proteins STRUCTURE O FUNCTION O BIOINFORMATICS

Structural genomics for drug design against the pathogen Coxiella burnetii Matthew C. Franklin,1* Jonah Cheung,1 Michael J. Rudolph,1 Fiana Burshteyn,1 Michael Cassidy,1 Ebony Gary,1 Brandan Hillerich,1 Zhong-Ke Yao,2 Paul R. Carlier,2 Maxim Totrov,3 and James D. Love1 1 Special Projects Division, New York Structural Biology Center, New York 2 Department of Chemistry, Virginia Tech, Blacksburg, Virginia 3 Molsoft, LLC, San Diego, California

ABSTRACT Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biological agents, and very few of its proteins have been structurally characterized. To address this situation, we undertook a study of critical metabolic enzymes in C. burnetii that have great potential as drug targets. We used high-throughput techniques to produce novel crystal structures of 48 of these proteins. We selected one protein, C. burnetii dihydrofolate reductase (CbDHFR), for additional work to demonstrate the value of these structures for structure-based drug design. This enzyme’s structure reveals a feature in the substrate binding groove that is different between CbDHFR and human dihydrofolate reductase (hDHFR). We then identified a compound by in silico screening that exploits this binding groove difference, and demonstrated that this compound inhibits CbDHFR with at least 25-fold greater potency than hDHFR. Since this binding groove feature is shared by many other prokaryotes, the compound identified could form the basis of a novel antibacterial agent effective against a broad spectrum of pathogenic bacteria. Proteins 2015; 00:000–000. C 2015 Wiley Periodicals, Inc. V

Key words: X-ray crystallography; dihydrofolate reductase; inhibitor; antifolate; antibiotic.

INTRODUCTION Coxiella burnetii is a gram-negative intracellular pathogenic bacterium, first identified in the late 1930s.1,2 It causes “Q fever,”3,4 a debilitating, sometimes fatal, flulike disease with possible complications including pneumonia, hepatitis, and endocarditis.4,5 C. burnetii is quite common in nature, and periodically causes significant outbreaks of Q fever, such as the epidemic that sickened >4000 people in The Netherlands.6 C. burnetii particles can be easily carried on dust and aerosols and are durable and resistant to disinfectants.7 It is one of the most infectious organisms ever characterized, capable of causing illness with a single viable bacterium.8 C. burnetii is therefore considered a potential weapon of bioterrorism, and has been placed on the Centers for Disease Control (CDC) high risk biological agent list (42 CFR part 73). The study of C. burnetii has been hampered by the difficulty of culturing this bacterium and the lack of good genetic tools, problems that have been solved only

C 2015 WILEY PERIODICALS, INC. V

recently.9,10 Very little work has been done on the structural basis for inhibition of C. burnetii proteins. Prior to our investigation, the structures of only four proteins from C. burnetii (PDB codes 3IJ3, 3K96, 3KQ5, 3Q7H) had been determined as compared to 121 proteins from Bacillus anthracis (anthrax), another bacterium on the CDC’s high risk biological agent list. We undertook a high-throughput study of critical C. burnetii soluble proteins, with the goal of quickly producing a large number Additional Supporting Information may be found in the online version of this article. Grant sponsor: U.S. Department of Defense, Edgewood Chemical and Biological Center; Grant number: W911SR-11-C-0014. Institution at which work was performed: New York Structural Biology Center Matthew C. Franklin, Jonah Cheung, and Michael J. Rudolph contributed equally to this manuscript. *Correspondence to: Matthew C. Franklin, Special Projects Division, New York Structural Biology Center, 89 Convent Avenue, NY 10027. E-mail: [email protected] Received 21 November 2014; Revised 1 May 2015; Accepted 19 May 2015 Published online 1 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.24841

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of X-ray crystal structures that could serve as the foundation for drug design against this pathogen, either by in silico screening of compound libraries against the novel structures, or by adapting drug leads against homologous proteins to be specific for C. burnetii. MATERIALS AND METHODS Materials

Oligonucleotides (PCR and sequencing primers) were synthesized by IDT DNA. C. burnetii RSA 493 Nine Mile (phase I) genomic DNA, screened to confirm the total absence of viable bacteria, was generously provided by Dr. James E. Samuel, Texas A&M College of Medicine. This DNA was then expanded using a whole-genome amplification kit (GenomiPhi, GE Healthcare) and used as template DNA for the PCR reactions. Bioinformatics and target list generation

The specific strain of C. burnetii selected for this project (C. burnetii RSA 493 Nine Mile phase I)11 contains 1817 protein coding genes in its primary genome,12 plus another 30 protein coding genes in an endogenous plasmid denoted QpH1.13 All of these genes were analyzed for the presence of transmembrane helices by TMHMM.14 The 1408 genes with no predicted transmembrane helices were submitted to a BLAST search15 against the sequences found in the Protein Data Bank. Four genes (CBU_0560, 0572, 0738, 1518) matched perfectly to a PDB entry, indicating that the structure of the C. burnetii protein had already been solved; these genes were removed from our target list. Four hundred and twenty-two sequences had good comparative sequence alignment in the BLAST search (sequence identity over 35%, no gaps over 30 residues long) to a protein from some other organism (for example, E. coli) along essentially the entire length of the C. burnetii gene. The median sequence identity score in this group was 53%, with only 1/5 of the sequences under 40% identity to the homologous protein. We considered an alignment to be “full length” if the endpoints of the aligned region were within 15 residues of both the N and C termini of the C. burnetii protein. An additional 544 genes had a good sequence alignment (sequence identity over 35% in the aligned region) covering only a portion of the C. burnetii gene. Truncation constructs of these “partial length” genes were designed by hand to be slightly longer than the ordered region of the PDB entry that gave the best sequence match. (See Supplementary Structural Data for details on truncation constructs.) The remaining genes had no significant alignment to any sequence in the Protein Data Bank. These genes were prioritized further using information from the SEED database16 of annotated genes for C. bur-

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netii, to identify the genes that lie at critical metabolic junctions (for example, dihydrofolate reductase) since inhibition of such a gene product would be highly effective at preventing C. burnetii growth. The Virulence Factor Database17 was also used to identify key virulence factors in related bacteria (C. burnetii was not in the database); the homologous C. burnetii virulence factors were then selected. 315 genes were placed on a "high priority" list in this manner, including 60 genes with no detectable PDB homology in the search described above. The remaining C. burnetii genes with full PDB matches were next in order of priority, followed by the truncation constructs of those genes having only partial PDB matches. PCR primers were designed using Primer-Prim’er18 to amplify the desired gene (full length or truncated) from the C. burnetii genomic DNA. PCR products were inserted by ligation-independent cloning (LIC)19 into three target vectors: pMCSG7, pMCSG9, and pMCSG24,20 which encode N terminal His, His-MBP, and His-GST fusion tags respectively, followed by a Tobacco Etch Virus (TEV) protease cleavage site. High throughput small scale expression

BL21(DE3) E. coli transformed with C. burnetii expression constructs were grown in 0.6 mL of 2xYT in a 96well deepwell block; protein expression was induced with 1 mM IPTG at a culture OD600 of 0.6. Bacteria were lysed by sonication in the 96-well growth block using a custom-designed robotic sonicator.21 The lysates were then centrifuged and the supernatants extracted from the 96-well block and applied to a 96-well filter block (Thomson Instrument) with 40 lL of 50% Ni-NTA resin slurry (Qiagen) added to each well. Samples were incubated, washed with a buffer containing 75 mM imidazole, then eluted at a volume of 35 lL into a 96-well plate. The eluted samples were loaded onto Criterion gels (Biorad), which can hold 24 samples, or 2 rows of a 96-well block per gel. The gels were run in parallel using a Dodeca Cell (Biorad); this apparatus can hold 12 gels, so three 96-well blocks can be processed at once. Gels were stained with Coomassie stain and visualized in the standard manner. Parallel large scale protein expression and purification

Large scale protein expression was carried out using a Lex airlift bioreactor (Harbinger Biotechnology) with a capacity of 24 3 2 L cultures. All large-scale bacterial culture used selenomethionine-containing defined media, specifically Studier PASM-5052.22 Protein was purified from bacterial lysates in an automated manner by NiNTA binding and gel filtration chromatography using A˚KTAxpress chromatography systems (GE Life Sciences).

Drug Design Against Coxiella Burnetii

Fusion tags were removed by cleavage with TEV protease, and the postcleavage products, in a buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 40 mM imidazole, were passed over a gravity-fed Ni-NTA column to remove uncleaved fusion protein, free His tag, and TEV protease (which was also His-tagged).23 Samples were then concentrated to 10–15 mg/mL for crystallization. High throughput crystallization

Purified proteins were prepared for crystallization using Gryphon crystallization robots (Art Robbins Instruments), combining 0.2 lL of protein solution with 0.2 lL of well solution in 96-well Intelliplates (Art Robbins Instruments). Up to four 96-well sparse matrix screens (JCSG Core 1-4, Qiagen) were prepared for each protein if the purification yield was sufficient; 130 lL of protein stock at 10–20 mg/mL were needed to prepare these four screens. If the protein was known to bind cofactors (for example, NADPH, GTP) these were added to the protein stock at 1 mM before crystallization setup. (See Supplementary Structural Data.) Crystallization plates were incubated and imaged using two RockImager robots (Formulatrix), one holding the plates at 258C, the other at 68C. If crystals were observed, follow-up crystallization screens varying the hit conditions were prepared in 96-well format using an Alchemist II liquid-handling robot (Rigaku). It was not necessary to prepare anything other than 96-well screens in order to obtain diffraction-quality crystals.

molecular replacement structures as well to aid in model building. Molecular replacement was carried out using Phaser24 as implemented in the CCP4 package25 or in Phenix.26 SAD phasing was carried out using Solve/ Resolve27 as implemented in Phenix. Initial models were built into SAD-phased maps using Autobuild (Phenix) or Arp/wARP.28 All structures were rebuilt using Coot,29 and refined using Phenix or Refmac.30 Final structure quality checks were done using Molprobity.31 CbDHFR inhibitor complex structures

CbDHFR (CBU_1993) was crystallized as described above, with 1 mM NADPH added to the protein solution before crystallization. Once high-quality crystals had grown (1–2 days), the crystals were transferred to a soaking solution, typically containing 0.1 M MES, pH 5; 30–35% PEG 6000; 5% ethylene glycol; 0.5–1 mM compound (folate, methotrexate, trimethoprim, or compound 3). After overnight soaking, crystals were frozen and data collected as described above. Folate, methotrexate, and trimethoprim were manually placed into clear difference electron density; however, there was no electron density corresponding to compound 3, in the active site groove or elsewhere on CbDHFR. CbDHFR inhibitor in silico screening

Crystals were harvested directly from the 96-well Intelliplates by simply cutting a hole in the plate sealing film over the well of interest. Crystals were cryoprotected and frozen in liquid nitrogen following standard techniques. Initial crystal screening for diffraction quality was performed on our home X-ray source, a Rigaku MicroMax 007HF generator with VariMax HF optics and a Saturn944 CCD detector. Many crystals diffracted well enough to collect full datasets on the same system, while others were taken to a synchrotron beamline, primarily X4C at the National Synchrotron Light Source (Brookhaven National Laboratory). Synchrotron datasets were collected as single-wavelength anomalous dispersion (SAD) experiments at the selenium peak wavelength as determined by fluorescence scans.

ChEMBL32 database queries were performed using MolCart (Molsoft). Queries were restricted to compounds with reported, but weak mammalian DHFR Ki, containing an aminopteridine or pyridopyridine scaffold. The resulting compound list was visually inspected, and several molecules that had features (bulk and/or rigidity) possibly conflicting with binding in the narrow groove of human dihydrofolate reductase (hDHFR) were selected for docking. Flexible ligand docking of potential selective inhibitors was performed in ICM using default settings (ICM-Docking module, Molsoft).33,34 Receptor structures were prepared for docking from PDB entries 3TQ8 (CbDHFR) and 3GYF (hDHFR) following the standard conversion procedure in ICM. To allow for induced fit, final docked structure was refined with flexible receptor relaxation also in ICM by performing five alternating cycles of restrained gradient minimizations of the receptor and unrestrained minimizations of ligand (1000 steps each, ECEPP/3 forcefield35 for the protein and MMFF9436 for the ligand minimizations).

Structure determination

Synthesis of compound 3

Thirty-seven of the 48 structures we determined could be solved by molecular replacement using closely related structures from the Protein Data Bank (see Supplementary Structural Data for details); 11 structures required SAD phasing to generate initial maps, although we generated SAD-phased maps for a large number of the

Compound 3 was synthesized according to the published route37 in 27% overall yield over 4 steps from 3bromomalonaldehyde, 2,4,6-triaminopyrimidine, pivalic anhydride, and 3,4,5-trimethoxystyrene. One significant improvement to the procedure was made: the di-pivaloyl protected precursor was converted to compound 3 by

Crystal screening and data collection

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treatment with saturated ammonia in methanol at 508C for 18 h (sealed tube), rather than by deprotection in liquid ammonia in a stainless steel pressure bomb. Activity testing of compound 3

Enzymatic inhibition assays were carried out using a commercial DHFR assay kit (Sigma-Aldrich CS0340), following the manufacturer’s instructions, with 0.6 mM dihydrofolate as the initial substrate concentration. CbDHFR, purified as described above, was diluted to match the activity of the hDHFR provided with the kit. Compound 3 (50 mM stock in 100% DMSO) was serially diluted in 13 assay buffer, starting at a concentration of 1 mM. A methotrexate dilution series was assayed in the same 96-well plate, to serve as a positive control, and to determine the 100 and 0% activity levels for CbDHFR and hDHFR. Reported activity levels represent the average of two or three independent measurements. Curve fitting and IC50 measurements were carried out using GraphPad Prism (GraphPad Software). RESULTS AND DISCUSSION High throughput structure determination

Our goal was to produce approximately 50 crystal structures in less than a year. The experience of other structural genomics projects38–41 suggested that this could be achieved with a target list of fewer than 1000 genes, well below the size of the C. burnetii genome (1847 protein-coding ORFs).12 Since time was a factor in this project, we needed to balance the scientific and drug discovery potential of a given protein target against the expected difficulty in determining that target’s structure. We identified 966 C. burnetii genes encoding soluble proteins with significant homology to a previously deposited structure in the Protein Data Bank (PDB), which suggested that these proteins would be more likely than average to crystallize and yield a structure. We also generated a high-priority target list of critical metabolic enzymes and predicted virulence factors based on the SEED16 and VFDB17 databases. These C. burnetii genes were thought to be the most promising targets for structure-based drug design. Some particularly interesting targets were included in our high-priority list even though they had no homology to any structure in the PDB. (See the Methods for more details on target selection.) We cloned 522 genes (full length or truncated as described in the Methods) into expression vectors (Table I); nearly 70% produced enough protein in a small-scale expression trial to warrant preparation at large scale; about half of the large-scale efforts yielded protein that could be used for crystallization. (None of the highpriority targets lacking PDB homology produced usable protein.) 94 of the proteins set up in crystallization trials

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Table I Structural Genomics Pipeline Progress Month 1 2 3 4 5 6 7 8 9 10 11

Cloning

Expression

Purification

Crystals

Structures

143 426 426 522 522 522 522 522 522 522 522

9 14 47 73 95 184 236 279 310 347 354

4 7 19 37 58 103 134 152 166 185 191

1 2 12 24 32 68 81 86 87 92 94

1 3 6 6 17 26 33 42 45 46a

Entries in this table represent the number of unique C. burnetii genes to reach the following milestones: Cloning: first expression clone prepared; Expression: first preparative-scale bacterial growth; Purification: first crystallization-quality purified protein; Crystals: first crystals observed in trays; Structures: first refined crystal structure. Numbers are cumulative from the start of the project. a An additional two structures were solved at a later date, using data collected during this 11-month period.

produced some form of crystal, and we were able to solve structures for 48 of these (Fig. 1, Table II). Initial crystallization hits for 22 proteins could not be optimized to produce usable crystals, and another 18 proteins produced crystals with very poor or no X-ray diffraction. Six proteins (CBU_0034, 0657, 0682, 1241, 1410, and 1709; CBU numbers refer specifically to the C. burnetii RSA 493 Nine Mile phase I genome12) produced crystals with problematic diffraction (low 3 - 4 A˚ resolution along with multiple lattices, extremely high mosaicity, or other pathologies). We chose not to pursue these six proteins further for structure determination, but they could potentially have yielded a structure with sufficient effort. All of the structures that we present here are novel*, greatly increasing the structural knowledge of the C. burnetii proteome. (See Supplementary Structural Data for details on each of the 48 structures.) The great majority of these structures (40 out of 48) are at a resolution better than 2.5 A˚, making them good foundations for structure-based drug design; 39 of the 48 have an active site capable of binding small molecule inhibitors: 23 of these 39 can be matched to PDB entries of homologous proteins bound to drug-like molecules (see Supplementary Structural Data), while the other 16 can be matched to structures with small substrates or products bound at the active site; 35 of the 48 structures are scored “druggable” by the DrugEBIlity server (part of the ChEMBL database).32 Therefore, our set of structures provides a fertile base for further drug discovery efforts. Selected C. burnetii structures

In this section, we present a few C. burnetii structures in more detail. Some of them have been well validated as *EPSP synthase (CBU_0526) was independently determined by the Center for Structural Genomics of Infectious Diseases, and deposited in the Protein Data Bank (PDB code 3ROI) about two months after our structure determination, but before our own deposition.

Drug Design Against Coxiella Burnetii

Figure 1 Montage of 48 C. burnetii structures. All of the structures presented in this article are shown in ribbon representation, with the biologically relevant assembly (monomer, dimer, up to dodecamer) depicted. Multiple copies of the protein generated by non-crystallographic symmetry are given different colors for each protein monomer; additional copies generated by crystallographic symmetry are colored in shades of gray. The black scale bar under each ribbon representation denotes a distance of 40 A˚.

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Table II Structural Summary Protein Name Purine NTP pyrophosphatase Met ABC transporter substrate binding 2-Amino-3-ketobutyrate CoA ligase Pantothenate kinase ssDNA binding protein Phosphopantetheine adenylyltransferase dUTP nucleotidylhydrolase Guanylate kinase CMP-KDO synthase Arg ABC transporter substrate binding Malonyl CoA-ACP transacylase EPSP synthase OMP decarboxylase BolA family protein Inorganic pyrophosphatase Nucleotide-sugar aminotransferase Hpr(Ser) kinase/phosphatase Ribosome-associated factor Y 2-Methylcitrate synthase GntR transcriptional regulator Peptide chain release factor 3 Polynucleotide phosphorylase O-methyltransferase Uracil-DNA glycosylase Quinone oxidoreductase Oligoribonuclease D-Alanine-D-alanine ligase GMP synthase Dihydropteroate synthase Cysteinyl-tRNA synthetase Addiction module antidote protein PEP-protein phosphotransferase YebC family protein SurE 5'-nucleotidase Phosphopyruvate dehydratase Spermidine N1-acetyltransferase Superoxide dismutase GAR formyltransferase Alpha/beta hydrolase Phosphoglycerate kinase Translation elongation factor P Shikimate kinase 16S rRNA dimethyladenosine transferase Dihydrofolate reductase Dihydrofolate reductase 1 folate Dihydrofolate reductase 1 trimethoprim Dihydrofolate reductase 1 methotrexate Acylphosphatase Methionyl-tRNA formyltransferase N5-CAIR mutase Pyrroline-5-carboxylate reductase

CBU number

PDB code

Resolution ()

R/Rfree (%)

Ramachand. outliers (%)

0043 0109 0111 0199 0271 0288 0293 0301 0479 0482 0494 0526 0531 0582 0628 0696 0744 0745 0772 0775 0811 0852 0924 0988 1023 1235 1338 1341 1351 1487 1490 1550 1566 1671 1674 1678 1708 1737 1769 1782 1816 1892 1982 1993 1993 1993 1993 1995 1997 2002 2090

3TQU 3TQW 3TQX 3TQC 3TQY 4F3R 3TQZ 3TR0 3TQD 3TQL 3TQE 3TR1 3TR2 3TR3 3TR4 3UWC 3TQF 3TQM 3TQG 3TQN 3TR5 4NBQ 3TR6 3TR7 3TQH 3TR8 3TQT 3TQI 3TR9 3TQO 3TRB 3TRC 4F3Q 3TY2 3TQP 3TTH 3TQJ 3TQR 3TRD 4NG4 3TRE 3TRF 3TQS 3TQA 3TQB 3TQ8 3TQ9 3TRG 3TQQ 3TRH 3TRI

1.9 2.0 2.3 2.3 2.6 2.2 1.7 1.8 1.8 1.6 1.5 2.0 2.0 2.4 2.0 1.8 2.8 2.4 2.3 2.8 2.1 2.9 2.7 2.2 2.4 2.5 1.9 2.8 1.9 2.3 2.0 1.6 2.1 1.9 2.2 3.3 2.0 2.0 1.5 2.8 2.9 2.6 2.0 2.3 2.4 1.9 2.3 1.6 2.0 2.2 2.5

20/24 17/20 21/27 21/27 20/27 21/24 17/19 19/23 19/23 16/21 15/17 18/20 18/22 19/22 18/23 17/19 25/27 24/27 19/25 26/30 22/26 21/26 20/25 19/24 20/25 21/28 18/22 25/28 17/21 21/26 20/24 19/21 22/26 20/24 18/22 24/29 23/27 19/23 17/20 27/31 24/27 21/27 19/25 25/31 28/34 18/24 21/27 17/19 20/25 19/23 18/24

0 0.2 0.5 0.5 1.1 0.5 0 0 0 0.4 0.7 0.2 0.2 0 0 0 1.3 0.6 0.5 3.9 0.1 0 0.5 0 0.3 0 0 6.0 0 0 0 0 0 0.8 1.6 1.0 0.5 0.5 0 0.4 0 0.3 0 0.6 0 0.6 0 0 1.3 0 0.4

drug targets in other organisms, indicating that a drug discovery effort targeting the C. burnetii protein is likely to be productive. Others have features that could not easily be predicted from other structures in the PDB, demonstrating the need to examine the C. burnetii structures specifically. Each structure is presented with a small amount of background information. Quinone oxidoreductases (QORs) fall into two broad enzyme families, one membrane-bound42 and one solu-

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ble.43 The two families have distinct enzymatic mechanisms, but both detoxify quinones by reduction using NADH or NADPH as a cofactor.42,43 C. burnetii QOR (CBU_1023) is a member of the soluble family, and has sequence homology to other bacterial QORs as well as to mammalian proteins such as zeta-crystallin.44 Only one protein from this family, an enone oxidoreductase from strawberries (Fragaria x ananassa), has been crystallized with a bound product.45 CBU_1023 is not closely related

Drug Design Against Coxiella Burnetii

Figure 2 Selected C. burnetii structural comparisons. A: The superimposed structures of C. burnetii QOR (CBU_1023) (brown) and the homologous Fragaria x ananassa enzyme (white) are shown in ribbon form. The NADPH cofactor and HDMF reaction product from the Fragaria structure (PDB code 4IDC) are shown in stick form, colored by element with white carbons, with the label “Fa.” Some of the C. burnetii residues mentioned in the text are indicated, labeled “Cb.” B: C. burnetii PPAT (CBU_0288) is shown in ribbon form, colored blue, with stick form depiction of Leu 69. The superimposed S. aureus PPAT (PDB code 4NAT) is shown in ribbon form, colored by element with white carbons, with stick form depiction of Phe 71 and the bound inhibitor. Selected protein residues are labeled with “Cb” for C. burnetii or “Sa” for S. aureus.

to any of the QORs with structures in the Protein Data Bank, having no more than 34% sequence identity to any of them. Although the overall fold of the protein is well conserved, the protein regions that make up the substrate binding environment (residues 45, 48, 53–56, 95–101, 241–244, and 263–265) are quite different compared to the strawberry enzyme, both structurally [Fig. 2(A)] and in sequence: only 5% identity. (This enzyme should still be catalytically active despite the low sequence identity: catalysis is carried out by the bound NADPH, not by protein residues45,46). The binding pocket defined by these residues is twice as large in CBU_1023 as in the strawberry enzyme (583 A˚3 vs. 257 A˚3 as measured by CASTp47) This binding region has been previously observed to be quite variable between different QOR homologues, contributing to different substrate preferences.48 Therefore, the substrate specificity of the C. burnetii QOR will be different from the homologous proteins, and further structural verification will be required to successfully design inhibitors to this enzyme. The enzyme 5-enoylpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes an essential step in the shikimate biosynthetic pathway, which produces chorismate as its end product. A number of pathogenic bacteria require chorismate for pathogenicity49,50 so EPSP synthase has been investigated as a target for broadspectrum antibiotics.51 EPSP synthase from C. burnetii (CBU_0526) falls into the less well studied “Class II” of this enzyme family.52 Class II enzymes are not strongly inhibited by glyphosate52 or other inhibitors designed against Class I EPSP synthases.51 It has been proposed that the Class II enzymes have a more rigid active site

than Class I, causing Class I-targeted inhibitors to be distorted upon binding to Class II.51 This EPSP synthase structure, along with its homologues in the PDB such as Streptococcus pneumoniae (1RF4, 43% identity), Agrobacterium CP4 (2GGD, 42% identity), or Vibrio cholerae (3NVS, 31% identity), will be valuable for designing inhibitors targeting the Class II enzymes. The enzymes pantothenate kinase (coaA) and phosphopantetheine adenylyltransferase (PPAT; coaD) catalyze consecutive steps in the biosynthetic pathway of the essential cofactor coenzyme A. The C. burnetii pantothenate kinase (CBU_0199) is a close homologue of the enzyme from M. tuberculosis, which has recently been the target of a structure-based drug design effort.53 C. burnetii PPAT (CBU_0288) is more distantly related to its homologue in Staphylococcus aureus, which has also been targeted for drug development.54 However, the compounds identified by this screen were not effective against Gram-negative bacteria such as C. burnetii,54 and a comparison of the drug binding sites in the two structures reveals substantial differences. The substitution of Gln 70 (S. aureus) by Pro 68 (C. burnetii) causes the neighboring residue (Leu 69 in C. burnetii) to reorient and protrude into the binding pocket used by the S. aureus inhibitors [Fig. 2(B)]. C. burnetii residues 36–38 are also displaced by this residue substitution, completely reshaping this part of the inhibitor binding pocket. Therefore, a separate drug design effort will be necessary to successfully inhibit the C. burnetii enzyme. The enzymes dihydropteroate synthase (FolP) and dihydrofolate reductase (DHFR; FolA) catalyze steps in PROTEINS

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the biosynthetic pathway of folate, an absolutely essential precursor in nucleic acid biosynthesis.55 Inhibition of this pathway is lethal to any living cell. Dihydropteroate synthase (CBU_1351 in C. burnetii) is the target of the sulfonamide class of antibiotics, the first effective antibacterial drugs ever synthesized.56 Dihydrofolate reductase (CBU_1993 in C. burnetii) has been widely studied as a target for antibacterial agents and cancer therapies.57,58 The combination of a FolP inhibitor such as sulfamethoxazole and a DHFR inhibitor such as trimethoprim has proven to be a very effective antibacterial combination, sometimes called cotrimoxazole.59 However, bacterial resistance to these drugs has developed,59 showing the need for new drugs targeting this pathway. Dihydrofolate reductase drug design

Figure 3 Chemical Structures of DHFR Ligands. DHFR substrate analogues and inhibitors mentioned in the manuscript are depicted. Compound 1a is folic acid, compound 1b is methotrexate, compound 2 is trimethoprim, compound 3 is the trimetrexate analogue discussed in the text, and compound 4 is trimetrexate.

We selected C. burnetii DHFR (CbDHFR) for additional structural analysis precisely because of the wealth of information available on drug development against this target. We intended that our structural work would build upon this base to more readily yield a viable candidate inhibitor. Therefore, we determined the structure of CbDHFR with the bound cofactor NADPH (Supporting Information Fig. S1), then added three ligands to the CbDHFR-NADPH crystals: the substrate analog folic acid (compound 1a) [Figs. 3 and 4(A)], and the inhibitors trimethoprim (compound 2) [Figs. 3 and 4(B)] and methotrexate (compound 1b; Figs. 3 and 5). All structures were solved to high resolution (2.4 2 1.9 A˚; see Table I and Supplementary Structural Data), and all ligands were clearly defined by the electron density (Supporting Information Fig. S2). Structures of human DHFR1NADPH bound to folate, trimethoprim, and methotrexate have been reported previously,60–62 and can be

Figure 4 Substrate binding to C. burnetii DHFR. A: CbDHFR is shown in ribbon form, with stick form depictions of bound NADPH (colored by element, gray carbon atoms) and folate (green carbon atoms). B: A close-up view of the CbDHFR active site shows the protein in ribbon representation, with bound NADPH (gray carbon atoms) and trimethoprim (blue carbon atoms). Trimethoprim from a superimposed hDHFR complex is shown as thin black lines.

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Drug Design Against Coxiella Burnetii

Figure 5 Comparison of C. burnetii and human DHFR binding grooves. A: CbDHFR (in gray) and superimposed hDHFR (in cyan, from PDB code 1U72) are shown as Ca backbone representations. Gly 52 from CbDHFR, and Pro 61, Lys 63, and Asn 64 from hDHFR are labeled. Bound methotrexate from the CbDHFR complex is shown in stick form. B: The same depiction as in panel A, but in a different orientation that matches the orientation in panels C and D. Ile 51 from CbDHFR and the homologous Ile 60 from hDHFR are also labeled. C: CbDHFR is shown as a solvent-accessible surface, with bound methotrexate in stick form (yellow carbon atoms). CbDHFR residues 51–54 are shown in stick form behind the semitransparent surface, with Ile 51 and Gly 52 labeled. D: The hDHFR:methotrexate structure (1U72) is shown in the same orientation and depiction as the CbDHFR structure in panel C. Human residues 60–66 are shown in stick form as in panel C. Ile 60 and Pro 61 (equivalent to C. burnetii residues 51 and 52) are labeled, as are Lys 63 and Asn 64 (no C. burnetii equivalent).

used for comparison. Overall, the human DHFR (hDHFR) and CbDHFR structures are quite similar (1.9 A˚ Ca RMSD, excluding 23 residues in divergent loops), and the residues around the active site superimpose very well (0.7 A˚ Ca RMSD for 16 residues). The NADPH binding is likewise almost identical to that seen in the hDHFR structures. The binding of trimethoprim (2) to CbDHFR shows substantial differences compared to hDHFR [Fig. 4(B)]. Although the molecule occupies the same binding site, and the surrounding protein residues superimpose well, the pose of the trimethoprim molecule is quite different due to changes in the torsion angles (usually denoted u1 and u2) between the 2,4-diaminopyrimidine ring and the trimethoxybenzene ring. The pose of trimethoprim bound to CbDHFR is quite similar to that seen for other

prokaryotic DHFR structures, including other pathogens such as S. aureus63 and M. tuberculosis.64 The altered binding to hDHFR explains why trimethoprim is quite selective for the bacterial enzyme and is therefore an effective, broad-spectrum antibiotic.65 However, resistance to trimethoprim has arisen from DHFR mutations in the bacterial genome59,66 or through the acquisition of a plasmid encoding a resistant DHFR.63,67 Methotrexate (1b) binds to CbDHFR in a very similar orientation as it does to hDHFR, with an all-atom RMSD of 0.65 A˚ when superimposed using the protein residues. The benzene ring and carboxylate tail of methotrexate lie in a groove on the surface of DHFR, defined on one side by helix A, and on the other by the linker between helix B and strand 4 [Fig. 5(A)] (see Supporting PROTEINS

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Figure 6 Compound 3 specifically inhibits CbDHFR. A: The docked position of compound 3 is shown in stick form, with the CbDHFR protein depicted in the same manner and orientation as Figure 5(C). There are no steric clashes to depict in this panel. B: The CbDHFR-docked coordinates of compound 3 are shown superimposed on hDHFR. Regions of DHFR that sterically clash with the compound (interatomic distances < 2.0 A˚) are colored red. C: CbDHFR inhibition by compound 3 was carried out as described in the Methods. Mean normalized activity values are shown; error bars represent SEM (n 5 3). Fitted curve corresponds to an IC50 5 43 lM. D: hDHFR inhibition assay was carried out in the same manner as panel C, although covering a wider concentration range of compound 3. Mean normalized activity values are shown; error bars represent SEM (n 5 2).

Information Fig. S1 for secondary structure element naming) The helix B-strand 4 linker adopts different conformations in hDHFR and CbDHFR [Fig. 5(A,B)] owing to a three-residue insertion in the hDHFR sequence relative to the CbDHFR sequence (Supporting Information Fig. S2). Residue 52 in CbDHFR is a glycine, which occupies the left-handed helical region of the Ramachandran plot. The corresponding residue in hDHFR is a proline, for which this conformation is forbidden; instead, human Pro 61, like the surrounding residues, adopts a right-handed alpha helical conformation. As a result, the binding groove is wide in CbDHFR [Fig. 5(C)] and narrow in hDHFR [Fig. 5(D)]. Nearly all eukaryotic DHFR structures in the PDB, from humans to fungi, have this same 3-residue insertion, with a proline at the same position as human Pro 61, producing essentially the same narrow binding groove (Supporting Information Fig. S4A). In contrast, the large majority of prokaryotic DHFR structures have a glycine at this position, and a binding groove as wide as

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CbDHFR (Supporting Information Fig. S4B). There are a handful of exceptions to this rule (Supporting Information Fig. S4C); the most significant one from a disease perspective is M. tuberculosis,64 which has the same proline, 3-residue insertion, and narrow groove conformation as the eukaryotes. The only archaeal DHFR structure determined to date, from Haloferax volcanii,68 has arginine at the homologous position and a completely different conformation of this region (Supporting Information Fig. S4C). DHFR structures containing the wider binding groove similar to CbDHFR come from a number of wellknown disease agents, including Y. pestis (plague; PDB code 3Q1H), B. anthracis (anthrax),69 S. aureus,63 and E. coli.70 The difference in binding groove width between human and most prokaryotic DHFRs suggests that an inhibitor capable of exploiting this difference could potentially serve as an antibacterial agent against a wide variety of bacterial pathogens in addition to C. burnetii while retaining low toxicity for humans. To the best of our

Drug Design Against Coxiella Burnetii

knowledge, no one has yet described a DHFR inhibitor designed to exploit the binding groove width difference. To begin the design process, we searched ChEMBL32 for previously synthesized antifolate compounds based on aminopteridine or pyridopyridine scaffolds that appeared to have features incompatible with binding in the narrow hDHFR groove. One candidate, a more rigid analog of trimetrexate (compound 6 in Gangjee et al. 1997, shown here as compound 3 in Fig. 3), seemed promising. Trimetrexate (compound 4, Fig. 3) is a hybrid molecule in which the trimethoxybenzyl group of trimethoprim is linked to a quinazoline isostere of the pteridine group of methotrexate.58 Trimetrexate binds and inhibits both mammalian and bacterial DHFRs.71 However, compound 3 features a trans-ethylene unit in place of the aminomethylene linker of trimetrexate, fixing the linkage between the two ring systems into a single rigid conformation. Docking in silico suggests that compound 3 is still able to fit into the wide CbDHFR binding groove [Fig. 6(A)], but it cannot be placed into the narrower human binding groove without large steric clashes, especially with Pro 61 and Asn 64 of hDHFR [Fig. 6(B)]. These clashes explain the reported 4300-fold reduction in affinity to mammalian (rat) DHFR of compound 3 compared to trimetrexate.37 To confirm that this compound can be selective for CbDHFR over hDHFR, we synthesized the compound, and assayed its inhibition potency against both enzymes. The compound inhibited CbDHFR with an IC50 of 43 lM [Fig. 6(C)], whereas it showed no detectable inhibition of hDHFR up to the compound’s solubility limit of 1 mM [Fig. 6(D)]. This result suggests that the rigidification of the linker has produced the desired selectivity for CbDHFR (at least 25fold compared with hDHFR), although it has come at the price of a substantial loss in affinity. Further modifications of this compound will be necessary to improve CbDHFR affinity and to increase aqueous solubility. We intend to continue developing inhibitors that exploit the groove width difference between CbDHFR and hDHFR, starting from the compound described here, or from other frameworks that rely on large, bulky groups placed into the wide CbDHFR groove. Any compounds that show promise in the enzymatic assay described here could then be tested against DHFRs from other pathogenic bacteria, and ultimately in growth inhibition tests using the pathogens themselves. Further structure-based refinement of the drug design would be performed, with the ultimate goal of producing a broad-spectrum antibiotic agent with minimal toxicity to humans. CONCLUSION The 48 structures of C. burnetii proteins presented here provide valuable information for drug discovery efforts against this highly infectious bacterium. Our follow-up work on C. burnetii dihydrofolate reductase identified a compound that is able to exploit a structural

difference in substrate binding grooves between the C. burnetii and human versions of this enzyme. This structural difference is also present in other bacteria, so a drug based on the compound we identified could have broad applicability to a wide range of pathogens that have already developed resistance to existing treatments. A novel broad-spectrum antibacterial agent is a critical medical need in a world of multidrug-resistant bacterial diseases. AUTHOR CONTRIBUTIONS MCF, JC, MJR, and JL designed the experiments. MCF, JC, MJR, FB, MC, EG, BH, and JL performed the experiments. MCF, PRC, and MT carried out the CbDHFR inhibitor design. ZKY and PRC performed the CbDHFR inhibitor synthesis. MCF wrote the manuscript with assistance from JC, MJR, PRC, and MT. ACKNOWLEDGMENTS The authors thank Wayne A. Hendrickson and Willa Appel for their comments on the manuscript. They thank the staff of beamlines X4 and X29 at the National Synchrotron Light Source for their assistance in data collection. Use of the National Synchrotron Light Source, Brookhaven National Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC0298CH10886. C. burnetii expression constructs prepared during this study have been deposited in the DNASU Plasmid Repository. REFERENCES 1. Burnet FM, Freeman M. Experimental studies on the virus of Q fever. Med J Aust 1937;2:299–302. 2. Cox HR. A filter-passing agent isolated from ticks. III. Description of organism and cultivation experiments. Public Health Rep 1938; 53:2270–2276. 3. Derrick EH. “Q” fever, a new fever entity: clinical features, diagnosis, and laboratory investigations. Med J Aust 1937;2:281– 299. 4. Maurin M, Raoult D. Q fever. Clin Microbiol Rev 1999;12:518–553. 5. Edouard S, Million M, Royer G, Giorgi R, Grisoli D, Raoult D. Reduction in incidence of Q fever endocarditis: 27 years of experience of a national reference center. J Infect 2014;68:141–148. 6. Delsing CE, Kullberg BJ, Bleeker-Rovers CP. Q fever in the Netherlands from 2007 to 2010. Neth J Med 2010;68:382–387. 7. Oyston PC, Davies C. Q fever: the neglected biothreat agent. J Med Microbiol 2011;60:9–21. 8. Jones RM, Nicas M, Hubbard AE, Reingold AL. The Infectious Dose of Coxiella Burnetii (Q Fever). Appl Biosafety 2006;11:32–41. 9. Beare PA, Heinzen RA. Gene inactivation in Coxiella burnetii. Methods Mol Biol 2014;1197:329–345. 10. Omsland A, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, Porcella SF, Heinzen RA. Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci USA 2009;106:4430–4434.

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Structural genomics for drug design against the pathogen Coxiella burnetii.

Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biolo...
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