Hugues Matile

Identification of an antimalarial synthetic trioxolane drug development candidate

The discovery of artemisinin more than 30 years ago provided a completely new antimalarial structural prototype; that is, a molecule with a pharmacophoric peroxide bond in a unique 1,2,4-trioxane heterocycle. Available evidence suggests that artemisinin and related peroxidic antimalarial drugs exert their parasiticidal activity subsequent to reductive activation by haem, released as a result of haemoglobin digestion by the malaria-causing parasite. This irreversible redox reaction produces carbon-centred free radicals, leading to alkylation of haem and proteins (enzymes), one of which—the sarcoplasmic-endoplasmic reticulum ATPase PfATP6 (ref. 7)—may be critical to parasite survival. Notably, there is no evidence of drug resistance to any member of the artemisinin family of drugs. The chemotherapy of malaria has benefited greatly from the semi-synthetic artemisinins artemether and artesunate as they rapidly reduce parasite burden, have good therapeutic indices and provide for successful treatment outcomes. However, as a drug class, the artemisinins suffer from chemical (semi-synthetic availability, purity and cost), biopharmaceutical (poor bioavailability and limiting pharmacokinetics) and treatment (non-compliance with long treatment regimens and recrudescence) issues that limit their therapeutic potential. Here we describe how a synthetic peroxide antimalarial drug development candidate was identified in a collaborative drug discovery project.

An assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials

Chloroquine is thought to exert its antimalarial activity by preventing the polymerisation of toxic haematin released during proteolysis of haemoglobin in the Plasmodium digestive vacuole. However, the molecular mechanisms by which this inhibition occurs and the universality of this mechanism for other quinoline antimalarials remain to be established. We demonstrate here a correlation for eight antimalarial quinolines between inhibition of haematin polymerisation in vitro and inhibition of P. falciparum growth in culture, confirming haematin polymerisation as the likely target of quinoline blood schizonticides. Furthermore, using isothermal titration microcalorimetry, a correlation was observed between the haematin binding constant of these compounds and their ability to inhibit haematin polymerisation, suggesting that these compounds mediate their activity through binding to haematin. It was also observed that the compounds bind primarily to the mu-oxo dimer form of haematin rather than the monomeric form. It is postulated that this binding inhibits haematin polymerisation by shifting the haematin dimerisation equilibrium to the mu-oxo dimer, thus reducing the availability of monomeric haematin for incorporation into haemozoin. These data reconcile the haematin polymerisation theory with the Fitch hypothesis, which states that chloroquine mediates its activity through binding to haematin.

Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria

Ozonide OZ439 is a synthetic peroxide antimalarial drug candidate designed to provide a single-dose oral cure in humans. OZ439 has successfully completed Phase I clinical trials, where it was shown to be safe at doses up to 1,600 mg and is currently undergoing Phase IIa trials in malaria patients. Herein, we describe the discovery of OZ439 and the exceptional antimalarial and pharmacokinetic properties that led to its selection as a clinical drug development candidate. In vitro, OZ439 is fast-acting against all asexual erythrocytic Plasmodium falciparum stages with IC50 values comparable to those for the clinically used artemisinin derivatives. Unlike all other synthetic peroxides and semisynthetic artemisinin derivatives, OZ439 completely cures Plasmodium berghei-infected mice with a single oral dose of 20 mg/kg and exhibits prophylactic activity superior to that of the benchmark chemoprophylactic agent, mefloquine. Compared with other peroxide-containing antimalarial agents, such as the artemisinin derivatives and the first-generation ozonide OZ277, OZ439 exhibits a substantial increase in the pharmacokinetic half-life and blood concentration versus time profile in three preclinical species. The outstanding efficacy and prolonged blood concentrations of OZ439 are the result of a design strategy that stabilizes the intrinsically unstable pharmacophoric peroxide bond, thereby reducing clearance yet maintaining the necessary Fe(II)-reactivity to elicit parasite death.

System for High-Level Production in Escherichia coli and Rapid Purification of Recombinant Proteins: Application to Epitope Mapping, Preparation of Antibodies, and Structure—Function Analysis

Publisher Summary This chapter presents an application of the system for high-level production in Escherichia coli and rapid purification of recombinant proteins. The advent of gene cloning, the engineering of vectors for efficient expression, and the application of fast and high-flux methods for protein purification made available many recombinant proteins of biological interest. This represented a breakthrough for the structure–function analysis of bioactive proteins and cell receptors and facilitated X-ray crystallographic studies for definition of the three-dimensional structures of these proteins. The E. coli expression system allows the high-level production of recombinant proteins in authentic form, as fusion proteins with the [His] 6 affinity tail and with mouse DHFR and the [His] 6 tail. Because of the presence of the affinity tail, proteins that are produced in a soluble form or can be solubilized with GuHCl or urea can be purified almost to homogeneity in one step by nickel chelate affinity chromatography. The purified recombinant proteins simplify the production of monoclonal and polyclonal antibodies directed against defined regions of the native proteins.

4-aminoquinoline analogs of chloroquine with shortened side chains retain activity against chloroquine-resistant Plasmodium falciparum.

We have synthesized several 4-aminoquinolines with shortened side chains that retain activity against chloroquine-resistant isolates of Plasmodium falciparum malaria (W. Hofheinz, C. Jaquet, and S. Jolidon, European patent 94116281.0, June 1995). We report here an assessment of the activities of four selected compounds containing ethyl, propyl, and isopropyl side chains. Reasonable in vitro activity (50% inhibitory concentration, < 100 nM) against chloroquine-resistant P. falciparum strains was consistently observed, and the compounds performed well in a variety of plasmodium berghei animal models. However, some potential drawbacks of these compounds became evident upon in-depth testing. In vitro analysis of more than 70 isolates of P. falciparum and studies with a mouse in vivo model suggested a degree of cross-resistance with chloroquine. In addition, pharmacokinetic analysis demonstrated the formation of N-dealkylated metabolites of these compounds. These metabolites are similarly active against chloroquine-susceptible strains but are much less active against chloroquine-resistant strains. Thus, the clinical dosing required for these compounds would probably be greater for chloroquine-resistant strains than for chloroquine-susceptible strains. The clinical potential of these compounds is discussed within the context of chloroquines low therapeutic ratio and toxicity.

Modulating cholesteryl ester transfer protein activity maintains efficient pre-β-HDL formation and increases reverse cholesterol transport

The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-β-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-β-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [3H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [3H]neutral sterols and [3H]bile acids, whereas all compounds increased plasma HDL-[3H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-β-HDL formation, which may be required to increase reverse cholesterol transport.

A Comparison and Analysis of Several Ways to Promote Haematin (Haem) Polymerisation and an Assessment of Its Initiation In Vitro

We compared several methods for producing haematin polymerisation at physiological temperatures (i.e., 37 degrees) and found that a trophozoite lysate-mediated reaction was inappropriate for measuring compound inhibition of haematin polymerisation. Using this method, we obtained significantly higher IC50 values (concentration inhibiting haematin polymerisation by 50%) for certain compounds than when other methods were used, including a food vacuole lysate-mediated reaction. This difference was probably due to the binding of these compounds to cytosolic parasite proteins, as proteinase K treatment of the trophozoite lysate reversed this effect. The initiation of haematin polymerisation was also investigated using several assays. It was found that haematin polymerisation occurred spontaneously, in the absence of preformed haemozoin, over a period of several days, but that the process was more rapid when an acetonitrile extract of malarial trophozoites was added. This extract contained no detectable protein, and its activity could be replicated using an extract from uninfected erythrocytes and by using lipids. We therefore postulate that no protein or parasite-specific material is absolutely required for the initiation of haematin polymerisation. The formation of beta-haematin de novo using the acetonitrile extract is more pH-dependent than the generation of newly synthesised beta-haematin from preformed haemozoin and cannot proceed much above pH = 6. We postulate that the initiation of haematin polymerisation is more sensitive to the equilibrium of haematin between its monomeric and mu-oxo dimer form and requires a higher concentration of monomer than for the elongation phase of polymerisation.

Characterisation and sequence of a protective rhoptry antigen from Plasmodium falciparum.

We have recently demonstrated that a non-polymorphic rhoptry antigen, RAP-1 (rhoptry associated protein-1), which is recognised by human immune serum, can successfully protect Saimiri monkeys from a lethal infection of Plasmodium falciparum malaria. In this report we further characterise the antigen, which consists of four major proteins of 80, 65, 42 and 40 kDa and two minor proteins of 77 and 70 kDa, and present the antigens gene sequence. Monoclonal antibody evidence, autocatalytic processing and immunological cross-reactivity suggest that all components of this antigen are derived from the same precursor protein. The antigen is lipophilic, and disulphide bonding plays an important role in its structure. We discuss the structure and function of RAP-1 in the light of its deduced amino acid sequence and consider the relationship of this antigen to other rhoptry antigens of similar subunit size and composition.

Artemether administered together with haemin damages schistosomes in vitro

We conducted experiments in vitro to assess the effect of artemether in combination with haemin on adult Schistosoma japonicum, S. mansoni and S. haematobium. When schistosomes were maintained in a medium containing artemether at concentrations of 20 micrograms/mL or less for 72 h, no apparent effect on the schistosomes was seen. When the medium contained 50 or 100 micrograms/mL haemin as well as artemether, the schistosomes showed decreased motor activity 2-24 h after exposure, which was followed by the staining of the whole worm body a reddish-yellow colour, dilatation of the intestine, and extensive vesiculation of the tegument. Some of the schistosomes died 24 h after exposure, and almost all died within 48-72 h. When schistosomes were exposed to the same concentrations of haemin alone, they were stained a light yellow colour but there was no apparent effect on their survival. Our findings suggest that artemether interacts with haemin to exert a toxic effect on the worms, which might be of importance in the further elucidation of the mechanism of action of artemether on schistosomes.

The Structure−Activity Relationship of the Antimalarial Ozonide Arterolane (OZ277)

The structure and stereochemistry of the cyclohexane substituents of analogues of arterolane (OZ277) had little effect on potency against Plasmodium falciparum in vitro. Weak base functional groups were not required for high antimalarial potency, but they were essential for high antimalarial efficacy in P. berghei-infected mice. Five new ozonides with antimalarial efficacy and ADME profiles superior or equal to that of arterolane were identified.