SARS-CoV-2 Spike protein peptides displayed in the Pyrococcus furiosus RAD system preserve epitopes antigenicity, immunogenicity, and virus-neutralizing activity of antibodies.

Amongst the potential contribution of protein or peptide-display systems to study epitopes with relevant immunological features, the RAD display system stands out as a highly stable scaffold protein that allows the presentation of constrained target peptides. Here, we employed the RAD display system to present peptides derived from the SARS-CoV-2 Spike (S) protein as a tool to detect specific serum antibodies and to generate polyclonal antibodies capable of inhibiting SARS-CoV-2 infectivity in vitro. 44 linear S-derived peptides were genetically fused with the RAD scaffold (RAD-SCoV-epitopes) and screened for antigenicity with sera collected from COVID-19-infected patients. In a second step, selected RAD-SCoV-epitopes were used to immunize mice and generate antibodies. Phenotypic screening showed that some of these antibodies were able to recognize replicating viral particles in VERO CCL-81 and most notably seven of the RAD-SCoV-epitopes were able to induce antibodies that inhibited viral infection. Our findings highlight the RAD display system as an useful platform for the immunological characterization of peptides and a potentially valuable strategy for the design of antigens for peptide-based vaccines, for epitope-specific antibody mapping, and for the development of antibodies for diagnostic and therapeutic purposes.

The evaluation of in vitro antichagasic and anti-SARS-CoV-2 potential of inclusion complexes of ß- and methyl-ß-cyclodextrin with naphthoquinone.

The compound 3a,10b-dihydro-1H-cyclopenta[b]naphtho[2,3-d]furan-5,10-dione (IVS320) is a naphthoquinone with antifungal and antichagasic potential, which however has low aqueous solubility. To increase bioavailability, inclusion complexes with ß-cyclodextrin (ßCD) and methyl-ß-cyclodextrin (MßCD) were prepared by physical mixture (PM), kneading (KN) and rotary evaporation (RE), and their in vitro anti-SARS-CoV-2 and antichagasic potential was assessed. The formation of inclusion complexes led to a change in the physicochemical characteristics compared to IVS320 alone as well as a decrease in crystallinity degree that reached 74.44% for the IVS320-MßCD one prepared by RE. The IVS320 and IVS320-MßCD/RE system exhibited anti-SARS-CoV-2 activity, showing half maximal effective concentrations (EC50) of 0.47 and 1.22 µg/mL, respectively. Molecular docking simulation suggested IVS320 ability to interact with the SARS-CoV-2 viral protein. Finally, the highest antichagasic activity, expressed as percentage of Tripanosoma cruzi growth inhibition, was observed with IVS320-ßCD/KN (70%) and IVS320-MßCD/PM (72%), while IVS320 alone exhibited only approximately 48% inhibition at the highest concentration (100 µg/mL).

Potential of plant extracts in targeting SARS-CoV-2 main protease: an in vitro and in silico study.

The deaths caused by the covid-19 pandemic have recently decreased due to a worldwide effort in vaccination campaigns. However, even vaccinated people can develop a severe form of the disease that requires ICU admission. As a result, the search for antiviral drugs to treat these severe cases has become a necessity. In this context, natural products are an interesting alternative to synthetic medicines used in drug repositioning, as they have been consumed for a long time through traditional medicine. Many natural compounds found in plant extracts have already been shown to be effective in treating viral and bacterial diseases, making them possible hits to exploit against covid-19. The objective of this work was to evaluate the antiviral activity of different plant extracts available in the library of natural products of the Universidade Estadual de Maringá, by inhibiting the SARS-CoV-2 main protease (Mpro), and by preventing viral infection in a cellular model. As a result, the extract of Cytinus hypocistis, obtained by ultrasound, showed a Mpro inhibition capacity greater than 90%. In the infection model assays using Vero cells, an inhibition of 99.6% was observed, with a selectivity index of 42.7. The in silico molecular docking simulations using the extract compounds against Mpro, suggested Tellimagrandin II as the component of C. hypocistis extract most likely to inhibit the viral enzyme. These results demonstrate the potential of C. hypocistis extract as a promising source of natural compounds with antiviral activity against covid-19.Communicated by Ramaswamy H. Sarma.

Antiviral activity of Cenostigma pluviosum var. peltophoroides extract and fractions against SARS-CoV-2.

Few extracts of plant species from the Brazilian flora have been validated from a pharmacological and clinical point of view, and it is important to determine whether their traditional use is proven by pharmacological effects. Cenostigma pluviosum var. peltophoroides is one of those plants, which belongs to the Fabaceae family that is widely used in traditional medicine and is very rich in tannins. Due to the lack of effective drugs to treat severe cases of Covid-19, the main protease of SARS-CoV-2 (Mpro) becomes an attractive target in the research for new antivirals since this enzyme is crucial for virus replication and does not have homologs in humans. This study aimed to prospect inhibitor candidates among the compounds from C. pluviosum extract, by virtual screening simulations using SARS-CoV-2 Mpro as target. Experimental validation was made by inhibitory proteolytic assays of recombinant Mpro and by antiviral activity with infected Vero cells. Docking simulations identify four compounds with potential inhibitory activity of Mpro present in the extract. The compound pentagalloylglucose showed the best result in proteolytic kinetics experiments, with suppression of recombinant Mpro activity by approximately 60%. However, in experiments with infected cells ethyl acetate fraction and sub-fractions, F2 and F4 of C. pluviosum extract performed better than pentagalloylglucose, reaching close to 100% of antiviral activity. The prominent activity of the extract fractions in infected cells may be a result of a synergistic effect from the different hydrolyzable tannins present, performing simultaneous action on Mpro and other targets from SARS-CoV-2 and host.Communicated by Ramaswamy H. Sarma.

A multi-species phenotypic screening assay for leishmaniasis drug discovery shows that active compounds display a high degree of species-specificity

High genetic and phenotypic variability between Leishmania species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse Leishmania species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: L. amazonensis, L. braziliensis, and L. donovani. The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any Leishmania species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.

A multi-species phenotypic screening assay for leishmaniasis drug discovery shows that active compounds display a high degree of species-specificity

High genetic and phenotypic variability between Leishmania species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse Leishmania species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: L. amazonensis, L. braziliensis, and L. donovani. The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any Leishmania species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.

Sofosbuvir inhibits yellow fever virus in vitro and in patients with acute liver failure

Introduction and objectives Direct antiviral agents (DAAs) are very efficient in inhibiting hepatitis C virus and might be used to treat infections caused by other flaviviruses whose worldwide detection has recently increased. The aim of this study was to verify the efficacy of DAAs in inhibiting yellow fever virus (YFV) by using drug repositioning (a methodology applied in the pharmaceutical industry to identify new uses for approved drugs). Materials and methods Three DAAs were evaluated: daclatasvir, sofosbuvir and ledipasvir or their combinations. For in vitro assays, the drugs were diluted in 100% dimethyl sulfoxide. Vaccine strain 17D and a 17D strain expressing the reporter fluorescent protein were used in the assays. A fast and reliable cell-based screening assay using Vero cells or Huh-7 cells (a hepatocyte-derived carcinoma ell line) was carried out. Two patients who acquired yellow fever virus with acute liver failure were treated with sofosbuvir for one week as a compassionate use. Results Using a high-content screening assay, we verified that sofosbuvir presented the best antiviral activity against YFV. Moreover, after an off-label treatment with sofosbuvir, the two female patients diagnosed with yellow fever infection displayed a reduction in blood viremia and an improvement in the course of the disease, which was observed in the laboratory medical parameters related to disease evolution. Conclusions Sofosbuvir may be used as an option for treatment against YFV until other drugs are identified and approved for human use. These results offer insights into the role of nonstructural protein 5 (NS5) in YFV inhibition and suggest that nonstructural proteins may be explored as drug targets for YFV treatment.

Expanding the biological application of fluorescent benzothiadiazole derivatives: a phenotypic screening strategy for anthelmintic drug discovery using caenorhabditis elegans

The current methodologies used to identify promising new anthelmintic compounds rely on subjective microscopic examination of worm motility or involve genetic modified organisms. We describe a new methodology to detect worm viability that takes advantage of the differential incorporation of the fluorescent molecular marker propidium iodide and the 2,1,3-benzothiadiazole core, which has been widely applied in light technology. The new assay developed could be validated using the "Pathogen Box" library. By use of this bioassay, it was possible to identify three molecules with activity against Caenorhabditis elegans that were previously described as effective in in vitro assays against other pathogens, such as Schistosoma mansoni, Mycobacterium tuberculosis, and Plasmodium falciparum, accelerating the identification of molecules with anthelmintic potential. The current fluorescence-based bioassay may be used for assessing C. elegans viability. The described methodology replaces the subjectivity of previous assays and provides an enabling technology that is useful for rapid in vitro screens of both natural and synthetic compound libraries. It is expected that the results obtained from these robust in vitro screens would select the most effective compounds for follow-up in vivo experimentation with pathogenic helminths.

Sofosbuvir inhibits yellow fever virus in vitro and in patients with acute liver failure

Introduction and objectives Direct antiviral agents (DAAs) are very efficient in inhibiting hepatitis C virus and might be used to treat infections caused by other flaviviruses whose worldwide detection has recently increased. The aim of this study was to verify the efficacy of DAAs in inhibiting yellow fever virus (YFV) by using drug repositioning (a methodology applied in the pharmaceutical industry to identify new uses for approved drugs). Materials and methods Three DAAs were evaluated: daclatasvir, sofosbuvir and ledipasvir or their combinations. For in vitro assays, the drugs were diluted in 100% dimethyl sulfoxide. Vaccine strain 17D and a 17D strain expressing the reporter fluorescent protein were used in the assays. A fast and reliable cell-based screening assay using Vero cells or Huh-7 cells (a hepatocyte-derived carcinoma ell line) was carried out. Two patients who acquired yellow fever virus with acute liver failure were treated with sofosbuvir for one week as a compassionate use. Results Using a high-content screening assay, we verified that sofosbuvir presented the best antiviral activity against YFV. Moreover, after an off-label treatment with sofosbuvir, the two female patients diagnosed with yellow fever infection displayed a reduction in blood viremia and an improvement in the course of the disease, which was observed in the laboratory medical parameters related to disease evolution. Conclusions Sofosbuvir may be used as an option for treatment against YFV until other drugs are identified and approved for human use. These results offer insights into the role of nonstructural protein 5 (NS5) in YFV inhibition and suggest that nonstructural proteins may be explored as drug targets for YFV treatment.

Expanding the biological application of fluorescent benzothiadiazole derivatives: a phenotypic screening strategy for anthelmintic drug discovery using caenorhabditis elegans

The current methodologies used to identify promising new anthelmintic compounds rely on subjective microscopic examination of worm motility or involve genetic modified organisms. We describe a new methodology to detect worm viability that takes advantage of the differential incorporation of the fluorescent molecular marker propidium iodide and the 2,1,3-benzothiadiazole core, which has been widely applied in light technology. The new assay developed could be validated using the "Pathogen Box" library. By use of this bioassay, it was possible to identify three molecules with activity against Caenorhabditis elegans that were previously described as effective in in vitro assays against other pathogens, such as Schistosoma mansoni, Mycobacterium tuberculosis, and Plasmodium falciparum, accelerating the identification of molecules with anthelmintic potential. The current fluorescence-based bioassay may be used for assessing C. elegans viability. The described methodology replaces the subjectivity of previous assays and provides an enabling technology that is useful for rapid in vitro screens of both natural and synthetic compound libraries. It is expected that the results obtained from these robust in vitro screens would select the most effective compounds for follow-up in vivo experimentation with pathogenic helminths.

Development of a focused library of triazole-linked privileged-structure-based conjugates leading to the discovery of novel phenotypic hits against protozoan parasitic infections

Protozoan infections caused by Plasmodium, Leishmania, and Trypanosoma spp. contribute significantly to the burden of infectious diseases worldwide, causing severe morbidity and mortality. The inadequacy of available treatments calls for cost- and time-effective drug discovery endeavors. To this end, we envisaged the triazole linkage of privileged structures as an effective drug design strategy to generate a focused library of high-quality compounds. The versatility of this approach was combined with the feasibility of a phenotypic assay, integrated with early ADME-tox profiling. Thus, an 18-membered library was efficiently assembled via Huisgen cycloaddition of phenothiazine, biphenyl, and phenylpiperazine scaffolds. The resulting 18 compounds were then tested against seven parasite strains, and counter-screened for selectivity against two mammalian cell lines. In parallel, hERG and cytochrome P450 (CYP) inhibition, and mitochondrial toxicity were assessed. Remarkably, 10-((1-(3-([1,1-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-5-yl)methyl)-10H-phenothiazine (7) and 10-(3-(1-(3-([1,1-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-4-yl)propyl)-10H-phenothiazine (12) showed respective IC50 values of 1.8 and 1.9gmL(-1) against T.cruzi, together with optimal selectivity. In particular, compound 7 showed a promising ADME-tox profile. Thus, hit 7 might be progressed as an antichagasic lead.

Aryl thiosemicarbazones for the treatment of trypanosomatidic infections

Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc). Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi. Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide (A14) yielding a comparable anti-parasitic activity against the three parasitic species (TbEC50=231 mu M, LiEC50 = 6.14 mu M, TcEC50 = 1.31 mu M) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4'-dimethoxy-[1.1'-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide (A12) and (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene)hydrazine carbothioamide (A14) were able to potentiate the anti parasitic activity of methotrexate (MTX) when evaluated in combination against T. brucei, yielding a 6 fold and 4-fold respectively Dose Reduction Index for MTX. The toxicity profile against four human cell lines and a panel of in vitro early-toxicity assays (comprising hERG, Aurora B, five cytochrome P450 isoforms and mitochondrial toxicity) demonstrated the low toxicity for the thosemicarbazones class in comparison with known drugs. The results confirmed thiosemicarbazones as a suitable chemical scaffold with potential for the development of properly decorated new anti-parasitic drugs.

Development of a focused library of triazole-linked privileged-structure-based conjugates leading to the discovery of novel phenotypic hits against protozoan parasitic infections

Protozoan infections caused by Plasmodium, Leishmania, and Trypanosoma spp. contribute significantly to the burden of infectious diseases worldwide, causing severe morbidity and mortality. The inadequacy of available treatments calls for cost- and time-effective drug discovery endeavors. To this end, we envisaged the triazole linkage of privileged structures as an effective drug design strategy to generate a focused library of high-quality compounds. The versatility of this approach was combined with the feasibility of a phenotypic assay, integrated with early ADME-tox profiling. Thus, an 18-membered library was efficiently assembled via Huisgen cycloaddition of phenothiazine, biphenyl, and phenylpiperazine scaffolds. The resulting 18 compounds were then tested against seven parasite strains, and counter-screened for selectivity against two mammalian cell lines. In parallel, hERG and cytochrome P450 (CYP) inhibition, and mitochondrial toxicity were assessed. Remarkably, 10-((1-(3-([1,1-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-5-yl)methyl)-10H-phenothiazine (7) and 10-(3-(1-(3-([1,1-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-4-yl)propyl)-10H-phenothiazine (12) showed respective IC50 values of 1.8 and 1.9gmL(-1) against T.cruzi, together with optimal selectivity. In particular, compound 7 showed a promising ADME-tox profile. Thus, hit 7 might be progressed as an antichagasic lead.

Aryl thiosemicarbazones for the treatment of trypanosomatidic infections

Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc). Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi. Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide (A14) yielding a comparable anti-parasitic activity against the three parasitic species (TbEC50=231 mu M, LiEC50 = 6.14 mu M, TcEC50 = 1.31 mu M) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4'-dimethoxy-[1.1'-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide (A12) and (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene)hydrazine carbothioamide (A14) were able to potentiate the anti parasitic activity of methotrexate (MTX) when evaluated in combination against T. brucei, yielding a 6 fold and 4-fold respectively Dose Reduction Index for MTX. The toxicity profile against four human cell lines and a panel of in vitro early-toxicity assays (comprising hERG, Aurora B, five cytochrome P450 isoforms and mitochondrial toxicity) demonstrated the low toxicity for the thosemicarbazones class in comparison with known drugs. The results confirmed thiosemicarbazones as a suitable chemical scaffold with potential for the development of properly decorated new anti-parasitic drugs.

Chroman-4-one derivatives targeting pteridine reductase 1 and showing anti-parasitic activity

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.

Synthesis and trypanocidal activity of a library of 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols

A library of 16 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols 17-32 has been synthesized for use in biological testing against Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. The 4-substituted 2-(1H-pyrrolo[3,2-c]pyridin-2-yl)propan-2-ols 17-32 were subjected to biological testing to evaluate their efficacy against intracellular Trypanosoma cruzi (Y strain) amastigotes infecting U2OS human cells, with benznidazole as a reference compound. The assay was performed in duplicate (two independent experiments) and submitted to High Content Analysis (HCA) for determination of trypanocidal activity. Three of the tested compounds presented relatively high trypanocidal activity (19, 22 and 29), however severe host cell toxicity was observed concomitantly. Chemical optimization of the highly active compounds and the synthesis of more compounds for biological testing against Trypanosoma cruzi will be required to improve selectivity and so that a structure-activity relationship can be generated to provide a more insightful analysis of both chemical and biological aspects.

High content screening of a kinase-focused library reveals compounds broadly-active against dengue viruses.

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.

Biochemical characterization of proline racemases from the human protozoan parasite Trypanosoma cruzi and definition of putative protein signatures.

Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.