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DOI: 10.1002/cbic.201300748

Inhibition of Mycobacterium tuberculosis TransaminaseBioA by Aryl Hydrazines and Hydrazides

Ran Dai,[a] Daniel J. Wilson,[b] Todd W. Geders,[a] Courtney C. Aldrich,[a] and Barry C. Finzel*[a]

Introduction

Mycobacterium tuberculosis (Mtb), the major pathogen causingtuberculosis in humans, is more prevalent in the world thanever before.[1] One-third of the world’s population harbora latent form of Mtb and endure a lifelong risk of reactivation.

Immune-compromised individuals, such as those co-infectedwith human immunodeficiency virus, are particularly at risk forMtb infection.[2] The emergence of multi- and extensively-drug-resistant (MDR-TB and XDR-TB) strains has complicated treat-

ment of Mtb infections worldwide. The failure rate with currentcombination therapy in the treatment of MDR-TB infections isalmost 30%, and therapeutic options for XDR-TB-infected pa-tients are very limited due to extensive drug resistance. [3] Sincethe approval of Rifampicin in 1970, no new anti-TB drugs wereapproved that act through a novel mechanism until Bedaqui-line in late 2012.[4] To control the global TB pandemic, there isan urgent need for additional antitubercular agents with newmechanisms of action that can act in synergy with existingtherapies to treat MDR-TB and XDR-TB and shorten the dura-tion of treatment.

The enzymes involved in biotin biosynthesis by Mtb repre-sent potential drug targets because the biotin synthetic path-

way is required for Mtb survival both in vitro[5] and in vivo[6]

and, unlike bacteria or plants, mammalian hosts do not haveenzymes for biotin cofactor synthesis. [7] Mtb utilizes four en-zymes to synthesize biotin from pimeloyl-CoA.[8] The enzyme

catalyzing the second step, 7,8-diaminopelargonic acid (DAPA)synthase (BioA) has been shown by Schnappinger and co-workers to be essential for persistence during chronic infec-tions in a murine model of TB by using a conditionally regulat-

ed Mtb knockdown strain.[6] Furthermore, biotin deprivation of the DbioA Mtb mutant leads to cell death in the presence of a carbon source in the growth media (i.e., bactericidal), whichis an unusual phenotype, as most auxotrophs simply undergo

growth arrest when deprived of their nutrient. Collectively,these findings make BioA a promising new TB drug target. [6]

BioA is a class I aminotransferase responsible for the conver-sion of 7-keto-8-aminopelargonic acid (KAPA) to DAPA, em-ploying S-adenosyl methionine (SAM) as the amino donoraccording to a ping-pong bi–bi mechanism with PLP as a co-factor[9] that cycles between the reduced pyridoxamine (PMP)and oxidized (PLP) states (Scheme 1). First, SAM reacts with theinternal aldimine of PLP and Lys283 to donate an amino groupto the PLP, forming pyridoxamine phosphate (PMP). KAPA thenreacts with the PMP-bound form of BioA, receiving the aminogroup to form the product DAPA. With this last step, BioA re-turns to its PLP bound holo form.

Generally, transaminases can use many endogenous aminoacids as amino donors, but BioA has an unusual preference forSAM, which normally serves as a methyl donor.[10] SAM is struc-turally very different from the amino accepter KAPA, and howthe enzyme retains specificity in the recognition of these twodifferent substrates is only partially understood. Mtb BioA hasbeen structurally characterized with bound PLP cofactor (PDBID: 3V0, 3TFT).[7,11] Based on a comparison of an H315R mutat-ed Mtb BioA complex with the unreactive SAM analogue sine-fungin (PDB ID: 3LV2)[7] and an E. coli BioA complex with KAPA(PDB ID: 1QJ3),[12] Sacchettini and co-workers proposed thatMtb BioA catalyzes transamination by an induced fit mecha-

nism that relies upon active site conformational changes that

7,8-Diaminopelargonic acid synthase (BioA) of Mycobacteriumtuberculosis is a recently validated target for therapeutic inter-vention in the treatment of tuberculosis (TB). Using biophysicalfragment screening and structural characterization of com-pounds, we have identified a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal-5’-phosphate (PLP)cofactor, forming a stable quinonoid. Analogous hydrazidesalso form covalent adducts that can be observed crystallo-graphically but are incapable of inactivating the enzyme. In

the X-ray crystal structures, small molecules induce unexpectedconformational remodeling in the substrate binding site. Wecompared these conformational changes to those inducedupon binding of the substrate (7-keto-8-aminopelargonic acid),and characterized the inhibition kinetics and the X-ray crystalstructures of BioA with the hydrazine compound and ana-logues to unveil the mechanism of this reversible covalentmodification.

[a] R. Dai, Dr. T. W. Geders, Prof. Dr. C. C. Aldrich, Prof. Dr. B. C. Finzel Department of Medicinal Chemistry, University of Minnesota

308 Harvard St. SE, Minneapolis, MN 55455 (USA)

E-mail: [email protected]

[b] D. J. WilsonCenter for Drug Design, Academic Health Center, University of Minnesota

516 Delaware St. SE, Minneapolis, MN 55455 (USA)

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cbic.201300748.

2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2014, 15, 575–586 575

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accommodate structurally different substrates.[7] This necessarycapacity for change in the active site conformation makes itchallenging to design Mtb BioA inhibitors and predict ligandbinding by using computational modeling methods, particular-ly based on homology models derived from structures from

other species.Limited efforts to identify selective inhibitors of Mtb BioA

have been reported. Amiclenomycin (ACM), a natural productextracted from Streptomyces strains, and a simplified derivativewere identified many years ago as mechanism-based inhibitorsof Mtb BioA.[9,13] ACM showed good activity against Mtb cellsbut failed in animal models,[13] likely due to its low chemicalstability.[14] More recently, mechanism-based inhibitors basedon ACM with improved chemical stability have been described,but the stability comes at the expense of lower potency. [11,15] Aneed remains, therefore, for more diverse chemical scaffoldsthat can serve as a starting point for inhibitor optimization and

the development of potential drugs targeting Mtb BioA.

We employed fragment-based drug discovery (FBDD) meth-ods to identify new inhibitors.[16] Fragment screening providesa means to empirically identify small molecules that bind toa protein target, and subsequent structural characterization of these compounds can provide information about alternate

conformational states that support that binding. Here, wereport our discovery and kinetic characterization of a series of reversible covalent inhibitors of Mtb BioA selected for studybased on their similarity to a fragment hit identified duringbiophysical fragment screening. Using X-ray crystallography,we also revealed the binding mode of the inactivated com-plexes and described unexpectedly diverse protein conforma-tional changes that occur near the ligand binding site uponbinding of a series of hydrazine and hydrazide analogues.

Scheme 1. Catalysis of DAPA synthesis by BioA.


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Results

KAPA binding

To provide a more reliable picture of different conformationalstates promoted by substrate binding, we determined thestructure of the KAPA-bound enzyme at 1.8 resolution(Table 1). At this resolution, we could confirm from carbon hy-bridization and stereochemistry that, as expected, KAPA is notcatalyzed in the active site to form DAPA (Figure 1C). We hadno difficulty preparing this complex by cocrystallization, once

the fully PLP-saturated holoenzyme was prepared and crystal-lized.[17] KAPA binds to the holoenzyme much as predicted,and induces no significant conformational changes to bindingsite residues, except for an inward shift of the guanidinium of Arg400, which moves to pair against the KAPA carboxylate andcap the binding pocket (Figure 1D). Such a shift had been pre-dicted[7] based on similarity to the E. coli enzyme structure.[12]

The KAPA amino group does not displace the lysine bond tothe PLP but instead resides between the Tyr25 hydroxy groupand the PLP phosphate, forming strong hydrogen bonds toboth. Other hydrogen bonds to the Tyr157 hydroxy group andthe carbonyl oxygen of Gly316’ complete the tetrahedral hy-

drogen bonding around the protonated amine (Gly316’ is con-

tributed by the alternate chain of the BioA homodimer). Theketone oxygen of KAPA forms no hydrogen bonds but is posi-tioned just 3.4 from the Nz of Lys283, which remains cova-lently bound to the PLP. The close packing of the b-methylgroup against the aromatic ring of the Trp64 indole addsa strong hydrophobic contact to enhance binding.

Fragment screening and characterization

In order to discover novel BioA inhibitor scaffolds, we screeneda fragment library of ~1000 small molecules to identify com-

pounds that bind to BioA. This screening employed biophysicalmeasurements using differential scanning fluorimetry[18] (alsoknown as DSF or ThermoFluor[19]) to identify compounds thatinduce a shift in the denaturation temperature (T m) upon expo-sure to small molecules at a concentration of 5 mm. Com-pounds that effected a T m shift greater than 28C were select-ed for subsequent characterization by macromolecular crystal-lography. Although 12 of the 21 compounds selected for crys-tallographic study caused a downward (destabilizing) shift inthe T m, only one of these was ultimately characterized structur-ally, the 2-(aminomethyl)-benzothiazole hereafter designatedcompound 1 (Table 2). The T m of the holoenzyme under similar

conditions (including 2.5 % DMSO vehicle) is 858C (Figure S1 in

Table 1. Summary of crystallographic data.

Ligand KAPA Compound 1 Compound 2 Compound 3 Compound 4

PDB ID 4MQN 4MQO 4MQP 4MQQ 4MQRsource APS 17-ID (IMCA) APS 17-ID (IMCA) APS 17-ID (IMCA) APS 17-ID (IMCA) Rigaku Micromax-007detector Dectris Pilatus 6M Dectris Pilatus 6M Dectris Pilatus 6M Dectris Pilatus 6M Saturn 944+ CCD l [] 1.000 1.000 1.000 1.000 1.541

Space group P 212121molecules per ASU 2Cell dimensionsa, b, c [] 62.55, 66.15, 205.29 62.94, 66.08, 201.90 63.02, 65.92, 201.96 62.95, 66.35, 203.97 63.13, 66.48, 203.68resolution (highest 55.60–1.80 100.95–1.70 201.96–1.83 63.05–1.55 63.20–2.10

shell) [] (1.806–1.800) (1.706–1.700) (1.93–1.83) (1.555–1.550) (2.18–2.10)Rmerge 0.075 (0.432) 0.060 (0.445) 0.063 (0.284) 0.088 (0.366) 0.055 (0.182)mean I /s (I ) 16.6 (4.0) 17.4 (3.8) 18.0 (5.5) 14.0 (2.7) 16.6 (2.8)no. observations 515 130 403 601 467 939 757 129 145 139completeness [%] 98.9 (98.3) 97.6 (96.2) 98.3 (96.6) 99.0 (86.0) 88.4 (73.4)multiplicity 6.5 (6.5) 4.4 (4.7) 6.4 (6.3) 6.2 (4.5) 3.22 (2.17)no. unique reflections 79 062 86 627 73 075 12 1718 45 088

Refinement

resolution [] 55.60–1.80 100.95–1.70 100.93–1.83 63.05–1.70 63.20–2.10Rwork /Rfree [%] 17.2/20.4 19.7/22.6 16.6/20.7 18.2/20.8 20.5/25.5

no. protein atoms 7184 6750 7106 7597 6885no. waters 527 232 474 747 375no. ligand molecules 2 1 2* 2* 2*no. PLP molecules 2 2 2 2 2no. other molecules 5 3 4 4 4Ramachandran plotfavored 765 (95.6 %) 797 (96.6 %) 758(95.0%) 707 (96.7 %) 784 (94.0 %)allowed 29 (3.6 %) 22 (2.7 %) 27(3.4 %) 20(2.7 %) 35 (4.2 %)disallowed 6 (0.7 %) 6 (0.7 %) 13(1.6 %) 4(0.5 %) 15 (1.8 %)RMSDbond length [] 0.009 0.008 0.009 0.009 0.01bond angle [8] 1.24 1.32 1.34 1.27 1.31

* The ligand forms a covalent adduct with the PLP cofactor in the active site.


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the Supporting Information). TheT m observed in the presence of

5 mm

of compound 1 is 788

C(Table 2).To show that binding of 1

occurs in solution and is notsimply a crystallographic artifact,a mixture of 1 and BioA was fol-lowed by saturation transfer dif-ference (STD) NMR.[20] In this ex-periment, the amplitude differ-ence of compound 1 resonancebetween the on-resonance spec-trum (in which the magnetiza-tion of the protein is saturated)

and off-resonance spectrum (inwhich the magnetization of theprotein is unsaturated) was measured (Figure S2). In the on-resonance spectrum, ligand NMR signals are attenuated by sat-uration transfer from the target protein to the bound ligand. [20]

A significant STD-NMR signal for 1 indicated that it binds toMtb BioA, which coincides with the DSF result (Table 2).

The binding site of 1 was unambiguously identified by thecrystallographic data, which extend to 2.1 resolution. Themolecule occupies only one of the two BioA active sites in theasymmetric unit. There is no obvious difference between thetwo active sites that would readily explain this, but we can

relate the empirical observation that we have frequently seen

differences in the sensitivitytoward binding of small mole-cules in the two “equivalent” po-sitions in this crystal form,[7] evenwith respect to the covalentadduct we previously de-scribed.[11] The two binding envi-ronments are not identical, andthey do not always behave so.

Compound 1 binds in apocket adjacent to the PLP co-factor but does not disrupt theinternal aldimine that definesthe resting state of the enzyme.Electron density (Figure 2A)clearly confirms that the cova-lent bond between Lys283 andthe PLP cofactor remains intact.Compound 1 can be positioned

without ambiguity, due tohigher density of the sulfur onthe thiazole ring. Compound 1binds in direct contact with thePLP, oriented so that the aminogroup is hydrogen bonded tothe phosphate of PLP, the Tyr25hdyroxy, the Tyr157 hydroxy, and

the Gly316’ oxygen (Figure 2B). The amino group of KAPA inthe complex described above makes these same interactions.The binding of 1 is accommodated with little change to theholoenzyme conformational state (Figure 1A). The benzothia-zole heterocycle lies adjacent to the Lys283 side chain and be-tween the side chains of Trp64 and Trp65. The two tryptophanindoles are oriented with a 908 angle between them, and thebenzothiazole plane almost perfectly bisects that angle, sothat comparable hydrophobic contacts are made with eachtryptophan. The benzothiazole is just long enough to stretch

across the binding pocket and to pack at right angles against

Figure 1. A comparison of the BioA complex with KAPA to other structures. A) The holo (PLP-bound) Mtb BioAstructure (PDB ID: 3TFT).[11] The enzyme resting state with PLP covalently linked to Lys283. B) Sinefungin (SFG)-bound structure of B. subtilis BioA (PDB ID: 3LV2) complex. [7] Electron density for KAPA in the active site. C) 2 F oF celectron density (1s ) for KAPA, PLP, and Lys383. D) Detail of KAPA binding vicinity.

Figure 2. Structure of complex with 1. A) Binding site vicinity including electron density (1s 2 F oF c) for bound1 (magenta). Density shows binding without disruption of the Schiff base formed between Lys383 and the PLP(cyan). B) Active site detail in the same orientation as shown in Figure 1. Compound 1 is labeled IN1.


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the face of the aromatic ring of Phe402. Hydrophobic contactsalso exist with Met174 andAla226 (not shown).

Structure-guided fragment

modification

In an effort to develop struc-ture–activity relationships (SARs)around the benzothiazole scaf-

fold, commercially available ana-logues were purchased and eval-uated for their ability to shift theT m of BioA (Table 2). When theT m shift was significant, com-pounds were further investigat-ed by crystallography. One compound from this set, the 2-(hy-drazinyl)benzothiazole (compound 2 ; Table 2) was particularlynotable. Compound 2 was selected because, from the com-pound 1 structure, it appeared that replacing the a-carbonwith nitrogen would result in an extra hydrogen bonding inter-action with the Thr318’ hydroxy group. This compound in-

duced a destabilizing shift of nineteen degrees (T m=678C; Fig-

ure S1). It did not escape our attention that this is nearly theT m assigned to apo BioA.

[17]

Upon initiation of crystal soaking experiments with com-pound 2 , it became clear that this analogue behaved different-ly in the presence of BioA. The PLP-bound holoenzyme crystalstypically grown under the same conditions are yellowish incolor.[11] Holo BioA crystals change color from yellow to redwithin 2 min of soaking in a solution of compound 2

(Figure 3). In cocrystallization experiments, a similar phenom-enon was observed. Within seconds of the addition of 2 tocrystallization drops, the solution develops a deep red colorsuggestive of a chemical reaction. The reaction of 2 with BioAcan be followed spectroscopically, with the time-dependentincrease in absorbance at 330 and 500 nm and a decrease at420 nm that suggests the reaction is complete in just a fewminutes (Figure 3). Cocrystals of the complex were preparedunder the same conditions as holo crystals, but the red colorwas clearly concentrated in the crystals. Diffraction data forcompound 2 cocrystals were collected to 1.90 resolution

(Table 1).The crystal structure of the complex with 2 is shown in

Figure 4. The hydrazine analogue forms a covalent adduct withthe PLP aldehyde to form an extended cis-azo quinonoid spe-cies. The bond between PLP and Lys283 is clearly broken, andthe lysine amino group is relocated to hydrogen bond withThr318’. A planar Schiff base is formed with the hydrazine, andthe entire benzothiazole is well-defined in unambiguous elec-

tron density (Figure 4A). Although the benzothiazole can stillbe considered as occupying the SAM binding subsite, it isrotated ~908 from the position occupied by 1, shifted signifi-cantly away from Tyr25, and out of direct contact with Phe402

(Figure 4A). Two localized enzyme conformational adjustmentsinvolving only the reorientation of side chains occur in con-

junction with adduct formation. The hydroxy group of Tyr407shifts 2.5 from the holoenzyme position to form a newhydrogen bond with the carbonyl oxygen of Arg400. If un-changed, a short contact to the benzothiazole C7 would have

existed. In the largest conformational change, the side chain of

Table 2. Binding of small molecules to BioA.

Compound Structure DSF T m [8C]

1 78

2 67

3 70

4 77

5 83

6 83

7 73

8 83

9 83

10 not available

Figure 3. A) UV/Vis spectrum of 0.16 mm PLP-bound BioA (Holo BioA) upon mixing with 0.4 mm compound 2 atdifferent time points. B) Time-lapse photos of a PLP-bound BioA crystal soaked in 2 (15% PEG 8000, 100 mmHEPES pH 7.5, 100 mm MgCl2, and 5 mm 2) over 2 min.


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Trp65 rotates from the holo position ( c1=608; c2=208) to

a new position ( c1=608; c2=908), in which the hydrogenon the indole Ne1 is positioned for optimal interaction withthe benzothiazole p system. Tyr25 occupies two different posi-tions in this complex with roughly equal occupancy. In one

conformation, the hydroxy is hydrogen bonded to Asp160 andthe hydroxy of Tyr157 as it is in the holo structure. In the otherconformation, the side chain is rotated so that the hydroxygroup forms a hydrogen bond with the alternate carboxylateoxygen of Asp160 (Figure 4 A).

To determine whether the inhibition of BioA by compound2 is reversible, the BioA–2 adduct was dialyzed, which resultedin loss of the deep red color and full restoration of enzyme ac-tivity. To further characterize the mode of inhibition, steady-state kinetic studies were performed under initial velocity con-ditions. Double reciprocal plots of initial reaction velocityunder conditions with varying inhibitor and reactant concen-trations (Figure 5A) show that 2 is a competitive reversible in-

hibitor of BioA with respect to SAM (K i=10.40.6 m m) and anuncompetitive inhibitor with respect to KAPA (K iu=85.4

3.4 m m). This is the expected behavior for an inhibitor of onestep of a ping-pong bi–bi mechanism.[21] In forming a reversibleadduct with the PLP cofactor, compound 2 inhibits the sameBioA enzyme form (PLP-BioA) with which SAM reacts. The in-hibition pattern toward KAPA is uncompetitive, because KAPAbinding is required to regenerate the PLP-bound form of BioA(Scheme 1).

In an effort to explore whether 2 possesses any selectivity, itwas evaluated as an inhibitor of alanine transaminase (ALT)and of aspartate transaminase (AST), two other important

mammalian PLP-dependent transaminases.[22]

Mode of inhibi-

tion studies revealed that com-pound 2 is an uncompetitivereversible inhibitor of ALT withrespect to a-ketoglutarate, witha K iu value of 74.08.2 m m, anda competitive inhibitor with re-spect to alanine (K i=18.9

1.6 m m ; Figure 5B). As with BioA,the inhibitor competes for bind-ing with the compound that isthe amino group donor respon-sible for converting the PLP-bound holo form to the PMPstate. We did not observe anydetectable inhibition of AST,however (data not shown). Al-though compound 2 might notbe a selective inhibitor of BioA,it is also not entirely nonspecific.

Armed with the knowledgethat 2 is a reversible competitiveinhibitor of BioA, we chose toinvestigate attributes of theanalogous hydrazide (3 ; Table 2).Compound 3 also gave rise toa large destabilizing T m shift

(15 8C; Figure S1). We were also able to characterize the com-

plex by cocrystallization (Table 1). No visible color change wasobserved upon addition of 3 to BioA.

The crystal structure of compound 3 also confirms the for-mation of a covalent adduct, but again, the bound adduct con-

formation is unique (Figure 4B). From high-resolution diffrac-tion data (1.7 resolution), we can assert that conjugationthrough the Schiff base appears complete; each of the atomsis sp2-hybridized and planar in its bonding, but there isa subtle curvature imposed throughout the adduct that likelyimplies some degree of electronic strain. Whereas the sidechain of Trp65 remains primarily in the rotated position re-quired to accommodate the adduct with 2, the benzothiazolein the complex with 3 is positioned well above and out of con-tact with this side chain but stacks instead against the indoleof Trp64. The hydrazide carbonyl is oriented toward the Trp65indole nitrogen, but it is 3.9 away from it—too far to be con-sidered a stabilizing hydrogen bond. A water-mediated interac-

tion is possible, but no well-ordered water molecule is ob-served. The benzothiazole also does not point directly towardPhe402 but instead reaches past it and into the larger pocketbounded by Arg400, the side chain that interacts directly withthe carboxylate of KAPA. Tyr25 is observed in the same twoconformations found in the complex with 2.

The structural similarity between 3 and isoniazid (4 inTable 2), one of several chemical agents that comprise thecocktail of drugs often used to treat Mtb infections, led us toevaluate this aryl hydrazide as a BioA inhibitor. In the samebattery of biophysical studies, isoniazid appeared to interactwith BioA (Table 2). It produced a more modest but still large

negative shift in the T m (88C), and crystallographic analysis

Figure 4. A)–C) A comparison of complexes of covalent adducts created by reaction of PLP with compounds 2, 3,and 4. Covalent adducts are labeled PLP-IN2, PLP-IN3, and PLP-IN4, respectively. 1 s 2 F oF c electron density forLys383, the PLP, and each ligand is shown (mesh). Electron density clearly shows that the bond to Lys383 is re-placed with a covalent bond to each compound. Tyr25 is shown in two conformations when so observed. Localbinding environment of each adduct (2–4) is shown in the same orientation in frames D)–F) to emphasize struc-tural similarities and differences.


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showed that it forms a covalent adduct with the PLP that isvery similar to that formed by compound 3 (Figure 4C). Never-theless, in the coupled BioA assay used to assess inhibition ki-netics,[23] both hydrazides 3 and 4 show no inhibitory activity

toward BioA (K i>100 m m). We concluded that the adductsseen crystallographically are so easily reversed upon exposureto substrates that no significant amount of competitive inhibi-tion can be observed by these low affinity compounds.

Discussion

Flexibility of the active site

As previously described,[7] Mtb BioA (also known as DAPA syn-thase or DAPAS) is a functional homodimer with two catalyticsites that lie about 18 apart. Each active site is comprised of

loops contributed by both polypeptide chains, including resi-

dues Pro24–Ser34, Ser62–Ala67, Arg156–Asp160, His171–Arg181, Gln224–Gly228, Arg400–Arg403, Met87’–His97’, andAla307’–Asn322’ (Figure 1 A). Residues tagged with a primeoriginate in the alternate chain. Ser125, Asp254, Lys283 and

Thr318’ all make specific interactions with the PLP, are strictlyconserved in all class I transaminases,[24] and adopt a conservedconformation in all BioA structures reported to date. Althoughresidues 25–33 are disordered in the original BioA structure re-ported by Dey et al. (PDB ID: 3V0),[7] Tyr25 is well ordered inthe structure we reported as a “pre-reaction” conformation inour study of an irreversible inhibitor of BioA,[11] and the side-chain hydroxy of the tyrosine lies hydrogen bonded to Asp160and poised to interact with substrates. This structure (PDB ID:3TFT) is used represent the holoenzyme (PLP-bound restingstate) conformation in the remainder of this discussion. In thisstate, the PLP is covalently bound to the side-chain amine of

Lys283, a well-known feature of transaminases (Figure 1A).

Figure 5. A)–B): Inhibition studies with 2. Initial rate data of variable amounts of inhibitor and either KAPA (A) or SAM (B). KAPA was varied from 0.94 to7.5 m m, and SAM was varied from 0.3 to 2.5 mm, with the fixed substrates held at 2.34 mm for SAM and 1.9 m m for KAPA. Compound 2 was used at concentra-tions of 0 m m (*), 31.3 m m (*), 62.5 m m (!), and 125 m m (! ). The inhibitor is uncompetitive with respect to KAPA and competitive with respect to SAM, withK i values of 85.43.4 m m and 10.40.6 m m, respectively. Inhibition of ALT by compound 2. C)–D): Initial rate data of variable amounts of inhibitor with respectto a-ketoglutarate (C) or alanine (D). The substrate a-ketoglutarate was varied from 6.25 to 50 m m, and alanine was varied from 0.625 to 10 mm, with thefixed substrates held at 10 mm for alanine or 75 m m for a-ketoglutarate. Compound 2 was used at concentrations of 0 m m (*), 29.6 m m (*), 44.4 m m (! ), and66.6 m m (!). The inhibitor is uncompetitive with respect to a-ketoglutarate and competitive with respect to alanine, with K i values of 74.08.2 m m and18.91.6 m m, respectively.


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Structures of Mtb BioA with substrates SAM and KAPA havenot been previously reported, but detailed homology-basedconceptions of substrate binding have been proposed basedon studies with substrate surrogates (e.g., SAM analogue sine-fungin; PDB ID: 3LV2) and homologous proteins (e.g., Bacillussubtilis BioA co-structure with KAPA, PDB ID: 3DU4).[7] Despitethe fact that the two substrates are quite different, bothappear likely to occupy the same binding site, comprisinga largely hydrophobic pocket between aromatic side chains of Trp64, Trp65, Tyr25 and Phe402. The adenosine of SAM likelyextends further (Figure 1B) but, apart from the side chain of Arg400, the specific groups that interact with the nucleobaseare not convincingly known because of the disorder observedin the surrounding loops (residues 23–34 and 309–317) inmany of the homologue and BioA crystal structures. This flexi-bility might be an important characteristic of an enzyme thatmust adapt to diverse substrates during the catalysis of transa-mination.

Our own attempts to obtain an experimental complex struc-

ture of Mtb BioA with SAM or sinefungin were unsuccessful. Itcan be seen, however, that the benzothiophene compoundseach occupy much of the same subsite (Figure 2B). In the sine-fungin complex, the amino group makes a cation–p interactionwith the Phe402 side chain conserved in both the B. subtilisand Mtb enzymes. In the complex with 1 , C6 of the benzothia-zole sits in exactly the same position. KAPA also binds againstthis phenylalanine side chain (Figure 1 D), providing an explan-

ation for why a phenylalanine at a comparable position is con-served in all homologue structures. Models for the binding of both KAPA and SAM substrates led to assertions of a significantrole for Tyr25 as a participant in hydrogen bonding that helps

orient substrate heteroatoms so they are poised to displacethe lysine and form adducts with the PLP.[7] This role is sup-ported by our complex with KAPA. The Tyr25 hydroxy is indirect contact with the KAPA amino group, as well as sidechains of Tyr157 and Asp160—residues that combine to holdthe KAPA amino group against the PLP. The exchange of hy-drogen bonds between the Tyr25 hydroxy, Asp160, and anypotential substrate amino groups is facilitated by the flexibilityof the Tyr25 side chain, which can move in and out of contactwith the substrate during catalysis by using small conforma-tional changes such as those observed in different complexeswith the covalent adducts of this structural study. This rolecould be sufficient to explain why mutation of Tyr25 in B. subti-

lis BioA leads to catalytic inactivation of the enzyme.[7]

In the B. subtilis BioA–sinefungin complex reported by Deyet al.,[7] Tyr25 is observed in a different conformation with theside chain rotated 180

to contact the adenosine base (Fig-ure 2B). They conclude that p-stacking between the adenosineand this tyrosine or the phenylalanine found in other bacterialspecies is an important feature stabilizing SAM binding in MtbBioA. Although we have no specific data to contradict thishypothesis, we do not find their arguments compelling. Thegeometry for effective p-stacking in the sinefungin complexstructure is poor, as the phenol ring of Tyr25 is tilted 458 fromthe plane of the adenosine; the Tyr25 hydroxy makes the clos-

est contact to sinefungin in this complex, and this is not indi-

cative of favorable p-stacking. In all complexes reported here,Tyr25 is oriented as seen in the KAPA or apo complexes, andnever as seen in the sinefungin complex (Figure 1). The bind-ing of most of the benzothiophene molecules is enhanced byp-stacking, but none of them precipitate such a dramaticchange so as to involve Tyr25. The B. subtilis BioA complex(PDB ID: 3LV2) is a cocrystal with an H315R mutant, and thehistidine occupies the position taken by the side chain of Tyr25 in the wild-type enzyme (Figure S3). It is possible thatthe shift in Tyr25 observed in the sinefungin–BioA complex isa consequence of this mutation, rather than of sinefunginbinding.

Stabilization of an azo-quinonoid intermediate

Compounds 2, 3, and 4 all react with the PLP cofactor reversi-bly to form stable covalent adducts. UV/Vis spectroscopy andcrystallography results for compound 2 confirm that the hydra-zine analogue forms an extensively conjugated cis-azo-quino-

noid species. Although similar quinonoids form transiently asintermediates in the catalytic mechanism of all transaminases,they are rarely captured.[25,26] We propose a mechanism for thestabilization of this adduct from 2 in Scheme 2A. By analogyto the reaction with substrates, the hydrazine first displacesthe lysine to form a PLP-coupled imine. Tautomerization of thisimine leads to the formation of an azo-quinonoid; delocalizedbonding is responsible for the sanguine color. Further reversi-

ble tautomerization of the pyridoxal ring is possible throughconjugation all the way to the benzothiophene (Scheme 2B).This is a unique chemical feature of compound 2 not sharedwith the hydrazides (3 or 4) that can only form less extensively

delocalized imines (Scheme 2 C). Although these hydrazide ad-ducts are evidently more stable than the PLP-lysine Schiff basethat they supplant, the fact that these compounds show nodetectable inhibition in competition with substrates suggeststhat their stability is low. The hydrazine adduct, however, is un-usually stable, with a K i of 10 m m, which is approximately 80times lower than the K m of SAM.

[9]

The reactivity of hydrazines and hydrazides with PLP-depen-dent transaminases has been known for decades,[27] and the ki-netics of inhibition were studied in detail long ago.[28] More re-cently, there have been a number of attempts to increase thepotency of inhibitors of PLP-dependent enzymes that containa reactive hydrazide. Ejim et al. confirmed the formation of a

stable adduct to PLP bound to cystathionine b-lyase with aseries of hydrazinocarbonylmethylbenzamides, and they wereable to improve inhibitor binding affinity by 50-fold with thepreparation of small analogue library.[29] Another hydrazide hasrecently been identified as an inhibitor of E. coli BioA bywhole-cell phenotypic screening.[30] Others have investigateda series of aryl hydrazides as inhibitors of LL-diaminopimelateaminotransferase.[31] Structure–activity relationships revealed inthe study of these systems have identified the reactive hydra-zide as a necessary, but not sufficient, molecular featureneeded for inhibition. Potent inhibition can only occur whenother binding site complementarity also exists. These examples

do not benefit from the extensive electronic delocalization


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that occurs upon reaction with the hydrazylbenzothiophene of 2. For this reason, we suggest that this compound might serveas a particularly effective starting point for further optimiza-tion. Whether selective inhibitors of BioA can be generatedfrom the 2-(hydrazinyl)benzothiophene lead remains to be de-termined, but complexes with compounds characterized aspart of this study have exposed a variety of different confor-mational states that are accessible to the BioA enzyme thatshould prove valuable in the structure-based design of morepotent inhibitors based on this scaffold.

The value of destabilizing fragment hits

Many studies evaluating the use of thermal shift methods foridentification of protein ligands focus only on molecules thatincrease the transition temperature.[18,19,32] All the compoundsthat were part of this study shifted T m significantly downward .This is true of the initial noncovalent fragment hit 1 (DT m=5 8C), as well as the reversible adducts with DT m shifts in therange of 6 to 16 8C. In prior DSF studies, we had tentativelyascribed a much lower T m (67 8C) to the apo (PLP-free) form of BioA,[17] and we were initially drawn to these fragment hits be-cause we expected to find that the molecules bound in place

of the PLP. Structural characterization of these compounds

clearly shows this is not the case. While fragment hits that shiftthe BioA T m to higher temperature may be described ina future report, we can confirm that other molecules havebeen identified that bind in the same subsites and shift the T mupward (data not shown). Why these compounds are so sharp-ly destabilizing is not known; it could be that they play a great-er role in the stabilization of the unfolded state. Empirically, itis useful to note, for the benefit of those hoping to conductsimilar studies with other proteins, that destabilizing com-pounds might also be worth structural investigation. Com-pounds that destabilize a protein are expected to lead to more

rapid protein degradation. In Mtb, damaged proteins are re-moved by the Pup-proteasome system.[33] Thus, small mole-cules that lead to protein destabilization could potentiallyresult in protein depletion, which might be advantageousunder non-replicating conditions where protein synthesis isnot occurring to replenish degraded proteins.

Conclusions

Fragment screening permitted an exploration of BioA confor-mational space. By coupling the screening of a small library of compounds for those capable of affecting the BioA tempera-

ture of unfolding with structural characterization of resulting

Scheme 2. A) Proposed mechanism of compound 2–PLP adduct formation; B) Tautomerization of compound 2–PLP adduct; C) Proposed mechanism of com-pound 3–PLP adduct formation.


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compounds, we identified an 2-(aminomethyl)benzothiazole(1) that binds in a position where it must compete with sub-strate SAM. Commercially available analogues were acquiredand characterized, leading to the identification of hydrazineand hydrazide analogues that are capable of forming covalentadducts with the PLP cofactor. The structural characterizationof adducts formed with these compounds has provided aglimpse of the different ways that BioA can adapt in responseto the binding of small molecules. The side chains of Tyr25,Trp65, Arg400, and Tyr407 are shown to be quite flexible. Al-though the movement of Arg400 and Tyr25 had been predict-ed, the flexibility of the other side chains was not anticipated.Reactive analogues, including the hydrazines and hydrazides,are useful tools for exploration of structure–activity relation-ships of fragment hits, as they provide a means to explorea broader sampling of conformational changes near the PLPbinding site.

Some aryl hydrazines and hydrazides appear to readily formcovalent complexes with the PLP cofactor in this enzyme. In

many cases, however, this adduct can be readily reversed sothat the formation of covalent bond is no guarantee of potentinhibition. Although the hydrazides we investigated can formcovalent adducts, no inhibition was observed by these com-pounds. Apparently, the resulting PLP-hydrazones formedupon reaction of these hydrazides are as reactive in the pres-ence of substrates as the Schiff base conjugate of the nativeprotein lysine. Isoniazid is one such compound that can form

an easily reversible adduct but shows no activity as a BioA in-hibitor.

The 2-(hydrazinyl)benzothiazole (2) does seem to have un-usually stable characteristics that distinguish it as a reversible

covalent inhibitor of BioA (K i=

10 m m

). The compound displayssome selectivity with respect to other PLP-dependent transa-minases (ALT K i=19 m m ; no effect on AST), so the benzothia-zole constitutes a viable scaffold for the development of moreselective inhibitors of biotin biosynthesis in Mtb.

Experimental Section

BioA protein expression and purification: BioA protein was ex-pressed and purified according to published procedures. [7,17,23] Es-cherichia coli Rosetta 2 (DE3) cells (EMD Millipore), transformedwith a plasmid (pCDD126) encoding N-terminally His-tagged BioA,were initially grown at 37 8C in lysogeny broth (LB; 5 mL) contain-

ing ampicillin (100 m g mL1

) and chloramphenicol (50 m g mL1

) untilreaching an OD600 of 0.6. The culture was transferred into four 2 Lbaffle flasks containing Terrific Broth (400 mL) with ampicillin(100 m g mL1) and chloramphenicol (50 m g mL1) and was allowedto grow for 16 h at 37 8C. The cells were harvested by centrifuga-tion (8000 g for 20 min at 4 8C). The cells were resuspended in lysisbuffer (70 mL, 50 mm HEPES, 500 mm NaCl, 1 mm PMSF, 0.4 mmPLP, 1 mm MgCl2, pH 7.5) and lysed by sonication. Next, TCEP(0.1 mm) and benzonase (3 units, EMD Millipore) were added tothe lysate, and clarified lysate was obtained by centrifugation(53300 g for 45 min at 4 8C). Clarified lysate was filtered and loadedonto a HisTrap HP Ni-NTA column (5 mL, GE Healthcare). Thecolumn was washed to baseline with buffer A containing HEPES(50 mm pH 7.5), NaCl (500 mm), PLP (0.1 mm), TCEP (0.1 mm), and

imidazole (40 mm) and eluted with a linear gradient to 100% buf-

fer B (buffer A containing 500 mm imidazole). BioA eluted asa single peak in 45% buffer B. This BioA fraction was pooled, con-centrated by centrifugation (Amicon Ultra), and loaded ontoa HiPrep 26/60 Sephacryl S-200 HR column (GE Healthcare) pre-equilibrated with SEC buffer (25 mm HEPES pH 7.5, 50 mm NaCl,0.1 mm TCEP, and 1 mm EDTA). Full PLP occupancy was ensured byadding PLP (1 mm) to the pooled BioA fraction and concentrating

by centrifugation to a low volume (2 mL). Unbound PLP was re-moved by repeated dilution in SEC buffer lacking PLP and recon-centration. The homogeneity of the protein was assessed by SDS-PAGE (4–20% Tris-HCl BioRad). Differential scanning fluorimetry(DSF) was used as previously described to ensure that the holoen-zyme was fully saturated with co-enzyme.[17]

Fragment library: The Maybridge Ro3 1000 Diversity Fragment Li-brary was obtained from Thermo Fischer Scientific. This library in-cludes ~ 1000 compounds that are “rule-of-three” compliant (MW300 D; c log P 3.0; H-bond acceptors3; H-bond donors3; ro-tatable bonds3; polar surface area60 2). Small molecules ob-tained in solid form were dissolved in 100% DMSO to a final com-pound concentration of 200 mm and stored at 20 8C.

Differential scanning fluorimetry (DSF) for fragment screening:DSF was used to assess the homogeneity of the BioA protein; itwas also used to identify small molecules that could cause a signifi-cant shift in the denaturation temperature (T m) of the protein. Toassess the homogeneity of the protein, purified BioA was dilutedat 4 8C to give a DSF solution (40 m L) consisting of BioA(0.05 mgmL1), HEPES (25 mm, pH 7.5), NaCl (50 mm) , and 5XSYPRO Orange (Life Technologies). To identify small molecule hits,small molecules from the Maybridge Ro3 library were added indi-vidually to each DSF solution to a final concentration of 5 mm. Thefluorescence response (melting curve) was measured across a tem-perature range following established procedures.[18] A T m value wasdetermined from the peak of the first derivative of each meltingcurve; calculations were performed by using Bio-Rad CFX Manag-

er software. The plots were generated by using R (http://www.R-project.org).

Saturation transfer difference NMR spectroscopy (STD-NMR):

STD-NMR spectra were obtained at 208C on a Bruker 700 MHzNMR spectrometer with a TCI cryoprobe, incorporating Z-axis gra-dients.[20] Samples contained BioA protein (30 m m) and com-pound 1 (200 m m). A one-dimensional pulse sequence incorporat-ing a T11 filter was used for the acquisition of STD-NMR spectra.The on-resonance frequency was set to 0.8 ppm, and the off-reso-nance frequency was set to 30 ppm. Irradiation was performed byusing 50-Gaussian pulses with a 1% truncation and a 49 ms dura-tion, separated by a delay of 1 ms, to give a total saturation timeof 2 s. The duration of the T 11 filter was 15 ms. STD-NMR spectra

were acquired with a total of 6144 transients, in addition to 32scans, to allow the sample to come to equilibrium. The spectralwidth was 8 kHz. A reference spectrum was taken under the sameconditions.

Crystallization and X-ray data collection: Crystallization condi-tions were as described previously.[7] BioA was cocrystallized witheach compound respectively by vapor diffusion in a hanging dropat 20 8C. Protein solution (10 mgmL1 in 25 mm HEPES pH 7.5,50 mm NaCl, and 0.1 mm TCEP) was mixed with reservoir solution(9–14% PEG 8000, 100 mm HEPES pH 7.5, 100 mm MgCl2, and5 mm compound) and a seed solution (a reservoir solution contain-ing crushed BioA crystals) in a 4:3:1 ratio. Crystals appeared in thedrop within 24 h and grew to their full size in 72 h. BioA-com-

pound cocrystals were cryoprotected by transferring to a cryo solu-


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tion (15 % PEG400, 15% PEG 8000, 100 mm HEPES pH 7.5, 100 mmMgCl2, and 5 mm compound) and then flash frozen in liquid nitro-gen. The diffraction data for KAPA and for cocrystals with com-pounds 1, 2, and 3 were collected at 100 K by using synchrotronradiation with a Dectris Pilatus 6M pixel detector on beamline 17-ID (IMCA-CAT) at APS (Chicago, USA). Data were processed, inte-grated, and scaled with XDS[34] and SCALA[35] by using the auto-

PROC scripts available at APS-17-ID (IMCAT-CAT). Data for the com-pound 4 cocrystal were collected at 100 K by using Cu K a radiationon a Rigaku HighFlux HomeLab rotating-anode system witha Saturn 944+ CDD detector in the Kahlert Structural Biology Lab-oratory at the University of Minnesota. These data were processed,integrated, and scaled with d*TREK.[36] Data collection and process-ing statistics are given in Table 1.

Structure determination: Structures were solved by molecular re-placement using Phaser[37] in the CCP4 package,[38] and atomic co-ordinates from PDB 3TFT as a search model.[11] Refinement andmodel building were performed with REFMAC5[39] and COOT.[40]

The figures were prepared with PyMOL (The PyMOL MolecularGraphics System, Version 1.5.0.4 Schrçdinger, LLC.). Structures weresuperimposed for analysis and display by using the shared BioA-

PLP overlay method of the DrugSite server.[41] Atomic coordinatesand diffraction data for the five crystal structures presented in thisreport have been deposited in the Protein Data Bank with acces-sion codes 4MQN, 4MQO, 4MQP, 4MQQ, and 4MQR (Table 1).

UV/Vis spectroscopy: A NanoDrop 1000 UV/Vis spectrophotome-ter (Thermo Scientific) was used for all UV-Vis spectroscopy. MtbBioA protein (2.0 m L, 0.16 mm) in HEPES (25 mm, pH 7.5), NaCl(50 mm, 1 mm EDTA), and TCEP (0.1 mm) was mixed with com-pound 2 (2.0 m L, 0.4 mm) in HEPES (25 mm, pH 7.5), NaCl (50 mm),EDTA (1 mm), and TCEP (0.1 mm), and its UV/Vis spectrum was im-mediately measured upon mixing. UV/Vis spectra were taken at 0,15, 30, 60, and 120 s after mixing.

Biochemical evaluation: Mode of inhibition studies were carriedout under initial velocity conditions in a total volume of 50 m L at25 8C in 384-well black plates (Corning 3575). Reactions were setup in triplicate and consisted of BioA (114 nm) in reaction buffer(100 mm Bicine pH 8.6, 50 mm NaHCO3, 1 mm MgCl2, 0.0025%Igepal CA-630, 5 mm ATP, 0.1 mm PLP, 320 nm E. coli BioD, 20 nmFl-DTB, 184 nm streptavidin, and 1 mm TCEP) with either variableamounts of KAPA (0.94–7.5 m m) with a fixed concentration of SAM(2.34 mm) or with a variable amount of SAM (0.3–2.5 mm) anda fixed concentration of KAPA (1.9 m m). Each substrate concentra-tion was run with 0, 31.25, 62.5, and 125 m m inhibitor. Reactionswere monitored on a microplate reader with lex=485 nm and lem=535 nm. A standard curve for dethiobiotin (2.7 nm–2 m m) inreaction conditions lacking only BioA was used to convert fluores-

cence into enzyme velocities as previously described.[23]

The datawere fit by using the enzyme kinetics module of SigmaPlot tocompetitive, uncompetitive, and non-competitive models, and themodel with the highest r 2 value was selected. The PLP-utilizing en-zymes alanine transaminase (ALT, EC 2.6.1.2) and aspartate transa-minase (AST, EC 2.6.1.1) were used to test off target inhibition of compound 2. Reactions were carried out in 100 m L in 96 well UVclear half-area plates (Corning 3679). For ALT, reactions consistedof enzyme (10 mU) in reaction buffer (60 mm Bicine pH 8.0, 0.1 mmNADH, 1 mm TCEP) containing 100 mU lactate dehydrogenase(LDH) and either a fixed concentration of a-ketoglutarate (75 m m)with 0.625–10 mm alanine or a fixed concentration of alanine(10 mm) with 6.25–50 m m a-ketoglutarate. Each concentration of substrate was run with DMSO or 29.6–66.7 m m compound 2. The

reactions (in duplicate) were monitored by the decrease in A340

that corresponds to the consumption of NADH by LDH upon theformation of pyruvate from ALT. Initial velocities were calculated byusing the molar absorptivity of NADH (6220 m1 cm1 at 340 nm)and fit as described above by using SigmaPlot. To test for inhibi-tion of AST, 2 mU of enzyme in reaction buffer containing 100 mUmalate dehydrogenase (MDH), 236 m m a-ketoglutarate and 0.156–2.5 mm aspartate was tested against compound 2 (100 m m).

Acknowledgements

This work was supported in part by a grant from the Minnesota

Department of Employment and Economic Development #SPAP-

06-0014-P-FY07 to B.C.F. and a grant from the National Institutes

of Health (AI091790) to Dirk Schnappinger (Weill Cornell Medical

College). Use of the IMCA-CAT beamline 17-ID (or 17-BM) at the

Advanced Photon Source was supported by the companies of the

Industrial Macromolecular Crystallography Association through

a contract with Hauptman–Woodward Medical Research Insti-

tute. The authors gratefully acknowledge the University of Minne-sota Supercomputing Institute for access to software and compu-

tational resources.

Keywords: hydrazine · reversible covalent inhibitors ·transaminase · tuberculosis · X-ray crystal structures

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