Published Ahead of Print 23 December 2013. 10.1128/AAC.01875-13.

2014, 58(3):1458. DOI:Antimicrob. Agents Chemother. and Donald D. LorimerUlrike Holzgrabe, Mitali Sarkar-Tyson, Thomas E. Edwards Myler, Bart L. Staker, Nicholas J. Harmer, Isobel H. Norville,Spencer O. Moen, Amy C. Raymond, Robin Stacy, Peter J. Safford, Vanessa Anderson, Kateri Atkins, Steve R. Barnes,Juli, Phillip G. Pierce, Jan Abendroth, Muigai Muruthi, Kris Darren W. Begley, David Fox III, Dominic Jenner, Christina Bacterial ImmunophilinsDevelopment of Antimicrobials Targeting A Structural Biology Approach Enables the

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A Structural Biology Approach Enables the Development ofAntimicrobials 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 andFood Chemistry, University of Würzburg, Würzburg, Germanyc; Seattle Biomedical Research Institute, Seattle, Washington, USAd; Departments of Global Health andMedical 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 pathogenicorganisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the caus-ative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macro-cyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency forBpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compoundseries with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. Thebest compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhib-ited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macro-phages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectiv-ity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new classof antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery.

Burkholderia 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 ofdeath by infectious disease in northeastern Thailand, after humanimmunodeficiency virus (HIV) and tuberculosis (1). Multiple fac-tors, including its frequent misdiagnosis as tuberculosis infectionand the ability of B. pseudomallei to evade the host immune re-sponse, have led to its designation as an emerging infectious dis-ease and a potential bioterrorism agent by the National Institute ofAllergy and Infectious Diseases (NIAID) (2). We recently identi-fied a macrophage infectivity potentiator (Mip) protein from B.pseudomallei (BpML1) that is important for intracellular replica-tion and required for full virulence (3). Homologous Mip proteinsin Legionella pneumophila and Trypanosoma cruzi also appear tobe involved in the infection machinery and are required for fullvirulence in these distinct pathogenic organisms (4, 5). BpML1 isan immunophilin, part of a well-conserved class of peptidyl-prolylisomerases (PPIases), which include FK506-binding proteins(FKBPs) (6–8). Binding of the potent fungal macrocyclic com-pounds rapamycin (Sirolimus) and FK506 (Tacrolimus) to PPI-ases in lymphocytes has a well-characterized immunosuppressiveeffect unrelated to their PPIase function and allows for modernorgan transplantation (9–11). Human PPIase cyclophilin A caninfluence the infectivity of HIV-1 virions (12–16), and cellularexpression levels of cyclophilin A can alter the replicative ability ofother viruses, in part via regulation of type 1 interferons (17–19).Thus, the roles of immunophilins in infection and the inhibitoryeffects on PPIases with known natural product inhibitors makethem attractive targets for novel drug discovery, if suitable host-pathogen selectivity can be achieved.

Descriptions of human FKBPs that have been targeted withsmall synthetics have been reported in the literature. These repre-

sent attempts to influence protein localization, signaling path-ways, and protein activation with smaller lead-like molecules dis-possessed of the immunosuppressive effects of FK506 and relatedmacrocycles (20–23). This work describes our initial efforts todesign and characterize small lead-like molecules that can influ-ence the virulence of B. pseudomallei through BpML1 inhibitionwithout compromising host immunity. Through synthetic varia-tion and testing, we replaced the anchoring moiety in known mac-rocyclic inhibitors with a select set of small-molecule pipecolicacid derivatives, forming an important anchoring scaffold (24,25). We obtained cocrystallization structures to demonstrate thatBpML1 binds to our pipecolic acid derivatives in a manner anal-ogous to the piperidine moieties of macrocyclic FK506 (26, 27)and rapamycin (28), allowing identification of the essential chem-ical scaffold necessary for binding. Nuclear magnetic resonance(NMR) studies on compounds from this series have revealed arange of micromolar affinities and confirmed binding along a hy-drophobic groove only minimally exploited by either macrocyclein solution (29, 30). We show here that these compounds inhibitthe in vitro PPIase activity of BpML1 as well as Mip proteins fromother pathogenic organisms. Most importantly, these compounds

Received 27 August 2013 Returned for modification 2 October 2013Accepted 26 November 2013

Published ahead of print 23 December 2013

Address correspondence to Darren W. Begley, dbegley@embios.com.

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 hu-man macrophages, with no significant cytotoxicity for macro-phage cell lines. The structural and functional data generated fromthis report thus provide a basis for rational, structure-based designof novel Mip inhibitors with the potential to minimize the viru-lence effects of B. pseudomallei and other pathogenic organisms.

MATERIALS AND METHODSProtein production. The BpML1 construct (clone BPSS1823; SSGCID IDBupsA.00130.a.D21) was engineered and optimized for expression inEscherichia coli by using Gene Composer software (31, 32). Syntheticgenes were purchased from DNA 2.0 and cloned into a modified pET28vector by using the polymerase incomplete primer extension (PIPE) clon-ing method (33). Insert PCR (iPCR) of the BpML1 synthetic gene wascarried out with chimeric primers having homology to gene termini (25bp of homology) and the vector junction (15 bp of homology). A pET28vector engineered to donate an amino-terminal 6His-Smt tag (MSHHHHHHSGEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGS)was amplified by vector PCR (vPCR) (31). The iPCR thermal cyclingparameters were as follows: initial denaturation for 2 min at 95°C, then 25cycles of 95°C for 30 s, 50°C for 45 s, and 68°C for 3 min, followed by a finalholding 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°Cfor 45 s, and 68°C for 14 min, followed by holding at 4°C. Neither reactionincluded a final extension step, allowing the products to remain variablysingle stranded at the termini. All PCRs were performed using PfuUltraHotstart master mix (Stratagene Agilent). Equal volumes of crude iPCRand vPCR products were merged and transformed into TOP10 chemicallycompetent cells according to the manufacturer’s specifications. Once se-quence-verified clones were identified, these expression plasmids weretransformed into BL21(DE3) cells. BpML1 was expressed in E. coliBL21(DE3) cells in both rich TB autoinduction medium with kanamycinand M9 minimal medium with 15NH4Cl and kanamycin with 2% (vol/vol) glycerol as a carbon source. Starter cultures were grown in TB me-dium (T7060; Teknova) for 18 h at 37°C and then used to inoculate 2-literflasks of minimal medium. After 6 h of growth at 37°C and 220 rpm, thetemperature was reduced to 20°C and cells were induced with 1 mMisopropyl-�-D-thiogalactopyranoside and grown for 24 h. To harvest, thecultures were centrifuged at 5,000 relative centrifugal force for 15 min at5°C, and the cell paste was frozen at �80°C.

Frozen cell paste was resuspended in the following lysis buffer: 25 mMTris, 200 mM sodium chloride, 50 mM L-arginine monohydrochloride, 10mM imidazole, 0.5% (vol/vol) glycerol, 1 mM Tris(2-carboxyethyl) phos-phine (TCEP), 0.02% (wt/vol) 3-[(3-cholamidopropyl)dimethylammo-nio]-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 bycentrifugation. Clarified lysate was first purified by immobilized metalaffinity chromatography (IMAC) with a HiTrap chelating high-perfor-mance (HP) column (PN 17-0409-03; GE Life Sciences) charged withNi2� using the ProteinMaker system (34). The column was equilibratedwith wash buffer (20 mM Tris, 200 mM sodium chloride, 50 mM L-argi-nine monohydrochloride, 10 mM imidazole, 1 mM TCEP; pH 8.0), andthe protein of interest was eluted with wash buffer containing 500 mMimidazole, excluding L-arginine. Some eluted protein was treated withubiquitin-like protease 1 (Ulp1) to cleave the 6His-Smt tag, leaving anN-terminal serine after the QIGG residue sequence, and was simultane-ously dialyzed into wash buffer. The target was further purified with asecond Ni2� IMAC step by using a HiTrap chelating HP column equili-brated with wash buffer. The flowthrough containing the cleaved targetprotein was then concentrated with centrifugal filters (PN VS2092; Sarto-rius Stedim), and injected over a HiPrep 16/60 Sephacryl S-100 HR (17-1165-01; GE Life Sciences) size exclusion chromatography (SEC) columnequilibrated with SEC running buffer (25 mM Tris [pH 8.0], 200 mM

sodium chloride, 1 mM TCEP, and 1% [vol/vol] glycerol). The purifiedprotein of interest was concentrated to a target concentration of 10 mg/mlas measured with a Nanodrop ND-1000 spectrophotometer and stored at�80°C. The uncleaved target was obtained by employing Ni2� IMAC andSEC; however, treatment with Ulp1 was not performed.

Cocrystallization and structure determinations of BpML1 com-plexes. Crystals of Smt-fused to BpML1 (wild type or D44G) were grownby sitting drop vapor diffusion at 295 K using 10 to 20 mg/ml protein inthe presence of 2 to 5 mM compound over the course of several weeks.Compound cocrystals with BpML1 were grown under the following con-ditions: FK-506 (0.1 M MIB buffer [pH 9.0], 25% [wt/vol] polyethyleneglycol 1500 [PEG 1500] cryoprotected with 30% [wt/vol] PEG 400), com-pound 37 (10% [wt/vol] PEG 20,000, 20% [vol/vol] PEG MME550, 0.03M 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 cat-ions [MgCl2, CaCl2], 0.1 M MES-imidazole [pH 6.5]) directly cryopre-served, compound 168 (50 mM ammonium formate, 24.55% [wt/vol]PEG 3350) cryopreserved with 10% (vol/vol) ethylene glycol, and com-pound 183 (50 mM ammonium formate, 30% [wt/vol] PEG 3350) cryo-protected with 25% (vol/vol) ethylene glycol. In each case, the drop con-tained 2 to 5% (vol/vol) dimethyl sulfoxide (DMSO) and 100 mMcompound.

All data sets were collected with the Advanced Light Source (ALS)beamlines 5.0.1 and 5.0.3 equipped with ADSC 315R charge-coupled-device detectors. Diffraction data were reduced and scaled by using XDS/XSCALE (35–37). Each structure was solved by molecular replacement viapreexisting structures of BpML1 and Smt. Structures were refined by us-ing iterative cycles of TLS and restrained refinement with REFMAC5 (38),part of the CCP4 program suite (39), and by model building using thecrystallographic object-oriented tool kit (Coot) (40). All structures werepeer reviewed internally and validated using Molprobity (41) prior todeposition in the Protein Data Bank (42, 43). Diffraction data and refine-ment statistics are listed for each structure in Tables S1 and S2 of thesupplemental material.

NMR spectroscopy. NMR samples were prepared for screening bydiluting concentrated, 15N-labeled BpML1 protein to 330 �M in NMRbuffer (50 mM NaCl, 50 mM K-Phos [pH 7.0], 20% [vol/vol] D2O, 10%[vol/vol] DMSO-d6). Samples prepared for titration experiments con-tained a protein concentration of 200 �M, with compound:protein ratiosof 0.25 to 7.50. All experiments were conducted on a Varian Inova 500-MHz NMR spectrometer equipped with a standard HCN probe at a tem-perature of 25°C. Screening was completed by using protein-observe,two-dimensional (1H-15N) band-selective optimized-flip-angle shorttransient– 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 recycledelay of 0.3 s. NMR spectra were processed using the Varian VnmrJ Bio-pack software and analyzed using either MestReNova (Mestrelab) oriNMR (http://www.inmr.net). Resonance assignments were transcribedfrom previously determined NMR structures, made available by the Bio-logical Magnetic Resonance Data Bank (BMRB) (46). Chemical shift per-turbation (CSP) values for each residue were calculated based on thefollowing equation: �� � [(�1H)2 � (�15N/5)2]1/2. CSP values were fit-ted for each resonance and titration by using GraphPad Prism version 5according to equation 1:

�� � (��max) �(Pt � L � KD) � �(Pt � L � KD)2 � 4PtL

2Pt(1)

where Pt is the total protein concentration, L is the concentration of com-pound, KD is the dissociation constant, and ��max is the maximum CSPobserved in the titration (47). Final KD values were calculated using datafrom three reporter residues (D48, V62, and G93) acquired from dupli-cate titrations.

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Peptidyl-prolyl cis/trans isomerase assay. For the peptidyl-prolyl cis/trans isomerase assay, inhibitors were dissolved in DMSO to a final con-centration 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 recombinantBPSS1823 was determined by using a protease-coupled assay as describedpreviously (48). Briefly, 50 nM BPSS1823 protein was incubated for 6min 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 finalconcentration of 0.8 mg/ml and mixed. Hydrolysis of the substrate wasmeasured at 390 nm by using a Shimadzu 1800 UV/visible spectropho-tometer at 1-s intervals until there was no further change in absorbance.For inhibition measurements, BPSS1823 was preincubated with 50 nM to50 �M compound for 6 min prior to the addition of the chymotrypsin.Three independent readings were taken at each concentration with eachinhibitor. Positive (no inhibitor) and negative (no BPSS1823) controlswere 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 substratebecame limiting) were fit to the equation, and kobs was calculated by linearregression.

In[A� � At] � �kobs t � In[A� � A0] (2)

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

kenz � kobs � kuncat (3)

The KI data were fit to equation 4 (50, 51):

v � v0

[E] � [I] � K � �([E] � [I] � K)2 � 4[E][K]

2[E](4)

Using least-square nonlinear fitting, 0 and KI were fit using initialestimates based on the raw data, and [E] was kept constant.

Bacterial strains. B. pseudomallei K96243 was routinely grown onLuria agar (L-agar) at 37°C overnight. Fresh bacterial streak plates wereused for all cell-based assays. B. pseudomallei �BPSS1823 was constructedas previously described (3).

B. pseudomallei cytotoxicity assays. J774A.1 cells (ECACC; HPAPorton Down, Salisbury, United Kingdom) were seeded onto a 24-welltissue culture plates and used at a density of 1 106 cells in Dulbecco’smodified Eagle medium (DMEM; Gibco, Paisley, United Kingdom) sup-plemented with 10% fetal bovine serum (FBS; Gibco, Paisley, UnitedKingdom) and 40 mM glutamine (Sigma-Aldrich, United Kingdom). B.

pseudomallei K96243 or B. pseudomallei �BPSS1823 was harvested into 1ml Leibovitz L-15 medium supplemented with 10% fetal calf serum (L15;Gibco, Paisley, United Kingdom) to an absorbance at 600 nm of 0.25 to0.3. B. pseudomallei K96243 was treated with 50 �M each inhibitor orDMSO alone for 1 h. J774A.1 cells were infected with treated B. pseu-domallei K96243 at a multiplicity of infection (MOI) of approximately400:1 for 40 min at 37°C. Postinfection, the bacteria were removed andreplaced with 1 ml L15 supplemented with 15 �g/ml gentamicin and theappropriate inhibitor at 50 �M. Infected J774A.1 cells were incubated for24 h at 37°C before the supernatant was tested for lactate dehydrogenase(LDH) release via the CytoTox 96 nonradioactive cytotoxicity assay (Pro-mega, Southampton, United Kingdom) per the manufacturer’s instruc-tions. All results are presented as the means of three independent experi-ments. Infected cells treated with DMSO or uninfected cells were includedas controls. To express the results as the percentage of wild-type cytotox-icity, the LDH activity in test compound wells was compared to that inwells infected with B. pseudomallei K96243 treated with DMSO. Sponta-neous release (LDH release following incubation of uninfected cells withthe corresponding inhibitor or DMSO) was subtracted from the finalvalues. All statistical analyses were carried out using a repeated measuresanalysis of variance with Bonferroni posttests and analyzed using Graph-Pad Prism version 5.

Protein structure accession numbers. The BpML1 mutant proteinsdescribed here (i.e., those that yielded high-quality diffracting crystals)were submitted to the Protein Data Bank and assigned the accession num-bers summarized in Table 1.

RESULTS AND DISCUSSIONGeneration of a crystal form suitable for BpML1–small-mole-cule complexes. In the published apo crystal form of BpML1, thepurification tag cleavage site peptide sequence on one moleculefills the FK506-binding site of a neighboring molecule in the crys-tal lattice (see Fig. S1 in the supplemental material) (51). Multiplecrystallization efforts with native sequence BpML1 constructswith tags removed failed to yield crystals. Therefore, to obtainalternative crystal forms not dependent on the cleavage site se-quence interaction with the protein, a series of BpML1 point mu-tations was designed using GeneComposer software (31, 32, 52).Via GeneComposer, specific residues were identified that couldalter the crystal lattice while minimizing the impact on bindingsite topology, based on crystal contacts observed in the apo form ofthe BpML1 structure. These residues were mutated according to

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

PDB IDb SSGCID ID Method Space group Mutation6His-Smt tagintact? Ligand (PDB ligand name)

3UF8 BupsA.00130.a.D242 XRD P 21 G95A Yes FK506 (FK5)3UQA BupsA.00130.a.D220 XRD C 2 A54E Yes FK506 (FK5)3UQB BupsA.00130.a.D214 XRD P 21 D44G Yes FK506 (FK5)3VAW BupsA.00130.a.D24 XRD P 21 V3I Yes FK506 (FK5)4DZ2 BupsA.00130.a.D239 XRD C 2 R92G No FK506 (FK5)4DZ3 BupsA.00130.a.D227 XRD C 2 2 21 M61H No FK506 (FK5)4FN2 BupsA.00130.a.D214 XRD P 1 D44G Yes Compound 37 (854)4G50 BupsA.00130.a.D214 XRD P 1 D44G Yes Compound 168 (861)4GGQ BupsA.00130.a.D21 XRD P 1 Native Yes Compound 40 (861)4GIV BupsA.00130.a.D214 XRD C 2 D44G Yes Compound 183 (4GI)2KE0* BupsA.00130.a NMR NA Native No apo2KO7* BupsA.00130.a NMR NA Native No Cycloheximide N-ethylethanoate (JZF)2L2S* BupsA.00130.a NMR NA Native No 1-{[(4-Methylphenyl)thio]acetyl}piperidine (L2S)2Y78* NA XRD P43 21 2 Native No apoa Abbreviations: XRD, X-ray diffraction; NMR, nuclear magnetic resonance; NA, not available.b * PDB ID information was previously reported for these structures (51).

Begley et al.

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genetic variants from related organisms, which were found byusing the basic local alignment search tool (BLAST) database ofthe 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 showed47 out of 51 constructs produced measurable amounts of protein,including both higher and lower bacterial expression levels rela-tive to the native sequence (see Fig. S2 in the supplemental mate-rial). A subset of these BpML1 point mutants was selected forscale-up, purification, and crystallization (see Materials andMethods).

Crystallization of 15 purified BpML1 mutants with and with-out cleavage of the 6His-Smt solubility tag (54) was attempted inthe presence and absence of FK506. Among the BpML1 mutantproteins tested, six yielded high-quality diffracting crystals, all inthe presence of FK506 (Table 1). Crystals of BpML1 mutantsR92G and M61H with the 6His-Smt tag removed exhibited li-gand-dependent dimerization; adjacent BpML1-bound mole-cules of FK506 make direct contact within the asymmetric unit(see Fig. S3 in the supplemental material). The extensive ligand-ligand interactions observed for FK506 suggest that this crystalform may not be suitable for crystallization experiments withnovel small molecules. The remaining four crystal structures wereproduced from uncleaved N-terminal 6His-Smt-tagged BpML1mutants (Table 1). The lattices in these crystal systems create anopen FK506-binding pocket and are stabilized by a continuous10-stranded �-sheet formed between BpML1 and Smt. This sta-bilization involves direct contact between BpML1-�1 and Smt-�2of a neighboring symmetry-related molecule (see Fig. S4 in thesupplemental material).

Of all the BpML1 variants screened, the D44G mutant was themost consistent in producing high-quality crystals, and it outper-formed the native sequence under a number of sparse matrix con-ditions. We validated the use of the D44G mutant for biophysicalstudies by comparing crystal structures of FK506-bound BpML1with and without the D44G mutation, and we found no significantdetriment to FK506 binding resulting from this mutation. Thenative sequence D44 side chain forms a hydrogen-bonding net-work with FK506 and the hydroxyl of Y33 and may participate ina weak salt bridge with R49, helping to stabilize this loop (see Fig.S5 in the supplemental material). In the D44G BpML1 mutant, awater molecule occupies the position of one D44 carboxylate ox-ygen, allowing polar contacts between FK506 and Y33, which re-capitulate the FK506-bound structure in the native sequence. Fur-thermore, previous solution-state NMR studies suggested theidentity of this residue to be unimportant for binding small-mol-ecule inhibitors, with a wide range of side chain conformationsdetected (51). Therefore, we prioritized cocrystallization of thenative and D44G mutant 6His-Smt–BpML1 proteins with se-lected pipecolic acid derivatives for structural characterization.

Identification of a key chemical scaffold for binding BpML1.Cocrystallization trials were set up with 16 different pipecolic acidderivatives (24) and BpML1, from which compounds 37, 40, 168,and 183 yielded crystals with sufficient X-ray data for structuredetermination (Fig. 1). Three of the four structures contained theD44G mutant, while the complex of BpML1 with compound 40was solved with the native sequence. The N-terminal 6His-Smt tagwas attached in all four cases, with a majority of the Smt residuesresolved in each structure (for complete crystallization data, see

Tables S1 and S2 in the supplemental material). Compounds 37,40, and 183 were added as racemates, while compound 168 was theS enantiomer of compound 40 (see Fig. S6 in the supplementalmaterial). In all four structures solved, only the S conformation ofeach pipecolic acid derivative was observed (Fig. 1). This struc-tural preference for the S conformation was reflected in the 4-fold-higher inhibitory strength observed with pure enantiomer com-pound 168 over the racemate compound 40 (see below).

The central pipecolic acid scaffold binding conformation waslargely conserved across the four complexes and demonstrated nostructural dependence on the presence of a D44 side chain. More-over, the lone structure solved for the native sequence showed thatthis aspartyl group rotates away from the FK506-binding site, notparticipating in any intra- or intermolecular interactions (Fig. 1).A sulfonyl oxygen for compounds 37, 40, and 168 each makeshydrogen bond contacts with the hydroxyl group of Y89, a closecontact also observed by solution-state NMR (see below). Thesulfonyl piperidine scaffolds of compounds 37, 40, and 168 gen-erated virtually identical binding poses in their respective asym-metric units, similar to that seen previously in computationaldocking studies, as well as with the chemically distinct biphenylpiperidine (PDB ID 1FKG) (24, 55). In contrast to the best con-formation determined from our previous docking analysis, thecarbonyl ester of compound 168 coordinated with the backboneamide of I63 in the crystal complex, and the terminal phenyl ringparticipated in the �-edge stacking interaction with F43 (24).Variability in the position of trimethoxybenzene was observedcrystallographically, with the most common conformation ob-served for this group overlapping the same subpocket as the me-thoxy-cyclohexanol of FK506 and rapamycin (Fig. 1).

Although the binding conformation of compound 183 is sim-ilar to that of the other pipecolic acid derivatives, the carbonylester of compound 183 is sufficiently rotated to abrogate any hy-drogen bond with the I63 backbone amide in the crystal. Theterminal phenyl group of compound 183 is also rotated �90°from its position in compound 168, with nearly 20° of rotationaldifference between the chain A and chain B conformations (Fig.1). The crystal lattice observed for compound 183 also differedfrom that of the other pipecolic acid derivatives and exhibited thesame type of ligand-induced dimerization pattern seen for BpML1bound to FK506 when the 6His-Smt tag was cleaved. Insufficientelectron density was observed to be able to model the nitro groupas well as most of the linked pyridine moiety of compound 183.However, the close proximity of the benzene ring to the shortF43-G44 �-strand makes it an unlikely direction for the meta nitrogroup to be positioned. The flexible outer loop from A94 to I98clearly shifts outward, relative to the same loop observed for theother three ligand-bound complexes, most likely to accommodatethe nitrobenzene scaffold.

Solution-state NMR characterization of pipecolic acid deriv-atives with BpML1. Compounds 37, 40, 168, and 183 weretitrated into samples of 15N-labeled, tag-cleaved, native sequenceBpML1 (clone BPSS1823) to calculate biophysical binding con-stants by NMR spectroscopy (Table 2). Overall, chemical shiftchanges to BpML1 observed during pipecolic acid titrations wereindicative of fast on/off rates relative to the NMR time scale, incontrast to slow on/off binding for analogous spectra acquiredwhen titrating with FK506 (Fig. 2). Using the SOFAST-HMQCpulse sequence (44, 45), chemical shift perturbations (CSPs) weremonitored for three equally spaced residues (D48, V62, and G93)

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

a result consistent with in vitro activity data and the low bindingpotential of the R enantiomer. Compounds 168 and 183 wereobserved to have essentially the same affinities based on NMR, aresult which deviated somewhat from the relative potencies deter-mined in the in vitro PPIase inhibition assay (Table 2). This may bedue to the higher concentrations required for NMR, limiting thesensitivity for KD determinations of the more potent molecules(56). An additional 8 compounds from the series that had failed incrystallization trials were also tested by HSQC. None of these gen-erated significant chemical shift perturbations for the protein insolution, despite showing inhibitory PPIase activities, and theywere excluded from further study by NMR.

SOFAST-HMQC spectra with and without saturating amountsof compound provided structural details on the solution statebinding interactions between pipecolic acid derivatives andBpML1. Strong CSPs for V62 and I63, and the relatively weakshifts for F53, suggest that the methoxy-cyclohexanol subpocketof FK506 is the favored solution-state binding region for the pyr-idine and trimethoxybenzene groups of compounds 40, 168, and183 (Fig. 3). The I63 amide signal for BpML1 generates the largestCSPs of any residue in the protein (see Fig. S8 in the supplemental

FIG 1 Structure ensemble for pipecolic acid derivative binding to BpML1. Four crystallographic poses were observed for compound 40 (lower left), while twoposes 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) andis 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 ineach PDB entry. Noncarbon atoms for each compound are colored as follows: red, oxygen; blue, nitrogen; gold, sulfur). The figure was generated using PyMol(57).

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

CompoundKI

a PPIase activity(�M)

KDb NMR

(�M) BEIc C(log P)d

37 10 3 190 15 12 3.140 0.7 0.2 51 6 8.7 4.4168 0.17 0.05 14 4 9.9 4.4183 3.6 0.7 10 2 12 3.2a For calculation of the KI, see equation 4 in Materials and Methods. Values reported asmean standard error of the mean.b For calculation of the KD, see equation 1 in Materials and Methods. Values reported asmean 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 wasdetermined via NMR (see reference 58).d The C(log P) is the water/octanol partition coefficient, calculated using ChemDrawUltra version 12.0.

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material), with the signal nearly abolished when any of the fourpipecolic acid derivatives was added. This result confirmed hydro-gen bond formation between I63 and the carbonyl ester of eachcompound, interactions which recapitulate the more sterically

constrained hydrogen bond formed when FK506 is bound. Me-dium to strong chemical shifts were also seen in the A94-to-I98loop for compounds 40, 168, and 183, due to proximity of theterminal phenyl group when bound. These interactions mapped

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 acquiredin 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 riseto duplicate peaks for FK506, indicative of a two-state slow exchange, versus the exchange-averaged Gly93 peak when compound 183 is added. The figure wasgenerated using iNMR.

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), and183 (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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to conformational changes observed crystallographically, as seenwith loop R92-to-I98, adapting to the benzene ring of compound168, relative to the loop conformation when bound to FK506 (Fig.4). The accommodation of these phenyl moieties by BpML1 rep-resent protein-ligand binding interactions in which FK506 andrapamycin do not participate, and may serve as a foothold in de-veloping species-specific protein inhibition compounds.

The CSP for BpML1 residue A54 was higher in the presence ofcompound 183 versus the others, indicating a greater likelihoodthat compound 183 samples alternate conformations in solution.This was reflected in the poor electron density observed for com-pound 183, which prevented accurate modeling of any pyridinering conformations. However, the binding efficiency for com-pound 183 (Table 2) was equivalent to that of compound 37,indicative of methyl pyridine being an effective moiety added tothe original scaffold on a per-atom basis. Furthermore, the NMRdata showed that the strongest interaction for the pyridine ring ofcompound 183 is with the methoxy-cyclohexanol binding pocket

and is indicated in our proposed full complex model (Fig. 2).CSPs for Y89 were also much higher for compound 183 thanfor compounds 40 and 168, while the CSPs were quite similarfor the extended A94-to-I98 loop region. This key CSP differ-ence for compound 183 validates our proposed model struc-ture, in which the preferred conformation permits contact be-tween the meta nitro group and the hydroxyl of Y89, withsimilar accommodation for the hydrophobic moiety in theA94-to-I98 flexible loop (Fig. 3).

PPIase activity inhibition by pipecolic acid derivatives. In aprotease-coupled assay, the specificity constant kcat/Km for theBpML1 construct selected for study (BPSS1823) was previouslydetermined to be 6.7 106 0.4 106 M�1 s�1, with inhibitionby the known PPIase inhibitor rapamycin at a KI of 3 2 nM(values are means standard errors of means) (3). Pipecolic acid-derived compounds have also been reported for inhibitory prop-erties against human FKBP12 and the Legionella pneumophila Mipprotein (24). We tested a wide selection of pipecolic acid deriva-tives, resulting in data for a select number of agents with inhibitoryproperties against BpML1 (Table 2). The racemic compounds 37and 183 inhibited the PPIase activity of BpML1 at low micromolarconcentrations, while the S enantiomer, compound 168, andracemic compound 40 inhibited the activity with a high-nanomo-lar KI. Compound 168 is the enantiomerically pure form of com-pound 40 (see Fig. S6 in the supplemental material) and possessesapproximately four times the inhibitory strength of the racemate,indicating the preferred recognition motif is the S conformationof this molecule. This result was anticipated based on a previouscomputational docking analysis (24) and confirmed by both NMRand X-ray results in the present study. In addition, the pipecolicacid-derived compounds were shown to inhibit PPIase activitiesfor recombinant Mip proteins from Francisella tularensis and Yer-sinia pestis (see Fig. S9 in the supplemental material). This resultdemonstrated that our pipecolic acid compound series possessesPPIase activity across a spectrum of pathogenic organisms thatcontain homologous Mip genes.

Inhibitors 40, 168, and 183 decreased B. pseudomallei viru-lence in a cell-based cytotoxicity assay. Previously, we observedreduced protease production and motility for B. pseudomallei�BpML1 deletion mutants; both of these phenotypes are typicallyassociated with virulence mechanisms. Inactivation of BpML1 inB. pseudomallei also reduced intracellular survival within macro-phages and virulence in mice (3). To determine whether BpML1 isrequired for cytotoxicity, B. pseudomallei �BpML1 was used toinfect J774A.1 cells, and LDH release was measured 24 h postin-fection. While infection with wild-type B. pseudomallei resulted inhigh cytotoxicity, LDH release from cells infected with the mutantstrain was similar to levels in uninfected control wells (P � 0.05)(Fig. 5). As B. pseudomallei-induced cytotoxicity is at least par-tially dependent on BpML1, this was used as a measure of BpML1activity in live bacteria. Compounds 37, 40, 168, and 183 werethen tested for inhibition of B. pseudomallei-induced cytotoxicitytoward J774A.1 macrophages in vitro. Spontaneous release ofLDH following incubation of uninfected J774A.1 cells with eitherinhibitor or DMSO was subtracted from the treatment data(Fig. 5) to account for any off-target effects that these compoundsmay exert in this cell-based assay.

Compounds 40, 168, and 183 significantly reduced cytotoxic-ity by 30 to 40% (P � 0.05) (Fig. 5). Compound 37 was observedto be less active than the other three compounds, but with a mea-

FIG 4 Crystal structures of BpML1 bound to FK506 (brown; PDB ID 3UQB)and compound 168 (cyan; PDB ID 4G50). The conformational change withinthe R92-I98 loop accommodates the terminal phenyl group of compound 168(bottom), relative to the FK506-bound structure (top). The R92 side chain isnot modeled in the FK506 structure (top) due to insufficient electron density.The figure was generated using PyMol (57)

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surable reduction in cytotoxicity against infected macrophagesgreater than that in DMSO control experiments. No statisticallysignificant difference was detected between compounds 40, 168,and 183 in B. pseudomallei-induced cytotoxicity. The apparenttrend in PPIase inhibitory activity (Table 2) between compounds40, 168, and 183 was not recapitulated with infected J774A.1 cells,suggesting no significant difference across the compound set atthe cellular level. However, the addition of a specific aromaticgroup to compound 37 does appear to increase both PPIase inhi-bition and cell-based activity when targeting the in vitro infectivityof B. pseudomallei. When J774A.1 cells were incubated with 50 �Mof each compound in the absence of bacteria, no cytotoxicity wasobserved for compounds 37 and 183, and �10% cytotoxicity wasobserved for compounds 40 and 168. The slightly higher cytotox-icity of compounds 40 and 168 toward noninfected macrophagesmay be due to increased cell permeability associated with higherlipophilicity (Table 2). Any such apparent differences are mini-mized in the B. pseudomallei infectivity assay data, since back-ground LDH levels for noninfected J774A.1 cells exposed to in-hibitor are subtracted. In any case, the minimal cytotoxicityobserved for pipecolic acid derivatives in noninfected macro-phages provides evidence that this compound series does not exertsignificant adverse effects on the biological function of healthymammalian macrophages in vitro.

Conclusions. This report summarizes the preliminary steps inthe process of adapting a macrocyclic inhibitor capable of sup-pressing the human immune system reaction to a small moleculewith antimicrobial properties (see Fig. S10 in the supplementalmaterial). Biophysical and structural biology trials with com-pounds from our design series led to a set of small molecules thatbind and inhibit the BpML1 enzyme. Leaving the solubility pro-tein tag on BpML1 led to a crystal form suitable for soak andcocrystallization experiments and allowed the determination ofcomplex structures for 4 pipecolic acid derivatives. The bindingconformations of these compounds with the native and the D44Gmutant mimic the pipecoline group of rapamycin when bindingFKBP12. NMR studies of these systems confirmed the binding

orientations of these compounds in aqueous solution and permit-ted a hypothetical model for compound 183, for which insuffi-cient electron density was observed in the crystal. NMR titrationdata were used to calculate bimolecular dissociation constants andyielded values for these compounds near those of their peptidyl-prolyl cis/trans isomerase inhibitory activity. The NMR-based af-finities to BpML1 also compared well with in vitro data generatedfor the ability of B. pseudomallei to infect macrophages in thepresence of these compounds. This series of compounds dem-onstrated reduced infectivity for B. pseudomallei in the macro-phage infection model and, thus, represent a class of compoundswith a novel mode of action for antiinfective small molecules andhave the potential for development into new antimicrobial drugs.

For this investigation, we sought a means to quickly generatestructural and biological information for a set of novel pipecolicacid derivatives synthesized to inhibit BpML1. This study testedthe ability of our high-throughput structural biology platform torapidly produce one or more protein constructs capable of bind-ing small molecules. By starting with a crystal form incompatiblewith ligand binding, our multitarget approach generated over 50constructs in parallel and quickly delivered several working crystalsystems for structural biology investigation. This approach dif-fered from an iterative structural biology process, by which infor-mation gained for an individual construct informs each subse-quent attempt to improve expression levels, crystal packing, andother characteristics important for X-ray crystallography. Al-though a stepwise approach is often necessary for novel biologicaltargets, designing and testing variations in sequences may greatlyextend the time required to achieve success. With strategic, target-dependent choices made at the outset, this parallelized workflowreduces the overall time required for success at the structure stageand greatly decreases the cost per experiment through efficienciesgained in batch processing. We believe this strategy will be criticalin pursuing structural information on pipecolic acid derivativestargeting Mip proteins from a range of other pathogenic organ-isms.

ACKNOWLEDGMENTS

We thank the entire team at The Seattle Structural Genomics Consortiumfor Infectious Disease (SSGCID), without whom this work would nothave been possible. The following authors are or were members of thisconsortium at the time the work was conducted: Darren W. Begley, DavidFox III, Phillip G. Pierce, Jan Abendroth, Muigai Muruthi, Kris Safford,Vanessa Anderson, Kateri Atkins, Steve R. Barnes, Spencer O. Moen, AmyC. Raymond, Robin Stacy, Peter J. Myler, Bart L. Staker, Thomas E. Ed-wards, and Donald D. Lorimer.

Purified Mip protein from Francisella tularensis was provided by ChadStratilo from Defense Research and Development, Medicine Hat, Alberta,Canada. Purified Mip protein from Yersinia pestis was provided by theCenter for Structural Genomics of Infectious Diseases (CSGID).

Part of this research was funded under federal contractsHHSN272200700057C and HHSN272201200025C from the NationalInstitute of Allergy and Infectious Diseases, National Institutes of Health,Department of Health and Human Services. Part of this study was alsofunded by the UK Ministry of Defence. We also thank the German Re-search Foundation (DFG) for financial support via the Collaborative Re-search Center 630 (SFB630). The Advanced Light Source is supported bythe Director, Office of Science, Office of Basic Energy Sciences, of the U.S.Department of Energy under contract DE-AC02-05CH11231. The Berke-ley Center for Structural Biology is supported in part by the NationalInstitutes of Health, National Institute of General Medical Sciences, andthe Howard Hughes Medical Institute.

FIG 5 Compounds 37, 40, 168, and 183 reduce B. pseudomallei-induced cy-totoxicity. J774A.1 cells were infected with Burkholderia pseudomallei K96243and treated with compounds 37, 40, 68, and 183 before overnight incubation at37°C. Cytotoxicity was measured based on LDH release, and the results arepresented as the percent cytotoxicity. Infected cells treated with DMSO (wild-type release) or uninfected cells treated with a corresponding inhibitor (spon-taneous release) were included as controls. *, statistically significant, P � 0.05,n � 3. The dotted line indicates 100% wild-type cytotoxicity. Error bars rep-resent standard errors of the means.

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