A Fragmenting Hybrid Approach for Targeted Delivery of Multiple Therapeutic Agents to the Malaria Parasite

Artemisinin combination therapies (ACT) represent the current standard of care in the treatment of uncomplicated malaria. The widespread adoption of ACT has been motivated by a desire to minimize the emergence of drug resistance and to address the problem of recrudescence associated with artemisinin monotherapy.1–4 We set out to explore a single-molecule ‘fragmenting hybrid’ strategy in which an artemisinin-like peroxide is employed to deliver a partner drug, only upon activation by ferrous iron in the parasite. In principle, iron(II)-dependent drug delivery from a fragmenting hybrid could alleviate unwanted off-target bioactivities of the partner drug, which would be inactive in its hybrid form.


Supplementary Information
where E, I, E I, and E-I represent free enzyme, inhibitor, inhibitor bound to the enzyme, and the enzyme-inhibitor covalent complex, respectively. t is the treatment time of lysates with inhibitor, Ki the dissociation constant of the non-covalent enzyme:inhibitor complex, and ki is the rate constant of covalent modification of the enzyme by the inhibitor.
Estimation of the rate of ML4118S release from hybrid 8 in living parasites. Three assumptions were taken to estimate the rate of the retro-Michael reaction for hybrid 8 in vivo: 1) The rate of DPAP1 inhibition by ML4118S inside the food vacuole is identical to that measured in vitro in acetate buffer.
2) The initial concentration of hybrid 8 inside the parasites is identical to the one in the media.
3) The kinetic of the β-elimination reaction can be approximated to a simple single exponential decay (eq 3).
where k r is the average rate of release of ML4118S in the food vacuole, which is much slower than the reaction between iron(II) and the trioxolane moiety of compound 8. The formation of ML4118S can therefore be described by (eq 4).

(eq 4)
where k r is the average rate of release of ML4118S in the food vacuole, and [8] 0 is the initial concentration of hybrid 8, i.e. 50 nM.
To obtain k r , the residual DPAP1 activity values, measured during the treatment of a parasite culture with 50 nM of hybrid 8 ( Figure  3c), were fitted to eq 2 where [I] was replace by [ML4118S] as it is defined in eq 4, and the k i and K i values were fixed to those determined in vitro for ML4118S ( Supplementary Fig 2a).

3) Experimental Procedures -Chemical Biology
Parasite culture, harvesting and lysate preparation. D10 P. falciparum clones were cultured with media containing Albumax (Invitrogen) using standard procedures 2, 3 . D10 parasites were synchronized every 48 h at ring stage by treatment with 5 % sorbitol. Parasite pellets were harvested at trophozoite stage by selectively lysing the RBC membranes with 0.15 % saponin (Calbiochem, San Diego, CA). Lysates were prepared by treating one volume of parasite pellet with two volumes of 1 % nonidet P40 in PBS for 1 h on ice. The soluble fraction was separated from the insoluble one by a 5 min microcentrifugation at 13000 rpm.
P. falciparum replication assay. 200 µL of synchronized cultures of D10 parasites (~ 2 % parasitemia and 0.5 % hematocrit) were treated at ring stage with increasing concentrations of compound and were left to grow in 96-well plates for ~75 h. Cells were fixed in 0.05 % glutaraldehyde (Sigma) in PBS for at least 12 h at 4 °C, permeabilized for 5 min with 0.25 % Triton X in PBS, and stained with 0.05 mg/mL of propidium iodide (Sigma) in water. Infected and uninfected RBCs were quantified by FACS as the populations with positive and negative fluorescence in the propidium iodide channel, respectively. 4 All FACS measurements were taken on a BD FACScan flow cytometer (Becton, Dickinson and Co.). All EC 50,Pot values for parasite death were obtained by fitting the percentage parasitemia to a dose response curve.
Labeling of DPAP1 activity with FY01. FY01 is a cell permeable BODIPY-TMR fluorescently-tagged probe containing a vinyl sulfone reactive group that covalently modifies the catalytic cysteine of DPAP1. 4 Parasite lysates were diluted 10-times in acetate buffer and treated with 1 µM FY01 for 1 h at room temperature. To measure the specificity of an inhibitor against DPAP1, parasite lysates were treated with increasing concentrations of inhibitor for 30 min prior to labeling with FY01. Samples were then boiled in SDS-loading buffer and run on a SDS-PAGE gel. DPAP1 labeled bands run as a doublet around 20 kDa and were directly detected in a 9410 Typhoon Scanner (Amersham Bioscience, GE Healthcare).

Kinetics of DPAP1 inhibition in living parasites.
A synchronous culture of parasites (~ 20 % parasitemia) at trophozoite stage was cultured with 50 nM of compounds 8, 9, 13, ML4118S or DMSO. 1mL aliquots of culture were taken after 0.5 to 6 h of treatment, and the RBC membranes were lysed with 0.15 % saponin. Parasite pellets were resuspended in acetate buffer containing 1% nonidet P40, and DPAP1 activity was labeled with 1 μM FY01 at room temperature for 1 h.
After stirring overnight at room temperature, the reaction was not complete so more selenium dioxide was added (140 mg) and the reaction mixture was stirred another 12 h. The mixture was loaded directly on a silica gel column and purified eluting with 5%-10% MeOH/CH 2 Cl 2 . The final product was isolated as an oil (108 mg, 0.42 mmol, 67%). 1