RICERCA ITALIANA     

The plant-type ferredoxin-NADP+ reductase/ferredoxin redox system as a potential drug target against apicomplexan parasites.

Università degli Studi di Milano

Abstract

The current knowledge of the biology of Apicomplexa points to the apicoplast as a major determinant of virulence in these protozoan parasites. This organelle can be considered a nonphotosynthetic plastid, and the redox system ferredoxin-NADP+ reductase/ferredoxin (FNR/Fd) localized in the apicoplast, is expected to be an important source of reducing equivalents for many biosynthetic pathways. We propose that the redox system FNR/Fd might be a novel target for drugs for treatment of infective diseases caused by Apicomplexa (malaria, toxoplasmosis, etc.). We have successfully expressed in Escherichia coli Fd and FNR of Toxoplasma gondii (TgFd and TgFNR). Both proteins turned out to be similar to the plant-type counterparts. The large knowledge about the biochemistry of the plant FNR/Fd systems has allowed us to obtain a detailed characterization of the recombinant redox system of T. gondii .
The unit, in collaboration with two international groups, will develop the following research plan. A major aim will be to obtain the three-dimensional structure of an apicomplexan FNRs, a very important requirement for drug design. In the case that our present efforts with TgFNR crystallization will be unsuccessful, we are considering to switch to other apicomplexan FNR, e.i., Plasmodium spp. enzymes, with the hope that the protein from a different source might yield to crystallization more easily. In the meantime, we plan to engineer TgFNR by removing the parasite-specific >>>

Principal Investigator

Giuliana ZANETTI Università degli Studi di MILANO

Research Objectives

Goal of this research project is the structural and functional characterization of the plant-type redox system FNR/Fd found in the apicoplast of the protozoan parasites of the phylum Apicomplexa. Our intent is to show that this redox system might be a novel target for drugs against these protozoan parasites. The major aim will be to obtain the three-dimensional structure of an apicomplexan FNR, either from T.gondii or Plasmodium spp.. This will allow us to get a better insight on the parasite FNR active site, a fundamental requirement for designing improved inhibitors of the enzyme. A search for potential inhibitors of the apicomplexan redox system will be accomplished on the basis of the vast knowledge already gathered on the plant redox system and on preliminary data obtained by us on the T. gondii system. Characterization of the enzyme system of Apicomplexa will be carried out by combining traditional enzymological approaches with the most recent developments of rapid reaction kinetics, site-directed mutagenesis and crystallography. Thus, we should be able to gather a large amount of information regarding the metabolic role and the structure-function relationship of the apicomplexan ferredoxin-NADP+ reductase system. In particular, inhibitors of the reductase or of the protein complex will be identified by classical methods to set the basis for the future use of the most advanced techniques of drug design, based on the enzyme three-dimensional structure and combinatorial >>>

First Results

1) Identification of structurally disordered surface regions of TgFNR. Identification of surface regions involved in substrate (NADP and Fd) interaction.
2) Plasmids for the overproduction of engineered TgFNR forms. Purified samples of mutant FNRs, where the main TgFNR insertion and possibly other disordered peptide regions have been replaced by shorter loops.
3) Plasmids for the overproduction of PfFNR.
4) Protocol for the purification of large amounts of recombinant PfFNR from E. coli. Samples of ultrapure PfFNR for crystallization trials.
5) Crystallization conditions for PfFNR and for mutant forms of TgFNR. Possible crystals of the same proteins for X-ray diffractometry studies.
6) Steady-state kinetic parameters of PfFNR for different substrates. Substrate specificity (NADH/NADPH) of PfFNR.
7) Identification of inhibitors active on apicomplexan FNRs.1) Characterization of complexes of PfFNR with various ligand (NAD+, NADP+, Fds from various sources). Dissociation constants for the same complexes.
2) Characterization of the presteady-state of TgFNR and PfFNR. Kinetic constants of some individual steps of the PfFNR catalytic mechanism.
3) Characterization of complexes between apicomplexan FNRs and their inhibitors. Dissociation constants of complexes with the reversible inhibitors. Rate constants for the FNR inactivation by irreversible inhibitors. Possible identification of amino acid residues modified by >>>

Timescale

24 months

National and international background

The phylum Apicomplexa comprises a large number of species which are obligate intracellular parasites of humans and livestock (1). They include pathogens like Plasmodium spp., the causative agent of malaria, one of the leading infectious diseases worldwide which causes more than one million deaths per year (2), and Toxoplasma gondii, which is an opportunistic pathogen of immunocompromised people and a cause of congenital infection (toxoplasmosis). T. gondii infection is very common, in some countries more than 50 % of the inhabitants are infected (3). Recently, T gondii infection has been suggested as a cause of psychiatric disorders like schizophrenia (4). Several other apicomplexa are to be mentioned as important pathogens: Cryptosporidium parvum, Eimeria spp., Cyclospora sp., Pneumocystis carinii, etc.. Therapy against these parasitic diseases is considered unsatisfactory, because of significant toxicity of the present drugs and of development of resistance to anti-protozoan agents. Development of new drugs is thus a matter of urgency before the current drugs become inefficient.
Nearly all the Apicomplexa possess a unique organelle, the apicoplast. Recent studies on its nature have established that it is evolutionarily related to a plastid of the non-photosynthetic type and have raised much interest (5,6). It has been proposed that it has arisen by secondary endosymbiosis of a cyanobacterial-like prokaryotic cell. The prokaryotic origin of the apicoplast is of >>>