The malaria parasite Plasmodium falciparum degrades host cell hemoglobin inside an acidic food vacuole during the blood stage of the infectious cycle. A number of aspartic proteinases called plasmepsins (PMs) have been identified to play important roles in this degradation process and therefore generated significant interest as new antimalarial targets. Several x-ray structures of PMII have been described previously, but thus far, structure-guided drug design has been hampered by the fact that only inhibitors comprising a statine moiety or derivatives thereof have been published. Our drug discovery efforts to find innovative, cheap, and easily synthesized inhibitors against aspartic proteinases yielded some highly potent non-peptidic achiral inhibitors. A highly resolved (1.6 Å) x-ray structure of PMII is presented, featuring a potent achiral inhibitor in an unprecedented orientation, contacting the catalytic aspartates indirectly via the "catalytic" water. Major side chain rearrangements in the active site occur, which open up a new pocket and allow a new binding mode of the inhibitor. Moreover, a second inhibitor molecule could be located unambiguously in the active site of PMII. These newly obtained structural insights will further guide our attempts to improve compound properties eventually leading to the identification of molecules suitable as antimalarial drugs.Malaria is a major public health issue in many areas of the world, with Plasmodium falciparum being the causative agent of the most severe and deadliest form of this disease. Each year, 500 million new infections resulting in up to 2 million deaths and enormous economic damage (1) are attributed to this parasite. Drug resistance in P. falciparum has been aggravating the problem in many parts of the world during the last two decades, and new antimalarial agents addressing new targets are desperately needed.The protozoan parasite resides in erythrocytes of infected individuals during the asexual part of its life cycle. Recent studies indicated that hemoglobin degradation in a parasitic acidic organelle represents a major metabolic pathway and is crucial for survival of the parasite. Multiple proteinases appear to be actively involved in hemoglobin degradation (2-5). In particular, three members of a family of P. falciparum aspartic proteinases (PMI, 1 PMII, and PMIV) have been localized in the food vacuole (4, 5) and shown to be able to degrade hemoglobin in vitro. Another sequence-related proteinase with a new catalytic apparatus called PMIII or histo-aspartic proteinase (6) is also involved in hemoglobin catabolism in vitro. A number of research groups have reported attempts to find potent inhibitors of plasmepsins (7-12). Many of the identified molecules are peptidomimetic in nature, a compound class often associated with relatively low bioavailability and, importantly for use in developing countries, unfeasible due to significant cost of goods. We have discovered and subsequently optimized a new class of potent PMII inhibitors that could potential...

Malaria is a very serious infectious disease against which the currently available drugs are loosing effectiveness. The main problem is the emergence and the spreading of resistant parasite strains. New treatments are needed in order to regain control over the disease. Drug discovery efforts towards this goal are likely to be more successful, if they focus towards novel mechanisms of action. Such efforts will result in drugs that are functionally and structurally different from the existing drugs and therefore will overcome existing resistances. Here we focus on the aspartic protease plasmepsin II, which is a promising new drug target. We review the drug discovery efforts that were published in the literature on this enzyme, and we present the compounds synthesized at Actelion Pharmaceuticals Ltd.

Starting from the structure of bosentan (1), we embarked on a medicinal chemistry program aiming at the identification of novel potent dual endothelin receptor antagonists with high oral efficacy. This led to the discovery of a novel series of alkyl sulfamide substituted pyrimidines. Among these, compound 17 (macitentan, ACT-064992) emerged as particularly interesting as it is a potent inhibitor of ET(A) with significant affinity for the ET(B) receptor and shows excellent pharmacokinetic properties and high in vivo efficacy in hypertensive Dahl salt-sensitive rats. Compound 17 successfully completed a long-term phase III clinical trial for pulmonary arterial hypertension.

The most dangerous of the malaria-causing parasites, Plasmodium falciparum, infects 300-660 million people each year.[1] The plasmepsins (PMs) are a family of Plasmodium aspartic proteases that digest human hemoglobin and deliver amino acids that are required for growth. [2,3] In the search for targets for new antimalarial therapies, the mutually redundant proteolytic activity of plasmepsins for hemoglobin [4,5] requires inhibitors that are broadly active against all hemoglobin-degrading plasmepsins (PM I, PM II, PM IV, and HAP) [6] while remaining inactive against the most closely related human aspartic proteases (cathepsins D and E; hCat D and E). Among the plasmepsins, only the structure of PM II has been extensively characterized.[2, 7-9] As a result of the similar sequence identity among the plasmepsins, we hypothesized that the renin-like flap-open conformation (Figure 1 a), observed in two [9,10] of the fourteen known structures of PM II, would be accessible by each of the other three members of the Plasmepsin family. On this basis, we initiated a structure-based design program to explore the activity and selectivity of Plasmepsin inhibitors that target the flap pocket.Our earliest inhibitors (Figure 1 b) displayed moderate activity (IC 50 = 3000-35 000 nm; IC 50 = concentration of inhibitor at which 50 % of the maximum initial rate is observed) for PM II. [11][12][13] Herein we report the creation and elaboration of a new core consisting of a bicyclic diamine that forms a hydrogen-bonded clamp around the catalytic aspartate residues (Figure 1 c). The exo amino group additionally provides an attachment point for a pocket-directed binding substitu- Figure 1. Plasmepsin II and the discussed inhibitors. a) Schematic diagram of the active site showing the catalytic dyad, the S1/S3 pocket, and the flap pocket. b) A bicyclic amine that engages the catalytic aspartate dyad of Plasmepsin II. c) A "diamine clamp" designed to provide extra hydrogen bonding to the catalytic residues.

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