Structure-based virtual screening and computational study towards identification of novel inhibitors of hypoxanthine-guanine phosphoribosyltransferase of Trypanosoma cruzi.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key regulatory enzyme of the purine salvage pathway present in the members of trypanosomatids. The parasite solely depends on this pathway for the synthesis of nucleotides due to the absence of the de novo pathway. This study intends to identify putative inhibitors towards Trypanosoma cruzi HGPRT (TcHGPRT). Initial virtual screening was performed with substructures of phosphoribosyl pyrophosphate (PRPP), an original substrate of HGPRT. Twenty compounds that had greater binding energy than the substrate was treated as hits and was further screened and narrowed down through induced fit docking which resulted in top five compounds which was distinguished into two groups based on the ligand occupancy within the PRPP binding site of TcHGPRT. Group-I compounds (PubChem CID 130316561 and 134978234) are analogous to PRPP structure with greater occupancy, were preferred over Group-II compounds which had lesser occupancy than the substrate. However, one compound (22404820) among Group II was chosen for further analysis considering its significant electrostatic interactions. Molecular docking studies revealed the requirement of an electronegative moiety like phosphate group to be present in the ligand due to the presence of metal ions in the substrate binding site. The three chosen compounds along with PRPP were subjected to molecular dynamics analysis, which indicated a strong presence of electrostatic interaction. Considering the dynamic stability of interactions as well as pharmacological properties of ligands based on absorption, distribution, metabolism, excretion prediction, Group-I compounds were selected as lead compounds and were subjected to molecular electrostatic potential analysis to determine the charge distribution of the compound. The overall analysis thus suggests both 130316561 and 134978234 can be used as TcHGPRT inhibitors. Furthermore, these computational results emphasize the requirement of phosphorylated ligands which are essential in mediating electrostatic interactions and to compete with the binding affinity of the original substrate.

Structure-Based Virtual Screening of New Benzoic Acid Derivatives as Trypanosoma cruzi Trans-sialidase Inhibitors.

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, represents a worldwide epidemiological, economic, and social problem. In the last decades, the trans-sialidase enzyme of Trypanosoma cruzi has been considered an attractive target for the development of new agents with potential trypanocidal activity. OBJECTIVE: In this work, the aim was to find new potential non-sugar trans-sialidase inhibitors using benzoic acid as a scaffold. METHODS: A structure-based virtual screening of the ZINC15 database was carried out. Additionally, the enzyme and trypanocidal activity of the selected compounds was determined. RESULTS: The results of this work detected 487 compounds derived from benzoic acid as potential transsialidase inhibitors with a more promising binding energy value (< -7.7 kcal/mol) than the known inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). In particular, two lead compounds, V1 and V2, turned out to be promising trans-sialidase inhibitors. Even though the trypanocidal activity displayed was low, these compounds showed trans-sialidase inhibition values of 87.6% and 29.6%, respectively. CONCLUSION: Structure-based virtual screening using a molecular docking approach is a useful method for the identification of new trans-sialidase inhibitors.

A structure-based approach towards the identification of novel antichagasic compounds: Trypanosoma cruzi carbonic anhydrase inhibitors.

Trypanosoma cruzi carbonic anhydrase (TcCA) has recently emerged as an interesting target for the design of new compounds to treat Chagas disease. In this study we report the results of a structure-based virtual screening campaign to identify novel and selective TcCA inhibitors. The combination of properly validated computational methodologies such as comparative modelling, molecular dynamics and docking simulations allowed us to find high potency hits, with KI values in the nanomolar range. The compounds also showed trypanocidal effects against T. cruzi epimastigotes and trypomastigotes. All the candidates are selective for inhibiting TcCA over the human isoform CA II, which is encouraging in terms of possible therapeutic safety and efficacy.

Chemical Constituents of Anacardium occidentale as Inhibitors of Trypanosoma cruzi Sirtuins.

Benznidazole and nifurtimox, the only drugs available for the treatment of Chagas disease, have limited efficacy and have been associated with severe adverse side effects. Thus, there is an urgent need to find new biotargets for the identification of novel bioactive compounds against the parasite and with low toxicity. Silent information regulator 2 (Sir2) enzymes, or sirtuins, have emerged as attractive targets for the development of novel antitrypanosomatid agents. In the present work, we evaluated the inhibitory effect of natural compounds isolated from cashew nut (Anacardium occidentale, L. Anacardiaceae) against the target enzymes TcSir2rp1 and TcSir2rp3 as well as the parasite. Two derivates of cardol (1, 2), cardanol (3, 4), and anacardic acid (5, 6) were investigated. The two anacardic acids (5, 6) inhibited both TcSir2rp1 and TcSir2rp3, while the cardol compound (2) inhibited only TcSir2rp1. The most potent sirtuin inhibitor active against the parasite was the cardol compound (2), with an EC50 value of 12.25 µM, similar to that of benznidazole. Additionally, compounds (1, 4), which were inactive against the sirtuin targets, presented anti-T. cruzi effects. In conclusion, our results showed the potential of Anacardium occidentale compounds for the development of potential sirtuin inhibitors and anti-Trypanosoma cruzi agents.

In silico identification and evaluation of new Trypanosoma cruzi trypanothione reductase (TcTR) inhibitors obtained from natural products database of the Bahia semi-arid region (NatProDB).

Trypanosoma cruzi Trypanothione Reductase (TcTR) is one of the therapeutic targets studied in the development of new drugs against Chagas' disease. Due to its biodiversity, Brazil has several compounds of natural origin that were not yet properly explored in drug discovery. Therefore, we employed the Virtual Screening against TcTR aiming to discover new inhibitors from the Natural Products Database of the Bahia Semi-Arid region (NatProDB). This database has a wide chemical diversity favoring the discovery of new chemical entities. Subsequently, we analyzed the best docking conformations using self-organizing maps (AuPosSOM) aiming to verify their interaction sites at TcTR. Finally, the Pred-hERG, the Aggregator Advisor, the FAF-DRUGS and the pkCSM results allowed us to evaluate, respectively, the cardiotoxicity, aggregation capacity, presence of false positives (PAINS) and its toxicity. Thus, we selected three molecules that could be tested in in vitro assays in the hope that the computational results reported here would favor the development of new anti-chagasic drugs.

Identification of potential trypanothione reductase inhibitors among commercially available ß-carboline derivatives using chemical space, lead-like and drug-like filters, pharmacophore models and molecular docking.

American trypanosomiasis or Chagas disease caused by the protozoan Trypanosoma cruzi (T. cruzi) is an important endemic trypanosomiasis in Central and South America. This disease was considered to be a priority in the global plan to combat neglected tropical diseases, 2008-2015, which indicates that there is an urgent need to develop more effective drugs. The development of new chemotherapeutic agents against Chagas disease can be related to an important biochemical feature of T. cruzi: its redox defense system. This system is based on trypanothione ([Formula: see text],[Formula: see text]-bis(glutathyonil)spermidine) and trypanothione reductase (TR), which are rather unique to trypanosomes and completely absent in mammalian cells. In this regard, tricyclic compounds have been studied extensively due to their ability to inhibit the T. cruzi TR. However, synthetic derivatives of natural products, such as [Formula: see text]-carboline derivatives ([Formula: see text]-CDs), as potential TR inhibitors, has received little attention. This study presents an analysis of the structural and physicochemical properties of commercially available [Formula: see text]-CDs in relation to compounds tested against T. cruzi in previously reported enzymatic assays and shows that [Formula: see text]-CDs cover chemical space that has not been considered for the design of TR inhibitors. Moreover, this study presents a ligand-based approach to discover potential TR inhibitors among commercially available [Formula: see text]-CDs, which could lead to the generation of promising [Formula: see text]-CD candidates.

Trypanocidal Effect of Isotretinoin through the Inhibition of Polyamine and Amino Acid Transporters in Trypanosoma cruzi.

Polyamines are essential compounds to all living organisms and in the specific case of Trypanosoma cruzi, the causative agent of Chagas disease, they are exclusively obtained through transport processes since this parasite is auxotrophic for polyamines. Previous works reported that retinol acetate inhibits Leishmania growth and decreases its intracellular polyamine concentration. The present work describes a combined strategy of drug repositioning by virtual screening followed by in vitro assays to find drugs able to inhibit TcPAT12, the only polyamine transporter described in T. cruzi. After a screening of 3000 FDA-approved drugs, 7 retinoids with medical use were retrieved and used for molecular docking assays with TcPAT12. From the docked molecules, isotretinoin, a well-known drug used for acne treatment, showed the best interaction score with TcPAT12 and was selected for further in vitro studies. Isotretinoin inhibited the polyamine transport, as well as other amino acid transporters from the same protein family (TcAAAP), with calculated IC50 values in the range of 4.6-10.3 µM. It also showed a strong inhibition of trypomastigote burst from infected cells, with calculated IC50 of 130 nM (SI = 920) being significantly less effective on the epimastigote stage (IC50 = 30.6 µM). The effect of isotretinoin on the parasites plasma membrane permeability and on mammalian cell viability was tested, and no change was observed. Autophagosomes and apoptotic bodies were detected as part of the mechanisms of isotretinoin-induced death indicating that the inhibition of transporters by isotretinoin causes nutrient starvation that triggers autophagic and apoptotic processes. In conclusion, isotretinoin is a promising trypanocidal drug since it is a multi-target inhibitor of essential metabolites transporters, in addition to being an FDA-approved drug largely used in humans, which could reduce significantly the requirements for its possible application in the treatment of Chagas disease.

Synthesis and biological evaluation of potential inhibitors of the cysteine proteases cruzain and rhodesain designed by molecular simplification.

Analogues of 8-chloro-N-(3-morpholinopropyl)-5H-pyrimido[5,4-b]indol-4-amine 1, a known cruzain inhibitor, were synthesized using a molecular simplification strategy. Five series of analogues were obtained: indole, pyrimidine, quinoline, aniline and pyrrole derivatives. The activity of the compounds was evaluated against the enzymes cruzain and rhodesain as well as against Trypanosoma cruzi amastigote and trypomastigote forms. The 4-aminoquinoline derivatives showed promising activity against both enzymes, with IC50 values ranging from 15 to 125µM. These derivatives were selective inhibitors for the parasitic proteases, being unable to inhibit mammalian cathepsins B and S. The most active compound against cruzain (compound 5a; IC50=15µM) is considerably more synthetically accessible than 1, while retaining its ligand efficiency. As observed for the original lead, compound 5a was shown to be a competitive enzyme inhibitor. In addition, it was also active against T. cruzi (IC50=67.7µM). Interestingly, the pyrimidine derivative 4b, although inactive in enzymatic assays, was highly active against T. cruzi (IC50=3.1µM) with remarkable selectivity index (SI=128) compared to uninfected fibroblasts. Both 5a and 4b exhibit drug-like physicochemical properties and are predicted to have a favorable ADME profile, therefore having great potential as candidates for lead optimization in the search for new drugs to treat Chagas disease.

Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.

BACKGROUND: The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate. METHODOLOGY/PRINCIPAL FINDING: A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low µM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 µM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds. CONCLUSIONS/SIGNIFICANCE: Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors.

Molecular Docking and Binding Mode Analysis of Plant Alkaloids as in vitro and in silico Inhibitors of Trypanothione Reductase from Trypanosoma cruzi.

Trypanothione reductase (TryR) is a key enzyme in the metabolism of Trypanosoma cruzi, the parasite responsible for Chagas disease. The available repertoire of TryR inhibitors relies heavily on synthetic substrates of limited structural diversity, and less on plant-derived natural products. In this study, a molecular docking procedure using a Lamarckian Genetic Algorithm was implemented to examine the protein-ligand binding interactions of strong in vitro inhibitors for which no X-ray data is available. In addition, a small, skeletally diverse, set of natural alkaloids was assessed computationally against T. cruzi TryR in search of new scaffolds for lead development. The preferential binding mode (low number of clusters, high cluster population), together with the deduced binding interactions were used to discriminate among the virtual inhibitors. This study confirms the prior in vitro data and proposes quebrachamine, cephalotaxine, cryptolepine, (22S,25S)-tomatidine, (22R,25S)-solanidine, and (22R,25R)-solasodine as new alkaloid scaffold leads in the search for more potent and selective TryR inhibitors.

Development of a Fluorescence-based Trypanosoma cruzi CYP51 Inhibition Assay for Effective Compound Triaging in Drug Discovery Programmes for Chagas Disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life threatening global health problem with only two drugs available for treatment (benznidazole and nifurtimox), both having variable efficacy in the chronic stage of the disease and high rates of adverse drug reactions. Inhibitors of sterol 14α-demethylase (CYP51) have proven effective against T. cruzi in vitro and in vivo in animal models of Chagas disease. Consequently two azole inhibitors of CYP51 (posaconazole and ravuconazole) have recently entered clinical development by the Drugs for Neglected Diseases initiative. Further new drug treatments for this disease are however still urgently required, particularly having a different mode of action to CYP51 in order to balance the overall risk in the drug discovery portfolio. This need has now been further strengthened by the very recent reports of treatment failure in the clinic for both posaconazole and ravuconazole. To this end and to prevent enrichment of drug candidates against a single target, there is a clear need for a robust high throughput assay for CYP51 inhibition in order to evaluate compounds active against T. cruzi arising from phenotypic screens. A high throughput fluorescence based functional assay using recombinantly expressed T. cruzi CYP51 (Tulahuen strain) is presented here that meets this requirement. This assay has proved valuable in prioritising medicinal chemistry resource on only those T. cruzi active series arising from a phenotypic screening campaign where it is clear that the predominant mode of action is likely not via inhibition of CYP51.

Different Therapeutic Outcomes of Benznidazole and VNI Treatments in Different Genders in Mouse Experimental Models of Trypanosoma cruzi Infection.

The lack of translation between preclinical assays and clinical trials for novel therapies for Chagas disease (CD) indicates a need for more feasible and standardized protocols and experimental models. Here, we investigated the effects of treatment with benznidazole (Bz) and with the potent experimental T. cruzi CYP51 inhibitor VNI in mouse models of Chagas disease by using different animal genders and parasite strains and employing distinct types of therapeutic schemes. Our findings confirm that female mice are less vulnerable to the infection than males, show that male models are less susceptible to treatment with both Bz and VNI, and thus suggest that male models are much more suitable for selection of the most promising antichagasic agents. Additionally, we have found that preventive protocols (compound given at 1 dpi) result in higher treatment success rates, which also should be avoided during advanced steps of in vivo trials of novel anti-T. cruzi drug candidates. Another consideration is the relevance of immunosuppression methods in order to verify the therapeutic profile of novel compounds, besides the usefulness of molecular diagnostic tools (quantitative PCR) to ascertain compound efficacy in experimental animals. Our study aims to contribute to the development of more reliable methods and decision gates for in vivo assays of novel antiparasitic compounds in order to move them from preclinical to clinical trials for CD.

Computational and Investigative Study of Flavonoids Active Against Typanosoma cruzi and Leishmania spp.

Flavonoid compounds active against Trypanosoma cruzi and Leishmania species were submitted to several methodologies in silico: docking with the enzymes cruzain and trypanothione reductase (from T. cruzi), and N-myristoyltransferase, dihydroorotate dehydrogenase, and trypanothiona reductase (from Leishmania spp). Molecular maps of the complexes and the ligands were calculated. In order to compare and evaluate the antioxidant activity of the flavonoids with their antiprotozoal activity, quantum parameters were calculated. Considering the energies, interactions, and hydrophobic surfaces calculated, the flavonoids chrysin dimethyl ether against T. cruzi, and ladanein against Leishmania sp. presented the best results. The antioxidant activity did not show any correlation with anti-parasitic activity; only chrysin and its dimethyl ether showed favorable anti-parasitic results. This study hopes to contribute to existing research on these natural products against these tropical parasites.

Trypanothione reductase: a target protein for a combined in vitro and in silico screening approach.

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC50 values of <1 µM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC50 values down to 2 µM.

A novel role of Rab11 in trafficking GPI-anchored trans-sialidase to the plasma membrane of Trypanosoma cruzi.

Trypanosoma cruzi, the causative agent of Chagas disease, is a unicellular parasite that possesses a contractile vacuole complex (CVC). This organelle is usually present in free-living protists and is mainly involved in osmoregulation. However, in some organisms, like for example Dictyostelium discoideum, other roles include calcium homeostasis and transference of proteins to the plasma membrane. T. cruzi plasma membrane is very rich in glycosylphosphatidylinositol anchored proteins (GPI-AP) and a very important group of GPI-AP is that of the trans-sialidases. These enzymes catalyze the transfer of sialic acid from host glycoconjugates to mucins present in the surface of the parasite and are important for host cell invasion among other functions. We recently reported that a pathway dependent on the Rab GTPase Rab11 is involved in the traffic of trans-sialidases to the plasma membrane through the CVC of the infective stages of the parasite and that preventing this traffic results in considerable reduction in the ability of T. cruzi to infect host cells. We also found that traffic of other GPI-anchored proteins is also through the CVC but uses a Rab11-independent pathway. These represent unconventional pathways of GPI-anchored protein traffic to the plasma membrane.

Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits.

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization.

Virtual screening strategies in medicinal chemistry: the state of the art and current challenges.

Virtual screening (VS) techniques are well-established tools in the modern drug discovery process, mainly used for hit finding in drug discovery. The availability of knowledge of structural information, which includes an increasing number of 3D protein structures and the readiness of free databases of commercially available smallmolecules, provides a broad platform for VS. This review summarizes the current developments in VS regarding chemical databases and highlights the achievements as well as the challenges with an emphasis on a recent example of the successful application for the identification of new hits for sterol 14α-demethylase (CYP51) of Trypanosoma cruzi.

Squalene synthase as a target for Chagas disease therapeutics.

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

Scorpiand-like azamacrocycles prevent the chronic establishment of Trypanosoma cruzi in a murine model.

Chagas disease is today one of the most important neglected diseases for its upcoming expansion to non-endemic areas and has become a threat to blood recipients in many countries. In this study, the trypanocidal activity of ten derivatives of a family of aza-scorpiand like macrocycles is evaluated against Trypanosoma cruzi in vitro and in vivo murine model in which the acute and chronic phases of Chagas disease were analyzed. The compounds 4, 3 and 1 were found to be more active against the parasite and less toxic against Vero cells than the reference drug benznidazole, 4 being the most active compound, particularly in the chronic phase. While all these compounds showed a remarkable degree of inhibition of the Fe-SOD enzyme of the epimastigote forms of T. cruzi, they produced a negligible inhibition of human CuZn-SOD and Mn-SOD from Escherichia coli. The modifications observed by (1)H NMR and the amounts of excreted catabolites by the parasites after treatment suggested that the mechanism of action could be based on interactions of the side chains of the compounds with enzymes of the parasite metabolism. The ultrastructural alterations observed in treated epimastigote forms confirmed that the compounds having the highest activity are those causing the largest cell damage. A complementary histopathological analysis confirmed that the compounds tested were significantly less toxic to mammals than the reference drug.

Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i) in the low micromolar range (3-60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol(-1) atom(-1) (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.