Small and lethal: searching for new antibacterial compounds with novel modes of action.

The discovery of drugs used to combat infectious diseases is in the process of constant change to address the ever-worsening problem of antibiotic resistance in pathogens and a lack of recent success in discovering new antibacterial drugs. In the past 2 decades, research in both academia and industry has made use of molecular biology, genetics, and comparative genomics, which has led to the development of key technologies for the discovery of novel antibacterial agents. Genome-scale efforts have led to the identification of numerous molecular targets. Chemical diversity from synthetic combinatorial libraries and natural products is being used to screen for new molecules. A wide variety of approaches are being used in the search for novel antibiotics, and these can be categorized as being either biochemically focused or cell based. The over-riding goal of all methods in use today is to discover new chemical matter with novel mechanisms of action against drug-resistant pathogens.     

A unique right end-enhancer complex precedes synapsis of Mu ends: the enhancer is sequestered within the transpososome throughout transposition

Assembly of the Mu transpososome is dependent on interactions of transposase subunits with the left (L) and right (R) ends of Mu and an enhancer (E). We have followed the order and dynamics of association of these sites within a series of transpososomes prior to and during formation of a three-site complex (LER), engagement of Mu ends by the transposase active site (type 0 complex), cleavage of the ends (type I complex) and their transfer to target DNA (type II complex). LER appears to be preceded by a two-site complex (ER) where E and R are interwrapped twice, as in the mature transpososome. At each stage thereafter, the overall topology of five DNA supercoils is retained: two between E and R, one between E and L and two between L and R. However, L-R interactions within LER appear to be flexible. Unexpectedly, the enhancer was seen to persist within the transpososome through cleavage and strand transfer of Mu ends to target DNA.     

Immunomodulatory and cytoprotective role of RP-1 in γ-irradiated mice

RP-1 has been reported to provide protection against lethal -irradiation in mice. The present study was undertaken to understand its mechanism of action, especially with respect to modulation of radiation-induced changes in immune cell function, plasma antioxidant potential, cell cycle perturbations, apoptosis in mouse bone marrow cells, and micronuclei frequency in mice reticulocytes. 2 Gy reduced mitogenic response of splenic lymphocytes significantly at 48 h. Pre-irradiation RP-1 treatment significantly countered the radiation-induced loss of splenocyte proliferation. RP-1 treatment, with or without radiation, suppressed macrophage activation as compared to control. Irradiation decreased plasma antioxidant status significantly (p < 0.05) at 1 and 2 h (4.8 ± 0.224 and 4.9 ± 0.057 mM Fe²⁺) as compared to control (6.29 ± 0.733 mM Fe²⁺) that was countered by RP-1 pre-treatment significantly (p < 0.05). RP-1 and irradiation individually caused G₂ delay in bone marrow cells. RP-1 pre-treatment augmented radiation-induced G₂ delay and elicited significant (p < 0.05) recovery in S phase fraction at 48 h in comparison to irradiated group. Radiation-induced apoptosis (3%) was significantly higher than the control. RP-1 pre-treatment further enhanced apoptosis frequency (7.2%) in bone marrow cells. RP-1 pre-treatment significantly (p < 0.05) reduced (1.23%) the radiation-induced MN frequency (2.9%) observed at 48 h post-irradiation interval. Since the radioprotective manifestation of RP-1 is mediated through multiple mechanisms, needs further investigation.     

Design and synthesis of 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-ones by Huisgen 1,3-dipolar cycloaddition with PI3Kγ isoform selective activity

A strategy for construction of medicinally important 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-ones has been devised and presented here. The compounds have been synthesized using one-pot multicomponent strategy under microwave assisted conditions. Triazolyl-quinazolinone based D-ring modified analogs are designed based on IC87114 scaffold, which is first known isoform selective inhibitor of PI3Kδ. Herein, we identified two triazolyl-quinazolinone compounds (5a and 5l) based on same scaffold with PI3Kγ specific inhibitory potential, the selectivity towards this isoform is well supported by in silico results, wherein, these compounds show better interaction and affinity and inhibitory activity for PI3Kγ rather than PI3Kδ. This repositioning of scaffold from PI3Kδ to PI3Kγ isoform can be very useful from medicinal chemistry and drug discovery perspective to unravel molecular interactions of this new scaffold in different cellular pathways.     

A general model for site-specific recombination by the integrase family recombinases.

We present here a general model for integrase family site-specific recombination using the geometric relationships of the cleavable phosphodiester bonds and the disposition of the recombinase monomers (defined by their binding planes) with respect to them. The 'oscillation model' is based largely on the conformations of the recombinase-bound DNA duplexes and their dynamics within Holliday junctions. The duplex substrate or the Holliday junction intermediate is capable of 'oscillating' between two cleavage-competent asymmetric states with respect to corres-ponding chemically inert 'equilibrium positions'. The model accommodates several features of the Flp system and predicts two modes of DNA cleavage during a normal recombination event. It is equally applicable to other systems that mediate recombination across 6, 7 or 8 bp long strand exchange regions (or spacers). The model is consistent with approximately 0-1, 1-2 and 2-3 bp of branch migration during recombination reactions involving 6, 7 and 8 bp spacers, respectively.     

Molecular Mechanism of SLC5A8 Inactivation in Breast Cancer

SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRAS^G12V) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.     

Reversal of boswellic acid analog BA145 induced caspase dependent apoptosis by PI3K inhibitor LY294002 and MEK inhibitor PD98059

PI3K/Akt and ERK pathways are important for growth and proliferation of many types of cancers. Therefore, PI3K inhibitor LY294002 (LY) and MEK1/2 inhibitor PD98059 (PD) are used to sensitize many types of cancer cell lines to chemotherapeutic agents, where AKT and ERK pathways are over activated. However, in this study, we show for the first time that PD could protect the leukemia cells independent of ERK pathway inhibition, besides, we also report a detailed mechanism for antiapoptotic effect of LY in HL-60 cells against the cytotoxicity induced by a boswellic acid analog BA145. Apoptosis induced by BA145 is accompanied by downregulation of PI3K/Akt and ERK pathways in human myelogenous leukemia HL-60 cells, having activating N-Ras mutation. Both LY and PD protected the cells against mitochondrial stress caused by BA145, and reduced the release of cytochrome c and consequent activation of caspase-9. LY and PD also diminished the activation of caspase-8 without affecting the death receptors. Besides, LY and PD also reversed the caspase dependent DNA damage induced by BA145. Further studies revealed that LY and PD significantly reversed the inhibitory effect of BA145 on cell cycle regulatory proteins by upregulating hyperphosphorylated retinoblastoma, pRB (S795) and downregulating p21 and cyclin E. More importantly, all these events were reversed by caspase inhibition by Z-VAD-fmk, suggesting that both LY and PD act at the level of caspases to diminish the apoptosis induced by BA145. These results indicate that inhibitors of PI3K/Akt and ERK pathways can play dual role and act against chemotherapeutic agents.     

miR-132 enhances dendritic morphogenesis, spine density, synaptic integration, and survival of newborn olfactory bulb neurons

An array of signals regulating the early stages of postnatal subventricular zone (SVZ) neurogenesis has been identified, but much less is known regarding the molecules controlling late stages. Here, we investigated the function of the activity-dependent and morphogenic microRNA miR-132 on the synaptic integration and survival of olfactory bulb (OB) neurons born in the neonatal SVZ. In situ hybridization revealed that miR-132 expression occurs at the onset of synaptic integration in the OB. Using in vivo electroporation we found that sequestration of miR-132 using a sponge-based strategy led to a reduced dendritic complexity and spine density while overexpression had the opposite effects. These effects were mirrored with respective changes in the frequency of GABAergic and glutamatergic synaptic inputs reflecting altered synaptic integration. In addition, timely directed overexpression of miR-132 at the onset of synaptic integration using an inducible approach led to a significant increase in the survival of newborn neurons. These data suggest that miR-132 forms the basis of a structural plasticity program seen in SVZ-OB postnatal neurogenesis. miR-132 overexpression in transplanted neurons may thus hold promise for enhancing neuronal survival and improving the outcome of transplant therapies.