Macrocyclic Peptidomimetic Plasmepsin X Inhibitors with Potent In Vitro and In Vivo Antimalarial Activity

Abstract

The Plasmodium falciparum aspartic protease plasmepsin X (PMX) is essential for the egress of invasive merozoite forms of the parasite. PMX has therefore emerged as a new potential antimalarial target. Building on peptidic amino alcohols originating from a phenotypic screening hit, we have here developed a series of macrocyclic analogues as PMX inhibitors. Incorporation of an extended linker between the S1 phenyl group and S3 amide led to a lead compound that displayed a 10-fold improved PMX inhibitory potency and a 3-fold improved half-life in microsomal stability assays compared to the acyclic analogue. The lead compound was also the most potent of the new macrocyclic compounds in in vitro parasite growth inhibition. Inhibitor 7k cleared blood-stage P. falciparum in a dose-dependent manner when administered orally to infected humanized mice. Consequently, lead compound 7k represents a promising orally bioavailable molecule for further development as a PMX-targeting antimalarial drug.

Figure 1

Representative PMIX and/or PMX inhibitors.

Figure 2

Development of phenotypic screening hit 5 to macrocyclic peptidic inhibitor series 7.

Scheme 1. Synthesis of Building Blocks 16, 18, and 22 for the Preparation of Inhibitors 7a–k

Scheme 2. Synthesis of Inhibitors 7a,e,g,h

Scheme 3. Synthesis of Inhibitors 7b–d,f

Scheme 4. Synthesis of Inhibitor 7i

Scheme 5. Synthesis of Inhibitors 7j,k

Figure 3

Macrocycles 7a and 7k docked in the PMX active site. (A) Chemical structures of 7a and 7k. (B) The molecular surface of PMX (PDB: 7TBC) shown as a semi-transparent envelope, illustrating the size and shape of the enzyme active site pocket containing both 7a and 7k docked inhibitors. The inhibitors are shown as sticks, yellow for 7a (C: yellow, N: blue, O: red) and green for 7k (C: green, N: blue, O: red). The ICM-pro docking scores (dimensionless) are −8 for 7a and −12 for 7k, with an RMSD value of 3.3 Å between the poses of the two inhibitors. (C) Vicinity of the PMX (PDB: 7TBC) active site in cartoon representation with the inhibitors and side chain residues shown as sticks, colored and labeled according to their properties. The catalytic aspartic acid dyad (Asp266 and Asp457) is labeled in red. Docked inhibitors are shown as sticks, 7a in yellow (C: yellow, N: blue, O: red) and 7k in green (C: green, N: blue, O: red). Hydrogen bonds are represented by dashed lines, yellow for 7a and green for 7k.

Figure 4

Macrocycle PMX inhibitors have no effect on intraerythrocytic parasite maturation but prevent egress and maturation of SUB1. (A) Light microscopic images of Giemsa-stained P. falciparum 3D7 parasites allowed to mature for 44 h in the presence of vehicle only (dimethyl sulfoxide (DMSO), control) or macrocycle compounds 7a and 7k (20 nM). Development of ring-stage parasites to the multinucleated schizont stage occurred similarly in all cultures. However, while new ring-stage parasites arising from successful egress and invasion were beginning to become evident by this time point in control cultures (DMSO panel, white arrows), schizont rupture and appearance of new rings did not occur in the cultures containing the PMX inhibitors, even following extended further incubation. Scale bar, 20 μm. (B) Simplified schematic of proteolytic maturation of the egress effector SUB1. Conversion of the precursor form to p54 is through autocatalytic removal of the prodomain region (PD), whereas conversion of p54 to p47 is mediated by PMX.^,, (C) Left, Western blot analysis of P. falciparum 3D7 schizonts allowed to mature in the presence of the indicated macrocycle compounds (20 nM), showing defective SUB1 maturation in parasites treated with the indicated macrocycle compounds. Right, Western blot of the same extracts probed with a monoclonal antibody (mAb) specific to the rhoptry protein RAP1, which is a substrate for cleavage by the related P. falciparum protease PMIX. The compounds had no discernible effect on the maturation of RAP1. The results shown are typical of 3 independent experiments.

Figure 5

Washout experiments indicate low reversibility of PMX and egress inhibition mediated by macrocycle 7k. (A) Washout of the macrocycle inhibitors allows restoration of SUB1 maturation in the case of compounds 7a and 7j but not in the case of 7k. (B) Washout of the macrocycle inhibitors allows restoration of parasite egress (schizont rupture) and new ring formation in the case of compounds 7a and 7j but not in the case of 7k. The plot shows both new ring formation and levels of residual unruptured schizonts after 3.5 h of further culture following washout.

Figure 6

Plasma concentrations (±SEM) of inhibitor 7k after p.o. administration in mice at a dose of 50 mg/kg.

Figure 7

Therapeutic efficacy of inhibitor 7k against P. falciparumin vivo. (a) Parasitemia in peripheral blood of PfalcHuMice: untreated (QC1 and QC2), treated with chloroquine, or treated with inhibitor 7k at 40 mg/kg, p.o., UID, BID, or TID. (b) Distribution of stages of P. falciparum in peripheral blood of untreated and 7k-treated PfalcHuMice after one parasite cycle of drug exposure (day 3 of the assay). The figure shows flow cytometry plots of human erythrocytes (not stained with the anti-mouse erythrocyte mAb TER-119 conjugated with phycoerythrine) stained with the nucleic acid dye SYTO-16. The red rectangles inside flow cytometry plots indicate the area of viable parasites. The photographs show Giemsa-stained blood smears of the very same cytometry plots representative of the parasite cells found in the corresponding regions of viability. (c) Pharmacokinetic modeling of the concentrations of inhibitor 7k during the in vivo testing. Experimental data are represented by open symbols. The red lines indicate the preliminary estimate of minimal parasiticidal concentration (MPC) calculated from the experimental results. (d) PK/PD analysis of parasite killing in vivo induced by inhibitor 7k in comparison with chloroquine as a standard reference antimalarial drug. The data shown in the plot are the day of recrudescence (DoR) versus the total exposure in the blood of 7k of each individual mouse normalized by their respective individual parasite burdens as described. Data for Chloroquine are from historical data available at TAD for a set of PfalcHuMice treated p.o. UID, for 4 days, with different dose levels of the drug. In this plot, mapping to similar areas of the plot indicates equipotency for parasite killing in the PfalcHuMouse model.