Plasmodium dihydrofolate reductase is a second enzyme target for the antimalarial action of triclosan

Malaria, caused by parasites of the genus Plasmodium, leads to over half a million deaths per year, 90% of which are caused by Plasmodium falciparum. P. vivax usually causes milder forms of malaria; however, P. vivax can remain dormant in the livers of infected patients for weeks or years before re-emerging in a new bout of the disease. The only drugs available that target all stages of the parasite can lead to severe side effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency; hence, there is an urgent need to develop new drugs active against blood and liver stages of the parasite. Different groups have demonstrated that triclosan, a common antibacterial agent, targets the Plasmodium liver enzyme enoyl reductase. Here, we provide 4 independent lines of evidence demonstrating that triclosan specifically targets both wild-type and pyrimethamine-resistant P. falciparum and P. vivax dihydrofolate reductases, classic targets for the blood stage of the parasite. This makes triclosan an exciting candidate for further development as a dual specificity antimalarial, which could target both liver and blood stages of the parasite.

falciparum in erythrocytes and so inhibition of PfENR cannot explain the effect of triclosan on blood-stage parasites.
Efforts to optimize the compound for use as an antimalarial showed no correlation between PfENR inhibition in vitro and triclosan's activity against the living parasite 21,22 . Since it has been demonstrated that PfENR is important for the liver stage of the parasite, but not for the erythrocyte stage 22,23 , action against a different target must be responsible for triclosan's inhibition of the growth of blood-stage P. falciparum.
We have developed an automated yeast-based assay for use in high-throughput screens for compounds that are selectively active against target enzymes from parasites 24,25 . Our assay is based on replacing yeast genes essential for growth, with coding sequences specifying orthologous proteins from either parasites or humans, making the strains dependent on the activity of the parasite or the human enzyme in order to grow 25 . These strains are then labeled with different fluorescent proteins and pooled to allow monitoring of the growth, in real time, of each strain in competition with the others.
We carried out growth competition experiments between 3 yeast strains dependent on a DHFR enzyme from P. falciparum, P. vivax, or the human DHFR (these three strains were differentiated from one another by each expressing either the fluorescent proteins mCherry, Venus, or Sapphire 26,27 ). These competitions took place in single wells of microtiter trays, enabling a Robot Scientist to screen thousands of different compounds and identify candidates with no general cytotoxicity, but which inhibited the parasite target without affecting its human counterpart 24,28 .

Results and Discussion
Dihydrofolate reductase (DHFR) is an enzyme that catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. This reaction is essential for the de novo synthesis of purines and certain amino acids, making DHFR essential for rapid growth, and is the target for the action of the important antimalarial drugs pyrimethamine and proguanil. Employing our yeast-based high-throughput screening approach 24 , we screened the Johns Hopkins Library of FDA-approved compounds against yeast strains expressing human DHFR (HsDHFR), P. falciparum DHFR (PfDHFR), P. vivax DHFR (PvDHFR) as well as the pyrimethamine-resistant P. vivax and P. falciparum DHFRs (PvRdhfr and PfRdhfr) 28 . The automated screen identified triclosan (Eve ID 21658, JHU-10450) as a specific inhibitor of P. vivax and P. falciparum DHFRs, including the pyrimethamine-resistant variants of the two enzymes. The Robot Scientist Eve prepared titration experiments with 1 to 20 μM of triclosan and successfully confirmed the hits. We prepared serial dilutions of yeast cultures expressing the human or Plasmodium DHFRs and spotted those onto agar plates containing 10 μM of triclosan, further confirming the specificity of the drug for the parasite target (Fig. 1).
We expressed the human and P. vivax DHFRs in E. coli, and purified the proteins using methotrexate affinity columns. We then performed in vitro enzyme assays and observed that the P. vivax DHFR is >20× more sensitive to triclosan (IC 50 of 775 ± 384 nM) than the human enzyme (IC 50 of 17.2 ± 4.9 μM) (Fig. 2).
To further validate DHFR as a target of triclosan in Plasmodium, we transfected blood-stage P. falciparum NF54 with a plasmid expressing the human DHFR enzyme (HsDHFR). If the blood-stage target of triclosan is indeed the Plasmodium DHFR, expression of HsDHFR (which our yeast and in vitro assays showed to be resistant to triclosan) should protect the parasite against the compound.
We found that our transfected Plasmodium strain was more tolerant to exposure to triclosan than the wild type ( Fig. 3), consistent with DHFR being the compound's target when the parasite is within the erythrocyte. The difference in the rate of progression of the parasitemia in wild-type and transfected strains was compared at a range of triclosan concentrations and found to be statistically significant (SEM). It should be noted that, in contrast to the in vitro enzyme inhibition kinetics, those for growth inhibition are complex. This is likely to be due to the fact that our parasite lines have been transfected with episomal constructs bearing the coding sequence for human DHFR. The resulting range of plasmid copy numbers would result in differences in the levels of human DHFR between individual Plasmodium cells in the population 29 . The consequence is that a considerable proportion of cells do not express human DHFR at levels that are sufficiently high to confer drug resistance. Finally, triclosan was docked into the X-ray structures of human and Plasmodium DHFRs (Fig. 4). Although triclosan displayed slightly altered binding poses in the different parasitic DHFRs, the inhibitor showed the same interactions with all four DHFRs, indicating comparable affinity (binding free energies in the range −7.24 and −7.56 kcal/mol). In contrast, triclosan was not interacting with the HsDHFR through hydrogen bonding, resulting in reduced affinity (∆G = −6.57 kcal/mol) and supporting the contention that triclosan discriminates between the Plasmodium and human enzymes.
DHFR inhibitors are routinely used as prophylactic drugs and are given to over a million children during the malaria season. However, DHFR inhibitors are no longer a standard treatment for the disease because of the evolution of drug-resistant variants of Plasmodium. Extensive efforts to discover a DHFR-targeted antimalarial that is effective against pyrimethamine-resistant strains have yet to produce a clinically approved entity, thus a novel Triclosan-derived compound with activity against drug-resistant forms of DHFR is of high potential value.
The presence of triclosan in consumer products in concentrations 2500-fold above the present IC 50 30 , suggests that it can be administered in quantity and short-term toxicity effects can be disregarded, notwithstanding possible long-term hazards 31 . This tolerance is important, as delivering effective concentrations in vivo may be challenging since data on the effect of administering triclosan to mice infected with P.berghei are contradictory 11,18 , with sub-cutaneous delivery being more effective than oral administration 18 . It may be that further engineering of triclosan by synthetic chemistry is necessary to improve delivery and half-life, but with regard to affinity triclosan is a viable non-toxic lead with a mechanism based on hitherto undiscovered selectivity. A number of triclosan analogs have already been synthesized 16,21 , demonstrating the ample opportunity for its improvement for use as an antimalarial.
In this work, we have provided four different lines of evidence (screens using recombinant yeast strains, enzyme assays, growth assays with blood-stage Plasmodium, and in silico simulations of drug-enzyme interactions) demonstrating that triclosan specifically targets the DHFR enzyme of both P. vivax and P. falciparum, and that it is capable of selectively inhibiting both the wild-type and pyrimethamine-resistant enzymes compared to human DHFR. Our conclusion that triclosan targets Plasmodium DHFR also explains much of the inconsistency in the literature regarding the efficacy of triclosan 22 : HsDHFR is commonly used as a Plasmodium selective marker as it provides pyrimethamine resistance, and controls for this confounding factor are not always performed. Given the earlier evidence that triclosan inhibits ENR, it may be that triclosan is an example of "polypharmacology" 32 , poised for multitarget therapies. The totality of data on this compound suggests that it is capable of inhibiting blood-stage Plasmodium, via its action against DHFR, and the liver stage of the parasite, via the inhibition of a key enzyme in fatty-acid synthesis 20 . Interestingly, polyphenol compounds such as epigallocathechin gallate (EGCG), also inhibit fatty acid synthesis and have antiplasmodial activity 33 , and have demonstrated antifolate activity 34 . Given the recent success in developing drug candidates with multi-stage activity 35 , it is realistic to envision the development of a new dual-action drug, based on triclosan, that is effective against both the blood and liver stages of wild-type and pyrimethamine-resistant Plasmodium species. The fact that triclosan has two distinct enzyme targets in different domains of metabolism may militate against the development of resistance. All of this engenders the hope that our discovery of a second enzyme target for triclosan may prove to be the first step in the development of a novel class of antimalarials.

Methods
Yeast strains and fluorescent plasmids. All yeast strains and marker plasmids used in this work are described in Bilsland et al. 24,25 . Briefly: plasmids, bearing genes for the expression of fluorescent proteins, were constructed by replacing the coding region of yEmRFP from yEpGAP-Cherry with Venus or Sapphire, and replacing the URA3 marker with LEU2. These three fluorescent proteins have very distinct excitation and emission wavelengths: mCherry (λ ex 580 nm; λ em 612 nm), Sapphire (λ ex 405 nm; λ em 510 nm) and Venus (λ ex 500 nm; λ em 540 nm).
A strain expressing the drug-resistant P. vivax DHFR (yPvRdhfr) was constructed by making the following site-directed mutations in the coding sequence for the P. vivax enzyme: S58R, S117N and I173L. This plasmid was transformed into dfr1Δ/DFR1 a yeast strain with the BY4743 genetic background. The strain was sporulated and MATα haploids were selected and used in drug screens. The drug-resistant yPfRdhfr strain, which is a triple mutant for residues N51I, C59R, and S108N, was generated in a similar manner.
Library-screening assays and hit confirmation using the Robot Scientist Eve. Automated screens were performed as described in Williams et al. 28 . Starter cultures of individual strains, labeled with different fluorescent proteins, were grown to stationary phase in YNB-glucose (0.67% yeast nitrogen base without amino acids, 2% ammonium sulphate and 2% glucose) with the appropriate auxotrophic supplements. An aliquot (1 mL) of each pre-culture was inoculated into 100 mL of fresh medium. Pools of three strains were incubated for 4 h at 30 °C, with shaking, to ensure exponential growth. Doxycycline (Sigma-Aldrich) (5 μg/mL) was then added to  24 . The culture was attached to a Thermo Combi multidrop within the Robot Scientist Eve work cell 24 . The culture was stirred continuously and maintained at 23 °C during assay plate set-up.
High-throughput drug screens were performed by the Robot Scientist Eve using the mixed cultures described above and the ~1,600 FDA-and foreign-approved drugs from the Johns Hopkins University Clinical Compound Library. Strains were grown in competition in the presence of a library compound, and the relative growth rates used to estimate the activity of the drug against the parasite target. For hit confirmation assays, the Robot Scientist Eve prepared plates with eight replicates of eight different compounds, at six different concentrations (0, 1, 2.5, 5, 10, 20 μM), and 64 negative control wells. . We attempted to express and purify P. falciparum DHFR and pyrimethamine-resistant P.vivax DHFR under multiple conditions, but were unable to recover stable proteins for enzyme assays. Enzyme Assays. DHFR catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), coupled to the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). This reaction was followed in vitro by absorbance at λ abs = 340 nm by spectrophotometry (SpectraMax 190, Molecular Devices) in UV-transparent 96-well plates (Costar, Corning) at 21 °C. NADPH and DHF absorb at 340 nm with a combined extinction coefficient of ε 340nM = 12,300 M −1 cm −1 , which takes into consideration the oxidation of NADPH and the reduction of DHF.

Recombinant protein expression and purification. Synthetic genes encoding versions of the
H. sapiens DHFR (HsDHFR) and P. vivax DHFR (PvDHFR) were thawed and stored at 4 °C for use in the enzyme assay. DHF and NADPH stocks were made to 1 mM in assay buffer ( Initial rates of reaction at various concentrations of triclosan were monitored by spectrophotometry (SpectraMax 190, Molecular Devices) and data were analyzed using Excel (Microsoft). Initial rates were determined by following the linear absorbance decrease and used to derive IC 50 values. Reaction turnover was assayed in UV-transparent 96-well plates (Costar, Corning) with a total volume of 100 μL. DHF and NADPH stocks were made up to 1 mM in assay buffer (0.1 M Tris, pH 7.4) and stored at −20 °C until use.
To deal with variations in enzyme activity in different preparations, kinetic data were normalized as relative activities and fitted to the following equation: Culture of wild-type and transgenic P. falciparum. P. falciparum strain NF54 was transfected with a pDC10 derivate 37 and cultivated as described previously 38 . Wild-type and transgenic parasites were cultivated in the presence of increasing concentrations of triclosan (0-2.6 µM) or pyrimethamine (0-35 nM). Drug treatment of cultures was started at 0.5-1% parasitemia (trophozoite stage) and run for 48 h. Parasitemias were quantified using the SYBR method 39  All the enzymatic structures were also checked for missing atoms, bonds, and contacts. The structure of triclosan was first constructed using Maestro's building tool and energetically minimized for processing using LigPrep, which allowed the acquisition of a valid low-energy 3D structure for this inhibitor with correct protonation state in the pH range of 7 ± 2. Molecular Docking was initiated by generating a Grid file using Receptor Grid Generation tool of Glide. Grid files containing receptor and binding site information required for molecular docking were prepared using the default options of the Receptor Grid Generation tool with the grid box being centered at the coordinates average of the co-crystallized ligand for each crystallographic structure. To test the reliability of the docking procedure, all the co-crystallized ligands were first docked into the corresponding crystallographic structure using the Extra Precision (XP) Glide algorithm. Subsequently, triclosan was also successfully docked in each active site and the lowest energy conformation provided by Glide Score was chosen.
Ethics statement. Human blood and plasma were obtained from the local blood bank and ethical clearance for using this blood for this research was granted by the Ethics Committee of the Institute of Biomedical Sciences at the University of São Paulo (No. 842/2016).