Plasmodium Gametocyte Inhibition Identified from a Natural-Product

Sep 30, 2013 - AbstractsICBS 3rd Annual Conference Driving Biology with Chemistry November 17–19, 2014San Francisco, California. ASSAY and Drug ...
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Plasmodium Gametocyte Inhibition Identified from a NaturalProduct-Based Fragment Library Hoan Vu,† Catherine Roullier,† Marc Campitelli,† Katharine R. Trenholme,‡,§ Donald L. Gardiner,‡ Katherine T. Andrews,† Tina Skinner-Adams,† Gregory J. Crowther,# Wesley C. Van Voorhis,# and Ronald J. Quinn*,† †

Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia Queensland Institute of Medical Research, Herston, Queensland, Australia § School of Medicine, University of Queensland, Brisbane, Queensland, Australia # Department of Medicine, University of Washington, Seattle, Washington, United States ‡

S Supporting Information *

ABSTRACT: Fragment-based screening is commonly used to identify compounds with relatively weak but efficient localized binding to protein surfaces. We used mass spectrometry to study fragment-sized three-dimensional natural products. We identified seven securinine-related compounds binding to Plasmodium falciparum 2′-deoxyuridine 5′-triphosphate nucleotidohydrolase (PfdUTPase). Securinine bound allosterically to PfdUTPase, enhancing enzyme activity and inhibiting viability of both P. falciparum gametocyte (sexual) and blood (asexual) stage parasites. Our results provide a new insight into mechanisms that may be applicable to transmission-blocking agents.

T

match to protein binding sites.5,7−9 FBS has proven to be a successful drug discovery strategy with 13 fragment-derived compounds in the clinic in 200910 and the approval of the first fragment-derived drug, vemurafenib, in 2011.11 We developed a fragment library of natural products in order to sample regions of biologically relevant chemical space that are generally absent from existing fragment libraries. We used Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to detect the weak binding between fragments and malaria proteins. Plasmodium falciparum deoxyuridine 5′-triphosphate nucleotidohydrolase (PfdUTPase), a potential drug target,12 was identified as protein that bound to members of the fragment library. An identified compound series was studied for antimalarial properties and mechanism of enzyme action. A natural-product-based fragment library was assembled from an existing library of drug-like natural products.13 A large number of privileged motifs have been identified to date and are suggested as a basis for fragment design.14,15 However, many of the cores used in FBS are aromatic compounds leading to synthetic libraries that are populated with planar structures exhibiting low chemodiversity and poorly matched to drug chemical space. Natural products and their analogues, on the other hand, have had high impact as drugs due to the embedded biosynthetic molecular recognition characteristics

he identification of small molecules that interact with proteins of therapeutic interest is a key step in drug discovery. Conventional molecular library screening methods such as high-throughput screening (HTS) typically identify lead compounds exhibiting binding affinities in the low micromolar range. These methods have proven to be successful in identifying starting points for drugs that are now clinically available.1 However, superfluous chemical functionality incorporated into these relatively complex molecules may lead to suboptimal binding interactions that are detrimental to binding affinity.2 A complementary molecular screening technology is fragment-based screening (FBS), which can identify relatively simple compounds with lower binding affinity resulting from fewer binding interactions. The FBS idea originated from Jencks in 1981, who suggested that large molecules can be considered as the combination of two or more ‘fragments’ that contain all of the features necessary for binding to the target protein.3 Since the implementation of FBS at Abbott Laboratories in 1996,4 it has matured into a remarkably efficient complementary approach to HTS lead generation, both in terms of reduced screening library size and the rational approaches for subsequent follow-up and optimization of the initial hits.5 A much greater proportion of chemical space can be sampled in FBS compared to drug-like molecules used in HTS, resulting in the delivery of higher hit rates.2 The smaller size of fragments and the resulting reduction in mismatched protein binding interactions result in high ligand efficiency (i.e., binding energy per atom).6 This approach delivers starting points with a better © 2013 American Chemical Society

Received: August 2, 2013 Accepted: September 30, 2013 Published: September 30, 2013 2654

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Figure 1. Construction of a structurally diverse fragment-screening library of natural products. (A) Physicochemical property profiles of the library composed of 331 natural product fragments. The orange line and number indicates the mean value for each property, molecular weight (MW), calculated octanol−water partition coefficient (log P), count of hydrogen bond donors (HBD), count of hydrogen bond acceptors (HBA), topological polar surface area (tPSA), percent polar surface area (%PSA), and count of rotatable bonds (RtB). (B) Library examples. From left to right: lepistine, deoxynupharidine, camoensidine, lycopodine, securinine, elaeokanine E, elaeokanine C, anabasine, N-demethyldarlingine, (−)-erycibelline, (−)-12-cytisineacetic acid, huperzine A.

too large (250−400 Da) to be considered fragments. However, this study provided some evidence that weak protein-fragment binding may be observable by MS analysis. MS detection methods have an advantage in that modifications or labeling of the protein target or the fragment library compounds are not required. The ESI-FTICR-MS screening revealed strong binding from securinine (1) and weaker binding from the hydroxylated analog (2) (Figure 2). Securinine had already been reported to have weak antimalarial activity (IC50 ∼25 μM against P. falciparum strain K1), but the mechanism of action was not elucidated.22 Hit expansion was undertaken with a set of seven securinine-related compounds including the two hits from the initial FTMS screening (Figures 2 and 3). Four fragments (1, 3, 4, and 5) reduced ATP production in P. falciparum stage V gametocytes by more than 50% with a corresponding reduction in gametocyte viability (Figure 3A). Five fragments (1, 3, 4, 5, and 6) demonstrated greater than 50% inhibition against the chloroquine-sensitive P. falciparum line 3D7 at 100 μM (Figure 3A). The compounds were shown to enhance dUTPase activity (Figure 3B) using two independent techniques, although the observed enhancement was somewhat variable between experiments (see Supporting Information). Dose−response studies with securinine (1) suggested that concentrations above 10 μM led to nearmaximal stimulation of enzyme activity (Figure 3C).

that are favorably matched to therapeutic targets as described by protein fold topology (PFT).16,17 PFT describes binding site recognition points unrelated to fold or sequence similarity and explains a natural product’s ability to recognize biology space. Molecular recognition patterns used during biosynthesis (the biosynthetic imprint) have been shown to carry through to interactions with similar binding motifs within therapeutic targets. The incorporation of these biosynthetic imprints has resulted in nature inspiring around half of all new small molecule drugs approved on the market in the past 30 years.18 Our drug-like natural product library was filtered in silico according to the following parameter set: molecular weight ≤ 250 Da; calculated log P < 4; hydrogen bond donors ≤ 4; hydrogen bond acceptors ≤ 5; rotatable bonds ≤ 6; polar surface area < 45%. Accordingly, 331 natural product fragments were present in sufficient quantity for the initial FBS and follow-up assays. The physicochemical profile of the natural product fragment library and some typical compounds are shown in Figure 1. We have previously reported that direct bioaffinity screening using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) can detect a protein−ligand interaction in complex natural product extracts.19,20 While another study has shown that weak binders can be detected by mass spectrometry using electrospray ionization (ESI) techniques,21 all compounds in the study were 2655

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Figure 2. ESI-FTICR-MS analysis demonstrates noncovalent binding of the securinine-related compounds to PfdUTPase. (A) Folded PfdUTPase was usually observed as three peaks representing the 10+, 9+, and 8+ charge states. (B) Pools of 8 fragments were mixed with the PfdUTPase, incubated, and subsequently analyzed by ESI-FTICR-MS. (C) Individual securinine-related fragments were incubated with PfdUTPase. The 10+ charged native protein and the protein-fragment complex appear on the left- and right-hand side of each individual mass spectrum, respectively. Ratios of bound to unbound protein are shown in parentheses. (D) Chemical structures of the securinine fragment series shown to bind PfdUTPase: securinine (1), 4-α-hydroxy-allosecurinine (2), virosecurinine (3), allosecurinine (4), viroallosecurinine (5), norsecurinine (6), and securinine-Noxide (7). See Supplementary Table 1 for quantification.

mono- and bifunctional dCTP deaminases and dUTPase, the C-terminal is important for the catalytically active complex by binding at the active site while allowing substrate binding. It has been shown that dTTP inhibition of E. coli dCTP deaminase is achieved by stabilizing the inactive form of the enzyme after binding to the C-terminal. We postulate that the securinine compounds may activate dUTPase by stabilizing the active form of dUTPase. Growing resistance of the malaria parasite to current drug therapies demands new approaches and new targets.27 To date, the only clinically approved drug that is effective against latestage gametocytes is primaquine, and P. falciparum stage V gametocytes have previously been shown to be refractory to most antimalarial compounds.28 Compounds able to target the gametocytes and also the asexual stage parasites would allow the discovery of a radical cure that will not only kill the parasite in the patient but also reduce transmission. Here we report the use of bioaffinity mass spectrometry applied to fragment-based screening of a natural product fragment library. Our results suggest that compounds acting by activating dUTPase may allow development of transmission blocking agents.

Previous studies reported that the binding affinities and equilibrium dissociation constants (KD) of ligands could be determined by ESI-MS titration methods in the gas phase, and they were in good agreement with the values derived from solution-phase experiments using alternative technologies.23,24 Relative peak intensities between the native protein and the protein-fragment complex gave the rank order of binding affinity 1 ≈ 3 > 6 ≈ 4 > 5 > 7 > 2 (Figure 2). The trend observed from MS binding experiments was consistent with the percentage inhibition of either the P. falciparum asexual erythrocytic stage or stage V gametocytes. The ligand efficiency of ∼0.4 kcal/mol per non-hydrogen atom of the most active compounds is a promising starting point for further synthetic elaboration of the molecules. dUTPase is an essential enzyme found prolifically in prokaryotic and eukaryotic organisms. In P. falciparum, dUTPase is the only biosynthetic route leading to deoxyuridine 5′-monophosphate (dUMP), which is then converted to deoxythymidine 5′-triphosphate (dTTP). It also serves an important role in the maintenance of DNA integrity by catalyzing the hydrolysis of deoxyuridine 5′-triphosphate (dUTP) into dUMP in order to maintain a low concentration of dUTP that can be misincorporated in DNA.25 It has been suggested that this enzyme was associated with parasite forms undergoing active DNA replication and was stage-specific. Inhibition of the enzyme has been proposed as the mechanism of action.12 dUTPase is structurally closely related to the trimeric dCTP deaminases.26 They belong to the same superfamily and form trimers of identical subunits. For both



METHODS

Reagents. Agilent tuning mixture (P/N G2421A) was used to calibrate the instrument. Ammonium bicarbonate (P/N A6141) was purchased from Sigma. Buffer exchange used an Illustra Nap 5 column (Sephadex G25) from GE Healthcare. Protein Production. Recombinant full-length dUTPase from P. falciparum with an N-terminal 6xHis tag was cloned, expressed, and purified using standard methods, as described previously.29 2656

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stages of differential pumping to achieve a factor of 104 pressure reduction between the source and the analyzer region. Prior to MS acquisition, PfdUTPase was buffer-exchanged to 10 mM ammonium bicarbonate buffer (pH 7.8). Mass spectra were recorded in the positive ion mode with a mass range from 50 to 6000 m/z for broadband low resolution acquisition. Each spectrum was an average of 16 transients (scans) composed of 256,000 data points. For the fragment library screening, pools of eight fragments (each fragment 1 μL, 5 mM in MeOH) were incubated with dUTPase (80 μL, 11.2 μM) overnight at 4 °C and analyzed by ESI-FTMS. The fragment:protein ratio was 5.5:1. When a noncovalent complex was found, the molecular weight of the binding fragment was deducted from the spectrum. On the basis of this mass information, a binding confirmation on the selected ligand was performed. For the single fragment binding assays on the 7 securinine-related compound library, each fragment (5 μL, 1 mM in MeOH) was incubated with dUTPase (50 μL, 11.2 μM) overnight at 4 °C and analyzed by ESI-FTMS. The final fragment:protein ratio was ∼9:1. Blood-Stage P. falciparum Growth Inhibition Assays. In vitro antimalarial growth inhibition assays were carried out using the chloroquine-sensitive P. falciparum line 3D7,30 essentially as previously described.31 Asynchronous asexual stage infected erythrocytes (1% parasitemia and 1% hematocrit) were incubated for 48 h in 96-well culture dishes with test fragments or controls and 0.5 μCi [3H]hypoxanthine. Cultures were harvested onto 1450 MicroBeta filter mats (Wallac), and [3H]-hypoxanthine incorporation was determined using a 1450 MicroBeta liquid scintillation counter. Primary screens were carried out at 100 μM in two independent experiments, each in triplicate wells. Fragments giving greater than 50% inhibition compared to DMSO vehicle controls (0.5%) in the primary screen were then tested in an 8 point dose−response, in triplicate wells, in three independent experiments. Percentage inhibition compared to DMSO controls was determined and mean IC50 values (±SD) were calculated using linear interpolation of inhibition curves.32 P. falciparum Gametocyte Inhibition Assay. P falciparum line 3D7 was cultured in RPMI-HEPES supplemented with 10% human serum under standard conditions.33 Gametocytes were obtained using a modification of a published protocol.34 Cultures of synchronous 3D7 ring stage parasites were established at 2−3% parasitaemia and cultured through one invasion cycle to between 6% and 8% parasitaemia. Following treatment with 5% sorbitol35 parasites were cultured until they reached trophozoite stage and were then diluted to 2% parasitaemia and 5% hematocrit with fresh uninfected RBC. The cultures were maintained under standard conditions with daily media change but without the addition of fresh RBC and treated with 5% sorbitol to remove asexual stage parasites. Gametocyte maturation was monitored daily by microscopic examination of Giemsa stained thin smears. After approximately 10−12 days mature stage V gametocytes were enriched by magnetic separation34 using MACS columns. Testing was performed essentially as described.28 Approximately 5,000 gametocytes were dispensed into each well of a 96-well plate. The test compounds were added to give a final volume of 100 μL, and the plate was incubated at 37 °C for 24 h under standard culture conditions. BacTiter-Glo reagents (Promega G8231) were added to a final volume of 200 μL, and the assay was read using a GloMax 96 Microplate Luminometer [cat. no. E6501] with an integration constant of 0.5 s. Sample readouts were compared to the control ATP levels, and percentage inhibition was calculated. All compounds were dissolved in DMSO and tested in triplicate on 2 separate occasions at a concentration of 100 μM (1% DMSO). The assay included a vehicle control of standard culture media with 1% DMSO (no drug). Enzymatic Assays. dUTPase cleaves dUTP into dUMP + PPi. PPi may be converted to 2Pi with pyrophosphatase, and Pi detected with a malachite green kit (R&D Systems, Minneapolis, MN, USA). Assays were conducted in a buffer of 25 mM MES, pH 8.0, with 5 mM MgCl2, 100 mM KCl, 10 ng/mL dUTPase, 25 μM dUTP, and 1 μg/mL pyrophosphatase from S. cerevisiae (Sigma, St. Louis, MO, USA). Samples were incubated at RT (∼20 °C) for up to 60 min; dUTPase activity was stopped after 0, 15, 30, 45, and 60 min by addition of the

Figure 3. Activity of fragments against Plasmodium parasites and PfdUTPase activity. (A) Anti-Plasmodial activity of fragments at 100 μM against in vitro cultured asexual-stage (black bars; n = 2, each in triplicate) and gametocyte-stage (white bars; n = 2, each in triplicate) P. falciparum parasites. The average percentage inhibition (±SD) was compared to no compound DMSO (≤0.5%) vehicle controls. Secondary asexual-stage assays (n = 2) resulted in IC50 values of 17.1 (±0.9), 20.7 (±4.5), 33.9 (±3.7), 45.9 (±2.2), and 82.4 (±10.8) for 1, 3, 4, 5, and 6, respectively. Secondary gametocyte-stage assays (n = 1 in triplicate) resulted in IC50 values of 36.7 (±3.0), 29.7 (±1.3), 23.6 (±3.4), and 81.1 (±2.1) for 1, 3, 4, and 5, respectively. (B) The effect of fragments at 33.3 μM on PfdUTPase activity was compared to no compound 2% DMSO vehicle control (none; set to 100% activity) and to activity obtained in the presence of 25 μM 5′-DMTdeoxyuridine (DMT-dU), a known dUTPase inhibitor. Data shown are averages (±SD) of three independent experiments. (C) Response of dUTPase to increasing concentrations of 1 where each data point represents the slope of a best-fit line of catalytic rate versus time from one representative experiment of three performed. Natural Product Fragment Library. The library of 331 fragments was built from an analysis of our natural product pure compound collection, retaining available molecules with molecular weight ≤ 250 Da; calculated log P < 4; hydrogen bond donors ≤ 4; hydrogen bond acceptors ≤ 5; rotatable bonds ≤ 6; and polar surface area < 45%. FTMS Binding Experiments. All experiments were performed on a Bruker Apex III 47e external ESI source FTICR mass spectrometer, which employ an actively shielded 4.7-T superconducting magnet. All aspects of pulse sequence control and data acquisition were performed on Bruker’s Xmass software. Samples were injected manually by a Cole-Parmer syringe pump. A nebulizing N2 gas with a pressure of 60 psi and a countercurrent drying N2 gas with a flow rate of 60 L/min were employed. The drying gas temperature was maintained at 125 °C. The capillary exit voltage was tuned at 120 V to preserve the weak noncovalent complexes, and skimmer 1 voltage was 23 V. Ions were accumulated in an external ion reservoir composed of an rf-only hexapole, a skimmer cone (skimmer 2) with a tuning voltage of 5 V, and an auxiliary gate electrode prior to injection into the cylindrical Infinity analyzer cell, where they were mass analyzed. There were two 2657

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acidic “Reagent A” of the malachite green kit. The final readout was absorbance at 600 nm, reflecting [Pi] production, as read on an ELx800 microplate reader (BioTek Instruments, Winooski, VT, USA). Slopes of lines fit to absorbance readings between 0 and 45−60 min were taken as indicators of relative dUTPase catalytic rates. The unexpected finding of enhanced dUTPase activity in the presence of compounds 1−7 was verified semiquantitatively via a luciferase-based PPiLight kit (Lonza, Rockland, ME, USA) that detects PPi directly (i.e., without converting it to Pi); luminescence at 528 nm was read on a FLx800 microplate reader (BioTek).



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ASSOCIATED CONTENT

S Supporting Information *

Compound characterization and table. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail: r.quinn@griffith.edu.au. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors thank D. Leibly and S. Hewitt for producing and purifying the recombinant PfdUTPase, J. Mo for the work on the enzymatic assays, and M. Simpson and N. Pham for plating out the library. We also acknowledge the Australian Red Cross Blood Service for the provision of human blood and sera. The work was supported by Bill & Melinda Gates Foundation Grand Challenges Explorations Grants OPP1008376 and OPP1035218 and the Australian Research Council (ARC) Linkage Project (LP120100485). We thank the Australian Research Council (ARC) for support toward NMR and MS equipment (Grant LE0668477 and LE0237908). K.T.A. acknowledges the Australian Research Council for a Future Fellowship.



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