article: 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 (Ki) 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.
  Chagas disease, widespread in Latin America, is caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. Despite efforts to reduce the transmission of the parasite by controlling the hematophagous triatomine insect vector, the World Health Organization estimates that 10 million people are infected worldwide, with another 25 million at risk. Most cases are in Latin America, where Chagas disease is endemic, but it is also found in Canada, the United States, Europe (mainly in Spain and Portugal), Japan and Australia [1]–[3]. T. cruzi' s complex life cycle involves two replicative forms: the epimastigote, in the gut of the insect vector, and the amastigote, an intracellular form in the infected mammal. The two infective non-replicative forms are the metacyclic trypomastigote in the insect vector and the bloodstream trypomastigote released from infected cells into the blood of the mammal [4]. Chagas disease has an acute phase and a chronic latent phase. The acute phase, which occurs shortly after infection, lasts for a few weeks or months, whereas the chronic phase develops over many years. The acute phase may not be noticed because it is symptom-free or exhibits only mild symptoms that are not unique to Chagas disease. These can include fever, fatigue, body aches, headache, and rash, loss of appetite, diarrhea and vomiting. Chronic phase symptoms appear between 10 and 20 years after infection and affect the heart, nervous and digestive systems. The treatment of Chagas disease involves the front-line drugs nifurtimox and benznidazole. These two old drugs are effective at curing the infection mostly in the acute phase, with successful cure up to 80%, but are almost ineffective in chronically infected patients [5]. Moreover, due to its collateral effects nifurtimox is no longer available in most Latin American countries. In addition to the severe side effects of the available chemotherapy, drug-resistance has been observed in some trypanosome strains. Thus, the discovery of new, safer and more effective drugs to treat Chagas disease is of utmost importance. Cruzain (a recombinant form of cruzipain, EC 3. 4. 22. 51) has excellent pre-clinical validation evidence as a druggable target. Cruzain belongs to the family of cysteine proteases (papain-like enzymes known as clan CA) and is closely related to cathepsins L and S, which are also associated with other pathologies in humans [6]. It is the major cysteine protease in T. cruzi and is essential for the development and survival of the parasite within the host cells. Numerous protease inhibitors with different scaffolds and catalytic mechanisms show activity against the parasite in culture and animal models of the disease [7]. Clan CA cysteine proteases are effectively inhibited by several classes of peptide inhibitors including transition state-based, reversible and irreversible inhibitors [8]. Examples of reversible transition state-based inhibitors are peptide aldehydes, α-diketones, α-ketoesters, α-ketoamides and α-ketoacids [9]. Clan CA proteases are also irreversibly inhibited by peptidyldiazomethyl ketones, fluoromethyl ketones, peptide epoxides (E-64, E-64-c, E-64-d) and vinyl sulfones [10]. Recently, non-covalent inhibitors have been discovered through high-throughput screening (HTS) platforms and, despite their lower potency relative to previously reported covalent compounds, they represent important breakthroughs in the development of non-peptidic compounds with drug-like features [11], [12]. A promising molecular class acting with antiparasitic activity can be found in vinyl sulfones. In pre-clinical trials, the inhibitor K11777 (Figure 1A) has been shown to be non-mutagenic, well tolerated, to have an acceptable pharmacokinetic profile and demonstrated efficacy in models of acute and chronic Chagas disease both in mice and dogs [13]. Additional studies of vinyl sulfone compounds have led to the identification of an arginine variant of K11777, named WRR-483 (Figure 1B) with remarkable biological properties [14]. The aim of this study was to identify new molecular classes of cruzain inhibitors by focusing on non-peptidic non-covalent ligands. To this end, we have carried out virtual screening of the ZINC Database [15], a free database of commercially-available compounds for virtual screening, utilizing ligand- and target-based virtual screening methods [16], [17], followed by enzymatic assays, X-ray crystallography and SAR studies of the most promising hits. Of nine cruzain inhibitors, five show trypanocidal activity against the trypomastigote infective form of the Tulahuen lacZ strain. We also expect that a newly identified fragment of the 2-acetamidothiophene-3-carboxamide class can advance the search for new non-covalent cruzain inhibitors.
  A variety of methods are available to virtually screen small organic compound databases. A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was applied as illustrated in Figure 2. The Ethics Committee on Animal Experimentation of the Faculty of Medicine of Ribeirão Preto – University of Sao Paulo approved the cytotoxicity assays (approval no. 076/2010). This Committee adheres to Conselho Nacional de Controle de Experimentação Animal – CONCEA, created by Brazilian Law number 11794 of 8 October 2008. Assays were run according to the guidelines of the Ministry of Science, Technology and Innovation of Brazil.
  Chagas disease is a neglected trypanosomiasis with enormous social and economic impact in most countries of Latin America. It is of utmost importance to develop new and more effective drugs with fewer side effects than the currently available chemotherapy. Hitherto, significant efforts have been made focusing on cruzain enzyme as a promising target and compound K11777, a cruzain inhibitor set to enter clinical studies as a new antichagasic drug. Here, we successfully used integrated in silico and in vitro approaches, with X-ray crystallography as an orthogonal tool, to discover new non-peptidic hits with trypanocidal activity against cruzain. Thus, we identified new trypanocidal agents that bear the 2-acetamidothiophene-3-carboxamide as the group responsible for enzyme inhibition and trypanocidal activity. The 2-acetamidothiophene-3-carboxamide binds non-covalently to cruzain, does not violate the rule of five and actually is a fragment with proper ligand efficiency (0. 33 kcal mol−1 atom−1), with a low molecular mass (283. 3 g mol−1) and CLogP of 0. 7, properties that illuminate the way ahead for maneuvering toward a lead-like molecule. In summary, we present a new hit, 2-acetamidothiophene-3-carboxamide, that non-covalently inhibits cruzain, has trypanocidal activity and manageable structure-activity and structure-toxicity relationships. We anticipate that this compound will advance the lead optimization process for Chagas disease chemotherapy. Detailed information on cruzain EC 3. 4. 22. 51 (also known as cruzipain) can be found at http: //www. brenda-enzymes. org/php/result_flat. php4? ecno=3. 4. 22. 51&Suchword=&organism%5B%5D=Trypanosomacruzi&show_tm=0 and also at https: //www. ebi. ac. uk/chembldb/target/inspect/CHEMBL3563. For Cathepsin L, this site is also of interest: https: //www. ebi. ac. uk/chembldb/target/inspect/CHEMBL3837.
summary: Chagas disease (American trypanosomiasis) is a parasitic infection that kills millions of mostly poverty-stricken people in Latin America. In recent years it has also spread to nonendemic countries – the United States, Canada, Europe, Australia and Japan – as a result of immigration. The only available drugs for its treatment were introduced more than forty years ago, have low efficacy, and cause various severe side effects. This dire public health situation has prompted us to search for new small molecules to act as drug candidates to treat Chagas disease. The T. cruzi enzyme cruzain, a key biological catalyst used by the protozoan to digest host proteins, is a validated drug target for Chagas disease. By combining in silico molecular design, X-ray crystallography and biological screening, we found a new class of non-covalent small molecules that inhibit cruzain in low micromolar concentrations.
keywords: physics
  biochemistry
  medicinal chemistry
  organic chemistry
  x-rays
  chemistry
  biology
  computational chemistry
  organic compounds
  electromagnetic radiation
  drug discovery
title: Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by Virtual Screening and In Vitro Assay