Abstract 3097: Prodrugs of a 1-hydroxy-2-oxopiperidin-3-yl phosphonate enolase inhibitor for the treatment of ENO1-deleted cancers
Authors: Yan Victoria C. , Pham Cong-Dat , Ballato Elliot S. , Yang Kristine L. , Khadka Sunada , Barektain Yasaman , Shrestha Prakriti , Tran Theresa , Poral Anton H. , Washington Mykia , Raghavan Sudhir , Czako Barbara , Pisaneschi Federica , Lin Yu-Hsi , Satani Nikunj , Hammoudi Naima , Ackroyd Jeffrey J. , Georgiou Dimitra K. , Millward Steven W. , Muller Florian L. 
Published: April 2023

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). We previously identified and characterized a competitive, small molecule phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX), and its lipophilic bis-ester prodrug (POMHEX) in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma. Treatment with either HEX (150 mg/kg IV/IP) or POMHEX (20 mg/kg IV/IP) yielded tumor regression even after drug discontinuation. However, due to the poor pharmacokinetics of esterase-labile POMHEX, we synthesized a library of novel phosphonate prodrugs with distinct mechanisms of bioactivation and assessed their potency in D423 (ENO1-/-) cells. By conducting a prodrug structure activity relationship (SAR) study, we found that phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, while canonical McGuigan (ProTide) prodrugs were not. Other strategies, including salicylic alcohol (cycloSal) and lipid prodrugs of HEX, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over POMHEX. En route, we developed a novel class of aliphatic amine/ester prodrugs that can be broadly applied to efficiently deliver phosph(on)ate pharmacophores in cells. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation. In sum, we have developed a novel class of phosph(on)ate prodrugs that is efficiently bioactivated in cells in vitro. Our prodrug SAR study disputes the common notion that ProTides are universally advantageous promoieties on phosph(on)ate pharmacophores and we provide mechanistic rationale for this observation with HEX. Finally, we show that the cycloSal prodrug yields efficient intracellular delivery of HEX in vitro, with a mechanism of bioactivation consistent with the GBM microenvironment, making this promoiety promising for further evaluation in vivo.

Citation Format: Victoria C. Yan, Cong-Dat Pham, Elliot S. Ballato, Kristine L. Yang, Sunada Khadka, Yasaman Barektain, Prakriti Shrestha, Theresa Tran, Anton H. Poral, Mykia Washington, Sudhir Raghavan, Barbara Czako, Federica Pisaneschi, Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Jeffrey J. Ackroyd, Dimitra K. Georgiou, Steven W. Millward, Florian L. Muller. Prodrugs of a 1-hydroxy-2-oxopiperidin-3-yl phosphonate enolase inhibitor for the treatment of ENO1-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3097.