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Bioorganic and Medicinal Chemistry Letters
Volumn 16, Issue 8, 2006, Pages 2283-2292
Specific inhibitors of Plasmodium falciparum thioredoxin reductase as potential antimalarial agents
Author keywords

Drug development; Enzyme inhibition; Malaria; Plasmodium falciparum; Thioredoxin reductase
Indexed keywords

ANTIMALARIAL AGENT; CHLOROQUINE; ENZYME INHIBITOR; PLASMODIUM FALCIPARUM THIOREDOXIN REDUCTASE INHIBITOR; THIOL; THIOREDOXIN REDUCTASE; UNCLASSIFIED DRUG;
EID: 33644790392     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2006.01.027     Document Type: Article
Times cited : (73)
References (55)
  • 25
    • 33644795036 scopus 로고    scopus 로고
    • note
    • R) are given in min. ESI-MS of the glutathionylation products was carried out in Johannes Lechner's group at the Biochemie-Zentrum der Universität Heidelberg.
  • 26
    • 33644814557 scopus 로고    scopus 로고
    • note
    • 8S: C, 39.35; H, 1.65; N, 15.30; S, 8.75. Found: C, 39.77; H, 1.59; N, 15.45; S, 8.99.
  • 27
    • 33644788907 scopus 로고    scopus 로고
    • note
    • 50 values, were tested further with varying inhibitor and substrate concentrations to determine the inhibition type. To test time-dependent enzyme inhibition, P. falciparum TrxR (20 μM) was allowed to react for 15 min with 300 μM NADPH in the presence or absence of 100 μM compound 7 in a final volume of 50 μL at 25°C. All reaction mixtures contained 2% DMSO. Five microliters of the reaction mixture was removed and the residual activity was measured in the Trx standard assay (see above). Both unreacted and 7-reacted enzymes were dialyzed overnight and then tested for residual activity in the Trx assay.
  • 35
    • 33644796350 scopus 로고    scopus 로고
    • note
    • Glutathionylation of 6,7-dinitroquinoxaline (7). The glutathionylation product of compound 7 was prepared by mixing 736 μL of a 10 mM solution of 7 in DMSO (442 μM final concentration) with an equal volume of 20 mM GSH in water (884 μM final concentration), in the presence of a 9.7 mM triethylamine solution in water (final pH 7.4). The suspension was allowed to react at 25°C. The glutathionylation products were analyzed by HPLC (Nucleosil C-18) by injecting 30 μL aliquots after different times (0 min to 27 h) and by electrospray mass spectroscopy (ESI-MS). The retention times were 16.8 and 16.9 min for the glutanionylated-7 products ('normal'- and cine-substitution products) and 21.3 min for the starting 6,7-dinitroquinoxaline 7. The percentage of the starting materials converted with time was calculated from the peak area ratio. The reaction mixture was also analyzed by ESI-MS after completion.
  • 39
    • 33644808832 scopus 로고    scopus 로고
    • note
    • 52 Nitrite solutions (10-100 μM) were used for the calibration curve.
  • 41
    • 33644804505 scopus 로고    scopus 로고
    • note
    • -1). Absorption spectra were recorded on a HP 8452A spectrophotometer. All spectra were corrected for slight turbidity by subtracting the absorbance between 800 and 820 nm. A typical experiment proceeded in three phases: first, 25.7 μM TrxR (in a cuvette with two sidearms similar to those described previously in Ref. 53) was reduced anaerobically in 100 mM sodium phosphate buffer, pH 7.0, by 1.1 equiv NADPH (contained in a 100 μL gastight Hamilton syringe) forming the FAD-thiolate CTC; next, 3 equiv of compound 7 per FAD-containing subunit (20 mM inhibitor solution in 100% DMSO contained in one sidearm of the cuvette) was added and the effect on the CTC was followed. In the described experiment, an undefined trace amount of compound 7 was already present in the enzyme solution before NADPH addition; this is, likely due to volatility of the compound revealed during the eight vacuum-argon cycles. Finally, the protein modified by 3 equiv of compound 7 was titrated with additional NADPH and again the effect of enzyme modification on the FAD-thiolate CTC was examined. The enzyme was reoxidized by opening the cuvette to air and tested for residual activity. In parallel, two controls were prepared according to the same titration conditions over the same period of time and assayed for residual Trx reduction at the end of the titration. The first control, that is, 'control TrxR-NADPH', consisted of TrxR incubated with the same amount of NADPH as in the titration. The second control, that is, 'control TrxR inhibitor', consisted of TrxR incubated with inhibitor but in the absence of NADPH. This study was performed using a recombinant enzyme with a specific activity of 18 U/mg which was >95% pure as determined by SDS-polyacrylamide gel electroporesis (data not shown). The specific activities of wild type TrxR and inhibitor-reacted enzyme from titration experiments were determined in the Trx reduction assay. It was carried out at 25°C in a 1 mL cuvette containing 100 mM phosphate buffer, pH 7.0, in the presence of 100 μM NADPH, 0.01 to 0.1 μM TrxR (unreacted and inhibitor-reacted TrxR), and 50 μM Trx. The change in absorbance was measured at 340 nm.
  • 42
    • 33644796213 scopus 로고    scopus 로고
    • note
    • 50 values were calculated using the software Prism 3.0.
  • 43
    • 33644796506 scopus 로고    scopus 로고
    • note
    • 50 value is <1, the two drugs act synergistically, if it is equal to 1 the two drugs act additively, and if it is >1 the drugs act antagonistically.

* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.