal inhibitor. Thus, the present study emphasizes these leads for an effective drug to treat Alzheimer disease.

Identification of potential PKC inhibitors through pharmacophore designing, 3D-QSAR and molecular dynamics simulations targeting Alzheimer’s disease

Protein kinases are ubiquitously expressed as Serine/Threonine kinases, and play a crucial role in cellular activities. Protein kinases have evolved through stringent regulation mechanisms. Protein kinases are also involved in tauopathy, thus are important targets for developing Anti-Alzheimer’s disease compounds. Structures with an indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors for human protein kinase C, here we report the generation of four point 3D geometric featured pharmacophore model. In order to identify novel and potent PKCθ inhibitors, the pharmacophore model was screened against 80,000,00 compounds from various chemical databases such as., ZINC, SPEC, ASINEX, which resulted in 127 compound hits, and were taken for molecular docking filters (HTVS, XP docking). After in-depth analysis of binding patterns, induced fit docking (flexible) was employed for six compounds along with the cocrystallized inhibitor. Molecular docking study reveals that compound 6F found to be tight binder at the active site of PKCθ as compared to the cocrystal and has occupancy of 90 percentile. MM-GBSA also confirmed the potency of the compound 6F as better than cocrystal. Molecular dynamics results suggest that compound 6F showed good binding stability of active sites residues similar to cocrystal 7G compound. Present study corroborates the pharmacophore-based virtual screening, and finds the compound 6F as a potent Inhibitor of PKC, having therapeutic potential for Alzheimer’s disease. Worldwide, 46.8 million people are believed to be living with Alzheimer’s disease. When elderly population increases rapidly and neurodegenerative burden also increases in parallel, we project the findings from this study will be useful for drug developing efforts targeting Alzheimer’s disease.

Structural analysis of β-prism lectin from Colocasia esculenta (L.) S chott

The Mannose-binding β-Prism Colocasia esculenta lectin (β-PCL) was purified from tubers using ion exchange chromatography. The purified β-PCL appeared as a single band of ∼12kDa on SDS-PAGE. β-PCL crystallizes in trigonal space group P3121 and diffracted to a resolution of 2.1Å. The structure was solved using Molecular replacement using Crocus vernus lectin (PDB: 3MEZ) as a model. From the final refined model to an R-factor of 16.5% and an Rfree of 20.4%, it has been observed that the biological unit consists of two β-Prism domains augmented through C-terminals swap over to form one of faces for each domain. Cα superposition of individual domains of β-PCL with individual domains of other related structures and superposition of whole protein structures were carried out. The higher RMS deviation for the superposition of whole structures suggest that β-prism domains assume different orientation in each structure.

Identification of novel Nicotinamide Phosphoribosyltransferase (NAMPT) inhibitors using computational approaches

Nicotinamide Phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the biosynthesis of NAD. Cancer cells have elevated poly [ADP-Ribose] polymerase 1 (PARP) activity as well as the immense necessity of ATP: thereby consuming NAD at a higher rate than normal tissues. The perturbation of these intracellular processes is more sensitive and highly dependent on NAMPT to maintain the required NAD levels. Functional inhibition of NAMPT is, therefore, a promising drug target in therapeutic oncology. In this study, the importance of intermolecular contacts was realized based on contact occupancy and favorable energetic from molecular dynamic simulation to discern non-critical contacts of four different classes of potential NAMPT inhibitor bound complexes. Further, pharmacophore modeling, molecular docking, a quantum mechanical properties and MD simulation, as well as active site residual network communication were employed to identify potential leads. Present studies identified two leads, 2 and 3 which have better binding free energy compared to known inhibitors and showed stable hydrogen bonding and hydrophobic contacts with β barrel cavity lining residues in the active site of the dimer interface (A′B). Lead 2 containing fluorene as central core and lead 3 having phenyl-benzamide as a core showed stable moiety which was observed from electronic property analysis. Active site residual communication in identified leads bound complex also showed similarity to known inhibitor complexes. Compounds containing these moieties were not reported until now against NAMPT inhibition and can be considered as novel cores for future development of drugs to inhibit NAMPT function.

A structural biology approach to understand human lymphatic filarial infection.

The presence of aspartic protease inhibitor in filarial parasite Brugia malayi (Bm-Aspin) makes it interesting to study because of the fact that the filarial parasite never encounters the host digestive system. Here, the aspartic protease inhibition kinetics of Bm-Aspin and its NMR structural characteristics have been investigated. The overall aim of this study is to explain the inhibition and binding properties of Bm-Aspin from its structural point of view. UV-spectroscopy and multi-dimensional NMR are the experiments that have been performed to understand the kinetic and structural properties of Bm-Aspin respectively. The human aspartic proteases that are considered for this study are pepsin, renin, cathepsin-E and cathepsin-D. The results of this analysis performed with the specific substrate [Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu (4-pyridylmethyl) ester] against aspartic proteases suggest that Bm-Aspin inhibits the activities of all four human aspartic proteases. The kinetics studies indicate that Bm-Aspin follows a competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin and mixed mode for cathepsin-D. The triple resonance NMR experiments on Bm-Aspin suggested the feasibility of carrying out NMR studies to obtain its solution structure. The NMR titration studies on the interactions of Bm-Aspin with the proteases indicate that it undergoes fast-exchange phenomena among themselves. In addition to this, the chemical shift perturbations for some of the residues of Bm-Aspin observed from 15N-HSQC spectra upon the addition of saturated amounts of aspartic proteases suggest the binding between Bm-Aspin and human aspartic proteases. They also provide information on the variations in the intensities and mode of binding between the proteases duly corroborating with the results from the protease inhibition assay method.

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases.

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable ‘enzyme-product’ complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23 × 103 and 6.52 × 103 M−1. The binding reactions were enthalpy driven with ΔHb values between −50.99 and −46.07 kJ mol−1. From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.

Crystal structure of a novel Kunitz type inhibitor, alocasin with anti-Aedes aegypti activity targeting midgut proteases: Structure and mechanism of alocasin

BACKGROUND The pesticidal properties of many Kunitz-type inhibitors have been reported previously; however, the mechanism of action is not well established. In this study, the activity of alocasin against Aedes aegypti is demonstrated and the structure-activity relationship of this Kunitz-type inhibitor is explained through X-ray structure analyses. RESULTS Alocasin was purified from mature rhizomes of Alocasia as a single polypeptide chain of ∼ 20 kDa. The structure at 2.5 Å resolution revealed a Kunitz-type fold, but variation in the loop regions makes this structure unique; one loop with a single disulfide bridge is replaced by a long loop with two bridges. Alignment of homologous sequences revealed that this long loop contains a conserved Arg residue and modeling studies showed interaction with the catalytic Ser residue of trypsin-like enzymes. The anti-Aedes aegypti activity of alocasin is examined and discussed in detail. The in vitro activity of alocasin against midgut proteases of Aedes aegypti showed profound inhibition. Further, morphological changes in larvae upon treatment with alocasin revealed its activity against Ae. aegypti. Docking studies of alocasin with trypsin (5G1), a midgut protease involved in the development cycle and blood meal digestion, illustrated its insecticidal activity. CONCLUSION The three-dimensional structure of alocasin was determined and its structure-function relationship established for its anti Ae. aegypti activity.