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There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.

Fluorescence decrease ratio was applied to determine of artemisinin (qinghaosu, QHS) based on the catalytic effect of hemoglobin (Hb) using tetraethyldiaminoxanthenyl chloride (Pryonine B, PB) as monitor in this work. Experimental result show that the catalytic characteristics of Hb for QHS, being expressed as Michaelis-Menten parameters,Km,Vmax, andKcat are 2.8×10−5 mol ·L−1, 4.2×106 mol · L−1 · s−1 and 280 s−1, respectively. Like hemin, enzymatic bioactivities of Hb is inhibited by both deactivated agents and high temperature where-as enhanced by ethanol. With the catalytic action of Hb, quantitative method of QHS can be established based on the fluorescence decrease of PB and linear relationship between the fluorescence decrease ratioF0/F and the concentration of QHS is in the range of (0.0−1.1)×105 mol · L−1 with detection limit (3σ) being 7.2 ×10−9 mol ·L−1. The concentration of QHS in the media of plasma or urine was detected using this method.

Objectives: Gene amplification/expression of G250 is a major event in human renal tumorigenesis. G250-based therapeutic agents and G250-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen G250 alterations in renal cell cancer (RCC) patients and investigate the relationship between G250 mRNA expression and RCC. Methods: We developed a quantitative RT-PCR assay for the measurement of G250 mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The method has been applied to the measurement of quantitative mRNA level of G250 in 31 cases RCC and 6 normal renal tissues. Results: The dynamic range was 103–108. The relationship between Ct and log starting concentration was linear (r=0.99). G250 expression was present in all RCCs with G250 amplification but was absent in normal ones. G250 mRNA expression ranged from 2.9×103 to 6.5×107 copy/μg RNA, with a mean value of 3.5×106 copy/μg RNA. The expression of G250 revealed an inverse correlation to tumor grade. G250 mRNA level did not correlate with the cell types and clinical stages (P>0.05). Conclusions: G250 has the potential to be used as a marker of diagnosis and increasing proliferation in RCC. This new simple, rapid, semi-automated assay was a major alternative to competitive PCR and Northern blot analysis for gene alteration analysis in human tumors and might be a powerful tool for large randomized, prospective cooperative group trials and supporting future G250-based biological and gene therapy approaches.