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A infecção por Ascaris lumbricoides decorre da ingestão de ovos embrionados deste parasita, o que justifica a pesquisa de substâncias que tenham efeito deletério sobre estes ovos. Nosso objetivo foi estudar a ação de 16 produtos detergentes e desinfetantes, de uso doméstico e laboratorial, sobre a embriogênese deste helminto. Crianças portadoras desta infecção foram tratadas com levamisol e os vermes fêmeas expelidos foram recolhidos e dissecados, para obtenção dos ovos intra-uterinos. Os ovos foram postos em contato com os produtos em diversas diluições e tempos, lavados e incubados a 28masculineC, por 20 dias, para teste da viabilidade e determinação da porcentagem de embrionamento. Apenas um produto inibiu completamente o embrionamento dos ovos, em todos os tempos e diluições testados. Cinco produtos inibiram o embrionamento dos ovos em mais de 50%, seis inibiram o embrionamento em menos de 50% e três não tiveram efeito sobre o embrionamento dos ovos. Por outro lado, com um produto observou-se aumento da porcentagem de embrionamento dos ovos em relação aos controles.

Cruzipain (CZP), the major cysteine protease present in T. cruzi, the ethiological agent of Chagas disease, has attracted particular attention as a therapeutic target for the development of targeted covalent inhibitors (TCI). The vast chemical space associated with the enormous molecular diversity feasible to explore by means of modern synthetic approaches allows the design of CZP inhibitors capable of exhibiting not only an efficient enzyme inhibition but also an adequate translation to anti-T. cruzi activity. In this work, a computer-aided design strategy was developed to combinatorially construct and screen large libraries of 1,4-disubstituted 1,2,3-triazole analogues, further identifying a selected set of candidates for advancement towards synthetic and biological activity evaluation stages. In this way, a virtual molecular library comprising more than 75 thousand diverse and synthetically feasible analogues was studied by means of molecular docking and molecular dynamic simulations in the search of potential TCI of CZP, guiding the synthetic efforts towards a subset of 48 candidates. These were synthesized by applying a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) centered synthetic scheme, resulting in moderate to good yields and leading to the identification of 12 hits selectively inhibiting CZP activity with IC50 in the low micromolar range. Furthermore, four triazole derivatives showed good anti-T. cruzi inhibition when studied at 50 μ M; and Ald-6 excelled for its high antitrypanocidal activity and low cytotoxicity, exhibiting complete in vitro biological activity translation from CZP to T. cruzi. Overall, not only Ald-6 merits further advancement to preclinical in vivo studies, but these findings also shed light on a valuable chemical space where molecular diversity might be explored in the search for efficient triazole-based antichagasic agents. [ABSTRACT FROM AUTHOR]

Chagas disease and Human African Trypanosomiasis, caused by Trypanosoma cruzi and T. brucei, respectively, pose relevant health challenges throughout the world, placing 65 to 70 million people at risk each. Given the limited efficacy and severe side effects associated with current chemotherapy, new drugs are urgently needed for both diseases. Here, we report the screening of the Pathogen Box collection against cruzain and TbrCatL, validated targets for Chagas disease and Human African Trypanosomiasis, respectively. Enzymatic assays were applied to screen 400 compounds, validate hits, determine IC50 values and, when possible, mechanisms of inhibition. In this case, 12 initial hits were obtained and ten were prioritized for follow-up. IC50 values were obtained for six of them (hit rate = 1.5%) and ranged from 0.46 ± 0.03 to 27 ± 3 µM. MMV687246 was found to be a mixed inhibitor of cruzain (Ki = 57 ± 6 µM) while MMV688179 was found to be a competitive inhibitor of cruzain with a nanomolar potency (Ki = 165 ± 63 nM). A putative binding mode for MMV688179 was obtained by docking. The six hits discovered against cruzain and TbrCatL are of great interest for further optimization by the medicinal chemistry community. [ABSTRACT FROM AUTHOR]

The cysteine protease cruzain is a Chagas disease target, exploited in computational studies. However, there is no consensus on the protonation states of the active site residues Cys25, His162, and Glu208 at the enzyme's active pH range. We evaluated the impact of different protonation states of these residues on docking calculations. Through a retrospective study with cruzain inhibitors and decoys, we compared the performance of virtual screening using four grids, varying protonation states of Cys25, His162, and Glu208. Based on enrichment factors and ROC plots, docking with the four grids affected compound ranking and the overall charge of top‐ranking compounds. Different grids can be complementary and synergistic, increasing the odds of finding different ligands with diverse chemical properties. [ABSTRACT FROM AUTHOR]

Cruzipains are the main papain-like cysteine proteases of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. Encoded by a multigenic family, previous studies have estimated the presence of dozens of copies spread over multiple chromosomes in different parasite strains. Here, we describe the complete gene repertoire of cruzipain in three parasite strains, their genomic organization, and expression pattern throughout the parasite life cycle. Furthermore, we have analyzed primary sequence variations among distinct family members as well as structural differences between the main groups of cruzipains. Based on phylogenetic inferences and residue positions crucial for enzyme function and specificity, we propose the classification of cruzipains into two families (I and II), whose genes are distributed in two or three separate clusters in the parasite genome, according with the strain. Family I comprises nearly identical copies to the previously characterized cruzipain 1/cruzain, whereas Family II encompasses three structurally distinct sub-types, named cruzipain 2, cruzipain 3, and cruzipain 4. RNA-seq data derived from the CL Brener strain indicates that Family I genes are mainly expressed by epimastigotes, whereas trypomastigotes mainly express Family II genes. Significant differences in the active sites among the enzyme sub-types were also identified, which may play a role in their substrate selectivity and impact their inhibition by small molecules. [ABSTRACT FROM AUTHOR]

Chagas disease, which is caused by Trypanosoma cruzi, affects more than six million people worldwide. Cruzain is the major cysteine protease involved in the survival of this parasite. Here, the expression, purification and crystallization of this enzyme are reported. The cruzain crystals diffracted to 1.2 Å resolution, yielding two novel cruzain structures: apocruzain and cruzain bound to the reversible covalent inhibitor S‐methyl thiomethanesulfonate. Mass‐spectrometric experiments confirmed the presence of a methylthiol group attached to the catalytic cysteine. Comparison of these structures with previously published structures indicates the rigidity of the cruzain structure. These results provide further structural information about the enzyme and may help in new in silico studies to identify or optimize novel prototypes of cruzain inhibitors. [ABSTRACT FROM AUTHOR]

Background: Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation and decreased production of meat, wool, and milk. Current diagnosis or treatment protocols are not fully effective and, thus, require further research of Cp pathogenesis. Results: Here, we mapped known protein-protein interactions (PPI) from various species to nine Cp strains to reconstruct parts of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous. Conclusions: The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing according wet-lab studies. The non-host homologous essential proteins are attractive targets for therapeutic and diagnostic proposes. They allow for searching of small molecule inhibitors of binding interactions enabling modern drug discovery. Overall, the predicted Cp PPI networks form a valuable and versatile tool for researchers interested in Corynebacterium pseudotuberculosis. [ABSTRACT FROM AUTHOR]

Background: The bacterium Corynebacterium pseudotuberculosis (Cp) causes caseous lymphadenitis (CLA), mastitis, ulcerative lymphangitis, and oedema in a number of hosts, comprising ruminants, thereby intimidating economic and dairy industries worldwide. So far there is no effective drug or vaccine available against Cp. Previously, a pan-genomic analysis was performed for both biovar equi and biovar ovis and a Pathogenicity Islands (PAIS) analysis within the strains highlighted a large set of proteins that could be relevant therapeutic targets for controlling the onset of CLA. In the present work, a structural druggability analysis pipeline was accomplished along 15 previously sequenced Cp strains from both biovar equi and biovar ovis. Methods and results: We computed the whole modelome of a reference strain Cp1002 (NCBI Accession: NC_017300.1) and then the homology models of proteins, of 14 different Cp strains, with high identity (≥ 85%) to the reference strain were also done. Druggability score of all proteins pockets was calculated and only those targets that have a highly druggable (HD) pocket in all strains were kept, a set of 58 proteins. Finally, this information was merged with the previous PAIS analysis giving two possible highly relevant targets to conduct drug discovery projects. Also, off-targeting information against host organisms, including Homo sapiens and a further analysis for protein essentiality provided a final set of 31 druggable, essential and non-host homologous targets, tabulated in table S4, additional file 1. Out of 31 globally druggable targets, 9 targets have already been reported in other pathogenic microorganisms, 3 of them (3-isopropylmalate dehydratase small subunit, 50S ribosomal protein L30, Chromosomal replication initiator protein DnaA) in C. pseudotuberculosis. Conclusion: Overall we provide valuable information of possible targets against C. pseudotuberculosis where some of these targets have already been reported in other microorganisms for drug discovery projects, also discarding targets that might be physiologically relevant but are not amenable for drug binding. We propose that the [ABSTRACT FROM AUTHOR]

Abstract The Serine/arginine-rich protein kinases (SRPK) are involved in pre-mRNA splicing control through the phosphorylation of the SR protein family of splicing factors. Over the last years, several studies have shown the relevance of SRPK for human cancers and their potential as promising drug targets. In this context, we have previously selected three trifluoromethyl arylamides (named here as SRVIC24, SRVIC30 and SRVIC36) with improved in vitro antileukemia effect and ability of impairing the cellular activity of SRPK. Given the increasing amount of reports on the implication of these kinases in metastatic cancers, in this study, we have evaluated the antimetastatic effect of these compounds and the known SRPK inhibitor (SRPIN340) on a murine model of metastatic melanoma. The compounds were able to impact the melanoma cell metastatic behavior by decreasing migration, invasion, adhesion, and colony formation in in vitro assays. Also, they presented antimetastatic in vivo activity, without apparent signs of systemic toxicity after treatments, as revealed by the histology of organs and analysis of key serum biochemical markers. Moreover, the effect of the treatments on SRPK1 nuclear translocation and SR protein phosphorylation was observed. Finally, molecular docking studies were carried out to gain structural information on the SRPK-compound complexes. Together, these data suggest that SRPK pharmacological inhibition should be considered as an interesting therapeutic strategy against metastatic cancers. Graphical abstract Unlabelled Image Highlights • Trifluoromethyl arylamides exhibit antimetastatic activity in vitro and in vivo. • The compounds impair SRPK cellular activity. • Molecular docking afforded structural information on the SRPK-compound complexes. [ABSTRACT FROM AUTHOR]

Introduction: Benznidazole, the drug of choice for treating Chagas Disease (CD), has significant limitations, such as poor cure efficacy, mainly in the chronic phase of CD, association with side effects, and parasite resistance. Understanding parasite resistance to benznidazole is crucial for developing new drugs to treat CD., Areas Covered: Here, the authors review the current understanding of the molecular basis of benznidazole resistance. Furthermore, they discuss the state-of-the-art methods and critical outcomes employed to evaluate the efficacy of potential drugs against T. cruzi , aiming to select better compounds likely to succeed in the clinic. Finally, the authors describe the different strategies employed to overcome resistance to benznidazole and find effective new treatments for CD., Expert Opinion: Resistance to benznidazole is a complex phenomenon that occurs naturally among T. cruzi strains. The combination of compounds that inhibit different metabolic pathways of the parasite is an important strategy for developing a new chemotherapeutic protocol.

Around three billion people are at risk of infection by the dengue virus (DENV) and potentially other flaviviruses. Worldwide outbreaks of DENV, Zika virus (ZIKV), and yellow fever virus (YFV), the lack of antiviral drugs, and limitations on vaccine usage emphasize the need for novel antiviral research. Here, we propose a consensus virtual screening approach to discover potential protease inhibitors (NS3 pro ) against different flavivirus. We employed an in silico combination of a hologram quantitative structure-activity relationship (HQSAR) model and molecular docking on characterized binding sites followed by molecular dynamics (MD) simulations, which filtered a data set of 7.6 million compounds to 2,775 hits. Lastly, docking and MD simulations selected six final potential NS3 pro inhibitors with stable interactions along the simulations. Five compounds had their antiviral activity confirmed against ZIKV, YFV, DENV-2, and DENV-3 (ranging from 4.21 ± 0.14 to 37.51 ± 0.8 μM), displaying aggregator characteristics for enzymatic inhibition against ZIKV NS3 pro (ranging from 28 ± 7 to 70 ± 7 μM). Taken together, the compounds identified in this approach may contribute to the design of promising candidates to treat different flavivirus infections.

Introduction: Chagas disease (CD) imposes social and economic burdens, yet the available treatments have limited efficacy in the disease's chronic phase and cause serious adverse effects. To address this challenge, target-based approaches are a possible strategy to develop new, safe, and active treatments for both phases of the disease., Areas Covered: This review delves into target-based approaches applied to CD drug discovery, emphasizing the studies from the last five years. We highlight the proteins cruzain (CZ), trypanothione reductase (TR), sterol 14 α-demethylase (CPY51), iron superoxide dismutase (Fe-SOD), proteasome, cytochrome b (Cyt b) , and cleavage and polyadenylation specificity factor 3 (CPSF3), chosen based on their biological and chemical validation as drug targets. For each, we discuss its biological relevance and validation as a target, currently related challenges, and the status of the most promising inhibitors., Expert Opinion: Target-based approaches toward developing potential CD therapeutics have yielded promising leads in recent years. We expect a significant advance in this field in the next decade, fueled by the new options for Trypanosoma cruzi genetic manipulation that arose in the past decade, combined with recent advances in computational chemistry and chemical biology.

The need to develop safer and more efficacious drugs to treat Chagas disease has motivated the search for cruzain inhibitors. Cruzain is the recombinant, truncated version of cruzipain, a cysteine protease from Trypanosoma cruzi with important roles during the parasite life cycle. Several computational techniques have been applied to discover and optimise cruzain inhibitors, providing a molecular basis to guide this process. Here, we review some of the most recent computational studies that provided important information for the design of cruzain inhibitors. Moreover, we highlight the diversity of applications of in silico techniques and their impact.

Free energy perturbation (FEP) calculations are now routinely used in drug discovery to estimate the relative FEB (RFEB) of small molecules to a biomolecular target of interest. Using enhanced sampling can improve the correlation between predictions and experimental data, especially in systems with conformational changes. Due to the large number of perturbations required in drug discovery campaigns, the manual setup of FEP calculations is no longer viable. Here, we introduce PyAutoFEP, a flexible and open-source tool to aid the setup of RFEB FEP. PyAutoFEP is written in Python3, and automates the generation of perturbation maps, dual topologies, system building and molecular dynamics (MD), and analysis. PyAutoFEP supports multiple force fields, incorporates replica exchange with solute tempering (REST) and replica exchange with solute scaling (REST2) enhanced sampling methods, and allows flexible λ values along perturbation windows. To validate PyAutoFEP, it was applied to a set of 14 Farnesoid X receptor ligands, a system included in the drug design data resource grand challenge 2. An 88% mean correct sign prediction was achieved, and 75% of the predictions had an error below 1.5 kcal/mol. Results using Amber03/GAFF, CHARMM36m/CGenFF, and OPLS-AA/M/LigParGen had Pearson's r values of 0.71 ± 0.13, 0.30 ± 0.27, and 0.66 ± 0.20, respectively. The Amber03/GAFF and OPLS-AA/M/LigParGen results were on par with the top grand challenge 2 submissions. Applying REST2 improved the results using CHARMM36m/CGenFF (Pearson's r = 0.43 ± 0.21) but had little impact on the other force fields. CHARMM36-YF and CHARMM36-WYF modifications did not yield improved predictions compared to CHARMM36m. Finally, we estimated the probability of finding a molecule 1 p K i better than a lead when using PyAutoFEP to screen 10 or 100 analogues. The probabilities, when compared to random sampling, increased up to sevenfold when 100 molecules were to be screened, suggesting that PyAutoFEP would likely be useful for lead optimization. PyAutoFEP is available on GitHub at https://github.com/lmmpf/PyAutoFEP.

Cysteine proteases are essential hydrolytic enzymes present in the majority of organisms, including viruses and unicellular parasites. Despite the high sequence identity displayed among these proteins, specific structural features across different species grant distinct functions to these biomolecules, frequently related to pathological conditions. Consequently, their relevance as promising targets for potential specific inhibitors has been highlighted and occasionally validated in recent decades. In this review, we discuss the recent outcomes of structure-based campaigns aiming the discovery of new inhibitor prototypes against cruzain and falcipain, as alternative therapeutic tools for Chagas disease and malaria treatments, respectively. Computational and synthetic approaches have been combined on hit optimization strategies and are also discussed herein. These rationales are extended to additional tropical infectious and neglected pathologies, such as schistosomiasis, leishmaniasis and babesiosis, and also to Alzheimer's Disease, a widespread neurodegenerative disease poorly managed by currently available drugs and recently linked to particular physiopathological roles of human cysteine proteases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Corynebacterium pseudotuberculosis can be classified into two biovars or biovars based on their nitrate-reducing ability. Strains isolated from sheep and goats show negative nitrate reduction and are termed biovar Ovis, while strains from horse and cattle exhibit positive nitrate reduction and are called biovar Equi. However, molecular evidence has not been established so far to understand this difference, specifically if these C. pseudotuberculosis strains are under an evolutionary process., Results: The ERIC 1 + 2 Minimum-spanning tree from 367 strains of C. pseudotuberculosis showed that the great majority of biovar Ovis strains clustered together, but separately from biovar Equi strains that also clustered amongst themselves. Using evolutionarily conserved genes (rpoB, gapA, fusA, and rsmE) and their corresponding amino acid sequences, we analyzed the phylogenetic relationship among eighteen strains of C. pseudotuberculosis belonging to both biovars Ovis and Equi. Additionally, conserved point mutation based on structural variation analysis was also carried out to elucidate the genotype-phenotype correlations and speciation. We observed that the biovars are different at the molecular phylogenetic level and a probable anagenesis is occurring slowly within the species C. pseudotuberculosis., Conclusions: Taken together the results suggest that biovar Equi is forming the biovar Ovis. However, additional analyses using other genes and other bacterial strains are required to further support our anagenesis hypothesis in C. pseudotuberculosis.

Reverse transcriptase (RT) is a multifunctional enzyme in the human immunodeficiency virus (HIV)-1 life cycle and represents a primary target for drug discovery efforts against HIV-1 infection. Two classes of RT inhibitors, the nucleoside RT inhibitors (NRTIs) and the nonnucleoside transcriptase inhibitors are prominently used in the highly active antiretroviral therapy in combination with other anti-HIV drugs. However, the rapid emergence of drug-resistant viral strains has limited the successful rate of the anti-HIV agents. Computational methods are a significant part of the drug design process and indispensable to study drug resistance. In this review, recent advances in computer-aided drug design for the rational design of new compounds against HIV-1 RT using methods such as molecular docking, molecular dynamics, free energy calculations, quantitative structure-activity relationships, pharmacophore modelling and absorption, distribution, metabolism, excretion and toxicity prediction are discussed. Successful applications of these methodologies are also highlighted.

The pressing need for better drugs against Chagas disease, African sleeping sickness, and schistosomiasis motivates the search for inhibitors of cruzain, rhodesain, and Schistosoma mansoni CB1 (SmCB1), the major cysteine proteases from Trypanosoma cruzi, Trypanosoma brucei, and S. mansoni, respectively. Thiosemicarbazones and heterocyclic analogues have been shown to be both antitrypanocidal and inhibitory against parasite cysteine proteases. A series of compounds was synthesized and evaluated against cruzain, rhodesain, and SmCB1 through biochemical assays to determine their potency and structure-activity relationships (SAR). This approach led to the discovery of 6 rhodesain, 4 cruzain, and 5 SmCB1 inhibitors with 50% inhibitory concentrations (IC50s) of ≤ 10 μM. Among the compounds tested, the thiosemicarbazone derivative of peracetylated galactoside (compound 4i) was discovered to be a potent rhodesain inhibitor (IC50 = 1.2 ± 1.0 μM). The impact of a range of modifications was determined; removal of thiosemicarbazone or its replacement by semicarbazone resulted in virtually inactive compounds, and modifications in the sugar also diminished potency. Compounds were also evaluated in vitro against the parasites T. cruzi, T. brucei, and S. mansoni, revealing active compounds among this series., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The study of protein-protein interactions (PPIs) can help researchers raise new hypotheses about an organism or disease and guide new experiments. Various methods for the identification and analysis of PPIs have been discussed in the literature. These methods are generally categorized as experimental or computational - each having its own advantages and disadvantages. Experimental methods provide insights into the real state of biological interactions but tend to be time-consuming and costly. Computational methods, on the other hand, can study thousands of PPIs at a very low cost and in much less time; however, the accuracy of such in silico prediction results heavily depends on the specific computational approach used. Furthermore, there is no gold standard for these computational methods; a method that works well for predicting one PPI may perform poorly (by generating false positives and false negatives) for a different PPI. Therefore, all such predictions must be carefully validated, preferably with experimental data. In this paper, we review the existing computational approaches and emphasize the use of biological data as inputs for accurate predictions of PPIs. We also discuss how such input datasets and approaches may influence the sensitivity and specificity of the predicted PPI networks.

Corynebacterium pseudotuberculosis (Cp) is a pathogenic bacterium that causes caseous lymphadenitis (CLA), ulcerative lymphangitis, mastitis, and edematous to a broad spectrum of hosts, including ruminants, thereby threatening economic and dairy industries worldwide. Currently there is no effective drug or vaccine available against Cp. To identify new targets, we adopted a novel integrative strategy, which began with the prediction of the modelome (tridimensional protein structures for the proteome of an organism, generated through comparative modeling) for 15 previously sequenced C. pseudotuberculosis strains. This pan-modelomics approach identified a set of 331 conserved proteins having 95-100% intra-species sequence similarity. Next, we combined subtractive proteomics and modelomics to reveal a set of 10 Cp proteins, which may be essential for the bacteria. Of these, 4 proteins (tcsR, mtrA, nrdI, and ispH) were essential and non-host homologs (considering man, horse, cow and sheep as hosts) and satisfied all criteria of being putative targets. Additionally, we subjected these 4 proteins to virtual screening of a drug-like compound library. In all cases, molecules predicted to form favorable interactions and which showed high complementarity to the target were found among the top ranking compounds. The remaining 6 essential proteins (adk, gapA, glyA, fumC, gnd, and aspA) have homologs in the host proteomes. Their active site cavities were compared to the respective cavities in host proteins. We propose that some of these proteins can be selectively targeted using structure-based drug design approaches (SBDD). Our results facilitate the selection of C. pseudotuberculosis putative proteins for developing broad-spectrum novel drugs and vaccines. A few of the targets identified here have been validated in other microorganisms, suggesting that our modelome strategy is effective and can also be applicable to other pathogens.

We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6-24. Compounds with a phenyl at position N3, 15-19, 22-24, exhibited better inhibitory properties for cruzain and against the parasite than 2-iminothiazolidin-4-one 5. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which inhibited the activity of cruzain and the proliferation of epimastigotes and was cidal for trypomastigotes but was not toxic for splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26-45 and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.

Ascaris lumbricoides infection is acquired via ingestion of embryonated eggs of the parasite, thus justifying the search for ovicidal compounds. We studied the effect of 16 household and laboratory detergents and disinfectants on the embryogenesis of this helminth. Children carrying this infection were treated with levamisole. Eliminated female worms were collected and dissected to obtain eggs from the uteri. The eggs were placed in contact with various products at different dilutions and for various periods of time. After washing, eggs were incubated at 28 grades C for 20 days, for viability tests and to determine the embryonation rate. Only one product completely inhibited the embryonation of eggs at every dilution and for every time period tested. Five products inhibited more than 50% of embryonation, while six inhibited less than 50%. Three products showed no effect on embryonation, while for one the percentage of embryonated eggs was higher than in the control tubes.