Isolation of Halomicroarcula pellucida strain GUMF5, an archaeon from the Dead Sea-Israel possessing cellulase.

A strain designated GUMF5 was isolated in Goa-India from sediments of Dead Sea-Israel  and identified as haloarchaeon Halomicroarcula pellucida based on 16S rRNA gene analysis similarity value of 99.84%. Strain GUMF5 grew on mineral salts medium with 20% NaCl and 0.5% carboxymethyl cellulose-sodium (CMC-Na) as a sole source of carbon and produced haloextremozyme cellulase. The enzyme was concentrated using Sephadex G20, precipitated with ethanol, dialyzed and retentate purified using Sephadex G200, the size exclusion chromatography. A yield of 78.53% cellulase with an activity of 131.13 U/mg and 1.24-fold purity was obtained. The purified cellulase had optimum activity at 20% NaCl, at 40 ºC, 0.5% CMC-Na, pH 7 and 150 rpm. SDS-PAGE combined with zymographic analysis revealed the molecular weight of cellulase as 240 kDa, 40 kDa and 17.4 kDa. The activity of the enzyme was stimulated by metallic cations in the order of Ca+2 > Mn+2 > Mg+2 > SO4 2- > NH4 + and was inhibited by Ag+ > Fe+2 > Cu+2. Methanol and ethanol enhanced the cellulase activity by 6% and 26%, respectively. The haloextremozyme cellulase degraded Whatman No. 1 filter paper indicated in scanning electron micrographs, exposure of open pores and fibers without any intra connectivity corresponding to paperase activity and implicating the possible use of enzyme to bio-convert cellulosic waste. Conclusively, Halomicroarcula pellucida GUMF5 (Accession number: MH244431), globally, is the only Halomicroarcula pellucida isolated from the sediments of Dead Sea producing haloextremozyme cellulase, and hence is an important biotechnological resource.

Haloferax sulfurifontis GUMFAZ2 producing xylanase-free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna, Goa-India.

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.

Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors.

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.

Synthesis and biological evaluation of novel phosphatidylinositol 3-kinase inhibitors: Solubilized 4-substituted benzimidazole analogs of 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474).

A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile.

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors.

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.

Novel pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors: Exploring the benzenesulfonohydrazide SAR.

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.