A Structural Biology Approach Enables the Development of Antimicrobials Targeting Bacterial Immunophilins Darren W. Begley,a David Fox III,a Dominic Jenner,b Christina Juli,c Phillip G. Pierce,a Jan Abendroth,a Muigai Muruthi,a Kris Safford,a Vanessa Anderson,a Kateri Atkins,a Steve R. Barnes,a Spencer O. Moen,a Amy C. Raymond,a Robin Stacy,a,d Peter J. Myler,d,e Bart L. Staker,a Nicholas J. Harmer,f Isobel H. Norville,b Ulrike Holzgrabe,c Mitali Sarkar-Tyson,b Thomas E. Edwards,a Donald D. Lorimera Emerald Bio, Bainbridge Island, Washington, USAa; Defence Science and Technology Laboratory, Porton Down, Salisbury, United Kingdomb; Institute of Pharmacy and Food Chemistry, University of Würzburg, Würzburg, Germanyc; Seattle Biomedical Research Institute, Seattle, Washington, USAd; Departments of Global Health and Medical Education and Biomedical Informatics, University of Washington, Seattle, Washington, USAe; School of Biosciences, University of Exeter, Exeter, United Kingdomf

Macrophage infectivity potentiators (Mips) are immunophilin proteins and essential virulence factors for a range of pathogenic organisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the causative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macrocyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency for BpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compound series with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. The best compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhibited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macrophages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectivity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new class of antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery.

B

urkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to southeast Asia and northern Australia. Melioidosis is believed to be the third most common cause of death by infectious disease in northeastern Thailand, after human immunodeficiency virus (HIV) and tuberculosis (1). Multiple factors, including its frequent misdiagnosis as tuberculosis infection and the ability of B. pseudomallei to evade the host immune response, have led to its designation as an emerging infectious disease and a potential bioterrorism agent by the National Institute of Allergy and Infectious Diseases (NIAID) (2). We recently identified a macrophage infectivity potentiator (Mip) protein from B. pseudomallei (BpML1) that is important for intracellular replication and required for full virulence (3). Homologous Mip proteins in Legionella pneumophila and Trypanosoma cruzi also appear to be involved in the infection machinery and are required for full virulence in these distinct pathogenic organisms (4, 5). BpML1 is an immunophilin, part of a well-conserved class of peptidyl-prolyl isomerases (PPIases), which include FK506-binding proteins (FKBPs) (6–8). Binding of the potent fungal macrocyclic compounds rapamycin (Sirolimus) and FK506 (Tacrolimus) to PPIases in lymphocytes has a well-characterized immunosuppressive effect unrelated to their PPIase function and allows for modern organ transplantation (9–11). Human PPIase cyclophilin A can influence the infectivity of HIV-1 virions (12–16), and cellular expression levels of cyclophilin A can alter the replicative ability of other viruses, in part via regulation of type 1 interferons (17–19). Thus, the roles of immunophilins in infection and the inhibitory effects on PPIases with known natural product inhibitors make them attractive targets for novel drug discovery, if suitable hostpathogen selectivity can be achieved. Descriptions of human FKBPs that have been targeted with small synthetics have been reported in the literature. These repre-

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sent attempts to influence protein localization, signaling pathways, and protein activation with smaller lead-like molecules dispossessed of the immunosuppressive effects of FK506 and related macrocycles (20–23). This work describes our initial efforts to design and characterize small lead-like molecules that can influence the virulence of B. pseudomallei through BpML1 inhibition without compromising host immunity. Through synthetic variation and testing, we replaced the anchoring moiety in known macrocyclic inhibitors with a select set of small-molecule pipecolic acid derivatives, forming an important anchoring scaffold (24, 25). We obtained cocrystallization structures to demonstrate that BpML1 binds to our pipecolic acid derivatives in a manner analogous to the piperidine moieties of macrocyclic FK506 (26, 27) and rapamycin (28), allowing identification of the essential chemical scaffold necessary for binding. Nuclear magnetic resonance (NMR) studies on compounds from this series have revealed a range of micromolar affinities and confirmed binding along a hydrophobic groove only minimally exploited by either macrocycle in solution (29, 30). We show here that these compounds inhibit the in vitro PPIase activity of BpML1 as well as Mip proteins from other pathogenic organisms. Most importantly, these compounds

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Received 27 August 2013 Returned for modification 2 October 2013 Accepted 26 November 2013 Published ahead of print 23 December 2013 Address correspondence to Darren W. Begley, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.01875-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.01875-13

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reduce the virulence of B. pseudomallei in its ability to infect human macrophages, with no significant cytotoxicity for macrophage cell lines. The structural and functional data generated from this report thus provide a basis for rational, structure-based design of novel Mip inhibitors with the potential to minimize the virulence effects of B. pseudomallei and other pathogenic organisms. MATERIALS AND METHODS Protein production. The BpML1 construct (clone BPSS1823; SSGCID ID BupsA.00130.a.D21) was engineered and optimized for expression in Escherichia coli by using Gene Composer software (31, 32). Synthetic genes were purchased from DNA 2.0 and cloned into a modified pET28 vector by using the polymerase incomplete primer extension (PIPE) cloning method (33). Insert PCR (iPCR) of the BpML1 synthetic gene was carried out with chimeric primers having homology to gene termini (25 bp of homology) and the vector junction (15 bp of homology). A pET28 vector engineered to donate an amino-terminal 6His-Smt tag (MSHHH HHHSGEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKR QGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGS) was amplified by vector PCR (vPCR) (31). The iPCR thermal cycling parameters were as follows: initial denaturation for 2 min at 95°C, then 25 cycles of 95°C for 30 s, 50°C for 45 s, and 68°C for 3 min, followed by a final holding temperature at 4°C. The vPCR cycling parameters were as follows: initial denaturation for 2 min at 95°C, then 25 cycles of 95°C for 30 s, 50°C for 45 s, and 68°C for 14 min, followed by holding at 4°C. Neither reaction included a final extension step, allowing the products to remain variably single stranded at the termini. All PCRs were performed using PfuUltra Hotstart master mix (Stratagene Agilent). Equal volumes of crude iPCR and vPCR products were merged and transformed into TOP10 chemically competent cells according to the manufacturer’s specifications. Once sequence-verified clones were identified, these expression plasmids were transformed into BL21(DE3) cells. BpML1 was expressed in E. coli BL21(DE3) cells in both rich TB autoinduction medium with kanamycin and M9 minimal medium with 15NH4Cl and kanamycin with 2% (vol/ vol) glycerol as a carbon source. Starter cultures were grown in TB medium (T7060; Teknova) for 18 h at 37°C and then used to inoculate 2-liter flasks of minimal medium. After 6 h of growth at 37°C and 220 rpm, the temperature was reduced to 20°C and cells were induced with 1 mM isopropyl-␤-D-thiogalactopyranoside and grown for 24 h. To harvest, the cultures were centrifuged at 5,000 relative centrifugal force for 15 min at 5°C, and the cell paste was frozen at ⫺80°C. Frozen cell paste was resuspended in the following lysis buffer: 25 mM Tris, 200 mM sodium chloride, 50 mM L-arginine monohydrochloride, 10 mM imidazole, 0.5% (vol/vol) glycerol, 1 mM Tris(2-carboxyethyl) phosphine (TCEP), 0.02% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; pH 8.0) containing 10 ␮l benzonase, 100 mg lysozyme, and one EDTA-free Complete protease inhibitor tablet (Roche); cells were then lysed by sonication. The lysate was clarified by centrifugation. Clarified lysate was first purified by immobilized metal affinity chromatography (IMAC) with a HiTrap chelating high-performance (HP) column (PN 17-0409-03; GE Life Sciences) charged with Ni2⫹ using the ProteinMaker system (34). The column was equilibrated with wash buffer (20 mM Tris, 200 mM sodium chloride, 50 mM L-arginine monohydrochloride, 10 mM imidazole, 1 mM TCEP; pH 8.0), and the protein of interest was eluted with wash buffer containing 500 mM imidazole, excluding L-arginine. Some eluted protein was treated with ubiquitin-like protease 1 (Ulp1) to cleave the 6His-Smt tag, leaving an N-terminal serine after the QIGG residue sequence, and was simultaneously dialyzed into wash buffer. The target was further purified with a second Ni2⫹ IMAC step by using a HiTrap chelating HP column equilibrated with wash buffer. The flowthrough containing the cleaved target protein was then concentrated with centrifugal filters (PN VS2092; Sartorius Stedim), and injected over a HiPrep 16/60 Sephacryl S-100 HR (171165-01; GE Life Sciences) size exclusion chromatography (SEC) column equilibrated with SEC running buffer (25 mM Tris [pH 8.0], 200 mM

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sodium chloride, 1 mM TCEP, and 1% [vol/vol] glycerol). The purified protein of interest was concentrated to a target concentration of 10 mg/ml as measured with a Nanodrop ND-1000 spectrophotometer and stored at ⫺80°C. The uncleaved target was obtained by employing Ni2⫹ IMAC and SEC; however, treatment with Ulp1 was not performed. Cocrystallization and structure determinations of BpML1 complexes. Crystals of Smt-fused to BpML1 (wild type or D44G) were grown by sitting drop vapor diffusion at 295 K using 10 to 20 mg/ml protein in the presence of 2 to 5 mM compound over the course of several weeks. Compound cocrystals with BpML1 were grown under the following conditions: FK-506 (0.1 M MIB buffer [pH 9.0], 25% [wt/vol] polyethylene glycol 1500 [PEG 1500] cryoprotected with 30% [wt/vol] PEG 400), compound 37 (10% [wt/vol] PEG 20,000, 20% [vol/vol] PEG MME550, 0.03 M halides [NaF, NaBr, NaI], 0.1 M morpholineethanesulfonic acid [MES]-imidazole [pH 6.5]) directly cryopreserved, compound 40 (10% [wt/vol] PEG 20,000, 20% [vol/vol] PEG MME550, 0.03 M divalent cations [MgCl2, CaCl2], 0.1 M MES-imidazole [pH 6.5]) directly cryopreserved, compound 168 (50 mM ammonium formate, 24.55% [wt/vol] PEG 3350) cryopreserved with 10% (vol/vol) ethylene glycol, and compound 183 (50 mM ammonium formate, 30% [wt/vol] PEG 3350) cryoprotected with 25% (vol/vol) ethylene glycol. In each case, the drop contained 2 to 5% (vol/vol) dimethyl sulfoxide (DMSO) and 100 mM compound. All data sets were collected with the Advanced Light Source (ALS) beamlines 5.0.1 and 5.0.3 equipped with ADSC 315R charge-coupleddevice detectors. Diffraction data were reduced and scaled by using XDS/ XSCALE (35–37). Each structure was solved by molecular replacement via preexisting structures of BpML1 and Smt. Structures were refined by using iterative cycles of TLS and restrained refinement with REFMAC5 (38), part of the CCP4 program suite (39), and by model building using the crystallographic object-oriented tool kit (Coot) (40). All structures were peer reviewed internally and validated using Molprobity (41) prior to deposition in the Protein Data Bank (42, 43). Diffraction data and refinement statistics are listed for each structure in Tables S1 and S2 of the supplemental material. NMR spectroscopy. NMR samples were prepared for screening by diluting concentrated, 15N-labeled BpML1 protein to 330 ␮M in NMR buffer (50 mM NaCl, 50 mM K-Phos [pH 7.0], 20% [vol/vol] D2O, 10% [vol/vol] DMSO-d6). Samples prepared for titration experiments contained a protein concentration of 200 ␮M, with compound:protein ratios of 0.25 to 7.50. All experiments were conducted on a Varian Inova 500MHz NMR spectrometer equipped with a standard HCN probe at a temperature of 25°C. Screening was completed by using protein-observe, two-dimensional (1H-15N) band-selective optimized-flip-angle short transient– heteronuclear multiple quantum coherence spectroscopy (SOFAST-HMQC) according to previously published methods (44, 45). Briefly, 8 scans and 2,048 by 48 points were collected, with a total recycle delay of 0.3 s. NMR spectra were processed using the Varian VnmrJ Biopack software and analyzed using either MestReNova (Mestrelab) or iNMR (http://www.inmr.net). Resonance assignments were transcribed from previously determined NMR structures, made available by the Biological Magnetic Resonance Data Bank (BMRB) (46). Chemical shift perturbation (CSP) values for each residue were calculated based on the following equation: ⌬␦ ⫽ [(⌬1H)2 ⫹ (⌬15N/5)2]1/2. CSP values were fitted for each resonance and titration by using GraphPad Prism version 5 according to equation 1: ⌬␦ ⫽ (⌬␦max) ⫻

(Pt ⫹ L ⫹ KD) ⫺ 兹(Pt ⫹ L ⫹ KD)2 ⫺ 4PtL 2Pt (1)

where Pt is the total protein concentration, L is the concentration of compound, KD is the dissociation constant, and ⌬␦max is the maximum CSP observed in the titration (47). Final KD values were calculated using data from three reporter residues (D48, V62, and G93) acquired from duplicate titrations.

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TABLE 1 Structures of BpML1 and variants bound to FK506 and select pipecolic acid derivativesa PDB IDb

SSGCID ID

Method

Space group

Mutation

6His-Smt tag intact?

Ligand (PDB ligand name)

3UF8 3UQA 3UQB 3VAW 4DZ2 4DZ3 4FN2 4G50 4GGQ 4GIV 2KE0* 2KO7* 2L2S* 2Y78*

BupsA.00130.a.D242 BupsA.00130.a.D220 BupsA.00130.a.D214 BupsA.00130.a.D24 BupsA.00130.a.D239 BupsA.00130.a.D227 BupsA.00130.a.D214 BupsA.00130.a.D214 BupsA.00130.a.D21 BupsA.00130.a.D214 BupsA.00130.a BupsA.00130.a BupsA.00130.a NA

XRD XRD XRD XRD XRD XRD XRD XRD XRD XRD NMR NMR NMR XRD

P 21 C2 P 21 P 21 C2 C 2 2 21 P1 P1 P1 C2 NA NA NA P43 21 2

G95A A54E D44G V3I R92G M61H D44G D44G Native D44G Native Native Native Native

Yes Yes Yes Yes No No Yes Yes Yes Yes No No No No

FK506 (FK5) FK506 (FK5) FK506 (FK5) FK506 (FK5) FK506 (FK5) FK506 (FK5) Compound 37 (854) Compound 168 (861) Compound 40 (861) Compound 183 (4GI) apo Cycloheximide N-ethylethanoate (JZF) 1-{[(4-Methylphenyl)thio]acetyl}piperidine (L2S) apo

a b

Abbreviations: XRD, X-ray diffraction; NMR, nuclear magnetic resonance; NA, not available. * PDB ID information was previously reported for these structures (51).

Peptidyl-prolyl cis/trans isomerase assay. For the peptidyl-prolyl cis/ trans isomerase assay, inhibitors were dissolved in DMSO to a final concentration of 10 mM and stored at ⫺20°C. Recombinant BPSS1823 protein was produced as described previously (3). The peptidyl-prolyl cis/trans isomerase activity of recombinant BPSS1823 was determined by using a protease-coupled assay as described previously (48). Briefly, 50 nM BPSS1823 protein was incubated for 6 min at 10°C in 1.2 ml 35 mM HEPES buffer (pH 7.8) with succinyl–Ala– Phe–Pro–Phe–p-nitroanilide (10 mg/ml stock; Bachem). Chymotrypsin (Sigma-Aldrich, United Kingdom) was added to the cuvette at a final concentration of 0.8 mg/ml and mixed. Hydrolysis of the substrate was measured at 390 nm by using a Shimadzu 1800 UV/visible spectrophotometer at 1-s intervals until there was no further change in absorbance. For inhibition measurements, BPSS1823 was preincubated with 50 nM to 50 ␮M compound for 6 min prior to the addition of the chymotrypsin. Three independent readings were taken at each concentration with each inhibitor. Positive (no inhibitor) and negative (no BPSS1823) controls were also included. The pseudo-first-order rate constant was calculated using equation 2 (49); data from 10 to 50 s (following the lag phase and before substrate became limiting) were fit to the equation, and kobs was calculated by linear regression. In[A⬁ ⫺ At] ⫽ ⫺kobs t ⫹ In[A⬁ ⫺ A0]

(2)

The enzymatic rate (kenz) was determined by comparing the observed rate (kobs) with the uncatalyzed rate (kuncat), using equation 3: kenz ⫽ kobs ⫺ kuncat

(3)

RESULTS AND DISCUSSION

The KI data were fit to equation 4 (50, 51): [E] ⫺ [I] ⫺ K ⫹ 兹([E] ⫺ [I] ⫺ K)2 ⫹ 4[E][K] v ⫽ v0 2[E]

(4)

Using least-square nonlinear fitting, ␯0 and KI were fit using initial estimates based on the raw data, and [E] was kept constant. Bacterial strains. B. pseudomallei K96243 was routinely grown on Luria agar (L-agar) at 37°C overnight. Fresh bacterial streak plates were used for all cell-based assays. B. pseudomallei ⌬BPSS1823 was constructed as previously described (3). B. pseudomallei cytotoxicity assays. J774A.1 cells (ECACC; HPA Porton Down, Salisbury, United Kingdom) were seeded onto a 24-well tissue culture plates and used at a density of 1 ⫻ 106 cells in Dulbecco’s modified Eagle medium (DMEM; Gibco, Paisley, United Kingdom) supplemented with 10% fetal bovine serum (FBS; Gibco, Paisley, United Kingdom) and 40 mM glutamine (Sigma-Aldrich, United Kingdom). B.

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pseudomallei K96243 or B. pseudomallei ⌬BPSS1823 was harvested into 1 ml Leibovitz L-15 medium supplemented with 10% fetal calf serum (L15; Gibco, Paisley, United Kingdom) to an absorbance at 600 nm of 0.25 to 0.3. B. pseudomallei K96243 was treated with 50 ␮M each inhibitor or DMSO alone for 1 h. J774A.1 cells were infected with treated B. pseudomallei K96243 at a multiplicity of infection (MOI) of approximately 400:1 for 40 min at 37°C. Postinfection, the bacteria were removed and replaced with 1 ml L15 supplemented with 15 ␮g/ml gentamicin and the appropriate inhibitor at 50 ␮M. Infected J774A.1 cells were incubated for 24 h at 37°C before the supernatant was tested for lactate dehydrogenase (LDH) release via the CytoTox 96 nonradioactive cytotoxicity assay (Promega, Southampton, United Kingdom) per the manufacturer’s instructions. All results are presented as the means of three independent experiments. Infected cells treated with DMSO or uninfected cells were included as controls. To express the results as the percentage of wild-type cytotoxicity, the LDH activity in test compound wells was compared to that in wells infected with B. pseudomallei K96243 treated with DMSO. Spontaneous release (LDH release following incubation of uninfected cells with the corresponding inhibitor or DMSO) was subtracted from the final values. All statistical analyses were carried out using a repeated measures analysis of variance with Bonferroni posttests and analyzed using GraphPad Prism version 5. Protein structure accession numbers. The BpML1 mutant proteins described here (i.e., those that yielded high-quality diffracting crystals) were submitted to the Protein Data Bank and assigned the accession numbers summarized in Table 1.

Generation of a crystal form suitable for BpML1–small-molecule complexes. In the published apo crystal form of BpML1, the purification tag cleavage site peptide sequence on one molecule fills the FK506-binding site of a neighboring molecule in the crystal lattice (see Fig. S1 in the supplemental material) (51). Multiple crystallization efforts with native sequence BpML1 constructs with tags removed failed to yield crystals. Therefore, to obtain alternative crystal forms not dependent on the cleavage site sequence interaction with the protein, a series of BpML1 point mutations was designed using GeneComposer software (31, 32, 52). Via GeneComposer, specific residues were identified that could alter the crystal lattice while minimizing the impact on binding site topology, based on crystal contacts observed in the apo form of the BpML1 structure. These residues were mutated according to

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genetic variants from related organisms, which were found by using the basic local alignment search tool (BLAST) database of the National Center for Biotechnology Information (NCBI) (53). To minimize disruption of the overall native fold of the protein, only single point mutations were selected for construct design, resulting in 51 gene variants. Small-scale expression tests showed 47 out of 51 constructs produced measurable amounts of protein, including both higher and lower bacterial expression levels relative to the native sequence (see Fig. S2 in the supplemental material). A subset of these BpML1 point mutants was selected for scale-up, purification, and crystallization (see Materials and Methods). Crystallization of 15 purified BpML1 mutants with and without cleavage of the 6His-Smt solubility tag (54) was attempted in the presence and absence of FK506. Among the BpML1 mutant proteins tested, six yielded high-quality diffracting crystals, all in the presence of FK506 (Table 1). Crystals of BpML1 mutants R92G and M61H with the 6His-Smt tag removed exhibited ligand-dependent dimerization; adjacent BpML1-bound molecules of FK506 make direct contact within the asymmetric unit (see Fig. S3 in the supplemental material). The extensive ligandligand interactions observed for FK506 suggest that this crystal form may not be suitable for crystallization experiments with novel small molecules. The remaining four crystal structures were produced from uncleaved N-terminal 6His-Smt-tagged BpML1 mutants (Table 1). The lattices in these crystal systems create an open FK506-binding pocket and are stabilized by a continuous 10-stranded ␤-sheet formed between BpML1 and Smt. This stabilization involves direct contact between BpML1-␤1 and Smt-␤2 of a neighboring symmetry-related molecule (see Fig. S4 in the supplemental material). Of all the BpML1 variants screened, the D44G mutant was the most consistent in producing high-quality crystals, and it outperformed the native sequence under a number of sparse matrix conditions. We validated the use of the D44G mutant for biophysical studies by comparing crystal structures of FK506-bound BpML1 with and without the D44G mutation, and we found no significant detriment to FK506 binding resulting from this mutation. The native sequence D44 side chain forms a hydrogen-bonding network with FK506 and the hydroxyl of Y33 and may participate in a weak salt bridge with R49, helping to stabilize this loop (see Fig. S5 in the supplemental material). In the D44G BpML1 mutant, a water molecule occupies the position of one D44 carboxylate oxygen, allowing polar contacts between FK506 and Y33, which recapitulate the FK506-bound structure in the native sequence. Furthermore, previous solution-state NMR studies suggested the identity of this residue to be unimportant for binding small-molecule inhibitors, with a wide range of side chain conformations detected (51). Therefore, we prioritized cocrystallization of the native and D44G mutant 6His-Smt–BpML1 proteins with selected pipecolic acid derivatives for structural characterization. Identification of a key chemical scaffold for binding BpML1. Cocrystallization trials were set up with 16 different pipecolic acid derivatives (24) and BpML1, from which compounds 37, 40, 168, and 183 yielded crystals with sufficient X-ray data for structure determination (Fig. 1). Three of the four structures contained the D44G mutant, while the complex of BpML1 with compound 40 was solved with the native sequence. The N-terminal 6His-Smt tag was attached in all four cases, with a majority of the Smt residues resolved in each structure (for complete crystallization data, see

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Tables S1 and S2 in the supplemental material). Compounds 37, 40, and 183 were added as racemates, while compound 168 was the S enantiomer of compound 40 (see Fig. S6 in the supplemental material). In all four structures solved, only the S conformation of each pipecolic acid derivative was observed (Fig. 1). This structural preference for the S conformation was reflected in the 4-foldhigher inhibitory strength observed with pure enantiomer compound 168 over the racemate compound 40 (see below). The central pipecolic acid scaffold binding conformation was largely conserved across the four complexes and demonstrated no structural dependence on the presence of a D44 side chain. Moreover, the lone structure solved for the native sequence showed that this aspartyl group rotates away from the FK506-binding site, not participating in any intra- or intermolecular interactions (Fig. 1). A sulfonyl oxygen for compounds 37, 40, and 168 each makes hydrogen bond contacts with the hydroxyl group of Y89, a close contact also observed by solution-state NMR (see below). The sulfonyl piperidine scaffolds of compounds 37, 40, and 168 generated virtually identical binding poses in their respective asymmetric units, similar to that seen previously in computational docking studies, as well as with the chemically distinct biphenyl piperidine (PDB ID 1FKG) (24, 55). In contrast to the best conformation determined from our previous docking analysis, the carbonyl ester of compound 168 coordinated with the backbone amide of I63 in the crystal complex, and the terminal phenyl ring participated in the ␲-edge stacking interaction with F43 (24). Variability in the position of trimethoxybenzene was observed crystallographically, with the most common conformation observed for this group overlapping the same subpocket as the methoxy-cyclohexanol of FK506 and rapamycin (Fig. 1). Although the binding conformation of compound 183 is similar to that of the other pipecolic acid derivatives, the carbonyl ester of compound 183 is sufficiently rotated to abrogate any hydrogen bond with the I63 backbone amide in the crystal. The terminal phenyl group of compound 183 is also rotated ⬃90° from its position in compound 168, with nearly 20° of rotational difference between the chain A and chain B conformations (Fig. 1). The crystal lattice observed for compound 183 also differed from that of the other pipecolic acid derivatives and exhibited the same type of ligand-induced dimerization pattern seen for BpML1 bound to FK506 when the 6His-Smt tag was cleaved. Insufficient electron density was observed to be able to model the nitro group as well as most of the linked pyridine moiety of compound 183. However, the close proximity of the benzene ring to the short F43-G44 ␤-strand makes it an unlikely direction for the meta nitro group to be positioned. The flexible outer loop from A94 to I98 clearly shifts outward, relative to the same loop observed for the other three ligand-bound complexes, most likely to accommodate the nitrobenzene scaffold. Solution-state NMR characterization of pipecolic acid derivatives with BpML1. Compounds 37, 40, 168, and 183 were titrated into samples of 15N-labeled, tag-cleaved, native sequence BpML1 (clone BPSS1823) to calculate biophysical binding constants by NMR spectroscopy (Table 2). Overall, chemical shift changes to BpML1 observed during pipecolic acid titrations were indicative of fast on/off rates relative to the NMR time scale, in contrast to slow on/off binding for analogous spectra acquired when titrating with FK506 (Fig. 2). Using the SOFAST-HMQC pulse sequence (44, 45), chemical shift perturbations (CSPs) were monitored for three equally spaced residues (D48, V62, and G93)

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FIG 1 Structure ensemble for pipecolic acid derivative binding to BpML1. Four crystallographic poses were observed for compound 40 (lower left), while two poses each were obtained for compounds 37 (top left), 168 (top right), and 183 (bottom right). A full model complex for compound 183 is postulated (gray) and is based on NMR data and available space in the crystal structure. All structures depict polar contacts between the small molecule and residues from chain A in each PDB entry. Noncarbon atoms for each compound are colored as follows: red, oxygen; blue, nitrogen; gold, sulfur). The figure was generated using PyMol (57).

around the binding site to calculate a mean dissociation constant (KD) (see Fig. S7 in the supplemental material). Compound 37 was found to be the weakest binder, and compound 168 was observed to have nearly 4 times the binding affinity of compound 40,

TABLE 2 NMR-based binding affinity, PPIase activity, and select properties for BpML1 and compounds 37, 40, 168, and 183 Compound

KIa PPIase activity (␮M)

KDb NMR (␮M)

BEIc

C(log P)d

37 40 168 183

10 ⫾ 3 0.7 ⫾ 0.2 0.17 ⫾ 0.05 3.6 ⫾ 0.7

190 ⫾ 15 51 ⫾ 6 14 ⫾ 4 10 ⫾ 2

12 8.7 9.9 12

3.1 4.4 4.4 3.2

a For calculation of the KI, see equation 4 in Materials and Methods. Values reported as mean ⫾ standard error of the mean. b For calculation of the KD, see equation 1 in Materials and Methods. Values reported as mean ⫾ standard error of the mean. c BEI, binding efficiency index. This value was calculated as follows: ⫺log(KD[M])/(molecular mass); the units for mass are kDa, and the KD was determined via NMR (see reference 58). d The C(log P) is the water/octanol partition coefficient, calculated using ChemDraw Ultra version 12.0.

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a result consistent with in vitro activity data and the low binding potential of the R enantiomer. Compounds 168 and 183 were observed to have essentially the same affinities based on NMR, a result which deviated somewhat from the relative potencies determined in the in vitro PPIase inhibition assay (Table 2). This may be due to the higher concentrations required for NMR, limiting the sensitivity for KD determinations of the more potent molecules (56). An additional 8 compounds from the series that had failed in crystallization trials were also tested by HSQC. None of these generated significant chemical shift perturbations for the protein in solution, despite showing inhibitory PPIase activities, and they were excluded from further study by NMR. SOFAST-HMQC spectra with and without saturating amounts of compound provided structural details on the solution state binding interactions between pipecolic acid derivatives and BpML1. Strong CSPs for V62 and I63, and the relatively weak shifts for F53, suggest that the methoxy-cyclohexanol subpocket of FK506 is the favored solution-state binding region for the pyridine and trimethoxybenzene groups of compounds 40, 168, and 183 (Fig. 3). The I63 amide signal for BpML1 generates the largest CSPs of any residue in the protein (see Fig. S8 in the supplemental

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FIG 2 BpML1 NMR titrations. An apo protein HSQC amide spectrum at 200 ␮M (black) is shown overlaid with spectra acquired in the presence of 80 ␮M (red), 120 ␮M (green), and 140 ␮M (blue) FK506 (left). An apo protein SOFAST-HMQC amide spectrum at 200 ␮M (black) is shown overlaid with spectra acquired in the presence of 50 ␮M (red), 100 ␮M (green), and 200 ␮M (blue) compound 183 (right). The insets (gray boxes) show a Gly93 N-H residue, which gives rise to duplicate peaks for FK506, indicative of a two-state slow exchange, versus the exchange-averaged Gly93 peak when compound 183 is added. The figure was generated using iNMR.

material), with the signal nearly abolished when any of the four pipecolic acid derivatives was added. This result confirmed hydrogen bond formation between I63 and the carbonyl ester of each compound, interactions which recapitulate the more sterically

constrained hydrogen bond formed when FK506 is bound. Medium to strong chemical shifts were also seen in the A94-to-I98 loop for compounds 40, 168, and 183, due to proximity of the terminal phenyl group when bound. These interactions mapped

FIG 3 Surface renderings of chain A from 4 different crystal structures of BpML1 complexed with compounds 37 (top left), 168 (top right), 40 (bottom left), and 183 (bottom right). Residues exhibiting large (orange) and moderate (green) CSPs upon addition of each compound in solution are mapped on each structure. The figure was generated using PyMol (57).

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.

FIG 4 Crystal structures of BpML1 bound to FK506 (brown; PDB ID 3UQB) and compound 168 (cyan; PDB ID 4G50). The conformational change within the R92-I98 loop accommodates the terminal phenyl group of compound 168 (bottom), relative to the FK506-bound structure (top). The R92 side chain is not modeled in the FK506 structure (top) due to insufficient electron density. The figure was generated using PyMol (57)

to conformational changes observed crystallographically, as seen with loop R92-to-I98, adapting to the benzene ring of compound 168, relative to the loop conformation when bound to FK506 (Fig. 4). The accommodation of these phenyl moieties by BpML1 represent protein-ligand binding interactions in which FK506 and rapamycin do not participate, and may serve as a foothold in developing species-specific protein inhibition compounds. The CSP for BpML1 residue A54 was higher in the presence of compound 183 versus the others, indicating a greater likelihood that compound 183 samples alternate conformations in solution. This was reflected in the poor electron density observed for compound 183, which prevented accurate modeling of any pyridine ring conformations. However, the binding efficiency for compound 183 (Table 2) was equivalent to that of compound 37, indicative of methyl pyridine being an effective moiety added to the original scaffold on a per-atom basis. Furthermore, the NMR data showed that the strongest interaction for the pyridine ring of compound 183 is with the methoxy-cyclohexanol binding pocket

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and is indicated in our proposed full complex model (Fig. 2). CSPs for Y89 were also much higher for compound 183 than for compounds 40 and 168, while the CSPs were quite similar for the extended A94-to-I98 loop region. This key CSP difference for compound 183 validates our proposed model structure, in which the preferred conformation permits contact between the meta nitro group and the hydroxyl of Y89, with similar accommodation for the hydrophobic moiety in the A94-to-I98 flexible loop (Fig. 3). PPIase activity inhibition by pipecolic acid derivatives. In a protease-coupled assay, the specificity constant kcat/Km for the BpML1 construct selected for study (BPSS1823) was previously determined to be 6.7 ⫻ 106 ⫾ 0.4 ⫻ 106 M⫺1 s⫺1, with inhibition by the known PPIase inhibitor rapamycin at a KI of 3 ⫾ 2 nM (values are means ⫾ standard errors of means) (3). Pipecolic acidderived compounds have also been reported for inhibitory properties against human FKBP12 and the Legionella pneumophila Mip protein (24). We tested a wide selection of pipecolic acid derivatives, resulting in data for a select number of agents with inhibitory properties against BpML1 (Table 2). The racemic compounds 37 and 183 inhibited the PPIase activity of BpML1 at low micromolar concentrations, while the S enantiomer, compound 168, and racemic compound 40 inhibited the activity with a high-nanomolar KI. Compound 168 is the enantiomerically pure form of compound 40 (see Fig. S6 in the supplemental material) and possesses approximately four times the inhibitory strength of the racemate, indicating the preferred recognition motif is the S conformation of this molecule. This result was anticipated based on a previous computational docking analysis (24) and confirmed by both NMR and X-ray results in the present study. In addition, the pipecolic acid-derived compounds were shown to inhibit PPIase activities for recombinant Mip proteins from Francisella tularensis and Yersinia pestis (see Fig. S9 in the supplemental material). This result demonstrated that our pipecolic acid compound series possesses PPIase activity across a spectrum of pathogenic organisms that contain homologous Mip genes. Inhibitors 40, 168, and 183 decreased B. pseudomallei virulence in a cell-based cytotoxicity assay. Previously, we observed reduced protease production and motility for B. pseudomallei ⌬BpML1 deletion mutants; both of these phenotypes are typically associated with virulence mechanisms. Inactivation of BpML1 in B. pseudomallei also reduced intracellular survival within macrophages and virulence in mice (3). To determine whether BpML1 is required for cytotoxicity, B. pseudomallei ⌬BpML1 was used to infect J774A.1 cells, and LDH release was measured 24 h postinfection. While infection with wild-type B. pseudomallei resulted in high cytotoxicity, LDH release from cells infected with the mutant strain was similar to levels in uninfected control wells (P ⬍ 0.05) (Fig. 5). As B. pseudomallei-induced cytotoxicity is at least partially dependent on BpML1, this was used as a measure of BpML1 activity in live bacteria. Compounds 37, 40, 168, and 183 were then tested for inhibition of B. pseudomallei-induced cytotoxicity toward J774A.1 macrophages in vitro. Spontaneous release of LDH following incubation of uninfected J774A.1 cells with either inhibitor or DMSO was subtracted from the treatment data (Fig. 5) to account for any off-target effects that these compounds may exert in this cell-based assay. Compounds 40, 168, and 183 significantly reduced cytotoxicity by 30 to 40% (P ⬍ 0.05) (Fig. 5). Compound 37 was observed to be less active than the other three compounds, but with a mea-

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FIG 5 Compounds 37, 40, 168, and 183 reduce B. pseudomallei-induced cytotoxicity. J774A.1 cells were infected with Burkholderia pseudomallei K96243 and treated with compounds 37, 40, 68, and 183 before overnight incubation at 37°C. Cytotoxicity was measured based on LDH release, and the results are presented as the percent cytotoxicity. Infected cells treated with DMSO (wildtype release) or uninfected cells treated with a corresponding inhibitor (spontaneous release) were included as controls. *, statistically significant, P ⬍ 0.05, n ⫽ 3. The dotted line indicates 100% wild-type cytotoxicity. Error bars represent standard errors of the means.

surable reduction in cytotoxicity against infected macrophages greater than that in DMSO control experiments. No statistically significant difference was detected between compounds 40, 168, and 183 in B. pseudomallei-induced cytotoxicity. The apparent trend in PPIase inhibitory activity (Table 2) between compounds 40, 168, and 183 was not recapitulated with infected J774A.1 cells, suggesting no significant difference across the compound set at the cellular level. However, the addition of a specific aromatic group to compound 37 does appear to increase both PPIase inhibition and cell-based activity when targeting the in vitro infectivity of B. pseudomallei. When J774A.1 cells were incubated with 50 ␮M of each compound in the absence of bacteria, no cytotoxicity was observed for compounds 37 and 183, and ⬍10% cytotoxicity was observed for compounds 40 and 168. The slightly higher cytotoxicity of compounds 40 and 168 toward noninfected macrophages may be due to increased cell permeability associated with higher lipophilicity (Table 2). Any such apparent differences are minimized in the B. pseudomallei infectivity assay data, since background LDH levels for noninfected J774A.1 cells exposed to inhibitor are subtracted. In any case, the minimal cytotoxicity observed for pipecolic acid derivatives in noninfected macrophages provides evidence that this compound series does not exert significant adverse effects on the biological function of healthy mammalian macrophages in vitro. Conclusions. This report summarizes the preliminary steps in the process of adapting a macrocyclic inhibitor capable of suppressing the human immune system reaction to a small molecule with antimicrobial properties (see Fig. S10 in the supplemental material). Biophysical and structural biology trials with compounds from our design series led to a set of small molecules that bind and inhibit the BpML1 enzyme. Leaving the solubility protein tag on BpML1 led to a crystal form suitable for soak and cocrystallization experiments and allowed the determination of complex structures for 4 pipecolic acid derivatives. The binding conformations of these compounds with the native and the D44G mutant mimic the pipecoline group of rapamycin when binding FKBP12. NMR studies of these systems confirmed the binding

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orientations of these compounds in aqueous solution and permitted a hypothetical model for compound 183, for which insufficient electron density was observed in the crystal. NMR titration data were used to calculate bimolecular dissociation constants and yielded values for these compounds near those of their peptidylprolyl cis/trans isomerase inhibitory activity. The NMR-based affinities to BpML1 also compared well with in vitro data generated for the ability of B. pseudomallei to infect macrophages in the presence of these compounds. This series of compounds demonstrated reduced infectivity for B. pseudomallei in the macrophage infection model and, thus, represent a class of compounds with a novel mode of action for antiinfective small molecules and have the potential for development into new antimicrobial drugs. For this investigation, we sought a means to quickly generate structural and biological information for a set of novel pipecolic acid derivatives synthesized to inhibit BpML1. This study tested the ability of our high-throughput structural biology platform to rapidly produce one or more protein constructs capable of binding small molecules. By starting with a crystal form incompatible with ligand binding, our multitarget approach generated over 50 constructs in parallel and quickly delivered several working crystal systems for structural biology investigation. This approach differed from an iterative structural biology process, by which information gained for an individual construct informs each subsequent attempt to improve expression levels, crystal packing, and other characteristics important for X-ray crystallography. Although a stepwise approach is often necessary for novel biological targets, designing and testing variations in sequences may greatly extend the time required to achieve success. With strategic, targetdependent choices made at the outset, this parallelized workflow reduces the overall time required for success at the structure stage and greatly decreases the cost per experiment through efficiencies gained in batch processing. We believe this strategy will be critical in pursuing structural information on pipecolic acid derivatives targeting Mip proteins from a range of other pathogenic organisms. ACKNOWLEDGMENTS We thank the entire team at The Seattle Structural Genomics Consortium for Infectious Disease (SSGCID), without whom this work would not have been possible. The following authors are or were members of this consortium at the time the work was conducted: Darren W. Begley, David Fox III, Phillip G. Pierce, Jan Abendroth, Muigai Muruthi, Kris Safford, Vanessa Anderson, Kateri Atkins, Steve R. Barnes, Spencer O. Moen, Amy C. Raymond, Robin Stacy, Peter J. Myler, Bart L. Staker, Thomas E. Edwards, and Donald D. Lorimer. Purified Mip protein from Francisella tularensis was provided by Chad Stratilo from Defense Research and Development, Medicine Hat, Alberta, Canada. Purified Mip protein from Yersinia pestis was provided by the Center for Structural Genomics of Infectious Diseases (CSGID). Part of this research was funded under federal contracts HHSN272200700057C and HHSN272201200025C from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services. Part of this study was also funded by the UK Ministry of Defence. We also thank the German Research Foundation (DFG) for financial support via the Collaborative Research Center 630 (SFB630). The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under contract DE-AC02-05CH11231. The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute.

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