Traditional photodynamic therapy for cancer relies on dye-photosensitized generation of singlet oxygen. However, therapeutically effective singlet oxygen generation requires well-oxygenated tissues, whereas many tumor environments tend to be hypoxic. We describe a platform for targeted enhancement of photodynamic therapy that produces singlet oxygen in oxygenated environments and hydroxyl radical, which is typically regarded as the most toxic reactive oxygen species, in hypoxic environments. The 24-subunit iron storage protein bacterioferritin (Bfr) has the unique property of binding 12 heme groups in its protein shell. We inserted the isostructural photosensitizer, zinc(II) protoporphyrin IX (ZnP), in place of the hemes and extended the surface-exposed N-terminal ends of the Bfr subunits with a peptide targeting a receptor that is hyperexpressed on the cell surface of many tumors and tumor vasculature. We then loaded the inner cavity with ∼2500 irons as a ferric oxyhydroxide polymer and finally conjugated 2 kDa polyethylene glycol to the outer surface. We showed that the inserted ZnP photosensitizes generation of both singlet oxygen and the hydroxyl radical, the latter via the reaction of photoreleased ferrous iron with hydrogen peroxide. This targeted iron-loaded ZnP-Bfr construct was endocytosed by C32 melanoma cells and localized to lysosomes. Irradiating the treated cells with light at wavelengths overlapping the ZnP Soret absorption band induced photosensitized intracellular Fe release and substantial lowering of cell viability. This targeted, light-triggered production of intracellular singlet oxygen and Fenton-reactive iron could potentially be developed into a phototherapeutic adjunct for many types of cancers.
Ferritins and bacterioferritins (Bfrs) utilize a binuclear non-heme iron binding site to catalyze oxidation of Fe(II), leading to formation of an iron mineral core within a protein shell. Unlike ferritins, in which the diiron site binds Fe(II) as a substrate, which then autoxidizes and migrates to the mineral core, the diiron site in Bfr has a 2-His/4-carboxylate ligand set that is commonly found in diiron cofactor enzymes. Bfrs could, therefore, utilize the diiron site as a cofactor rather than for substrate iron binding. In this study, we applied circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field MCD (VTVH-MCD) spectroscopies to define the geometric and electronic structures of the biferrous active site in Escherichia coli Bfr. For these studies, we used an engineered M52L variant, which is known to eliminate binding of a heme cofactor but to have very minor effects on either iron oxidation or mineral core formation. We also examined an H46A/D50A/M52L Bfr variant, which additionally disrupts a previously observed mononuclear non-heme iron binding site inside the protein shell. The spectral analyses define a binuclear and an additional mononuclear ferrous site. The biferrous site shows two different five-coordinate centers. After O2 oxidation and re-reduction, only the mononuclear ferrous signal is eliminated. The retention of the biferrous but not the mononuclear ferrous site upon O2 cycling supports a mechanism in which the binuclear site acts as a cofactor for the O2 reaction, while the mononuclear site binds the substrate Fe(II) that, after its oxidation to Fe(III), migrates to the mineral core.
Non‐heme diiron‐carboxylate ( NHDC ) active sites are found in several classes of monooxygenases, oxidases, and dioxygen transport or sensing proteins, as well as a few metallohydrolases. The unique and defining structural feature of NHDC sites consists of two non‐heme irons bridged by at least one carboxylate‐containing amino acid residue. A second characteristic feature is a bridging solvent ligand, either oxo or hydroxo in at least the diferric state. NHDC sites also feature terminal ligands derived from histidine and carboxylate residues. The hemerythrin superfamily functions have expanded from reversible dioxygen binding to include sensing of iron, dioxygen, or redox status. Substantial progress has been made in characterization of flavo‐diiron enzymes, including its nitric oxide reductase mechanism. A diiron urease is a recent addition to the metallohydrolase family. The variety of characterized NHDC O 2 ‐activating enzymes has increased substantially since the previous compilation in this encyclopedia. Novel monooxygenation mechanisms have been identified.
Targeted exogenous agents that rapidly elevate reactive oxygen species (ROS) above toxic threshold levels has been touted as a potentially effective cancer therapeutic strategy. The “traditional” ROS‐mediated cancer therapy, referred to as photodynamic therapy (PDT), relies on dye‐photosensitized generation of singlet oxygen. However, therapeutically effective singlet oxygen generation requires well‐oxygenated tissues, whereas many tumor environments tend to be hypoxic. We describe here a potential alternative to PDT that would generate hydroxyl radical rather than singlet oxygen as the ROS, and do so in a tumor‐targeted fashion. Hydroxyl radical (OH•) is typically regarded as the most toxic among ROS, and can be generated by the Fenton reaction of ferrous iron with relatively low levels of hydrogen peroxide. We provide proof‐of‐principle for a strategy to flood tumor cells with Fenton reactive iron. The iron storage protein bacterioferritin (Bfr) has the unique property of binding 12 heme groups in its protein shell. We substituted the hemes with the well‐known photosensitizer, zinc protoporphryin IX and loaded the inner cavity of Bfr with ~1000 irons as a ferric oxyhydroxide polymer. We also fused the RGD4C tumor‐targeting peptide to the outer surface of the 24 subunits comprising the Bfr protein shell. Irradiation with visible light released ferrous iron and led to light‐dependent killing of C32 melanoma cells. Iron is a relatively cheap pro‐drug and our approach should be generally applicable to many types of cancers.
An iron storage protein called bacterioferritin (Bfr) has been adapted for a novel approach to cancer therapy. Tumor‐targeting peptides (TTPs) have been fused to the protein and photosensitizers, like zinc protoporphyrin (ZnPPIX), have been substituted into the native heme binding sites. After binding to cancer cell receptors, photo‐excitation would trigger release of iron to generate a flux of toxic hydroxyl radicals that would overwhelm a cell's defenses.The gene encoding the TTP‐Bfr was synthesized and inserted into an E. coli expression plasmid and was readily expressed with ZnPPIX and purified by standard methods. The protein was then loaded by anaerobic incubation of iron. Bfr solutions were irradiated for various time points. Ferrozine was added to quantitate the iron release. These procedures reproducibly resulted in soluble fully loaded [Fe~1,000ZnPPIX12TTP24‐Bfr]. After 3 hours irradiation, ~30% of the iron was released. At longer irradiation times, 65–70% of the iron was released. No iron was released from dark identical control solutions. We have established that [Fe~1,000ZnPPIX12TTP24‐Bfr] releases significant amounts of iron upon irradiation. Current work is aimed at assessing binding of this protein to melanoma cells, and assessing inhibition of cell growth or apoptosis upon irradiation. Results of these investigations could provide proof of principle for a new approach to cancer therapy.
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