Abstract
Hypoxia-driven adenosinergic signaling suppresses antitumor immunity and enables many tumors to evade cancer immunotherapy. Alkaline phosphatase (ALP) contributes to this immunosuppression by hydrolyzing extracellular adenosine triphosphate (ATP) to adenosine. Rather than inhibiting this broadly essential enzyme, we exploit elevated ALP activity in tumors as a tumor-specific trigger for enzyme-instructed self-assembly (EISA). Using a potent naphthalene-capped phosphopeptide diester precursor (1P), which exhibits in vivo efficacy as a starting point, we designed and synthesized a series of analogs (2P-15P) that vary the N-terminal capping group and/or the position of the phosphotyrosine trigger to explore structure-activity relationships. We evaluated these analogs for anticancer activity in ALP-overexpressing cancer cells (e.g., Saos-2), using ALP-low cells (e.g., HEK-293) as controls. One analog (6P) exhibited potent, selective cytotoxicity, with submicromolar GI50 values (similar to 0.3 mu M) in ALP-high cancer cells. Notably, 6P demonstrated submicromolar GI90 values and surpassed the efficacy of cisplatin and paclitaxel against Saos-2 cells. These findings highlight the potential of rationally designed d-phosphopeptide diesters to enable ALP-responsive self-assembly and selectively inhibit the growth of ALP-overexpressing tumors. This strategy offers a promising platform for the development of supramolecular therapeutics targeting immunosuppressive tumor microenvironments.