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

Lead Research Organisation: Liverpool School of Tropical Medicine
Department Name: Parasitology

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description • UK management committee member for COST Action CM1307, 'Targeted chemotherapy towards diseases caused by endoparasites" - 2014- to date
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
URL http://www.costcm1307.org/CM1307/Home.html
 
Description Wellcome Trust - Multi-User Equipment Grant
Amount £600,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2014 
End 10/2019
 
Title Multi-User Equipment Grant entitled 'Supporting excellence in basic and clinical research: A flow cytometry/sorting and cell imaging platform for the genotypic and phenotypic analysis of Hazard Group 3 pathogens' 
Description Multi-User Equipment Grant entitled 'Supporting excellence in basic and clinical research: A flow cytometry/sorting and cell imaging platform for the genotypic and phenotypic analysis of Hazard Group 3 pathogens' We have set up to our knowledge the first dedicated HG3 imaging facility, able to sort and image HG3 pathogens, e.g. TB and HIV 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact It has just been set up, impact will be described next year 
 
Title OptiMal-PK: an internet-based, user-friendly interface for the mathematical-based design of optimized anti-malarial treatment regimens 
Description Background The search for highly effective anti-malarial therapies has gathered pace and recent years have seen a number of promising single and combined therapies reach the late stages of development. A key drug development challenge is the need for early assessment of the clinical utility of new drug leads as it is often unclear for developers whether efforts should be focused on efficacy or metabolic stability/exposure or indeed whether the continuation of iterative QSAR (quantitative structure-activity and relationships) cycles of medicinal chemistry and biological testing will translate to improved clinical efficacy. Pharmacokinetic and pharmacodynamic (PK/PD)-based measurements available from in vitro studies can be used for such clinical predictions. However, these predictions often require bespoke mathematical PK/PD modelling expertise and are normally performed after candidate development and, therefore, not during the pre-clinical development phase when such decisions need to be made. Methods An internet-based tool has been developed using STELLA® software. The tool simulates multiple differential equations that describe anti-malarial PK/PD relationships where the user can easily input PK/PD parameters. The tool utilizes a simple stop-light system to indicate the efficacy of each combination of parameters. This tool, called OptiMal-PK, additionally allows for the investigation of the effect of drug combinations with known or custom compounds. Results The results of simulations obtained from OptiMal-PK were compared to a previously published and validated mathematical model on which this tool is based. The tool has also been used to simulate the PK/PD relationship for a number of existing anti-malarial drugs in single or combined treatment. Simulations were predictive of the published clinical parasitological clearance activities for these existing therapies. Conclusions OptiMal-PK is designed to be implemented by medicinal chemists and pharmacologists during the pre-clinical anti-malarial drug development phase to explore the impact of different PK/PD parameters upon the predicted clinical activity of any new compound. It can help investigators to identify which pharmacological features of a compound are most important to the clinical performance of a new chemical entity and how partner drugs could potentially improve the activity of existing therapies. 
Type Of Material Computer model/algorithm 
Year Produced 2016 
Provided To Others? Yes  
Impact The software has been used by a number of users for both teaching and research purposes. The use of the model has lowered the number of animal experiments in my laboratory but it is difficult to estimate how many in vivo experiments it has reduced externally. We are currently working to promote the on-line tool. 
URL http://optimalpk.lstmed.ac.uk
 
Description AstraZeneca 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution A collaboration is underway for both malaria and TB drug discovery. LSTM research team are providing in vitro and in vivo PD and PK models and expertise in drug discovery for these specific diseases. Aspects of this collaboration are also within the WIPO (World intellectual Property Organisation) umbrella.
Collaborator Contribution AstraZeneca are providing starting points (hits) for testing against TB and malaria and they are also providing HTS expertise, chemoinformatics and ADMET screening. AZ collaborating partners are based both in the UK and India. Aspects of this collaboration are also within the WIPO (World intellectual Property Organisation) umbrella.
Impact multidisciplinary collaboration. The project is still on going and only hit molecules identified thus far. Lead series will be identified in the next 12 months.
Start Year 2011
 
Description Collaboration with GSK 
Organisation GlaxoSmithKline (GSK)
Department Tres Cantos Medicines Development Campus
Country Spain 
Sector Private 
PI Contribution The Liverpool research team undertakes early phase drug discovery of new anti-tuberculosis compounds which includes HTS screening, predictive in vitro models, medicinal chemistry, in vitro ADMET and in vivo DMPK
Collaborator Contribution The GSK team is part of the product development team and specifically carries out in vivo antitubercular testing in their acute and chronic models
Impact The team has generated early leads targeting a novel biological target in M. tuberculosis
Start Year 2011
 
Description Drug Discovery Project to develop combination partners targeting the respiratory chain of Mycobacterium tuberculosis 
Organisation Janssen Research & Development
Country Global 
Sector Private 
PI Contribution Novel strategy to improve the efficacy of bedaquiline and NCE
Collaborator Contribution Janssen have provided Materials e.g. bedaquiline. Discussions are taking place for a formal partnership and funding by Janssen of project as well as exclusive licensing of LSTM IP to Janssen.
Impact No outputs as yet
Start Year 2015
 
Description Guangdong University of Technology, China (GDUT) 
Organisation Guangdong University of Technology
Country China 
Sector Academic/University 
PI Contribution We have set up a Liverpool-Guangdong Drug Discovery Consortium. The consortium, made up of LSTM, University of Liverpool (UoL), Department of Chemistry and the Department of Pharmacy Engineering, Guangdong University of Technology, China (GDUT), is focussed on the development of new drug therapies for the treatment of TB, Malaria and other infectious diseases. A new laboratory has been opened known as the " Liverpool-Guangzhou drug discovery joint laboratory", located at GDUT. The laboratory will accommodate a drug discovery team made up of staff and students from GDUT and other parts of China. Pre-clinical projects targeting TB as well as malaria and NTD infections will be co-developed by the consortium.
Collaborator Contribution We have set up a Liverpool-Guangdong Drug Discovery Consortium. The consortium, made up of LSTM, University of Liverpool (UoL), Department of Chemistry and the Department of Pharmacy Engineering, Guangdong University of Technology, China (GDUT), is focussed on the development of new drug therapies for the treatment of TB, Malaria and other infectious diseases. A new laboratory has been opened known as the " Liverpool-Guangzhou drug discovery joint laboratory", located at GDUT. The laboratory will accommodate a drug discovery team made up of staff and students from GDUT and other parts of China. Pre-clinical projects targeting TB as well as malaria and NTD infections will be co-developed by the consortium.
Impact Compounds have been synthesised and teaching programs are being developed by the consortium
Start Year 2014
 
Description Wellcome Trust - Sanger Centre 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution In our MRC funded primaquine project we have identified potential genes of interest that are responsible for the mode of action of this important drug. The Sanger Centre are helping us with genetic confirmation studies in mouse models of malaria.
Collaborator Contribution In our MRC funded primaquine project we have identified potential genes of interest that are responsible for the mode of action of this important drug. The Sanger Centre are helping us with genetic confirmation studies in mouse models of malaria.
Impact Work is still on going
Start Year 2014
 
Title New drug against malaria 
Description New drug target against malaria identified. There are several projects under way from hit identification to late leads. These projects are in collaboration with GSK and AZ and involve MMV (malaria PDP). 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2008
Development Status Under active development/distribution
Impact New drug target against malaria identified. There are several projects under way from hit identification to late leads. These projects are in collaboration with GSK and AZ and involve MMV (malaria PDP). 
URL http://mmv.org
 
Description Press Release - New Centre for Drugs and Diagnostics 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release of the new LSTM Centre for Drugs and Diagnostics

A breakfast launch meeting was hosted at the British Society for Parasitology in Nest Gardens, 2013 with 20+ representatives of Small-Medium Enterprises
Year(s) Of Engagement Activity 2013
URL http://www.lstmliverpool.ac.uk/about-lstm/news-and-media/latest-news/launch-of-the-lstm-research-cen...
 
Description School Visit (Chester) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact 60 children attended - this was a presentation by me and interactive sessions with the children and teachers.

My visit was discussed by the headmaster at the whole school level and was reported to governors and in local news.
Year(s) Of Engagement Activity 2013