PARP1 and PD-L1 dual-targeted bifunctional small-molecule inhibitors: a novel strategy for dual immunotherapy of triple-negative breast cancer

Objectives Current single-agent PD-L1 immunotherapy and PARP inhibitor combination regimens have several drawbacks, including limited response rates, mismatched pharmacokinetics, and overlapping toxicities. This research aims to discover novel bifunctional small molecules that simultaneously block the PD-1/PD-L1 axis and PARP1, with prominent selectivity for PARP subtypes, to overcome bottlenecks in triple-negative breast cancer immunotherapy. Methods Computer-aided molecular docking, 100 ns all-atom molecular dynamics simulation, homogeneous time-resolved fluorescence (HTRF) binding assay, biolayer interferometry (BLI), differential scanning fluorimetry (DSF), and PD-1/PD-L1 NFAT reporter cell assay were integrated to characterize dual-target lead molecules. A panel of eight PARP isoforms was tested to evaluate compound subtype selectivity. Results The optimized molecule SP-1 potently disrupts the PD-1/PD-L1 protein-protein interaction (IC 50 = 630 nM) and inhibits PARP1 at nanomolar levels (IC 50 = 13 nM). Notably, SP-1 shows excellent PARP isoform selectivity: its inhibitory potency against PARP2 is nearly 18-fold weaker. At the same time, IC 50 values for PARP3, PARP5a, PARP5b, PARP7, PARP8, and PARP10 all exceed 500 nM, greatly reducing the off-target risks associated with pan-PARP suppression. Orthogonal biophysical characterizations confirm that SP-1 directly binds to both PD-L1 and PARP1, forming stable intermolecular interactions. Cell-based functional assays demonstrate that SP-1 produces robust anti-tumor effects in MDA-MB-231 triple-negative breast cancer cells (IC 50 = 1.01 μM) and markedly restrains tumor cell migration, invasion, and long-term clonogenic growth. Functional cellular assays further demonstrated that SP-1 effectively restored T cell function and enhanced T cell-mediated tumoricidal activity. Conclusions SP-1 serves as a balanced dual-target inhibitor of PD-L1 and PARP1, with potent PARP1 subtype selectivity, which can be prioritized as a lead scaffold for developing novel dual-functional anti-breast cancer agents.