Background: The carbon metabolism of the blood stages of Plasmodium falciparum, comprising rapidly dividing asexual stages and non-dividing gametocytes, is thought to be highly streamlined, with glycolysis providing most of the cellular ATP. However, these parasitic stages express all the enzymes needed for a canonical mitochondrial tricarboxylic acid (TCA) cycle, and it was recently proposed that they may catabolize glutamine via an atypical branched TCA cycle. Whether these stages catabolize glucose in the TCA cycle and what is the functional significance of mitochondrial metabolism remains unresolved. Results: We reassessed the central carbon metabolism of P. falciparum asexual and sexual blood stages, by metabolically labeling each stage with 13 C-glucose and 13 C-glutamine, and analyzing isotopic enrichment in key pathways using mass spectrometry. In contrast to previous findings, we found that carbon skeletons derived from both glucose and glutamine are catabolized in a canonical oxidative TCA cycle in both the asexual and sexual blood stages. Flux of glucose carbon skeletons into the TCA cycle is low in the asexual blood stages, with glutamine providing most of the carbon skeletons, but increases dramatically in the gametocyte stages. Increased glucose catabolism in the gametocyte TCA cycle was associated with increased glucose uptake, suggesting that the energy requirements of this stage are high. Significantly, whereas chemical inhibition of the TCA cycle had little effect on the growth or viability of asexual stages, inhibition of the gametocyte TCA cycle led to arrested development and death.
Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using 13C-stable isotope resolved metabolomics and 2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.
SUMMARY Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, have potential use as chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers, and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.
Leishmania parasites proliferate within nutritionally complex niches in their sandfly vector and mammalian hosts. However, the extent to which these parasites utilize different carbon sources remains poorly defined. In this study, we have followed the incorporation of various 13 C-labeled carbon sources into the intracellular and secreted metabolites of Leishmania mexicana promastigotes using gas chromatography-mass spectrometry and C]alanine uptake and catabolism. TCA cycle anaplerosis is apparently needed to sustain glutamate production under standard culture conditions. Specifically, inhibition of mitochondrial aconitase with sodium fluoroacetate resulted in the rapid depletion of intracellular glutamate pools and growth arrest. Addition of high concentrations of exogenous glutamate alleviated this growth arrest. These findings suggest that glycosomal and mitochondrial metabolism in Leishmania promastigotes is tightly coupled and that, in contrast to the situation in some other trypanosomatid parasites, the TCA cycle has crucial anabolic functions.Leishmania spp. are parasitic protozoa that cause a spectrum of disease in humans, ranging from self-limiting cutaneous infections to disseminating infections (mucocutaneous and visceral leishmaniasis) that can lead to severe morbidity and death (1). Approximately 12 million people are infected worldwide, resulting in more than 50,000 deaths each year (2). There are no vaccines against any of these diseases, and current drug therapies are both limited and, in many cases, are being undermined by widespread resistance (2). Although the genomes of several Leishmania species have now been sequenced and key aspects of metabolism intensively studied, major gaps exist in our understanding of central carbon metabolism in these divergent eukaryotes (3, 4). Given the importance of intermediary metabolism for parasite growth and protection against host microbicidal processes, detailed dissection of Leishmania carbon metabolism may reveal new therapeutic targets (4 -6).Leishmania develop as extracellular flagellated promastigote stages within the digestive tract of the sandfly vector. This stage is transmitted to the mammalian host when the female sandfly host takes a blood meal and is rapidly phagocytosed by neutrophils and macrophages (1). Promastigotes internalized into the phagolysosome compartment of macrophages differentiate into the obligate intracellular amastigote stage that perpetuates infection in the mammalian host. Promastigote stages are readily cultivated in vitro and have been the focus of most metabolic studies. Like the intensively studied insect (procyclic) stage of the related trypanosomatid, Trypanosoma brucei (7,8), Leishmania promastigotes are thought to catabolize glucose via glycolysis, with the initial enzymes in this pathway being largely or exclusively compartmentalized in modified peroxisomes, termed glycosomes (9, 10). The glycosomal catabolism of glucose to 1,3-bisphosphoglycerate requires an investment of both ATP and NAD ϩ , and the rate at whic...
The α-carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible hydration of CO 2 in forming HCO 3 -. The active site of an α-CA contains a catalytically essential Zn 2+ coordinated by three histidine residues at the bottom of a 15 Å deep cleft, and the tightest binding CA inhibitors developed to date contain a sulfonamide moiety that coordinates to Zn 2+ as a sulfonamidate anion. 1 Notably, human isozyme II (CAII) is an ideal model system for exploring new inhibitor designs, some of which can be exploited in biosensing applications. 2-4 Here, CAII is utilized for the structure-based design of a xenon ( 129 Xe) biosensor for potential use as a magnetic resonance imaging (MRI) contrast agent.The 129 Xe isotope has a spin-1/2 nucleus, a >200-ppm chemical shift window in water, and a natural isotopic abundance of 26% (commercially available up to 86%), which makes it an appealing biomolecular probe for MRI. Moreover, 129 Xe can be laser polarized to enhance MRI signals ∼10,000-fold. 5 Although current in vivo MRI applications are limited to functional lung imaging through the diffusion of Xe gas, 6 the encapsulation of 129 Xe within a cryptophane cage (K D ≈ 30 μM at 37 °C in phosphate-buffered solution) 7 facilitates its use as a biosensor that can be targeted to specific proteins using an appropriate affinity tag. 8,9 For example, racemic biosensor 1 (Figure 1a) has been designed to bind to the CA isozymes (K D = 60 ± 20 nM against CAII in solution), and yields a distinctive 129 Xe-MRI spectrum when bound to CAII. 10 Here, we report the X-ray crystal structure of the CAII-1-Xe complex at 1.70 Å resolution.For structure determination, CAII was overexpressed in E. coli and purified as described, 11 then incubated with a two-fold excess of 1, concentrated to 10 mg/mL, and crystallized by the hanging drop vapor diffusion method. Crystals were cryoprotected in 15% glycerol and subsequently pressurized under 20 atm Xe for 30 min prior to flash cooling and X-ray data collection. The structure was refined to final R work and R free values of 0.23 and 0.25, respectively.Biosensor 1 coordinates to the active site Zn 2+ ion as the sulfonamidate anion, displacing the zinc-bound hydroxide ion of the native enzyme as previously observed in other complexes of CAII with benzenesulfonamide derivatives. 1,2,12 The crystallographic occupancies of 1 and Zn 2+ are refined at 0.5. It is unusual to observe diminished Zn 2+ occupancy in a CAII-inhibitor complex, but the molecular origins of this effect are not clear.The encapsulation of Xe within the cryptophane cage of 1 is confirmed by inspection of the Bijvoet difference Fourier map calculated from anomalous scattering data (Figures 1b and S1.) X-ray diffraction data was collected at a wavelength λ = 0.9795 Å, which is far from the Xe L I edge of 2.27 Å. 13 Nevertheless, the anomalous scattering component f" is 3.4 e -for Xe, so Email: chris@sas.upenn.edu. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2009 June 4. NIH-PA A...
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