PfMDR1 Transport Rates Assessed in Intact Isolated Plasmodium falciparum Digestive Vacuoles Reflect Functional Drug Resistance Relationship with pfmdr1 Mutations.

Drug resistance often emerges from mutations in solute transporters. Single amino acid exchanges may alter functionality of transporters with 'de novo' ability to transport drugs away from their site of action. The PfMDR1 transporter (or P-glycoprotein 1) is located in the membrane of the digestive vacuole (DV), functions as an ATP-dependent pump, and transports substrates into the DV. In this study, four strains of Plasmodium falciparum, carrying various pfmdr1 gene mutations, were analysed for their transport characteristics of Fluo-4 in isolated DVs of parasites. To obtain quantitative estimates for PfMDR1 DV surface expression, PfMDR1 protein amounts on each strain's DV membrane were evaluated by quantitative ELISA. Fluo-4, acting as a substrate for PfMDR1, was applied in DV uptake assays ('reverse Ca2+ imaging'). Viable DVs were isolated from trophozoite stages with preserved PfMDR1 activity. This newly developed assay enabled us to measure the number of Fluo-4 molecules actively transported into isolated DVs per PfMDR1 molecule. The drug-resistant strain Dd2 presented the highest transport rates, followed by K1 and the drug-sensitive strain 3D7, compatible with their copy numbers. With this assay, an evaluation of the probability of resistance formation for newly developed drugs can be implemented in early stages of drug development.

Improved stability of polyclonal antibodies: A case study with lyophilization-conserved antibodies raised against epitopes from the malaria parasite Plasmodium falciparum.

Antibodies can be produced as polyclonal (pAb) or monoclonal (mAb) liquid formulations with limited shelf-life. For pAbs, unlike mAbs, only little is known about excipients and lyophilization affecting antibody stability upon reconstitution. We used a model pAb directed against Plasmodium falciparum (Pf) pyridoxal 5'-phosphate synthase 2 (Pdx2) to systemically study effects of bulking agents (amino acids, phosphate buffers, salt solutions), sugar(alcohols), surfactants and protein additions (bovine serum albumin, BSA) in liquid pAb formulations (isolated or in combinations) on the activity to detect the antigen in Pf extracts by Western blots. Repeated freeze-thaw cycles (20x) and extended room temperature storage markedly compromised pAb stability, the former being ameliorated by addition of cryoprotectants (glycerol, sucrose). Lyophilization of pure liquid pAb formulation markedly decreased antibody reactivity upon reconstitution which was not preserved by most bulking agents tested (e.g., histidine, arginine, acetate). Among the tested salt solutions (NaCl, Ringer, PBS), phosphate buffered saline had the largest lyoprotective potential, alongside sucrose, but not trehalose or maltitol. Among combinations of excipients, PBS, sucrose, low concentration BSA and Tween potently preserved PfPdx2 stability. Results for PBS were transferable to PfEnolase pAb, indicating that some of the formulations investigated here might be a low-cost solution for more general applicability to pAbs.

SYBR® Green I-Based Fluorescence Assay to Assess Cell Viability of Malaria Parasites for Routine Use in Compound Screening.

Owing to its fast and reliable assessment of parasite growth, the SYBR® Green I-based fluorescence assay is widely used to monitor drug susceptibility of malaria parasites. Its particular advantages are that it is a simple, one-step procedure and very cost-effective making it especially suited for high through put screening of newly developed drugs and drug combinations. Here we describe a SYBR® Green I-based fluorescence assay protocol to be used for routine screening of compounds and extracts in a research laboratory environment. A variation of the standard protocol is also provided allowing to address stage-specific effects of fast-acting drugs.

Highly potent artemisinin-derived dimers and trimers: Synthesis and evaluation of their antimalarial, antileukemia and antiviral activities.

New pharmaceutically active compounds can be obtained by modification of existing drugs to access more effective agents in the wake of drug resistance amongst others. To achieve this goal the concept of hybridization was established during the last decade. We employed this concept by coupling two artemisinin-derived precursors to obtain dimers or trimers with increased in vitro activity against Plasmodiumfalciparum 3D7 strain, leukemia cells (CCRF-CEM and multidrug-resistant subline CEM/ADR5000) and human cytomegalovirus (HCMV). Dimer 4 (IC50 of 2.6 nM) possess superior antimalarial activity compared with its parent compound artesunic acid(3) (IC50 of 9.0 nM). Dimer5 and trimers6 and 7 display superior potency against both leukemia cell lines (IC50 up to 0.002 µM for CCRF-CEM and IC50 up to 0.20 µM for CEM/ADR5000) and are even more active than clinically used doxorubicin (IC50 1.61 µM for CEM/ADR5000). With respect to anti-HCMV activity, trimer6 is the most efficient hybrid (IC50 0.04 µM) outperforming ganciclovir (IC50 2.6 µM), dihydroartemisinin(IC50 >10 µM) and artesunic acid (IC50 3.8 µM).