Olga Senkovich

Structure‐based approach to pharmacophore identification, in silico screening, and three‐dimensional quantitative structure–activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function

We have employed a structure‐based three‐dimensional quantitative structure–activity relationship (3D‐QSAR) approach to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase‐thymidylate synthase (DHFR‐TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate‐free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure‐based alignment used as input for the QSAR study. Contrary to indirect ligand‐based approaches the strategy described here employs a direct receptor‐based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D‐QSAR models were obtained for T. cruzi DHFR‐TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between experimental and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave‐one‐out method. The derived 3D‐QSAR models were tested against a few selected compounds (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D‐QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of additional activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability. Proteins 2008.

Structures of dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexate.

The flagellate protozoan parasite Trypanosoma cruzi is the pathogenic agent of Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. The parasite is usually transmitted by a blood-feeding insect vector, but can also be transmitted via blood transfusion. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. There is an urgent need for the development of chemotherapeutic agents for the treatment of T. cruzi infection and therefore for the identification of potential drug targets. The dihydrofolate reductase activity of T. cruzi, which is expressed as part of a bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), is a potential target for drug development. In order to gain a detailed understanding of the structure-function relationship of T. cruzi DHFR, the three-dimensional structure of this protein in complex with various ligands is being studied. Here, the crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain are reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). These structures reveal that the enzyme is a homodimer with substantial interactions between the two TS domains of neighboring subunits. In contrast to the enzymes from Cryptosporidium hominis and Plasmodium falciparum, the DHFR and TS active sites of T. cruzi lie on the same side of the monomer. As in other parasitic DHFR-TS proteins, the N-terminal extension of the T. cruzi enzyme is involved in extensive interactions between the two domains. The DHFR active site of the T. cruzi enzyme shows subtle differences compared with its human counterpart. These differences may be exploited for the development of antifolate-based therapeutic agents for the treatment of T. cruzi infection.

Trypanosoma cruzi genome encodes a pteridine reductase 2 protein.

Pteridine metabolism in Trypanosoma cruzi is poorly understood. The term ‘pteridine’ is used collectively for two classes of structurally-related compounds, folates and biopterins, which differ only in the nature of the side chain attached to the C6 atom of the pterin ring. Both folate and biopterin, in their reduced (tetrahydro) forms, serve as essential cofactors in a number of critical metabolic steps in many organisms [1]. While some microorganisms and parasites such as Plasmodium can synthesize folate, mammals and trypanosomatid parasites lack this ability. On the other hand, mammalian cells can synthesize tetrahydrobiopterin de novo from GTP, while these parasites cannot synthesize biopterin either [2–4]. In order to meet the need for these essential nutrients, folate and biopterin are transported from the host into the parasite, and are subsequently reduced to their respective dihydro and tetrahydro forms by parasitic dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) enzymes [1,2]. DHFR is one of the best characterized enzymes. Structure function relationships in DHFR from a variety of sources have been studied in detail and a number of inhibitors targeting DHFR has been successfully used in cancer chemotherapy and against some infectious pathogens including malaria parasite [1,5,6]. On the other hand, much of our current knowledge about pteridine reductase 1 (PTR1) is derived from research in Leishmania [7–12]. The Leishmania gene encoding PTR1 enzyme was found to be responsible for resistance to the classical antifolate drug methotrexate (MTX). The enzyme belongs to the family of short-chain dehydrogenases/reductases (SDR). Biochemical studies showed that it had broad substrate specificity and was able to reduce both folates and biopterins using NADPH as cofactor. PTR1 is considerably less sensitive (at

Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes.

Docking and biological activity of pteridine analogs: search for inhibitors of pteridine reductase enzymes from Trypanosoma cruzi

Abstract The program DOCK4.0 was used to predict and evaluate the binding mode for a set of inhibitors from a compound library of pteridine analogs against Trypanosoma cruzi pteridine reductase 2 ( Tc PTR2). The docked ligand conformations are, as expected, similar to the observed binding mode of MTX in the crystal structure of the Tc PTR2 inhibitor complex. Dock energy scores are correlated with experimental enzymatic activity data ( K i ) and calculated binding affinities ( K d ) for these inhibitors. The screened compounds showed activity comparable to MTX in enzyme assays. An initial attempt is made to correlate the structure of these compounds to their affinity.

Structure of Streptococcus agalactiae glyceraldehyde-3-phosphate dehydrogenase holoenzyme reveals a novel surface

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a conserved cytosolic enzyme, which plays a key role in glycolysis. GAPDH catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate using NAD or NADP as a cofactor. In addition, GAPDH localized on the surface of some bacteria is thought to be involved in macromolecular interactions and bacterial pathogenesis. GAPDH on the surface of group B streptococcus (GBS) enhances bacterial virulence and is a potential vaccine candidate. Here, the crystal structure of GBS GAPDH from Streptococcus agalactiae in complex with NAD is reported at 2.46 Å resolution. Although the overall structure of GBS GAPDH is very similar to those of other GAPDHs, the crystal structure reveals a significant difference in the area spanning residues 294-307, which appears to be more acidic. The amino-acid sequence of this region of GBS GAPDH is also distinct compared with other GAPDHs. This region therefore may be of interest as an immunogen for vaccine development.

Structure of the catalytic domain of Plasmodium falciparum ARF GTPase-activating protein (ARFGAP).

The crystal structure of the catalytic domain of the ADP ribosylation factor GTPase-activating protein (ARFGAP) from Plasmodium falciparum has been determined and refined to 2.4 Å resolution. Multiwavength anomalous diffraction (MAD) data were collected utilizing the Zn(2+) ion bound at the zinc-finger domain and were used to solve the structure. The overall structure of the domain is similar to those of mammalian ARFGAPs. However, several amino-acid residues in the area where GAP interacts with ARF1 differ in P. falciparum ARFGAP. Moreover, a number of residues that form the dimer interface in the crystal structure are unique in P. falciparum ARFGAP.