Targeting malaria protein kinases.

Malaria is one of the most impacting public health problems in tropical and subtropical areas of the globe, with approximately 200 million cases worldwide annually. In the absence of an effective vaccine, rapid treatment is vital for effective malaria control. However, parasite resistance to currently available drugs underscores the urgent need for identifying new antimalarial therapies with new mechanisms of action. Among potential drug targets for developing new antimalarial candidates, protein kinases are attractive. These enzymes catalyze the phosphorylation of several proteins, thereby regulating a variety of cellular processes and playing crucial roles in the development of all stages of the malaria parasite life cycle. Moreover, the large phylogenetic distance between Plasmodium species and its human host is reflected in marked differences in structure and function of malaria protein kinases between the homologs of both species, indicating that selectivity can be attained. In this review, we describe the functions of the different types of Plasmodium kinases and highlight the main recent advances in the discovery of kinase inhibitors as potential new antimalarial drug candidates.

Detection of atovaquone and Malarone resistance conferring mutations in Plasmodium falciparum cytochrome b gene (cytb).

Clinical treatment failures of the hydroxynaphthoquinone atovaquone or its combination with proguanil (Malarone) in Plasmodium falciparum malaria has been recently documented. These events have been associated to single nucleotide polymorphisms (SNPs) in the parasite cytochrome b gene (cytb). In this report we describe a set of nest PCR-RFLP methods developed for the fast detection of all known cytb mutations associated to resistance to these drugs. The methods were successfully applied for the analysis of phenol-chloroform extracted DNA samples from patients not cured by Malarone, and from an established parasite clone. Further, the protocol for the detection of the A803C mutation was applied to 164 DNA field samples extracted through crude methanol-based protocols, originated from several malaria settings. The PCR-RFLP methods here presented can be used as a valuable for the clinical detection and study of Malarone and atovaquone P. falciparum resistance.

Plasmodium falciparum: gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine.

Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22-46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred. Some resistant clones obtained by this method were then examined by pulsed-field gel electrophoresis, and the results indicated that there had been an increase in the size of chromosome 4. This was confirmed by hybridization of Southern blots with a chromosome 4-specific probe, the vacuolar ATPase subunit B gene, and a probe to DHFR. Dot-blotting with an oligonucleotide probe to DHFR confirmed that there had been increases up to 44-fold in copy number of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a period of nearly 2 years, and reversion nearly to the level of pyrimethamine sensitivity of the original clone T9/94RC17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population reverted to a size like that of the original sensitive clone T9/94RC17. The procedure of growing parasites in stepwise increases of pyrimethamine concentration was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2.3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by developing gradually increased resistance to pyrimethamine. However, in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mutation from Ala to Ser at position 16, but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of clone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude that whereas some mutations producing changes in the amino acid sequence of the DHFR molecule may occur occasionally in clones or populations of P. falciparum grown in vitro in the presence of pyrimethamine, amplification of the DHFR gene following adaptation to growth in medium containing pyrimethamine occurs as a regular feature. The bearing of these findings on the development of pyrimethamine-resistant forms of malaria parasites in endemic areas is discussed.

Of mice and malaria mutants: unravelling the genetics of drug resistance using rodent malaria models.

It is well recognized that drug resistance is the most significant obstacle to gaining effective malaria control. Despite the enormous advances in the knowledge of the biochemistry and molecular biology of malaria parasites, only a few genes determining resistance to the commonly used drugs have been identified. The idea that rodent malaria parasites should be exploited more widely for such work, in view of the practical problems of studying this subject experimentally in human malaria, is presented.