Molecular and Structural Discrimination of Proline Racemase and Hydroxyproline-2-Epimerase from Nosocomial and Bacterial Pathogens

The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for ‘proline racemase’ virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not ‘one way’ directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on contraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host

Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies

Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi , is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy

Proline racemases: insights into Trypanosoma cruzi peptides containing D-proline

Trypanosoma cruzi proline racemases (TcPRAC) are homodimeric enzymes that interconvert the L and D-enantiomers of proline. At least two paralogous copies of proline racemase (PR) genes are present per parasite haploid genome and they are differentially expressed during T. cruzi development. Non-infective epimastigote forms that overexpress PR genes differentiate more readily into metacyclic infective forms that are more invasive to host cells, indicating that PR participates in mechanisms of virulence acquisition. Using a combination of biochemical and enzymatic methods, we show here that, in addition to free D-amino acids, non-infective epimastigote and infective metacyclic parasite extracts possess peptides composed notably of D-proline. The relative contribution of TcPRAC to D-proline availability and its further assembly into peptides was estimated through the use of wild-type parasites and parasites over-expressing TcPRAC genes. Our data suggest that D-proline-bearing peptides, similarly to the mucopeptide layer of bacterial cell walls, may be of benefit to T. cruzi by providing resistance against host proteolytic mechanisms

Inhibition of Trypanosoma cruzi proline racemase affects host-parasite interactions and the outcome of in vitro infection

Proline racemase is an important enzyme of Trypanosoma cruzi and has been shown to be an effective mitogen for B cells, thus contributing to the parasite’s immune evasion and persistence in the human host. Recombinant epimastigote parasites overexpressing TcPRAC genes coding for proline racemase present an augmented ability to differentiate into metacyclic infective forms and subsequently penetrate host-cells in vitro. Here we demonstrate that both anti T. cruzi proline racemase antibodies and the specific proline racemase inhibitor pyrrole-2- carboxylic acid significantly affect parasite infection of Vero cells in vitro. This inhibitor also hampers T. cruzi intracellular differentiation

Two new compounds are more potent inhibitors of <i>Tc</i>PRAC than PYC.

<p>Two new compounds are more potent inhibitors of <i>Tc</i>PRAC than PYC.</p

Inhibition of <i>Tc</i>PRAC proline racemization measured by polarimetry.

<p>Inhibition of <i>Tc</i>PRAC proline racemization measured by polarimetry.</p

Transition path characteristics.

<p>(A) Energy profile and metric quantities for the <i>Tc</i>PRAC transition path. The energy profile (full line) shows that the intermediate states have low energy and do not present any energy barriers. Dotted and dashed lines show the distance from the first structure (d_1,i) and the cumulative distance covered from the first structure (l_1,i), respectively (RMS in Å, see Material and Methods). Little swerving was necessary to avoid the energy barriers. The points corresponding to the intermediate structures used in the screening are marked by crosses. (B) Scores of known ligands, synthesized analogues, and new inhibitors when docked in the selected binding site models. Br-OxoPA could not be docked in the crystallographic structure and its score in the fourth conformation is circled. The score threshold that was chosen in the subsequent virtual screening phase for ligand selection is indicated by a dashed line and the exclusion region is striped. Transition path characteristics. (A) Energy profile and metric quantities for the <i>Tc</i>PRAC transition path. The energy profile (full line) shows that the intermediate states have low energy and do not present any energy barriers. Dotted and dashed lines show the distance from the first structure (d_1,i) and the cumulative distance covered from the first structure (l_1,i), respectively (RMS in Å, see Material and Methods). Little swerving was necessary to avoid the energy barriers. The points corresponding to the intermediate structures used in the screening are marked by crosses. (B) Scores of known ligands, synthesized analogues, and new inhibitors when docked in the selected binding site models. Br-OxoPA could not be docked in the crystallographic structure and its score in the fourth conformation is circled. The score threshold that was chosen in the subsequent virtual screening phase for ligand selection is indicated by a dashed line and the exclusion region is striped. (C) Cavity volume and extension in transition path intermediates, and docked molecules properties. Volume and extension are calculated as explained in Material and Methods. The volume is displayed by the thin line curve. Cavity extension is displayed by the thick curve and crosses mark intermediates that were used for virtual screening. The extension of PYC is shown by the horizontal line, that of OxoPA in all-trans conformation is shown by the horizontal dashed line. The extension of BrOxoPA in all-trans conformation is given in dotted line for two extreme rotamers on the C4–C5 bond. The average molecular weight of the library compounds successfully docked in conformers 1, 4 and 10 is displayed by filled circles. For clarity, the average mass has been divided by 2 to fit the same scale as the cavity volume.</p