INTRODUCTION: Leishmaniasis comprise a group of neglected tropical diseases caused by an intracellular protozoa of the genus Leishmania sp., responsible for causing different clinical (cutaneous, mucocutaneous and visceral leishmaniasis), histopathological and immunopathological manifestations, according to the characteristics of the species in question and the molecular defense mechanism of the host [1,2]. Nevertheless, the chemotherapy treatment currently employed for the different clinical conditions of leishmaniasis is expressly limited, given that there are several limiting factors involving these medications, such as: low efficacy, high cost, increasing resistance, difficult administration, and high risk of systemic toxicity [3,4]. Therefore, there is a need to obtain new antileishmanial drugs and, based on this premise, acridine derivatives demonstrate chemotherapeutic potential and have shown good results for antileishmanial purposes, such as those obtained by Almeida et al. [5] by the investigation of the activity of spiro-acridine derivatives against species of L. infantum. AIMS: Therefore, this work proposes the in vitro evaluation of four different spiro-acridine derivatives (AMTAC-01, AMTAC-02, AMTAC-06 and AMTAC-22) against promastigote forms of L. infantum and L. amazonensis, and in silico target proposal through molecular docking. METHODS: The in vitro antileishmanial activity was determined based on the Neubauer chamber method [6,7]. Promastigotes of L. amazonensis and L. infantum were collected, counted, and diluted in Schneider medium (Sigma-Aldrich, St Louis, USA) supplemented with 20% FBS in a concentration of 1x106 cells/mL. The evaluated promastigotes were then incubated with different concentrations of the tested compounds (10, 5, 2.5, 1.25, and 0.625 ?g/mL). After 72h of incubation at room temperature, culture growth was observed by counting the viable cells using a Neubauer chamber. The positive control selected was Amphotericin B®. The IC50 was determined by linear regression analysis with SPSS 8.0 software for Windows. In addition to in vitro studies, in silico predictions were carried out. The energy minimization calculation for the spiro-acridine derivatives was defined through the ArgusLab software. Molecular docking was performed using GOLD 2020.2 (Genetic Optimization for Ligand Docking) Cambridge Crystallographic Data Center (CCDC). The selected biological targets for this virtual screening were: sterol 14-alpha demethylase (L. infantum) (CYP51, PDB ID: 3L4D, RMSD: 1.57 Å) and the heterodimeric topoisomerase I-vanadate-DNA complex (L. donovani) (TOP1, PDB ID: 2B9S). It was not possible to validate the methodology of the PDB ID: 2B9S, as this target does not present a co-crystallized ligand. The images of the interactions were viewed using Discovery Studio 2017 R2 Client. RESULTS AND DISCUSSION: The evaluation of the antipromastigote activity demonstrated the potential of the selected compounds, demonstrating the influence of the substituent on the activity according to the species evaluated. The prototype compound of the series, AMTAC-01 (-phenyl), along with the derivative AMTAC-06 (p-chlorophenyl), were previously evaluated by Almeida et al. [5] against L. infantum through a different methodology and showed IC50 values of 2.039 and 5.681 µg/mL. In the current assay, the values were similar (6.27 and 13.8 µM, respectively), and an improvement in the activity was observed due to the molecular modifications carried out to obtain the compounds AMTAC-02 (p-methoxyphenyl) and AMTAC-22 (-indole), which presented IC50 values of 2.22 and 2.00 µM. On the other hand, more satisfying results were obtained for these compounds against the promastigote forms of L. amazonensis, with IC50 values of 1.23, 0.73, 0.75, and 152.19 µM for AMTAC-01, -02, -06, and -22, respectively. These results show a higher selectivity of compounds with lower lipophilicity to the L. infantum forms, while the activity in L. amazonensis is favored by the substitution in the phenyl ring by electron acceptor groups, but expressly reduced by the insertion of the pyrrole ring fused to the phenyl ring. Therefore, we particularly highlight the greater selectivity between species presented by the compound AMTAC-22 (I.S. [amaz/inf] = 76.1). Therefore, in order to determine possible pharmacological targets that may be involved in the leishmanicidal activity executed in vitro, docking studies were performed. For this, the results previously obtained by Almeida et al. [5] were used as reference, and simulations with the AMTAC-02 and AMTAC-22 derivatives were carried out. Therefore, it was observed that, for the CYP51 target, the compound that presented the best fitscore was AMTAC-01 (75.39), followed by the co-crystallized compound (66.82), AMTAC-06 (56.73), AMTAC-02 (55.38) and AMTAC-22 (22.02). For the stabilization of these complexes, interactions with the amino acid residues Tyr115, Met459, and Leu399 appeared to be important. In the TOP1 target, the compound that presented the highest fitscore was AMTAC-22 (64.93). AMTAC-01, AMTAC-02, and AMTAC-06 presented a fitscore of 45.19, 58.37, and 57.04, respectively, while Camptothecin, used as standard, presented a score of 53.2. When analyzing the interactions carried out between the target-ligand complex, it is possible to visualize a remarkably polar binding site, with emphasis on interactions with the amino acid residues Lys211 and Arg190. CONCLUSION: Through the following study, the potential of spiro-acridine compounds as leishmanicidal agents was observed. In vitro studies demonstrated good results and the possibility of selectivity between species based on simple structural modifications, combined with molecular docking studies that assist in the mechanistic elucidation and suggested CYP51 and TOP1 as possible targets as they present more desirable fitscores than the reference compounds. Thus, these results promote further studies involving spiro-acridine derivatives with this direction. ACKNOWLEDGMENT: The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE), Fundação de Apoio à Pesquisa do Estado da Paraíba (FAPESQ), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their support to the Brazilian Postgraduate Programs..
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Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
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