Molecular characterization of G6PD mutations identifies new mutations and a high frequency of intronic variants in Thai females.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy caused by mutations in the G6PD gene. A medical concern associated with G6PD deficiency is acute hemolytic anemia induced by certain foods, drugs, and infections. Although phenotypic tests can correctly identify hemizygous males, as well as homozygous and compound heterozygous females, heterozygous females with a wide range of G6PD activity may be misclassified as normal. This study aimed to develop multiplex high-resolution melting (HRM) analyses to enable the accurate detection of G6PD mutations, especially among females with heterozygous deficiency. Multiplex HRM assays were developed to detect six G6PD variants, i.e., G6PD Gaohe (c.95A>G), G6PD Chinese-4 (c.392G>T), G6PD Mahidol (c.487G>A), G6PD Viangchan (c.871G>A), G6PD Chinese-5 (c.1024C>T), and G6PD Union (c.1360C>T) in two reactions. The assays were validated and then applied to genotype G6PD mutations in 248 Thai females. The sensitivity of the HRM assays developed was 100% [95% confidence interval (CI): 94.40%-100%] with a specificity of 100% (95% CI: 88.78%-100%) for detecting these six mutations. The prevalence of G6PD deficiency was estimated as 3.63% (9/248) for G6PD deficiency and 31.05% (77/248) for intermediate deficiency by phenotypic assay. The developed HRM assays identified three participants with normal enzyme activity as heterozygous for G6PD Viangchan. Interestingly, a deletion in intron 5 nucleotide position 637/638 (c.486-34delT) was also detected by the developed HRM assays. G6PD genotyping revealed a total of 12 G6PD genotypes, with a high prevalence of intronic variants. Our results suggested that HRM analysis-based genotyping is a simple and reliable approach for detecting G6PD mutations, and could be used to prevent the misdiagnosis of heterozygous females by phenotypic assay. This study also sheds light on the possibility of overlooking intronic variants, which could affect G6PD expression and contribute to enzyme deficiency.

Production of Lactobacillus plantarum ghosts by conditional expression of a prophage-encoded holin.

Bacterial ghosts (BGs) are nonviable empty bacterial cell envelopes with intact cellular morphology and native surface structure. BGs made from pathogenic bacteria are used for biomedical and pharmaceutical applications. However, incomplete pathogenic cell inactivation during BG preparation raises safety concerns that could limit the intended use. Therefore, safer bacterial cell types are needed for BG production. Here, we produced BGs from the food-grade Gram-positive bacterium Lactobacillus plantarum TBRC 2-4 by conditional expression of a prophage-encoded holin (LpHo). LpHo expression was regulated using the pheromone-inducible pSIP system and LpHo was localized to the cell membrane. Upon LpHo induction, a significant growth retardation and a drastic decrease in cell viability were observed. LpHo-induced cells also showed membrane pores by scanning electron microscopy, membrane depolarization by flow cytometry, and release of nucleic acid contents in the cell culture supernatant, consistent with the role of LpHo as a pore-forming protein and L. plantarum ghost formation. The holin-induced L. plantarum BG platform could be developed as a safer alternative vehicle for the delivery of biomolecules.

Mangiferin is a new potential antimalarial and anticancer drug for targeting serine hydroxymethyltransferase.

Mangiferin, a polyphenolic xanthone glycoside found in various botanical sources, including mango (Mangifera indica L.) leaves, can exhibit a variety of bioactivities. Although mangiferin has been reported to inhibit many targets, none of the studies have investigated the inhibition of serine hydroxymethyltransferase (SHMT), an attractive target for antimalarial and anticancer drugs. SHMT, one of the key enzymes in the deoxythymidylate synthesis cycle, catalyzes the reversible conversion of l-serine and (6S)-tetrahydrofolate (THF) into glycine and 5,10-methylene THF. Here, in vitro and in silico studies were used to probe how mangiferin isolated from mango leaves inhibits Plasmodium falciparum and human cytosolic SHMTs. The inhibition kinetics at pH 7.5 revealed that mangiferin is a competitive inhibitor against THF for enzymes from both organisms. Molecular docking and molecular dynamic (MD) simulations demonstrated the inhibitory effects of the deprotonated forms of mangiferin, specifically the C6-O- species and its resonance C9-O- species appearing at pH 7.5, combined with two docked poses, either a xanthone or glucose moiety, placed inside the THF-binding pocket. The MD analysis revealed that both C6-O- and its resonance-stabilized C9-O- species can favorably bind to SHMT in a similar fashion to THF, supporting the THF competitive inhibition of mangiferin. In addition, characterization of the proton dissociation equilibria of isolated mangiferin revealed that only three hydroxy groups of the xanthone moiety, C6-OH, C3-OH, and C7-OH, underwent varying degrees of deprotonation with pKa values of 6.38 ± 0.11, 8.21 ± 0.35, and 12.37 ± 0.30, respectively, while C1-OH remained protonated. Altogether, our findings demonstrate a new bioactivity of mangiferin and provide the basis for the future development of mangiferin as a potent antimalarial and anticancer drug.

Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure.

Background: Plasmodium vivax remains the malaria species posing a major threat to human health worldwide owing to its relapse mechanism. Currently, the only drugs of choice for radical cure are the 8-aminoquinolines (primaquine and tafenoquine), which are capable of killing hypnozoites and thus preventing P. vivax relapse. However, the therapeutic use of primaquine and tafenoquine is restricted because these drugs can cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. This study aimed to assess and understand the hemolytic risk of using 8-aminoquinolines for radical treatment in a malaria endemic area of Thailand. Methods: The prevalence of G6PD deficiency was determined using a quantitative test in 1,125 individuals. Multiplexed high-resolution meltinging (HRM) assays were developed and applied to detect 12 G6PD mutations. Furthermore, biochemical and structural characterization of G6PD variants was carried out to understand the molecular basis of enzyme deficiency. Results: The prevalence of G6PD deficiency was 6.76% (76/1,125), as assessed by a phenotypic test. Multiplexed HRM assays revealed G6PD Mahidol in 15.04% (77/512) of males and 28.38% (174/613) of females, as well as G6PD Aures in one female. G6PD activity above the 30% cut-off was detected in those carrying G6PD Mahidol, even in hemizygous male individuals. Two variants, G6PD Murcia Oristano and G6PD Songklanagarind + Viangchan, were identified for the first time in Thailand. Biochemical characterization revealed that structural instability is the primary cause of enzyme deficiency in G6PD Aures, G6PD Murcia Oristano, G6PD Songklanagarind + Viangchan, and G6PD Chinese 4 + Viangchan, with double G6PD mutations causing more severe enzyme deficiency. Conclusion: In western Thailand, up to 22% of people may be ineligible for radical cure. Routine qualitative tests may be insufficient for G6PD testing, so quantitative tests should be implemented. G6PD genotyping should also be used to confirm G6PD status, especially in female individuals suspected of having G6PD deficiency. People with double G6PD mutations are more likely to have hemolysis than are those with single G6PD mutations because the double mutations significantly reduce the catalytic activity as well as the structural stability of the protein.

Dual role of azo compounds in inhibiting Plasmodium falciparum adenosine deaminase and hemozoin biocrystallization.

Protein-ligand (GOLD) docking of the NCI compounds into the ligand-binding site of Plasmodium falciparum adenosine deaminase (PfADA) identified three most active azo compounds containing 4-[(4-hydroxy-2-oxo-1H-quinolin-3-yl) moiety. These compounds showed IC50 of 3.7-15.4 µM against PfADA, as well as inhibited the growth of P. falciparum strains 3D7 (chloroquine (CQ)-sensitive) and K1 (CQ-resistant) with IC50 of 1.8-3.1 and 1.7-3.6 µM, respectively. The identified compounds have structures similar to the backbone structure (4-N-(7-chloroquinolin-4-yl)) in CQ, and NSC45545 could mimic CQ by inhibiting the bioformation of hemozoin in parasitic food vacuole. The amount of in situ hemozoin in the ring-stage parasite was determined using a combination of synchrotron transmission Fourier transform infrared microspectroscopy and Principal Component Analysis. Stretching of the C-O bond of hemozoin propionate group measured at 1220-1210 cm-1 in untreated intraerythrocytic P. falciparum strains 3D7 and K1 was disappeared following treatment with 1.85 and 1.74 µM NSC45545, similar to those treated with 0.02 and 0.13 µM CQ, respectively. These findings indicate a novel dual function of 4-[(4-hydroxy-2-oxo-1H-quinolin-3-yl) azo compounds in inhibiting both PfADA and in situ hemozoin biocrystallization. These lead compounds hold promise for further development of new antimalarial therapeutics that could delay the onset of parasitic drug resistance.

Thermoresponsive C22 phage stiffness modulates the phage infectivity.

Bacteriophages offer a sustainable alternative for controlling crop disease. However, the lack of knowledge on phage infection mechanisms makes phage-based biological control varying and ineffective. In this work, we interrogated the temperature dependence of the infection and thermo-responsive behavior of the C22 phage. This soilborne podovirus is capable of lysing Ralstonia solanacearum, causing bacterial wilt disease. We revealed that the C22 phage could better infect the pathogenic host cell when incubated at low temperatures (25, 30 °C) than at high temperatures (35, 40 °C). Measurement of the C22 phage stiffness revealed that the phage stiffness at low temperatures was 2-3 times larger than at high temperatures. In addition, the imaging results showed that more C22 phage particles were attached to the cell surface at low temperatures than at high temperatures, associating the phage stiffness and the phage attachment. The result suggests that the structure and stiffness modulation in response to temperature change improve infection, providing mechanistic insight into the C22 phage lytic cycle. Our study signifies the need to understand phage responses to the fluctuating environment for effective phage-based biocontrol implementation.

Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.

Combined effects of double mutations on catalytic activity and structural stability contribute to clinical manifestations of glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans, affecting ~ 500 million worldwide. A detailed study of the structural stability and catalytic activity of G6PD variants is required to understand how different mutations cause varying degrees of enzyme deficiency, reflecting the response of G6PD variants to oxidative stress. Furthermore, for G6PD double variants, investigating how two mutations jointly cause severe enzyme deficiency is important. Here, we characterized the functional and structural properties of nine G6PD variants: G6PD Gaohe, G6PD Mahidol, G6PD Shoklo, G6PD Canton, G6PD Kaiping, G6PD Gaohe + Kaiping, G6PD Mahidol + Canton, G6PD Mahidol + Kaiping and G6PD Canton + Kaiping. All variants were less catalytically active and structurally stable than the wild type enzyme, with G6PD double mutations having a greater impact than single mutations. G6PD Shoklo and G6PD Canton + Kaiping were the least catalytically active single and double variants, respectively. The combined effects of two mutations were observed, with the Canton mutation reducing structural stability and the Kaiping mutation increasing it in the double mutations. Severe enzyme deficiency in the double mutants was mainly determined by the trade-off between protein stability and catalytic activity. Additionally, it was demonstrated that AG1, a G6PD activator, only marginally increased G6PD enzymatic activity and stability.

A novel bicyclic 2,4-diaminopyrimidine inhibitor of Streptococcus suis dihydrofolate reductase.

Streptococcus suis is a Gram-positive bacterial pathogen of pigs and an emerging zoonotic pathogen. It has become increasingly resistant to multiple classes of antibiotics. New drug candidates and knowledge of their targets are needed to combat antibiotic-resistant S. suis. In this study, the open-source Pathogen Box compound library was screened. Thirty hits that effectively inhibited S. suis growth at 10 µM were identified. Among the most potent hits, MMV675968 (a diaminoquinazoline analog) was shown to target S. suis dihydrofolate reductase (SsDHFR) via (1) growth inhibition of an E. coli surrogate whose growth is dependent on exogenously expressed SsDHFR and (2) inhibition of in vitro SsDHFR activity. Thymidine supplement is able to reverse growth inhibition by MMV675968 in both E. coli surrogate and S. suis, indicating that a thymidine-related pathway is a major target of MMV675968. Comparison of MMV675968 with seven DHFR inhibitors representing different core structures revealed that bicyclic 2,4-diaminopyrimidines with long and flexible side chains are highly effective in inhibiting SsDHFR and S. suis growth. MMV675968 and related compounds thus may serve as starting points for developing antibiotics against drug resistant S. suis.

C22 podovirus infectivity is associated with intermediate stiffness.

Bacteriophages have potential for use as biological control agents (biocontrols) of pathogenic bacteria, but their low stability is limiting for their utilization as biocontrols. Understanding of the conditions conducive to storage of phages in which infectivity is maintained over long periods will be useful for their application as biocontrols. We employed a nanomechanical approach to study how external environmental factors affect surface properties and infectivity of the podovirus C22 phage, a candidate for biocontrol of Ralstonia solanacearum, the agent of bacterial wilt in crops. We performed atomic force microscopy (AFM)-based nano-indentation on the C22 phage in buffers with varying pH and ionic strength. The infectivity data from plaque assay in the same conditions revealed that an intermediate range of stiffness was associated with phage titer that remained consistently high, even after prolonged storage up to 182 days. The data are consistent with the model that C22 phage must adopt a metastable state for maximal infectivity, and external factors that alter the stiffness of the phage capsid lead to perturbation of this infective state.

Real-time detection of changes in yeast plasma membrane potential using genetically encoded voltage indicator proteins.

In yeast, adaptation to varying conditions often requires proper regulation of the plasma membrane potential. To determine yeast membrane potential change, optical methods involving potentiometric dyes have been supplemental to the direct electrode-based method. However, the hydrophobic nature of the dyes and their slow distribution across the membrane still limits their utilization. Genetically encoded voltage indicator (GEVI) proteins employed in neuroscience offer a tantalizing alternative for monitoring yeast membrane potential change. In this work, several widely used GEVI proteins were assessed in Saccharomyces cerevisiae for their expression and function as a voltage reporter. Among them, only ArcLight and Accelerated Sensor of Action Potential (ASAP) proteins could be expressed and transported to the plasma membrane. While the voltage-sensing capability was demonstrated for both ArcLight and ASAP, ArcLight fluorescence was sensitive to the intracellular pH change concurrently with the voltage change. Therefore, we established that ASAP is the more suitable GEVI protein for reporting yeast membrane potential change. This voltage-sensing reporter for yeast based on ASAP offers a new effective strategy for real-time optical detection of yeast membrane potential change, which potentially facilitates many areas of yeast research including optimizing growth conditions for industrial use and investigating yeast ion transport system.

Functional analysis of BPSS2242 reveals its detoxification role in Burkholderia pseudomallei under salt stress.

A bpss2242 gene, encoding a putative short-chain dehydrogenase/oxidoreductase (SDR) in Burkholderia pseudomallei, was identified and its expression was up-regulated by ten-fold when B. pseudomallei was cultured under high salt concentration. Previous study suggested that BPSS2242 plays important roles in adaptation to salt stress and pathogenesis; however, its biological functions are still unknown. Herein, we report the biochemical properties and functional characterization of BPSS2242 from B. pseudomallei. BPSS2242 exhibited NADPH-dependent reductase activity toward diacetyl and methylglyoxal, toxic electrophilic dicarbonyls. The conserved catalytic triad was identified and found to play critical roles in catalysis and cofactor binding. Tyr162 and Lys166 are involved in NADPH binding and mutation of Lys166 causes a conformational change, altering protein structure. Overexpression of BPSS2242 in Escherichia coli increased bacterial survival upon exposure to diacetyl and methylglyoxal. Importantly, the viability of B. pseudomallei encountered dicarbonyl toxicity was enhanced when cultured under high salt concentration as a result of BPSS2242 overexpression. This is the first study demonstrating that BPSS2242 is responsible for detoxification of toxic metabolites, constituting a protective system against reactive carbonyl compounds in B. pseudomallei..

Diaminoquinazoline MMV675968 from Pathogen Box inhibits Acinetobacter baumannii growth through targeting of dihydrofolate reductase.

Antibiotic resistance in Acinetobacter baumannii is a major global health threat. New drugs with novel chemical structures are needed to overcome a myriad of resistance mechanisms in A. baumannii. In this study, we screened an open-source Pathogen Box library for anti-A. baumannii compounds. Compound MMV675968 (a diaminoquinazoline analog) was the only non-reference compound found to inhibit the growth of all four A. baumannii test strains with IC50 of 0.6-2.7 µM, IC90 of 0.7-3.9 µM, and MIC of 1.6-10 µM. We showed that MMV675968 targeted A. baumannii dihydrofolate reductase (AbDHFR) as determined by an E. coli surrogate whose growth was dependent on AbDHFR function and by an in vitro DHFR activity assay. Additionally, chemical scaffolds of DHFR inhibitors that are effective as antibiotics against A. baumannii were identified using an in vitro DHFR activity assay and A. baumannii growth inhibition. MMV675968 was the most potent among DHFR inhibitors tested in inhibiting A. baumannii growth. This study shows for the first time that MMV675968 inhibits A. baumannii growth via selective inhibition of AbDHFR and is therefore a promising scaffold for further antibiotic development against A. baumannii.

A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release.

Human cytosolic serine hydroxymethyltransferase (hcSHMT) is a promising target for anticancer chemotherapy and contains a flexible "flap motif" whose function is yet unknown. Here, using size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering (SAXS), molecular dynamics (MD) simulations, and ligand-binding and enzyme-kinetic analyses, we studied the functional roles of the flap motif by comparing WT hcSHMT with a flap-deleted variant (hcSHMT/Δflap). We found that deletion of the flap results in a mixture of apo-dimers and holo-tetramers, whereas the WT was mostly in the tetrameric form. MD simulations indicated that the flap stabilizes structural compactness and thereby enhances oligomerization. The hcSHMT/Δflap variant exhibited different catalytic properties in (6S)-tetrahydrofolate (THF)-dependent reactions compared with the WT but had similar activity in THF-independent aldol cleavage of ß-hydroxyamino acid. hcSHMT/Δflap was less sensitive to THF inhibition than the WT (Ki of 0.65 and 0.27 mm THF at pH 7.5, respectively), and the THF dissociation constant of the WT was also 3-fold lower than that of hcSHMT/Δflap, indicating that the flap is important for THF binding. hcSHMT/Δflap did not display the burst kinetics observed in the WT. These results indicate that, upon removal of the flap, product release is no longer the rate-limiting step, implying that the flap is important for controlling product release. The findings reported here improve our understanding of the functional roles of the flap motif in hcSHMT and provide fundamental insight into how a flexible loop can be involved in controlling the enzymatic reactions of hcSHMT and other enzymes.

Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays.

BACKGROUND: The reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. However, the application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. In this study, we reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. The assay is performed in a microplate format, where a single endpoint is measured at 450 nm. RESULTS: The optimized dehydrogenase-WST-8 assay conditions, the limit of detection (LOD), accuracy, and precision for measuring NAD(P)H, were demonstrated. The sensitivity of the WST-8 assay for detecting NAD(P)H was 5-fold greater than the spectrophotometric measurement of NAD(P)H absorption at 340 nm (LOD of 0.3 nmole vs 1.7 nmole, respectively). In the dehydrogenase assay, the colorimetric WST-8 method exhibits excellent assay reproducibility with a Z' factor of 0.9. The WST-8 assay was also used to determine dehydrogenase activity in biological samples, and for screening the substrate of uncharacterized short-chain dehydrogenase/oxidoreductase from Burkholderia pseudomallei. CONCLUSION: The results suggest that the WST-8 assay is a sensitive and rapid method for determining NAD(P)H concentration and dehydrogenase enzyme activity, which can be further applied for the high-throughput screening of dehydrogenases.

Crystal structure of Plasmodium falciparum adenosine deaminase reveals a novel binding pocket for inosine.

Plasmodium falciparum (Pf), a malarial pathogen, can only synthesize purine nucleotides employing a salvage pathway because it lacks de novo biosynthesis. Adenosine deaminase (ADA), one of the three purine salvage enzymes, catalyzes the irreversible hydrolytic deamination of adenosine to inosine, which is further converted to GMP and AMP for DNA/RNA production. In addition to adenosine conversion, Plasmodium ADA also catalyzes the conversion of 5'-methylthioadenosine, derived from polyamine biosynthesis, into 5'-methylthioinosine whereas the human enzyme is not capable of this function. Here we report the crystal structure of a surface engineered PfADA at a resolution of 2.48 Å, together with results on kinetic studies of PfADA wild-type and active site variants. The structure reveals a novel inosine binding pocket linked to a distinctive PfADA substructure (residues 172-179) derived from a non-conserved gating helix loop (172-188) in Plasmodium spp. and other ADA enzymes. Variants of PfADA and human (h) ADA active site amino acids were generated in order to study their role in catalysis, including PfADA- Phe136, -Thr174, -Asp176, and -Leu179, and hADA-Met155, equivalent to PfADA-Asp176. PfADA-Leu179His showed no effect on kinetic parameters. However, kinetic results of PfADA-Asp176Met/Ala mutants and hADA-Met155Asp/Ala showed that the mutation reduced adenosine and 5'-methylthioadenosine substrate affinity in PfADA and kcat in hADA, thereby reducing catalytic efficiency of the enzyme. Phe136Leu mutant showed increased Km (>10-fold) for both substrates whereas Thr174Ile/Ala only affected 5'-methylthioadenosine binding affinity. Together, the structure with the novel inosine binding pocket and the kinetic data provide insights for rational design of inhibitors against PfADA.

Characterization of Plasmodium knowlesi dihydrofolate reductase-thymidylate synthase and sensitivity to antifolates.

Malaria caused by an infection of Plasmodium knowlesi can result in high parasitemia and deaths. Therefore, effective and prompt treatment is necessary to reduce morbidity and mortality. The study aims to characterize P. knowlesi dihydrofolate reductase-thymidylate synthase enzyme (PkDHFR-TS) and its sensitivity to antifolates. The putative Pkdhfr gene was PCR amplified from field isolates collected from the Southern Thailand. Molecular analysis showed 11 polymorphisms in the dhfr domain of the bifunctional dhfr-ts gene. Of these, 1 polymorphism was a non-synonymous substitution (R34L) that had previously been reported but not associated with antifolate resistance. The recombinant PkDHFR-TS enzyme was found to be sensitive to standard antifolates-pyrimethamine and cycloguanil-as well as P218, a registered candidate drug currently first in human clinical trial. Results suggest that antifolates class of compounds should be effective against P. knowlesi infection.

Potent Inhibitors of Plasmodial Serine Hydroxymethyltransferase (SHMT) Featuring a Spirocyclic Scaffold.

With the discovery that serine hydroxymethyltransferase (SHMT) is a druggable target for antimalarials, the aim of this study was to design novel inhibitors of this key enzyme in the folate biosynthesis cycle. Herein, 19 novel spirocyclic ligands based on either 2-indolinone or dihydroindene scaffolds and featuring a pyrazolopyran core are reported. Strong target affinities for Plasmodium falciparum (Pf) SHMT (14-76 nm) and cellular potencies in the low nanomolar range (165-334 nm) were measured together with interesting selectivity against human cytosolic SHMT1 (hSHMT1). Four co-crystal structures with Plasmodium vivax (Pv) SHMT solved at 2.2-2.4 Šresolution revealed the key role of the vinylogous cyanamide for anchoring ligands within the active site. The spirocyclic motif in the molecules enforces the pyrazolopyran core to adopt a substantially more curved conformation than that of previous non-spirocyclic analogues. Finally, solvation of the spirocyclic lactam ring of the receptor-bound ligands is discussed.

Conformational Aspects in the Design of Inhibitors for Serine Hydroxymethyltransferase (SHMT): Biphenyl, Aryl Sulfonamide, and Aryl Sulfone Motifs.

Malaria remains a major threat to mankind due to the perpetual emergence of resistance against marketed drugs. Twenty-one pyrazolopyran-based inhibitors bearing terminal biphenyl, aryl sulfonamide, or aryl sulfone motifs were synthesized and tested towards serine hydroxymethyltransferase (SHMT), a key enzyme of the folate cycle. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target, as well as PfNF54 strains in cell-based assays in the low nanomolar range (18-56 nm). Seven co-crystal structures with P. vivax (Pv) SHMT were solved at 2.2-2.6 Šresolution. We observed an unprecedented influence of the torsion angle of ortho-substituted biphenyl moieties on cell-based efficacy. The peculiar lipophilic character of the sulfonyl moiety was highlighted in the complexes with aryl sulfonamide analogues, which bind in their preferred staggered orientation. The results are discussed within the context of conformational preferences in the ligands.

Human and Plasmodium serine hydroxymethyltransferases differ in rate-limiting steps and pH-dependent substrate inhibition behavior.

Serine hydroxymethyltransferase (SHMT), an essential enzyme for cell growth and development, catalyzes the transfer of -CH2OH from l-serine to tetrahydrofolate (THF) to form glycine and 5,10-methylenetetrahydrofolate (MTHF) which is used for nucleotide synthesis. Insights into the ligand binding and inhibition properties of human cytosolic SHMT (hcSHMT) and Plasmodium SHMT (PvSHMT) are crucial for designing specific drugs against malaria and cancer. The results presented here revealed strong and pH-dependent THF inhibition of hcSHMT. In contrast, in PvSHMT, THF inhibition and the influence of pH were not as pronounced. Ligand binding experiments performed at various pH values indicated that the hcSHMT:Gly complex binds THF more tightly at lower pH conditions, while the binding affinity of the PvSHMT:Gly complex for THF is not pH-dependent. Pre-steady state kinetic (rapid-quench) analysis of hcSHMT showed burst kinetics, indicating that glycine formation occurs fastest in the first turnover relative to the subsequent turnovers i.e. glycine release is the rate-limiting step in the hcSHMT reaction. All data suggest that excess THF likely binds E:Gly binary complex and forms the E:Gly:THF dead-end complex before glycine is released. A unique flap motif found in the structure of hcSHMT may be the key structural feature that imparts these described characteristics of hcSHMT.