Discovery of novel anti-inflammatory drug-like compounds by aligning in silico and in vivo screening: The nitroindazolinone chemotype

Abstract In this report, we propose the combination of computational methods and in vivo primary screening in zebrafish larvae and confirmatory in mice models as a novel strategy to accelerate anti-inflammatory drug discovery. Initially, a database of 1213 organic chemicals with great structural variability – 587 of them anti-inflammatory agents plus 626 compounds with other clinical uses – was divided into training and test groups. Atom-based quadratic indices – a TOMOCOMD - CARDD molecular descriptors family – and linear discriminant analysis (LDA) were used to develop a total of 13 models to describe the anti-inflammatory activity. The best model (Eq. (13) ) shows an accuracy of 87.70% in the training set, and values of Matthews correlation coefficient ( C ) of 0.75. The robustness of the models was demonstrated using an external test set as validation method, i.e ., Eq. (13) revealing classification of 88.44% ( C  = 0.77) in this series. All models were employed to develop ensemble a QSAR classification system, in which the individual QSAR outputs are the inputs of the aforementioned fusion approach. The fusion model was used for the identification of novel anti-inflammatory compounds using virtual screening of 145 molecules available in our in - house library of indazole, indole, cinnoline and quinoxaline derivatives. Out of these, 34 chemicals were selected, synthesized and tested in a lipopolysaccharide (LPS)-induced leukocyte migration assay in zebrafish larvae. This activity was evaluated based on leukocyte migration to the injury zone of tail-transected larvae. Compounds 18 (3 μM), 24 (10 μM), 25 (10 μM), 6 (10 μM), 15 (30 μM), 11 (30 μM) and 12 (30 μM) gave the best results displaying relative leukocyte migration (RLM) values of 0.24, 0.27, 0.35, 0.41, 0.17, 0. 26 and 0.27 respectively, date that suggest an anti-inflammatory activity of 76, 73, 65, 59, 83, 84 and 73%, respectively. Compound 18 was the most potent but showed high toxicity together with compound 6 . Next, we used the tetradecanoylphorbol acetate (TPA)-induced mouse ear oedema model to evaluate the most potent compounds in the zebrafish larvae tail transection assay. All assayed compounds, with the exception of chemical 15 , showed anti-inflammatory activity in mice. Compound 12 ( VA5 - 13l , 2-benzyl-1-methyl-5-nitro-1,2-dihydro-3 H -indazol-3-one) was the most active and completely abolished the oedema. Compounds 6 , 11 and 24 showed inhibition percentages in the range of the reference drug (indomethacin), whereas compounds 18 and 25 reduced the oedema in a lesser extent (inhibition of 73 and 80%, respectively). In addition, all compounds except chemical 15 , significantly reduced neutrophil infiltration, measured as myeloperoxidase activity on TPA application test. Compounds 6 , 11 , 12 and 18 showed values comparable to indomethacin (inhibition percentage of 61), but compounds 6 and 18 were toxic in zebrafish and showed unspecific cytotoxicity in murine macrophages at 100 μg/mL, while the remaining compounds 11 , 12 and 25 were inactive at most levels. Evidently, this study suggests a new support structure ( 12 , 11 and 24 ; a nitroindazolinone chemotype ) that constitutes a novel promising lead and may represent an important therapeutic alternative for the treatment of inflammatory conditions.