Autophagy-related gene (Atg) 5 is a gene product required for the formation of autophagosomes. Here, we report that Atg5, in addition to the promotion of autophagy, enhances susceptibility towards apoptotic stimuli. Enforced expression of Atg5-sensitized tumour cells to anticancer drug treatment both in vitro and in vivo. In contrast, silencing the Atg5 gene with short interfering RNA (siRNA) resulted in partial resistance to chemotherapy. Apoptosis was associated with calpain-mediated Atg5 cleavage, resulting in an amino-terminal cleavage product with a relative molecular mass of 24,000 (Mr 24K). Atg5 cleavage was observed independent of the cell type and the apoptotic stimulus, suggesting that calpain activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the anti-apoptotic molecule Bcl-xL and triggered cytochrome c release and caspase activation. Taken together, calpain-mediated Atg5 cleavage provokes apoptotic cell death, therefore, represents a molecular link between autophagy and apoptosis--a finding with potential importance for clinical anticancer therapies.

The understanding of molecular recognition processes of small ligands and biological macromolecules requires a complete characterization of the binding energetics and correlation of thermodynamic data with interacting structures involved. A quantitative description of the forces that govern molecular associations requires determination of changes of all thermodynamic parameters, including free energy of binding (deltaG), enthalpy (deltaH), and entropy (deltaS) of binding and the heat capacity change (deltaCp). A close insight into the binding process is of significant and practical interest, since it provides the fundamental know-how for development of structure-based molecular design-strategies. The only direct method to measure the heat change during complex formation at constant temperature is provided by isothermal titration calorimetry (ITC). With this method one binding partner is titrated into a solution containing the interaction partner, thereby generating or absorbing heat. This heat is the direct observable that can be quantified by the calorimeter. The use of ITC has been limited due to the lack of sensitivity, but recent developments in instrument design permit to measure heat effects generated by nanomol (typically 10-100) amounts of reactants. ITC has emerged as the primary tool for characterizing interactions in terms of thermodynamic parameters. Because heat changes occur in almost all chemical and biochemical processes, ITC can be used for numerous applications, e.g., binding studies of antibody-antigen, protein-peptide, protein-protein, enzyme-inhibitor or enzyme-substrate, carbohydrate-protein, DNA-protein (and many more) interactions as well as enzyme kinetics. Under appropriate conditions data analysis from a single experiment yields deltaH, K(B), the stoichiometry (n), deltaG and deltaS of binding. Moreover, ITC experiments performed at different temperatures yield the heat capacity change (deltaCp). The informational content of thermodynamic data is large, and it has been shown that it plays an important role in the elucidation of binding mechanisms and, through the link to structural data, also in rational drug design. In this review we will present a comprehensive overview to ITC by giving some historical background to calorimetry, outline some critical experimental and data analysis aspects, discuss the latest developments, and give three recent examples of studies published with respect to macromolecule-ligand interactions that have utilized ITC technology.

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD ؉ , and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for C␣s, 0.30 Å). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.Treatment of Plasmodium falciparum malaria has depended for decades on the use of the aminoquinoline chloroquine or the synergistic antifolate combination pyrimethamine-sulfadoxine. These drugs were characterized by their potency against the P. falciparum asexual intra-erythrocytic stages (responsible for malaria pathogenesis), their affordability and their safety. The occurrence and spread of drug-resistant strains of P. falciparum have largely contributed to a recent resurgence of malaria, which claims 1 to 3 million lives annually and which is endemic in inter-tropical areas representing 40% of the world's population (1). The current situation of antimalarial chemotherapy and resistance, in conjunction with the reappearance of malaria in formerly well-controlled areas, reinforces the need for new, highly potent antimalarials.Recent investigations into Apicomplexan parasites, including Plasmodium and Toxoplasma, have uncovered the existence of a unique organelle, the apicoplast (2-4). The finding that ciprofloxacin-mediated inhibition of plastid replication in Toxoplasma gondii tachyzoites blocked parasite propagation provided evidence for the indispensable nature of this nonphotosynthetic plastid organelle (5). Studies of plastid inhibitors and apicoplast mis-segregation mutants confirmed the essential requirement of this organelle for normal parasite development and indicated that inhibition of apicoplast function or loss of this organelle resulted in parasite death following reinvasion of host cells (5-7). This organelle appears to derive ultimately from a cyanobacterial endosymbiont (4,8,9) and as such was postulated to contain prokaryotic-type metabolic pathways, of significant interest from the perspective of developing antiparasitic drugs (10). Recent studies indicate that these pathways include fatty acid and isoprenoid bios...

Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD ؉ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.

Understanding ligand-protein recognition and interaction processes is of primary importance for structure-based drug design. Traditionally, several approaches combining docking and molecular dynamics (MD) simulations have been exploited to investigate the physicochemical properties of complexes of pharmaceutical interest. Even if the geometric properties of a modeled protein-ligand complex can be well predicted by computational methods, it is challenging to rank a series of analogues in a consistent fashion with biological data. In the unique beta-hydroxyacyl-ACP dehydratase of Plasmodium falciparum (PfFabZ), the application of standard molecular docking and MD simulations was partially sufficient to shed light on the activity of previously discovered inhibitors. Complementing docking results with atomistic simulations in the steered molecular dynamics (SMD) framework, we devised an in silico approach to study molecular interactions and to compare the binding characteristics of ligand analogues. We hypothesized an interaction model that both explained the biological activity of known ligands, and provided insight into designing novel enzyme inhibitors. Mimicking single-molecule pulling experiments, we used SMD-derived force profiles to discern active from inactive compounds for the first time. A new compound was designed and its biological activity toward the PfFabZ enzyme predicted. Finally, the computational predictions were experimentally confirmed, highlighting the robustness of the drug design approach presented herein.

After the discovery of a potent natural flavonoid glucoside as a potent inhibitor of FabI, a large flavonoid library was screened against three important enzymes (i.e., FabG, FabZ, and FabI) involved in the fatty acid biosynthesis of P. falciparum. Although flavones with a simple hydroxylation pattern (compounds 4-9) showed moderate inhibitory activity toward the enzymes tested (IC50 10-100 microM), the more complex flavonoids (12-16) exhibited strong activity toward all three enzymes (IC50 0.5-8 microM). Isoflavonoids 26-28 showed moderate (IC50 7-30 microM) but selective activity against FabZ. The most active compounds were C-3 gallic acid esters of catechins (32, 33, 37, 38), which are strong inhibitors of all three enzymes (IC50 0.2-1.1 microM). Kinetic analysis using luteolin (12) and (-)-catechin gallate (37) as model compounds revealed that FabG was inhibited in a noncompetitive manner. FabZ was inhibited competitively, whereas both compounds behaved as tight-binding noncompetitive inhibitors of FabI. In addition, these polyphenols showed in vitro activity against chloroquine-sensitive (NF54) and -resistant (K1) P. falciparum strains in the low to submicromolar range.

In the resolution of infl ammatory responses, neutrophils rapidly undergo apoptosis. We describe a new proapoptotic pathway in which cathepsin D directly activates caspase-8. Cathepsin D is released from azurophilic granules in neutrophils in a caspase-independent but reactive oxygen species -dependent manner. Under infl ammatory conditions, the translocation of cathepsin D in the cytosol is blocked. Pharmacological or genetic inhibition of cathepsin D resulted in delayed caspase activation and reduced neutrophil apoptosis. Cathepsin D defi ciency or lack of its translocation in the cytosol prolongs innate immune responses in experimental bacterial infection and in septic shock. Thus, we identifi ed a new function of azurophilic granules that is in addition to their role in bacterial defense mechanisms: to regulate the life span of neutrophils and, therefore, the duration of innate immune responses through the release of cathepsin D.

SummaryThe two-component regulatory system CitA /CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain¯anked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE±CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327±547) showed constitutive autokinase activity and transferred the g-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB±D56N formed a stable complex with MalE±CitAC.