DNA-encoded chemical libraries (DELs) are powerful drug discovery tools, enabling the parallel screening of millions of DNA-barcoded compounds. We investigated how the DEL input affects the hit discovery rate in DEL screenings. Evaluation of selection fingerprints revealed that the use of approximately 10⁵ copies of each library member is required for the confident identification of nanomolar hits, using generally applicable methodologies.

Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein that is overexpressed on the surface of cancerous prostate cells both in primary tumors and in metastases. Small organic ligands targeting PSMA have been broadly and successfully used to deliver radionuclide payloads to prostate cancer lesions. 177Lu-PSMA-617 (Pluvicto, a Novartis product) is a PSMA-targeted product that has been recently approved for the treatment of metastatic castration-resistant prostate cancer. By contrast, no small molecule–drug conjugates (SMDC) directed against PSMA have gained marketing authorization yet. In this article, we present the development of novel SMDCs generated by conjugating the tumor-targeting moiety of Pluvicto (here named “OncoPSMA”) to highly cytotoxic auristatin payloads through cleavable linkers, including valine–citrulline, disulfide bridges, and a recently described postprolyl peptidase–cleavable linker [glycine–proline (Gly-Pro)]. The efficiency of payload release at the cancer site and in healthy tissues was assessed via biodistribution studies using mass spectrometry quantification upon systemic administration in tumor-bearing mice. SMDCs based on the Gly-Pro linker mediated the highest payload release in solid tumors compared with widely utilized cathepsin B–cleavable and disulfide linkers. The in vivo efficacy of OncoPSMA-Gly-Pro-monomethyl auristatin E and OncoPSMA-Gly-Pro-monomethyl auristatin F was tested in therapy studies alone and in combination with an antibody–IL2 fusion protein, capable of preferential homing to solid tumors. Combination treatments resulted in complete and durable responses, highlighting the potential benefit of this therapeutic modality to patients with metastatic prostate cancer.

Two-way ANOVA multiple comparisons (Bonferroni test) of tumor volume of mice bearing HT1080.hPSMA tumor xenografts after treatment. Tumor growth is depicted in Figure 3.