Far, E. | Search bioRxiv

bioRxiv2026

The coumarin derivative X6632 is a pan-ID protein inhibitor that suppresses tumor growth by targeting cancer cells and the tumor-associated microvasculature

Inhibitor of DNA binding (ID) proteins are key regulators of tumor cell stemness, therapy resistance and pathological angiogenesis in multiple cancer types and other diseases. Here, we characterize the coumarin-derived compound X6632 as a pan-ID inhibitor with dual activity against tumor cells and the tumor-associated microvasculature in a number of human and murine models. X6632 efficiently suppressed ID protein expression, inhibited the proliferation, migration, invasion of melanoma cells, and impaired multiple endothelial cell functions, including proliferation, migration, invasion, tube formation and sprouting in vitro. In back-to-back comparisons, X6632 exhibited an approximately ten-fold higher efficacy compared to the first-generation ID antagonist AGX51. In vivo, X6632 potently reduced pathological (neo)vascularization in established angiogenesis models, including oxygen-induced retinopathy and in Matrigel plug assays. It also significantly decreased blood vessel density in syngeneic melanoma models, delayed tumor growth and, when combined with immune checkpoint blockade, achieved superior tumor control compared with either monotherapy. Moreover, X6632 inhibited clonogenic growth in several breast cancer models, and robustly suppressed the growth of triple negative breast cancer in vivo, both in the highly aggressive 4T1 syngeneic model and in patient-derived xenografts. Collectively, these data establish X6632 as a second-generation, pan-ID protein inhibitor that can simultaneously target malignant cells and the tumor-supporting vasculature, and support the further pre-clinical development of the compound for the treatment of melanoma, breast cancer and potentially additional ID-dependent malignancies, as well as diseases driven by pathological neoangiogenesis.

cancer biology↗

bioRxiv2026

A fungal hemophore relay mediates heme transfer via transient protein interactions

The fungal pathobiont Candida albicans acquires heme from host proteins via a set of soluble and anchored extracellular CFEM-type hemophores that can capture the bound heme and exchange it, eventually delivering it to the cell membrane for endocytosis into the yeast cell. Yet the molecular mechanism by which the heme is transferred through this protein cascade across the cell envelope remains unclear. To address this mechanism, we developed a set a fusions of three C. albicans hemophores with fluorescent proteins. Fluorescence of these fusion proteins is strongly quenched when heme is bound to the hemophore moiety, enabling to measure heme transfer instantaneously. Kinetic analysis of the different transfer reactions reveals that heme transfer from the host protein to the CFEM hemophores and between the CFEM hemophores are governed by different regimes. Kinetics of heme transfer from hemoglobin or serum albumin to the CFEM hemophores is mainly first-order, suggesting that heme stochastically released from host proteins is captured by the hemophores. In contrast, transfer of heme between the hemophores is near second-order, consistent with a mechanism requiring protein-protein interactions. To confirm this, we show that CFEM hemophores can interact in homodimeric and heterodimeric complexes. Furthermore, while dimerization-defective mutants of the soluble hemophore Csa2 are proficient in heme binding and extraction, they are defective in heme transfer. This supports a model of heme transfer by direct interaction between the members of the fungal hemophore cascade. SIGNIFICANCEAcquisition of extracellular heme as iron or heme sources is common in microorganisms, and particularly prevalent among pathogenic organisms that must contend with an iron-poor host environment. To extract heme from host proteins, microorganisms deploy various systems that include extracellular soluble and cell-anchored hemophores. Here we describe a new approach for monitoring heme binding and transfer in real time, based on the development of fluorescent derivatives of fungal hemophores. These novel reagents open a new window on the study of a common virulence factor of microbial pathogens.

microbiology↗