Background
Malaria is estimated to be responsible for 435,000 deaths and over 200 million cases of infection per year, primarily in Africa and Southeast Asia [
1]. While huge advancements have been made in reducing both malaria incidence and mortality rates over the last 15 years, more recent data from 2015 to 2017 suggest progress has stalled, highlighting the need for further concentrated research. Particular challenges emerging are resistance to the most effective and commonly used artemisinin-based combination therapy, and deletions of the parasite gene encoding histidine rich protein II, which is a biomarker used in rapid diagnostic tests [
1]. There is an ever-growing need for effective new anti-malarials and diagnostic tools to be developed, alongside the goal of a vaccine.
Human-infective parasites of the genus
Plasmodium invade erythrocytes (e.g.,
Plasmodium falciparum) or reticulocytes (
Plasmodium vivax) during the malaria blood stage, when symptoms, such as fever, anaemia and inflammation, and lethality occur [
2]. While in the intra-erythrocytic stage,
Plasmodium parasites feed by consuming 60–80% of haemoglobin in red blood cells, breaking it down and using the amino acids as both an energy source and for protein synthesis [
3,
4]. An additional purpose for haemoglobin breakdown may be to provide the space needed for the parasite to grow and replicate in the erythrocyte [
5]. The breakdown of haemoglobin is carried out in a specialized parasite organelle, the acidic food vacuole, by a ~ 200 kDa protein complex containing cysteine (falcipain-2/2′, FP2/2′), aspartate (plasmepsin II and IV), and histo-aspartic proteases, and a dedicated enzyme (haem detoxification protein) for converting toxic haem into an inert crystalline form (haemozoin) [
6]. Genetic analysis has shown many of these components, including FP2, plasmepsin II and the haem detoxification protein, to be essential for parasite viability [
7]. Combined, the toxic nature of free haem in solution, the need of the parasite to feed on haemoglobin and to create space for its replication, and the essentiality of many protein components, make the haemoglobin breakdown pathway a strong candidate for anti-malarial therapeutics, as demonstrated by its targeting by current and historic drugs. For example, chloroquine, discovered in 1934 and still used against sensitive malaria strains, binds FP2 and interferes with initial haemoglobin proteolysis [
6], as well as haemozoin, where it prevents incorporation of additional haem molecules thereby increasing free haem concentration [
8]. Furthermore, in past studies parasite development in cultures was arrested, and malaria treated in murine models, by direct blocking of the haemoglobin breakdown pathway using broad-spectrum cysteine-protease inhibitors [
9‐
11].
Mature FP2 is a 27 kDa papain-type protease responsible for cleaving haemoglobin to small peptides [
12], with 93% sequence identity to FP2′ and 68% to parasite falcipain-3. In vitro, FP2/2′ and falcipain-3 are active, at least in part, against the same substrates and can be inhibited by the same small molecule compounds [
13]; in addition, genetic studies suggested that in vivo the roles of these proteases may overlap, as FP2 knock-outs could be compensated by increased expression of FP2′ and/or falcipain-3 [
9]. As well as having haemoglobinase activity, FP2 is involved in degrading the erythrocyte skeletal protein ankyrin [
14] in a process necessary for red blood cell rupture at the end of the parasite intra-erythrocytic cycle. Intriguingly, the optimum pH for FP2-mediated degradation of specific substrates differs, with haemoglobinase activity favoured at pH 5–6 as found in the food vacuole, while ankyrin degradation and FP2 self-activation by autoproteolysis are favoured at neutral or slightly alkaline pH [
14]. This suggests that FP2 activity may depend on the local cellular environment, thereby providing a mechanism for substrate discrimination.
A large number of FP2 inhibitors have been identified (e.g., [
15‐
19]) the majority of which are peptide based, although a number of peptidomimetic (e.g., [
20]) and non-peptidic inhibitors (e.g., [
21,
22]) have also been found. Despite this proliferation of potential therapeutics, no anti-malarials specifically targeting FP2 are currently available. This is in part due to poor selectivity of these inhibitors against human cysteine-proteases of the cathepsin family, which are structurally homologous to FP2. In addition, some of the strongest FP2 inhibitors known are peptides, which limits their potential as drug candidates since they degrade rapidly in vivo and cannot be administered orally. There is substantial interest in understanding the mechanisms of action and binding of non-peptidic FP2 inhibitors, as this knowledge could help in the design of more potent and specific compounds.
In this study, the crystallographic structure of FP2 is determined in complex with an inhibitor from the (E)-chalcone family of molecules [
21]. In contrast to previously resolved FP2 or falcipain-3 complexes with small molecules, all of which bound exclusively at the catalytic site [
23,
24], this (E)-chalcone inhibitor binds to the rear of the substrate-binding cleft, thereby mimicking interactions seen in FP2 when bound to other proteins [
25‐
27]. As (E)-chalcones can be easily synthesized and derivatised, and possess broadly understood biological properties [
28,
29], it is likely that combinations of (E)-chalcone scaffolds with inhibitors targeting the FP2 catalytic site may provide good starting points for anti-malarial drug design.
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