Visible Light Control over the Cytolytic Activity of a Toxic Pore-Forming Protein.

Enabling control over the bioactivity of proteins with light, along with the principles of photopharmacology, has the potential to generate safe and targeted medical treatments. Installing light sensitivity in a protein can be achieved through its covalent modification with a molecular photoswitch. The general challenge in this approach is the need for the use of low energy visible light for the regulation of bioactivity. In this study, we report visible light control over the cytolytic activity of a protein. A water-soluble visible-light-operated tetra-ortho-fluoro-azobenzene photoswitch was synthesized by utilizing the nucleophilic aromatic substitution reaction for installing a solubilizing sulfonate group onto the electron-poor photoswitch structure. The azobenzene was attached to two cysteine mutants of the pore-forming protein fragaceatoxin C (FraC), and their respective activities were evaluated on red blood cells. For both mutants, the green-light-irradiated sample, containing predominantly the cis-azobenzene isomer, was more active compared to the blue-light-irradiated sample. Ultimately, the same modulation of the cytolytic activity pattern was observed toward a hypopharyngeal squamous cell carcinoma. These results constitute the first case of using low energy visible light to control the biological activity of a toxic protein.

Reversible Photocontrolled Nanopore Assembly.

Self-assembly is a fundamental feature of biological systems, and control of such processes offers fascinating opportunities to regulate function. Fragaceatoxin C (FraC) is a toxin that upon binding to the surface of sphingomyelin-rich cells undergoes a structural metamorphosis, leading to the assembly of nanopores at the cell membrane and causing cell death. In this study we attached photoswitchable azobenzene pendants to various locations near the sphingomyelin binding pocket of FraC with the aim of remote controlling the nanopore assembly using light. We found several constructs in which the affinity of the toxin for biological membranes could be activated or deactivated by irradiation, thus enabling reversible photocontrol of pore formation. Notably, one construct was completely inactive in the thermally adapted state; it however induced full lysis of cultured cancer cells upon light irradiation. Selective irradiation also allowed isolation of individual nanopores in artificial lipid membranes. Photocontrolled FraC might find applications in photopharmacology for cancer therapeutics and has potential to be used for the fabrication of nanopore arrays in nanopore sensing devices, where the reconstitution, with high spatiotemporal precision, of single nanopores must be controlled.

Modular Pore-Forming Immunotoxins with Caged Cytotoxicity Tailored by Directed Evolution.

Immunotoxins are proteins containing a cell-targeting element linked to a toxin that are under investigation for next-generation cancer treatment. However, these agents are difficult to synthesize, chemically heterogeneous, expensive, and show toxicity toward healthy cells. In this work, we describe the synthesis and characterization of a new type of immunotoxin that showed exquisite selectivity toward targeted cells. In our construct, targeting molecules were covalently attached or genetically fused to oligomeric pore-forming toxins. The activity of the immunotoxin was then caged by fusing a soluble protein to the transmembrane domain and activated via cleavage with furin, which is a protease that is overexpressed in many cancer cells. During the several coupling steps, directed evolution allowed the efficient synthesis of the molecules in E. coli cells, as well as selection for further specificity toward targeted cells. The final construct showed no off-target activity, while acquiring an additional degree of specificity toward the targeted cells upon activation. The pore-forming toxins described here do not require internalization to operate, while the many protomeric subunits can be individually modified to refine target specificity.