PubMed · 2654888
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L Dulau, A Cheyrou, D Dubourdieu, M Aigle. 1989-04-11. Directed mutagenesis using PCR.. https://doi.org/10.1093/nar%2F17.7.2873
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Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment, but emerging resistance threatens their efficacy. The potential for the development of drug resistance against plasmepsin X (PMX)-selective inhibitors and dual plasmepsin IX/X (PMIX/X) inhibitors was investigated in Plasmodium falciparum. A series of PMX-selective (WM4, WM76, WM92) and PMIX/X dual inhibitors (WM382, WM09, WM42) were characterised for potency against parasite growth and enzyme inhibition. In vitro selection experiments showed that all compounds had a high barrier to resistance, although parasites with reduced sensitivity to PMX‑selective inhibitors could still be selected. Resistance mechanisms involved pmx gene amplification and point mutations (D245N, S315P, S359P, I363L) that alter inhibitor binding. Recombinant expression and Michaelis-Menten kinetics demonstrated that these mutations impair drug binding whilst preserving PMX catalytic function. Reverse genetics confirmed that introducing these mutations into the pmx gene resulted in decreased potency of the inhibitors. In this study, resistance to the PMIX/X dual inhibitors evaluated here could not be selected, despite prolonged selection pressure. Antimalarial Resistome Barcoding (AReBar) assays confirmed the absence of pre-existing resistance to either inhibitor class. Critically, PMIX/X dual inhibitors maintained efficacy against parasites with decreased sensitivity to PMX-selective compounds. These findings demonstrate that dual PMIX/X inhibitors present a substantially higher barrier to resistance than PMX-selective inhibitors, informing antimalarial drug development strategies and highlighting dual-target inhibition as a promising approach to mitigate resistance risks.
Eosinophil cationic protein (ECP) is a major component of eosinophil granule protein that is used as a clinical bio-marker for asthma and allergic inflammatory diseases. Previously, it has been reported that the signal peptide of human ECP (ECPsp) inhibits the cell growth of Escherichia coli (E. coli) and Pichia pastoris (P. pastoris), but not mammalian A431 cells. The inhibitory effect is due to the lack of human signal peptide peptidase (hSPP), a protease located on the endoplasmic reticulum (ER) membrane, in the lower organisms. In this study, we show that the epidermal growth factor receptor (EGFR) is upregulated by the exogenous ECPsp-eGFP as a result of the increased expression of the transforming growth factor-alpha (TGF-alpha) at both transcriptional and translational levels in A431 and HL-60 clone 15 cell lines. Furthermore, the N-terminus of ECPsp fragment generated by the cleavage of hSPP (ECPspM1-G17) gives rise to over threefold increase of TGF-alpha protein expression, whereas another ECPsp fragment (ECPspL18-A27) and the hSPP-resistant ECPsp (ECPspG17L) do not show similar effect. Our results indicate that the ECPspM1-G17 plays a crucial role in the upregulation of TGF-alpha, suggesting that the ECPsp not only directs the secretion of mature ECP, but also involves in the autocrine system.
Neutrophils isolated from human peripheral blood added to a monolayer of human endothelial cells (ECV-304 cell line) stimulated with LPS (100 ng ml(-1)) resulted in: (a) neutrophil activation, measured by spreading and release of leukotriene B(4) (LTB(4)); (b) neutrophil degranulation, measured by release of matrix pro-metalloproteinase-9 (proMMP-9) and (c) loss of the monolayer integrity due to detachment of the endothelial cells. Stimulation of endothelial cells with tumor necrosis factor-alpha (TNF-alpha 10 ng ml(-1)) or interleukin-1 (IL-1; 10 ng ml(-1)) induced a similar dose-dependent increase in the neutrophil activation and endothelial cell detachment. Pre-treatment of LPS-activated ECV-304 cells with [Phe22]BigET-1(19-37) (10(-9) M; an inhibitor of endothelin converting enzyme (ECE)) or addition of BQ-123 (10(-6) M; a selective endothelin A (ET(A)) receptor antagonist) to the co-cultures, significantly reduced neutrophil spreading (50-70% inhibition) as well as the levels of LTB(4) (70-100% inhibition) and proMMP-9 (40-50% inhibition) in the co-culture supernatants. In addition, the detachment of endothelial cells was also reduced (60-75% inhibition). Moreover, the exogenous addition of ET-1 (10(-9) M) to neutrophil suspensions induced neutrophil spreading and release of LTB(4) and proMMP-9. Taken together, these findings indicate that neutrophils added to stimulated endothelial cells in the co-culture system employed in this study, get activated by products of these cells and degranulate. In parallel, the detachment of endothelial cell monolayer from the culture plates, possibly by the action of neutrophil granule-derived gelatinases, is observed. Endothelins (ETs) produced by the endothelial cells are suggested to play an essential role in these phenomena.