Exploring DOXP-reductoisomerase binding limits using phosphonated N-aryl and N-heteroarylcarboxamides as DXR inhibitors

DOXP-reductoisomerase (DXR) is a validated target for the development of antimalarial drugs to address the increase in resistant strains of Plasmodium falciparum. Series of aryl- and heteroarylcarbamoylphosphonic acids, their diethyl esters and disodium salts have been prepared as analogues of the potent DXR inhibitor fosmidomycin. The effects of the carboxamide N-substituents and the length of the methylene linker have been explored using in silico docking studies, saturation transfer difference NMR spectroscopy and enzyme inhibition assays using both EcDXR and PfDXR. These studies indicate an optimal linker length of two methylene units and have confirmed the importance of an additional binding pocket in the PfDXR active site. Insights into the constraints of the PfDXR binding site provide additional scope for the rational design of DXR inhibitors with increased ligand–receptor interactions.     

Kinetic characterization of Synechocystis sp. PCC6803 1-deoxy-D-xylulose 5-phosphate reductoisomerase mutants

The methylerythritol phosphate pathway to isoprenoids has been firmly established as an alternate to the mevalonate pathway in many bacteria, plants, algae, and the malaria parasite Plasmodium falciparum. The second enzyme in this pathway, deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; E.C. 1.1.1.267), has been the focus of many investigations since it was found to be the target of the antibacterial and antimalarial compound, fosmidomycin. Several x-ray crystal structures of the Escherichia coli and Zymomonas mobilis DXR enzymes have provided important structural information about the residues potentially involved in substrate binding and catalysis. Site-directed mutagenesis studies can be used to complement the structural studies, providing kinetic data for specific changes of active site residues. Active site mutants were prepared of the recombinant Synechocystis sp. PCC6803 DXR, targeting residues D152, S153, E154, H155, M206, and E223. Alteration of the three acidic residues had major effects on catalysis, changes to S153 and M206 had variable effects on binding and catalysis, and a H155A mutation had only minimal effects on the kinetic parameters.     

Inhibition of doxorubicin-induced mutagenicity by Baccharis dracunculifolia

Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, have been used as an antipyretic, stomachic and health tonic in Brazil. The objective of the present study was to investigate the potential mutagenic effect of B. dracunculifolia ethyl acetate extract (Bd-EAE) and its influence on the mutagenicity induced by the chemotherapeutic agent doxorubicin (DXR) using the rat bone marrow and peripheral blood micronucleus test. Wistar rats were divided into 10 treatment groups. Five groups received DXR (90 mg/kg body weight, b.w., intraperitoneally) to induce mutagenicity and three of these groups received a single oral dose of Bd-EAE at a concentration of 6, 12 or 24 mg/kg b.w. prior to DXR administration. A vehicle-treated control group and Bd-EAE control groups were also included. The results showed that Bd-EAE itself was not mutagenic, in the rat micronucleus assay. In animals treated with Bd-EAE and DXR, the number of MNPCEs was significantly decreased compared to animals receiving DXR alone. HPLC analysis of the extract obtained permitted the identification of the following phenolic compounds: caffeic acid, p-coumaric acid, aromadendrin-4'O-methyl ether, 3-prenyl-p-coumaric acid (drupanin), 3,5-diprenyl-p-coumaric acid (artepillin C) and baccharin. The putative antioxidant activity or the interference of one or more of the active compounds of Bd-EAE with mutagenic metabolic pathways may explain its effect on DXR mutagenicity.     

Mutation in the flexible loop of 1-deoxy-D-xylulose 5-phosphate reductoisomerase broadens substrate utilization

The second enzyme in the methylerythritol phosphate pathway to isoprenoids, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; EC 1.1.1.267) mediates the transformation of 1-deoxy-D-xylulose 5-phosphate (DXP) into 2-C-methyl-D-erythritol 4-phosphate. Several DXR mutants have been prepared to study amino acid residues important in binding or catalysis, but in-depth studies of many conserved residues in the flexible loop portion of the enzyme have not been conducted. In the course of our studies of this enzyme, an analog of DXP, 1,2-dideoxy-D-threo-3-hexulose 6-phosphate (1-methyl-DXP), was found to be a weak competitive inhibitor. Using the X-ray crystal structures of DXR as a guide, a highly conserved tryptophan residue in the flexible loop was identified that potentially blocks the use of this analog as a substrate. To test this hypothesis, four mutants of the Synechocystis sp. PCC6803 DXR were prepared and a W204F mutant was found to utilize the analog as a substrate.     

Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis

Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I(50) values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.     

Inhibition of doxorubicin-induced mutagenicity by Baccharis dracunculifolia

Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, have been used as an antipyretic, stomachic and health tonic in Brazil. The objective of the present study was to investigate the potential mutagenic effect of B. dracunculifolia ethyl acetate extract (Bd-EAE) and its influence on the mutagenicity induced by the chemotherapeutic agent doxorubicin (DXR) using the rat bone marrow and peripheral blood micronucleus test. Wistar rats were divided into 10 treatment groups. Five groups received DXR (90 mg/kg body weight, b.w., intraperitoneally) to induce mutagenicity and three of these groups received a single oral dose of Bd-EAE at a concentration of 6, 12 or 24 mg/kg b.w. prior to DXR administration. A vehicle-treated control group and Bd-EAE control groups were also included. The results showed that Bd-EAE itself was not mutagenic, in the rat micronucleus assay. In animals treated with Bd-EAE and DXR, the number of MNPCEs was significantly decreased compared to animals receiving DXR alone. HPLC analysis of the extract obtained permitted the identification of the following phenolic compounds: caffeic acid, p-coumaric acid, aromadendrin-4′O-methyl ether, 3-prenyl-p-coumaric acid (drupanin), 3,5-diprenyl-p-coumaric acid (artepillin C) and baccharin. The putative antioxidant activity or the interference of one or more of the active compounds of Bd-EAE with mutagenic metabolic pathways may explain its effect on DXR mutagenicity.     

The crystal structure of E.coli 1-deoxy-D-xylulose-5-phosphate reductoisomerase in a ternary complex with the antimalarial compound fosmidomycin and NADPH reveals a tight-binding closed enzyme conformation

The key enzyme in the non-mevalonate pathway of isoprenoid biosynthesis, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) has been shown to be the target enzyme of fosmidomycin, an antimalarial, antibacterial and herbicidal compound. Here we report the crystal structure of selenomethionine-labelled Escherichia coli DXR in a ternary complex with NADPH and fosmidomycin at 2.2 A resolution. The structure reveals a considerable conformational rearrangement upon fosmidomycin binding and provides insights into the slow, tight binding inhibition mode of the inhibitor. Although the inhibitor displays an unusual non-metal mediated mode of inhibition, which is an artefact most likely due to the low metal affinity of DXR at the pH used for crystallization, the structural data add valuable information for the rational design of novel DXR inhibitors. Using this structure together with the published structural data and the 1.9 A crystal structure of DXR in a ternary complex with NADPH and the substrate 1-deoxy-D-xylulose 5-phosphate, a model for the physiologically relevant tight-binding mode of inhibition is proposed. The structure of the substrate complex must be interpreted with caution due to the presence of a second diastereomer in the active site.     

Crystal structures of MAP kinase p38 complexed to the docking sites on its nuclear substrate MEF2A and activator MKK3b

The structures of the MAP kinase p38 in complex with docking site peptides containing a phi(A)-X-phi(B) motif, derived from substrate MEF2A and activating enzyme MKK3b, have been solved. The peptides bind to the same site in the C-terminal domain of the kinase, which is both outside the active site and distinct from the "CD" domain previously implicated in docking site interactions. Mutational analysis on the interaction of p38 with the docking sites supports the crystallographic models and has uncovered two novel residues on the docking groove that are critical for binding. The two peptides induce similar large conformational changes local to the peptide binding groove. The peptides also induce unexpected and different conformational changes in the active site, as well as structural disorder in the phosphorylation lip.     

The influence of physical characteristics of liposomes containing doxorubicin on their pharmacological behavior

We have investigated the behavior of two populations of doxorubicin (DXR)-containing phospholipid vesicles with regard to various physical and pharmacological parameters. DXR-containing liposomes were prepared by ultrasonic irradiation, the lipid composition being phosphatidylglycerol (or phosphatidylserine), phosphatidylcholine and cholesterol. The vesicles were fractionated into oligolamellar vesicles (OLV) and small unilamellar vesicles (SUV) by preparative differential ultracentrifugation (150,000 x g for 1 h). Unentrapped DXR was removed by gel exclusion chromatography. OLV and SUV liposomes differed in size (mean diameters, 247 +/- 113 nm and 61 +/- 16 nm, respectively) and number of lamellae (two for OLV, one for SUV). Drug entrapment per unit of lipid was three to 5-fold higher in OLV than in SUV. In both liposome populations more than 95% of the entrapped drug was membrane-associated. Physical studies on these two vesicle populations revealed higher motional restriction and greater susceptibility to iodide-mediated fluorescence collisional quenching of DXR in the small vesicles. OLV showed superior stability in the presence of plasma as determined by the fraction of DXR retained by the vesicles. It was also found that the tissue distribution of DXR in SUV follows a pattern different from that of DXR in OLV and resembling that of soluble DXR. In accordance with these differences in patterns of tissue distribution, animal studies demonstrated that DXR in OLV is significantly less toxic than DXR in SUV and more effective in a tumor model with predominant involvement of the liver. These results indicate that vesicle size and/or number of lamellae play an important role in optimizing liposome-mediated delivery of DXR, and that oligolamellar liposomes are distinctively superior to small unilamellar liposomes when fluid phase formulations (Tm less than 37 degrees C) with bilayer-associated DXR are considered.     

Modeling GPCR active state conformations: the β(2)-adrenergic receptor

The recent publication of several G protein-coupled receptor (GPCR) structures has increased the information available for homology modeling inactive class A GPCRs. Moreover, the opsin crystal structure shows some active features. We have therefore combined information from these two sources to generate an extensively validated model of the active conformation of the β(2)-adrenergic receptor. Experimental information on fully active GPCRs from zinc binding studies, site-directed spin labeling, and other spectroscopic techniques has been used in molecular dynamics simulations. The observed conformational changes reside mainly in transmembrane helix 6 (TM6), with additional small but significant changes in TM5 and TM7. The active model has been validated by manual docking and is in agreement with a large amount of experimental work, including site-directed mutagenesis information. Virtual screening experiments show that the models are selective for β-adrenergic agonists over other GPCR ligands, for (R)- over (S)-β-hydroxy agonists and for β(2)-selective agonists over β(1)-selective agonists. The virtual screens reproduce interactions similar to those generated by manual docking. The C-terminal peptide from a model of the stimulatory G protein, readily docks into the active model in a similar manner to which the C-terminal peptide from transducin, docks into opsin, as shown in a recent opsin crystal structure. This GPCR-G protein model has been used to explain site-directed mutagenesis data on activation. The agreement with experiment suggests a robust model of an active state of the β(2)-adrenergic receptor has been produced. The methodology used here should be transferable to modeling the active state of other GPCRs.     

Activation of Bak,Bax, and BH3-only proteins in the apoptotic response to doxorubicin

The anthracyclin doxorubicin (DXR) is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. It is not clear, however, how the subsequent lesions may lead to the apoptotic death of the cell. We have here examined the effects of DXR on activation of pro-apoptotic members of the Bcl-2 family, all of which are connected to the mitochondrial events of apoptosis. In two human cell lines (lymphoma and myeloma), clinically relevant concentrations of DXR were found to induce apoptosis, first observed after 24 h of treatment. Apoptosis correlated with modulation of Bak and Bax to their active conformations. bax- as well as bak-deficient mouse embryo fibroblasts were resistant to DXR compared with wild-type mouse embryo fibroblasts further supporting a role for these proteins as main DXR-induced apoptosis regulators. Furthermore, using immunocytochemistry as well as chemical blocking of putative apical pathways we could demonstrate that Bak is activated prior to Bax. In the human cell lines, DXR was furthermore found to induce high protein levels of Bik, another BH3-only protein. DXR-induced apoptosis was completely blocked in Bcl-2-overexpressing U266 cells. Interestingly, in Bcl-2-transfected cells Bak activation was also blocked, while Bax was still partially active in agreement with differential regulation of these two proteins. Furthermore, co-incubation of the phosphatidylinositol 3-kinase (PI3K)-inhibitor LY294002 potentiated the apoptotic response to DXR. This enhanced apoptosis was preceded by enhanced Bak and Bax activation, and both responses as well as apoptosis were blocked in transfectants overexpressing Bcl-2. In summary, several pieces of evidence suggest that DXR induces apoptosis through a sequential and differential activation of Bak and Bax.     

Docking-based 3D-QSAR study for 11beta-HSD1 inhibitors

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) enzymes catalyze the conversion of biologically inactive 11-ketosteroids into their active 11beta-hydroxy derivatives and vice versa. 11beta-HSD1 has been studied as a potential treatment for metabolic disease such as diabetes and obesity. To find correlation between 11beta-HSD1 and inhibitors, three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were performed on 70 inhibitors, based on molecular docking conformations obtained by using FlexX-Pharm. The studies include comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Based on the docking results, highly predictive 3D-QSAR models were developed with q(2) values of 0.543 and 0.519 for CoMFA and CoMSIA, respectively. A comparison of the 3D-QSAR field contributions with the structural features of the binding site showed good correlation between the two analyses. Therefore, these results should be useful to the prediction of the activities of new 11beta-HSD1 inhibitors.     

Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein

The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10–100 μM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3±1.11% and 68.8±1.52% at 80 μM, respectively. Importantly, OLA (80 μM) or MLA (30 μM) enhanced the antitumoral effect of DXR (0.5–10 μM) by up to 2.3-fold. On the molecular level, efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells. Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment.     

Identification of Phytochemicals Targeting c-Met Kinase Domain using Consensus Docking and Molecular Dynamics Simulation Studies

c-Met receptor tyrosine kinase is a proto-oncogene whose aberrant activation is attributed to a lower rate of survival in most cancers. Natural product-derived inhibitors known as “fourth generation inhibitors” constitute more than 60% of anticancer drugs. Furthermore, consensus docking approach has recently been introduced to augment docking accuracy and reduce false positives during a virtual screening. In order to obtain novel small-molecule Met inhibitors, consensus docking approach was performed using Autodock Vina and Autodock 4.2 to virtual screen Naturally Occurring Plant-based Anti-cancer Compound–Activity–Target database against active and inactive conformation of c-Met kinase domain structure. Two hit molecules that were in line with drug-likeness criteria, desired docking score, and binding pose were subjected to molecular dynamics simulations to elucidate intermolecular contacts in protein–ligand complexes. Analysis of molecular dynamics simulations and molecular mechanics Poisson–Boltzmann surface area studies showed that ZINC08234189 is a plausible inhibitor for the active state of c-Met, whereas ZINC03871891 may be more effective toward active c-Met kinase domain compared to the inactive form due to higher binding energy. Our analysis showed that both the hit molecules formed hydrogen bonds with key residues of the hinge region (P1158, M1160) in the active form, which is a hallmark of kinase domain inhibitors. Considering the pivotal role of HGF/c-Met signaling in carcinogenesis, our results propose ZINC08234189 and ZINC03871891 as the therapeutic options to surmount Met-dependent cancers.     

Methodology for multi-site ligand-protein docking identification developed for the optimization of spirostenol inhibition of beta-amyloid-induced neurotoxicity

Spirostenol steroids have been found to inhibit beta-amyloid-induced neurotoxicity. We have evaluated in parallel experimental and molecular-modeling studies the relative effectiveness of 17 (22R)-hydroxycholesterol derivatives in binding to the target peptide. Our results support the previous evidence that beta-amyloid offers multiple docking sites for these steroids. Molecular modeling allowed for the correlation of spirostenol candidate structural differences with a choice of proposed active sites. A multi-site identification technique based on a Site-Identifier Matrix (SIM) was developed that clearly showed the uniqueness of our lead (maximum neurotoxicity inhibition) candidate SP233, with a nearly equal docking affinity for two sites.     

Outside‐binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1

The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA‐oseltamivir complex (PDB ID: 3NSS) was used as a wild‐type structure. After selecting the target NA sequences, their three‐dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free‐energy analysis using the MM‐PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM‐PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature. © 2012 Wiley Periodicals, Inc.     

Dexrazoxane Diminishes Doxorubicin-Induced Acute Ovarian Damage and Preserves Ovarian Function and Fecundity in Mice

Advances in cancer treatment utilizing multiple chemotherapies have dramatically increased cancer survivorship. Female cancer survivors treated with doxorubicin (DXR) chemotherapy often suffer from an acute impairment of ovarian function, which can persist as long-term, permanent ovarian insufficiency. Dexrazoxane (Dexra) pretreatment reduces DXR-induced insult in the heart, and protects in vitro cultured murine and non-human primate ovaries, demonstrating a drug-based shield to prevent DXR insult. The present study tested the ability of Dexra pretreatment to mitigate acute DXR chemotherapy ovarian toxicity in mice through the first 24 hours post-treatment, and improve subsequent long-term fertility throughout the reproductive lifespan. Adolescent CD-1 mice were treated with Dexra 1 hour prior to DXR treatment in a 1:1 mg or 10:1 mg Dexra:DXR ratio. During the acute injury period (2–24 hours post-injection), Dexra pretreatment at a 1:1 mg ratio decreased the extent of double strand DNA breaks, diminished γH2FAX activation, and reduced subsequent follicular cellular demise caused by DXR. In fertility and fecundity studies, dams pretreated with either Dexra:DXR dose ratio exhibited litter sizes larger than DXR-treated dams, and mice treated with a 1:1 mg Dexra:DXR ratio delivered pups with birth weights greater than DXR-treated females. While DXR significantly increased the “infertility index” (quantifying the percentage of dams failing to achieve pregnancy) through 6 gestations following treatment, Dexra pretreatment significantly reduced the infertility index following DXR treatment, improving fecundity. Low dose Dexra not only protected the ovaries, but also bestowed a considerable survival advantage following exposure to DXR chemotherapy. Mouse survivorship increased from 25% post-DXR treatment to over 80% with Dexra pretreatment. These data demonstrate that Dexra provides acute ovarian protection from DXR toxicity, improving reproductive health in a mouse model, suggesting this clinically available drug may provide ovarian protection for cancer patients.     

Crystal structure of 1-deoxy-d-xylulose-5-phosphate reductoisomerase from Zymomonas mobilis at 1.9-A resolution

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) is the second enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. The structure of the apo-form of this enzyme from Zymomonas mobilis has been solved and refined to 1.9-A resolution, and that of a binary complex with the co-substrate NADPH to 2.7-A resolution. The subunit of DXR consists of three domains. Residues 1-150 form the NADPH binding domain, which is a variant of the typical dinucleotide-binding fold. The second domain comprises a four-stranded mixed beta-sheet, with three helices flanking the sheet. Most of the putative active site residues are located on this domain. The C-terminal domain (residues 300-386) folds into a four-helix bundle. In solution and in the crystal, the enzyme forms a homo-dimer. The interface between the two monomers is formed predominantly by extension of the sheet in the second domain. The adenosine phosphate moiety of NADPH binds to the nucleotide-binding fold in the canonical way. The adenine ring interacts with the loop after beta1 and with the loops between alpha2 and beta2 and alpha5 and beta5. The nicotinamide ring is disordered in crystals of this binary complex. Comparisons to Escherichia coli DXR show that the two enzymes are very similar in structure, and that the active site architecture is highly conserved. However, there are differences in the recognition of the adenine ring of NADPH in the two enzymes.