The Past, Present, and Future in the Diagnosis of a Neglected Sexually Transmitted Infection: Trichomoniasis.

More than one million curable sexually transmitted infections occur every day. Trichomonas vaginalis is one of the main infections responsible for these epidemiological data; however, the diagnosis of this protozoan is still mainly based on microscopic and culture identification. The commercialization of immunological tests and the development of molecular techniques have improved the sensitivity of classical methods. Nevertheless, the fact that trichomoniasis is a neglected parasitic infection hinders the development of novel techniques and their implementation in routine diagnosis. This review article shows the different methods developed to identify T. vaginalis in population and the difficulties in diagnosing male and asymptomatic patients. The importance of including this parasite in routine gynecological screening, especially in pregnant women, and the importance of considering T. vaginalis as an indicator of high-risk sexual behavior are also discussed.

Promising hit compounds against resistant trichomoniasis: Synthesis and antiparasitic activity of 3-(ω-aminoalkoxy)-1-benzyl-5-nitroindazoles.

A series of 11 3-(ω-aminoalkoxy)-1-benzyl-5-nitroindazoles (2-12) has been prepared starting from 1-benzyl-5-nitroindazol-3-ol 13, and evaluated against sensitive and resistant isolates of the sexually transmitted protozoan Trichomonas vaginalis. Compounds 2, 3, 6, 9, 10 and 11 showed trichomonacidal profiles with IC50 < 20 µM against the metronidazole-sensitive isolate. Moreover, all these compounds submitted to cytotoxicity assays against mammalian cells exhibited low non-specific cytotoxic effects, except compounds 3 and 9 which displayed moderate cytotoxicity (CC50 = 74.7 and 59.1 µM, respectively). Those compounds with trichomonacidal effect were also evaluated against a metronidazole-resistant culture. Special mention deserve compounds 6 and 10, which displayed better IC50 values (1.3 and 0.5 µM respectively) than that of the reference drug (IC50 MTZ = 3.0 µM). The high activity of these compounds against the resistant isolate reinforces the absence of cross-resistance with the reference drug. The remarkable trichomonacidal results against resistant T. vaginalis isolates suggest the interest of 3-(ω-aminoalkoxy)-1-benzyl-5-nitroindazoles to be considered as good prototypes to continue in the development of new drugs with enhanced trichomonacidal activity, aiming to increase the non-existent drugs to face clinical resistance efficiently for those patients in whom therapy with 5-nitroimidazoles is contraindicated.

Antichagasic, Leishmanicidal, and Trichomonacidal Activity of 2-Benzyl-5-nitroindazole-Derived Amines.

Three different series of new 5-nitroindazole derivatives-1-(ω-aminoalkyl)-2-benzylindazolin-3-ones (series A; ten compounds), 3-(ω-aminoalkoxy)-2-benzylindazoles (series B; four compounds) and 3-alkylamino-2-benzylindazoles (series C; five compounds)-have been synthesized and evaluated against the protozoan parasites Trypanosoma cruzi, Leishmania amazonensis, and Trichomonas vaginalis: etiological agents of Chagas disease, cutaneous leishmaniasis, and trichomoniasis, respectively. Many indazoles of series A, B, and C were efficient against T. cruzi. Some compounds in series A, after successfully passing the preliminary screening for epimastigotes, exhibited activity values against amastigotes of several T. cruzi strains that were better than or similar to those shown by the reference drug benznidazole and displayed low nonspecific toxicity against mammalian cells. On the other hand, preliminary studies against promastigotes of L. amazonensis showed high leishmanicidal activity for some derivatives of series A and C. With regard to activity against T. vaginalis, some indazoles of series B and C were rather efficient against trophozoites of a metronidazole-sensitive isolate and showed low nonspecific toxicities toward Vero cell cultures. Additionally, some of these compounds displayed similar activity against metronidazole-sensitive and resistant isolates, showing the absence of cross-resistance between these derivatives and the reference drug.

Antichagasic and trichomonacidal activity of 1-substituted 2-benzyl-5-nitroindazolin-3-ones and 3-alkoxy-2-benzyl-5-nitro-2H-indazoles.

Two series of new 5-nitroindazole derivatives, 1-substituted 2-benzylindazolin-3-ones (6-29, series A) and 3-alkoxy-2-benzyl-2H-indazoles (30-37, series B), containing differently functionalized chains at position 1 and 3, respectively, have been synthesized starting from 2-benzyl-5-nitroindazolin-3-one 5, and evaluated against the protozoan parasites Trypanosoma cruzi and Trichomonas vaginalis, etiological agents of Chagas disease and trichomonosis, respectively. Many indazolinones of series A were efficient against different morphological forms of T. cruzi CL Brener strain (compounds 6, 7, 9, 10 and 19-21: IC50 = 1.58-4.19 µM for epimastigotes; compounds 6, 19-21 and 24: IC50 = 0.22-0.54 µM for amastigotes) being as potent as the reference drug benznidazole. SAR analysis suggests that electron-donating groups at position 1 of indazolinone ring are associated with an improved antichagasic activity. Moreover, compounds of series A displayed low unspecific toxicities against an in vitro model of mammalian cells (fibroblasts), which were reflected in high values of the selectivity indexes (SI). Compound 20 was also very efficient against amastigotes from Tulahuen and Y strains of T. cruzi (IC50 = 0.81 and 0.60 µM, respectively), showing low toxicity towards cardiac cells (LC50 > 100 µM). In what concerns compounds of series B, some of them displayed moderate activity against trophozoites of a metronidazole-sensitive isolate of T. vaginalis (35 and 36: IC50 = 9.82 and 7.25 µM, respectively), with low unspecific toxicity towards Vero cells. Compound 36 was also active against a metronidazole-resistant isolate (IC50 = 9.11 µM) and can thus be considered a good prototype for the development of drugs directed to T. vaginalis resistant to 5-nitroimidazoles.

In vitro trichomonacidal activity and preliminary in silico chemometric studies of 5-nitroindazolin-3-one and 3-alkoxy-5-nitroindazole derivatives.

A selection of 1,2-disubstituted 5-nitroindazolin-3-ones (1-19) and 3-alkoxy-5-nitroindazoles substituted at positions 1 (20-24) or 2 (25-39) from our in-house compound library were screened in vitro against the most common curable sexually transmitted pathogen, Trichomonas vaginalis. A total of 41% of the studied molecules (16/39) achieved a significant activity of more than 85% growth inhibition at the highest concentration assayed (100 µg mL(-1)). Among these compounds, 3-alkoxy-5-nitroindazole derivatives 23, 24, 25 and 27 inhibited parasite growth by more than 50% at 10 µg mL(-1). In addition, the first two compounds (23, 24) still showed remarkable activity at the lowest dose tested (1 µg mL(-1)), inhibiting parasite growth by nearly 40%. Their specific activity towards the parasite was corroborated by the determination of their non-specific cytotoxicity against mammalian cells. The four mentioned compounds exhibited non-cytotoxic profiles at all of the concentrations assayed, showing a fair antiparasitic selectivity index (SI > 7·5). In silico studies were performed to predict pharmacokinetic properties, toxicity and drug-score using Molinspiration and OSIRIS computational tools. The current in vitro results supported by the virtual screening suggest 2-substituted and, especially, 1-substituted 3-alkoxy-5-nitroindazoles as promising starting scaffolds for further development of novel chemical compounds with the main aim of promoting highly selective trichomonacidal lead-like drugs with adequate pharmacokinetic and toxicological profiles.

Sequestration of host-CD59 as potential immune evasion strategy of Trichomonas vaginalis.

Trichomonas vaginalis is known to evade complement-mediated lysis. Because the genome of T. vaginalis does not possess DNA sequence with homology to human protectin (CD59), a complement lysis restricting factor, we tested the hypothesis that host CD59 acquisition by T. vaginalis organisms mediates resistance to complement killing. This hypothesis was based on the fact that trichomonads are known to associate with host proteins. No CD59 was detected on the surface of T. vaginalis grown in serum-based medium using as probe anti-CD59 monoclonal antibody (MAb). We, therefore, infected mice intraperitoneally with live T. vaginalis, and trichomonads harvested from ascites were tested for binding of CD59. Immunofluorescence showed that parasites had surface CD59. Furthermore, as mouse erythrocytes (RBCs) possess membrane-associated CD59, and trichomonads use RBCs as a nutrient source, organisms were co-cultured with murine RBCs for one week. Parasites were shown to have detectable surface CD59. Importantly, live T. vaginalis with bound CD59 were compared with batch-grown parasites without surface-associated CD59 for sensitivity to complement in human serum. Trichomonads without surface-bound CD59 had a higher level of killing by complement than did parasites with surface CD59. These data show that host CD59 acquired onto the surface by live T. vaginalis may be an alternative mechanism for complement evasion. We describe a novel strategy by T. vaginalis consistent with host protein procurement by this parasite to evade the lytic action of complement.

Synthesis and in vitro and in vivo biological evaluation of substituted nitroquinoxalin-2-ones and 2,3-diones as novel trichomonacidal agents.

Two series of ten novel 7-nitroquinoxalin-2-ones and ten 6-nitroquinoxaline-2,3-diones with diverse substituents at positions 1 and 4 were synthesized and evaluated against the sexually transmitted parasite Trichomonas vaginalis. Furthermore, diverse molecular and drug-likeness properties were analyzed to predict the oral bioavailability following the Lipinski's "rule of five". 7-Nitroquinoxalin-2-one derivatives displayed moderate to high in vitro activity while the efficiency of most nitroquinoxaline-2,3-diones was rather low; both kinds of compounds did not show cytotoxic effects in mammalian cells. 7-Nitro-4-(3-piperidinopropyl)quinoxalin-2-one 9 achieved the highest trichomonacidal activity (IC50 = 18.26 µM) and was subsequently assayed in vivo in a murine model of trichomonosis. A 46.13% and a 50.70% reduction of pathogenic injuries were observed in the experimental groups treated orally during 7 days with 50 mg/kg and 100 mg/kg doses. The results obtained in the biological assays against T. vaginalis indicate that compounds with ω-(dialkylamino)alkyl substituents and a keto group at positions 4 and 2 of quinoxaline ring, respectively, provide interesting structural cores to develop novel prototypes to enhance the nitroquinoxalinones activity as trichomonacidal agents with interesting ADME properties according to virtual screening analysis.

A sequential procedure for rapid and accurate identification of putative trichomonacidal agents.

In the current report, a sequential step-wise methodology based on in silico, in vitro and in vivo experimental procedures for the prompt detection of potential trichomonacidal drugs is proposed. A combinatorial of 12 QSAR (Quantitative Structure-Activity Relationship) models based on Linear Discrimination Analysis (LDA) are suggested for the rational identification of new trichomonacidal drugs from virtual screening of in house chemical libraries and drug databases. Subsequently, compounds selected as potential anti-trichomonas are screened in vitro against Trichomonas vaginalis. Finally, molecules with specific trichomonacidal activity are evaluated in vivo. Herein, different molecules were exposed to the proposed methodology. Firstly, the agents were virtually screened and two of the eight molecules (G-1 and dimetridazole) were classified as trichomonacidals by the 12 models. Subsequently both drugs were proved in vitro and in vivo following the workflow procedure. Although a remarkable in vitro activity was observed in both cases, dimetridazole achieved higher MIC100 activity than metronidazole against the resistant isolate. Furthermore, the in vivo models showed a remarkable reduction of lesions of more than 55% in both compounds. These observations support the current flowchart screening and suggest the use of dimetridazole as a promising drug-like scaffold for novel therapeutic alternatives against T. vaginalis resistant infections.

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species.

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~83%. There was >40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.

Synthesis and evaluation of 1,1'-hydrocarbylenebis(indazol-3-ols) as potential antimalarial drugs.

Bis(indazol-3-ol) derivatives (5, 30-38) were prepared by alkylation of 3-alkoxyindazoles with alpha,omega-dibromides, followed by removal of the O-protecting groups. These compounds were subsequently evaluated as inhibitors of biocrystallization of ferriprotoporphyrin IX (heme) to hemozoin, a Plasmodium detoxification specific process. Most bis(5-nitroindazol-3-ols) were good inhibitors, however, a denitro analogue (38), the intermediate bis(3-alkoxyindazoles) (15-29) as well as bis(indazolin-3-ones) (39-42) were not active, showing the importance of the NO(2) and OH groups in the inhibition process.

Immunomodulation of the response to excretory/secretory antigens of Fasciola hepatica by Anapsos in Balb/C mice and rat alveolar macrophages.

It is known that excretory/secretory antigens of Fasciola hepatica (ESFh) trigger a Th2-like immune response. Anapsos (A) is an aqueous hydrosoluble extract obtained from the rhizomes of the fern Polypodium leucotomos that has shown immunomodulator effects in some parasitic infections and immunological disorders. In this work we assess the effect of Anapsos and ESFh and Quillaja saponaria extract (Qs) on BALB/c mice and rat alveolar macrophages. Anapsos modulates the response of mice immunized with ESFh, decreasing IgG antibodies in A+ESFh- and A+Qs+ESFh-treated mice and triggering high levels of gammaIFN in spleen cell culture in comparison with ESFh- and Qs + ESFh--treated groups. Moreover, Anapsos showed statistically significant inhibitory effects on the nitrite production by rat alveolar macrophages prestimulated with lipopolysaccharide (LPS) as well as ESFh antigen in comparison with macrophages stimulated only with LPS. The application of ESFh and Anapsos combined avoids this inhibitory effect. Thus, Anapsos modulates the immune response against ESFh in naive mice and on the nitrite production in prestimulated rat aveolar macrophages.

Trichinella: differing effects of antigens from encapsulated and non-encapsulated species on in vitro nitric oxide production.

Trichinellosis is a cosmopolitan zoonotic disease affecting a wide variety of animals, including man. Non-encapsulated and encapsulated species diverge with respect to their developmental strategies. Little is known at the molecular level about parasite-derived mediators responsible for host muscle cell transformation occurring during trichinellosis. In this context, host-parasite relationships in Trichinella-infected animals could be related to different host-immune and cell mediators, e.g. nitric oxide (NO). Here, we investigate the stimulatory/inhibitory role of L1 antigens from four encapsulated (T. spiralis, T. britovi, T. nelsoni and T. nativa) and one non-encapsulated (T. pseudospiralis) Trichinella species on NO production from rat macrophages in vitro. Our results demonstrate that encapsulated and non-encapsulated Trichinella species differ in their capacity to stimulate the secretion of NO from host macrophages. Biological significance of these differences should be further assessed in the available experimental models.

Predicting antitrichomonal activity: a computational screening using atom-based bilinear indices and experimental proofs.

Existing Trichomonas vaginalis therapies are out of reach for most trichomoniasis people in developing countries and, where available, they are limited by their toxicity (mainly in pregnant women) and their cost. New antitrichomonal agents are needed to combat emerging metronidazole-resistant trichomoniasis and reduce the side effects associated with currently available drugs. Toward this end, atom-based bilinear indices, a new TOMOCOMD-CARDD molecular descriptor, and linear discriminant analysis (LDA) were used to discover novel, potent, and non-toxic lead trichomonacidal chemicals. Two discriminant functions were obtained with the use of non-stochastic and stochastic atom-type bilinear indices for heteroatoms and H-bonding of heteroatoms. These atomic-level molecular descriptors were calculated using a weighting scheme that includes four atomic labels, namely atomic masses, van der Waals volumes, atomic polarizabilities, and atomic electronegativities in Pauling scale. The obtained LDA-based QSAR models, using non-stochastic and stochastic indices, were able to classify correctly 94.51% (90.63%) and 93.41% (93.75%) of the chemicals in training (test) sets, respectively. They showed large Matthews' correlation coefficients (C); 0.89 (0.79) and 0.87 (0.85), for the training (test) sets, correspondingly. The result of predictions on the 15% full-out cross-validation test also evidenced the robustness and predictive power of the obtained models. In addition, canonical regression analyses corroborated the statistical quality of these models (R(can) of 0.749 and of 0.845, correspondingly); they were also used to compute biological activity canonical scores for each compound. On the other hand, a close inspection of the molecular descriptors included in both equations showed that several of these molecular fingerprints are strongly interrelated with each other. Therefore, these models were orthogonalized using the Randic orthogonalization procedure. These classification functions were then applied to find new lead antitrichomonal agents and six compounds were selected as possible active compounds by computational screening. The designed compounds were synthesized and tested for in vitro activity against T. vaginalis. Out of the six compounds that were designed, and synthesized, three molecules (chemicals VA5-5a, VA5-5c, and VA5-12b) showed high to moderate cytocidal activity at the concentration of 10 microg/ml, other two compounds (VA5-8pre and VA5-8) showed high cytocidal and cytostatic activity at the concentration of 100 microg/ml and 10 microg/ml, correspondingly, and the remaining chemical (compound VA5-5e) was inactive at these assayed concentrations. Nonetheless, these compounds possess structural features not seen in known trichomonacidal compounds and thus can serve as excellent leads for further optimization of antitrichomonal activity. The LDA-based QSAR models presented here can be considered as a computer-assisted system that could potentially significantly reduce the number of synthesized and tested compounds and increase the chance of finding new chemical entities with antitrichomonal activity.

A linear discrimination analysis based virtual screening of trichomonacidal lead-like compounds: outcomes of in silico studies supported by experimental results.

A computational (virtual) screening test to identify potential trichomonacidals has been developed. Molecular structures of trichomonacidal and non-trichomonacidal drugs were represented using stochastic and non-stochastic atom-based quadratic indices and a linear discrimination analysis (LDA) was trained to classify molecules regarding their antiprotozoan activity. Validation tests revealed that our LDA-QSAR models recognize at least 88.24% of trichomonacidal lead-like compounds and suggest using this methodology in virtual screening protocols. These classification functions were then applied to find new lead antitrichomonal compounds. In this connection, the biological assays of eight compounds, selected by computational screening using the present models, give good results (87.50% of good classification). In general, most of the compounds showed high activity against Trichomonas vaginalis at the concentration of 100 microg/ml and low cytotoxicity to this concentration. In particular, two heterocyclic derivatives (VA7-67 and VA7-69) maintained their efficacy at 10 microg/ml with an important trichomonacidal activity (100.00% of reduction), but it is remarkable that the compound VA7-67 did not show cytotoxic effects in macrophage cultivations. This result opens a door to a virtual study considering a higher variability of the structural core already evaluated, as well as of other chemicals not included in this study.

A computer-based approach to the rational discovery of new trichomonacidal drugs by atom-type linear indices.

Computational approaches are developed to design or rationally select, from structural databases, new lead trichomonacidal compounds. First, a data set of 111 compounds was split (design) into training and predicting series using hierarchical and partitional cluster analyses. Later, two discriminant functions were derived with the use of non-stochastic and stochastic atom-type linear indices. The obtained LDA (linear discrimination analysis)-based QSAR (quantitative structure-activity relationship) models, using non-stochastic and stochastic descriptors were able to classify correctly 95.56% (90.48%) and 91.11% (85.71%) of the compounds in training (test) sets, respectively. The result of predictions on the 10% full-out cross-validation test also evidenced the quality (robustness, stability and predictive power) of the obtained models. These models were orthogonalized using the Randic orthogonalization procedure. Afterwards, a simulation experiment of virtual screening was conducted to test the possibilities of the classification models developed here in detecting antitrichomonal chemicals of diverse chemical structures. In this sense, the 100.00% and 77.77% of the screened compounds were detected by the LDA-based QSAR models (Eq. 13 and Eq. 14, correspondingly) as trichomonacidal. Finally, new lead trichomonacidals were discovered by prediction of their antirichomonal activity with obtained models. The most of tested chemicals exhibit the predicted antitrichomonal effect in the performed ligand-based virtual screening, yielding an accuracy of the 90.48% (19/21). These results support a role for TOMOCOMD-CARDD descriptors in the biosilico discovery of new compounds.