Natural aromatic compounds as scaffolds to develop selective G-quadruplex ligands. From previously reported berberine derivatives to new palmatine analogues

In this paper, the selective interactions of synthetic derivatives of two natural compounds, berberine and palmatine,with DNA G-quadruplex structureswere reported. In particular, the previous works on this subject concerning berberine were further presented and discussed, whereas the results concerning palmatine are presented here for the first time. In detail, these palmatine derivatives were developed by inserting seven different small peptide basic chains, giving several new compounds that have never been reported before. The preliminary studies of the interactions of these compounds with various G-quadruplex-forming sequences were carried out by means of various structural and biochemical techniques, which showed that the presence of suitable side chains is very useful for improving the interaction of the ligands with G-quadruplex structures. Thus, these new palmatine derivatives might act as potential anticancer drugs

Experimental study and computational modelling of cruzain cysteine protease inhibition by dipeptidyl nitriles

Chagas disease affects millions of people in Latin America. This disease is caused by the protozoan parasite Trypanossoma cruzi. The cysteine protease cruzain is a key enzyme for the survival and propagation of this parasite lifecycle. Nitrile-based inhibitors are efficient inhibitors of cruzain that bind by forming a covalent bond with this enzyme. Here, three nitrile-based inhibitors dubbed Neq0409, Neq0410 and Neq0570 were synthesized, and the thermodynamic profile of the bimolecular interaction with cruzain was determined using isothermal titration calorimetry (ITC). The result suggests the inhibition process is enthalpy driven, with a detrimental contribution of entropy. In addition, we have used hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) and Molecular Dynamics (MD) simulations to investigate the reaction mechanism of reversible covalent modification of cruzain by Neq0409, Neq0410 and Neq0570. The computed free energy profile shows that the nucleophilic attack of Cys25 on the carbon C1 of inhibitiors and the proton transfer from His162 to N1 of the dipeptidyl nitrile inhibitor take place in a single step. The calculated free energy of the inhibiton reaction is in agreement with covalent experimental binding. Altogether, the results reported here suggests that nitrile-based inhibitors are good candidates for the development of reversible covalent inhibitors of cruzain and other cysteine proteases

Design, Synthesis and Trypanocidal Activity of Cruzain Reversible-Covalent Inhibitors

A doença de Chagas, causada pelo parasito protozoário Trypanosoma cruzi, continua sendo um grave problema de saúde devido à terapia inadequada e à falta de uma vacina eficaz. Novos fámacos que sejam seguros e eficazes são, portanto, criticamente necessários. A cruzaína (Cz), principal protease cisteínica (CP) do T. cruzi, é uma enzima essencial para o ciclo de vida do parasito e tem sido utilizada como alvo viável para a busca e identificação de novas entidades químicas (NCE) como agentes tripanossomicidas. Alguns resultados recentes demonstram que os inibidores covalentes reversíveis da Cz podem bloquear os passos de diferenciação celular no parasito e levá-lo à morte. Nesta tese, por emprego de abordagem racional baseada em hipóteses, foram planejados e sintetizados diferentes séries de inibidores da Cz baseadas em dipeptidil nitrilas. Em especial, o esqueleto molecular foi modificado para compreender as interações S3/P3, o papel do grupo ciclopropila no processo de reconhecimento bimolecular e o mapeamento S1/S1´ para melhorar a afinidade e avaliar a seletividade frente a outras CPs presentes em células de mamíferos. A partir do protótipo Neq0570 (pKi = 6,3), a afinidade para Cz foi aumentada em 1,6 unidades log para o Neq0922, pKi = 7,9, por uma combinação de modificações estruturais em P3, P2 e P1. Dos resultados relevantes obtidos, destacam-se três novos inibidores da Cz (Neq0940, Neq0938 e Neq0877) equipotentes ao Benzonidazol - Bz, (o fármaco atualmente usado no Brasil para o tratamento da doença de Chagas) em ensaios in vitro para a avaliação da atividade antitripanossomal sobre o T. cruzi. Considerando-se que o índice de seletividade (SI) do Bz é > 20, como no caso desses inibidores, testes in vivo serão necessários para avaliar a potência frente à forma aguda da doença de Chagas. Inibidores bioisostéricos de dipeptidil nitrilas estão sob investigação pré-clínica como agentes tripanossomicidas. Destarte, realizamos a síntese e os estudos de relação estrutura-atividade (SAR) de inibidores da Cz por substituição da amida em P3/P2 por trifluoroetilamina. Foram identificados três novos inibidores de Cz com afinidade em concentração nanomolar de um dígito (Neq0642, Neq0643 e Neq0659) com alta seletividade sobre a catepsina L de humanos (CatL). Os efeitos da configuração do centro estereogênico em P3/P2 associados à substituição de diferentes grupos em P1 levam a fortes efeitos não-aditivos nas SARs. A troca bioisostérica pelo grupo sulfonamida em P3/P2 resultou em uma nova série de inibidores que foi avaliada em um painel de CPs de agentes infecciosos e de seres humanos. Há uma reconhecida SAR na inibição da Cz, ao mesmo tempo em que revelam uma inibição na ordem nanomolar para a CatS, com elevada seletividade para os outras CPs de mamíferos (CatB, CatK e CatL). A exploração da SAR também incorpora os efeitos do grupo eletrofílico nitrila por inclusão de \"warheads\" alternativos. As constantes de inibição representadas por aldeídos e nitrilo-azadipeptídios são duas ordens de grandeza maiores para os melhores inibidores de Cz. Além disso, o inibidor de Cz nitrilo-azadipeptídico (Neq0690) foi usado na primeira geração de inibidores da CPB de Leishmania mexicana (LmCPB) obtida na forma cristalina com alta resolução da estrutura cristalográfica.Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a serious health problem due to inadequate therapy and lack of an effective vaccine. New drugs that are safe and efficient are therefore critically needed. Cruzain (Cz), the main T. cruzi cysteine protease (CP), is an essential enzyme for the life cycle of the parasite and has been used as a viable target for the search and identification of new chemical entities (NCE) acting as trypanocidal agents. Recent literature results demonstrate that reversible covalent Cz inhibitors can block the cell differentiation steps in the parasite and kill it.To this end, by applying a knowledge-based design approach, we have designed and synthesized a new series of dipeptidyl nitrile inhibitors of Cz aimed at leveraging the S3/P3 interactions, understand the role of the cyclopropyl in the bimolecular recognition process and map the S1/S1´ to enhance affinity and selectivity over other CPs. Herein, we report three new Cz inhibitors (Neq0940, Neq0938 and Neq0877)being equipotent with Benznidazole (the only current drug used to treat Chagas disease in Brazil) with SI (selective index) ratio over 20 (respect to the host cell) and therefore making them attractive synthetic targets for further in vivo testing against the acute form of Chagas disease. Dipeptidyl nitrile bioisostere inhibitors are under preclinical investigation as trypanocidal agents. Therefore, we performed the synthesis and structure-activity relationship (SAR) investigation of nitrile-based Cz inhibitors incorporating a trifluoroethylamine moiety as P3/P2 amide replacement. Results display three new one-digit (Neq0642, Neq0643, and Neq0659) nanomolar Cz inhibitors with high selectivity over CatL. Also, this works reveals that the effects of configuration at the P3/P2 amide replacement and different substitution in P1 lead to strong nonadditivity outcome. The bioisosteric exchange by a sulfonamide moiety on P3/P2 resulted in a new series of inhibitors that was evaluated on a panel of infectious and human CPs. Data displays an interesting flat SAR for Cz while disclosing strong CatS inhibition with high selectivity for other mammalian CPs (CatB, CatK, and CatL). SAR investigation also includes the effects of the nitrile electrophilic group by including alternative warheads. Inhibition constants represented by aldehydes and nitrile azadipeptides are two orders of magnitude larger for the best Cz inhibitors. Besides, the nitrile azadipeptide Cz inhibitor (Neq0690) was used in the first generation of Leishmania mexicana CPB (LmCPB) high-resolution x-Ray crystal structure

Perylene and coronene derivatives binding to G-rich promoter oncogene sequences efficiently reduce their expression in cancer cells

A novel approach to cancer therapeutics is emerging in the field of G-quadruplex (G4) ligands, small molecules designed to stabilize four-stranded structures that can form at telomeres as well as in other genomic sequences, including oncogene promoter sequences, 50-UTR regions and introns. In this study, we investigated the binding activity of perylene and coronene derivatives PPL3C, CORON and EMICORON to G4 structures formed within the promoter regions of two important cancer-related genes, c-MYC and BCL-2, and their biochemical effects on gene and protein expression. In order to fully characterize the ability of the selected ligands to bind and stabilize the G4 structures originated by the c-MYC and BCL-2 promoter sequences, we performed electrospray ionization mass spectrometry (ESI-MS), Fluorescence Resonance Energy Transfer (FRET) measurements, Circular Dichroism (CD) spectra and polymerase stop assay. Altogether our results showed that the ligands had a high capacity in binding and stabilizing the G4 structures within the c-MYC and BCL-2 promoter sequences in vitro. Notably, when we evaluated by quantitative real-time PCR and western blotting analysis, the effects of treatment with the different G4 ligands on c-MYC and BCL2 expression in a human melanoma cell line, EMICORON appeared the most effective compound in reducing the mRNA and protein levels of both genes. These results encourage to consider EMICORON as a promising example of multimodal class of an antineoplastic drug, affecting different tumor crucial pathways simultaneously: telomere maintenance (as previously described), cell proliferation and apoptosis via down-regulation of both c-MYC and BCL-2 (this paper)

Leveraging the cruzain S3 subsite to increase affinity for reversible covalent inhibitors

Cruzain is themajor cysteine protease of Trypanosoma cruzi, the etiological agent of Chagas disease. Reversible covalent cruzain inhibitors can block the steps of cell differentiation in the parasite and kill the organism. To this end, the description of how inhibitors modified at the P2/P3 positions lead to analogs with greater cruzain affinity to the S2/S3 subsites is of fundamental importance. Albeit many efforts are being employed in the characterization of the interaction processes with S2 subsite, little is known about the cruzain S3 subsite. In this work, we show a brief but consistent study to identify favorable substitutions in P3 of dipeptidyl nitriles that increase cruzain affinity. Using molecular dynamics simulations, we have identified some dipeptidyl nitrile analogs with modifications at P3 position that had higher cruzain inhibition than the original un-substituted compound. A matched molecular pair analysis shows the importance of including a chlorine atom in the P3-meta position. The modifications implemented in P3 are confirmed when profiling the thermodynamic parameters via isothermal titration calorimetry. The classical enthalpy-entropy compensation phenomenon, in which enthalpy changes are counterbalanced by entropy results in a small modification of.G. The inclusion of the chlorine atom in the P3-meta position results in the highest reduction of the detrimental entropic contribution observed in P3

Assessment of the cruzain cysteine protease reversible and Irreversible covalent inhibition mechanism

Reversible and irreversible covalent ligands are advanced cysteine protease inhibitors in the drug development pipeline. K777 is an irreversible inhibitor of cruzain, a necessary enzyme for the survival of the Trypanosoma cruzi (T. cruzi) parasite, the causative agent of Chagas disease. Despite their importance, irreversible covalent inhibitors are still often avoided due to the risk of adverse effects. Herein, we replaced the K777 vinyl sulfone group with a nitrile moiety to obtain a reversible covalent inhibitor (Neq0682) of cysteine protease. Then, we used advanced experimental and computational techniques to explore details of the inhibition mechanism of cruzain by reversible and irreversible inhibitors. The isothermal titration calorimetry (ITC) analysis shows that inhibition of cruzain by an irreversible inhibitor is thermodynamically more favorable than by a reversible one. The hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) simulations were used to explore the mechanism of the reaction inhibition of cruzain by K777 and Neq0682. The calculated free energy profiles show that the Cys25 nucleophilic attack and His162 proton transfer occur in a single step for a reversible inhibitor and two steps for an irreversible covalent inhibitor. The hybrid QM/MM calculated free energies for the inhibition reaction correspond to -26.7 and -5.9 kcal mol-1 for K777 and Neq0682 at the MP2/MM level, respectively. These results indicate that the ΔG of the reaction is very negative for the process involving K777, consequently, the covalent adduct cannot revert to a noncovalent protein-ligand complex, and its binding tends to be irreversible. Overall, the present study provides insights into a covalent inhibition mechanism of cysteine proteases

N-Sulfonyl Dipeptide Nitriles as Inhibitors of Human Cathepsin S: In silico Design, Synthesis and Biochemical Characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S