Narayana Nagesh

Characterization of an alkaline active – thiol forming extracellular serine keratinase by the newly isolated Bacillus pumilus

Aims:  The aim of the study was to optimize microbial degradation of keratinous waste and to characterize the alkaline active keratinase showing its biotechnological importance.

Genetic Locus Encoding Functions Involved in Biosynthesis and Outer Membrane Localization of Xanthomonadin in Xanthomonas oryzae pv. oryzae

Xanthomonadins are membrane-bound, brominated, aryl-polyene pigments specific to the genus Xanthomonas. We have characterized a genetic locus (pig) from Xanthomonas oryzae pv. oryzae which contains four open reading frames (ORFs) that are essential for xanthomonadin production. Three of these ORFs are homologous to acyl carrier proteins, dehydratases, and acyl transferases, suggesting a type II polyketide synthase pathway for xanthomonadin biosynthesis. The fourth ORF has no homologue in the database. For the first time, we report that a putative cytoplasmic membrane protein encoded in the pig locus is required for outer membrane localization of xanthomonadin in X. oryzae pv. oryzae. We also report the identification of a novel 145-bp palindromic Xanthomonas repetitive intergenic consensus element that is present in two places in the pig locus. We estimate that more than 100 copies of this element might be present in the genome of X. oryzae pv. oryzae and other xanthomonads.

Design and Synthesis of C3-Pyrazole/Chalcone-Linked Beta-Carboline Hybrids: Antitopoisomerase I, DNA-Interactive, and Apoptosis-Inducing Anticancer Agents

A series of β‐carboline hybrids bearing a substituted phenyl and a chalcone/(N‐acetyl)‐pyrazole moiety at the C1 and C3 positions, respectively, was designed, synthesized, and evaluated for anticancer activity. These new hybrid molecules showed significant cytotoxic activity, with IC50 values ranging from <2.0 μM to 80 μM, and the structure–activity relationships (SAR) associated with substitutions at positions 1 and 3 of these hybrids was clearly addressed. Further, induction of apoptosis was confirmed by Annexin V‐FITC, Hoechst staining, and DNA fragmentation analysis. In addition, DNA photocleavage studies proved that two of the hybrids, (E)‐1‐(furan‐2‐yl)‐3‐(1‐(4‐(trifluoromethyl)phenyl)‐9H‐pyrido[3,4‐b]indol‐3‐yl)prop‐2‐en‐1‐one (7 d) and 1‐(3‐(furan‐2‐yl)‐5‐(1‐(4‐(trifluoromethyl)phenyl)‐9H‐pyrido[3,4‐b]indol‐3‐yl)‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethanone (8 d) could effectively cleave pBR322 plasmid DNA upon irradiation with UV light. Active hybrid 8 d inhibited DNA topoisomerase I activity efficiently and preserved DNA in the supercoiled form. To further corroborate the biological activities, as well as to understand the nature of the interaction of these hybrids with DNA, spectroscopic studies were also performed. Unlike simple β‐carboline alkaloids, the binding mode of these new hybrid molecules with DNA was not similar, and both biophysical as well as molecular docking studies speculated a combilexin‐type of interaction with DNA. Further, an in silico study of these β‐carboline hybrids revealed their drug‐like properties.

DNA-binding affinity and anticancer activity of β-carboline–chalcone conjugates as potential DNA intercalators: Molecular modelling and synthesis

A new series of DNA-interactive β-carboline-chalcone conjugates have been synthesized and evaluated for their in vitro cytotoxicity and DNA-binding affinity. It has been observed that most of these new hybrids have shown potent cytotoxic activities on A-549 (lung adenocarcinoma) cell lines with IC50 values lower than 10 μM. The hybrid 7b is more effective against some of the selected cancer cell lines with IC50 values less than 50 μM. In addition, compounds 7e, 7k, 7p-u has displayed significant elevation in ΔTm of DNA in comparison to Adriamycin, suggesting significant interaction and remarkable DNA stabilization. The DNA intercalation of these new hybrids has been investigated by fluorescence titration, DNA viscosity measurements, molecular docking as well as molecular dynamics and the results are in agreement with the thermal denaturation studies.

Ammonium ion at low concentration stabilizes the G-quadruplex formation by telomeric sequence

A synthetic analog of telomeric DNA, d(T6G16) or d(G4T2G4T2G4T2G4) was found to form G-quadruplex structure in NH4-ion environments as low as 1 mM. Other counter ions like K(I) or Sr(II) known to have a stabilizing effect on G-tetrad require much higher concentrations. The multiconformational form of G-tetrad could be identified in NH4(I) upon heating the sample up to 100 degrees C. Circular dichroism spectral studies indicated that on thermally treating the complex NH4(I) ion helps in intramolecular G-tetrad formation over intermolecular association. However the G-tetrad structure formed in NH4(I) was found to be unstable in the presence of intercalator Actinomycin D or Tb(III) ion. It appears that the ionic radius of NH4(I) influence tight fitting of this ion inside the cavity of the tetrad. The stability of G-tetrad in the presence of very low concentration of NH4(I) may have potential applications in future.

Design and synthesis of dithiocarbamate linked β-carboline derivatives: DNA topoisomerase II inhibition with DNA binding and apoptosis inducing ability.

A series of new β-carboline-dithiocarbamate derivatives bearing phenyl, dithiocarbamate and H/methyl substitutions at position-1, 3 and 9, respectively, were designed and synthesized. These derivatives 8a-l and 13a-l and their starting precursors (7 a-d and 12 a-d) have been evaluated for their in vitro cytotoxic activity on selected human cancer cell lines. Among the derivatives tested, 7 c, 12 c, 8 a, 8 d, 8 i, 8 j, 8 k, 8l and 13 d-l exhibited considerable cytotoxicity against most of the tested cancer cell lines (IC50<10μM). Interestingly, most of the derivatives (8 a-l and 13a-l) exhibited enhanced activity than their precursors (7 a-d and 12 a-d), which indicates that the combination of dithiocarbamate with β-carboline enhances the cytotoxicity of 8 a-l and 13 a-l. Moreover, the derivatives 8 j and 13 g exhibited significant cytotoxic activity with IC50 values of 1.34 μM and 0.79 μM on DU-145 cancer cells, respectively. Further, the induction of apoptosis by these derivatives was confirmed by Annexin V-FITC and Hoechst staining assays. However, both biophysical as well as molecular docking studies suggested a combilexin-type of interaction between these derivatives and DNA, unlike simple β-carbolines. With a view to understand their mechanism of action, DNA topoisomerase II (topo II) inhibition assay was also performed. Overall, the present study emphasizes the importance of linking a dithiocarbamate moiety to the β-carboline scaffold for exhibiting profound activity.

A dihydroindolizino indole derivative selectively stabilizes G-quadruplex DNA and down-regulates c-MYC expression in human cancer cells.

BACKGROUND Telomeric and NHE III1, a c-MYC promoter region is abundant in guanine content and readily form G-quadruplex structures. Small molecules that stabilize G-quadruplex DNA were shown to reduce oncoprotein expression, initiate apoptosis and they may function as anticancer molecules. METHODS Electrospray ionization mass spectrometry, spectroscopy, isothermal titration calorimetry, Taq DNA polymerase stop assay, real time PCR and luciferase reporter assay. Cell migration assay to find out the effect of derivatives on normal as well as cancer cell proliferation. RESULTS Among three different dihydroindolizino indole derivatives, 4-cyanophenyl group attached derivative has shown maximum affinity, selective interaction and higher stability towards G-quadruplex DNA over dsDNA. Further, as a potential G-quadruplex DNA stabilizer, 4-cyanophenyl linked dihydroindolizino indole derivative was found to be more efficient in inhibiting in vitro DNA synthesis, c-MYC expression and cancer cell proliferation among human cancer cells. CONCLUSION The present study reveals that dihydroindolizino indole derivative having 4-cyanophenyl group has potential to stabilize G-quadruplex DNA and exhibit anticancer activity. GENERAL SIGNIFICANCE These studies are useful in the identification and synthesis of lead derivatives that will selectively stabilize G-quadruplex DNA and function as anticancer agents.

Biological studies of chalcogenolato-bridged dinuclear half-sandwich complexes.

A series of cationic chalcogenolato-bridged diruthenium complexes [(η(6)-p-MeC6H4Pr(i))2Ru2(μ-EC6H5)3](+) (E = S, 1; E = Se, 2; E = Te, 3) has been obtained in ethanol from the reaction of (η(6)-p-MeC6H4Pr(i))2Ru2(μ-Cl)2Cl2 with benzenethiol, benzeneselenol, and sodium tellurophenolate, respectively. The thiolato and selenolato derivatives are isolated in good yield as the chloride salts, while the tellurolato analogue is isolated as the hexafluorophosphate salt. Similarly, the dinuclear pentamethylcyclopentadienyl (C5Me5) rhodium and iridium complexes (η(5)-C5Me5)2M2(μ-Cl)2Cl2 react with benzenethiol, benzeneselenol, and sodium tellurophenolate in ethanol to give the corresponding cationic dinuclear complexes of the general formula [(η(5)-C5Me5)2M2(μ-EC6H5)3](+) (M = Rh, E = S, 4; E = Se, 5; E = Te, 6; M = Ir, E = S, 7; E = Se, 8; E = Te, 9). In addition, cationic dinuclear complexes with mixed thiolato-selenolato and thiolato-tellurolato bridges have been prepared, [(η(6)-p-MeC6H4Pr(i))2Ru2(μ-EC6H5)(μ-SCH2C6H4-p-Bu(t))2](+) (E = Se, 10; E = Te, 11) and [(η(5)-C5Me5)2M2(μ-EC6H5)(μ-SCH2C6H5)2](+) (M = Rh, E = Se, 12; E = Te, 13; M = Ir, E = Se, 14; E = Te, 15), starting from the neutral dinuclear complexes (η(6)-p-MeC6H4Pr(i))2Ru2Cl2(μ-SCH2C6H4-p-Bu(t))2 and (η(5)-C5Me5)2M2Cl2(μ-SCH2C6H5)2. All complexes are highly cytotoxic showing activity in the submicromolar range. The nature of the chalcogenolato bridges seems to have an impact on the activity, while the nature of the metal center plays a minor role. Among the complexes tested, the dinuclear complexes 1, 4, and 7 with the thiolato bridges show the highest activity on cancer cells and the best affinity for CT-DNA as demonstrated by cell biology and biophysical experiments.

In situ immobilization of acid protease on mesoporous activated carbon packed column for the production of protein hydrolysates.

The mesoporous activated carbon (MAC) was used as a support material for in situ immobilization of acid protease (AP). The optimum temperature for the activities of both free and immobilized AP was found to be 50 degrees C. The catalytic efficiency of AP-MAC system has significantly been maintained for more than ten consecutive reaction cycles. The functional groups and surface morphology of the AP, MAC and AP-MAC were observed by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). The production of protein hydrolysates was carried out from bovine serum albumin (BSA) using AP-MAC packed column and its properties were studied.

Bcl-2 Promoter Sequence G-Quadruplex Interactions with Three Planar and Non-Planar Cationic Porphyrins: TMPyP4, TMPyP3, and TMPyP2

The interactions of three related cationic porphyrins, TMPyP4, TMPyP3 and TMPyP2, with a WT 39-mer Bcl-2 promoter sequence G-quadruplex were studied using Circular Dichroism, ESI mass spectrometry, Isothermal Titration Calorimetry, and Fluorescence spectroscopy. The planar cationic porphyrin TMPyP4 (5, 10, 15, 20-meso-tetra (N-methyl-4-pyridyl) porphine) is shown to bind to a WT Bcl-2 G-quadruplex via two different binding modes, an end binding mode and a weaker mode attributed to intercalation. The related non-planar ligands, TMPyP3 and TMPyP2, are shown to bind to the Bcl-2 G-quadruplex by a single mode. ESI mass spectrometry experiments confirmed that the saturation stoichiometry is 4:1 for the TMPyP4 complex and 2:1 for the TMPyP2 and TMPyP3 complexes. ITC experiments determined that the equilibrium constant for formation of the (TMPyP4)1/DNA complex (K1 = 3.7 × 106) is approximately two orders of magnitude greater than the equilibrium constant for the formation of the (TMPyP2)1/DNA complex, (K1 = 7.0 × 104). Porphyrin fluorescence is consistent with intercalation in the case of the (TMPyP4)3/DNA and (TMPyP4)4/DNA complexes. The non-planar shape of the TMPyP2 and TMPyP3 molecules results in both a reduced affinity for the end binding interaction and the elimination of the intercalation binding mode.