Efficient synthesis of benzamide riboside, a potential anticancer agent

An efficient five step synthesis of benzamide riboside (BR) amenable for a large scale synthesis has been developed. It allows for extensive pre-clinical studies of BR as a potential anticancer agent.     

DNA-energetics-based analyses suggest additional genes in prokaryotes

We present here a novel methodology for predicting new genes in prokaryotic genomes on the basis of inherent energetics of DNA. Regions of higher thermodynamic stability were identified, which were filtered based on already known annotations to yield a set of potentially new genes. These were then processed for their compatibility with the stereo-chemical properties of proteins and tripeptide frequencies of proteins in Swissprot data, which results in a reliable set of new genes in a genome. Quite surprisingly, the methodology identifies new genes even in well-annotated genomes. Also, the methodology can handle genomes of any GC-content, size and number of annotated genes.     

Identification of a Novel Abscisic Acid-Regulated Farnesol Dehydrogenase from Arabidopsis

In Arabidopsis (Arabidopsis thaliana), farnesylcysteine is oxidized to farnesal and cysteine by a membrane-associated thioether oxidase called farnesylcysteine lyase. Farnesol and farnesyl phosphate kinases have also been reported in plant membranes. Together, these observations suggest the existence of enzymes that catalyze the interconversion of farnesal and farnesol. In this report, Arabidopsis membranes are shown to possess farnesol dehydrogenase activity. In addition, a gene on chromosome 4 of the Arabidopsis genome (At4g33360), called FLDH, is shown to encode an NAD⁺-dependent dehydrogenase that oxidizes farnesol more efficiently than other prenyl alcohol substrates. FLDH expression is repressed by abscisic acid (ABA) but is increased in mutants with T-DNA insertions in the FLDH 5′ flanking region. These T-DNA insertion mutants, called fldh-1 and fldh-2, are associated with an ABA-insensitive phenotype, suggesting that FLDH is a negative regulator of ABA signaling.Isoprenylated proteins are modified at the C terminus via cysteinyl thioether linkage to either a 15-carbon farnesyl or a 20-carbon geranylgeranyl group (Clarke, 1992; Zhang and Casey, 1996; Rodríguez-Concepción et al., 1999; Crowell, 2000; Crowell and Huizinga, 2009). These modifications mediate protein-membrane and protein-protein interactions and are necessary for the proper localization and function of hundreds of proteins in eukaryotic cells. In Arabidopsis (Arabidopsis thaliana), the PLURIPETALA (PLP; At3g59380) and ENHANCED RESPONSE TO ABA1 (At5g40280) genes encode the α- and β-subunits of protein farnesyltransferase (PFT), respectively (Cutler et al., 1996; Pei et al., 1998; Running et al., 2004). These subunits form a heterodimeric zinc metalloenzyme that catalyzes the efficient transfer of a farnesyl group from farnesyl diphosphate to protein substrates with a C-terminal CaaX motif, where “C” is Cys, “a” is an aliphatic amino acid, and “X” is usually Met, Gln, Cys, Ala, or Ser (Fig. 1). The PLP and GERANYLGERANYL-TRANSFERASE BETA (At2g39550) genes encode the α- and β-subunits of protein geranylgeranyltransferase type 1 (PGGT1), respectively (Running et al., 2004; Johnson et al., 2005). These subunits form a distinct heterodimeric zinc metalloenzyme that catalyzes the efficient transfer of a geranylgeranyl group from geranylgeranyl diphosphate to protein substrates with a C-terminal CaaL motif, where “C” is Cys, “a” is an aliphatic amino acid, and “L” is Leu. A third protein prenyltransferase, called protein geranylgeranyltransferase type II or RAB geranylgeranyltransferase, catalyzes the dual geranylgeranylation of RAB proteins with a C-terminal XCCXX, XXCXC, XXCCX, XXXCC, XCXXX, or CCXXX motif, where “C” is Cys and “X” is any amino acid. However, RAB proteins must be associated with the RAB ESCORT PROTEIN to be substrates of RAB geranylgeranyltransferase. Plant protein prenylation has received considerable attention in recent years because of the meristem defects of Arabidopsis PFT mutants and the abscisic acid (ABA) hypersensitivity of Arabidopsis PFT and PGGT1 mutants (Cutler et al., 1996; Pei et al., 1998; Running et al., 1998, 2004; Johnson et al., 2005).Open in a separate windowFigure 1.Proposed metabolism of farnesal and farnesol as it relates to protein prenylation. The portion of the cycle shown in red is the subject of this article.Proteins that are prenylated by either PFT or PGGT1 undergo further processing in the endoplasmic reticulum (Crowell, 2000; Crowell and Huizinga, 2009). First, the aaX portion of the CaaX motif is removed by proteolysis (Fig. 1). This reaction is catalyzed by one of two CaaX endoproteases, which are encoded by the AtSTE24 (At4g01320) and AtFACE-2 (At2g36305) genes (Bracha et al., 2002; Cadiñanos et al., 2003). Second, the prenylated Cys residue at the new C terminus is methylated by one of two isoprenylcysteine methyltransferases (Fig. 1), which are encoded by the AtSTE14A (At5g23320) and AtSTE14B (ICMT; At5g08335) genes (Crowell et al., 1998; Crowell and Kennedy, 2001; Narasimha Chary et al., 2002; Bracha-Drori et al., 2008). A specific isoprenylcysteine methylesterase encoded by the Arabidopsis ICME (At5g15860) gene has also been described, demonstrating the reversibility of isoprenylcysteine methylation (Deem et al., 2006; Huizinga et al., 2008).Like all proteins, prenylated proteins have a finite half-life. However, unlike other proteins, prenylated proteins release farnesylcysteine (FC) or geranylgeranylcysteine (GGC) upon degradation. Mammals possess a prenylcysteine lyase enzyme that catalyzes the oxidative cleavage of FC and GGC (Zhang et al., 1997; Tschantz et al., 1999; Tschantz et al., 2001; Beigneux et al., 2002; Digits et al., 2002). This FAD-dependent thioether oxidase consumes molecular oxygen and generates hydrogen peroxide, Cys, and a prenyl aldehyde product (i.e. farnesal or geranylgeranial). In Arabidopsis, a similar lyase exists. However, the Arabidopsis enzyme, which is encoded by the FCLY (At5g63910) gene, is specific for FC (Fig. 1; Crowell et al., 2007; Huizinga et al., 2010). GGC is metabolized by a different mechanism.Plant membranes have been shown to contain farnesol kinase, geranylgeraniol kinase, farnesyl phosphate kinase, and geranylgeranyl phosphate kinase activities (Fig. 1; Thai et al., 1999). These membrane-associated kinases differ with respect to nucleotide specificity, suggesting that they are distinct enzymes (i.e. farnesol kinase and geranylgeraniol kinase can use CTP, UTP, or GTP as a phosphoryl donor, whereas farnesyl phosphate kinase and geranylgeranyl phosphate kinase exhibit specificity for CTP as a phosphoryl donor). However, it remains unclear if farnesol kinase is distinct from geranylgeraniol kinase or if farnesyl phosphate kinase is distinct from geranylgeranyl phosphate kinase. Nonetheless, it is clear that these kinases convert farnesol and geranylgeraniol to their monophosphate and diphosphate forms for use in isoprenoid biosynthesis, including sterol biosynthesis and protein prenylation.Because plants have the metabolic capability to generate farnesal from FC and farnesyl diphosphate from farnesol, we considered the possibility that plant membranes also contain an oxidoreductase capable of catalyzing the reduction of farnesal to farnesol and/or the oxidation of farnesol to farnesal (Fig. 1; Thai et al., 1999; Crowell et al., 2007). To date, the only reports of such an oxidoreductase are from the corpora allata glands of insects, where it participates in juvenile hormone synthesis, and black rot fungus-infected sweet potato (Ipomoea batatas; Baker et al., 1983; Inoue et al., 1984; Sperry and Sen, 2001; Mayoral et al., 2009). Insect farnesol dehydrogenase is an NADP⁺-dependent oxidoreductase that is encoded by a subfamily of short-chain dehydrogenase/reductase (SDR) genes (Mayoral et al., 2009). Farnesol dehydrogenase from sweet potato is a 90-kD, NADP⁺-dependent homodimer with broad specificity for prenyl alcohol substrates and is induced by wounding and fungus infection of potato roots (Inoue et al., 1984).Here, we extended previous work in which [1-³H]FC was shown to be oxidized to [1-³H]farnesal, and [1-³H]farnesal reduced to [1-³H]farnesol, in the presence of Arabidopsis membranes (Crowell et al., 2007). The reduction of [1-³H]farnesal to [1-³H]farnesol was abolished by pretreatment of Arabidopsis membranes with NADase, suggesting that sufficient NAD(P)H is present in Arabidopsis membranes to support the enzymatic reduction of farnesal to farnesol. In this report, we demonstrate the presence of farnesol dehydrogenase activity in Arabidopsis membranes using [1-³H]farnesol as a substrate. Moreover, we identify a gene on chromosome 4 of the Arabidopsis genome (At4g33360), called FLDH, that encodes an NAD⁺-dependent dehydrogenase with partial specificity for farnesol as a substrate. FLDH expression is repressed by exogenous ABA, and fldh mutants exhibit altered ABA signaling. Taken together, these observations suggest that ABA regulates farnesol metabolism in Arabidopsis, which in turn regulates ABA signaling.     

Restoration of catalytic functions in Cre recombinase mutants by electrostatic compensation between active site and DNA substrate

Two conserved catalytic arginines, Arg-173 and Arg-292, of the tyrosine site-specific recombinase Cre are essential for the transesterification steps of strand cleavage and joining in native DNA substrates containing scissile phosphate groups. The active site tyrosine (Tyr-324) provides the nucleophile for the cleavage reaction, and forms a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group formed during cleavage provides the nucleophile for the joining reaction between DNA partners, yielding strand exchange. Previous work showed that substitution of the scissile phosphate (P) by methylphosphonate (MeP) permits strand cleavage by a Cre variant lacking Arg-292. We now demonstrate that MeP activation and cleavage are not blocked by substitution of Arg-173 or even simultaneous substitutions of Arg-173 and Arg-292 by alanine. Furthermore, Cre(R173A) and Cre(R292A) are competent in strand joining, Cre(R173A) being less efficient. No joining activity is detected with Cre(R173A, R292A). Consistent with their ability to cleave and join strands, Cre(R173A) and Cre(R292A) can promote recombination between two MeP-full-site DNA partners. These findings shed light on the overall contribution of active site electrostatics, and tease apart distinctive contributions of the individual arginines, to the chemical steps of recombination. They have general implications in active site mechanisms that promote important phosphoryl transfer reactions in nucleic acids.     

Electrostatic Suppression Allows Tyrosine Site-specific Recombination in the Absence of a Conserved Catalytic Arginine

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3′-5′ phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5′-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.     

Studies on the antitumor activity and biochemical actions of cyclopentenyl cytosine against human colon carcinoma HT-29 in vitro and in vivo

Cyclopentenyl cytosine (CPEC) is cytotoxic to several tumor cell lines. CPEC inhibits CTP synthesis resulting in depletion of cytidylate pools. The aim of this study was to examine CPEC's cytotoxic and antitumor activity in vitro and in vivo against human colon carcinoma HT-29, and to relate its action on CTP synthesis. CPEC exhibits potent cytotoxicity in vitro to HT-29 cells with an LC50 (concentration that is lethal to the survival of 50% cell colonies) of 2.4 microM and 0.46 microM following 2 h and 24 h exposure, respectively. Incubation of cells with CPEC for 2 h resulted in a dose-dependent decrease in cytidylate pools. The in vivo antitumor activity of CPEC in athymic mice transplanted subcutaneously (s.c.) with 3 million HT-29 cells was examined. Antitumor activity of CPEC was elucidated in early-staged tumor, wherein CPEC (1.5 mg/kg, QD x 9 or 3 mg/kg, QOD x 9) was administered intraperitoneally (i.p.) 24 h after tumor implantation and it resulted in a significant reduction in tumor weight to 48% of control. The effect of CPEC on established solid tumors in vivo was examined in athymic mice transplanted s.c. 14 days earlier with HT-29 cells and treated i.p. with 1.5 mg/kg CPEC, QD x 5 for 4 courses, with a 10 day-interval between courses. This treatment resulted in a significant reduction in tumor weight (72%) in the treated group. HPLC analysis of HT-29 tumor obtained from mice after treatment with CPEC showed a depletion of the CTP concentration reaching a nadir at 8 h. In conclusion, the present studies demonstrate potent antitumor activity of CPEC against freshly transplanted and established human colon carcinoma which can be corroborated with the drug's biochemical actions.     

Role of the histidine triad-like motif in nucleotide hydrolysis by the rotavirus RNA-packaging protein NSP2

Octamers formed by the nonstructural protein NSP2 of rotavirus are proposed to function as molecular motors in the packaging of the segmented double-stranded RNA genome. The octamers have RNA binding, helix unwinding, and Mg(2+)-dependent NTPase activities and play a crucial role in assembly of viral replication factories (viroplasms). Comparison of x-ray structures has revealed significant structural homology between NSP2 and the histidine triad (HIT) family of nucleotidyl hydrolases, which in turn has suggested the location of the active site for NTP hydrolysis in NSP2. Consistent with the structural predictions, we show here using site-specific mutagenesis and ATP docking simulations that the active site for NTP hydrolysis is localized to residues within a 25-A-deep cleft between the C- and N-terminal domains of the NSP2 monomer. Although lacking the precise signature HIT motif (H?H?H?? where ? is a hydrophobic residue), our analyses demonstrate that histidines (His(221) and His(225)) represent critical residues of the active site. Similar to events occurring during nucleotide hydrolysis by HIT proteins, NTP hydrolysis by NSP2 was found to produce a short lived phosphorylated intermediate. Evaluation of the biological importance of the NTPase activity of NSP2 by transient expression in mammalian cells showed that such activity has no impact on the ability of NSP2 to induce the hyperphosphorylation of NSP5 or to interact with NSP5 to form viroplasm-like structures. Hence the NTPase activity of NSP2 probably has a role subsequent to the formation of viroplasms, consistent with its suspected involvement in RNA packaging and/or replication.     

Coding of sweet,bitter, and umami tastes: different receptor cells sharing similar signaling pathways

Mammals can taste a wide repertoire of chemosensory stimuli. Two unrelated families of receptors (T1Rs and T2Rs) mediate responses to sweet, amino acids, and bitter compounds. Here, we demonstrate that knockouts of TRPM5, a taste TRP ion channel, or PLCbeta2, a phospholipase C selectively expressed in taste tissue, abolish sweet, amino acid, and bitter taste reception, but do not impact sour or salty tastes. Therefore, despite relying on different receptors, sweet, amino acid, and bitter transduction converge on common signaling molecules. Using PLCbeta2 taste-blind animals, we then examined a fundamental question in taste perception: how taste modalities are encoded at the cellular level. Mice engineered to rescue PLCbeta2 function exclusively in bitter-receptor expressing cells respond normally to bitter tastants but do not taste sweet or amino acid stimuli. Thus, bitter is encoded independently of sweet and amino acids, and taste receptor cells are not broadly tuned across these modalities.     

A free energy based computational pathway from chemical templates to lead compounds: a case study of COX-2 inhibitors

Automation of lead compound design in silico given the structure of the protein target and a definition of its active site vies for the top of the wish list in any drug discovery programme. We present here an enumeration of steps starting from chemical templates and propose a solution at the state of the art, in the form of a system independent comprehensive computational pathway. This methodology is illustrated with cyclooxygenase-2 (COX-2) as a target. We built candidate molecules including a few Non Steroidal Anti-inflammatory Drugs (NSAIDs) from chemical templates, passed them through empirical filters to assess drug-like properties, optimized their geometries, derived partial atomic charges via quantum calculations, performed Monte Carlo docking, carried out molecular mechanics and developed free energy estimates with Molecular Mechanics Generalized Born Solvent Accessibility (MMGBSA) methodology for each of the candidate molecules. For the case of aspirin, we also conducted molecular dynamics on the enzyme, the drug and the complex with explicit solvent followed by binding free energy analysis. Collectively, the results obtained from the above studies viz. sorting of drugs from non-drugs, semi-quantitative estimates of binding free energies, amply demonstrate the viability of the strategy proposed for lead selection/design for biomolecular targets.