Review
Drugging the Plasmodium kinome: the benefits of academia–industry synergy

https://doi.org/10.1016/j.tips.2008.02.005Get rights and content

Malaria remains a major killer in many parts of the world. Recently, the development of nonprofit organisations aimed at fighting this deadly scourge incited academic and industrial scientists to merge their expertise in drug-target validation and lead discovery. Expectations are clear: identification and characterisation of new molecules showing high efficacy, low toxicity and little propensity to induce resistance in the parasite. In this context, protein kinase inhibitors represent an attractive possibility. Here, we compare traditional target-based drug-discovery approaches with innovative exploratory paths (parallel screening, cell-based assays, integrated systems biology and allosteric inhibition) and discuss the benefits of acadaemia–industry cooperation. Early characterisation of distribution, metabolism, pharmacokinetic (DMPK) and toxicology parameters are considered as well.

Introduction

Four to eight thousand people, most of them children in sub-Saharan Africa, die every day from malaria. The mortality and morbidity burden inflicted by this disease represents a serious hindrance to the socioeconomic development of afflicted countries [1]. The spread of drug resistance in Plasmodium falciparum, the parasitic protozoan responsible for the vast majority of lethal cases of malaria, is a cause for grave concern with respect to disease control and renders the development of novel chemotherapeutic agents an urgent task [2].

P. falciparum infection of the human host is initiated by injection of sporozoites into the bloodstream by an infected Anopheles mosquito. The sporozoites rapidly gain access to the liver and invade hepatocytes, where asymptomatic asexual multiplication (exoerythrocytic schizogony) occurs, leading to the production of several thousand merozoites. These are released into the bloodstream and invade erythrocytes that become the site of another round of asexual multiplication, producing 8 to 24 new merozoites. This phase of the infection (erythrocytic schizogony) is responsible for malaria pathogenesis and therefore is the target for most compounds in the antimalarial chemotherapeutic arsenal. Some of the merozoites, upon red blood cell invasion, arrest their cell cycle and differentiate into male or female gametocytes instead of initiating a new round of schizogony. Ingestion of male and female gametocytes by a mosquito is required for transmission into the insect vector, in the midgut of which these cells mature into male and female gametes. These gametes then fuse into a zygote, the only diploid stage in the parasite's life cycle. Meiotic reduction occurs in the ookinete, the motile form into which the zygote rapidly develops. After crossing the midgut epithelium, the ookinete undergoes further development into an oocyst, the site of intense asexual multiplication producing several thousands of sporozoites that then accumulate in the mosquito's salivary glands and become ready to be injected into a vertebrate host during a subsequent blood meal [see the Malaria Foundation International website (http://www.malaria.org) for general information on malaria].

Modern drug discovery relies on a variety of complementary strategies to optimise identification rates of potent compounds and subsequent qualification of hit and lead molecules. The latest breakthroughs in genomic, proteomic and metabolomic studies have been applied fairly systematically to identify human targets in major disorders such as cancer and neurodegenerative or inflammation-based diseases and now are available for identifying parasite-specific targets (Figure 1). The sequencing of the P. falciparum genome was a major milestone in this respect 3, 4.

Among many drug-target classes, protein kinases (PKs) are considered as particularly attractive [5] for several reasons. First, the catalysis mechanism and overall structure of PKs are conserved, and it is well established that small molecules can bind to their catalytic cleft [6]. Second, reversible protein phosphorylation is one of the most important and pleiotropic modes of regulation of cell physiology and is a major mediator in the control of cell proliferation, differentiation, migration and homeostasis. In the context of drug discovery for diseases such as cancer, diabetes, inflammation and neurodegenerative disorders, most pharmaceutical and biotechnology companies run active programs aimed at identifying novel PKs as potential drug targets. As a result, many drug candidates targeting PKs are in development, and some inhibitors of PK function are already on the market (reviewed in Ref. [7]). Third, the development of the analogue-sensitive kinase allele (ASKA) strategy, also known as the ‘chemical genetics’ approach 8, 9, 10, allows in vivo validation of specific PKs as potential drug targets. ASKA technology is based on an engineered ATP binding site in the kinase of interest that enables it to accommodate bulky inhibitors that do not affect wild-type PKs (selected Plasmodium PKs carrying such mutations and expressed as active enzymes in E. coli actually do display the expected hypersensitivity to bulky inhibitors; allelic replacement in live parasites is in progress (C. Doerig et al., unpublished).

In this article we first briefly review P. falciparum kinomics as well as the opportunities that divergences between parasite and host PKs offer in the search of selective inhibitors. Then we consider various drug-discovery strategies aimed at identifying kinase inhibitors as antimalarial leads, emphasising the benefits that can be derived from academia–industry cooperation.

Section snippets

Targeting the Plasmodium kinome

The human kinome comprises approximately 500 sequences belonging to the eukaryotic protein kinase (ePK) family (reviewed in Ref. [11]). These sequences are classified into seven large groups: CK1 (casein kinase 1), CMGC (CDK-, MAPK-, GSK3- and CDK-like), TKL (tyrosine kinase-like), AGC (PKA, PKG, PKC), CamK (calcium/calmodulin-dependent kinases), STE (PKs acting as regulators of MAPKs, first identified in a genetic screen of sterile yeast mutants) and TyrK (tyrosine kinases), with the PKs not

Conventional target-based screening

After publication of the P. falciparum genome [4], databases such as PlasmoDB [3] and the WHO/TDR database of targets in neglected diseases (http://TDRtargets.org) have provided research teams with efficient in silico tools for the identification, comparison, evaluation and selection of potential drug targets (Figure 1). A crucial determinant for selection of a PK as a drug target is the demonstration of its essential role in the erythrocytic asexual cycle (curative drug targets) or in the

Academia and industry: a joint venture to kill the killer

As previously detailed by Nwaka and Ridley [72], the last decade witnessed a significant increase in interactions between academic laboratories working on neglected diseases on the one hand and lead discovery departments of private companies on the other hand. Organisations dedicated to public–private partnerships (such as Medicines for Malaria Venture), joint projects between the pharmaceutical industry and international institutions [e.g. the World Health Organization (WHO)], and

Concluding remarks

Over the recent years, nonprofit organisations, European Framework Programmes and other funding agencies have strongly encouraged connections and exchanges between academic and industrial partners. This led to the creation of small exploratory teams in which scientists contribute to opening a new era of antimalarial drug discovery.

Today, fighting against malaria or any other disease requires new approaches and criteria, from target selection to lead identification. However, the quest for

Acknowledgements

The authors are members of the AntiMal project, funded by the Framework Programme 6 of the European Commission and work collaboratively on Plasmodium kinase inhibition, implementing some of the approaches described in this review. We apologise to those colleagues whose work is not mentioned here because of space constraints. We thank A. Dorr (Merck-Serono, Geneva) for advice on the manuscript and J. Chevalier (Service Scientifique de l’Ambassade de France à Londres) for continuing interest and

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