Abstract

Tumor-associated macrophages (TAMs) play a pivotal role in shaping the tumor microenvironment (TME) and influencing the outcomes of immunotherapy. However, most drug screening strategies emphasize tumor cell cytotoxicity and neglect immune effector modulation. Here, we describe a macrophage-centric phenotypic screening platform to identify selective HDAC6 inhibitors that reprogram TAMs toward an antitumor phenotype. Building on the HDAC6 inhibitor SS-208, we synthesized a novel class of tetrazolone-based compounds with potent selectivity and minimal cytotoxicity. Among these, SM-06-09 emerged as a lead candidate, showing subnanomolar HDAC6 inhibition, enhanced macrophage phagocytosis, antigen presentation, and T-cell activation in vitro. In a syngeneic melanoma model, SM-06-09 suppressed tumor growth and promoted M1-like TAM polarization. Combination with anti-PD-1 therapy further enhanced immune infiltration, increased effector memory and central memory T-cells, and improved antitumor efficacy. This study establishes a functional screening framework for identifying immunomodulatory compounds and supports the clinical potential of macrophage-targeted HDAC6 inhibitors as adjuncts to immune checkpoint blockade.