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Chagas disease is a neglected tropical disease, endemic in Latin America and caused by the protozoan parasite Trypanosoma cruzi. Available treatments show low cure efficacy during the chronic phase of the disease and cause a series of side effects, reinforcing the need to develop new drugs against Chagas disease. In this work, we describe the optimization of a trypanocidal hit compound recently reported in phenotypic high-throughput screening studies against Trypanosoma cruzi. A hit-to-lead process was initiated and a structure-activity relationship against Trypanosoma cruzi was obtained after the synthesis and biological evaluation of 22 new benzenesulfonylpiperazine derivatives. From this structure-activity relationship study, we identified three compounds with a promising predicted ADMET profile and potency comparable to the reference drug benznidazole, which are candidates for further development towards therapies for Chagas disease.

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Cálculos de Perturbações de Energia Livre (Free Energy Perturbation, FEP) são, na atualidade, rotineiramente aplicados na descoberta de fármacos para estimar a energia de Gibbs relativa de ligação de pequenas moléculas a alvos biomoleculares de interesse. A aplicação de técnicas de amostragem estendida pode melhorar a correlação entre predições e dados experimentais, especialmente em sistemas com mudanças conformacionais. Devido ao grande número de perturbações necessárias em uma campanha de descoberta de fármacos, a preparação manual de cálculos de FEP não é viável. Neste trabalho, apresentamos o PyAutoFEP, uma ferramenta flexível e de código aberto para automatizar a preparação de cálculos de FEP para energia de Gibbs relativa de ligação. PyAutoFEP foi escrito em Python3 e automatiza a geração de mapas de perturbação, topologias duplas, construção de sistemas para simulações de Dinâmica molecular e análise. O PyAutoFEP suporta diversos campos de força, incorpora diferentes versões de métodos de amostragem REST2 e esquemas flexíveis de janelas de λ para representar a perturbação. Para validar a ferramenta PyAutoFEP, esta foi aplicada para uma série de 14 ligantes do receptor Farnesóide X. Este sistema fez parte do desafio para a comunidade Grand Challenge 2, organizado pelo grupo Drug Design Data Resources. Uma média de 88% de sinais corretos das predições foi observado e 75% das predições alcançaram erros menores do que 1,5 kcal/mol. Resultados utilizando os campos de força Amber03/GAFF, CHARMM36m/CGenFF e OLSA-AA/M/LigParGen obtiveram r de Pearson de 0,71 ± 0,13, 0,30 ± 0,27 e 0,66 ± 0,20, respectivamente. Os resultados utilizando o Amber03/GAFF e OPSA-AA/M/LigParGen foram comparáveis às melhores submissões originais ao Grand Challenge 2. A aplicação da REST2 melhorou os resultados utilizando o CHARMM36m/CGenFF (r de Pearson = 0,43 ± 0,21), porém pouco impacto foi observado para os outros campos de força. Finalmente, utilizando um modelo estatístico, foi estimada a probabilidade de descoberta de moléculas com potência ao menos uma unidade de 1 pKi melhor do que um protótipo hipotético, quando uma ferramenta como o PyAutoFEP é aplicada para triar 10 ou 100 análogos. As probabilidades são até 7 vezes maiores se comparadas àquela obtida usando seleção aleatória, quando 100 moléculas são triadas, indicando que o PyAutoFEP pode ser útil em problemas de otimização de protótipos. O PyAutoFEP está disponível no repositório do grupo, https://github.com/lmmpf/PyAutoFEP. Free Energy Perturbation (FEP) calculations are now routinely used in drug discovery to estimate the relative free energy of binding (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, 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 two flavors of REST2 enhanced sampling method, 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. A mean 88% 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 OLSA-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 OLSA-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. Finally, we estimated the probability of finding a molecule 1 pKi better than a lead when using PyAutoFEP to screen 10 or 100 analogs. The probabilities, when comparing to random sampling, increased up 7-fold 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.

Aim: Cysteine proteases are important molecular targets involved in the replication, virulence and survival of parasitic organisms, including Trypanosoma and Leishmania species. Methodology & results: Analogs of the 7-chloro- N-[3-(morpholin-4-yl)propyl]quinolin-4-amine were synthesized and their inhibitory activity against the enzymes cruzain and rhodesain as well as against promastigotes forms of Leishmania species and epimastigotes forms of Trypanosoma cruzi were evaluated. Five compounds showed activity against both enzymes with IC50 values ranging from 23 to 123 μM. Among these, compounds 3 and 4 displayed leishmanicidal activity; compound 4 was the most promising with IC50 values

Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC50 = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure–activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.

Background The N-terminal domain of Tetracenomycin aromatase/cyclase (TcmN), an enzyme derived from Streptomyces glaucescens, is involved in polyketide cyclization, aromatization, and folding. Polyketides are a diverse class of secondary metabolites produced by certain groups of bacteria, fungi, and plants with various pharmaceutical applications. Examples include antibiotics, such as tetracycline, and anticancer drugs, such as doxorubicin. Because TcmN is a promising enzyme for in vitro production of polyketides, it is important to identify conditions that enhance its thermal resistance and optimize its function. Methods TcmN unfolding, stability, and dynamics were evaluated by fluorescence spectroscopy, circular dichroism, nuclear magnetic resonance 15N relaxation experiments, and microsecond molecular dynamics (MD) simulations. Results TcmN thermal resistance was enhanced at low protein and high salt concentrations, was pH-dependent, and denaturation was irreversible. Conformational dynamics on the μs-ms timescale were detected for residues in the substrate-binding cavity, and two predominant conformers representing opened and closed cavity states were observed in the MD simulations. Conclusion Based on the results, a mechanism was proposed in which the thermodynamics and kinetics of the TcmN conformational equilibrium modulate enzyme function by favoring ligand binding and avoiding aggregation. General significance Understanding the principles underlying TcmN stability and dynamics may help in designing mutants with optimal properties for biotechnological applications.

Chagas disease, Human African Trypanosomiasis, and schistosomiasis are neglected parasitic diseases for which new treatments are urgently needed. To identify new chemical leads, we screened the 400 compounds of the Open Access Malaria Box against the cysteine proteases, cruzain (Trypanosoma cruzi), rhodesain (Trypanosoma brucei) and SmCB1 (Schistosoma mansoni), which are therapeutic targets for these diseases. Whereas just three hits were observed for SmCB1, 70 compounds inhibited cruzain or rhodesain by at least 50% at 5 μM. Among those, 15 commercially available compounds were selected for confirmatory assays, given their potency, time-dependent inhibition profile and reported activity against parasites. Additional assays led to the confirmation of four novel classes of cruzain and rhodesain inhibitors, with potency in the low-to mid-micromolar range against enzymes and T. cruzi. Assays against mammalian cathepsins S and B revealed inhibitor selectivity for parasitic proteases. For the two competitive inhibitors identified (compounds 7 and 12), their binding mode was predicted by docking, providing a basis for structure-based optimization efforts. Compound 12 also acted directly against the trypomastigote and the intracellular amastigote forms of T. cruzi at 3 μM. Therefore, through a combination of experimental and computational approaches, we report promising hits for optimization in the development of new trypanocidal drugs. A doença de Chagas, a tripanossomíase humana africana e a esquistossomose são doenças parasitárias negligenciadas para as quais são urgentemente necessários novos tratamentos. Para identificar novas pistas químicas, selecionamos os 400 compostos do Open Access Malaria Box contra as proteases de cisteína, cruzaína (Trypanosoma cruzi), rodesaína (Trypanosoma brucei) e SmCB1 (Schistosoma mansoni), que são alvos terapêuticos para essas doenças. Enquanto apenas três acertos foram observados para SmCB1, 70 compostos inibiram cruzaína ou rodesaína em pelo menos 50% a 5μM. Entre esses, 15 compostos disponíveis comercialmente foram selecionados para ensaios confirmatórios, devido à sua potência, perfil de inibição dependente do tempo e atividade relatada contra parasitas. Ensaios adicionais levaram à confirmação de quatro novas classes de inibidores de cruzaína e rodesaína, com potência na faixa de micromolar baixa a média contra enzimas e T. cruzi. Ensaios contra catepsinas S e B de mamíferos revelaram seletividade de inibidores para proteases parasitárias. Para os dois inibidores competitivos identificados (compostos 7 e 12), seu modo de ligação foi previsto por docking, fornecendo uma base para esforços de otimização baseados em estrutura. O composto 12 também agiu diretamente contra as formas tripomastigotas e amastigotas intracelulares de T. cruzi a 3μM. Portanto, através de uma combinação de abordagens experimentais e computacionais, relatamos acertos promissores para otimização no desenvolvimento de novas drogas tripanocidas.

Aim: Limitations in available therapies for trypanosomiases indicate the need for improved medicines. Cysteine proteases cruzain and rhodesain are validated targets for treatment of Chagas disease and human African trypanosomiasis. Previous studies reported a benzimidazole series as potent cruzain inhibitors. Results & methodology: Considering the high similarity between these proteases, we evaluated 40 benzimidazoles against rhodesain. We describe their structure-activity relationships (SAR), revealing trends similar to those observed for cruzain and features that lead to enzyme selectivity. This series comprises noncovalent competitive inhibitors (best Ki = 0.21 μM against rhodesain) and micromolar activity against Trypanosoma brucei brucei. A cheminformatics analysis confirms scaffold novelty, and the inhibitors described have favorable predicted physicochemical properties. Conclusion: Our results support this series as a starting point for new human African trypanosomiasis medicines.

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 125 µM. 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 = 15 µM) 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 = 67.7 µM). Interestingly, the pyrimidine derivative 4b, although inactive in enzymatic assays, was highly active against T. cruzi (IC50 = 3.1 µM) 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.

Este estudo relata a síntese, estudos in vitro e computacionais de aminoquinolinas potenciais inibidoras das enzimas cruzaína e rodesaína com possivel atividade tripanossomicida. A doença de Chagas, cujo agente etiológico é o Tripanossoma cruzi, afeta cerca de oito milhões de pessoas em todo o mundo. A tripanossomíase africana humana, cujo agente etiológico é o Tripanossoma brucei, é um grave problema de saúde pública na África. A quimioterapia atualmente disponível para estas doenças apresenta eficiência limitada e toxicidade significativa. As cisteíno proteases cruzaína e rodesaína, principais proteases do T. cruzi e T. brucei, respectivamente, são alvos farmacológicos validados. Uma série de possíveis inibidores da cruzaína e da rodesaína foram planejados com base em protótipo inibidor da cruzaína (CI50 = 2,5 M) da classe das indol-pirimidinas. As substâncias sintetizadas possuem um grupo 7-cloro-4-aminoquinolina e variações no comprimento, basicidade e substituintes das cadeias laterais foram exploradas. Dezesseis substâncias foram obtidas utilizando mótodos eficientes com rendimentos variando de 16% a 99%. Os ensaios enzimáticos demonstraram que os compostos desta série inibem a cruzaína com CI50 de até 13,2 M e a rodesaína com CI50 de até 33,3 M. O modo de inibição da substância mais potente contra a cruzaína foi determinado como competitivo. Foi avaliada também a atividade tripanossomicida da série contra formas amastigotas intracelulares de T. cruzi e a citotoxicidade sobre fibroblastos não infectados. Foram identificadas três substâncias com CI50 menor do que 50 µM, porém com baixo índice de seletividade. Estudos de ancoragem molecular utilizando estruturas cristalográficas da cruzaína e rodesaína foram realizados. Os resultados de ancoragem na cruzaína sugerem que o anel quinolínico pode ocupar o subsítio hidrofóbico S2 e a cadeia lateral, mais flexível, pode interagir com resíduos dos subsítios S3 ou S1. Estudos de dinâmica molecular foram conduzidos e sugerem que o modo de ligação da substância mais potente contra a cruzaína pode ser dissimilar ao modo de ligação de ligantes cristalográficos possuindo grupos semelhantes. Os resultados sugerem que aminoquinolinas podem ser promissoras na busca por inibidores da cruzaína e rodesaína. In this work a series of aminoquinolines, potential inhibitors of cruzain and rhodesain, was synthesized, evaluated on in vitro assays and studied by in silico methods. Chagas disease is caused by Tripanossoma cruzi and about 8 million are estimated to be infected worldwide. Human African trypanosomiasis caused by Tripanossoma brucei is a major health problem in Africa. Current drugs used in treatment of both diseases shows high toxicity and low efficacy. Cysteine proteases cruzain and rhodesain, major proteases of T. cruzi and T. brucei respectively, are validated drug targets. A series of potential inhibitors of cruzain and rhodesain was planned based on a lead indolepyrimidine cruzain inhibitor (IC50 = 2.5 M). The synthesized series contains a 7chloro-4-aminoquinoline motif and various side chain lengths and basicity was explored. Sixteen compounds were prepared employing straightforward procedures with yields from 16% to 99%. Enzymatic assays showed that this series inhibited cruzain with CI50 as low as 13,2 M and rhodesain with IC50 as low as 33,3 M. The most potent cruzain inhibitor, aminoquinoline 20b, was found to be a competitive inhibitor. The activity of the compounds was evaluated in vitro against intracellular amastigotes T. cruzi and cytotoxicity on the normal fibroblast was evaluated. Among the compounds evaluated, three of them showed an IC50 lower than 50 µM, however low selectivity indexes were found. Docking studies against crystallographic structures of cruzain and rhodesain were conducted. The results of these studies suggest that the quinoline moiety can occupy the S2 pocket while the more flexible side chain can explore either S3 or S1 pockets. In order to gain further insight on binding mode of the aminoquinoline 20b, molecular dynamics were performed. The analysis of results suggests that the binding mode of this coumpound may differ from crystallographic binding modes of ligands containg similar groups. The results as a whole show that aminoquinolines may be a promising class in further investigations of cruzain and rhodeain inhibitors.