Catalytic Activity of the Archetype from Group 4 of the FTR-like Ferredoxin:Thioredoxin Reductase Family Is Regulated by Unique S = 7/2 and S = 1/2 [4Fe-4S] Clusters.

Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.

Cellular Effects of 2',3'-Cyclic Nucleotide Monophosphates in Gram-Negative Bacteria.

Organismal adaptations to environmental stimuli are governed by intracellular signaling molecules such as nucleotide second messengers. Recent studies have identified functional roles for the noncanonical 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) in both eukaryotes and prokaryotes. In Escherichia coli, 2',3'-cNMPs are produced by RNase I-catalyzed RNA degradation, and these cyclic nucleotides modulate biofilm formation through unknown mechanisms. The present work dissects cellular processes in E. coli and Salmonella enterica serovar Typhimurium that are modulated by 2',3'-cNMPs through the development of cell-permeable 2',3'-cNMP analogs and a 2',3'-cyclic nucleotide phosphodiesterase. Utilization of these chemical and enzymatic tools, in conjunction with phenotypic and transcriptomic investigations, identified pathways regulated by 2',3'-cNMPs, including flagellar motility and biofilm formation, and by oligoribonucleotides with 3'-terminal 2',3'-cyclic phosphates, including responses to cellular stress. Furthermore, interrogation of metabolomic and organismal databases has identified 2',3'-cNMPs in numerous organisms and homologs of the E. coli metabolic proteins that are involved in key eukaryotic pathways. Thus, the present work provides key insights into the roles of these understudied facets of nucleotide metabolism and signaling in prokaryotic physiology and suggest broad roles for 2',3'-cNMPs among bacteria and eukaryotes. IMPORTANCE Bacteria adapt to environmental challenges by producing intracellular signaling molecules that control downstream pathways and alter cellular processes for survival. Nucleotide second messengers serve to transduce extracellular signals and regulate a wide array of intracellular pathways. Recently, 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) were identified as contributing to the regulation of cellular pathways in eukaryotes and prokaryotes. In this study, we define previously unknown cell processes that are affected by fluctuating 2',3'-cNMP levels or RNA oligomers with 2',3'-cyclic phosphate termini in E. coli and Salmonella Typhimurium, providing a framework for studying novel signaling networks in prokaryotes. Furthermore, we utilize metabolomics databases to identify additional prokaryotic and eukaryotic species that generate 2',3'-cNMPs as a resource for future studies.

Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer.

Mouse double minute 2 (MDM2) protein functionally inactivates the tumor suppressor p53 in human cancer. Conventional MDM2 inhibitors provide limited clinical application as they interfere only with the MDM2-p53 interaction to release p53 from MDM2 sequestration but do not prevent activated p53 from transcriptionally inducing MDM2 expression. Here, we report a rationally synthesized chalcone-based pyrido[ b ]indole, CPI-7c, as a unique small-molecule inhibitor of MDM2, which not only inhibited MDM2-p53 interaction but also promoted MDM2 degradation. CPI-7c bound to both RING and N-terminal domains of MDM2 to promote its ubiquitin-mediated degradation and p53 stabilization. CPI-7c-induced p53 directly recruited to the promoters of DR4 and DR5 genes and enhanced their expression, resulting in sensitization of TNF-related apoptosis-inducing ligand (TRAIL)-resistant cancer cells toward TRAIL-induced apoptosis. Collectively, we identified CPI-7c as a novel small-molecule inhibitor of MDM2 with a unique two-prong mechanism of action that sensitized TRAIL-resistant cancer cells to apoptosis by modulating the MDM2-p53-DR4/DR5 pathway.

Synthesis of novel ß-carboline based chalcones with high cytotoxic activity against breast cancer cells.

A series of novel ß-carboline based chalcones was synthesized and evaluated for their cytotoxic activity against a panel of human cancer cell lines. Among them we found that two of the compounds 7c and 7d, showed marked anti-proliferative activity in a panel of solid tumor cell lines with highest effect in breast cancer. The compounds 7c and 7d showed an IC50 of 2.25 and 3.29 µM, respectively against human breast cancer MCF-7 cell line. Further, the compound 7c markedly induced DNA fragmentation and apoptosis in breast cancer cells.

Generation of nucleotide-linked resins for identification of novel binding proteins.

Organisms use numerous nucleotide-containing compounds as intracellular signals to control behavior. Identifying the biomolecules responsible to sensing and responding to changes in signaling molecule concentration is an important area of research. However, identifying the binding proteins can be challenging when there is no prior information available about binding motifs. In this chapter, we describe a straightforward method to generate nucleotide-linked resins for use in pull-down experiments to identify binding proteins. The protocol outlined in this chapter also can be adapted to generate custom resins linked to other molecules of interest.

Binding of 2',3'-Cyclic Nucleotide Monophosphates to Bacterial Ribosomes Inhibits Translation.

The intracellular small molecules 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) have recently been rediscovered within both prokaryotes and eukaryotes. Studies in bacteria have demonstrated that 2',3'-cNMP levels affect bacterial phenotypes, such as biofilm formation, motility, and growth, and modulate expression of numerous genes, suggesting that 2',3'-cNMP levels are monitored by cells. In this study, 2',3'-cNMP-linked affinity chromatography resins were used to identify Escherichia coli proteins that bind 2',3'-cNMPs, with the top hits including all of the ribosomal proteins, and to confirm direct binding of purified ribosomes. Using in vitro translation assays, we have demonstrated that 2',3'-cNMPs inhibit translation at concentrations found in amino acid-starved cells. In addition, a genetically encoded tool to increase cellular 2',3'-cNMP levels was developed and was demonstrated to decrease E. coli growth rates. Taken together, this work suggests a mechanism for 2',3-cNMP levels to modulate bacterial phenotypes by rapidly affecting translation.

Trioxaquines: hybrid molecules for the treatment of malaria.

Artemisinin, with its 1,2,4-trioxane as active motif, is now the first-line treatment for multidrug-resistant malaria. The endoperoxide ring is essential for the antimalarial activity of artemisinin. Based on its mechanism of action, new hybrid molecules named trioxaquines with a dual mode of action have been designed. Trioxaquines are made by the covalent attachment of a trioxane, having alkylating ability, to a quinoline, known to easily penetrate within infected erythrocytes. This review discusses the importance of various hybrid molecules of artemisinin and 4-aminoquinoline in the treatment of malaria and the evolution of a trioxaquine hybrid as a promising antimalarial drug candidate.

Synthesis and biological evaluation of indolyl glyoxylamides as a new class of antileishmanial agents.

A series of indolylglyoxylamide derivatives have been synthesized and evaluated in vitro against amastigote form of Leishmania donovani. Compound 8c has been identified as the most active analog of the series with IC(50) value of 5.17µM and SI value of 31.48, and is several folds more potent than the standard drugs sodium stilbogluconate and pentamidine.

Life on the thermodynamic edge: Respiratory growth of an acetotrophic methanogen.

Although two-thirds of the nearly 1 billion metric tons of methane produced annually in Earth's biosphere derives from acetate, the in situ process has escaped rigorous understanding. The unresolved question concerns the mechanism by which the exceptionally marginal amount of available energy supports acetotrophic growth of methanogenic archaea in the environment. Here, we show that Methanosarcina acetivorans conserves energy by Fe(III)-dependent respiratory metabolism of acetate, augmenting production of the greenhouse gas methane. An extensively revised, ecologically relevant, biochemical pathway for acetotrophic growth is presented, in which the conservation of respiratory energy is maximized by electron bifurcation, a previously unknown mechanism of biological energy coupling. The results transform the ecological and biochemical understanding of methanogenesis and the role of iron in the mineralization of organic matter in anaerobic environments.

Identification of a diverse indole-2-carboxamides as a potent antileishmanial chemotypes.

A novel series of highly diverse indole-2-carboxamides was synthesized utilizing the isocyanide based multicomponent reaction (IMCR)-post modification approach and were identified as potential antileishmanial chemotype. Among the synthesized 18 analogues, 12 analogues exhibited better antileishmanial activity against intracellular amastigotes form of Leishmania donovani (IC50 values of 0.6-7.5 µM) as compared to standard drugs miltefosine and sodium stibogluconate. The compounds were also non-toxic towards Vero cells. Compounds 2b, 2m and 2p with significant in vitro activity were then evaluated for their in vivo efficacy following intraperitoneal route. These three compounds at a concentration of 50 mg/kg/day for 5 consecutive days showed 70.0, 63.5 and 63.4% inhibition of Leishmania amastigotes, respectively at day 7 post treatment in hamster model of visceral leishmaniasis.

Discovery of novel antileishmanial agents in an attempt to synthesize pentamidine-aplysinopsin hybrid molecule.

In an attempt to synthesize pentamidine-aplysinopsin hybrid molecule 25, a lead molecule 8 (containing Z-configured aplysinopsin moiety) was identified for antileishmanial activity. Optimization of lead 8 provided 24 (containing E-configured aplysinopsin) possessing 10 times more activity and 401-fold less toxicity than the drug pentamidine in cell based assays. Synthesis of 24 was possible, surprisingly, because of two innate reactivities of indole-3-carbaldehyde which provided it in diastereo- and regio-selectively pure form without recourse to the long reaction pathway.