RESUMEN
Improvement of biomass utilization productivity following cascading strategy is a priority for the biorefinery-based circular bioeconomy. In recent years, the field of energy research has seen an increasing interest in bio-products from paddy-based biorefinery, but the utilization of the entire value of paddy biomass to guide the commercial viability of its products has not been got feasible outcomes. Here we propose a potential pathway for a conceptual paddy biorefinery framework by addressing wastes for producing more products. The feasibility of the integrated biorefinery was demonstrated by the conversion of wastes into value-added products such as nano-silica and lignin. In particular, this is the first time that silica recovered from bioethanol system was continued to be reused to produce ZSM-5 and Ni/ZSM-5 as catalysts of rice straw lignin depolymerization achieving high conversion of lignin up to 95% and fair yield of phenolic products up to 41%. Material flow of an integrated biorefinery model was reported to give a future outlook for making most of the processing routes of rice residues. We also established a life cycle that follows the circular bioeconomy concept and discussed the relationship between each of potential bioproducts and their market opportunities.
Asunto(s)
Biocombustibles , Lignina , Animales , Biomasa , Catálisis , Estadios del Ciclo de VidaRESUMEN
Ricin is a lethal protein toxin produced by the castor bean plant. Ricin is known to possess significant heat resistance. Therefore, we placed it in a variety of foods to study the influence of the food matrix on behavior of a thermally stable protein toxin. First order rate constants for the thermal inactivation of ricin in foods and simple buffers were measured using cytotoxicity assays. We observed greater thermal stability at 75 °C for the cytotoxic activity of ricin when it was placed in a yogurt-containing fruit drink compared to its stability when placed in the other foods tested. We found that galactose and high molecular weight exopolysaccharides present in various dairy products contributed to the thermal stability of ricin. Differential scanning calorimetry also showed enhanced thermal stability for ricin at pH 4.5. Our results demonstrate the importance of considering pH and the presence of stabilizing ligands in the thermal inactivation of protein toxins in foods.
Asunto(s)
Carbohidratos/química , Calor , Concentración de Iones de Hidrógeno , Ricina/antagonistas & inhibidores , Animales , Línea Celular , Cinética , Ratones , Ricina/química , TermodinámicaRESUMEN
We compared the kinetics and efficacies of sodium hypochlorite, peracetic acid, phosphoric acid-based detergent, chlorinated alkaline detergent, quaternary ammonium-based sanitizer, and peracetic acid-based sanitizer for inactivating the potential bioterrorism agents ricin and abrin in simple buffers, food slurries (infant formula, peanut butter, and pancake mix), and in dried food residues on stainless steel. The intrinsic fluorescence and cytotoxicity of purified ricin and abrin in buffers decreased rapidly in a pH- and temperature-dependent manner when treated with sodium hypochlorite but more slowly when treated with peracetic acid. Cytotoxicity assays showed rapid and complete inactivation of ricin and crude abrin in food slurries and dried food residues treated 0-5 min with sodium hypochlorite. Toxin epitopes recognized by ELISA decayed more gradually under these conditions. Higher concentrations of peracetic acid were required to achieve comparable results. Chlorinated alkaline detergent was the most effective industrial agent tested for inactivating ricin in dried food residues.
Asunto(s)
Abrina/antagonistas & inhibidores , Compuestos de Cloro/farmacología , Detergentes/farmacología , Contaminación de Alimentos/prevención & control , Proteínas de Plantas/antagonistas & inhibidores , Ricina/antagonistas & inhibidores , Animales , Bioterrorismo , Línea Celular , Macrófagos , Ratones , Ácido Peracético/farmacología , Ácidos Fosfóricos/farmacología , Hipoclorito de Sodio/farmacología , Acero InoxidableRESUMEN
Fourteen quinolone-resistant Pseudomonas putida isolates were recovered from imported frozen shrimp sold in the United States. Two isolates harbored plasmids with qnrA and qnrB genes. PCR and DNA sequencing of quinolone resistance-determining regions identified novel substitutions in GyrA (His139âGlu and Thr128âAla) and GyrB (Thr442âAsn, Gly470âAla, and Ile487âPro) and previously reported substitutions in GyrB (Asp489âGlu) and ParC (Thr105âPro).
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Plásmidos , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/genética , Quinolonas/farmacología , Alimentos Marinos/microbiología , Sustitución de Aminoácidos/genética , Técnicas de Tipificación Bacteriana , Análisis por Conglomerados , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Pruebas de Sensibilidad Microbiana , Tipificación Molecular , Mutación Missense , Reacción en Cadena de la Polimerasa , Pseudomonas putida/clasificación , Pseudomonas putida/aislamiento & purificación , Análisis de Secuencia de ADN , Estados UnidosRESUMEN
Conjugal transfer of chromosomal DNA between strains of Mycobacterium smegmatis occurs by a novel mechanism. In a transposon mutagenesis screen, three transfer-defective insertions were mapped to the lsr2 gene of the donor strain mc(2)155. Because lsr2 encodes a nonspecific DNA-binding protein, mutations of lsr2 give rise to a variety of phenotypes, including an inability to form biofilms. In this study, we show that efficient DNA transfer between strains of M. smegmatis occurs in a mixed biofilm and that the process requires expression of lsr2 in the donor but not in the recipient strain. Testing cells from different strata of standing cultures showed that transfer occurred predominantly at the biofilm air-liquid interface, as other strata containing higher cell densities produced very few transconjugants. These data suggest that the biofilm plays a role beyond mere facilitation of cell-cell contact. Surprisingly, we found that under standard assay conditions the recipient strain does not form a biofilm. Taking these results together, we conclude that for transfer to occur, the recipient strain is actively recruited into the biofilm. In support of this idea, we show that donor and recipient cells are present in almost equal numbers in biofilms that produce transconjugants. Our demonstration of genetic exchange between mycobacteria in a mixed biofilm suggests that conjugation occurs in the environment. Since biofilms are considered to be the predominant natural microhabitat for bacteria, our finding emphasizes the importance of studying biological and physical processes that occur between cells in mixed biofilms.
Asunto(s)
Biopelículas/crecimiento & desarrollo , ADN Bacteriano/genética , Transferencia de Gen Horizontal/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/genética , Conjugación Genética/genética , Conjugación Genética/fisiología , Prueba de Complementación Genética , Mutagénesis , Mutagénesis Sitio-DirigidaRESUMEN
We have previously described a novel conjugal DNA transfer process that occurs in Mycobacterium smegmatis. To identify donor genes required for transfer, we have performed a transposon mutagenesis screen; we report here that LpqM, a putative lipoprotein-metalloproteinase, is essential for efficient DNA transfer. Bioinformatic analyses predict that LpqM contains a signal peptide necessary for the protein's targeting to the cell envelope and a metal ion binding motif, the likely catalytic site for protease activity. Using targeted mutagenesis, we demonstrate that each of these motifs is necessary for DNA transfer and that LpqM is located in the cell envelope. The requirement for transfer is specific to the donor strain; an lpqM knockout mutant in the recipient is still proficient in transfer assays. The activity of LpqM is conserved among mycobacteria; homologues from both Mycobacterium tuberculosis and Mycobacterium avium can complement lpqM donor mutants, suggesting that the homologues recognize and process similar proteins. Lipoproteins constitute a significant proportion of the mycobacterial cell wall, but despite their abundance, very few have been assigned an activity. We discuss the potential role of LpqM in DNA transfer and the implications of the conservation of LpqM activity in M. tuberculosis.
Asunto(s)
Conjugación Genética , ADN Bacteriano/metabolismo , Metaloproteasas/fisiología , Mycobacterium smegmatis/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Elementos Transponibles de ADN , Eliminación de Gen , Orden Génico , Genes Bacterianos , Prueba de Complementación Genética , Metaloproteasas/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Mycobacterium avium/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Señales de Clasificación de Proteína , Alineación de SecuenciaRESUMEN
The emergence of bacterial pathogens resistant to current antibiotics has caused an urgent demand for new treatments. Peptide deformylase (PDF) has become an exciting target for designing novel antibiotics. To facilitate the screening of PDF inhibitors, three robust, coupled assays have been developed. The first method couples the PDF reaction with that of formate dehydrogenase. Formate dehydrogenase oxidizes formate into CO2 with a concomitant reduction of NAD+ to NADH, which can be monitored spectrophotometrically. The second method involves Aeromonas aminopeptidase (AAP) as the coupling enzyme and an artificial substrate, f-Met-Leu-p-nitroanilide. The sequential action of PDF and AAP releases p-nitroanilide as a highly chromogenic product. In the third method, f-Met-Lys-7-amino-4-methylcoumarin is used as the substrate. Deformylation by PDF gives an excellent substrate for dipeptidyl peptidase I, which releases the dipeptide Met-Lys and fluorogenic 7-amino-4-methylcoumarin. The combination of these assay methods should meet the needs of most laboratories.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Inhibidores Enzimáticos/aislamiento & purificación , Amidohidrolasas/aislamiento & purificación , Amidohidrolasas/metabolismo , Aminopeptidasas/metabolismo , Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Catepsina C/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Formiato Deshidrogenasas/metabolismo , Hidrogenasas/metabolismo , Cinética , Complejos Multienzimáticos/metabolismo , Especificidad por SustratoRESUMEN
Peptide deformylase (PDF, E.C. 3.5.1.88) catalyzes the removal of N-terminal formyl groups from nascent ribosome-synthesized polypeptides. PDF contains a catalytically essential divalent metal ion, which is tetrahedrally coordinated by three protein ligands (His, His, and Cys) and a water molecule. Previous studies revealed that the metal cofactor is a Fe2+ ion in Escherichia coli and many other bacterial PDFs. In this work, we found that PDFs from two iron-deficient bacteria, Borrelia burgdorferi and Lactobacillus plantarum, are stable and highly active under aerobic conditions. The native B. burgdorferi PDF (BbPDF) was purified 1200-fold and metal analysis revealed that it contains approximately 1.1 Zn2+ ion/polypeptide but no iron. Our studies suggest that PDF utilizes different metal ions in different organisms. These data have important implications in designing PDF inhibitors and should help address some of the unresolved issues regarding PDF structure and catalytic function.
Asunto(s)
Amidohidrolasas/química , Borrelia burgdorferi/enzimología , Zinc/química , Catálisis , Coenzimas/química , Activación Enzimática , Estabilidad de EnzimasRESUMEN
N-Formyl peptides are derived from proteolytic degradation/processing of bacterial and mitochondrial proteins and serve as potent chemoattractants for mammalian phagocytic leukocytes. A response to the chemotactic N-formyl peptides released by commensal bacteria in the gut region could be detrimental, leading to unwanted inflammation. Here, two enzymes that act sequentially to degrade N-formyl peptides were purified from the rat intestinal mucosal layer and biochemically characterized. The first enzyme cleaves chemotactic peptide f-MLF to release N-formylmethionine (f-Met) and dipeptide leucylphenylalanine, with a k(cat) value of 14 s(-)(1), a K(M) value of 0.60 mM, and a k(cat)/K(M) value of 22 500 M(-)(1) s(-)(1). In-gel tryptic digestion followed by mass spectral fingerprinting identified the protein as the alpha-N-acylpeptide hydrolase (or acylamino acid-releasing enzyme, EC 3.4.19.1). The second enzyme hydrolyzes N-formylmethionine into formate and methionine with a k(cat) value of 7.9 s(-)(1), a K(M) value of 3.1 mM, and a k(cat)/K(M) value of 2550 M(-)(1) s(-)(1). This protein was identified as the N-acylase IA (or N(alpha)-acyl-l-amino acid amidohydrolase, EC 3.5.1.14). Together, these two enzymes play a protective role in degrading bacterial and mitochondrial N-formylated peptides.
Asunto(s)
Amidohidrolasas/química , Amidohidrolasas/aislamiento & purificación , Aminopeptidasas/química , Aminopeptidasas/aislamiento & purificación , Quimiotaxis de Leucocito , N-Formilmetionina Leucil-Fenilalanina/química , Péptido Hidrolasas/química , Péptido Hidrolasas/aislamiento & purificación , Amidohidrolasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Aminopeptidasas/antagonistas & inhibidores , Animales , Catálisis , Hidrólisis , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismo , Datos de Secuencia Molecular , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
Peptide deformylase (PDF) catalyzes the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria. Its essential role in bacterial cells but not in mammalian cells makes it an attractive target for antibacterial drug design. We have previously reported an N-formylhydroxylamine-based, metal-chelating macrocyclic PDF inhibitor, in which the P(1)' and P(3)' side chains are covalently joined. In this work, we have carried out a structure-activity relationship study on the size of the macrocycle and found that 15-17-membered macrocycles are optimal for binding to the PDF active site. Unlike the acyclic compounds, which are simple competitive inhibitors, the cyclic compounds all act as slow-binding inhibitors. As compared to their acyclic counterparts, the cyclic inhibitors displayed 20-50-fold higher potency against the PDF active site (K(I) as low as 70 pM), improved selectivity toward PDF, and improved the metabolic stability in rat plasma. Some of the macrocyclic inhibitors had potent, broad spectrum antibacterial activity against clinically significant Gram-positive and Gram-negative pathogens. These results suggest that the macrocyclic scaffold provides an excellent lead for the development of a new class of antibiotics.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/química , Antibacterianos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos/química , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Animales , Sitios de Unión , Evaluación Preclínica de Medicamentos/métodos , Estabilidad de Medicamentos , Humanos , Pruebas de Sensibilidad Microbiana , Ratas , Relación Estructura-ActividadRESUMEN
A new spectrophotometric/fluorimetric assay for peptide deformylase (PDF) has been developed by coupling the PDF reaction with that of dipeptidyl peptidase I (DPPI) and using N-formyl-Met-Lys-AMC as substrate. Removal of the N-terminal formyl group by PDF renders the dipeptide an efficient substrate of DPPI, which subsequently removes the dipeptidyl units to release 7-amino-4-methylcoumarin as the chromophore/fluorophore. The PDF reaction is conveniently monitored on a UV-Vis spectrophotometer or a fluorimeter in a continuous fashion. The utility of the assay was demonstrated by determining the catalytic activity of PDF and the inhibition constants of PDF inhibitors. These studies revealed the slow-binding behavior of a previously reported macrocyclic PDF inhibitor. This method offers several advantages over the existing PDF assays and should be particularly useful for screening PDF inhibitors in the continuous fashion.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Espectrometría de Fluorescencia/métodos , Espectrofotometría Ultravioleta/métodos , Amidohidrolasas/metabolismo , Materiales Biomiméticos/química , Catepsina C/química , Catepsina C/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Cinética , Especificidad por SustratoRESUMEN
An altered version of peptide deformylase from Plasmodium falciparum (PfPDF), the organism that causes the most devastating form of malaria, has been cocrystallized with a synthesized inhibitor that has submicromolar affinity for its target protein. The structure is solved at 2.2 A resolution, an improvement over the 2.8 A resolution achieved during the structural determination of unliganded PfPDF. This represents the successful outcome of modifying the protein construct in order to overcome adverse crystal contacts and other problems encountered in the study of unliganded PfPDF. Two molecules of PfPDF are found in the asymmetric unit of the current structure. The active site of each monomer of PfPDF is occupied by a proteolyzed fragment of the tripeptide-like inhibitor. Unexpectedly, each PfPDF subunit is associated with two nearly complete molecules of the inhibitor, found at a protein-protein interface. This is the first structure of a eukaryotic PDF protein, a potential drug target, in complex with a ligand.
Asunto(s)
Amidohidrolasas/química , Inhibidores Enzimáticos/química , Plasmodium falciparum/enzimología , Estructura Terciaria de Proteína , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Animales , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Plasmodium falciparum/química , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
A macrocyclic, peptidomimetic inhibitor of peptide deformylase was designed by covalently cross-linking the P1' and P3' side chains. The macrocycle, which contains an N-formylhydroxylamine side chain as the metal-chelating group, was synthesized from a diene precursor via olefin metathesis using Grubbs's catalyst. The cyclic inhibitor showed potent inhibitory activity toward Escherichia coli deformylase (K(I) = 0.67 nM) and antibacterial activity against both Gram-positive and Gram-negative bacteria (MIC = 0.7-12 microg/mL).
Asunto(s)
Amidohidrolasas , Aminopeptidasas/antagonistas & inhibidores , Antibacterianos/síntesis química , Quelantes/síntesis química , Inhibidores Enzimáticos/síntesis química , Metaloendopeptidasas/antagonistas & inhibidores , Péptidos Cíclicos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Quelantes/química , Quelantes/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Imitación Molecular , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Relación Estructura-ActividadRESUMEN
Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes, making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencing has revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding Homo sapiens PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-amino-acid targeting sequence was overexpressed in Escherichia coli. The recombinant, Co(2+)-substituted protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhanced green fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion. However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation for the apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is at least partially due to mutation of a highly conserved residue (Leu-91 in E. coli PDF) in mammalian PDF. PDF inhibitors had no detectable effect on two different human cell lines. These results suggest that HsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylation and validates PDF as an excellent target for antibacterial drug design.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/química , Diseño de Fármacos , Escherichia coli/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Antibacterianos/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Clonación Molecular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Proteínas Fluorescentes Verdes , Humanos , Riñón/enzimología , Cinética , Leucina/química , Proteínas Luminiscentes/metabolismo , Mitocondrias/enzimología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria and the organelles of certain eukaryotes. PDF is a novel class of amide hydrolase, which utilizes an Fe2+ ion to effect the hydrolysis of an amide bond. The ferrous ion is tetrahedrally coordinated by two histidines from a conserved HEXXH motif, a cysteine, and a water molecule. In this work, the function of the conserved glutamate (Glu-133 in Escherichia coli PDF) is evaluated by difference FTIR spectroscopic analysis of a Co(II)-substituted E. coli wild-type and E133D mutant PDF. At pH <6, the wild-type enzyme exhibited a relatively sharp C=O stretch band at 1742 cm(-1), which is assigned to the COOH group of Glu-133. The pH titration study and curve fitting to the data revealed a pK(a) of 6.0 for Glu-133 (in the presence of 500 mM NaCl). For the E133D mutant, which is only approximately 10-fold less active than the wild-type enzyme, a similar pH titration study of the Asp-133 C=O stretch band at 1740 cm(-1) revealed a pK(a) of 10.1. This unusually high pK(a) for a carboxyl group is likely due to its hydrophobic environment and electrostatic repulsion from the metal-bound hydroxide. These results argue that in the active form of E133D PDF, Asp-133 is protonated and therefore acts as a general acid during the decomposition of the tetrahedral intermediate by donating a proton to the leaving amide ion perhaps through a water molecule in the cavity created by the E133D mutation. In contrast, Glu-133 is deprotonated in the active form of wild-type PDF. We propose that Glu-133 acts as a proton shuttle accepting a proton from the metal-bound water and subsequently acts as a general acid during the decomposition of the tetrahedral intermediate.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/química , Ácido Glutámico/química , Metaloproteínas/química , Protones , Absorción , Alanina/genética , Amidas/química , Sustitución de Aminoácidos/genética , Aminopeptidasas/genética , Ácido Aspártico/genética , Tampones (Química) , Catálisis , Cloruros , Cobalto/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ácido Glutámico/genética , Concentración de Iones de Hidrógeno , Metaloproteínas/genética , Mutagénesis Sitio-Dirigida , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.