MINIREVIEW Helicases ) feasible antimalarial drug target for Plasmodium falciparum Renu Tuteja Malaria Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India The universal presence of helicases in eukaryotes and prokaryotes, including parasites, reflects their funda- mental importance in DNA and RNA metabolic pro- cesses and the maintenance of genomic stability [1–4]. The emerging evidence demonstrates that helicases are indispensable enzymes because a growing number of human genetic disorders are attributed to mutations in helicase genes [1–4]. Helicases act on double-stranded nucleic acid substrate and thus can be designated DNA–DNA, RNA–DNA or RNA–RNA helicases depending on the composition of the substrate. They are also known as motor proteins because to unwind the duplexes they require energy, which is provided by their intrinsic nucleic acid-dependent ATPase activity. These enzymes act as necessary molecular tools for cel- lular machinery and significantly contribute to normal cellular metabolism. In general, helicases require a sin- gle-stranded nucleic acid region to bind and start their action of strand separation and once loaded onto the strand, they display a directional bias and translocate in either a 3¢ to 5¢ or 5¢ to 3¢ direction, however, a few bidirectional helicases have also been reported [5,6]. A typical helicase reaction occurs in three successive steps: (a) binding of the enzyme to the nucleic acid substrate, (b) NTP binding and hydrolysis, and (c) NTP-hydrolysis-dependent unwinding of the duplex substrate (Fig. 1). Various studies have shown that the unwinding activity of a helicase is tightly coupled to its intrinsic NTP-hydrolyzing (NTPase) activity [7]. There- fore, if the NTPase activity is inhibited, this will inhibit the helicase activity. An alternative approach, i.e. the reduction of NTP binding by blocking the NTP-bind- ing site with NTP analogs may also be a possible way to inhibit the NTPase and subsequently the helicase activity (Fig. 1). This binding results in the uncoupling of NTPase and helicase activities and hence functions through interaction with the enzyme. It is important to mention here that because the substrate- and NTP-binding regions are probably highly similar and conserved between various helicases, specifying the blockade through these sites will be immensely tough, although probably not impossible. Helicases form part of macromolecular complexes and contain discrete domains responsible for protein–protein interactions, Keywords DEAD-box; DNA unwinding; DNA-dependent ATPase; DNA-interacting compounds; drug target; helicase; inhibitors; malaria parasite; molecular motor; Plasmodium falciparum Correspondence R. Tuteja, Malaria Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India Fax: +91 11 2674 2316 Tel: +91 11 2674 1358 E-mail: renu@icgeb.res.in (Received 23 April 2007, revised 23 May 2007, accepted 19 July 2007) doi:10.1111/j.1742-4658.2007.06000.x Of the four Plasmodium species that cause human malaria, Plasmodium fal- ciparum is responsible for the most severe form of the disease and this par- asite is developing resistance to the major antimalarial drugs. Therefore, in order to control malaria it is necessary to identify new drug targets. One feasible target might be helicases, which are important unwinding enzymes and required for almost all the nucleic acid metabolism in the malaria parasite. FEBS Journal 274 (2007) 4699–4704 ª 2007 The Author Journal compilation ª 2007 FEBS 4699 therefore, in some cases strategies to block this inter- action will also lead to inhibition of the activity. Helicase signature motifs and Plasmodium falciparum helicases Based on sequence comparison and the presence of characteristic ‘helicase motifs’, three helicase superfam- ilies (SF1–3) have been identified [7–9]. SF1 and SF2 contain helicases that share a set of nine and SF3 con- tains only a set of three highly conserved ‘helicase motifs’, respectively [7–9]. The DEAD (Asp-Glu-Ala- Asp), DEAH (Asp-Glu-Ala-His), DExH and DExD- box helicases are ubiquitous and are the most common members of SF2 [8,9]. The various ‘helicase motifs’ have been named Q, I, Ia, Ib, II, III, IV, V and VI and, based on the mutational analysis and structural data in a variety of systems, specific roles have been suggested for a number of the conserved motifs [10– 12]. For example, motif I (A ⁄ GxxGxGKT), motif II (VLDEAD), motif III (SAT) and motif VI (HRIGRxxR) are responsible for ATP binding and hydrolysis, nucleic acid binding and ATP-hydrolysis- dependent nucleic acid unwinding, respectively [7]. It has been reported that Arabidopsis thaliana contains 55 members of the DEAD-box family of helicases, humans contain 38 and Saccharomyces cerevisiae con- tains 25 [13]. In addition to the ‘helicase core region’, which harbors the conserved motifs and functions as an ATP-dependent motor or switch, most helicases contain divergent amino and ⁄ or C-terminal extensions that confer substrate specificity and provide the basis for protein–protein interaction [14]. These structurally different domains are also responsible for targeting the helicase to a specific cellular process. Although helicases have been reported from a vari- ety of other systems there are very few reports of helicases from P. falciparum. Since completion of the P. falciparum genome in 2002, new opportunities for research have arisen [15]. The P. falciparum genome has 14 chromosomes, a linear mitochondrial genome and a circular plastid-like genome [15,16]. A full set of helicases was identified in the original genome sequence of P. falciparum during annotation (http:// www.plasmodb.org), but detailed analysis using a bioinformatic approach revealed that the genome con- tains at least 22 full-length putative DEAD-box heli- cases, as well as a few other putative helicases [15–18]. These 22 P. falciparum helicases contain all the con- served domains, but the length and sequence of the N- and C-terminal extensions and the intervening sequences are variable [17]. Based on the crystal struc- ture of human DEAD-box helicase, a model for the structure of p68 (a well-characterized 68 kDa protein of the DEAD-box protein family, which is conserved from yeast to human) homolog of P. falciparum (P. falciparum DNA helicase 60, PfDH60) was created, which suggests that although there are minor variations in length and sequence between the conserved domains these two structures are highly superimposable (Fig. 2). These observations further suggest that although these proteins most likely act through related mechanisms the parasite-specific sequences could still be specifically targeted because the antibodies to PfDH60 do not cross-react with the human p68 [17,19–21]. To the best of our knowledge only a few helicases have been characterized from P. falciparum. These include two members of the DEAD-box family namely PfDH60 and P. falciparum DNA helicase 45 (PfDH45) [20,21] (A. Pradhan and R. Tuteja, unpublished) and PfDHA, a 90 kDa DNA helicase which has been puri- fied from P. falciparum [18]. Our studies indicated that PfDH60 contains helicase and ssDNA-dependent Fig. 1. Schematic representation of the three successive steps involved in a typical helicase reaction. The details of steps a–c are written above the arrows. A particular helicase inhibitor ⁄ drug most probably acts at the substrate or enzyme level via one or more of the following processes: (i) modulates enzyme–substrate binding, (ii) inhibits helicase activity by obstructing NTP binding, (iii) inhibits NTPase activity via an undefined or allosteric mechanism, (iv) inhibits the coupling of NTP hydrolysis with the unwinding reaction, and (v) inhibits translocation of the helicase on the nucleic acid substrate due to the steric blockade. Helicases as an antimalarial drug target R. Tuteja 4700 FEBS Journal 274 (2007) 4699–4704 ª 2007 The Author Journal compilation ª 2007 FEBS ATPase activities and is expressed in schizont stages of the development of parasite [20,21]. It has also been reported that PfDH60 is a unique dual, bipolar heli- case and its enzyme activities are modulated by phos- phorylation [21]. PfDH45 is a homolog of eukaryotic initiation factor 4A contains helicase and ssDNA- dependent ATPase activities and is expressed in all the developmental stages of the parasite (A. Pradhan and R. Tuteja, unpublished). PfDHA moves in the 3¢ to 5¢ direction and prefers a fork-like substrate for its unwinding activity [18]. Helicases as drug and therapeutic target Resistance to the most efficient, reasonably priced and safe antimalarials has called for the search for new drug targets and ultimately new drugs. Because heli- cases contain multiple functional domains and a vari- ety of enzymatic activities, and have essential roles in the metabolism of DNA and RNA, helicase inhibitors might offer a feasible route towards the development of novel drugs. Various studies have shown that heli- cases are indispensable enzymes and in yeast the loss of one DEAD-box gene cannot be supplemented by overexpression of another family member, which fur- ther suggests that each helicase gene is independently essential [22,23]. Some helicases are required for the proliferation of bacteria and viruses, therefore, inhibi- tion of the unwinding activity of various helicases results in a decrease in virus replication in cell cultures as well as in animal models and this suggests a novel antiviral strategy [24–27]. Potent antihelicase agents have been reported for a number of helicases from dif- ferent viruses [28]. The detailed characterization of two related DEAD-box helicases, hepatitis C virus NS3 and human eIF-4A has provided evidence for design- ing specific inhibitors that can be used to target the viral NS3 helicase and inhibit the viral replication [5]. Table 1 shows a comparison of the inhibitory poten- tial of some of the helicases from P. falciparum and helicases from the human host. The comparison clearly indicates that the IC 50 value for various compounds tested, including daunorubicin and nogalamycin, is lowest for helicases from P. falciparum compared with the other helicases [29,30]. It is interesting to note that inhibition by nogalamycin is highly variable and depends on the source of the enzyme [31–34]. In a pre- vious study it was reported that the IC 50 value for this compound varied between 0.1 and >650 lm for heli- cases from different viruses such as hepatitis C virus, dengue fever virus, Japanese encephalitis virus and west Nile virus [35,36]. It is possible that some of these Fig. 2. Structural modeling of PfDH60. The protein sequence of PfDH60 (GenBank accession number AY700082; PlasmoDB No. PFL1310c) was subjected to the 3 D-JIGSAW program (version 2.0) in http://www.expasy.org. This server builds 3D models of proteins based on known structural homologs [37–40]. The model for PfDH60 was built based on the solved crystal structure of human DEAD-box helicase (Protein data bank Id:2I4I; Molecular Modeling Database Id:41213) [39]. The conserved helicase motifs of both proteins are displayed in different colors using a molecular visualization program to display, animate and analyze large biomolecule systems using 3D graphics and built-in scripting ( VMD software; http://www.ks.uiu- c.edu). (A) Template structure, (B) PfDH60 structure. The colors used for various motifs are: motif I, yellow; motif Ia, green; motif Ib, red; motif II, light blue; motif III, white; motif VI, white. R. Tuteja Helicases as an antimalarial drug target FEBS Journal 274 (2007) 4699–4704 ª 2007 The Author Journal compilation ª 2007 FEBS 4701 compounds, which inhibit helicase activity, could be utilized to inhibit parasite growth. In fact, it has been shown that some of these compounds inhibited the growth of P. falciparum in culture, which further con- firms that inhibition of the activity of parasite helicase inhibits the parasite growth [30]. Furthermore, these helicases can be specifically targeted using the specific antibody and dsRNA approach. Previous observations have shown that anti- PfDH60 sera, which recognize only this protein in par- asite lysate, inhibit parasite growth in culture [30]. Similar results were also obtained for anti-PfDH45 sera (A. Pradhan and R. Tuteja, unpublished). Regarding the antisense approach, it has been shown that the specific dsRNA against PfDH60 inhibited par- asite growth in culture [30]. This inhibition is due to the degradation of its cognate mRNA, which results in inhibition of PfDH60 protein synthesis and in turn inhibition of the parasite growth [30]. These results collectively indicate that the helicases can be specifi- cally targeted to inhibit their function. Although these results are encouraging but overall the data on inhibi- tor studies of malarial helicases are very limited. Because helicases belong to a large gene family exten- sive validation is required before the studies can focus on a specific malarial helicase that could be used as a specific target to control malaria. A comparative study of available inhibitors may help to identify a com- pound to specifically target and inhibit the parasite helicase without affecting the host, and thereby could be used as the potential drug ⁄ drugs to treat malaria. Conclusions and future perspectives Antimalarial drug resistance poses a major obstacle to the control of malaria. Therefore, the development of suitable and cost-effective drugs for the treatment of malaria is a significant endeavor. Detailed studies regarding the mechanism and function of all the helicases of P. falciparum (including the DEAD-box helicases) will help to establish their validity as a suitable target. 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