Applying the Fe(III) Binding Property of a Chemical Transferrin Mimetic to Ti(IV) Anticancer Drug Design

As an endogenous serum protein binder of Ti­(IV), transferrin (Tf) serves as an excellent vehicle to stabilize the hydrolysis prone metal ion and successfully transport it into cells. This transporting role is thought to be central to Ti­(IV)’s anticancer function, but efforts to synthesize Ti­(IV) compounds targeting transferrin have not produced a drug. Nonetheless, the Ti­(IV) transferrin complex (Ti₂Tf) greatly informs on a new Ti­(IV)-based anticancer drug design strategy. Ti₂Tf interferes with cellular uptake of Fe­(III), which is particularly detrimental to cancer cells because of their higher requirement for iron. Ti­(IV) compounds of chemical transferrin mimetic (cTfm) ligands were designed to facilitate Ti­(IV) activity by attenuating Fe­(III) intracellular levels. In having a higher affinity for Fe­(III) than Ti­(IV), these ligands feature the appropriate balance between stability and lability to effectively transport Ti­(IV) into cancer cells, release Ti­(IV) via displacement by Fe­(III), and deplete the intracellular Fe­(III) levels. The cTfm ligand <i>N,N</i>′-di­(<i>o</i>-hydroxybenzyl)­ethylenediamine-<i>N,N</i>′-diacetic acid (HBED) was selected to explore the feasibility of the design strategy. Kinetic studies on the Fe­(III) displacement process revealed that Ti­(IV) can be transported and released into cells by HBED on a physiologically relevant time scale. Cell viability studies using A549 cancerous and MRC5 normal human lung cells and testing the cytotoxicity of HBED and its Ti­(IV), Fe­(III), and Ga­(III) compounds demonstrate the importance of Fe­(III) depletion in the proposed drug design strategy and the specificity of the strategy for Ti­(IV) activity. The readily derivatized cTfm ligands demonstrate great promise for improved Ti­(IV) anticancer drugs

Metallopeptidase inhibitors potently block SP degradation in spinal cord lysates.

<p>A) Different class-selective inhibitors were tested for their ability to slow SP degradation in spinal cord membrane lysates. The most effective compound at inhibiting SP degradation in this assay is O-phenanthroline, a metalloprotease inhibitor. B) O-phenanthroline was also the most potent inhibitor of SP_1–9 production in these experiments. C) Multiple class-selective inhibitors regulate SP1-7 production including O-phenanthroline, pepstatin A and PMSF. (Statistical significance calculated by a Student's t-test; p-value <0.05, *; p-value <0.01, **; p-value <0.001, ***, N = 4).</p

SP is regulated by proteolysis but not by NEP.

<p>A) Phosphoramidon slows SP_1–9 production in tissue lysates, which suggests that NEP might have a role in SP processing. Experiments in NEP^+/+ and NEP^–/– spinal cord lysates reveals no significant difference in SP degradation. B) Likewise, no difference in endogenous SP levels is observed in spinal cords from NEP^+/+ and NEP^–/– mice. C) Acute treatment of mice with GM6001 results in a 3-fold elevation of SP in the spinal cord to reveal a GM6001-sensitive pathway for SP regulation. (Statistical significance calculated by a Student's t-test; p-value <0.001, ***, N = 4).</p

Absolute quantities of SP and SP fragments in the spinal cord as measured by isotope dilution mass spectrometry (IDMS).

<p>Absolute quantities of SP and SP fragments in the spinal cord as measured by isotope dilution mass spectrometry (IDMS).</p

GM6001 is significantly more effective than other metallopeptidase inhibitors at preventing SP degradation and conversion of SP to SP_1–9.

<p>A) Utilizing the MEROPS and Allen Brain Map databases a number of candidate metallopeptidases in the nervous system that are capable of cleaving SP to produce SP_1–9 are identified. Inhibitors against these peptidases were then used in lysates to evaluate their affect on SP degradation and SP_1–9 production. B) The matrix metalloprotease (MMP) inhibitor GM6001 was the most effective compound at preventing SP degradation. C) GM6001 is also the best inhibitor of SP_1–9 production. (Statistical significance calculated by a Student's t-test; p-value <0.05, *; p-value <0.01, **; p-value <0.001, ***, N = 4).</p

C-terminal processing is the primary mode of SP degradation.

<p>A) An integrated approach that combines chemical screening and peptide profiling provides a new strategy to determine whether proteolysis plays a role in the regulation of endogenous SP levels. B) Initial experiments begin in tissue lysates and the data clearly shows that SP is processed by membrane proteases to generate a series of C-terminally truncated fragments, while the soluble proteome has little impact on SP processing.</p

Endosomal localization of Cytc-Tf conjugate in A549 cells using confocal imaging.

<p>(A) A549 cells were treated with the Cyt c-Tf conjugate for 12 h and endosome localization was probed with an endosomal marker antibody (primary antibody against EEA1 and a secondary antibody conjugated with Alexafluor-555) and Cyt c was localized with an anti-Cyt c FITC labeled antibody. The upper panel shows untreated cells and the lower panel the cells treated with Cyt c-Tf. (B) Merged image of A549 cells treated with conjugate enlarged to show nonover lapping green (Cyt c) and red (endosomes) signal.</p

Inducing cell death in vitro in cancer cells by targeted delivery of cytochrome c via a transferrin conjugate

<div><p>One of the major drawbacks of many of the currently used cancer drugs are off-target effects. Targeted delivery is one method to minimize such unwanted and detrimental events. To actively target lung cancer cells, we have developed a conjugate of the apoptosis inducing protein cytochrome c with transferrin because the transferrin receptor is overexpressed by many rapidly dividing cancer cells. Cytochrome c and transferrin were cross-linked with a redox sensitive disulfide bond for the intra-cellular release of the protein upon endocytosis by the transferrin receptor. Confocal results demonstrated the cellular uptake of the cytochrome c-transferrin conjugate by transferrin receptor overexpressing A549 lung cancer cells. Localization studies further validated that this conjugate escaped the endosome. Additionally, an in vitro assay showed that the conjugate could induce apoptosis by activating caspase-3. The neo-conjugate not only maintained an IC₅₀ value similar to the well known drug cisplatin (50 μM) in A549 cancer cells but also was nontoxic to the normal lung (MRC5) cells. Our neo-conjugate holds promise for future development to target cancers with enhanced transferrin receptor expression.</p></div

Size determination (hydrodynamic radii) using dynamic light scattering measurements.

<p>(A) Tf (B) Cyt c-Tf conjugate (C) Reduced Cyt c-Tf conjugate after incubation with 10 mM of TCEP.</p

Three step synthesis of the Cyt c-Tf conjugate.

<p>In the first step, the proteins (molar ratio of 6 Cyt c:Tf) were reacted with the crosslinker (Sulfo-LC SPDP). In the second step, the LC-SPDP bound Cyt c was reduced to expose the–SH group. In the last step reduced Cyt c-LC-SPDP and Tf-LC-SPDP were incubated to obtain the final conjugate. The final protein conjugate was purified on a Superdex-200 10/300 column. The final conjugate contained a population of Cyt c-Tf conjugates with molar ratios of Cyt c to Tf varying from 1 to 5 Cyt c molecules attached to one Tf molecule.</p