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Target Elucidation by Co-crystal Structures of NADHUbiquinone Oxidoreductase of Plasmodium Falciparum (PfNDH2) with Small Molecule to Eliminate Drug-Resistant Malaria Yiqing Yang, You Yu, Xiaolu Li, Jing Li, Yue Wu, Jie Yu, Jingpeng Ge, Zhenghui Huang, Lubin Jiang, Yu Rao, and Maojun Yang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01733 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Target Elucidation by Co-crystal Structures of NADH-Ubiquinone Oxidoreductase of Plasmodium Falciparum (PfNDH2) with Small Molecule to Eliminate Drug-Resistant Malaria Yiqing Yang1,2,†, You Yu2,†, Xiaolu Li4,†, Jing Li2, Yue Wu1, Jie Yu2, Jingpeng Ge2, Zhenghui Huang3, Lubin Jiang3,*, Yu Rao1,*, Maojun Yang2,* 1

MOE Key Laboratory of Protein Sciences, School of Pharmaceutical Sciences, Tsinghua

University, Beijing 100084, China. 2

MOE Key Laboratory of Protein Sciences, Tsinghua-Peking Center for Life Sciences, School of

Life Sciences, Tsinghua University, Beijing 100084, China. 3

Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology,

Chinese Academy of Sciences, Shanghai 200031, China. 4

Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical

Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China.

ACS Paragon Plus Environment


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ABSTRACT: Drug-resistant malarial strains have been continuously emerging recently, which posts a great challenge for the global health. Therefore, new anti-malarial drugs with novel targeting mechanisms are urgently needed for fighting drug-resistant malaria. NADH-ubiquinone oxidoreductase of Plasmodium falciparum (PfNDH2) represents a viable target for anti-malarial drug development. However, the absence of structural information of PfNDH2 limited rational drug design and further development. Herein, we report high resolution crystal structures of the PfNDH2 protein for the first time in Apo-, NADH- and RYL-552 (a new inhibitor)-bound states. The PfNDH2 inhibitor exhibits excellent potency against both drug-resistant strains in vitro and parasite-infected mice in vivo via a potential allosteric mechanism. Furthermore, it was found that the inhibitor can be used in combination with dihydroartemisinin (DHA) synergistically. These findings are not only important for malarial PfNDH2 protein-based drug development, but could also have broad implications for other NDH2-containing pathogenic microorganisms such as Mycobacterium tuberculosis.

INTRODUCTION Nobel Prize in Physiology or Medicine of 2015 was awarded to Youyou Tu for her contributions in discovering artemisinin, which had been broadly used to treat malaria. Today malaria still remains a major challenge to global health, which causes more than 212 million new cases and over 429,000 deaths annually1. Although anti-malarial drugs have successfully mitigated the epidemics in the past few decades, drug resistance has been emerging rapidly2,3. In fact, clinical evidence has reported the drug resistance for all existing classes of anti-malarial medicines including chloroquine4 and artemisinin5. For instance, chloroquine-resistant P. falciparum has spread to most malaria-manifested areas up to date. Even for the most effective anti-malarial


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drug artemisinin, its resistance was first reported in 2008, which has become a major issue in parts of Southeast Asia3,6-8. To tackle these important problems, there is an urgent requirement for new anti-malarial drugs. The electron transport chain (ETC), which comprises oxidative phosphorylation Complex-I to IV located at the inner membrane of mitochondria, serves as the core for oxidation to produce ATP. Atovaquone, which has been used as competitive inhibitor for more than 20 years in clinics, targets the cytochrome bc1 complex (Complex-III) of the ETC to prevent the proton pumping and causes a loss of mitochondrial membrane potential leading to organelle dysfunction9,10. However, the utilization of atovaquone was limited severely after the emergence of its drug resistance. The bc1 complex activity requires the ubiquinol, which is produced by NADH dehydrogenase (NDH, and also named Complex-I)11 through the transfer of two electrons from NADH to ubiquinone. This ubiquinol-producing reaction is the rate-limiting step in respiration and is central to energy metabolism. Single-subunit type-II NADH dehydrogenase (NDH2), without the proton pumping activity, acts as an alternative to the multi-subunit respiratory Complex-I12,13. NDH2 exists in many pathogenic microorganisms, such as P. falciparum, Toxoplasma gondi and Mycobacterium tuberculosis14-16, and is essential for the viability and long-term survival of M. tuberculosis17 and P. falciparum14,18,19. The inhibition of their NDH2 could efficiently disrupt their respiration chain, so it could represent a viable target for combatting P. falciparum, the main malaria causing parasite20,21. Although previous literatures reported compounds like 1 (CK-2-68) (Fig. 1A) that had been shown to target PfNDH2 with a high potency18,19, the absence of high-resolution structure of PfNDH2 and inhibitor-bound PfNDH2 complex presents difficulties for molecular mechanism elucidation and structure-based rational drug design. Given the significance of structural



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information of NDH2 protein for both fundamental molecular mechanism understanding and anti-malarial drug discovery, herein, we reported the high resolution crystal structures of PfNDH2 in Apo-, NADH- and 2 (RYL-552)-bound states at 2.16, 2.70 and 2.05 Å, respectively (Table S1). These structures revealed a possible allosteric mechanism of compound 2 with two binding sites in PfNDH2. Allosteric ligands usually offer distinct advantages over competitive inhibitors22,23 and have been explored increasingly in various families of proteins in recent years, which paves a way to develop new pharmaceutical agents and increases our understanding of the fundamental biological processes as well24-27. The potential of PfNDH2 inhibitors were evaluated further through a series of in vitro and in vivo studies, which reflected the promising perspective for anti-malarial drug development based on PfNDH2 and its inhibitors. RESULTS AND DISCUSSION Overall structure of PfNDH2. The structures of Apo-, NADH- and the inhibitor-bound PfNDH2 complexes are solved for the first time. It was found that PfNDH2 forms a stable homodimer in solution and crystal structures (Fig. 1B and Fig. S1) with each monomer forms a globular structure comprising four domains (Fig. S2A). Residues of the C-terminal domain (CTD) that support self-dimerization in situ and membrane attachment28,29 are well conserved in different Plasmodium species (Fig. 1C-D). Between the two canonical Rossmann fold domains (domain A and domain B), which bind the substrates FAD and NADH (Fig. S2B), respectively, PfNDH2 contains a domain C in the middle of the sequence (Fig. 1B and Fig. S2A). Dali searches30 with domain C only returned entries with the highest Z-score of 5.2, suggesting that no known structure was identified to share significant homology with this domain. Evaluation of the most similar structure from this search, the EF-hand domain in calcium binding protein 40


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(CBP40) (PDB ID: 1IJ6) showed that none of the residues responsible for the Ca2+-binding occur in the extended PfNDH2 domain (Fig. S2C) and the function of domain C is undisclosed so far. The binding mode of PfNDH2 with compound 2. To understand the mechanism underlying the inhibitory function, we tried to co-crystallize PfNDH2 with its inhibitors. The previously reported compound 1 displays poor water solubility because of the strong intermolecular π-π stacking interactions18, which always results in the precipitation of compound 1 from the cocrystallization solution. To solve this challenge, new inhibitors with both excellent potency and improved aqueous solubility should be obtained as tool compounds. The quinolin-4-one compounds could be synthesized under three different reaction conditions with related starting materials and intermediates which were reported previously (Scheme 1, d, j and i)18,19. Some derivatives of 1-(2-hydroxyphenyl)ethanone such as compound 42 could react with aromatic aldehydes via aldol condensation to generate intermediates of α, β-unsaturated ketones that were used further as starting materials to prepare chromen-4-one by iodine catalyzed intramolecular cyclization (Scheme 1, g and h). To improve the aqueous solubility and meet the needs for our co-crystallization study, we modified the molecules with hydrophilic functional groups including –F, -C(=O)OH, -C(=O)NHNH2, and -NHC(=O)CF3, etc. (Scheme 1, Table S3), which led to the identification of some highly effective small molecules including compound 2 and 3 (RYL-552S) (Fig. 1A). It was found that only compound 2 could be co-crystallized with PfNDH2 successfully after many attempts, which might be due to a better combination of the aqueous solubility (CLogP=5.8) and inhibitory potency (IC50=3.73 nM) of compound 2 (Fig. 1A). The results may indicate the 5-F in compound 2 can contribute more than the 7-Cl in compound 1 in terms of binding affinity. Unexpectedly, compound 2 inserts into two different deep pockets in each PfNDH2 molecule (Fig. 2A). Two compound 2 molecules insert into the pocket between



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PfNDH2 homo-dimer interface (Fig. 2B) which exists in situ28,29. The 4-oxo-5-fluoro element of the quinolone scaffold grips the backbone carbonyl of G87 and side chain amino group of K523 with the help of two bridge water molecules. And the carbonyl oxygen atom also forms another hydrogen bond with the side chain of N92. The bisaryl part linked by methylene is clamped by the hydrophobic surface produced from V91, I170, L174 and I532. The terminal trifluoromethoxy forms two hydrogen bonds with Y74 and K533. More interestingly, instead of the predicted Q binding sites (catalytic domains), the other two molecules are deeply buried in the pocket formed by the helix-17 and 18 at the C-terminal separately (Fig. 2C). It is found that the 4-oxo-5-fluoro element of quinolone has two hydrogen bonds with the side chain of K501. The nitrogen atom from quinolone ring forms a hydrogen bond network with one water molecule, which further bridges with terminal carboxylic acid group of E218 and guanidine group of R529. And two different π-π stacking interactions around the quinolone ring (one likes T shape with edge to face style between 6-H of quinolone and W500; the other is face to face between quinolone and Y475) contributes more binding affinity. We designed single and double mutations of the residues that could interfere with the binding of compound 2 in one or the other binding pocket (N92R, D171R, Y475R, S497R, N92R/Y475R or N92R/S497R; Fig. 2D and Fig S3). Each of these engineered single mutations significantly reduced the ability of compound 2 to interfere with PfNDH2 activity, as reflected in the increased IC50 value by 3- to 10-fold. Despite the strong influences of the mutations inhibition, these single mutations in themselves showed only small effects on the native catalytic ability of the enzyme (Fig. 2E). Although the N92R/S497R double mutant lost about 50% of the enzyme activity, the compound 2 greatly reduce the inhibition activity by 34-fold, while the N92R/Y475R mutant protein lost 95% of the


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enzyme activity. These findings are consistent with the single mutant results which indicates the Y475R has a greater effect than S497R (IC50, 31 vs 17 nM). Further ligand-based SAR study verified the cocrystal structure results. The ligand-based SAR (structure-activity relationship) studies are consistent well with the co-crystal structure results of compound 2 (Fig. 3A-B and Table S3). For example: 1) 5-F of compound 2 can form a hydrogen bond with K501 (Fig. 3C). In contrast, if the F atom is installed at other sites including 6, 7 or 8 position, it can not help the carbonyl group of quinolone accommodating the H2O bridge (Fig. 3A). In addition, the 6-F substitution may decrease the π-π stacking interaction with W500 (Fig. 3A) and eject the compound out of the pocket. These structural observations are also consistent well with our experimental results that the overall inhibitory order for R1 is 5-F>7F>8-F>6-F (Fig. 3B); 2) the N-H of the quinolone is essential for the inhibitory activity, as it serves as a hydrogen bond donor (Fig. 3A). However, the flavone compounds would lost this critical interaction, which results in less effective inhibition (Fig. 3B, -NH- is better than -O- for X); 3) The R2 group is oriented towards the hydrophobic clamp composed of V91, I170, L174 and I532 (Fig. 2B). As a result, the polar and hydrophilic functional groups like -C(=O)OMe, C(=O)OH, -C(=O)NHNH2 (Fig. 3B) are not compatible for binding interactions. Because the linker Z is located at the gap of the same hydrophobic pocket as well, it is not surprising that methylene is better than -O- (Fig. 3B). 4) 4-OCF3 or 4-SCF3 is the best substituent group for R3 (Fig. 3B), which can be explained by the hydrogen bonding formations between them and residues Y74 and K533 via the fluoride atoms (Fig. 3A). The proposed allosteric inhibitory mechanism by compound 2. In the enzyme kinetic studies of PfNDH2 with different compound 2 concentrations (Fig. S4), the effective Vmax decreases with inhibition but the Km remains constant, which indicates that compound 2 is a non-



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competitive inhibitor. Then we compared the structure of compound 2 bound PfNDH2 with the Apo- as well as NADH-bound forms and searched for conformational changes that could explain the inhibitory effect of compound 2 on enzyme function (Fig. 4A-C). In these structural comparisons, the Rossmann fold involved in FAD-binding (amino acids 41-152) superposed with root mean square deviation values (RMSDs) of less than 0.18 Å. In the NADH-bound form of PfNDH2, the main chain extending from helix-7 was shifted to a more closed configuration, toward the NADH molecule, than in the Apo-form (Fig. 4A); However, in the compound 2 bound form, the main chain of the NADH binding region was moved away by about 1.1 Å from the position of the NADH molecule in the NADH-bound form (Fig. 4B). At the dimer interface, binding of the compound 2 pushed the main chain of the each monomer away from the interface by up to 1.0 Å (Fig. 4C). Our observations that 1) most mutations at the compound 2 binding sites impaired compound 2 inhibition but produced only a small effect on the catalytic ability of PfNDH2, 2) compound 2 behaved as a non-competitive inhibitor and 3) binding of compound 2 to distant regions of the protein produced structural shifts at the PfNDH2 catalytic site, together suggested a potential allosteric effect of compound 2 on NADH binding and PfNDH2 activity. To test this hypothesis, we performed Surface Plasmon Resonance (SPR) assays for the NADH binding affinity of PfNDH2 in the presence or absence of compound 2. Results from these assays and calculated dissociation constants (KDs) confirmed that compound 2 decreased the binding affinity of the NADH for PfNDH2 by about 7 fold in these experiments (Fig. 4D and Fig. S5). We also found that compound 2 was hard to come off from PfNDH2 in the SPR studies (Fig. S6) which could explain the high inhibitory effect of compound 2 to PfNDH2 in terms of the binding affinity. In the PfNDH2 enzyme, FAD binds deeply within the first of two Rossmann domains of NDH2 monomer, where the isoalloxazine ring of this cofactor lies in parallel closely from the


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nicotinamide ring of NADH in the second Rossmann domain. This close proximity is thought to support the transfer of electrons from NADH to FAD, forming FADH2 which interacts and converts ubiquinone to ubiquinol. The effect of compound 2’s binding is to transmit a shift of helix 7 in the opposite direction, forcing NADH to dissociate more readily from PfNDH2 (Fig. 4E). We suggest that these and possibly other allosteric effects of compound 2 impair electron transfer from NADH to FAD, compromising subsequent electron transfer through FADH2ubiquinone complex and blocking the reduction of ubiquinone to ubiquinol, which might affect the dynamic of the homo-dimerization of the proteins finally. Compound 2 and 3 kill drug-resistant strains of P. falciparum. To further evaluate the antimalarial potential of PfNDH2 inhibitors, we tested compound 2 and 3 against ten P. falciparum strains that are from different malaria-endemic areas worldwide and carry various phenotypes of resistance to major antimalarial drugs in clinical use31. Dose-response data from these experiments yielded characteristic sigmoid response curves and a narrow range of EC50 values (2 to 12 nM; Fig. 5A and Fig. S7) which, in this low nM range, are desirable for drug discovery and are characteristic of first-line antimalarials such as artemisinin derivatives and the spiroindolones32. There was no correlation of compound 2 or 3 EC50 with resistance to any particular anti-malarial drug, reinforcing the potential of these two compounds. We also tested whether parasites were completely killed or some could persist through an exposure to high concentration levels of drug. After 72 hr exposure to 10 × EC50 concentrations of compound 2 or 3, no recrudescence of 3D7, Dd2 or 803 parasites was observed in cultures maintained for 28 days post treatment (Fig. 5B). Transcripts of the PfNDH2 gene were detected at highest level in late asexual stages (Fig. 5C) and suggested experiments to determine the stage-specific inhibitory activity of compound 2 or 3



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against P. falciparum. We first examined morphological changes of drug-treated parasites by microscopy. Treatments initiated in the ring stage did not cause obvious alterations of parasite morphology until 36 h post invasion (Fig. 5D). Further experiments was conducted, in which compound 2 or 3 was applied to synchronized early ring stages and then removed from culture at 2 h intervals after the parasites entered mature stages of development (34 h trophozoite and older). It was found that these compounds reduced parasitemia in the subsequent cycle by ≥ 50% only if drug pressure was maintained for more than 40 h post invasion (Fig. 5E). Much mature stage parasites may therefore be particularly susceptible to the effects of compound 2 and 3 during the intraerythrocytic life cycle of P. falciparum. The anti-malarial potential of PfNDH2 inhibitors. MTT cell proliferation assays showed no reduced viability of HeLa cells at compound 2 and 3 concentrations of up to 2 µM (Fig. 6A), suggesting a good therapeutic window for these compounds. To evaluate the compound 2 and 3 against malaria infection in vivo, we took advantage of the high sequence conservation of NDH2 proteins among different Plasmodium species (Fig. S8) and observed the outcomes after treatment of P. yoelii infection in mice (Fig. 6B-E). Twenty-four hours after inoculation of P. yoelii parasites (1×106), we treated the groups of three mice with single intraperitoneal doses of 10 mg/kg, 30 mg/kg or 90 mg/kg of compound 2 or 3. Pyrimethamine and dihydroartemisinin served as two anti-malarial drugs for control experiments. Like pyrimethamine and dihydroartemisinin, compound 2 eliminated P. yoelii infection at all tested dosages, whereas compound 3 failed to cure infected mice at the dose of 10 mg/kg (Fig. 6B). Mice given no treatment (DMSO only) did not survive after 9 days of infection. To test whether the new PfNDH2 inhibitors were also able to cure mice with high parasitemia, we administered four daily subcutaneous doses of compound 2 (10 mg/kg/d) or 3 (30 mg/kg/d)


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to mice with ~30% infected erythrocytes four days after inoculation. These treatments achieved dramatic reductions of parasitemia by day 10 (Fig. 6C, D). Mice treated with 10 mg/kg/d compound 2 were completely clear of parasites by day 17 and showed 100% survival at day 40 (Fig. 6E). However, the compound 3 treatments of 30 mg/kg/d resulted in only 67% survival of the mice (Fig. 6E). These in vivo data from mice, in contrast to the higher activity of compound 3 than of compound 2 against P. falciparum in vitro, suggest reduced bioavailability or more rapid elimination of compound 3 (or active metabolites) than of compound 2. We also tested potential drug interactions of compound 2 with a number of major anti-malarial drugs against P. falciparum in in vitro dose-response assays33,34. Isobologram analysis showed strong synergistic interaction of compound 2 with dihydroartemisinin (ΣFICs=0.56±0.07) (Fig. 6F). Conversely, antagonism occurred between compound 2 and quinine (ΣFICs=2.33±0.48), and a potential antagonistic/additive interaction was evident with chloroquine (ΣFICs=1.52±0.19) (Fig. 6F). SUMMARY AND CONCLUSIONS In the present study, we solved the crystal structures of PfNDH2 and complexes with the small molecule that could inhibit the enzyme activity and parasites growth both in vitro and in vivo. In the most reported cases, the allosteric ligands exert their effects by inducing conformational changes of a target protein that are then transduced from the allosteric binding pocket to the orthosteric domain and/or directly to effect protein catalytic sites. In our case, the compound 2 seemed to bind to the two allosteric binding sites, which could influence the NADH binding (Fig. 4). Interestingly, both proposed allosteric binding sites are associated with the CTD of PfNDH2 that enabled the NDH2 to form a homo-dimer in situ and attach on the membrane28,29. Binding of compounds to this domain can result in greater potential selectivity that could provide maximal benefit with minimal side effects. The new compound 2 displays a strong synergistic interaction



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with dihydroartemisinin in killing parasites (Fig. 6F), which is consistent with the previous studies that the artemisinin might act on mitochondrial ETC to inhibit parasite growth35. It has been demonstrated that in P. falciparum the complete F-type ATP synthase appears to be imperfect and it could not synthesize ATP by itself36,37, in consistence with a more recent report that indicated the glycolysis as the main ATP generating process of the blood-stage parasites38. The mitochondrial ETC mainly functions as the regeneration of ubiquinone for pyrimidine biosynthesis by dihydroorotate dehydrogenase, which is important for the development of the blood-stage P. falciparum11. This could explain why both compound 2 and 3 mainly inhibit trophozoite/schizont stages (Fig. 5D-F). It is important to note that PfNDH2 is also expressed in gametocyte/ookinete/sporozoite stages beyond the late blood-stage39, suggesting a potential activity of PfNDH2 inhibitors against both sexual and exo-erythrocytic forms of parasites. The recommended treatment for both uncomplicated and severe malaria is a combination of two or more anti-malarials with different mechanisms of action40-42. Such as the atovaquone whose monotherapy would give rapid rise drug resistance corresponding to the bc1 mutations43, it was used as a combination component with proguanil in the drug Malarone. Considering the emergence of artemisinin-resistant P. falciparum strains, compound 2 based molecules may provide an additional choice for the clinical use of ACT against malaria due to its synergistic activity with dihydroartemisinin. In summary, PfNDH2 inhibitors such as compound 2 could eliminate malaria both in vitro and in vivo with a possible allosteric mechanism. The new mechanism could be suggested to attack pathogens that rely on NDH2 for mitochondrial respiratory chain function in drug discovery. So it not only is important for malarial PfNDH2 protein-based drug development, but also could has broad implications for the treatment of other NDH2-containing pathogenic microorganisms.


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EXPERIMENTAL SECTION Chemistry. Synthesis of compounds. All commercial chemical materials (Energy, Ouhe, Aladdin, J&K Chemical Co. Ltd) were used without further purification. All solvents were analytical grade. The 1H NMR and

13

C NMR spectra were recorded on a Bruker AVANCE III

400 MHz spectrometer in CDCl3, CD3OD, or d6-DMSO using tetramethylsilane (TMS) or solvent peak as a standard. All 13C NMR spectra were recorded with complete proton decoupling. Low-resolution mass spectral analyses were performed with an Waters AQUITY UPLCTM/MS. Analytical thin-layer chromatography (TLC) was performed on Yantai Chemical Industry Research Institute silica gel 60 F254 plates, and flash column chromatography was performed on Qingdao Haiyang Chemical Co. Ltd silica gel 60 (200-300 mesh). A BUCHI Rotavapor R-3 was used to remove solvents by evaporation. The purity of all the final tested compounds is more than 95% confirmed by NMR and UPLC. The general procedures for synthesis of target molecules compound 2 and 3 was described as below. Synthesis of other intermediates and compounds was followed as procedure for compound 2 or achieved as shown in Supporting Information. 5-Fluoro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-4(1H)-one (2). To a 50 ml round-bottom flask, was added 554 mg (1.8 mmol, 1.0 eq) compound 9, 370 mg (1.8 mmol, 1.0 eq) compound 6, 120 mg (0.63 mmol, 0.35 eq) PTSA.H2O and 10 ml n-BuOH. After reflux at 130 ˚C for 16 h, the solvent was removed by evaporation, 100 ml EtOAc was added and the mixture washed with 50 ml saturated NaHCO3 solution, then 100 ml saturated sodium chloride solution. The organic later was dried over 1 g anhydrous Na2SO4 and purified by 200-300 mesh silica gel flash column chromatography (Hexane:EtOAc= 1:1), yielding 200 mg compound 2 (I.Y. =30%). 1H-NMR(400MHz, d6-DMSO, ppm): 1.82(s, 3H), 4.07(s, 2H) , 6.92(dd, J = 8.0 Hz,



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J = 11.8 Hz, 1H), 7.30(d, J = 8.1 Hz, 1H), 7.36-7.55(m, 8H), 11.55(s, 1H). 13C-NMR(100MHz, d6-DMSO, ppm): 175.61, 160.55(d, JC-F = 258.4 Hz), 146.90, 142.46, 141.86(d, JC-F = 4.3 Hz), 140.60, 132.62, 131.70(d, JC-F = 10.1 Hz), 130.58, 129.20, 128.89, 121.22, 120.18(d, JC-F = 254.2 Hz), 116.18, 114.16(d, JC-F = 4.2 Hz), 113.04(d, JC-F = 8.7 Hz), 108.35(d, JC-F = 20.9 Hz), 40.07, 12.10. LC-MS: calculated for C24H18F4NO2 [M+H]+: 428.12, found 428.28. 5-Fluoro-3-methyl-2-(4-(4-((trifluoromethyl)thio)benzyl)phenyl)quinolin-4(1H)-one (3). To a 50 ml round-bottom flask, was added 1.0 g (3.0 mmol, 1.0 eq) compound 10, 650 mg (3.0 mmol, 1.0 eq) compound 6, 240 mg (1.26 mmol, 0.4 eq) PTSA.H2O and 10 ml n-BuOH. After reflux at 130 ˚C for 16 h，most solvent was removed by evaporation, 50 ml EtOAc were added and the mixture was washed with 50 ml saturated NaHCO3 solution, then 100 ml saturated sodium chloride solution. The organic layer was dried over 1 g anhydrous Na2SO4 and purified by 200-300 mesh silica gel flash column chromatography (Hexane:EtOAc= 1:1), yielding 320 mg compound 3 (I.Y. = 22%). 1H-NMR(400 MHz, d6-DMSO, ppm): 1.82(s, 3H), 4.11(s, 2H) , 6.93(m，1H), 7.37-7.46(m, 8H), 7.65-7.67(m, 2H), 11.55(s,1H). 13C-NMR(100MHz, d6-DMSO, ppm): 175.49, 160.51(d, JC-F = 257.6 Hz), 146.71, 144.94, 141.95, 141.81(d, JC-F = 4.1 Hz), 136.49,132.69, 131.58(d, JC-F = 10.7 Hz), 130.35, 129.65(q, JC-F = 305.7 Hz), 129.17, 128.91, 120.51, 116.10 , 114.08(d, JC-F = 4.00 Hz), 113.00(d, JC-F = 8.7 Hz), 108.24(d, JC-F = 20.8 Hz), 40.40, 12.02. LC-MS: calculated for C24H18F4NOS [M+H]+: 444.10, found 444.18. 5-Fluoro-1H-benzo[d][1,3]oxazine-2,4-dione (4). To a 100 ml round-bottom flask, was added 4.7 g (30 mmol, 1.0 eq) 2-amino-6-fluorobenzoic acid, 5 g (36 mmol,1.2 eq) K2CO3, 40 ml EtOAc and 9 g (30 mmol 1.0 eq) triphosgene. After stirring at room temperature for 24 h, the


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mixture was poured into 100 ml saturated NaHCO3 solution and filtered to collect the solid, compound 4 (4.9g, I.Y. = 86%). 2-(4,4-Dimethyl-4,5-dihydrooxazol-2-yl)-3-fluoroaniline (6). To a suspension of 4.7 g (23 mmol, 1.0 eq) compound 4 in 25 ml chlorobenzene, was added 6 ml (60 mmol, 2.5 eq) 2-amino2-methylpropan-1-ol and 820 mg (6 mmol, 0.25 eq) ZnCl2. After reflux at 140 ˚C for 18 h, solvent was removed by evaporation, 100 ml DCM was added and the mixture was washed with 100 ml water. The organic layer was dried over 1.0 g anhydrous Na2SO4 and purified by 200-300 mesh silicon-gel flash column chromatography (Hexane: EtOAc= 10:1) to yield 1.9 g compound 6 (I.Y. = 35%). 1H-NMR(400 MHz, CDCl3, ppm): 7.04(m, 1H), 6.41(d, J = 8.3 Hz, 1H), 6.33(m, 1H), 6.00(b, 1H), 4.02(s, 2H), 1.35(s, 6H). LC-MS: calcd for C11H14N2O [M+H]+: 209.10, found 209.11. 1-(4-(Bromomethyl)phenyl)propan-1-one (7). To a solution of 2.22 g (15 mmol, 1.0 eq) 1-(ptolyl)propan-1-one in 20 ml anhydrous MeCN, was added 3.0 g (17 mmol, 1.1 eq) Nbromosuccinimide (NBS) and 246 mg 2,2'-azobis-(2-methylpropionitrile) (AIBN) under argon. After reflux at 90 ˚C for 7 h，the solvent was removed by evaporation, and 100 ml DCM were added followed by washing with 100 ml water. The organic layer was dried over 1.0 g anhydrous Na2SO4 and

purified

by

200-300

mesh

silica

gel

flash

column

chromatography

(Hexane:EtOAc=30:1) to get 3.1g compound 7 as colorless oil (I.Y. =85%). 1H-NMR(400 MHz, CDCl3, ppm): 7.93(d, J = 8.3 Hz, 2H), 7.47(d, J = 8.3 Hz, 2H), 4.49(s, 2H), 2.98(q, J = 7.2 Hz, 2H), 1.22(t, J = 7.2 Hz, 3H). LC-MS: calcd for C10H12BrO [M+H]+: 226.00, found 226.11. 1-(4-(4-Ttrifluoromethoxy)benzyl)phenyl)propan-1-one (9). To a 100ml round-bottom flask, was added 3.1 g (14 mmol, 1.0 eq) compound 7, 63 mg (0.28 mmol, 0.02 eq) Pd(OAc)2, 147 mg



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(0.56 mmol, 0.04 eq) PPh3, 6 g (28 mmol, 2.0 eq) K3PO4, 3.2 g (15.5 mmol, 1.1 eq) (4(trifluoromethoxy)phenyl)boronic acid and 30 ml toluene under argon. After stirring at 80 ˚C for 12 h，the solvent was removed by evaporation，100 ml water were added and the mixture was extracted with 100 ml DCM. The organic layer was purified by 200-300 mesh silica gel flash column chromatography (Hexane:EtOAc=30:1), yielding 3.0 g compound 9 as colorless oil (I.Y. =71%). 1-(4-(4-((Trifluoromethyl)thio)benzyl)phenyl)propan-1-one (10). To a 100 ml round-bottom flask, was added 1.1g (5 mmol, 1.0 eq) compound 7, 198 mg (0.25 mmol, 0.05 eq) Pd(dppf)2Cl2.DCM (CAS: 95464-05-4), 2 g (10 mmol, 2.0 eq) K3PO4, 1.2 g (5.5 mmol, 1.1 eq) 4,4,5,5-tetramethyl-2-(4-((trifluoromethyl)thio) phenyl)-1,3,2-dioxaborolane (CAS: 1005206-256) and 30 ml toluene under argon. After stirring at 100 ˚C for 12 h，the solvent was removed by evaporation，100 ml water were added and the mixture was extracted with 70 ml DCM. The organic layer was purified by 200-300 mesh silica gel flash column chromatography (Hexane:EtOAc = 30:1), yielding 1.0 g compound 10 as colorless oil (I.Y. =64%). 1H-NMR(400 MHz, CDCl3, ppm): 7.80(d, J = 8.2 Hz, 2H), 7.56(d, J = 8.04 Hz, 2H), 7.23(m, 4H), 4.05(s, 2H), 2.96(q, J = 7.2 Hz, 2H), 1.21(d, J = 7.2 Hz, 2H). LC-MS: calcd for C17H15F3OS [M+H]+: 325.08, found 325.11. Biology. Crystallization, data collection and structure determination. Crystals were grown at 18 ˚C by hanging-drop method by mixing 1 µl protein (10 mg/ml) with 1 µL reservoir solution (2.7 M sodium acetate trihydrate, pH 4.8 or 10.0 to 10.6). The crystals appeared overnight and grew to full size in about 2 to 3 days. To obtain the NADH-bound PfNDH2 crystals, the crystals were soaked at 18 ˚C in cryo-protectant (reservoir solution with 25% glycerol) with 10 mM


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NADH for about 1-3 minutes. Inhibitors were co-crystallized at a concentration of 0.5 mM with PfNDH2. The crystals and their complexes were then transferred to liquid nitrogen for subsequent analysis. Crystallographic data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U, integrated and scaled using the HKL2000 package44. The molecular replacement method was employed to solve the phase problem using the program Phaser45 from the CCP4 suite46, with the crystal structure of Ndi1 from Saccharomyces cerevisae (PDB code 4G6G; 36 % sequence identity) as the initial search model. Data collection statistics are summarized in Table S1. The final model rebuilding was performed using COOT47 and the protein structure was refined with PHENIX48 against the high-resolution native data using NCS49 and stereochemistry information as restraints. Full data collection and structure statistics are summarized in Table S1. Enzyme activity assays for PfNDH2. The activity of PfNDH2 was tested firstly. The Kcat=664.3±31.7 S-1, Km=34.9±3.9 µM (Fig. S4). The Km was similar to the published data50. The enzymatic activity of wild type and mutant PfNDH2 proteins was measured spectrophotometrically using NADH and Ubiquinone-1 (UQ1) as substrates. Standard assays were carried out at 25 ˚C in a 1.6 ml reaction mixture containing 50 mM MOPS buffer pH 7.0, 150 mM NaCl, 1 mM EDTA, 0.01% Triton-X-100, 50 µM NADH, 30 µM UQ1, 0.5 nM enzymes and selected concentrations of inhibitors. Reactions were initiated by the enzyme addition. Progress of the reaction was monitored continuously by following the decrease of signal from NADH at 340 nm, in a Lambda 45 spectrophotometer (PerkinElmer Life Sciences) equipped with a magnetic stirrer in the cuvette holder. Activities were calculated using an NADH extinction of 6220 cm-1M-1 at 340 nm.



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Parasites growth inhibition assays. For synchronized assay, asynchronous cultures of schizont stage parasites were pretreated with 5% sorbital. Plasmodium falciparum strains as mentioned in the text (1% parasitemia, 2% hematocrit) at the mid ring stage (~6-10 hours post-invasion) were used to test antimalarial effects in 96-well plates. Parasites were incubated in triplicate in a 96 well plate compound 2, 3, or artemisinin at 200 nM. Parasites were further diluted at equal ratio containing 1% parasitemia, 2% hematocrit from the maxim concentration of 100 nM. Parasites were allowed to grow for 72-hour incubation period under an atmosphere of gas mixture containing 5% CO2, 5% O2, and 90% N2 at 37 °C. After 72 hours, the media were replaced with 100 µl lysis buffer (10 mM KHCO3, 150 mM NH4Cl, 0.1 mM EDTA) consisting of 5×SYBR Green I (Invitrogen; supplied in 10,000×concentration). The plates were then incubated for 20 min in the dark. The fluorescence signal was quantitated at 485 nm excitation and 538 nm emission by a microplate fluorometer. Stage of action studies were carried out by immediately adding 10×EC50 inhibitor and parasites were visualized at 16, 24, 32, 40, 48, 60 h after synchronization. Giemsa staining of fixed smears was for comparison of parasite morphology at each time point. Parasitemia was quantified by a light microscope (100 × objective lens) equipped with a PowerShot G6 digital camera (Leica). To test parasite maturation throughout a single (~48 h) asexual blood-stage cycle, compounds were removed every 2 h starting at life cycle t=34 h (trophozoite stage). The growth inhibition was determined by SYBR Green I DNA ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Other experimental details including: protein expression and purification; the DNA point mutants; in vivo efficacy studies on mice; MTT assay; SPR study; Figure S1-S8, Table S1-S3 and synthesis of compounds.


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Accession Codes. The atomic coordinates and structure factors (code 5JWA for Apo, 5JWB for NADH and 5JWC for compound 2) have been deposited in the Protein Data Bank (http://www.pdb.org/). Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION Corresponding Author Maojun Yang, [email protected], +86-010-62789400; Yu Rao, [email protected], +86-010-62782025; Lubin Jiang, [email protected], +86-021-54923072. Author Contributions †These authors contributed equally. Y.R., M.Y., and L.J. conceived the study. X.L., Y.Y., J.L., J.Y. and J.G. purified the proteins, grew the crystals and performed the enzyme activity analyses. YQ.Y. and Y.R. designed and synthesized inhibitors. Y.Y. and M.Y. collected synchrotron diffraction data and solved the crystal structures. Z.H. and L.J. performed the in vitro and in vivo studies. Y.R., Y.M., L.J., Y.Y and YQ.Y. analyzed the data and wrote the paper. Funding Sources This work was supported by grants from the Ministry of Science and Technology of China (2016YFA0501100), the National Science Fund for Distinguished Young Scholars (31625008), the National Natural Science Foundation of China (21302106, 81573277, 81622042, 81361120405, 21532004 and 31570733), Tsinghua University Initiative Scientific Research Program and National Institutes of Health (RO1AI116466).



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Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENTS We would like to thank the staff at the SSRF BL17U beamline for their assistance in data collection and thank Dr. Heng Wang of Peking Union Medical Sciences for helpful discussion. ABBREVIATIONS NADH,

Reduced

form

of

nicotinamide-adenine

dinucleotid;

PfNDH2,

Ubiquinone

oxidoreductase of Plasmodium falciparum; ETC, Electron transport chain; CTD, C-terminal domain; AA, amino acids; FAD, Flavin adenine dinucleotide; RMSDs, Root-mean square deviation values; SPR, Surface Plasmon Resonance; ATP, Adenosine triphosphate; SAR, Structure-activity relationship; PYR, Pyrimethamine; DHA, Dihydroartemisinin; DMSO, Dimethyl sulfoxide; FICs, fractional inhibitory concentrations.

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(32) Rottmann, M.; Mcnamara, C.; Yeung, B. K. S.; Lee, M. C. S.; Zou, B.; Russell, B.; Seitz, P.; Plouffe, D. M.; Dharia, N. V.; Tan, J. Spiroindolones, a potent compound class for the treatment of malaria. Science 2010, 329, 1175-1180. (33) Fidock, D. A.; Rosenthal, P. J.; Croft, S. L.; Brun, R.; Nwaka, S. Antimalarial drug discovery: efficacy models for compound screening. Nat. Rev. Drug Discovery 2004, 3, 509-520. (34) Hegreness, M.; Shoresh, N.; Damian, D.; Hartl, D.; Kishony, R. Accelerated evolution of resistance in multidrug environments. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 13977-13981. (35) Moon, A. M.; Biggs, H. M.; Rubach, M. P.; Crump, J. A.; Maro, V. P.; Saganda, W.; Reddy, E. A. Artemisinin directly targets malarial mitochondria through its specific mitochondrial activation. PLoS One 2014, 9, e89814. (36) Gardner, M. J.; Hall, N.; Fung, E.; White, O.; Berriman, M.; Hyman, R. W.; Carlton, J. M.; Pain, A.; Nelson, K. E.; Bowman, S.; Paulsen, I. T.; James, K.; Eisen, J. A.; Rutherford, K.; Salzberg, S. L.; Craig, A.; Kyes, S.; Chan, M. S.; Nene, V.; Shallom, S. J.; Suh, B.; Peterson, J.; Angiuoli, S.; Pertea, M.; Allen, J.; Selengut, J.; Haft, D.; Mather, M. W.; Vaidya, A. B.; Martin, D. M.; Fairlamb, A. H.; Fraunholz, M. J.; Roos, D. S.; Ralph, S. A.; McFadden, G. I.; Cummings, L. M.; Subramanian, G. M.; Mungall, C.; Venter, J. C.; Carucci, D. J.; Hoffman, S. L.; Newbold, C.; Davis, R. W.; Fraser, C. M.; Barrell, B. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 2002, 419, 498-511. (37) Vaidya, A. B.; Mather, M. W. A post-genomic view of the mitochondrion in malaria parasites. Curr. Top. Microbiol. Immunol. 2005, 295, 233-250.


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(38) Boysen, K. E.; Matuschewski, K. Arrested oocyst maturation in Plasmodium parasites lacking type II NADH:ubiquinone dehydrogenase. J. Biol. Chem. 2011, 286, 32661-32671. (39) Le, R. K.; Zhou, Y.; Blair, P. L.; Grainger, M.; Moch, J. K.; Haynes, J. D.; De, L. V. P.; Holder, A. A.; Batalov, S.; Carucci, D. J. Discovery of gene function by expression profiling of the malaria parasite life cycle. Evaluation of in-hospital management for febrile illness in Northern Tanzania before and after 2010 World Health Organization Guidelines for the treatment of malaria. Science 2003, 301, 1503-1508. (40) Moon, A. M.; Biggs, H. M.; Rubach, M. P.; Crump, J. A.; Maro, V. P.; Saganda, W.; Reddy, E. A. Evaluation of in-hospital management for febrile illness in Northern Tanzania before and after 2010 World Health Organization Guidelines for the treatment of malaria. PLoS One 2014, 9, e89814. (41) Reyburn, H. New WHO guidelines for the treatment of malaria. BMJ 2010, 340, c2637. (42) Khan, M. A.; Smego, R. A., Jr.; Razi, S. T.; Beg, M. A. merging drug--resistance and guidelines for treatment of malaria. J. Coll. Physicians Surg. Pak. 2004, 14, 319-324. (43) Srivastava, I. K.; Vaidya, A. B. A mechanism for the synergistic antimalarial action of atovaquone and proguanil. Antimicrob. Agents Chemother. 1999, 43, 1334-1339. (44) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, 307-326. (45) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658-674.



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(46) Bailey, S. The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760-763. (47) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126-2132. (48) Adams, P. D.; Grosse-Kunstleve, R. W.; Hung, L. W.; Ioerger, T. R.; McCoy, A. J.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.; Sauter, N. K.; Terwilliger, T. C. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 1948-1954. (49) Terwilliger, T. Rapid automatic NCS identification using heavy-atom substructures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 2213-2215. (50) Biagini, G. A.; Viriyavejakul, P.; O'Neill, P. M.; Bray, P. G.; Ward, S. A. Functional characterization and target validation of alternative complex I of Plasmodium falciparum mitochondria. Antimicrob. Agents. Chemother. 2006, 50, 1841-1851.


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Figure 1. Inhibitors and overall structure of PfNDH2. (A) Three effective inhibitors of PfNDH2. The IC50 and EC50 values of new compounds 2 and 3 are from three separate experiments. The CLogP values are calculated by the software ChemDraw. (B) PfNDH2 forms a homo-dimer. The CTDs (from 465 to 533AA) of two monomers of PfNDH2 are colored in blue and hot pink, respectively. The domain C (from 359 to 430AA) is colored in purple. The remaining parts of the two monomers of PfNDH2 are colored in green and cyan, respectively. FAD is shown as stick representation. (C) Detail of interactions between the α18 helices of the two PfNDH2 monomers. (D) The amino acids associated with dimerization are indicated by red stars. Secondary structural elements of NDH2s are indicated above the sequences. Conserved residues in the sequence alignment are shaded by the color scheme of the CLC (Cake-loving



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Company) main workbench software (Table S2) and highlighted by background colors when the identity ≥ 50%.


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Scheme 1. The synthetic routes for quinolin-4-one and chromen-4-one.

Reagents and conditions: (a) K2CO3, Triphosgene, EtOAc; (b) ZnCl2, 2-amino-2-methylpropan1-ol,

Chlorobenzene;

(c)

K2CO3, DMF;

or

Pd(OAc)2,

PPh3,

K3PO4,

Toluene; or

Pd(dppf)Cl2.DCM, K3PO4, Toluene; (d) PTSA.H2O, n-BuOH; (e) EtMgBr, THF; (f) BBr3, DCM; (g) KOH, EtOH; (h) I2, DMSO; (i), NaH, Dimethyl carbonate, THF; (j) NaH, Isatoic anhydrides, DMF; (k) AcOH, Anilines, EtOH; (l) Dowtherm A.



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Figure 2. Compound 2 binds to two different sites in PfNDH2. (A) Ribbon representation showing the binding sites of compound 2 in PfNDH2. Compound 2 and FAD molecules are shown as stick structures colored in yellow and orange, respectively. (B) The binding mode of compound 2 in the pocket between the dimer interface of PfDNH2. The Fo-Fc omit density map is colored in blue. (C) The binding mode of compound 2 in another allosteric pocket. The Fo-Fc omit density map is colored in blue. (D) Point mutations at the binding sites reduce the ability of compound 2 to inhibit PfNDH2 activity. IC50 values are from three separate experiments. (E) Point mutations at the compound 2binding sites have little effect on the inherent ability of PfNDH2 to convert NADH in activity assays. Histogram bars represent enzyme activity of the four recombinant PfNDH2 mutant enzymes relative to wild-type. Experiments were repeated three times; error bars represent s.e.m.


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Figure 3. The ligand-based SAR verified the co-crystal structure results. (A) The 2D display of interactions between compound 2 and PfNDH2. The blue interactions correspond to binding mode I (Fig. 2B); The red interactions correspond to binding mode II (Fig. 2C). (B) The structure-activity relationship (SAR) of related compounds. The compound numbers and the retention ratios of PfNDH2 enzyme activity in the presence of compound at certain concentration are in parentheses. More details see Table S3.



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Figure 4. Compound 2 inhibits PfNDH2 activity and alters the NADH site by proposed allosteric mechanism. (A) Ribbon representations of the Apo-, NADH- and compound 2 bound forms are colored in green, purple and yellow, respectively. Structural shifts bring elements of the NADH-binding site closer to more closed configuration in NADH-bound than in the Apoform of PfNDH2. In the compound 2 bound PfNDH2, structural elements of the NADH binding site are shifted further away than in Apo-PfNDH2. Carbon atoms of secondary structural elements in the FAD binding domain (amino acids 41–152) superposed in these three structures with RMSDs of less than 0.18 Å. (B) Comparison of NADH binding site of the compound 2 bound to the NADH-bound form of PfNDH2. In the compound 2 bound form, the NADHbinding domain opened away from the more closed positions of the NADH-bound structure. NADH- and compound 2 bound forms are colored in purple and yellow respectively. Stick structures of NADH and FAD are in purple and yellow respectively. (C) Structural differences at the PfNDH2 dimer interface between the compound 2 bound enzyme (yellow) and the NADHbound form (purple). The red arrow shows the direction of the shift in the compound 2 bound


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form. (D) Compound 2 influence the KD values of NADH from the SPR (Surface Plasmon Resonance, Fig. S5) study. (E) The model summary of proposed allosteric binding sites and mechanism of compound 2. The red bidirectional arrows mean conformational change induced by compound 2.



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Figure 5. PfNDH2 Inhibitors kill the asexual blood-stage of various drug-resistant parasites. (A) Summary of compound 2 and 3 activities against asexual blood-stage parasites with EC50 values indicated by the listed 95% Confidence Intervals (CI). DHA was used as positive control (EC50=4.32±0.3 nM on 3D7 strain) (B) Compound 2 and 3 can efficiently kill asexual bloodstage parasites in vitro at 10×EC50. (C) qPCR analyses of the expression profile of the PfNDH2 gene normalized by the actin gene in 3D7 at given time points. (D) Microscopy of asexual stage 3D7 under the treatment of 10×EC50 of different compounds. (E) Compound-free media was supplemented with compounds at concentration of ~10×EC50 at 2 h intervals starting at the


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mid/late trophozoite stage (t ~34 h after invasion). Parasite growth in the following life cycle was normalized to untreated parasites. Experiments were repeated three times. Error bars represent s.e.m.



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Figure 6. The anti-malarial potential of PfNDH2 inhibitors. (A) Cytotoxic effects of compounds on HeLa cells by MTT assay. (B)(C) In vivo study. Various dosages were given to mice at 1 day (b) or 4 days (c) post infection. Black arrows indicated the starting point of


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treatment. Red arrowheads indicated that the group of mice died at the same day or the next day. Dosage information for each tested mice group is listed in panel E. (D) Microscopy of blood smears of treated mice in panel C. (E) Summary of activities of compounds against P. yoelii infection in mice. *PYR, pyrimethamine; DHA, dihydroartemisinin. αInhibition of parasite growth determined at 24 h post treatment; βMinimum period to cure all mice of each group (n=3); γ

Percentage of survived mice determined at 40 days post infection. All mice in the DMSO

control groups died at 7 or 8 days post infection; †Subcutaneous injection once daily for 4 days starting at 2 h post infection; ‡Subcutaneous injection once daily for 4 days starting at 96 h post infection; ₸Subcutaneous injection twice daily for 4 days starting at 96 h post infection, 8 h apart; ∆

Not applicable because mice died due to malarial anaemia. (F) In vitro drug interaction analyses

of compound 2 and other anti-malarials. Experiments were repeated three times. Error bars represent s.e.m.



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Table of Contents Graphic


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Target Elucidation by Cocrystal Structures of ... - ACS Publications

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