Defining the mechanism of action of the 8-aminoquinolines: A pre-requisite to rationally designed safe antimalarials for the elimination era

Malaria affects some 0.5 billion people and results in nearly 1 million deaths each year, mainly African children under the age of five. Malaria elimination programmes have been successful in some countries and the ambition is to roll these out to many more countries in the coming years. An important element in malaria elimination programmes is to have drugs that are able to cure relapse malaria (by killing the malaria parasite that infects and persists in the liver) as well as drugs that are able to block the transmission of the disease (by killing the stages that are transmitted to and live in the mosquito host).

There is only one class of antimalarial drugs (known an 8-aminoquinolines) with these desired properties, with one registered drug from this class known as primaquine and a new derivative called tafenoquine (with an improved dosing regimen) that is currently in clinical trials. Unfortunately however, both drugs are potentially lethal to people with a genetic disorder (known as Glucose-6-phosphate dehydrogenase deficiency) that affects some 400 million people word-wide, especially people from malaria endemic countries.

A safer alternative to primaquine is therefore urgently required and this issue as been identified as a priority by the Medicines for Malaria Venture (MMV, the global organization that funds new antimalarial drugs), the malERA global study consortium (a consortium of international scientists and policy makers that set out the priorities towards malaria elimination) and by the World Health Organization (WHO).

An often cost-effective and rapid way of improving the safety or efficacy of drugs is to understand the mechanism of action and toxicity of current drugs and then generate derivatives with improved properties, e.g. such as in the generation of second-generation antibiotics. However, despite the fact that primaquine has been used for over 60 years, we hitherto do not know how this drug works, severely hampering efforts to generate safer derivatives.

Here we present our initial data that indicates the involvement of two parasite enzymes (known as PfCPR and PfFNR) in the mode of action of primaquine. We are confident that we will be able to use this knowledge to generate safer derivatives of primaquine. However, a new drug discovery programme is very expensive and labour intensive and before we can go ahead we need definitive proof for the role of these two enzymes in the mode of action of primaquine. This MRC grant application therefore proposes to use the very latest molecular biology techniques and experimental disease models (using mice with humanized livers) to generate the definitive proof that will then allow follow-on research for a drug discovery project.

The researchers on this application have extensive experience in the techniques and approaches set out in the schedule of work, and we will also collaborate with industry (GSK) and academia to provide support with the very latest advancements and technologies in the field. The Liverpool team have a track record in translating basic research such as that described here into the generation of improved therapies. The project has been discussed with MMV and GSK, both of who are fully supportive of the need of the study, the approach used and the potential for translation to meet this urgent medical need.

Despite its development some 60 years ago, the mode of action of primaquine, the only drug proven to be clinically effective against liver and sexual stage malaria parasites, is unknown. This knowledge gap is a major barrier to the development of safer alternative therapies based on rational re-design of molecules that share this mechanism of action.

We have new data demonstrating that hydroxylated primaquine metabolites are redox cycled in vitro by P. falciparum ferredoxin-NADP+ reductase (PfFNR) and a novel diflavin reductase (PfCPR). Furthermore spontaneous oxidation of the resulting quinoneimines generates hydrogen peroxide and hydroxyl radicals. Both these enzymes are up-regulated in gametocytes and in liver stages and we hypothesise that the reactive oxygen species generated through these enzymes leads to parasite kill. Significantly, this data is the first demonstration of specific parasite enzymes involved in the mode of action of the 8-aminoquinolines and represents a starting point for detailed mechanism of action studies.

The aim of this project is generate definitive evidence of the extent to which PfFNR and PfCPR are involved in the mode of action of the 8-aminoquinolines and to describe the mechanisms underpinning gametocidal and liver-stage activity. PfFNR and PfCPR transgenics (e.g. knock-outs/knockdowns/overexpressing) will be generated to determine the effect of primaquine/tafenoquine metabolites (with support from GSK) against gametocytes and liver stages using the humanised-liver mouse model (Kappe laboratory). Concurrently, we will study the mechanism of enzyme-dependent redox cycling of hydroxylated primaquine and tafenoquine metabolites, towards informing follow-on medicinal chemistry QSAR studies.

This information will form the foundation for future rational drug re-design strategies aimed at delivering the next generation of transmission blocking/radical curative antimalarials with utility for the malaria elimination agenda.

In this project we aim to generate definitive evidence to support our hypothesis of the mode of action of primaquine and related 8-amino quinolines. Primaquine is the only registered drug that has (i) the ability to cure relapse malaria and (ii) has the ability to block malaria transmission. This is because the drug is uniquely able to kill dormant hypnozoite stages (that cause relapse malaria) and kill late stage gametocytes (the stage that passes the disease back to the mosquito). For this reason primaquine is currently the only drug with utility in malaria elimination programmes in countries/regions wishing to eliminate malaria. In 2010, 18 countries were either in the pre- or elimination phase and 7 countries are working to prevent re-introduction of malaria and the ambition of the WHO is to expand this effort year on year.

Despite the use of primaquine for over 60 years, the mode of action of this drug and its class has not been defined and this knowledge gap significantly affects the ability to design safer derivatives. Alternatives to primaquine are urgently required because a genetic deficiency in a specific enzyme (glucose 6 phosphate dehydrogenase, G6PDH) in persons taking this drug can lead to acute toxicity (hemolytic anemia) and death.

G6PDH deficiency is the most common human enzyme defect affecting ~400 million people. This genetic disease confers some innate immunity against malaria and is therefore extensively prevalent in people from malaria endemic regions such as Africa, the Middle East and Southern Asia.

Despite concerted control efforts malaria remains a global health issue with an estimated ~216 million cases (2011) and some 0.65 million deaths (2010). The economic cost in Africa alone is estimated at US$12 billion/annum. In 2007, a new agenda was set by the Bill and Melinda Gates Foundation (endorsed by the WHO) with the final goal to eradicate malaria. This step-change in policy has caught the drug community off-guard as for the past 50 years, therapeutic strategies have focused on treating blood stages of malaria, responsible for the clinical symptoms, and the clinically "silent" stages of infection, e.g. the liver stages and sexual stages, have largely been ignored.

UK plc fully endorse the WHO objectives, on World malaria Day 2012, the International Development Minister, Stephen O'Brien made the UK's commitment clear: "Tackling malaria is a key priority for the UK Government. Our continued investment in malaria prevention, diagnosis, treatment and research will help to sustain the progress that has been made in fighting this terrible disease, and ensure British aid will help to halve malaria deaths in ten of the worst affected countries by 2015."

A strategy that is often successfully used to generate new drugs is to improve the efficacy or safety of current therapies. For this reason both GSK Pharmaceuticals (whom are currently developing tafenoquine, a primaquine related drug) and the Medicine for Malaria Venture, a public-private partnership focused on delivering new malaria therapies, fully support our proposed project. Furthermore, the Liverpool team has a strong track record in translating basic scientific knowledge towards improved therapeutics. In a previous programme for amodiaquine, we were able to divorce toxicity from antimalarial potency once we understood the mechanisms underpinning both (O'Neill et al., 2003, J Med Chem. 46, 4933) and as a direct result developed (with GSK pharmaceuticals) a candidate drug that entered clinical testing (O'Neill et al., 2009, J Med Chem. 52, 1408).

This example demonstrates our ability as a team to take pharmacological data to better develop drugs. If our hypotheses are proved correct this information will form the foundation for future rational drug re-design strategies aimed at delivering the next generation of safe 8-aminoquinoline-like transmission blocking/radical curative antimalarials.