Browsing by Author "Leartsakulpanich U."

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    Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure
    (2022-10-20) Sudsumrit S.; Chamchoy K.; Songdej D.; Adisakwattana P.; Krudsood S.; Adams E.R.; Imwong M.; Leartsakulpanich U.; Boonyuen U.; Mahidol University
    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.
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    Mangiferin is a new potential antimalarial and anticancer drug for targeting serine hydroxymethyltransferase
    (2023-09-01) Maenpuen S.; Mee-udorn P.; Pinthong C.; Athipornchai A.; Phiwkaow K.; Watchasit S.; Pimviriyakul P.; Rungrotmongkol T.; Tinikul R.; Leartsakulpanich U.; Chitnumsub P.; Mahidol University
    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.
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    Mechanisms and applications of bacterial luciferase and its auxiliary enzymes
    (2025-03-01) Kantiwiriyawanitch C.; Leartsakulpanich U.; Chaiyen P.; Tinikul R.; Kantiwiriyawanitch C.; Mahidol University
    Bacterial luciferase (LuxAB) catalyzes the conversion of reduced flavin mononucleotide (FMNH⁻), oxygen, and a long-chain aldehyde to oxidized FMN, the corresponding acid and water with concomitant light emission. This bioluminescence reaction requires the reaction of a flavin reductase such as LuxG (in vivo partner of LuxAB) to supply FMNH⁻ for the LuxAB reaction. LuxAB is a well-known self-sufficient luciferase system because both aldehyde and FMNH⁻ substrates can be produced by the associated enzymes encoded by the genes in the lux operon, allowing the system to be auto-luminous. This makes it useful for in vivo applications. Structural and functional studies have long been performed in efforts to gain a better understanding of the LuxAB reaction. Recently, continued exploration of the LuxAB reaction have elucidated the mechanisms of C4a-hydroperoxyflavin formation and identified key catalytic residues such as His44 that facilitates the generation of flavin intermediates important for light generation. Advancements in protein engineering and synthetic biology have improved the bioluminescence properties of LuxAB. Various applications of LuxAB for bioimaging, bioreporters, biosensing in metabolic engineering and real-time monitoring of aldehyde metabolites in biofuel production pathways have been developed during the last decade. Challenging issues such as achieving red-shifted emissions, optimizing the signal intensity and identifying mechanisms related to the generation of light-emitting species remain to be explored. Nevertheless, LuxAB continues to be a promising tool for diverse biotechnological and biomedical applications.
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    Molecular characterization of G6PD mutations identifies new mutations and a high frequency of intronic variants in Thai females
    (2023-11-20) Chamchoy K.; Sudsumrit S.; Wongwigkan J.; Petmitr S.; Songdej D.; Adams E.R.; Edwards T.; Leartsakulpanich U.; Boonyuen U.; Mahidol University
    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.
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    PlyCYU endolysin targeting Streptococcus agalactiae exhibits a CHAP activity and a glucosaminidase domain mediating multimerization
    (2025-09-17) Ubonprasert S.; Wachiradusit W.; Pornthanakasem W.; Songsungthong W.; Jaruwat A.; Premjaichon S.; Uengwetwanit T.; Suntivich R.; Thananon K.; Suksomjaisaman K.; Sucharitakul J.; Puyprom C.; Lotangchanintra T.; Salamteh K.; Wangkanont K.; Rodkhum C.; Visessanguan W.; Chaiyen P.; Chitnumsub P.; Leartsakulpanich U.; Ubonprasert S.; Mahidol University
    Bacteriophage endolysins are attractive alternatives to antibiotics owing to their rapid action, host specificity, and unlikeliness of resistance development. Here, bioinformatic analysis of Streptococcus suis prophage sequences identified an endolysin, named PlyCYU, containing two putative catalytic domains-an N-terminal amidase_5 and a C-terminal glucosaminidase (Lyz2) domain-with two CW_7 family cell wall binding motifs. PlyCYU exhibited bactericidal activity against Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis, with a minimum bactericidal concentration (MBC) range of 1.25 µM-40 µM, and retained bactericidal activity against S. agalactiae serotype II in ultra-high-temperature-processed milk (MBC 2.5 µM). Site-directed mutagenesis indicated that the amidase_5 domain was catalytically active and exhibited a cysteine-, histidine-dependent amidohydrolase/peptidase (CHAP) activity with the catalytic residues Cys34 and His99. Subdomain truncation analysis showed that PlyCYU214 and PlyCYU277, comprising the CHAP domain with one and two CW_7 motifs, respectively, conferred bactericidal activity, but lower than that of PlyCYU, while cyuLyz2 alone showed no activity. Notably, the bacteriolytic activity of PlyCYU277 was enhanced when cyuLyz2 was present. Agreeably, reducing sugars were detected in S. agalactiae lysis by PlyCYU and PlyCYU277 combined with cyuLyz2, but not by CHAP-inactive variants (PlyCYU-Cys34Ala/Ser and PlyCYU-His99Ala), PlyCYU277, and cyuLyz2 alone. This implied that cyuLyz2 action is CHAP dependent. Size exclusion chromatography (SEC) coupled with multi-angle light scattering and SEC-UV revealed PlyCYU and cyuLyz2 are homomultimers, whereas PlyCYU214 and PlyCYU277 are monomers. Therefore, the cyuLyz2 domain is important for the quaternary structure and the maximal activity of PlyCYU. Altogether, this study established PlyCYU endolysin as a potential antibiotic alternative against Streptococcus.IMPORTANCEStreptococcus agalactiae is a major pathogen responsible for severe neonatal infections, bovine mastitis, and streptococcosis in fish. The increasing prevalence of multidrug-resistant bacteria presses the urgent need to discover antibiotic alternatives. Bacteriophage-derived endolysins represent a promising solution due to their ability to specifically and rapidly kill target bacteria and be less likely to develop resistance. Here, we identified and characterized a novel endolysin, PlyCYU, with potent bactericidal activity against different Streptococcus species, including S. agalactiae, S. dysgalactiae, and S. uberis, isolated from bovine and fish sources. This study also demonstrated the relationships between the structure assembly and activity of PlyCYU. PlyCYU forms a multimer, facilitated by its glucosaminidase (cyuLyz2) domain, for maximal activity. Altogether, we revealed that PlyCYU is a promising candidate for development as an antibiotic alternative for Streptococcus infection treatment and food safety applications, as well as for advancing our understanding of endolysin.
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    Rapid and reliable detection of G6PD mutations using recombinase polymerase amplification coupled with lateral flow strip
    (2025-06-15) Jacob B.A.C.; Songsungthong W.; Leartsakulpanich U.; Boonyuen U.; Jacob B.A.C.; Mahidol University
    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy, affecting approximately 500 million people worldwide. It results from inherited mutations in the G6PD gene, causing increased susceptibility to drug-induced hemolytic anemia and severe neonatal jaundice. While phenotypic tests are commonly used, genetic testing is increasingly recognized for its value in the accurate diagnosis of G6PD deficiency, especially in heterozygous females and newborns. This study aimed to develop and evaluate a rapid, field-deployable genetic test for the detection of four common G6PD variants in Thailand: G6PD Gaohe (c.95A > G), G6PD Mahidol (c.487G > A), G6PD Viangchan (c.871G > A), and G6PD Canton (c.1376G > T). The assays utilize recombinase polymerase amplification with allele-specific primers incorporating locked nucleic acids to enhance specificity, followed by lateral flow strip detection for visual readout. The assays deliver results within 45 min at 37 ˚C. Singleplex detection demonstrated 100 % diagnostic sensitivity (Confidence interval (CI): 95.01–100.0 %) and specificity (CI: 95.49–100.0 %). Duplex assays (Gaohe + Canton and Mahidol + Viangchan) also demonstrated 100 % diagnostic sensitivity (CI: 94.87–100.0 %) and specificity (CI: 91.19–100.0 %). Limits of detection (LOD) for singleplex assays were 0.25, 1.00, 0.50, and 0.50 ng/µL, for Gaohe, Mahidol, Viangchan, and Canton, respectively. Duplex assays showed LODs of 0.10 ng/μL for Mahidol + Viangchan and 10.00 ng/μL for Gaohe + Canton. Band intensity differences ranged from 5.25 to 19.61 pixels between mutant, wild-type, and nontarget alleles, enabling clear allele discrimination. This innovative diagnostic tool offers a rapid, reliable, and accessible solution for point-of-care genetic testing, with the potential to improve clinical management and healthcare outcomes in regions with a high burden of G6PD deficiency.
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    Structural dynamics and in silico design of pyrazolopyran-based inhibitors against Plasmodium serine hydroxymethyltransferases
    (2022-09-15) Mee-udorn P.; Nutho B.; Chootrakool R.; Maenpuen S.; Leartsakulpanich U.; Chitnumsub P.; Rungrotmongkol T.; Mahidol University
    The clinical efficacy of antimalarial drugs has been reduced due to resistance spreading over many parts of the world. Target-based approaches on attractive drug targets, such as Plasmodium serine hydroxymethyltransferases (SHMTs) exhibiting distinct structure and function as well as kinetic mechanisms from the human enzyme homologue, are highly useful methods to be used for bypassing the present resistance in the field. Herein, 500-ns molecular dynamics (MD) simulations were carried out to investigate the mode of action of pyrazolopyran(+)-85 and pyrazolopyran(+)-86 with the most attractive inhibition efficiency in Plasmodium falciparum and P. vivax SHMTs (in the Schiff base form of PLP-L-serine (PLS) bound enzyme). The binding free energy results indicated the binding affinity of pyrazolopyran(+)-86 to Plasmodium SHMTs that is more favorable than pyrazolopyran(+)-85 by ∼ 2 kcal⋅mol−1, supported by the stronger ligand–protein hydrogen bonding and the lower solvent accessibility within the enzyme active site. According to the per-residue decomposition free energy analysis, residues L124, G128, H129, L130, K139, N356, and T357 are essential for inhibitors binding. By the rational structure-based drug design, the isopropyl moiety on the pyrazolopyran core should be changed to the negatively charged group (e.g., carboxylate group) for interacting with the positively charged residue R371. Alternatively, the phenolic compounds could be substituted with a phenyl or piperidine ring to promote hydrogen bond formation with the surrounding residues. Therefore, our findings presented here provide insights into the mode of inhibition of pyrazolopyran-based inhibitors and rational ideas for designing novel antimalarial drugs targeting Plasmodium SHMTs.