Structural basis for the recognition of α-1,6-branched α-glucan by GH13_47 α-amylase from Rhodothermus marinus.

Glycoside hydrolase (GH) family 13 is among the main families of enzymes acting on starch; recently, subfamily 47 of GH13 (GH13_47) has been established. The crystal structure and function of a GH13_47 enzyme from Bacteroides ovatus has only been reported to date. This enzyme has α-amylase activity, while the GH13_47 enzymes comprise approximately 800-900 amino acid residues which are almost double those of typical α-amylases. It is important to know how different the GH13_47 enzymes are from other α-amylases. Rhodothermus marinus JCM9785, a thermophilic bacterium, possesses a gene for the GH13_47 enzyme, which is designated here as RmGH13_47A. Its structure has been predicted to be composed of seven domains: N1, N2, N3, A, B, C, and D. We constructed a plasmid encoding Gly266-Glu886, which contains the N3, A, B, and C domains and expressed the protein in Escherichia coli. The enzyme hydrolyzed starch and pullulan by a neopullulanase-type action. Additionally, the enzyme acted on maltotetraose, and saccharides with α-1,6-glucosidic linkages were observed in the products. Following the replacement of the catalytic residue Asp563 with Ala, the crystal structure of the variant D563A in complex with the enzymatic products from maltotetraose was determined; as a result, electron density for an α-1,6-branched pentasaccharide was observed in the catalytic pocket, and Ile762 and Asp763 interacted with the branched chain of the pentasaccharide. These findings suggest that RmGH13_47A is an α-amylase that prefers α-1,6-branched parts of starch to produce oligosaccharides.

Hepatic phosphatidylcholine catabolism driven by PNPLA7 and PNPLA8 supplies endogenous choline to replenish the methionine cycle with methyl groups.

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.

PNPLA1 has a crucial role in skin barrier function by directing acylceramide biosynthesis.

Mutations in patatin-like phospholipase domain-containing 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, but the mechanism involved remains unclear. Here we show that PNPLA1, an enzyme expressed in differentiated keratinocytes, plays a crucial role in the biosynthesis of ω-O-acylceramide, a lipid component essential for skin barrier. Global or keratinocyte-specific Pnpla1-deficient neonates die due to epidermal permeability barrier defects with severe transepidermal water loss, decreased intercellular lipid lamellae in the stratum corneum, and aberrant keratinocyte differentiation. In Pnpla1-/- epidermis, unique linoleate-containing lipids including acylceramides, acylglucosylceramides and (O-acyl)-ω-hydroxy fatty acids are almost absent with reciprocal increases in their putative precursors, indicating that PNPLA1 catalyses the ω-O-esterification with linoleic acid to form acylceramides. Moreover, acylceramide supplementation partially rescues the altered differentiation of Pnpla1-/- keratinocytes. Our findings provide valuable insight into the skin barrier formation and ichthyosis development, and may contribute to novel therapeutic strategies for treatment of epidermal barrier defects.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

Altered sterol profile induced in Leishmania amazonensis by a natural dihydroxymethoxylated chalcone.

OBJECTIVES: The effects of the antileishmanial chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC) on Leishmania amazonensis sterol composition and biosynthesis were investigated to obtain information about the mechanism of growth inhibition by DMC on this parasite. METHODS: The interference of sterol biosynthesis by DMC was studied in drug-treated promastigotes by two different methods. (i) Newly synthesized sterols from parasites grown in the presence of [(3)H]mevalonate were analysed by thin layer chromatography (TLC)/fluorography. (ii) Total sterols extracted from the parasites grown with or without DMC were characterized by gas chromatography coupled to mass spectroscopy (GC/MS). RESULTS: TLC and GC/MS analyses of sterols extracted from DMC-treated promastigotes revealed the accumulation of early precursors and a reduction in the levels of C-14 demethylated and C-24 alkylated sterols, as well as a reduction in exogenous cholesterol uptake. CONCLUSIONS: This study demonstrates that the natural chalcone DMC alters the sterol composition of L. amazonensis and suggests that the parasite target is different from other known sterol inhibitors.

2-Oxo-tetrahydro-1,8-naphthyridines as selective inhibitors of malarial protein farnesyltransferase and as anti-malarials.

A new class of 2-oxo-tetrahydro-1,8-naphthyridine-based protein farnesyltransferase inhibitors were synthesized and found to inhibit protein farnesyltransferase from the malaria parasite with potencies in the low nanomolar range. The compounds were much less potent on mammalian protein prenyltransferases. Two of the compounds block the growth of malaria in culture with potencies in the sub-micromolar range. Some of the compounds were found to be much more metabolically stable than previously described tetrahydroquinoline-based protein farnesyltransferase inhibitors.

Protein geranylgeranyltransferase-I of Trypanosoma cruzi.

Protein geranylgeranyltransferase type I (PGGT-I) and protein farnesyltransferase (PFT) occur in many eukaryotic cells. Both consist of two subunits, the common alpha subunit and a distinct beta subunit. In the gene database of protozoa Trypanosoma cruzi, the causative agent of Chagas' disease, a putative protein that consists of 401 amino acids with approximately 20% amino acid sequence identity to the PGGT-I beta of other species was identified, cloned, and characterized. Multiple sequence alignments show that the T. cruzi ortholog contains all three of the zinc-binding residues and several residues uniquely conserved in the beta subunit of PGGT-I. Co-expression of this protein and the alpha subunit of T. cruzi PFT in Sf9 insect cells yielded a dimeric protein that forms a tight complex selectively with [(3)H]geranylgeranyl pyrophosphate, indicating a key characteristic of a functional PGGT-I. Recombinant T. cruzi PGGT-I ortholog showed geranylgeranyltransferase activity with distinct specificity toward the C-terminal CaaX motif of protein substrates compared to that of the mammalian PGGT-I and T. cruzi PFT. Most of the CaaX-containing proteins with X=Leu are good substrates of T. cruzi PGGT-I, and those with X=Met are substrates for both T. cruzi PFT and PGGT-I, whereas unlike mammalian PGGT-I, those with X=Phe are poor substrates for T. cruzi PGGT-I. Several candidates for T. cruzi PGGT-I or PFT substrates containing the C-terminal CaaX motif are found in the T. cruzi gene database. Among five C-terminal peptides of those tested, a peptide of a Ras-like protein ending with CVLL was selectively geranylgeranylated by T. cruzi PGGT-I. Other peptides with CTQQ (Tcj2 DNAJ protein), CAVM (TcPRL-1 protein tyrosine phosphatase), CHFM (a small GTPase like protein), and CQLF (TcRho1 GTPase) were specific substrates for T. cruzi PFT but not for PGGT-I. The mRNA and protein of the T. cruzi PGGT-I beta ortholog were detected in three life-cycle stages of T. cruzi. Cytosol fractions from trypomastigotes (infectious mammalian stage) and epimastigotes (insect stage) were shown to contain levels of PGGT-I activity that are approximately 100-fold lower than PFT activity. The CaaX mimetics known as PGGT-I inhibitors show very low potency against T. cruzi PGGT-I compared to the mammalian enzyme, suggesting the potential to develop selective inhibitors against the parasite enzyme.

Second generation tetrahydroquinoline-based protein farnesyltransferase inhibitors as antimalarials.

Substituted tetrahydroquinolines (THQs) have been previously identified as inhibitors of mammalian protein farnesyltransferase (PFT). Previously we showed that blocking PFT in the malaria parasite led to cell death and that THQ-based inhibitors are the most potent among several structural classes of PFT inhibitors (PFTIs). We have prepared 266 THQ-based PFTIs and discovered several compounds that inhibit the malarial enzyme in the sub- to low-nanomolar range and that block the growth of the parasite (P. falciparum) in the low-nanomolar range. This body of structure-activity data can be rationalized in most cases by consideration of the X-ray structure of one of the THQs bound to mammalian PFT together with a homology structural model of the malarial enzyme. The results of this study provide the basis for selection of antimalarial PFTIs for further evaluation in preclinical drug discovery assays.

Efficacy, pharmacokinetics, and metabolism of tetrahydroquinoline inhibitors of Plasmodium falciparum protein farnesyltransferase.

New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Zn-binding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation.

C-terminal proteolysis of prenylated proteins in trypanosomatids and RNA interference of enzymes required for the post-translational processing pathway of farnesylated proteins.

The C-terminal "CaaX"-motif-containing proteins usually undergo three sequential post-translational processing steps: (1) attachment of a prenyl group to the cysteine residue; (2) proteolytic removal of the last three amino acids "aaX"; (3) methyl esterification of the exposed alpha-carboxyl group of the prenyl-cysteine residue. The Trypanosoma brucei and Leishmania major Ras converting enzyme 1 (RCE1) orthologs of 302 and 285 amino acids-proteins, respectively, have only 13-20% sequence identity to those from other species but contain the critical residues for the activity found in other orthologs. The Trypanosoma brucei a-factor converting enzyme 1 (AFC1) ortholog consists of 427 amino acids with 29-33% sequence identity to those of other species and contains the consensus HExxH zinc-binding motif. The trypanosomatid RCE1 and AFC1 orthologs contain predicted transmembrane regions like other species. Membranes from Sf9 cells expressing the RCE1 ortholog of T. brucei or L. major showed proteolytic activity against farnesylated RAS-CVIM, whereas membranes containing T. brucei AFC1 ortholog were inactive. The results suggest that RCE1 is responsible for proteolytic removal of the C-terminal aaX from prenyl-CaaX proteins in these parasites. All the three enzymatic post-translational processes are thought to be required for proper cellular functioning of CaaX-proteins in eukaryotic cells. We carried out RNA interference experiments in Trypanosoma brucei of the enzymes involved in farnesyl protein post-translational modification to evaluate their importance in cell proliferation. Knockdown of T. brucei PFT beta subunit and RCE1 mRNAs resulted in >20-fold suppression of cell growth and dramatic morphologic changes. Knockdown of PPMT mRNA caused less dramatic effects on growth but induced noticeable changes in cell morphology.

Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase.

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. This enzymatic reaction is carried out by protein farnesyltransferase (PFT), which catalyzes the transfer of a 15-carbon isoprenoid lipid unit, a farnesyl group, from farnesyl pyrophosphate to the C-termini of proteins containing a CaaX motif. Inhibition of PFT is lethal to the pathogenic protozoa Plasmodium falciparum. Previously, we have shown that parasites resistant to a tetrahydroquinoline (THQ)-based PFT inhibitor BMS-388891 have mutations leading to amino acid substitutions in PFT that map to the peptide substrate binding domain. We now report the selection of parasites resistant to another THQ PFT inhibitor BMS-339941. In whole cell assays sensitivity to BMS-339941 was reduced by 33-fold in a resistant clone, and biochemical analysis demonstrated a corresponding 33-fold increase in the BMS-339941 K(i) for the mutant PFT enzyme. More detailed kinetic analysis revealed that the mutant enzyme required higher concentration of peptide and farnesyl pyrophosphate substrates for optimum catalysis. Unlike previously characterized parasites resistant to BMS-388891, the resistant parasites have a mutation which is predicted to be in a distinct location of the enzymatic pocket, near the farnesyl pyrophosphate binding pocket. This is the first description of a mutation from any species affecting the farnesyl pyrophosphate binding pocket with reduced efficacy of PFT inhibitors. These data provide further support that PFT is the target of THQ inhibitors in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents to minimize the development of resistant parasites.

Structurally simple, potent, Plasmodium selective farnesyltransferase inhibitors that arrest the growth of malaria parasites.

Third world nations require immediate access to inexpensive therapeutics to counter the high mortality inflicted by malaria. Here, we report a new class of antimalarial protein farnesyltransferase (PFT) inhibitors, designed with specific emphasis on simple molecular architecture, to facilitate easy access to therapies based on this recently validated antimalarial target. This novel series of compounds represents the first Plasmodium falciparum selective PFT inhibitors reported (up to 145-fold selectivity), with lead inhibitors displaying excellent in vitro activity (IC(50) < 1 nM) and toxicity to cultured parasites at low concentrations (ED(50) < 100 nM). Initial studies of absorption, metabolism, and oral bioavailability are reported.

Thematic review series: lipid posttranslational modifications. Fighting parasitic disease by blocking protein farnesylation.

Protein farnesylation is a form of posttranslational modification that occurs in most, if not all, eukaryotic cells. Inhibitors of protein farnesyltransferase (PFTIs) have been developed as anticancer chemotherapeutic agents. Using the knowledge gained from the development of PFTIs for the treatment of cancer, researchers are currently investigating the use of PFTIs for the treatment of eukaryotic pathogens. This "piggy-back" approach not only accelerates the development of a chemotherapeutic agent for protozoan pathogens but is also a means of mitigating the costs associated with de novo drug design. PFTIs have already been shown to be efficacious in the treatment of eukaryotic pathogens in animal models, including both Trypanosoma brucei, the causative agent of African sleeping sickness, and Plasmodium falciparum, one of the causative agents of malaria. Here, current evidence and progress are summarized that support the targeting of protein farnesyltransferase for the treatment of parasitic diseases.

Isothiazole dioxide derivative 6n inhibits vascular smooth muscle cell proliferation and protein farnesylation.

Isothiazole dioxides have been shown to inhibit Trypanosoma brucei protein farnesyltransferase (PFTase) in isolated enzyme, but elicited only a minor effect on mammalian PFTase. In the present study we have evaluated the effect of 3-diethylamino-4-(4-methoxyphenyl)-isothiazole 1,1-dioxides with different substituents at C5, on rat PFTase and protein geranylgeranyltransferase-I (PGGTase-I) with the final aims to improve the potency against mammalian PFTase and to identify new compounds with antiproliferative properties. For these purposes, in vitro and cell culture models have been utilized. The results showed that isothiazole dioxides with C4-C5 double bond and sulfaryl substituted at the C5 position but none of the dihydro-derivatives, were able to inhibit in vitro PFTase in a concentration dependent manner (IC50 ranging from 8.56 to 1015 microM). Among those, compound 6n (C5; methyl-S) displayed 500-fold higher inhibitory potency on PFTase than PGGTase-I. Compound 6n was shown to affect rat smooth muscle cell (SMC) proliferation at concentrations similar (IC50 = 61.4 microM) to those required to inhibit [3H]-farnesol incorporation into cellular proteins (-44.1% at 100 microM). Finally, compound 6n interferes with rat SMC proliferation by blocking the progression of G0/G1 phase without inducing apoptosis, as assessed by [3H]-thymidine incorporation assay and flow cytometry analysis. Taken together, we described a new PFTase inhibitor containing the isothiazole dioxide moiety that affects mammalian protein farnesylation and SMC proliferation by inhibiting G0/G1 phase of the cell cycle.

Protein farnesyl transferase inhibitors for the treatment of malaria and African trypanosomiasis.

Protein farnesyl transferase inhibitors (PFTIs) have been developed as oncology therapeutics but recent studies have supported the use of PFTIs for the treatment of eukaryotic pathogens. Data supporting PFTIs for the treatment of African sleeping sickness caused by Trypanosoma brucei sp, and for the therapy of malaria caused by Plasmodium spp is reviewed. Protein prenylation in T. brucei and P. falciparum has been studied using a variety of techniques, including recombinant and native enzyme assays. Studies have demonstrated farnesylation and geranylgeranylation in these parasites. A variety of PFTIs have shown growth inhibition activity and killing of T. brucei and P. falciparum, yet not all mammalian PFTIs are active on parasitic PFTs. Protein farnesyl transferase as well as protein geranylgeranyl transferase type II enzymatic activities have been demonstrated in T brucei and P. falciparum, but protein geranylgeranyl transferase type I activity may be lacking from these parasites, perhaps explaining the extreme sensitivity of these organisms to PFTIs compared with mammalian cells. Given that PFTIs are relatively non-toxic in short-term administration to humans, PFTIs specific to parasites are not required for therapy. Thus, the challenge in PFTI drug development is not to identify selective antiparasite compounds, but to identify compounds with sufficient potency and pharmacokinetic properties to produce satisfactory drugs for malaria and African sleeping sickness.

Protein farnesyltransferase inhibitors exhibit potent antimalarial activity.

New therapeutics to combat malaria are desperately needed. Here we show that the enzyme protein farnesyltransferase (PFT) from the malaria parasite Plasmodium falciparum (P. falciparum) is an ideal drug target. PFT inhibitors (PFTIs) are well tolerated in man, but are highly cytotoxic to P. falciparum. Because of their anticancer properties, PFTIs comprise a highly developed class of compounds. PFTIs are ideal for the rapid development of antimalarials, allowing "piggy-backing" on previously garnered information. Low nanomolar concentrations of tetrahydroquinoline (THQ)-based PFTIs inhibit P. falciparum PFT and are cytotoxic to cultured parasites. Biochemical studies suggest inhibition of parasite PFT as the mode of THQ cytotoxicity. Studies with malaria-infected mice show that THQ PFTIs dramatically reduce parasitemia and lead to parasite eradication in the majority of animals. These studies validate P. falciparum PFT as a target for the development of antimalarials and describe a potent new class of THQ PFTIs with antimalaria activity.

Resistance to a protein farnesyltransferase inhibitor in Plasmodium falciparum.

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. Inhibition of protein farnesyltransferase (PFT) is lethal to the pathogenic protozoa Plasmodium falciparum. Parasites were isolated that were resistant to BMS-388891, a tetrahydroquinoline (THQ) PFT inhibitor. Resistance was associated with a 12-fold decrease in drug susceptibility. Genotypic analysis revealed a single point mutation in the beta subunit in resistant parasites. The resultant tyrosine 837 to cysteine alteration in the beta subunit corresponded to the binding site for the THQ and peptide substrate. Biochemical analysis of Y837C-PFT demonstrated a 13-fold increase in BMS-388891 concentration necessary for inhibiting 50% of the enzyme activity. These data are consistent with PFT as the target of BMS-388891 in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents for effective therapy.

In vitro and in vivo antimalarial activity of peptidomimetic protein farnesyltransferase inhibitors with improved membrane permeability.

A series of protein farnesyltransferase inhibitor ester prodrugs of FTI-2148 (17) were synthesized in order to evaluate the effects of ester structure modification on antimalarial activity and for further development of a farnesyltransferase inhibitor with in vivo activity. Evaluation against P. falciparum in red blood cells showed that all the investigated esters exhibited significant antimalarial activity, with the benzyl ester 16 showing the best inhibition (ED50=150 nM). Additionally, compound 16 displayed in vivo activity and was found to suppress parasitemia by 46.1% at a dose of 50 mg kg(-1) day(-1) against Plasmodium berghei in mice. The enhanced inhibition potency of the esters is consistent with improved cell membrane permeability compared to that of the free acid. The results of this study suggest that protein farnesyltransferase is a valid antimalarial drug target and that the antimalarial activity of these compounds derives from a balance between the hydrophobic character and the size and conformation of the ester moiety.

Design and synthesis of peptidomimetic protein farnesyltransferase inhibitors as anti-Trypanosoma brucei agents.

On the basis of the structure of the CVIM tetrapeptide substrate of mammalian protein farnesyltransferase, a series of imidazole-containing peptidomimetics was designed and synthesized, and their inhibition activity against Trypanosoma brucei protein farnesyltransferase (TbPFT) was evaluated. Peptidomimetics where the 5-position of the imidazole ring was linked to the hydrophobic scaffold showed over 70% inhibition activity at 50 nM in the enzyme assay, whereas the corresponding C-4 regioisomers were less potent. The ester prodrug 23 was found to be a potent inhibitor against cultured Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense cells with ED(50) values of 0.025 and 0.0026 microM, respectively. Furthermore, introducing a second imidazole group into 23 led to 31, which showed the highest inhibition activity against the parasite with an ED(50) of 0.0015 microM. The potency of the TbPFT inhibitors and the cytotoxicity of the corresponding esters to T. brucei cells were shown to be highly correlated. These studies validate TbPFT as a target for the development of novel therapeutics against African sleeping sickness.