Specificity in structure-based drug design: Identification of a novel,selective inhibitor of Pneumocystis carinii dihydrofolate reductase

Specificity is an important aspect of structure-based drug design. Distinguishing between related targets in different organisms is often the key to therapeutic success. Pneumocystis carinii is a fungal opportunist which causes a crippling pneumonia in immunocompromised individuals. We report the identification of novel inhibitors of P. cariniidihydrofolate reductase (DHFR) that are selective versus inhibition of human DHFR using computational molecular docking techniques. The Fine Chemicals Directory, a database of commercially available compounds, was screened with the DOCK program suite to produce a list of potential P. carinii DHFR inhibitors. We then used a postdocking refinement directed at discerning subtle structural and chemical features that might reflect species specificity. Of 40 compounds predicted to exhibit anti-PneumocystisDHFR activity, each of novel chemical framework, 13 (33%) show IC₅₀ values better than 150 μM in an enzyme assay. These inhibitors were further assayed against human DHFR: 10 of the 13 (77%) bind preferentially to the fungal enzyme. The most potent compound identified is a 7 μM inhibitor of P. carinii DHFR with 25-fold selectivity. The ability of molecular docking methods to locate selective inhibitors reinforces our view of structure-based drug discovery as a valuable strategy, not only for identifying lead compounds, but also for addressing receptor specificity. Proteins 29:59–67, 1997. © 1997 Wiley-Liss, Inc.     

Cloning,recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly,Lucilia cuprina

While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non‐specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC₅₀ values at µg levels. Trimethoprim and pyrimethamine were 5–30‐fold less active. Relative IC₅₀ values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active‐site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.     

Analysis of Coxiela burnetti dihydrofolate reductase via in silico docking with inhibitors and molecular dynamics simulation

Coxiella burnetii is a gram-negative bacterium able to infect several eukaryotic cells, mainly monocytes and macrophages. It is found widely in nature with ticks, birds, and mammals as major hosts. C. burnetii is also the biological warfare agent that causes Q fever, a disease that has no vaccine or proven chemotherapy available. Considering the current geopolitical context, this fact reinforces the need for discovering new treatments and molecular targets for drug design against C. burnetii. Among the main molecular targets against bacterial diseases reported, the enzyme dihydrofolate reductase (DHFR) has been investigated for several infectious diseases. In the present work, we applied molecular modeling techniques to evaluate the interactions of known DHFR inhibitors in the active sites of human and C. burnetii DHFR (HssDHFR and CbDHFR) in order to investigate their potential as selective inhibitors of CbDHFR. Results showed that most of the ligands studied compete for the binding site of the substrate more effectively than the reference drug trimethoprim. Also the most promising compounds were proposed as leads for the drug design of potential CbDHFR inhibitors.     

Multicomplex-based pharmacophore modeling in conjunction with multi-target docking and molecular dynamics simulations for the identification of PfDHFR inhibitors

Abstract

Plasmodium falciparum dihydrofolate reductase enzyme (PfDHFR) is counted as one of the attractive and validated antimalarial drug targets. However, the point mutations in the active site of wild-type PfDHFR have developed resistance against the well-known antifolates. Therefore, there is a dire need for the development of inhibitors that can inhibit both wild-type and mutant-type DHFR enzyme. In the present contribution, we have constructed the common feature pharmacophore models from the available PfDHFR. A representative hypothesis was prioritized and then employed for the screening of natural product library to search for the molecules with complementary features responsible for the inhibition. The screened candidates were processed via drug-likeness filters and molecular docking studies. The docking was carried out on the wild-type PfDHFR (3QGT); double-mutant PfDHFR (3UM5 and 1J3J) and quadruple-mutant PfDHFR (1J3K) enzymes. A total of eight common hits were obtained from the docking calculations that could be the potential inhibitors for both wild and mutant type DHFR enzymes. Eventually, the stability of these candidates with the selected proteins was evaluated via molecular dynamics simulations. Except for SPECS14, all the prioritized candidates were found to be stable throughout the simulation run. Overall, the strategy employed in the present work resulted in the retrieval of seven candidates that may show inhibitory activity against PfDHFR and could be further exploited as a scaffold to develop novel antimalarials.

Communicated by Ramaswamy H. Sarma     

The influence of glycerol on the binding of methotrexate to Mycobacterium tuberculosis dihydrofolate reductase: a molecular modelling study

The enzyme, dihydrofolate reductase (DHFR), from Mycobacterium tuberculosis (mt-DHFR) is believed to be a potential drug target for the treatment of tuberculosis. The co-crystal structure of mt-DHFR bound with glycerol (GOL), NAPDH and methotrexate (MTX) reveals a GOL binding site on the enzyme. This GOL binding site could be very important for the design of novel, selective mt-DHFR inhibitors, because this binding site is absent on human DHFR (h-DHFR). We have performed molecular dynamic simulations and free energy calculations to evaluate the binding affinity of GOL and its free energy contribution to the binding of MTX to mt-DHFR. The results showed that GOL does not bind tightly to mt-DHFR. Although GOL itself contributed free energy on MTX binding to mt-DHFR, GOL also increased the flexibilities of MTX, so that MTX cannot maintain strong electronic interactions with ARG32 and ARG60, which caused the total binding free energy to decrease. These data suggest that GOL binding is weak and it could be expelled from the binding site, to allow inhibitors containing appropriate side chains to bind. This observation can be used to inform future drug design studies, especially those aimed at improving drug selectivity against h-DHFR.     

Purification and properties of recombinant Pneumocystis carinii dihydrofolate reductase

Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography. The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence. The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7. The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl. Urea at 2.5 M also increased the enzyme activity twofold. Kinetic analysis of the purified enzyme revealed that the K_m values for dihydrofolate and NADPH were 1.8 and 1.4 μM, respectively, and that the k_cat was 70 s⁻¹. Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P. carinii DHFR and showed little selectivity over the human DHFR. Trimetrexate and piritrexim were much more potent inhibitors of the P. carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR.     

Origins of the specificity of inhibitor P218 toward wild-type and mutant PfDHFR: a molecular dynamics analysis

Molecular dynamics simulations were performed to evaluate the origin of the antimalarial effect of the lead compound P218. The simulations of the ligand in the cavities of wild-type, mutant Plasmodium falciparum Dihydrofolate Reductase (PfDHFR) and the human DHFR revealed the differences in the atomic-level interactions and also provided explanation for the specificity of this ligand toward PfDHFR. The binding free energy estimation using Molecular Mechanics Poisson-Boltzmann Surface Area method revealed that P218 has higher binding affinity (~ ?30 to ?35 kcal/mol) toward PfDHFR (both in wild-type and mutant forms) than human DHFR (~ ?22 kcal/mol), corroborating the experimental observations. Intermolecular hydrogen bonding analysis of the trajectories showed that P218 formed two stable hydrogen bonds with human DHFR (Ile7 and Glu30), wild-type and double-mutant PfDHFR’s (Asp54 and Arg122), while it formed three stable hydrogen bonds with quadruple-mutant PfDHFR (Asp54, Arg59, and Arg122). Additionally, P218 binding in PfDHFR is stabilized by hydrogen bonds with residues Ile14 and Ile164. It was found that mutant residues do not reduce the binding affinity of P218 to PfDHFR, in contrast, Cys59Arg mutation strongly favors inhibitor binding to quadruple-mutant PfDHFR. The atomistic-level details explored in this work will be highly useful for the design of non-resistant novel PfDHFR inhibitors as antimalarial agents.     

Design,synthesis, biological evaluation and computational investigation of novel inhibitors of dihydrofolate reductase of opportunistic pathogens

The present work deals with design, synthesis and biological evaluation of novel, diverse compounds as potential inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii (tg) and Mycobacterium avium (ma). A set of 14 structurally diverse compounds were designed with varying key pharmacophoric features of DHFR inhibitors, bulky distal substitutions and different bridges joining the distal part and 2,4-diaminopyrimidine nucleus. The designed compounds were synthesized and evaluated in enzyme assay against pc, tg and ma DHFR. The rat liver (rl) DHFR was used as mammalian standard. As the next logical step of the project, flexible molecular docking studies were carried out to predict the binding modes of these compounds in pcDHFR active site and the obtained docked poses were post processed using MM-GBSA protocol for prediction of relative binding affinity. The predicted binding modes were able to rationalize the experimental results in most cases. Of particular interest, both the docking scores and MM-GBSA predicted ΔG_bind were able to distinguish between the active and low active compounds. Furthermore, good correlation coefficient of 0.797 was obtained between the IC₅₀ values and MM-GBSA predicted ΔG_bind. Taken together, the current work provides not only a novel scaffold for further optimization of DHFR inhibitors but also an understanding of the specific interactions of inhibitors with DHFR and structural modifications that improve selectivity.     

Targeting plant DIHYDROFOLATE REDUCTASE with antifolates and mechanisms for genetic resistance

The folate biosynthetic pathway and its key enzyme dihydrofolate reductase (DHFR) is a popular target for drug development due to its essential role in the synthesis of DNA precursors and some amino acids. Despite its importance, little is known about plant DHFRs, which, like the enzymes from the malarial parasite Plasmodium, are bifunctional, possessing DHFR and thymidylate synthase (TS) domains. Here using genetic knockout lines we confirmed that either DHFR‐TS1 or DHFR‐TS2 (but not DHFR‐TS3) was essential for seed development. Screening mutated Arabidopsis thaliana seeds for resistance to antimalarial DHFR‐inhibitor drugs pyrimethamine and cycloguanil identified causal lesions in DHFR‐TS1 and DHFR‐TS2, respectively, near the predicted substrate‐binding site. The different drug resistance profiles for the plants, enabled by the G137D mutation in DHFR‐TS1 and the A71V mutation in DHFR‐TS2, were consistent with biochemical studies using recombinant proteins and could be explained by structural models. These findings provide a great improvement in our understanding of plant DHFR‐TS and suggest how plant‐specific inhibitors might be developed, as DHFR is not currently targeted by commercial herbicides.     

Structural Characterisation of FabG from Yersinia pestis,a Key Component of Bacterial Fatty Acid Synthesis

Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.     

Targeting species specific amino acid residues: Design,synthesis and biological evaluation of 6-substituted pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors and potential anti-opportunistic infection agents

To combine the potency of trimetrexate (TMQ) or piritrexim (PTX) with the species selectivity of trimethoprim (TMP), target based design was carried out with the X-ray crystal structure of human dihydrofolate reductase (hDHFR) and the homology model of Pneumocystis jirovecii DHFR (pjDHFR). Using variation of amino acids such as Met33/Phe31 (in pjDHFR/hDHFR) that affect the binding of inhibitors due to their distinct positive or negative steric effect at the active binding site of the inhibitor, we designed a series of substituted-pyrrolo[2,3-d]pyrimidines. The best analogs displayed better potency (IC₅₀) than PTX and high selectivity for pjDHFR versus hDHFR, with 4 exhibiting a selectivity for pjDHFR of 24-fold.     

Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells

Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation in vivo, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an in silico model of human DHFR–TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.     

Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K_i 49 nM) compared to hDHFR (K_i 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K_i values of 593 and 617 nM, respectively; these K_i values are well above both the K_i for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes.     

Design,synthesis and biological activity of novel substituted 3-benzoic acid derivatives as MtDHFR inhibitors

The enzyme dihydrofolate reductase from M. tuberculosis (MtDHFR) has a high unexploited potential to be a target for new drugs against tuberculosis (TB), due to its importance for pathogen survival. Preliminary studies have obtained fragment-like molecules with low affinity to MtDHFR which can potentially become lead compounds. Taking this into account, the fragment MB872 was used as a prototype for analogue development by bioisosterism/retro-bioisosterism, which resulted in 20 new substituted 3-benzoic acid derivatives. Compounds were active against MtDHFR, with IC₅₀ values ranging from 7 to 40 μM, where compound 4e not only had the best inhibitory activity (IC₅₀ = 7 μM), but also was 71-fold more active than the original fragment MB872. The 4e inhibition kinetics indicated an uncompetitive mechanism, which was supported by molecular modeling which suggested that the compounds can access an independent backpocket from the substrate and competitive inhibitors. Thus, based on these results, substituted 3-benzoic acid derivatives have strong potential to be developed as novel MtDHFR inhibitors and also anti-TB agents.     

Biosynthesis of Drosophila yolk polypeptides

The three yolk polypeptides (YPs) of Drosophila are synthesized and secreted by female fat body and ovarian follicle cells, sequestered by pinocytosis into oocytes, and finally deposited into yolk granules. The biosynthesis of the YPs was studied using two-dimensional gels. Labeling the YPs with [³⁵S]-cysteine, an amino acid found only near the amino terminus of YP1 and YP2, showed that an amino terminal peptide is removed from YP1 and YP2 shortly after or during translation. Intermediates in YP biosynthesis corresponding in electrophoretic mobility to pancreatic membrane-processed primary translation products were also detected in a 5-min pulse label with [³⁵S]-methionine. Genetic variants that alter YP structure were used to identify which YP precursor comes from which Yp gene. Pulse labeling with [³⁵S]-methionine revealed that all three YPs becomes more negatively charged, that YP1 and YP2 become heterogeneously charged, and that YP1 gains in apparent molecular weight within 15 min after translation. Injecting female flies with radioiabeled sugars or orthophosphate revealed that the YPs are glycosylated and phosphorylated. Treating hemolymph proteins with phosphatase showed that phosphorylation is responsible for much of the change in charge and increase in molecular weight of the maturing YPs. These experiments with wild-type flies provide a basis for the analysis of mutations at the Yp genes which alter the structure of individual YPs.     

Ligand binding to a high‐energy partially unfolded protein

The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP⁺ as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP⁺ and found that NADP⁺ binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP⁺ is diminished upon partial unfolding. Based on known crystallographic structures of NADP⁺‐bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine‐binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP⁺. Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high‐energy non‐native forms.     

Development of a fluorescently labeled thermostable DHFR for studying conformational changes associated with inhibitor binding

A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C_MDCC DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently-attached fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR conformers that bind to MTX at different rates (20.2 and 0.067 μM⁻¹ s⁻¹), similar to previously published findings for Escherichia coli DHFR. Probe fluorescence of C73A/S131C_MDCC DHFR predominantly reports on MTX binding to one of the conformers while intrinsic tryptophan fluorescence of C73A/S131C DHFR reports on binding to the other conformer. This study demonstrates the use of an extrinsic fluorophore attached to a distal region to investigate ligand binding interactions that are not experimentally accessible via intrinsic tryptophan fluorescence alone. The thermostability of C73A/S131C_MDCC DHFR provides an important new tool with applications for investigating the temperature dependence of DHFR conformational changes associated with binding and catalysis.     

Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant

Summary Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vg ^B/+ flies are reared on these substances (vg^B is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies.Our data suggest that the vg ⁺ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.     

Molecular docking studies on DMDP derivatives as human DHFR inhibitors

Molecular docking is routinely used for understanding drug‐receptor interaction in modern drug design. Here, we describe the docking of 2, 4-diamino-5-methyl-5-deazapteridine (DMDP) derivatives as inhibitors to human dihydrofolate reductase (DHFR). We docked 78 DMDP derivates collected from literature to DHFR and studied their specific interactions with DHFR. A new shape-based method, LigandFit, was used for docking DMDP derivatives into DHFR active sites. The result indicates that the molecular docking approach is reliable and produces a good correlation coefficient (r² = 0.499) for the 73 compounds between docking score and IC₅₀ values (Inhibitory Activity). The chloro substituted naphthyl ring of compound 63 makes significant hydrophobic contact with Leu 22, Phe 31 and Pro 61 of the DHFR active site leading to enhanced inhibition of the enzyme. The docked complexes provide better insights to design more potent DHFR inhibitors prior to their synthesis.     

Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas’ disease. We have undertaken a detailed structure–activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme–ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60–70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.