Background. It has been estimated that more than 50% of cancers are related to the dysregulation of the PI3K-PDK1-Akt signaling axis. Within this pathway, phosphoinositide-dependent kinase-1 (PDK1) and Aurora kinase A (AurA) have key roles. PDK1 is a phosphorylation-mediated activator of Akt, the main apoptotic regulator, thereby acting at the crossroads of several oncogenic pathways. Overexpression and/or aberrant activation of AurA have been functionally linked to neoplastic transformation principally through the development of centrosome amplification and chromosomal instability. In this study, the molecular mechanisms involved in the dual inhibitory capacity of PDK1- and AurA-targeting OXID-pyridonyl compounds were explored in a panel of tumor cell lines carrying PI3KCA, K-RAS, PTEN, RB-1, EGFR and/or TP53 mutations

Materials and Methods. To evaluate the enzymatic inhibitory activity, the oxindole-based derivatives were subjected to FRET-based Z′-Lyte assay (Invitrogen™) against PDK1 and AurA. All in vitro assays for ADME-Tox profiling were performed as published previously[1]. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72-h exposures with increasing concentrations (from 0.014 to 30 µM) in a panel of 15 cell lines. Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK-1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds.

Results. Among the four evaluated compounds, SA16 displayed nanomolar inhibitory potency against both PDK1 and AurA. In vitro, drugs SA16 and VI8 showed to exert anti-tumor activity in the 6647, TC71, SKNMC Ewing sarcoma (ES) cells lines and in the HCT116 colon carcinoma-derived cells (IC50s values between 2.6 and 25 µM after 72-h exposures), when tested in a panel of 15 cancer cell lines. These results were confirmed by the evaluation of 3D-clonogenic capacity in soft agar. The effect of 1 µM SA16 at the protein level was evaluated after 5 to 120 min exposure in the ES cell line SKNMC. No modulation of Aur-A protein or PDK-1 phosphorylation (Ser241) were observed in SKNMC cells, whereas a decrease of downstream PDK1 targets such as p-AKT, p-RSK2, and p-p44/42 MAPK was observed after 30 min of exposure. In addition, SA16 displayed acceptable in vitro ADME-Tox properties. Our results on structural insights into the mode of binding[2], in vitro drug-intake kinetics and in vivo PK appear to indicate that SA16 presents adequate features as a scaffold for the design of PDK1/AurA dual-target molecules.

Conclusion. We report the synthesis of novel first-in-class 2-oxindole-based derivatives as dual PDK1-AurA kinase inhibitors as a novel scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma.[1] Runfola M. et al. Data Brief 29 (2020) 105206.[2] Sistito S. et al. Eu J Med Chem 226 (2021) 11389.

Citation Format: Maria Eugenia Riveiro, Samuel Huguet, Ramiro Vazquez, Roberta Frapolli, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai, Gianpiero Garau, Simona Rapposelli. Development of potent dual PDK1/AurA kinase inhibitors for Ewing sarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2569.