Studies on plasmodium falciparum enoyl-ACP reductase (Fabl)

Abstract

Inhibition of type-II fatty acid biosynthesis has recently been vahdated as an appropriate target for the development of newer antimalarials. The enzyme target of most of the known FAS-II antimicrobial compounds is enoyl-ACP reductase (FabI), which catalyzes the final enzymatic step in the elongation cycle of the FAS-II pathway, converting trans-2-enoyl-ACP to acyl-ACP in a NADH-dependent reaction. Specific inhibitors of the FAS-II pathway include triclosan and thiolactomycin. Triclosan, a specific inhibitor of FAS-II trans-2-enoyl-ACP reductase (FabI) is effective against a broad spectrum of bacteria including E. coli, mycobacteria, and multidrug-resistant Staphylococcus aureus and is widely used as an antimicrobial in household formulations, including soaps and toothpaste. Recently, triclosan was found to inhibit Plasmodium falciparum growth with an IC50 of ~1 |iM. It was fiirther demonstrated by the finding that precursor (acetate and malonyl-CoA) incorporation is sensitive to triclosan. First part of this thesis describes the cloning and expression of FabI fi-om Plasmodium falciparum. Various constructs generated include the one each for fiill-length FabI protein, mature protein and truncated protein. Full-length gene was cloned in pRSETB and pET20b expression vectors which could not result in any expression. Cloning of the mature protein nucleotide sequence in pET28a expression vector resulted in the overexpression of the protein of expected size. Truncated version of the same protein was subsequently generated to enhance the stability and crystallization of this protein. These expressed proteins were further purified to homogeneity fi-om E. coli cultures by passing through the His-bind resin column. Here we also report the inhibition studies of recombinant PfENR of Plasmodium falciparum purified to homogeneity fi"om E. coli cultures with triclosan and triclosan analogs by means of a spectrophotometric assay. We report that 2, 2'Dihydroxy, 5, 5'-Dichloro-diphenybnethane(D3M), 2, 2'-Dihydroxydiphenylmethane(2, 2'-DE), 4, 4' -Dihydroxydiphenylether(4, 4'-DE), 4-Hydroxyphenol(4-pOPhe), 2 ,4'- Dihydroxydiphenylmethane (2, 4'-DM) and 2, 2'-Dihydroxydiphenylmethane (2, 2'- DM) inhibit PffiNR with an IC50 value of 6.25, 21, 26, 29, 38 and 32^M respectively, hi separate assays triclosan was found to inhibit this enzyme with an IC50 value of 50 and 68 \xM. in presence or absence of NAD respectively. These studies led us to identify an analog of triclosan, 2, 2'dihydroxy, 5, 5'-Dichlorodiphenylmethane (D3M) with moderate inhibitory activity (Ki = 4.9 |xM compared with triclosan which shows Kj = 45 nM and 88 nM in presence and absence of NAD respectively). hi vitro whole cell studies with triclosan and its analogs indicated the killing of chloroquine resistant and sensitive strains with IC5oof 11, 65, 90, 110, 140 and 125 vM for 2, 2'-Dihydroxy. 5, S'-Dichloro-diphenylmethane (D3M), 2, 2'- Dihydroxycliphenylmethane(2, 2'-DE), 4, 4'-Dihydroxydiphenylether (4, 4'-DE), 4-Hydroxyphenol(4-pOPhe), 2, 4'-Dihydroxydiphenylmethane (2, 4'-DM), and 2, 2'-Dihydroxydiphenylmethane (2, 2'-DM) respectively. These concentrations are very high when compared to triclosan, which shows an IC50 of 1.25nM. We have used site directed mutagenesis approach to find out the role of few residues, which are considered crucial for the binding of triclosan to this enzyme. These residues have been foimd to be crucial in all ENRs studied across the species and have been found to be conserved throughout. We have generated two mutants of Ala217 (Ala217 changed to Glycine or Valine), which we believe should reduce the affinity of triclosan towards this enzyme. Similarly we have generated two more mutants of Phe368 (Phe368 changed to Alanine or Isoleucine), which is again very crucial for its interaction with the triclosan. All these mutants have been generated through overlap extension PCR base strategy and cloned in pET28a expression vector. On overexpression these mutants were found to be predominantly going into inclusion bodies even though there were no differences in the expression conditions of the mutants and wild type protein. Various parameters were changed to get soluble mutant proteins, which include the expression at lower temperatures as well, but we were unsuccessful to get these mutants in soluble form. We then went further to purify these mutants fi-om the inclusion bodies using various standard procedures for protein folding and purification fi-om inclusion bodies. The refolded purified proteins failed to show any activity in a spectrophotometric assays. Inactive forms of these enzymes did not allow characterization of inhibitor-enzyme interactions. Finally, polyclonal antibodies have been raised against this enzyme (PfENR) both in mice as well as rabbit and also cross reactivity of these antibodies were checked against few of the proteins available in the laboratory, and the antibodies were foimd to be FabI specific.