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Plasmodium Geranylgeranyl Diphosphate Synthase (GGPPS) and Exhibit Potent Antimalarial Activity

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Plasmodium Geranylgeranyl Diphosphate Synthase (GGPPS) and Exhibit Potent Antimalarial Activity Lipophilic analogs of zoledronate and risedronate inhibit Plasmodium geranylgeranyl diphosphate synthase (GGPPS) and exhibit potent antimalarial activity Joo Hwan Noa,1,2, Fernando de Macedo Dossinb,1, Yonghui Zhangc,1, Yi-Liang Liua, Wei Zhua, Xinxin Fengc, Jinyoung Anny Yooc, Eunhae Leed, Ke Wangc, Raymond Huie, Lucio H. Freitas-Juniorb,3, and Eric Oldfielda,c,3 aCenter for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; bCenter for Neglected Diseases Drug Discovery, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do 463-400, South Korea; cDepartment of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801; dDepartment of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801; and eStructural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L7 Edited by J. Andrew McCammon, University of California at San Diego, La Jolla, CA, and approved January 9, 2012 (received for review November 5, 2011) We report the results of an in vitro screening assay targeting nate had an IC50 of approximately 790 nM in the enzyme assay but the intraerythrocytic form of the malaria parasite Plasmodium an IC50 ¼ 120 μM in cells, and similar results were found with falciparum using a library of 560 prenyl-synthase inhibitors. Based several other systems with the average R2 value being 0.30 (17) for on “growth-rescue” and enzyme-inhibition experiments, geranyl- 10 diverse assays. This observation strongly suggested that zoledro- geranyl diphosphate synthase (GGPPS) is shown to be a major nate had poor permeability in the P. falciparum/red-cell assay due target for the most potent leads, BPH-703 and BPH-811, lipophilic to its highly polar nature (log P ¼ −2.9). However, when incorpor- analogs of the bone-resorption drugs zoledronate and risedronate. ating mathematical descriptors (such as logP) to begin to account We determined the crystal structures of these inhibitors bound to a for permeability, we found that good (R2 ¼ 0.7) correlations Plasmodium GGPPS finding that their head groups bind to the between cell and enzyme activity could be obtained (17). These ½ 2þ Mg 3 cluster in the active site in a similar manner to that found results indicated, at least for these types of compounds, that simply with their more hydrophilic parents, whereas their hydrophobic screening for good enzyme inhibitors might not be particularly tails occupy a long-hydrophobic tunnel spanning both molecules informative because many such inhibitors would be unable to get in the dimer. The results of isothermal-titration-calorimetric experi- into cells, and that cell assays would be much more desirable. ments show that both lipophilic bisphosphonates bind to GGPPS A second set of problems with the bisphosphonate class of −1 −1 with, on average, a ΔGof−9 kcal mol , only 0.5 kcal mol worse molecules is that they bind very tightly to bone mineral (18, 19), than the parent bisphosphonates, consistent with the observation resulting in their rapid removal from the bloodstream. This is, of that conversion to the lipophilic species has only a minor effect on course, a desirable feature of a bone drug but not of an antiin- enzyme activity. However, only the lipophilic species are active in fective-drug lead; and in recent work, we have been developing a cells. We also tested both compounds in mice, finding major de- class of compounds called “lipophilic bisphosphonates” (20, 21) creases in parasitemia and 100% survival. These results are of broad in which the 1-OH group on the bisphosphonate backbone, part general interest because they indicate that it may be possible to of the tridentate “bone-hook,” is removed and in which a variety overcome barriers to cell penetration of existing bisphosphonate of hydrophobic side chains are attached to the molecules to drugs in this and other systems by simple covalent modification increase logP values, typically from approximately −2 or −3 to to form lipophilic analogs that retain their enzyme-inhibition activity approximately 2 or 3. These lipophilic bisphosphonates have and are also effective in vitro and in vivo. far more potent activity both in vitro and in vivo than do conven- tional bisphosphonates in tumor cell-growth inhibition and γδ alaria, caused by Plasmodium spp., causes approximately T-cell-activation assays (20, 21) as well as against malaria para- M1 million deaths each year (1), and there are ever-present sites (14). In this work, we elected to screen our in-house library problems due to drug resistance (2). There is, therefore, a need for of 560 prenyl-synthase inhibitors, developed over the past decade new drugs and drug leads. In earlier work, we and others found as anticancer drug leads and as antibacterials (20–24), for their that the bisphosphonate class of drugs (3) used to treat bone- activity in P. falciparum growth inhibition inside red cells. We dis- related diseases—osteoporosis, Paget disease, and hypercalcemia covered two potent leads, BPH-703 and BPH-811 (Scheme 1), due to malignancy—also inhibited the growth of a range of para- lipophilic analogs of the commercial drugs zoledronate and rise- sitic protozoa, including Trypanosoma cruzi (4, 5), Trypanosoma dronate, and determined their crystal structures bound to P. vivax brucei (4, 6), Leishmania spp. (4, 7, 8), Toxoplasma gondii (4, 9), Cryptosporidium parvum (10, 11), Entamoeba histolytica (4, 12, 13), and Plasmodium spp. (4, 13–15). In the case of Plasmodium spp., Author contributions: J.H.N., F.d.M.D., Y.Z., Y.-L.L., L.H.F.-J., and E.O. designed research; the most potent inhibitors were not, however, the nitrogen- J.H.N., F.d.M.D., Y.Z., Y.-L.L., W.Z., X.F., J.A.Y., E.L., K.W., and E.O. performed research; containing bisphosphonates such as zoledronate or risedronate R.H. analyzed data; and L.H.F.-J. and E.O. wrote the paper. (Scheme 1) used to treat bone diseases, but more lipophilic n-alkyl The authors declare no conflict of interest. bisphosphonates (13). Their target in Plasmodium falciparum was *This Direct Submission article had a prearranged editor. not determined. However, more recently, a Plasmodium vivax Data deposition: The atomic coordinates and structure factors have been deposited in the geranylgeranyl diphosphate synthase (PvGGPPS) has been cloned, Research Collaboratory for Structural Bioinformatics Protein Data Bank, www.pdb.org for geranylgeranyl diphosphate synthase complexed with BPH-703 (PDB ID code 3RBM) and expressed, purified, and crystallized, and its three-dimensional for geranylgeranyl diphosphate synthase complexed with BPH-811 (PDB ID code 3RYW). structure determined (16). The enzyme is inhibited by bisphospho- 1J.H.N., F.d.M.D., and Y.Z. contributed equally to this work. nates (16), so it seemed possible that it might be a target for the 2Present address: Center for Neglected Diseases Drug Discovery, Institut Pasteur Korea, inhibitors discovered earlier. To investigate this possibility, we re- Seongnam-si, Gyeonggi-do 463-400, South Korea. cently determined the IC50 values for 25 bisphosphonates against 3To whom correspondence may be addressed. E-mail: [email protected] or eo@ PvGGPPS and compared the results for enzyme inhibition with chad.scs.uiuc.edu. ð¼ − Þ P. falciparum growth-inhibition pIC50 log10 IC50 values (17). This article contains supporting information online at www.pnas.org/lookup/suppl/ 2 The correlation was very poor: R ¼ 0.06. For example, zoledro- doi:10.1073/pnas.1118215109/-/DCSupplemental. 4058–4063 ∣ PNAS ∣ March 13, 2012 ∣ vol. 109 ∣ no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1118215109 Downloaded by guest on September 30, 2021 The screening library consisted primarily of bisphosphonates that might inhibit Plasmodium GGPPS. In addition, we included phosphonosulfonates and related systems known to inhibit head-to-head prenyl synthases (22–24) such as dehydrosqualene synthase and squalene synthase (31), which could also inhibit Plasmodium phytoene synthase, and cationic species (such as Ro 48-8071 and quinuclidines), some of which are known to inhibit Plasmodium cell growth (28, 32). The structures of all 560 com- pounds are shown in Fig. S1. Scheme 1. Chemical structures of selected bisphosphonates. We first screened all compounds at 10 μM to find possible hits, ¼ 20 GGPPS, in addition to testing them in vivo, finding potent activity. using artemisinin (IC50 nM) as a positive control. There ≥70% μ This finding opens up the possibility that other commercial bispho- were 78 hits (defined as giving inhibition at 10 M) as shown in Fig. 2A, and the Z0 factor for the control wells was 0.72 sphonates, inactive themselves against Plasmodium spp. as well Z0 as other organisms, may be converted to species that are highly as shown in Fig. 2B. The factor is defined (33) as 0 þ − þ − active both in vitro and in vivo, via simple chemical modification. Z ¼ 1–3ðσc þ σc Þ∕jμc − μc j Results and Discussion þ − where σc ∕σc are the standard deviations of the positive (arte- þ High-Throughput Screening (HTS). In Plasmodium spp., the initial misinin)/negative (phosphate-buffered saline) controls and μc ∕ − 0 steps in isoprenoid biosynthesis are carried out by the so-called μc are the corresponding mean values (33), with Z ¼ 0.72 methylerythritol phosphate (MEP) pathway, which produces indicating an excellent assay. Full-assay details are given in SI isopentenyl diphosphate (IPP) and dimethylallyl diphosphate Methods. We then performed dose-response assays for these hits (DMAPP) from pyruvate and glyceraldehyde-3-phosphate (25) (structures shown in Fig. S2) to determine their IC50 values; dose- (Fig. 1). DMAPP then condenses sequentially with three mole- response curves are shown in Fig. S3. Todetermine which of these cules of IPP to form geranyl diphosphate (GPP), farnesyl dipho- compounds might be worth further investigation, we next deter- sphate (FPP), and geranylgeranyl diphosphate (GGPP), which is mined their IC50 values for inhibiting the growth of three human then used to prenylate proteins (26).
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