Trypanosomatid Diseases (Molecular Routes to Drug Discovery) || Targeting the Trypanosomatidic Enzymes Pteridine Reductase and Dihydrofolate Reductase

24Targeting the Trypanosomatidic Enzym es Pteridine Reductaseand Dihydrofolate Reduc taseStefania Ferrari, Valeria Losasso, Puneet Saxena, and Maria Paola Costi �

AbstractDrugs currently in use against Leishmania and Trypanosoma infections have limita-tions in terms of ef ficacy, safety, duration of treatment, toxicity, and resistance. It istherefore mandatory to identify molecular targets to be speci fically inhibited. Folateis an essential cofactor in the biosynthesis of DNA and amino acids. The inhibitionof its metabolism leads to alterations of cell replication and function. Only a fewtrypanosomatid enzymes of the folate pathway are presently discussed as potentialtargets, among them the bifunctional enzyme dihydrofolate reductase-thymidylatesynthase (DHFR-TS) and pteridine reductase (PTR1). The identi fication of a speci ficenzyme such as PTR1, able to reduce folates other than biopterins, allowed theunderstanding of the resistance of trypanosomatids against known anti-folate drugs.In most cases only the inhibition of both enzymes, DHFR-TS and PTR1, would fullyarrest the pathway ’s metabolic function. The proposed combination therapy opensup a novel approach: repositioning of the well-established anti-folate strategy for thetreatment of trypanosomatid diseases by the discovery of novel anti-folates thatcomplement the ef ficacy pro file of known drugs. The present chapter compiles theexisting medicinal chemistry approaches speci fically targeting the folate pathway intrypanosomatids, in particular PTR1 and the DHFR activity of DHFR-TS. It coversthe structural biology of the targets, related computational studies, core structuresynthesis, and biological inhibitor characterization.

Introduction

Enzymes belonging to the folate pathway are among the most-studied biologicaltargets not only in the field of anti-parasitic, but also for anti-microbial and anti-tumoral drugs. They are among the best-ranking targets within the TDR Targetsdatabase (www.dndi.org and www.who.int) [1]. Some of the enzymes belonging tothis pathway, such as thymidylate synthase (TS), dihydrofolate reductase (DHFR),and pteridine reductase (PTR1), are of interest as targets for the design of newinhibitors, because they are involved in the biosynthesis of reduced folate, which is

� Corresponding Author

Trypanosomatid Diseases: Molecular Routes to Drug Discovery, First edition. Edited by T. Jäger, O. Koch, and L. Flohé.# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

j445

an essential cofactor for the synthesis of the 20-deoxythymidine-50-monophosphate(dTMP) necessary for DNA synthesis (Figure 24.1). TS catalyzes the reductivemethylation of 20-deoxyuridine-50-monophosphate (dUMP) to dTMP, using thecofactor N5,N10-methylenetetrahydrofolate (mTHF) as a carbon donor and reducingagent. DHFR restores the 5,6,7,8-tetrahydrofolate (THF) pool through the NADPH-dependent reduction of the 7,8-dihydrofolate previously produced. In trypanoso-matids the DHFR and TS activities are achieved by a single bifunctional enzyme,DHFR-TS. Folate analogs acting as DHFR inhibitors were ineffective in the controlof infections caused by the Leishmania and Trypanosoma parasites, due to differentresistance mechanisms, including reduced uptake mediated by folate transporters,modulation of the level of polyglutamylated folates, and the presence of PTR1 [2].PTR1 belongs to the family of short-chain dehydrogenases/reductases (SDRs), andreduces both folate and unconjugated pterin (biopterin) [3]. Reduced biopterins areessential for several cellular metabolic cycles, whereas the ability of PTR1 to reducefolates acts as a metabolic bypass when DHFR is inhibited. Under physiologicalconditions PTR1 is responsible for the reduction of 10% of the folic acid required bythe cell, but when classic anti-folate drugs inhibit DHFR-TS, PTR1 is upregulated,thus providing the amount of reduced folates necessary for parasite survival. In T.brucei, PTR1 was demonstrated to be a promising drug target by itself or whenDHFR is simultaneously inhibited [4]. However, a L. major PTR1-null mutant wasshown to be viable; consequently in L. major, PTR1 is not a drug target on its own, ifDHFR-TS is not inhibited [5,6], whereas a combination strategy has been demon-strated to work [7,8] in vitro. Therefore, identifying a specific inhibitor of PTR1 andusing that inhibitor in combination with known anti-folates to optimize anti-parasitic efficacy appears to be a valid concept [7]. Many anti-folates are availableas approved or investigational drugs or lead compounds from previous discoveryprograms. Many pteridine and pyrimidine derivatives have also been tested againstthe Trypanosoma and Leishmania parasites, and often exhibited anti-parasitic activi-ties. However, the usually poor selectivity for the parasitic enzyme(s) and relatedtoxicity has remained a matter of concern. In this chapter, medicinal chemistryapproaches to generate anti-folates targeting PTR1 and DHFR proteins will becompiled.

X-Ray Crystal Structures of DHFR and PTR1

Structural Studies of PTR1

The primary sequences of PTR1 show identity percentages in the range of 72–95%among Leishmania species, and 41–46% between the Leishmania and Trypanosomaparasites. The PTR1 sequences from Trypanosoma species share a 53% identity,whereas the sequences of PTR1 and PTR2 in T. cruzi are almost identical (95%identity), with only nine residues differing (Table 24.1 and Figure 24.2). The firstthree-dimensional (3-D) structure of a PTR, the ternary complex of LmPTR1,NADPH, and methotrexate (MTX), was deposited in the Protein Data Bank

446j 24 Targeting the Trypanosomatidic Enzymes Pteridine Reductase and Dihydrofolate Reductase

(PDB) ( www.rcsb.org) in September 2001. Since then, 38 other X-ray structures fromfive different kinetoplastid parasites have been released (Table 24.2). The functionalenzyme is a tetramer without any evidence for cooperativity in the mechanism. Twoactive sites colocalize on each side of the tetramer, separated by less than 25A

�. Each

monomer has a a/b-domain with typical SDR topology based on the Rossmann fold:a seven-stranded parallel b-sheet sandwiched between three helices on either side(Figure 24.3). The catalytic center is mainly constructed from a single chain exceptfor the side-chain of Arg 287 (LmPTR1 numbering), which is located in the activesite of the partner subunit, where it interacts with the substrates through a bridgingwater molecule (Figure 24.4).The catalytic center lies in a curved cleft – an L-shaped depression that is

approximately 30A�long, 22A

�wide, and 15A

�deep. The loop between b6 and a6

(residues 225–233 in LmPTR1), known as the substrate-binding loop (Figure 24.3b),is a common structural feature of the SDR family. This loop is located at the entranceof the active site and makes contacts with substrates or ligands present at the activesite. A comparison of different structures suggests that this loop is flexible and thatits conformation depends on its interactions with the substrate or inhibitors,respectively [5,11]. However, the comparison of different crystallographic 3-Dstructures shows that there is a high level of structural conservation of the protein,irrespective of whether PTR1 is in a binary or a ternary complex or whether asubstrate, product or inhibitor is bound. The PTR1 catalytic center appears to berelatively rigid, with the correct alignment of functional groups to support catalysis.Seven residues (Arg17, Ser111, Phe113, Asp181, Tyr194, Lys198, and Arg2870;LmPTR1 numbering) are important for creating the active site or substrate bindingor have been implicated in catalysis [5] (Figure 24.2 and Figure 24.4a). A network ofhydrogen bonds organizes the active site and serves to position the cosubstrateNADPH [10]. The interaction between PTR1 and NADPH differs from that of otherSDRs in two respects: (i) in the consensus coenzyme-binding motif, GxxxGxG(where x is any amino acid), which is involved in cofactor recognition, and (ii) in the

Table 24.1 Percentage of identity (and similarity) among the PTR1 sequencesfrom 10 trypanosomatidic parasites.

Lb Ld Li Lm Lme Lt Ltr Tb Tc (1) Tc (2)

L. amazonensis 72 (83) 91 (94) 91 (94) 91 (94) 88 (91) 79 (86) 95 (96) 41 (58) 45 (60) 45 (60)L. braziliensis 74 (84) 74 (84) 72 (83) 74 (83) 73 (83) 75 (85) 45 (58) 46 (60) 46 (60)L. donovani 99 (99) 90 (94) 90 (94) 82 (87) 95 (97) 42 (58) 45 (60) 46 (60)L. infantum 90 (94) 90 (94) 82 (87) 95 (97) 42 (58) 45 (60) 46 (60)L. major 87 (91) 80 (87) 93 (96) 43 (59) 45 (60) 45 (60)L. mexicana 82 (87) 92 (93) 43 (58) 45 (60) 45 (59)L. tarentolae 82 (88) 44 (60) 45 (60) 46 (60)L. tropica 42 (59) 45 (60) 45 (60)T. brucei 53 (69) 54 (69)T. cruzi (PTR1) 95 (97)


pattern determining the nucleotide (NADH versus NADPH) specificity. PTR1 hasan Arg residue (Arg17 in LmPTR1) instead of the second Gly residue (Figure 24.2),and it confers specificity using three residues of the main-chain (His38, Arg39, andSer40 in Lm PTR1) to generate a 20 -phosphate-binding pocket instead of having twobasic side-chains interacting with NADPH [4] (Figure 24.2). In substrates binding,

Figure 24.2 Alignment of 11 PTR1 sequencesfrom 10 different trypanosomatid species.Residues conserved (identity or strongsimilarity) among all the 11 sequences arerepresented with black background; residuesconserved among at least nine out of 11sequences are represented with graybackground. Important motifs and regions citedin the text are labeled: �residues form the

catalytic triad; þother residues important forcreating the active site, substrate binding, orimplicated in catalysis. Source: NCBI-Proteindatabase (http://www.ncbi.nlm.nih.gov/protein); accession codes: 2196544,134062149, 317455034, 134069808, 16975355,322491891, 34810072, 83701129, 270346627,2842819, and 38492443.

X-Ray Crystal Structures of DHFR and PTR1 j449

Table 24.2 List of crystallographic structures of PTR1 available in the PDB.

Source Cofactor/substrate/ligand present in the structure PDB ID

L. donovani – 2XOX [9]L. major NADPH, MTX 1E7W [4]

NADPþ, DHB 1E92 [4]NADPþ, TAQ 1WOC [10]NADPþ, DHB 2BF7 [5]NADPH, CB3717 2BFA [5]NADPH, trimethoprim 2BFM [5]NADPH 2BFO [5]NADPþ, tetrahydrobiopterin 2BFP [5]NADPþ, methyl 1-(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}benzoyl)piperidine-4-carboxylate

2QHX [7]

NADPþ, methyl 1-(4-{[(2,4-diaminopteridin-6-yl)methyl]amino}benzoyl)piperidine-4-carboxylate

3H4V [7]

L. tarentolae NADPH, MTX 1P33 [11]T. brucei NADPþ, 6,7-bis(1-methylethyl)pteridine-2,4-diamine 3JQ6 [8]

NADPþ, 6-phenylpteridine-2,4,7-triamine 3JQ7 [8]NADPþ, 6,7,7-trimethyl-7,8-dihydropteridine-2,4-diamine 3JQ8 [8]NADPþ, 2-amino-6-(1,3-benzodioxol-5-yl)-4-oxo-4,7-dihydro-H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile

3JQ9 [8]

NADPþ, 2-amino-1,9-dihydro-6H-purine-6-thione 3JQA [8]NADPþ, 2-amino-5-(2-phenylethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one

3JQB [8]

NADPþ, 2-amino-6-bromo-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile

3JQC [8]

NADPþ, 2-amino-4-oxo-6-phenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile

3JQD [8]

NADPþ, 2-amino-6-(4-methoxyphenyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile

3JQE [8]

NADPþ, 1,3,5-triazine-2,4,6-triamine 3JQF [8]NADPþ, 6-[(4-methoxybenzyl)sulfanyl]pyrimidine-2,4-diamine 3JQG [8]NADPþ, MTX 2C7V [12]NADPþ, folic acid 3BMC [8]NADPþ, N2-cyclopropyl-1,3,5-triazine-2,4,6-triamine 3BMN [8]NADPþ, 6-[(4-methylphenyl)sulfanyl]pyrimidine-2,4-diamine 3BMO [8]NADPþ, 6-(benzylsulfanyl)pyrimidine-2,4-diamine 3BMQ [8]NADPþ, 6-chloro-1H-benzimidazol-2-amine 2WD7 [13]NADPþ, 1-(3,4-dichlorobenzyl)-7-phenyl-1H-benzimidazol-2-amine 2WD8 [13]NADPþ, 6-(4-methylphenyl)quinazoline-2,4-diamine 2VZ0 [14]NADPþ, 1H-benzimidazol-2-amine 3GN1 [13]NADPþ, 1-(3,4-dichlorobenzyl)-1H-benzimidazol-2-amine 3GN2 [13]NADPþ, 5-[2-(2,5-dimethoxyphenyl)ethyl]thieno[2,3-d]pyrimidine-2,4-diamine

3MCV [15]

NADPþ, 2-{4-[2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)-ethyl]-benzoylamino}-pentanedioic acid

2X9G [15]

NADPþ, TMQ 2X9V [15]NADPþ, N2-cyclopropyl-1,3,5-triazine-2,4,6-triamine 2X9N [15]

T. cruzi NADPH, MTX 1MXF [16]NADPþ, dihydrofolic acid 1MXH [16]


the pterin is sandwiched between Phe113 (LmPTR1 numbering) and nicotinamideand all eight functional groups are involved in hydrogen -bond interactions, five ofwhich directly with the cosubstrate. Such extensive interaction between the sub-strate and cosubstrate is unique to PTR1 among the SDR family members [4]. In thebinary complex (LmPTR1) and (NADPH), the pterin-binding site is occupied bywater molecules, and ethylene glycol binds to Asp181 and Arg2870 [5]. As much of

Figure 24.3 LmPTR1 structure (PDB ID: 1E92).(a) Tetrameric structure of LmPTR1. Eachsubunit is colored with a different color. Thecofactor (in gray) and the substrate (in yellow)are represented in spheres. (b) Monomer of

LmPTR1 in ribbon representation (Rossmannfold). The substrate-binding loop is highlightedin green. The cosubstrate (in cyan) and thesubstrate (in yellow) are represented in ball andstick.

Figure 24.4 Residues forming the LmPTR1active site (PDB ID: 1E92). Each protein residueand molecule is colored by atom: N, blue; O,red; S, yellow; C, gray. (a) LmPTR1 active sitewith bound NADPþ (C atom in cyan) and DHB

(C atom in yellow). (b) Comparison of substrate(DHB, C atom in yellow) and MTX (C atom inmagenta, from PDB ID: 1E7W) bindingorientation.


the pterin-binding site is formed by nicotinamide, the substrate can only bindeffectively after the formation of the protein–cofactor complex. Such an ordered,sequential mechanism is common in SDRs [4].In PTR, both reductions (to the dihydro derivative and tetrahydro derivative) have

been shown to utilize the same substrate orientation. PTR1 accomplishes twomodesof reduction in a single active site. The first reduction, similar to that of other SDRfamily members, utilizes a catalytic triad formed by a Tyr, a Lys, and an Asp (Tyr194,Lys198, and Asp181 in LmPTR1) (Figure 24.2 and Figure 24.4a) to (i) position thenicotinamide of NADPH for hydride transfer (Lys), (ii) acquire a proton fromthe solvent (Asp), and (iii) pass the proton over onto the substrate (Tyr). The Lysresidue with its basic side-chain may also reduce the pKa of Tyr and thereby assistcatalysis. The second reduction presents similarities with DHFR: the proton source isa suitably positionedwatermolecule and the pKa has beenproposed to be decreased byan interaction with the acidic 4-hydroxy group of the enolized substrate. Theenolization is favored by the interaction between the NADPH diphosphate and anArg residue (Arg17 in LmPTR1, Figure 24.2 and Figure 24.4a) [4].PTR1s from different species display different activities: LmPTR1 is known to be

able to catalyze all four reductions (folates and biopterins), whereas TbPTR1 isequally active with biopterin and dihydrobiopterin (DHB) as substrates but relativelyinefficient with folate and dihydrofolate (DHF). The slower catalysis of folates andweaker inhibition displayed byMTX against TbPTR1may be due to the presence of aless flexible and more restricted binding pocket in that enzyme compared with thatof LmPTR1. TbPTR1 and LmPTR1 show 51% identity and a closely related topology;however, few structural differences are responsible for the different enzymaticactivities and inhibition profiles of these enzymes [5,8,12]. In 2003, Senkovich et al.expressed a recombinant PTR from T. cruzi (TcPTR2) that can reduce only dihy-dropteridines but not oxidized pteridine [17]. This protein differs from the previ-ously reported TcPTR1 [3] at only nine amino acid positions in the primary sequenceand the comparison of the two crystallographic structures could not explain theinability of TcPTR2 to catalyze the first reduction step [16].A number of structures of PTR1 complexed with different ligands have been

obtained, (Table 24.2). The binding of MTX to PTR1/NADPH is dominated byinteractions with the cosubstrate through five hydrogen bonds. The pteridine ring ofMTX binds in a different orientation, rotated about theN2–N5 axis by 180� relative tothe binding orientation observed for DHB (Figure 24.4b). CB3717 is an N10-substituted, conjugated, pterin-like molecule similar to MTX but with two signifi-cant differences: it is a 2-amino-4-oxoquinazoline and it has a prop-2-inyl (propargyl)group as the N10 substituent. The 2-amino-4-oxoquinazoline adopts a pterin-likebinding mode [5], and is sandwiched between the cosubstrate’s nicotinamide andPhe113 (LmPTR1 numbering), a position at which all of its functional groupsparticipate in hydrogen bonding. In both crystallographic structures, an ethyleneglycol molecule is present, which replaces several of the highly conserved andordered water molecules observed in the other structures [5]. The p-aminobenzoate(pABA)-glutamate tail of these inhibitors is poorly ordered, as reflected by theirthermal parameters and the less well-defined electron density associated with this


group [4]. This feature is likely a consequence of the shape of the PTR1 ligand-binding cavity, which is relatively wide just above the catalytic center with aconcomitant lack of specific interactions formed between the ligand and theenzyme. A biological consequence of this wide entry into the active site is thatPTR1 is a broad-spectrum enzyme that can process a range of pteridine compounds,including conjugated pteridines such as DHF. However, the shape of the cavity alsoexplains why ligands such as MTX and CB3717 are more potent inhibitors of DHFRand TS, respectively [5]. The lack of affinity of PTR1 for the pABA-Glu tail of MTX isdemonstrated indirectly by the inhibitor 2,4,6-triaminoquinazoline (TAQ), whichsimply is the pterin component of MTX. Notably, TAQ, despite being a muchsmaller molecule thanMTX, displays a comparable level of inhibition (IC50 values of2.0 and 1.1mM, respectively, towards LmPTR1). The overlay of the crystal structuresof these molecules in complex with LmPTR1 indicates that their pterin-like headgroups bind to the active site of the enzyme in a very similar fashion [10]. Theinteraction of trimethoprim (IC50 of 12 mM) with PTR1 is very different. Thediaminopyrimidine is displaced by approximately 2.5 A

�from the pterin-equivalent

binding position, which can be explained by steric restrictions imposed on thetrimethoxyphenyl tail of the inhibitor [5]. Few aminobenzimidazole derivatives,crystallized in complex with TcPTR1, showed distinct binding modes in the PTR1active site depending on their substituents [13]. Only one of these resembles thepreviously observed binding modes, suggesting a further synthetic elaboration ofknown ligands to exploit the alternative observed binding modes.

Structural Studies of DHFR

In the bifunctional enzyme DHFR-TS of trypanosomatids the N-terminal DHFRdomain (residues 1–232 in TcDHFR-TS) is joined by a linker to the TS domain(residues 235–521 in TcDHFR-TS). The TcDHFR domain possesses only 36%identity with its human counterpart and has an additional 20 amino acid residuesat the N-terminus. Moreover, several key residues involved in binding anti-folates inhuman DHFR (HsDHFR) are replaced by other amino acids in equivalent positionsin the T. cruzi protein [18]. The active site in the TcDHFR domain appears to bemorehydrophobic than the site in HsDHFR, suggesting that the former should favor thebinding of lipophilic inhibitors. The overall structure of TcDHFR-TS is very similarto that of L. major DHFR-TS (LmDHFR-TS), with a 68.8% sequence identity [18].Ten structures of trypanosomatid DHFRs have been deposited in the PDB since

2008 (Table 24.3). Other crystallographic structures of TcDHFR and LmDHFR areknown from the literature, but are not publically available [19,20].The overall structure of TcDHFR-TS is very flexible. There is evidence of functional

interactions between the domains, via conformational changes of the two domains, asrevealed in the rotation angles between different subunits [18]. Owing to the shortlinker connecting the two domains, the orientation of the DHFR domains relative tothe TS domains is restricted and the two active sites are on the same side of thestructure [18] (Figure 24.5). The fold of the DHFR domain is characterized by amixed


Table 24.3 List of crystallographic structures of DHFR available from the PDB (the structures ofT. cruzi are the complete bifunctional DHFR-TS enzyme).

Source Cofactor/substrate/ligand present in the structure PDB ID:

T. brucei NADPH, 5-(4-chloro-phenyl)-6-ethyl-pyrimidine-2,4-diamine 3QFX [21]NADPH, 6,6-dimethyl-1-[3-(2,4,5-trichlorophenoxy)propoxy]-1,6-dihydro-1,3,5-triazine-2,4-diamine

3RG9 [21]

T. cruzi NADPþ, dUMP 2H2Q [18,19]NADPþ, MTXþ dUMP 3CL9 [18,19]NADPþ, TMQ 3CLB/3HBBa)

[18,19]NADPþ, ethyl 4-(5-{[(2,4-diaminoquinazolin-6-yl)methyl]amino}-2-methoxyphenoxy)butanoate

3KJS [22]

NADPH, 1-[3-(2,3-dichlorophenoxy)propoxy]-6,6-dimethyl-1,6-dihydro-1,3,5-triazine-2,4-diamineþ dUMP

3INV [23]

1-(4-chlorophenyl)-6,6-dimethyl-1,6-dihydro-1,3,5-triazine-2,4-diamine

3IRM [23]

NADPH, 1-(4-chlorophenyl)-6,6-dimethyl-1,6-dihydro-1,3,5-triazine-2,4-diamine

3IRN [23]

NADPH, 5-[3-(3-fluorophenoxy)propoxy]quinazoline-2,4-diamine 3IRO [23]

a) These refer to two different refinements of the same structure.

Figure 24.5 Crystallographic structure ofTcDHFR-TS (PDB ID: 3CL9) in complex withdUMP (purple), NADPþ (gray), and MTX(green). The two TS domains are colored in

orange and red; the two DHFR domains arecolored in cyan and blue; the short linkingregion between the two domains is shown inblack.


b-sheet of eight strands (order 43251687), with strand 8 being antiparallel to the rest[18]. The cosubstrate specificity is determined by several contacts between the adeninephosphate O atoms and particular protein residues (Thr102, Ser100, Ser101, andArg78). Other hydrogen bonds between the cosubstrate and the protein involve thefollowing residues: Ala28, Thr80, Ser158, and Gly157. Hydrophobic interactions areformed between the adenine ring and the protein [18].The N-terminal extension (residues 1–20) of the TcDHFR domain folds back on

the TS domain; its modulation of the catalytic activity of the two domains has beenshown in L. major and Plasmodium falciparum enzymes [18,24] (Figure 24.5).Senkowich et al. [18] suggested a possible role of the C-terminal end of this loopin transmitting the information due to its contact with two helices of the DHFRdomain (residues 132–145 and 156–171 in TcDHFR), the conformation of whichmay be sensitive to the presence of the bound cosubstrate. A comparison of theTcDHFR domain in the binary and ternary complexes demonstrates a closure of thebinding pocket upon inhibitor binding; there are movements in Met49, Asp48,Phe52, Ile84, Phe88, Leu91, and the nicotinamide moiety of the cosubstrate [18,19].A study by Vanichtanankul et al. [21] highlighted the similarity of TbDHFR to a

mutant of the P. falciparum enzyme (PfDHFR) that is associated with pyrimeth-amine (PYR) resistance. This similarity has been suggested to explain the substan-tially higher inhibition constants of TbDHFR for rigid anti-folates, including PYR.The crystal structure of TbDHFR complexed with PYR has shown that Thr86 (inTbDHFR corresponding to Ser108 in PYR-sensitive PfDHFR) is responsible for adisplacement of the rigid PYR ligand, which then causes movements in the Leu90and the Pro91–Phe94 loop. The same study showed that this displacement does nothappen with more flexible inhibitors [21]. The search for effective trypanosomalDHFR inhibitors should benefit from the development of new PfDHFR inhibitorsthat are being developed to overcome PYR-resistance.

Discovery and Development of PTR1 Inhibitors

Pteridine-Like Compounds

The pteridine-like compounds resemble the substrate structure. Modulation of thefolate core can significantly change the activity profile. Beverley et al. [25] describedthe inhibition profile and anti-leishmanial activity of three series of compounds:diaminopteridines, quinazolines, and 5-deazapteridines. Some of the compoundsvery effectively inhibited PTR1 (Table 24.4 , compounds 1 and 2) but showed limitedefficacy against the L. major strain LT252 clone CC1. They were also tested againstmutants of Leishmania lacking DHFR-TS (dhfr-ts�) or PTR1 (ptr1�) to gain infor-mation regarding their mechanism of action.Compounds with similar structures (diaminopteridines and quinoxalines) have

also been studied by Cavazzuti et al. [7]. A medium-throughput screening approachwas performed against LmPTR1 and TcPTR1. The trypanosomatid studies wereenlarged to a panel of microbial enzymes and the corresponding human enzymes

Discovery and Development of PTR1 Inhibitors j455

Table 24.4 Chemical structures and activity values for PTR1 inhibitors.

ID Structure Activities

1

N

N Z

Y R1

R2W

XW¼NH2, NH-CH3, NH-Ac, OH,H, CH3

X¼NH2, NH-Ac, OH, HY¼N, CH, CH2

Z¼N, N-CH3, CH, CH2

best compounds of this seriesshowed IC50 versusLmPTR1¼ 0.4mM

2

N

N

NH2

H2N R2

R1

best compounds of this seriesshowed IC50 versusLmPTR1¼ 0.4mM

3

N

N N

NNH

NH2

H2N

N

O

O

O

Ki versus LmPTR1: 0.1 mMKi versus TcPTR1: 7mMKi versus HsTS: no inhibition at190mMKi versus HsDHFR: 10mM

4

N

N N

NN

N

O

O

O

NH2

H2N

Ki versus LmPTR1: 0.037mMKi versus HsDHFR: 0.8mM

5

N

NH

S

O

H

O

F

a)IC50 versus L. donovanipromastigote: 0.101Ma)IC50 versus L. donovaniamastigote: 0.0231M

6

O

O O

O

OO

O

N

O

a)IC50 versus L. donovanipromastigote: 0.0908Ma)IC50 versus L. donovaniamastigote: 0.018M


7

O NH

N SH

O

HO

Ki versus LdPTR1: 0.428mMIC50 versus L. donovaniamastigote: 10mM

8

OO

O O

N

O O

O O

R3 R3

R1

R2

best compounds of this seriesshowed:IC50 versus L. donovanipromastigote: 0.22mg/mlIC50 versus L. donovaniamastigote: 0.75mg/ml

9

O

N

O

O

R3

R2

COO

best compounds of this seriesshowed:IC50 versus L. donovanipromastigote: 4.17mg/mlIC50 versus L. donovaniamastigote: 14.35mg/ml

10

NHR1COOEt

R2

Nbest compounds of this seriesshowed:IC50 versus L. donovanipromastigote: 121mM

11NH

NR1

R2O

best compounds of this seriesshowed:IC50 versus L. donovanipromastigote: 29 mMIC50 versus L. donovaniamastigote: 3mM

12

N

N N

N

NH2

H N2

N

NH

OO OH

OH

O

IC50 versus LmPTR1¼ 1.1mMIC50 versusTcDHFR¼ 0.0038 nMEC50 versus L. majorpromastigote: 0.3mMEC50 versus T. cruzi amastigote:9.2mM

(continued )


Table 24.4 (Continued)

ID Structure Activities

13

N

N

NH2

H2N

NH2IC50 versus LmPTR1¼ 2.0mM

14S

N N

H2NIC50 versus LmPTR1¼ 5.6mM

15S

NO

H2N

FF

F

IC50 versus LmPTR1¼ 50 mM

16

N

N

HH2N Cl

Kiapp versus TbPTR1: 10.6mM

17

N

NNH2

HKi

app versus TbPTR1: 288mM

18Cl

Cl

N

N

H2N


19

Cl

Cl

N

N

H2NPh


Kiapp versus TbDHFR: >50 mM

Kiapp versusHsDHFR: >50 mM

EC50 versus T. brucei¼ 9.9mMEC50 versus MRC5� 30 mM

20

Cl

Cl

N

N

H2NO


Kiapp versus TbDHFR: >30 mM

Kiapp versusHsDHFR: >50 mM

EC50 versus T. brucei¼ 9.6mMEC50 versus MRC5¼ 21 mM

a) These data are reported in [26]. The numbers are obtained through flow cytometry test.


(HsTS, HsDHFR) to gain information on compound selectivity. The most activecompounds bear a glutamic acid chain embedded in a rigid piperidine nucleus(Table 24.4, compounds 3 and 4). Compounds 3 and 4 were also active against T.cruzi amastigote form. When tested in combination with PYR, a known DHFRinhibitor, the compounds showed additive activity, suggesting a potential use incombination therapy. The pteridine and quinoxaline scaffolds, therefore, deservefurther interest for the development of PTR1 inhibitors.

From DHFR to PTR1 Inhibitors

In 2008, Kumar et al. [26] tried to understand the role of pteridine metabolism in thechemotherapy of L. donovani infections by integrating experimental studies withcomputational techniques. The unavailability of the X-ray crystallographic structureof L. donovani PTR1 (LdPTR1) led these researchers to build a homology modelbased on LmPTR1 (PDB ID: 1E7W), which has a sequence identity of 91%. Theysynthesized 20 pyrimidine thiones, a kind of scaffold already known to generateinhibitors of DHFR. Two compounds: ((4-flour-phenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahedron-pyrimidine-5-carboxylic acid ethyl ester) (Tables 24.4, compound 5) and(4-(3-O-benzyl-1,2-O-isopropylidene-b-L-threo-furano-4-yl)-2,6-dimethyl-1,4-dihy-dro-pyridine-3,5-dicarboxylic acid ethyl ester) (Table 24.4, compound 6) showedgood activity. Docking studies on compounds 5 and 6 indicated that their carboxylicacid ethyl ester group of the pyridine moiety plays an important role in binding toPTR1. None of these inhibitors directly interacted with NADPH. However, dockingstudies without NADPH in the PTR1 active site were not successful. The resultssuggest that NADPH affects the conformation of the enzyme in a way that facilitatesbinding of these inhibitors.In 2010, Kaur et al. [27,28] reported on analogous docking studies with monastrol

(4-[3-hydroxyphenyl]-6-methyl-2-thioxo-1,2,3,4-tetrahedron-4H-pyramidine-5-carboxylicacid ethyl ester; Table 24.4, compound 7), a compound inhibiting recombinant LdPTR1and growth of amastigotes with no host cytotoxicity and now considered a drugcandidate in pre-clinical development.When it was docked into the active site of thehomologymodelofLdPTR1,dockingposescompatiblewithsimilaraffinitiesforbothenantiomericformsofmonastrolwereobtained.Asintheearlierstudy,thecarboxylicacid ethyl ester group of the pyridinemoiety showed an important role in binding toPTR1: the carbonyl oxygen atom acts as hydrogen bond acceptor interacting withArg17 and Ala230. The hydroxyl group of the phenyl ring interacts with PTR1 viahydrogen bonding with Tyr194. The binding of monastrol to the LdPTR1 is furtherstabilized by a p-stacking between its phenyl ring and the nicotinamide moiety ofNADPH [27,28].Also in 2010,Pandey et al. [29], starting fromcompound6 (Table 24.4), synthesized a

series of 1-phenyl-4-glycosyl-dihydropyridines of which inter alia compounds 8 and 9(Table 24.4)were active againstL. donovani in vitro and in vivo.Docking studies revealedthat all highly active dihydropyrimidine derivatives occupy similar spatial arrange-ments. Modeling studies on two classes of compounds, the dihydropyridines 8 and 9


(Table 24.4) and 12 compounds from Baylis–Hillman chemistry (Table 24.4, com-pounds 10 and 11) [29,30], showed that the phenyl ring from the former as well as thehexahydropyrimidopyrimidinone ringof11fitwell in thehydrophobic pocket formedby residues Ala230, Tyr191, Tyr194, Phe113, Pro224 and Leu18. Further, Phe113possibly acts as a p-donor site involved in edge to face p–p-stacking interaction.

Non-Pteridine-Like PTR1 Inhibitors

In 2004, McLuskey et al. solved the crystal structure of TAQ (Table 24.4, compound13) in complex with PTR1 [10]. TAQ shows an inhibition constant very similar to thatof MTX (Table 24.4, compound 12) and occupies the pteridine binding pocket,sandwiched between the cosubstrate’s nicotinamide and Phe113, with five func-tional groups participating in hydrogen-bond interactions. The 6-amino groupinteracts with highly conserved water molecules. The binding modes thus obtainedsuggested that the main anchor of both inhibitors is the pteridine ring. Therefore,fragment-based drug design approaches were undertaken to generate new PTR1inhibitors.Accordingly, a virtual screening approach has been performed to identify a new

scaffold for the inhibition of LmPTR1 [31]. Fifty-six compounds were selected fortesting, and 2-ammino-thiadiazole (Table 24.4, compound 14) was chosen forchemical development because of its chemical tractability, low molecular weight,and suitability to incremental fragment-based approach. Twenty-six thiazole deriv-atives were further studied together with a few more compounds selected to extendthe hydrophobic interaction. Riluzole (Table 24.4, compound 15) emerged as apotential new lead. It inhibits the enzyme with a Ki of 7 mM [31].In 2009, Brenk et al. [13] started a new program aiming at inhibitors of TbPTR1.

Most of the known PTR1 inhibitors were derived from DHFR inhibitors; it istherefore not surprising that many of them also inhibit human and parasitic DHFRwith inhibition constants in the low micromolar to nanomolar range. This broadspectrum activity is, of course, undesirable with respect to safe therapy. Untilrecently, though, conventional assays did not discriminate between inhibition ofPTR1 and DHFR. Therefore a sequential assay has been set up to meet this goal(Guerrieri, personal communication). Also, known core structures mostly have arelatively high polar surface area (PSA), which prevents permeation of the blood–brain barrier, as is required to treat the second stage of African sleeping sickness.The virtual screening campaign for fragments inhibiting PTR1 that considered thepossible complications yielded two novel chemical series: aminobenzothiazole andaminobenzimidazole derivatives. One of the hits (2-amino-6-chloro-benzimidazole;Table 24.4, compound 16) was subjected to crystal structure analysis in complex withPTR1, confirming the predicted binding mode. However, the crystal structures oftwo analogs (2-aminobenzimidazole and 1-(3,4-dichloro-benzyl)-2-amino-benz-imidazole; Table 24.4, compounds 17 and 18) in complex with PTR1 revealedtwo alternative binding modes due to previously unobserved protein movementsand the occurrence of water-mediated protein-ligand contacts. On the basis of the


alternative binding mode of 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole, furtherderivatives were designed, and selective PTR1 inhibitors with efficacies in the lownanomolar range and favorable physicochemical properties were obtained [32]. Themost potent compounds of this series have appropriate drug-like properties and arehighly selective (above 7000-fold) for PTR1 over human or trypanosomal DHFR.Two compounds (Table 24.4, compounds 19 and 20) are the most potent andselective TbPTR1 inhibitors so far disclosed in the literature.

Inhibition of DHFR

Anti-folates are roughly grouped into two classes: (i) classical anti-folates, i.e.structural analogs of folic acid with a polar glutamate side-chain, which requirea carrier-mediated active transport system for entering the cell, and (ii) non-classicalanti-folates that lack the glutamate side-chain, called lipophilic anti-folates. They areknown to enter the cell via passive diffusion.Most clinically used DHFR inhibitors (such as MTX, trimethoprim, cycloguanil,

and PYR; Tables 24.4 and 24.5 , compounds 12 and 21–23) are either not selective ornot active at all against the parasitic DHFR [33]. Only few inhibitors that selectivelyinhibit the parasitic bifunctional DHFR-TS (Table 24.5, compounds 24–28) havebeen reported in the literature [25,34–37]. Sirawaraporn et al. reported a milestonework on some 2,4-diaminopyrimidines as selective inhibitors of LmDHFR. Thesecompounds also showed some in vitro activity against L. donovani amastigotes. Themost active compound that inhibited the parasite enzyme with a selectivity factor of130 was the 3-octyloxy derivative (Table 24.5, compound 26) [36].Gilbert et al. obtained and tested 5-benzyl-2,4-diaminopyrimidines (Table 24.5,

compound 27) against recombinant DHFR from L. major, T. cruzi, T. brucei, andhumans [38]. An interaction between the 2,4-diaminopyrimidine moiety and anaspartic acid residue in the enzyme’s active site could be identified to be of criticalimportance. Along the same line, a new series of 2,4-diaminopyrimidines (Table 24.5,compound 29) were synthesized by Gilbert to optimize potency, selectivity, and thePSA [39]. These series showed broad potency against L. major and T. cruzi DHFR.Having two substituents on the aromatic ring generally did not yield selectivecompounds.In 2005, Senkovich et al. expressed and purified the TcDHFR-TS protein, thus

providing an essential tool for any related drug design strategy. Then they chosetrimetrexate (TMQ, Table 24.5, compound 30) as a template for the design ofderivatives with improved selectivity. TMQ is a lipophilic anti-folate, which is a USFood and Drug Administration-approved drug for the treatment of Pneumocystiscarinii infection in AIDS patients, but also a potent inhibitor of T. cruzi DHFRactivity and highly effective against T. cruzi [40]. In fact, the activity of TMQ againstT. cruzi is about 100- to 200-fold higher than that of the currently used drugs,benznidazole (LD50¼ 6 mMagainst trypomastigotes) and nifurtimox (LD50¼ 3.4mMagainst amastigotes) [41,42]. However, TMQ has not yet been tested in an animalmodel of Chagas disease. TMQ is also a good inhibitor of human DHFR; further

Inhibition of DHFR j467

improvement of the selectivity of this drug would therefore be necessary [40]. Basedon the differences in the active site of T. cruzi and human enzymes and the bindingmode of MTX, six new compounds (Table 24.5, compound 31) have been designedand synthesized by Zuccotto et al. as inhibitors of TcDHFR. However, none of thecompounds proved to be reasonably selective for the parasite enzyme or sufficientlyactive against T. cruzi amastigotes [37].In 2009, finally, 3-D structures of the entire TcDHFR-TS were reported: (i) the

folate-free state of the enzyme, (ii) the complex with the lipophilic anti-folate TMQ,and (iii) the complex with the classical anti-folateMTX. They show subtle differencescompared with the human counterpart. The differences between the DHFR domainof the TcDHFR-TS and HsDHFR (see above) could now be systematically exploitedfor the development of more specific anti-folates [18,19]. Schormann et al. used thestructural information to generate 3-D quantitative structure–activity relationshipmodels of TcDHFR-TS (30 inhibitors in the learning set) and HsDHFR (36inhibitors in the learning set) which showed good agreement between experimentaland predicted enzyme inhibition data. Following the medicinal chemistry workstarted in 2005, they designed and synthesized six novel TMQ derivatives [22]. Oneof these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (Table 24.5, compound 32), was co-crystallized withthe bifunctional TcDHFR-TS and the crystal structure of the ternary enzyme–cosubstrate–inhibitor complex was determined. Molecular docking was used toanalyze the potential interactions of all inhibitors with TcDHFR-TS and HsDHFR.Binding affinities of each inhibitor for the respective enzymes were calculated,based on the experimental or docked binding mode. An estimated 60–70% of thetotal binding energy turned out to be contributed by the 2,4-diaminoquinazolinescaffold. In consequence, these compounds showed low nanomolar affinity (Ki inthe low nanomolar range) for the parasitic enzyme, but unfortunately low specificitywith respect to the human enzyme (selectivity index¼ 1–3), thus revealing the needto improve the selectivity and also the difficulties in transforming a human DHFRinhibitor used as an anti-cancer drug into a useful anti-parasitic drug.Another series of 2,4-diaminoquinazolines (Table 24.5, compounds 33–35) eval-

uated as inhibitors of leishmanial DHFR [43] was, however, more promising.All compounds showed potent activity against recombinant LmDHFR. Those com-pounds that have a benzylidene group instead of a benzyl group at the terminalposition (e.g.34 inTable 24.5)were less effective than thebenzyl analog33 (Table 24.5),since the rigid alkene group in 34 likely prevents the compound from adopting anoptimal conformation. The compounds were all quite selective for the parasite enzyme.Also non-folate-related DHFR inhibitors have been designed and tested against

T. cruzi. They bear a heterocyclic pyrimidine or triazine [44]. Virtual screeningmethods were applied to discover novel parasite DHFR inhibitors not based onthe 2,4-diamminopyrimidine motif. Zuccotto et al. [45] identified in particulartwo compounds (Table 24.5, compounds 36 and 37) active against T. cruzi enzymeand also T. brucei trypomastigote. Chowdhury et al. [46] used the L. major activesite for docking and identified compounds with weak activity (Table 24.5,compounds 38–41).


Conclusions and Perspectives

The unusual primary resistance of trypanosomatids against anti-folates can beexplained by overlapping activities of PTR1 and DHFR-TS, which guarantee asufficient supply of THF as long as only one of the enzymes is inhibited. Exper-imentally, a combined inhibition of PTR1 and of the DHFR activity of DHFR-TSleads to efficient eradication of the parasites and is therefore considered to be apromising approach to treat trypanosomatid diseases. In view of the broad expe-rience in the design of anti-folates developed as anti-cancer drugs or anti-bacterialagents and the substantial structural knowledge on the pivotal trypanosomatidenzymes, the development of a combination therapy appear to be a realistic concept.The structural differences between trypanosomatid PTR1 and DHFR-TS and theirmammalian congeners are pronounced enough to enable selective inhibition and,thus, render the proposed combination therapy a perspective towards an efficaciousand safe therapy.

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Trypanosomatid Diseases (Molecular Routes to Drug Discovery) || Targeting the Trypanosomatidic Enzymes Pteridine Reductase and Dihydrofolate Reductase