e15021 Background: Protein tyrosine phosphatase non-receptor type 2 (PTPN2), a negative regulator of JAK–STAT pathways that plays a crucial role in the immune system, has been identified as a promising target for immunotherapy. Genetic deletion of PTPN2 in either tumor cells or host immune cells promotes the anti-tumor immunity. In addition, PTPN2 inhibitors, alone or in combination with anti-PD1 therapy, significantly reduced tumor growth and enhanced survival in several subcutaneous tumour models. These findings demonstrate that PTPN2 inhibitors may have clinical benefits for immunotherapy-resistant tumors. Herein, the discovery of a series of novel PTPN2 inhibitors and their implications for immunotherapy are reported. Methods: We have successfully developed a series of small molecule inhibitors that specifically target PTPN2/N1. Molecular design was based on a novel backbone and incorporating both structure-based drug design (SBDD) and computational chemistry techniques. To evaluate the efficacy of our inhibitors, PTPN2 phosphatase activity was monitored using the small-molecule substrate DiFMUP in a fluorescence assay format in vitro. Tumor cells were treated with IFNγ and the cell viability was measured. Pharmacokinetic studies were conducted in mice, with serial blood samples analyzed by liquid chromatography−mass spectrometry (LC−MS)/MS. Pharmacokinetic parameters were calculated by noncompartmental analysis. Results: Based on the co-crystal structure of this target reported and multiple previous research projects, we have designed a multitude of novel compounds with the employment of computer-aided drug design (CADD) and scaffold hopping. Through a comprehensive evaluation of the in vitro and in vivo parameters, we have identified a series of compounds displaying potent PTPN2 inhibition (IC50 < 10 nM). Moreover, the compounds displayed remarkable efficacy in killing tumor cells to IFNγ treatment (IC50 < 100 nM). Artificial intelligence was utilized to conduct assessment and prediction of ADME properties, improving the drug-ability including permeability and physicochemical properties. Currently, a series of compounds demonstrating outstanding potency and good pharmacokinetic profiles, with lower hERG blockage (hERG IC50 > 30 µM), were synthesized and characterized. Among these hit compounds, a leading compound with PTPN2 IC50 = 1.1 nM and tumor cells IC50 = 79.5 nM was identified for further optimization and evaluation. Conclusions: We reported our work on discovering HDM2010, in which a series of potent PTPN2 inhibitors with prominent PK and safety profile were synthesized and characterized. Further optimization including in vivo experiments will lead to a preclinical candidate (PCC) selection.