structures of leishmania major pteridine reductase complexes reveal the active site features

doi:10.1016/j.jmb.2005.06.076 J. Mol. Biol. (2005) 352, 105–116

Structures of Leishmania major Pteridine ReductaseComplexes Reveal the Active Site Features Importantfor Ligand Binding and to Guide Inhibitor Design

Alexander W. Schuttelkopf1, Larry W. Hardy2, Stephen M. Beverley3 andWilliam N. Hunter1*

1Division of BiologicalChemistry and MolecularMicrobiology, School of LifeSciences, University of DundeeDundee DD1 5EH, UK

2Sepracor Inc., 84 WaterfordDrive, Marlborough, MA 01752USA

3Department of MolecularMicrobiology, WashingtonUniversity School of MedicineCampus Box 8230, 660 S. EuclidAvenue, St. Louis, MO63110-1093, USA

0022-2836/$ - see front matter q 2005 E

Abbreviations used: ASA, accessidiffraction-component precision ind8-dihydro-6-(1,2-dihydroxypropyl)p7,8-dihydrobiopterin; DHFR, dihyE.S.R.F., European Synchrotron Rad4-amino-N10-methyl-pteroylglutamtrexate; NCS, non-crystallographicpara-aminobenzoic acid; PDB, Protepteridine reductase; SDR, short-chareductase; S.R.S., Synchrotron Radia6-triaminoquinazoline; THB, 2-amin6-(1,2-dihydroxypropyl)pteridin-4(3tetrahydrobiopterin; TOP, 5-((3,4,5-tmethyl)-2,4-diaminopyrimidine or tthymidylate synthase.E-mail address of the correspond

[email protected]

Pteridine reductase (PTR1) is an NADPH-dependent short-chain reductasefound in parasitic trypanosomatid protozoans. The enzyme participates inthe salvage of pterins and represents a target for the development ofimproved therapies for infections caused by these parasites. A series ofcrystallographic analyses of Leishmania major PTR1 are reported. Structuresof the enzyme in a binary complex with the cofactor NADPH, and ternarycomplexes with cofactor and biopterin, 5,6-dihydrobiopterin, and 5,6,7,8-tetrahydrobiopterin reveal that PTR1 does not undergo any majorconformational changes to accomplish binding and processing ofsubstrates, and confirm that these molecules bind in a single orientationat the catalytic center suitable for two distinct reductions. Ternarycomplexes with cofactor and CB3717 and trimethoprim (TOP), potentinhibitors of thymidylate synthase and dihydrofolate reductase, respect-ively, have been characterized. The structure with CB3717 reveals that thequinazoline moiety binds in similar fashion to the pterin substrates/products and dominates interactions with the enzyme. In the complex withTOP, steric restrictions enforced on the trimethoxyphenyl substituentprevent the 2,4-diaminopyrimidine moiety from adopting the pterin modeof binding observed in dihydrofolate reductase, and explain the inhibitionproperties of a range of pyrimidine derivates. The molecular detailprovided by these complex structures identifies the important interactionsnecessary to assist the structure-based development of novel enzymeinhibitors of potential therapeutic value.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: antifolates; dihydrofolate reductase; Leishmania; pterin; short-chain reductase
*Corresponding author
lsevier Ltd. All rights reserve

ble surface area; DPI,ex; DHB, 2-amino-7,teridin-4(3H)-one ordrofolate reductase;iation Facility; MTX,ic acid or metho-symmetry; pABA,in Data Bank; PTR1,in dehydrogenase/tion Source; TAQ, 2,4,o-5,6,7,8-tetrahydro-H)-one or 5,6,7,8-rimethoxyphenyl)rimethoprim; TS,

ing author:

Introduction

Infection by trypanosomatid protozoans, e.g.Trypanosoma and Leishmania species, causes arange of serious human diseases.1 Current treat-ments of these infections involve drugs such assodium stibogluconate and nifurtimox, whichinduce serious side-effects and this, together withan increase in drug-resistant parasites, has createdan urgent requirement for new and more effectivetreatments.2 The ideal enzyme targets for thedevelopment of such new antimicrobial drugs arethose that are essential for the survival of theparasite, and either absent from the human host ordisplay markedly differing substrate specificities.Our improved knowledge of trypanosomatid

d.

Figure 1. The two-stage reduction of biopterin (2-amino-6-(1,2-dihydroxypropyl)pteridin-4(3H)-one) to 7,8-dihydrobiopterin (2-amino-7,8-dihydro-6-(1,2-dihydroxy-propyl)pteridin-4(3H)-one), and then to 5,6,7,8-tetrahydro-biopterin (2-amino-5,6,7,8-tetrahydro-6-(1,2-dihydroxy-propyl)pteridin-4(3H)-one) catalyzed by PTR1. Each stagerequires one reducing equivalent provided by the cofactorNADPH.The carbonatoms (C6 andC7) that accept hydridefrom the cofactor are marked with an asterisk (*).

106 Structures of PTR1 Ligand Complexes

biology and biochemistry, greatly facilitated bydevelopments in molecular genetics and genomesequencing efforts, promotes understanding of howexisting drugs function and is helping to identifypotential targets for chemotherapeutic attack.3,4

Whilst there are increased opportunities to targetrecently discovered enzymes there are also well-studied systems that still have much to offer. Ourpresent study concerns the Leishmania pteridinereductase (PTR1; EC 1.5.1.33), which is involvedin folate/pterin metabolism and, as will beexplained, presents a link to the well-knowntherapeutic targets dihydrofolate reductase(DHFR; EC 1.5.1.3) and thymidylate synthase (TS;EC 2.1.1.45).

Folate metabolism, in particular DHFR and TS,have been targeted successfully for the treatment ofcancer and microbial infection.5–10 TS catalyses theconversion of dUMP to dTMP using the cofactorN5, N10-methylene tetrahydrofolate (THF) as boththe C-donor and reductant, whilst DHFR maintainsthe THF pool by the NADPH-dependent reductionof dihydrofolate (DHF). Inhibition of either enzymelimits the supply of dTMP required for DNAsynthesis, thus curtailing replication and leadingto cell death. In most organisms, DHFR and TS areseparate entities. Trypanosomatid protozoanspossess a bifunctional DHFR-TS, which has beenstructurally characterised.11

Since folates and pterins are essential for trypano-somatid growth, antifolates targeting DHFR, inprinciple, should provide an ideal treatment.However, DHFR inhibitors are largely ineffectivefor the control of trypanosomatid infections, partlydue to the presence of PTR1.12 This short-chaindehydrogenase/reductase (SDR) family memberexhibits a broad NADPH-dependent pteridinereductase activity, capable of reducing unconju-gated (biopterin) and conjugated (folate) pterinsfrom either the oxidized or dihydro-state (Figure 1).This activity is essential for parasite growthin vitro.12 The biochemical activities of PTR1 overlapthose of DHFR but PTR1 is less susceptible toinhibition by classical antifolates such as metho-trexate (MTX); thus, it can function as a metabolicby-pass to alleviate DHFR inhibition.13 However, aninhibitor of PTR1 has the potential to act inconcert with known antifolates to provide a novelapproach to the treatment of trypanosomatidinfection.14

The kinetics and stereochemical course of thereductions catalyzed by PTR1 have been studiedtogether with analysis of a library of inhibitors.14,15

Crystal structures are available of the enzyme fromLeishmania major (LmPTR1) in ternary complexeswith cofactor and 7,8-dihydrobiopterin (DHB),16

the archetypal antifolate drug MTX (4-amino-N10-methyl-pteroylglutamic acid; Figure 2)16 and 2,4,6-triaminoquinazoline (TAQ).17 Other laboratorieshave reported the 2.7 A resolution crystal struc-ture of PTR1 from Leishmania tarentolae18 and thecrystallization of the enzyme from Trypanosomacruzi.19

We set out to characterize PTR1–ligandcomplexes to derive important molecular detailsnecessary to understand the enzyme and to under-pin a structure-based approach to inhibitordevelopment. Structural analyses of discrete stepsalong the two-stage PTR1 catalytic process(Figure 1) are reported. The binary complex withcofactor (PTR1:NADPH) corresponds to the stateprior to binding substrate. The ternary complexeswith biopterin, and DHB represent the initialsubstrate complexes for stages I and II, respectively.The DHB complex also represents the productcomplex at the end of stage I, and finally the5,6,7,8-tetrahydrobiopterin (THB) complexrepresents the product complex at the end ofstage II.

The potent TS inhibitor CB3717 (N10-propargyl-5,8-dideazafolic acid, Figure 2), resembles DHF,though with the addition of a propargyl group, andwe reasoned that it might bind to PTR1, thereby

Figure 2. The chemical structures of three PTR1inhibitors: methotrexate, CB3717 and trimethoprim. Themolecules are depicted in a similar orientation in allFigures. The methotrexate molecule is depicted with thepterin-ring flipped about the N2–N5 axis relative to otherpterins as actually observed in the PTR1 active site.

Structures of PTR1 Ligand Complexes 107

raising the potential to design a molecule capableof inhibiting both PTR1 and TS. Structures ofCB3717 in complex with TS from Escherichia coli,20

Lactobacillus casei,21 Pneumocystis carinii TS22 and thebifunctional DHFR-TS of L. major11 are available forcomparative purposes.

One of the most successful antimicrobial anti-folates is trimethoprim (5-((3,4,5-trimethoxyphenyl)methyl)-2,4-diaminopyrimidine, TOP; Figure 2).9

TOP is a potent inhibitor of DHFR23 and structureswith the enzyme from Mycobacterium tuberculosis,24

Staphylococcus aureus,25 E. coli and chicken26 areknown. A structure of PTR1 in complex with TOPwas sought to provide a different molecular frame-work in the enzyme active site, one that is necessaryto understand PTR1 inhibition by a series of 2,4-diaminopyrimidine derivatives.

Results and Discussion

Crystallographic details

Five medium-resolution crystal structures ofLmPTR1 ligand complexes have been determinedwith diffraction data recorded using synchrotronradiation. The structures include the binarycomplex with cofactor NADPH (2.65 A resolution),ternary complexes of oxidized cofactor andbiopterin (2.40 A) and THB (2.55 A) and two ternarycomplexes with the inhibitors CB3717 (2.70 A) andTOP (2.60 A). The structures all display space groupP212121 and are isomorphous. A homotetramer, ofmolecular mass approximately 120 kDa, constitutesthe asymmetric unit and gives a Matthews coeffi-cient, VM, of 2.8 A3 DaK1 and 55% (v/v) solventvolume. The PTR1 tetramer displays 222 pointgroup symmetry. Most of the protein residues arewell defined by the electron density, with only twosegments disordered in all subunits; these are thesurface loops comprising residues 75–80 and120–130. In addition, residues 230–240 are dis-ordered in two subunits. This is discussed later. Inthe structures, the cofactor, biopterin, THB andpteridine component of CB3717 are well ordered ineach of the four active sites in the asymmetric unit.The remainder of CB3717 is poorly ordered. Theinhibitor TOP is observed in two active sites only.Further information is given in Table 1.

Overall structure and cofactor binding

PTR1 is a member of the SDR super family ofenzymes, of which there are over 3000 memberswith sequence identities at the 15–30% level.27,28

The subunit displays the extended double-Rossmann fold typical of the SDR family, which isbased on a central seven-stranded parallel b-sheetwith three a-helices on either side (Figure 3(a)). Inaddition, PTR1 has a short helix a6 following b6,and two short strands between b3 and a3 framing adisordered loop (residues 75–80). Another,frequently disordered section of the PTR1 structureoccurs between b4 and a4 (residues 120–130).Residues 230–240 form the so-called substrate-binding loop29 between b6 and a6, a commonstructural feature of the SDR family. Generally, thisloop is disordered or in an open conformation incrystal structures of the apo form or binarycomplexes of family members. Once substrate orinhibitors bind, the loop adopts an ordered andclosed conformation. In the present structures, thissubstrate-binding loop is ordered in two of the foursubunits that constitute the asymmetric unit, due tointeractions within the crystal lattice, in particularthe associations of Met233 and Pro234 with Tyr114from a symmetry mate (not shown).A comparison of seven LmPTR1 structures, the

five reported here plus the MTX and DHBcomplexes,16 by a least-squares fit of all super-imposable Ca atoms in the functional tetramers

Table 1. Data collection and refinement statistics

Structure PTR1:NADPHPTR1:NADPC:

biopterinPTR1:NADPC:

THBPTR1:NADPH:

CB3717PTR1:NADPH:

TOP

Wavelength (A) 0.933 0.890 0.933 1.033 0.890Unit cell:

a (A) 94.31 95.27 93.10 94.54 94.70b (A) 103.49 104.55 103.57 103.83 104.44c (A) 137.98 138.07 137.43 137.05 138.06

Resolution range (A) 25.00–2.65 25.00–2.40 25.00–2.55 30.00–2.70 25.00–2.60Unique reflections 40607 47504 43624 35456 41645Redundancy 3.7 4.3 4.8 3.9 4.2Completeness (%) 96.8 (92.6) 86.9 (76.5) 98.2 (98.0) 92.7 (85.0) 98.2 (96.4)I/s(I) 13.6 (5.5) 12.9 (3.7) 17.2 (5.6) 9.8 (2.9) 14.4 (5.5)Rsym (%) 8.8 (22.3) 9.9 (29.3) 7.6 (25.9) 13.7 (40.8) 9.4 (26.2)Wilson B (A2) 41.8 37.8 51.1 37.4 38.0

Protein residues 1044 1036 1046 1039 1042Molecules of solvent,cofactor, ligand andethylene glycol

378/4/0/6 268/4/4/5 152/4/4/6 211/4/4/6 185/4/2/0

R/Rfree (%) 22.6/26.9 21.9/24.2 19.8/23.8 20.3/23.6 25.3/28.8Average B (A2):

Overall 36.3 33.3 46.6 28.9 29.3Protein 36.3 33.3 46.7 28.3 29.0Cofactor 35.0 28.9 42.3 29.4 41.2Ligand – 42.2 48.6 69.4 51.9Solvent 35.5 31.4 44.6 23.2 21.3Ethlyene glycol 31.5 44.6 52.2 41.8

RMSD bond lengths (A) 0.012 0.008 0.008 0.007 0.010RMSD bond angles (8) 1.3 1.4 1.5 1.0 1.4Cruickshank’s DPI (A) 0.32 0.25 0.27 0.31 0.34Ramachandran plot:percentage of residues in

Most favoured region 88.6 88.6 89.1 88.2 87.4Add. allowed regions 10.8 10.5 10.1 10.6 11.6Gen. allowed regions 0.6 0.9 0.8 1.2 1.0

PDB accession codes 2BFO 2BF7 2BFP 2BFA 2BFM

Values in parentheses pertain to the highest resolution shell (widthz0.1 A). DPIZdiffraction-component precision index.52


(approximately 1035 atoms for each structure),gives a root-mean-square deviation (r.m.s.d.)range of 0.17–0.58 A with a mean of 0.34 A. Thesmallest deviation is observed for the overlay of theternary DHB and THB complexes, and the largest isobserved between the binary cofactor complexpaired with the ternary MTX complex. Since thestructures were refined independently, this indi-cates a high level of structural conservation of theprotein, irrespective of whether PTR1 is in a binaryor a ternary complex, or whether substrate, productor an inhibitor is bound. Inspection of the super-imposed molecules (not shown) confirms this highdegree of structural conservation, which extends tothe positions of side-chains, the conformation of thecofactor, and even the positions of numerous watermolecules. We also note structural conservationwithin the asymmetric unit of each complex. Suchconsistency indicates that PTR1 does not undergolarge conformational changes upon binding andprocessing substrate, and that it is necessary todescribe only one enzyme active site, in this casechosen arbitrarily as that formed mostly bysubunit A.

The PTR1 active site is an elongated L-shapedcleft (Figure 3(b)) approximately 22 A!15 A,created mainly by C-terminal sections of strandsb1 to b6, and sections contributed from a1, a4 and

a5 together with the residues on the loop betweenb6 and a6. The C terminus of a partner subunitblocks one end of the active site, with Arg287 0 (theprime ( 0) identifies a contribution from anothersubunit) directed towards the catalytic center andnear Asp181. The cofactor binds in the active site inan extended conformation, with the nicotinamidecreating the floor of the catalytic center; Phe113forms an overhang under which the pterin-bindingpocket is formed. The adenine residue adopts ananti conformation and the nicotinamide moietyadopts a syn conformation with respect to theirribose groups. In the binary complex, the pterin-binding site is occupied by water molecules with amolecule of ethylene glycol binding to Asp181 andArg2870 (Figure 4). The MTX complex also showsethylene glycol binding in this region of the activesite.

A complex network of hydrogen bonds organizethe enzyme active site and position the cofactor (notshown).16,17 Notably, Asn147 forms interactionswith Lys198 and Ser111, positioning the latter tworesidues to bind the nicotinamide ribose. The amidegroup of Ser111 is also able to donate a hydrogenbond to O4 0 of the adenine ribose. Arg17 interactswith the main-chain carbonyl group of Val228 andplays a critical role in binding the cofactorpyrophosphate. The main-chain amide and

Figure 4. Electron density associated with an active siteof (a) the binary cofactor complex, (b) the ternarybiopterin complex, (c) the ternary DHB complex16 and(d) the ternary THB complex. Themaps are drawn as bluechicken-wire and have been calculated with coefficients(jFoKFcj), ac and contoured at 2.6s. Fo represents theobserved structure factors, Fc represents the calculatedstructure factors, ac the calculated phases for which theligand contributions have been omitted. The atoms aredepicted in ball-and-stick mode and colored according totype. Spheres represent: N, blue; P, pink; O, red; and cyan,water. Green and yellow mark the PTR1 residues andcofactor, respectively. (a) An ethylene glycol molecule thatreplaces some ordered water molecules is colored purpleand the position of DHB is shown as a semitransparentobject.

Figure 3. (a) A ribbon diagram of the LmPTR1 subunit.Helices are colored cyan, b-strands are colored purple.Cofactor bonds are drawn as sticks, colored according toatom type: N, blue; P, purple; O, red; C, yellow. (b) Surfacerepresentation of the cofactor and active site. Subunit A isgray, blue identifies Arg2870 contributed from subunit D,and the cofactor is depicted in stick mode with theassociated surface colored as above. Selected residues areshown, and the position of Val230 is labeled.


carbonyl groups of Ser227 form hydrogen bondswith the carboxyamide group of the nicotinamide,which in turn interacts with the nearby cofactorphosphate group. The C2, C4, and C6 groups of thenicotinamide participate in three C–H/O hydro-gen bonds, interacting with a phosphodiester O of

the cofactor, and the carbonyl oxygen atoms ofGly225 and Val180, respectively (not shown).Though weak,27,30 these interactions assist theassociation of protein with cofactor and help toalign the nicotinamide to facilitate hydride transferfrom C4. These C–H/O associations are commonlyobserved in SDR structures.27

Substrate and product complexes

The ternary complex structures with biopterin,DHB and THB all have the pterin ligands bound inthe same orientation and participating in virtuallyidentical interactions with the enzyme and cofactor(Figures 4 and 5) as described for the DHBcomplex.16 An interesting observation from earlystudies on DHFR is that the pteridine moiety ofMTX binds in the active site in an orientationdifferent from that adopted by the substrate DHF.31

Likewise, in PTR1, the substrates bind with thepteridine rotated about the N2–N5 axis by 1808relative to MTX.Average surface-accessible areas have been

determined using a solvent probe of radius 1.4 A.

Figure 5. A representation ofbiopterin binding to the LmPTR1active site. Broken lines representpotential hydrogen bonds and theblocks of color are the same as inFigure 3.

Figure 6. CB3717 binding to LmPTR1. (a) Stereoview showing the omit difference density map (contoured at 2.4s) asdescribed for Figure 3. The same color scheme as Figure 3 is used with the addition that the glutamate tail of theinhibitor is red and broken lines represent potential hydrogen bonds. Phe113 is depicted as a semitransparent object.(b) A representation of the active site with the ligand in similar fashion to Figure 4.



The cofactor, when removed from the proteinmodel has an accessible surface area (ASA) of915 A2. In the binary complex the ASA drops toabout 110 A2, and a further decrease to 65 A2

accompanies binding of substrate/product. Therelatively small pterin substrates or products haveASA values of about 410 A2 in isolation and around50 A2 when bound in the active site formed byPTR1:NADPC. Almost 90% of the molecular sur-face is occluded by interactions head-on with thecofactor, sideways with Arg17 and Tyr181, andabove and belowwith the nicotinamide and Phe113,respectively (Figures 4 and 5).

These crystal structures confirm that the sub-strates bind in a single orientation and, in conjunc-tion with biochemical data, clearly define asequential two-step reduction mechanism.14,16 Thefirst catalytic step resembles that of other SDRfamily members and exploits three residues to (a)position the nicotinamide moiety of the cofactorNADPH for hydride transfer (Lys198), (b) acquire aproton from the solvent (Asp181), and (c) pass thison to the substrate (Tyr194). The second reductionstep, which occurs on the opposite side of thepterin, is similar to that postulated for DHFR.Nicotinamide again provides a hydride ion and anactivated water molecule supplies the proton.

The PTR1 catalytic center appears to be relativelyrigid, with the correct alignment of functionalgroups to support catalysis. Seven residues(Arg17, Ser111, Phe113, Asp181, Tyr194, Lys198and Arg287 0) are important for creating the activesite, substrate binding, or implicated in catalysis(Figure 5). These residues and the cofactor areamongst the most ordered parts of the structure, asindicated by the lower than average temperaturefactors (or B-factors, where BZ8p2 �U

2, and �U is the

mean displacement of atoms along the normal tothe reflecting planes, data not shown).

CB3717 binding to PTR1

CB3717 is an N10-substituted conjugated pterin-like molecule similar to MTX, but with twosignificant differences, 2-amino-4-oxoquinazolineand prop-2-inyl (propargyl) replacing the 2,4-diaminopteridine and N10-methyl substituents ofMTX, respectively (Figure 2). The presence of the4-oxo group on CB3717 suggested that the quinazo-line head group should adopt the pterin-likebinding mode as opposed to the MTX bindingmode and this has indeed proven to be the case(Figure 6).

The quinazoline ring is sandwiched between thecofactor nicotinamide and Phe113 and positionedsuch that all functional groups participate inhydrogen bonding interactions. These interactionsare with the O2 0 hydroxyl group and one of thephosphate oxygen atoms of the cofactor togetherwith side-chains of Arg17 and Ser111 (Figure 6). TheN1 atom of CB3717 is 3.6 A distant from Tyr194 OH,and this may represent a weak interaction. Thepropargyl moiety forms only a few van der Waals

interactions with the side-chains of Phe113 andLeu188, in contrast to the extensive van der Waalsinteractions observed for this moiety when CB3717is bound in the active site of TS. The benzyl group,again using van der Waals interactions, associateswith Leu188, Leu226, Leu228 and His241. At theglutamate tail there is a single weak potentialhydrogen bond accepted from Tyr283. Ethyleneglycol is observed binding between the inhibitor,with which it makes van der Waals contacts, and apolar section of the active site formed by Asp181and Arg287 0 at the same site noted in the binarycofactor complex and the MTX complex.16 Thisethylene glycol replaces several of the highlyconserved and ordered water molecules observedin the other structures (Figure 4). Replacement ofN5 and N8 of the ligand, as present on thesubstrates, with carbon atoms ablates polar inter-actions with Tyr194 and a water molecule adjacentto the Arg17 guanidine and the backbone amidenitrogen atom at Val230.The para-aminobenzoate (pABA)-glutamate tail

of the inhibitor is poorly ordered, as reflected by thethermal parameters, which exceed 80 A2, and lesswell-defined electron density associated with thisgroup. This feature is shared with MTX and is likelya consequence of the shape of the PTR1 ligand-binding cavity, which is relatively wide just abovethe catalytic center with a concomitant lack ofspecific interactions formed between the ligand andthe enzyme. A biological consequence of thisspacious entry into the active site is that PTR1 canprocess a wide range of pteridine compounds,including conjugated pteridines such as DHF.MTX is a more potent inhibitor of DHFR (IC50

5 nM) than of PTR1 (IC50 1.1 mM),16 mainly becausethe pABA group makes extensive interactions,including two direct salt-bridge associations withbasic residues in the DHFR active site.11 The lack ofaffinity of PTR1 for the pABA-Glu tail of MTX isdemonstrated indirectly by the inhibitor TAQ,which is essentially just the pterin-component ofMTX. TAQ adopts an identical orientation in theactive site of PTR1 and binds with an IC50

comparable to that of MTX.17

In a similar fashion, CB3717 is a highly potentinhibitor of TS with IC50 values in the range of30–60 nM.32 When bound to E. coli TS, for example,the glutamate moiety binds directly to His51 andSer54, and exploits numerous solvent-mediatedhydrogen bonding links to the enzyme to form astable complex (Protein Data Bank (PDB) code1AN5).33 In addition, the propargyl and pABAgroups participate in extensive van der Waalsinteractions with hydrophobic side-chains in TS.The molecular conformation of CB3717 in the

PTR1 ternary complex has an ASA of 200 A2,increasing to 765 A2 when the inhibitor is con-sidered in isolation. When bound in the E. coli TSternary complex the inhibitor in isolation, with aslightly different conformation, has an ASA of710 A2, which decreases to only 90 A2 uponbinding. The inhibitor is bound more tightly by

Figure 7. TOP binding to LmPTR1. Stereoview showing the omit difference density map (contoured at 2.6s). DHB,Phe113 and Leu188 are depicted as semitransparent objects. The orientation and conventions used in previous Figuresare reproduced here.


E. coli TS, assisted by large-scale adjustments ofprotein secondary structure to form a closedconformation about the drug. The increased ASAobserved for the drug in the PTR1 complex isprincipally due to the tail of the molecule stretchingout onto the surface of the ligand-binding site. Thenew structure and comparisons with the MTX andTAQ complexes suggests that the pABA-Glu moietycontributes little to binding PTR1 and could bedispensed with in the design of novel and potentinhibitors.

4-Oxo-2-amino quinazolines like CB3717 havepoor affinity for PTR1, with IC50 values O10 mM inall cases that have been examined.15 This affinity issimilar to that of the pteridine substrates for PTR1.The loss of the polar interactions with N5 and N8 inthe quinazolines cannot fully account for the pooraffinity of PTR1 for CB3717, because several6-substituted 2,4-diaminoquinazolines bind to thePTR1-NADPH complex with values of Kd!20 nM.15 Replacement of the 4-oxo with 2-aminofunctionality seems to be a necessary but notsufficient feature for potent inhibition of PTR1 byquinazolines.

Trimethoprim binding to PTR1

TOP is a potent DHFR inhibitor and structuralstudies have revealed that it binds to the enzymewith the diaminopyrimidine moiety mimicking apterin ring system with the TOP N4 replacing thepterin N8.24,34 The association of TOP with PTR1,with an IC50 of 12 mM,15 is very different. Here, thediaminopyrimidine is displaced by approximately2.5 A from the pterin-equivalent binding position(Figure 7). This can be explained by steric restric-tions imposed on the trimethoxyphenyl tail of theinhibitor, in particular by Phe113 and Leu188. Thisprevents the diaminopyrimidine group from insert-ing deeply enough into the binding pocket toexploit the hydrogen bonding opportunities withthe protein and cofactor, and to maximize van derWaals interactions with Phe113 and the nicotina-

mide. Only one well-defined direct hydrogenbond is formed between TOP and PTR1 viadonation from N2 to the hydroxyl group ofTyr194. A water molecule is trapped between thediaminopyrimidine and cofactor participating inhydrogen bonds with TOPN2, a cofactor phosphateand Ser111. This water molecule occupies theposition of N2 observed in the pterin complexes.TOP N1 is approximately equidistant (3.6 A)from Asp181 OD1 and Tyr194 OH, and thismay reflect the presence of a weak three-centerhydrogen bond.

The 2,4-diaminopyrimidine ring of TOP carriesfour functional groups, two hydrogen bond donor(N2 and N4) and two acceptor groups (N1 and N3).When TOP binds to DHFR it makes use of allfunctional groups to form hydrogen bonds directlywith the protein or mediated by a well-orderedwater molecule.24 The pyridine N3 atom is worthfurther comment about an interaction, which so farhas been overlooked. This group is 3.4 A distantfrom Trp6 Ca placed to participate in a C–H/Ninteraction similar to the C–H/O type of inter-actions discussed previously. Although this is not astrong attractive force, it would reduce thesignificant destabilization likely to occur in thepresence of an unfulfilled hydrogen bondacceptor.30

There is rotational freedom about the C5–C7 andC7–C8 bonds in TOP (Figure 2), and conformationalvariability contributes to this molecule’s enhancedinhibition of bacterial DHFR, compared to theeukaryotic enzymes, as the drug adapts to bindingin the active site.5,35 Also, in common with TS,conformational changes of DHFR (in particular of aloop at the edge of the catalytic center and the twosub-domains of the enzyme) accompany the bind-ing of ligands in the active site.36,37 In contrast, theresidues that form the substrate-binding site ofPTR1 are well ordered andwhen cofactor is present,the amount of space available where a ligand canbind is highly restricted. This suggests that thelimited conformational flexibility displayed by TOP


would not improve binding to PTR1, due inparticular to the side-chain positions of Leu188,Leu226, Leu229 and His241.

The molecular conformation of TOP in the PTR1ternary complex has an average ASA of 80 A2

and 500 A2 when considered in isolation. In theM. tuberculosis DHFR ternary complex (PDB code1DG5), the inhibitor in isolation adopts a slightlydifferent conformation (not shown) but with asimilar ASA of 500 A2, which decreases to 70 A2

upon binding. Although the ASA for TOP bound toPTR1 or DHFR is comparable, the loss of hydrogenbonding interactions together with reduced van derWaals associations contribute to the differences ininhibition against the two enzymes.

The inhibitory properties of 15 2,4-diamino-pyrimidine derivatives against PTR1 have beenassayed15 and the TOP complex now provides aclear explanation of the trends observed. Most 2,4-diaminopyrimidines with phenyl substituents at C5and bulky substituents at C6 (Figure 2) are verypoor inhibitors, with IC50 values ranging from50 mM to in excess of 300 mM, most likely becauseof steric restrictions in the active site imposed byPhe113 and Leu188.

Two 2,4-diaminopyrimidines with IC50 values ofw3 mM have chlorophenyl substituents at C5 andethyl groups at C6. Some of the moleculespreviously tested would, in addition to stericrepulsion, suffer by virtue of positioning a hydro-phobic group in a polar region of the active site nearAsp181 and Arg2870. However, 2,4-diaminopyrimi-dines with 5-propylphenyl, or 5-butylphenyl sub-stituents were improved inhibitors with IC50 values!1.5 mM, with the most potent of this series havinga Kd value of w30 nM for the PTR1-NADPHcomplex. Flexible C3 or C4 tethers attached to eitherC5 or C6 (Figure 2) might circumvent the stericrestrictions imposed by the PTR1 active site andallow the 2,4-diaminopyrimidine to bind further intoward the cofactor, perhaps adopting the pterin-like binding mode. It would be of interest toinvestigate if adjustment of the substituent confor-mations to optimize van derWaals interactions withthe enzyme could also occur. Such a mode ofinhibition offers scope for future design of morepotent inhibitors.

Concluding Remarks and Implicationsfor Inhibitor Design

The use of combinations of drugs, each withindependent modes of action, has been shown toimprove efficacy in some treatments withoutincreasing toxicity, and with the additional andsignificant benefit of providing mutual protectionagainst drug resistance. Of particular note withrespect to parasitic infections are combinations ofdapsone with chloroproguanil or pyrimethamine tocombat malaria.38 For the treatment of bacterialinfections, the combination of TOP with sulfona-mide drugs is often used.39 With respect to the

development of antifolate drugs to treat trypano-somatid infections, we note three valuable enzymeactivities, DHFR-TS and PTR1. A determination ofwhether it is reasonable to expect that a singlemolecule can be obtained that is a potent inhibitorof two or more of these enzymes, or whetherdifferent compounds will be needed for thedifferent enzymes, would indicate how to mostproductively pursue inhibitor discovery.Gangjee et al. have shown that dual inhibition of

DHFR and TS is feasible40 and, as discussed, MTX isan inhibitor of both DHFR and PTR1. Furthermore,a 2,4-diaminoquinazoline identified previouslyinhibited both PTR1 and DHFR with potency ofw30 nM, although that compound, like MTX, alsoinhibited human DHFR.15 On the basis of stericconsiderations and arrangement of functionalgroups, the TS, DHFR, and PTR1 active sites allhave some similarities, yet each has distinctivefeatures. In structures of ternary complexes of TSand PTR1, the substrates or inhibitors displayextensive interactions with the enzyme and withthe other ligand. For example, in PTR1 complexesthere are often hydrogen bonds and strongp-stacking associations involving Phe113 and thenicotinamide cofactor. These structural featuresmandate the ordered binding seen both with TSand PTR1. DHFR has different structural featuresthat allow it to bind its ligands in either order, albeitwith a kinetic preference. DHFR and TS undergoextensive conformational changes upon ternarycomplex formation, whereas PTR1 is more rigid.DHFR and TS structures show strong associationswith the pABA group of various ligands, whilst inPTR1 this group is bound only loosely. PTR1 offerssteric restrictions preventing access, for somecompounds, to the optimum pterin-like bindingposition. Both DHFR and PTR1 can each bindtightly the 2,4-diaminopteridine, 2,4-diaminoquina-zolines, or 2,4-diaminopyrimidine scaffolds, thoughin a reverse orientation from that observed for thecorresponding 4-oxo-2-amino molecules. TS, on theother hand, has no or very poor affinity for 2,4-diaminopteridine and 2,4-diaminoquinazolines.There are already a significant number of

inhibitors of DHFR-TS with well-characterizedpharmacokinetics and impressive affinity for theirtargets. Since TS is one of the most highly conservedof enzymes, it might seem daunting to considertrying to inhibit a pathogen enzyme, given thedeleterious affects likely to occur on human TS.Nevertheless, recent studies have shown con-clusively that specificity towards microbial TSinhibition can be achieved,32,33 thereby providingencouragement for such an approach. Certainly,less toxic inhibitors of human TSmight prove usefulfor antimicrobial drug development. However, atthis stage we suggest that the priority should bedevelopment of PTR1 inhibitors likely to comple-ment existing drugs that target DHFR, or that arecapable of inhibiting both PTR1 and DHFR, withrequisite selectivity against human DHFR.Future work developing such PTR1 inhibitors can


be based on our enzyme–ligand complexes byextending from those sections of the moleculesshown to be important for binding. The presence ofa solvent-filled cavity, occupied by conserved watermolecules (or ethylene glycol), and lined byhydrophilic residues (Asp181 and Arg287 0)suggests a suitable region to target by modificationof the pterin, quinazoline or 2,4-diaminopteridineframework.

† http://pymol.sourceforge.net/

Experimental

Materials

Biopterin and THB were obtained from SchirksLaboratories (Jona, Switzerland) and other reagentswere purchased from Sigma-Aldrich. CB3717 was pro-vided by Professor A. Jackman.

Crystallization and data collection

Recombinant L. major PTR1 was purified and crystal-lized following published methods.16,17,41 Briefly, ligandsolutions were prepared fresh (1 mM cofactor, 1 mMligand in 20 mM dithiothreitol buffered with 20 mMsodium acetate (pH 5.3)) and mixed in tenfold excesswith the enzyme solution in the same buffer. The enzyme–ligand mixtures were incubated on ice for an hour, andconcentrated to 15 mg mlK1 of protein. Crystals wereobtained using the hanging-drop method for vapordiffusion at 293 K from drops containing 2 ml of theenzyme–ligand complex plus 2 ml of a reservoir, com-prising 6–20% (w/v) monomethoxy-polyethylene glycol5000, 100 mM sodium acetate buffer (pH 5.5) and40–300 mM calcium acetate. Crystals formed mainly asclusters of fragile orthorhombic rods and grew up to0.5 mm in length over several days. All crystals in thispresent study display space group P212121 and areisomorphous with the PTR1:NADPC:DHB complex.16

Crystals were cryo-protected with ethylene glycol andcooled to 100 K in a stream of nitrogen gas and diffractiondata were recorded at the Synchrotron Radiation Source(S.R.S.) Daresbury Laboratory, UK (biopterin and TOPcomplexes) and the European Synchrotron RadiationFacility, (E.S.R.F.), Grenoble, France (CB3717, binary andTHB complexes). Data were processed and scaled usingHKL,42 and then truncated and converted with CCP4programs.43 Details are given in Table 1.

Refinement

The asymmetric unit contains a tetramer (subunits arelabeled A to D). The refinement protocol started with thePTR1:NADPC:DHB complex (PDB code 1E92), andapplied rigid-body refinement (REFMAC)44 andsimulated annealing (CNS)45 to that model. Cycles ofmodel-map inspection (O),46 identification of solventmolecules and restrained least-squares refinement(initially with CNS then REFMAC) followed. Coordinatesand topologies for ligands were obtained from HIC-Up.47

Throughout the refinement, non-crystallographic sym-metry (NCS) restraints were applied to the four subunitsin the asymmetric unit, excluding residues aroundseveral flexible loops and those that occupied clearlydifferent conformations in some chains. Completelydisordered residues have been omitted from the models,

while partially ordered side-chains were built in achemically sensible conformation and assigned lowoccupancies. Some density features have been interpretedas molecules of the cryo-protectant ethylene glycol. Forthe TOP complex, convincing electron density for theinhibitor was associated with two out of the four subunitsin the asymmetric unit. Model geometries were analyzedwith PROCHECK48 and WHAT_CHECK,49 and statisticsare given in Table 1. Figures were produced usingMOLSCRIPT,50 PyMOL†, and Raster3D.51

Protein Data Bank accession codes

Coordinates and structure factors have been depositedwith the Protein Data Bank (with accession codes 2BF0,2BF7, 2BFP, 2BFA, and 2BFM) and are available forimmediate release upon acceptance.

Acknowledgements

This work was funded by grants from theWellcome Trust (WNH) and the NIH (AI 21903,SMB). We thank S.R.S. Daresbury laboratory andE.S.R.F. for synchrotron beam time and their staff forexcellent support, Charlie Bond, Alice Dawson,David Gourley, and James Luba for advice and AnnJackman for a sample of CB3717.

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Edited by R. Huber

(Received 16 May 2005; received in revised form 29 June 2005; accepted 30 June 2005)Available online 14 July 2005

structures of leishmania major pteridine reductase complexes reveal the active site features

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