Discovery of Novel Chemical Series of OXA-48 ß-Lactamase Inhibitors by High-Throughput Screening.

The major cause of bacterial resistance to ß-lactams is the production of hydrolytic ß-lactamase enzymes. Nowadays, the combination of ß-lactam antibiotics with ß-lactamase inhibitors (BLIs) is the main strategy for overcoming such issues. Nevertheless, particularly challenging ß-lactamases, such as OXA-48, pose the need for novel and effective treatments. Herein, we describe the screening of a proprietary compound collection against Klebsiella pneumoniae OXA-48, leading to the identification of several chemotypes, like the 4-ideneamino-4H-1,2,4-triazole (SC_2) and pyrazolo[3,4-b]pyridine (SC_7) cores as potential inhibitors. Importantly, the most potent representative of the latter series (ID2, AC50 = 0.99 µM) inhibited OXA-48 via a reversible and competitive mechanism of action, as demonstrated by biochemical and X-ray studies; furthermore, it slightly improved imipenem's activity in Escherichia coli ATCC BAA-2523 ß-lactam resistant strain. Also, ID2 showed good solubility and no sign of toxicity up to the highest tested concentration, resulting in a promising starting point for further optimization programs toward novel and effective non-ß-lactam BLIs.

Caffeine extends yeast lifespan by targeting TORC1.

Dietary nutrient limitation (dietary restriction) is known to increase lifespan in a variety of organisms. Although the molecular events that couple dietary restriction to increased lifespan are not clear, studies of the model eukaryote Saccharomyces cerevisiae have implicated several nutrient-sensitive kinases, including the target of rapamycin complex 1 (TORC1), Sch9, protein kinase A (PKA) and Rim15. We have recently demonstrated that TORC1 activates Sch9 by direct phosphorylation. We now show that Sch9 inhibits Rim15 also by direct phosphorylation. Treatment of yeast cells with the specific TORC1 inhibitor rapamycin or caffeine releases Rim15 from TORC1-Sch9-mediated inhibition and consequently increases lifespan. This kinase cascade appears to have been evolutionarily conserved, suggesting that caffeine may extend lifespan in other eukaryotes, including man.

A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast.

Saccharomyces cerevisiae must reach a carbon source-modulated critical cell size, protein content per cell at the onset of DNA replication (Ps), in order to enter S phase. Cells grown in glucose are larger than cells grown in ethanol. Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type. Cln3 Delta, far1 Delta, and strains overexpressing Far1 do not delay budding during an ethanol glucose shift-up as wild type does. Together, these findings indicate that Cln3 has to overcome Far1 to trigger Cln-Cdc28 activation, which then turns on SBF- and MBF-dependent transcription. We show that a second threshold is required together with the Cln3/Far1 threshold for carbon source modulation of Ps. A new molecular network accounting for the setting of Ps is proposed.

A platform for high-throughput expression of recombinant human enzymes secreted by insect cells.

Functional genomics and proteomics have been fields of intense investigation, since the disclosure of the sequence of the human genome. To contribute to the assignment of a physiological role to the vast number of coding genes with unknown function, we have undertaken a program to clone, express, purify and determine the catalytic activity of those enzymes predicted to enter the secretory pathway, focusing our efforts on human peptidases. Our strategy to promote high-throughput expression and purification of recombinant proteins secreted by insect cells relies on the expression of the target enzymes with their native leader sequences and on the carboxyl-terminal fusion with a poly-histidine tag. Growth of host cells were optimized in 24-well format to achieve highly paralleled culture conditions with production yields comparable to shake flask. The purification was performed by a robotic system in 96-well format using either magnetic beads or minicolumns. In a pilot study using reference peptidases and lipases, the high-throughput approach demonstrated to support the secretion in the insect cell medium of 85% of the sample enzymes. Of them, 66% have been proven to be catalytically active using fluorescent homogeneous assays in 384-well format compatible with the high-throughput screening criteria. The implications of these results are discussed in light of the application of this procedure to genomic-predicted peptidases.

Checking cell size in budding yeast: a systems biology approach.

The regulation of cell cycle progression via the attainment of a critical cell size is a conserved feature from simpler unicellular organisms to mammalian cells that is obtaining much attention recently. Genome wide analysis of Saccharomyces cerevisiae deletion strains, genetic epistasis, DNA microarray analysis have recently revealed an increasingly complex network of cell size modulation mechanisms. A systems biology-based approach, that is needed to structure the underlying complexity of cell cycle regulatory mechanisms, is described.

Sch9 is a major target of TORC1 in Saccharomyces cerevisiae.

The Target of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intracellular and extracellular cues. Here we report that the AGC kinase Sch9 is a substrate of yeast TORC1. Six amino acids in the C terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin treatment as well as carbon or nitrogen starvation and transiently reduced following application of osmotic, oxidative, or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity, and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1 independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation, and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt.

Rim15 and the crossroads of nutrient signalling pathways in Saccharomyces cerevisiae.

In recent years, the general understanding of nutrient sensing and signalling, as well as the knowledge about responses triggered by altered nutrient availability have greatly advanced. While initial studies were directed to top-down elucidation of single nutrient-induced pathways, recent investigations place the individual signalling pathways into signalling networks and pursue the identification of converging effector branches that orchestrate the dynamical responses to nutritional cues. In this review, we focus on Rim15, a protein kinase required in yeast for the proper entry into stationary phase (G0). Recent studies revealed that the activity of Rim15 is regulated by the interplay of at least four intercepting nutrient-responsive pathways.

Regulation of G0 entry by the Pho80-Pho85 cyclin-CDK complex.

Eukaryotic cell proliferation is controlled by growth factors and essential nutrients. In their absence, cells may enter into a quiescent state (G0). In Saccharomyces cerevisiae, the conserved protein kinase A (PKA) and rapamycin-sensitive TOR (TORC1) pathways antagonize G0 entry in response to carbon and/or nitrogen availability primarily by inhibiting the PAS kinase Rim15 function. Here, we show that the phosphate-sensing Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) complex also participates in Rim15 inhibition through direct phosphorylation, thereby effectively sequestering Rim15 in the cytoplasm via its association with 14-3-3 proteins. Inactivation of either Pho80-Pho85 or TORC1 causes dephosphorylation of the 14-3-3-binding site in Rim15, thus enabling nuclear import of Rim15 and induction of the Rim15-controlled G0 program. Importantly, we also show that Pho80-Pho85 and TORC1 converge on a single amino acid in Rim15. Thus, Rim15 plays a key role in G0 entry through its ability to integrate signaling from the PKA, TORC1, and Pho80-Pho85 pathways.

The TOR and EGO protein complexes orchestrate microautophagy in yeast.

The rapamycin-sensitive TOR signaling pathway in Saccharomyces cerevisiae positively controls cell growth in response to nutrient availability. Accordingly, TOR depletion or rapamycin treatment causes regulated entry of cells into a quiescent growth phase. Although this process has been elucidated in considerable detail, the transition from quiescence back to proliferation is poorly understood. Here, we describe the identification of a conserved member of the RagA subfamily of Ras-related GTPases, Gtr2, which acts in a vacuolar membrane-associated protein complex together with Ego1 and Ego3 to ensure proper exit from rapamycin-induced growth arrest. We demonstrate that the EGO complex, in conjunction with TOR, positively regulates microautophagy, thus counterbalancing the massive rapamycin-induced, macroautophagy-mediated membrane influx toward the vacuolar membrane. Moreover, large-scale genetic analyses of the EGO complex confirm the existence of a growth control mechanism originating at the vacuolar membrane and pinpoint the amino acid glutamine as a key metabolite in TOR signaling.

TOR and PKA signaling pathways converge on the protein kinase Rim15 to control entry into G0.

The highly conserved Tor kinases (TOR) and the protein kinase A (PKA) pathway regulate cell proliferation in response to growth factors and/or nutrients. In Saccharomyces cerevisiae, loss of either TOR or PKA causes cells to arrest growth early in G(1) and to enter G(0) by mechanisms that are poorly understood. Here we demonstrate that the protein kinase Rim15 is required for entry into G(0) following inactivation of TOR and/or PKA. Induction of Rim15-dependent G(0) traits requires two discrete processes, i.e., nuclear accumulation of Rim15, which is negatively regulated both by a Sit4-independent TOR effector branch and the protein kinase B (PKB/Akt) homolog Sch9, and release from PKA-mediated inhibition of its protein kinase activity. Thus, Rim15 integrates signals from at least three nutrient-sensory kinases (TOR, PKA, and Sch9) to properly control entry into G(0), a key developmental process in eukaryotic cells.