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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.