The Aminopeptidase Inhibitor CHR-2863 Is an Orally Bioavailable Inhibitor of Murine Malaria

Abstract

g Malaria remains a significant risk in many areas of the world, with resistance to the current antimalarial pharmacopeia an everincreasing problem. The M1 alanine aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) are believed to play a role in the terminal stages of digestion of host hemoglobin and thereby generate a pool of free amino acids that are essential for parasite growth and development. Here, we show that an orally bioavailable aminopeptidase inhibitor, CHR-2863, is efficacious against murine malaria. Malaria is a major cause of morbidity and mortality worldwide, with as many as 1 million deaths each year (3). While four Plasmodium species commonly infect humans, malaria caused by Plasmodium falciparum is responsible for most deaths, particularly in children under the age of 5 and pregnant women (2, 24). Both prevention and treatment of P. falciparum malaria are under threat because of the spread of drug-resistant parasites. Resistance to chloroquine is now considered almost universal (36), while the efficacy of affordable antimalarial drugs, such as sulfadoxine-pyrimethamine, the major drug used for intermittent preventative therapy, is declining (19,22). Artemisinin and its derivatives may be our last line of drug defense, although recent reports have indicated signs of reduced effectiveness of artemisinin combination therapies (ACTs) (23,28). For the development of the next generation of antimalarial agents, new targets and pathways susceptible to interruption by chemotherapy need to be identified.The asexual intraerythrocytic stages of Plasmodium development are responsible for the clinical symptoms attributable to malaria, and most antimalarial drugs target these stages of the parasite's life cycle (15,30). Many laboratories have focused on the hydrolytic process of hemoglobin (Hb) digestion within the parasite's specialized digestive vacuole as a target for drug discovery (4,7,16,18,32). During the asexual development phase, malaria parasites degrade 65 to 75% of their host cell's Hb, a process that ultimately results in the release of amino acids that are used by the parasite for protein anabolism (29) and the maintenance of osmotic pressure within the infected erythrocyte (14). Two metalloaminopeptidase enzymes, the P. falciparum M1 alanine aminopeptidase (PfM1AAP) and the P. falciparum M17 leucine aminopeptidase (PfM17LAP), may be critical in the terminal stages of Hb degradation and the release of free amino acids (6, 21). We have shown that inhibitors of these aminopeptidases, such as the natural Phe-Leu dipeptide analog bestatin, derived from the fungus Streptomyces olivoretticuli, and several synthetic phosphinate dipeptide analogs, can kill P. falciparum parasites in culture. These compounds are also effective against the rodent malaria parasite Plasmodium chabaudi chabaudi in vivo when administered intraperitoneally (i.p.) (32,33). We have also recently reported the production and characterization of functionally active recombinant P. falciparum M1 alanine aminopeptidas...

X‐ray crystal structures of an orally available aminopeptidase inhibitor, Tosedostat, bound to anti‐malarial drug targets PfA‐M1 and PfA‐M17

“…The hydroxamate group additionally forms tight metallobonds with 2Zn 2+ , and the ordered catalytic carbonate ion. This is in contrast to the docked binding pose of CHR‐2863 to Pf A‐M17, which suggested that only the hydroxyl group would coordinate 2Zn 2+ …”

“…We were interested in examining the mechanism of binding of Tosedostat to Pf A‐M1 and Pf A‐M17. Previous work on a closely related compound, CHR‐2863, showed that both the ester (CHR‐2863) and acid (CHR‐6768) forms of the molecule have similar inhibition constants for Pf A‐M1 (1.4 μ M and 2.4 μ M , respectively) and Pf A‐M17 (76 n M and 26 n M , respectively) . We determined the inhibition constants of Tosedostat for Pf A‐M1 and Pf A‐M17 to be 6.0 μ M and 78.5 n M respectively, comparable to those reported for CHR‐2863 .…”

“…Previous work on a closely related compound, CHR‐2863, showed that both the ester (CHR‐2863) and acid (CHR‐6768) forms of the molecule have similar inhibition constants for Pf A‐M1 (1.4 μ M and 2.4 μ M , respectively) and Pf A‐M17 (76 n M and 26 n M , respectively) . We determined the inhibition constants of Tosedostat for Pf A‐M1 and Pf A‐M17 to be 6.0 μ M and 78.5 n M respectively, comparable to those reported for CHR‐2863 . Since the pharmacologically active acid form of Tosedostat (CHR‐79888) was not commercially available, we chose to investigate the binding mechanism of its prodrug, Tosedostat‐ester, which would allow us to obtain structural insights into the mechanism of inhibition by the acid metabolite, CHR‐79888.…”

“…Ligands that bind Pf A‐M1 by coordination of the catalytic zinc ion (Zn 2+ ) do so via two mechanisms: monoanionic, wherein the compound forms a single metallobond to the tetrahedrally coordinated Zn 2+ , or dianionic, where two metallobonds between the compound and Zn 2+ impart pentahedral coordination. Docking studies had suggested that the CHR‐2863‐ester interacts via monoanionic binding, with only the hydroxyl group of the hydroxamate moiety coordinating Zn 2+ . In contrast, the structure of Pf A‐M1 bound to Tosedostat clearly shows the Zn 2+ coordinated by both oxygens of the hydroxamate moiety.…”

“…(c)] and its acid product, CHR‐6768, were both shown to be capable of Pf A‐M1 and Pf A‐M17 inhibition. Excitingly, CHR‐2863 was also shown to be an effective oral treatment of murine malaria . Tosedostat is bioavailable and thus far has demonstrated an excellent safety profile, making it an ideal starting point for the development of specific Pf A‐M1 and Pf A‐M17 inhibitors.…”