Actinium chelation and crystallization in a macromolecular scaffold

Abstract

Targeted alpha therapy (TAT) pairs the specificity of antigen targeting with the lethality of alpha particles to eradicate cancerous cells. Actinium-225 [²²⁵Ac; t_1/2 = 9.920(3) days] is an alpha-emitting radioisotope driving the next generation of TAT radiopharmaceuticals. Despite promising clinical results, a fundamental understanding of Ac coordination chemistry lags behind the rest of the Periodic Table due to its limited availability, lack of stable isotopes, and inadequate systems poised to probe the chemical behavior of this radionuclide. In this work, we demonstrate a platform that combines an 8-coordinate synthetic ligand and a mammalian protein to characterize the solution and solid-state behavior of the longest-lived Ac isotope, ²²⁷Ac [t_1/2 = 21.772(3) years]. We expect these results to direct renewed efforts for ²²⁵Ac-TAT development, aid in understanding Ac coordination behavior relative to other +3 lanthanides and actinides, and more broadly inform this element's position on the Periodic Table.

Fig. 1. Generalized scheme of the work presented herein that aims to understand the coordination of an actinium hydroxypyridinone complex contained within a macromolecular scaffold.

a Synthetic route to an actinium small molecule complex encapsulated in a macromolecule through chelation of ²²⁷Ac^III with the 3,4,3-LI(1,2-HOPO) (HOPO) chelator and the siderocalin (Scn) protein. Under buffered conditions, HOPO binds Ac^III to yield a monoanionic coordination complex, nominally termed [Ac^III(HOPO)]^1–. Through electrostatic interactions with ligand aryl groups and amino acid residues, [Ac^III(HOPO)]^1– is then contained in the binding pocket of Scn to yield a ternary Ac^III–HOPO–Scn protein complex. The binding between HOPO, Ac^III, and Scn was probed using spectrofluorimetric competition titrations and fluorescence quenching binding assays. b The ternary Ac^III–HOPO–Scn protein complex was crystallized to structurally characterize actinium coordination within Scn. After Ac^III–HOPO–Scn was formed in solution, further purification was provided through protein concentration filters wherein superfluous ions, such as the radioactive daughters that had grown in since purification of the Ac^III stock solution, were filtered out. After one week, diffraction-quality crystals of the ternary Ac^III–HOPO–Scn complex were observed and structurally characterized with protein crystallography.

Fig. 2. Spectrofluorometric competition titrations used to assess the stability constants of trivalent lanthanides and actinides with the HOPO chelator.

a–c, e Surrogate experiments with La^III were successively scaled down to repeat the experiment with limited mass quantities of Ac^III. Competition against the [Eu^III(HOPO)]^1– complex at concentrations of (a) 3 $\mu$M [Eu^III(HOPO)]^1–, (b) 50 nM [Eu^III(HOPO)]^1–, and (c) 10 nM [Eu^III(HOPO)]^1– were performed with La^III to demonstrate the merit of the methodology, resulting in similar conditional stability constants (log ${\beta }_{\mathrm{ML}}^{\prime }$) tabulated in e. The error reported is the standard deviation corresponding to the last digit in triplicate measurements. d Spectrofluorimetric competition titrations with ²²⁷Ac^III and [Eu^III(HOPO)]^1– resulting in a conditional stability constant (log ${\beta }_{\mathrm{ML}}^{\prime }$) tabulated in e. ([Eu^III(HOPO)]^1– = 10 nM, I = 0.5 M KCl, pH = 7.36, T = 25 °C, λ_ex = 325 nm. f Stability constants of trivalent lanthanide and actinide ions with HOPO to yield the respective [M^III(HOPO)]^1– complex plotted as a function of ionic radii. The log ${\beta }_{\mathrm{ML}}^{\prime }$ values for Ac and La are reported herein (c, d), whereas the remaining values are from refs. ^–. The error reported is the standard deviation corresponding to the last digit in triplicate measurements, except for americium, which is the standard deviation corresponding to the last digit in duplicate measurements. The ionic radii are from ref. . (Ac) and ref. . for CN = 6. Note, the stability constants increase across the 4f and 5f series.

Fig. 3. Fluorescence quenching binding assays as a function of titrant concentration and subsequent determination of dissociation constants and cumulative stability constants.

a Luminescent behavior of tryptophan residues within the siderocalin (Scn) protein. Fluorescence ($\lambda$_ex = 281 nm; blue trace) is quenched upon titrant recognition in the Scn binding pocket ($\lambda$_ex = 281 nm; red trace), which can be used to quantify the interaction between the protein and the titrant to yield a dissociation constant (K_D). Fluorescence quenching analyses of Scn with HOPO (b), [La^III(HOPO)^1– (c), and [Ac^III(HOPO)^1– (d) at pH 7.4. Blue squares, purple triangles, and green diamonds denote the normalized fluorescence intensities at 340 nm ($\lambda$_ex = 281 nm) and the error bars represent the standard deviation of three separate trials. The dotted gray fit lines were calculated with a one-binding site model. e Tabulated K_D values from fluorescence quenching analyses in b–d. The Am^III value is from ref. . The log ${\beta }_2^{\prime }$ value, which represents the binding constant of the ligand or metal complex with Scn, was calculated by taking the inverse log of the K_D. The error reported is the standard deviation corresponding to the last digit in triplicate measurements. f The cumulative stability constant (log ${\beta }_{\mathrm{1,2}}^{\prime }$) of HOPO–Scn with Ac^III compared to other chelators, including DOTA, BATA, and EDTA. The asterisk denotes a conditional stability constant.

Fig. 4. Protein crystallographic renderings of [Ac^III(HOPO)]^1– and [La^III(HOPO)]^1–complexes inside the calyx of siderocalin (Scn).

a Overlay of [Ac^III(HOPO)]^1– and [La^III(HOPO)]^1–complexes inside the calyx of Scn. The protein is shown in a molecular surface representation (gray), the chelating groups are shown in a licorice-stick representation, colored by atom type (carbon = gray, nitrogen = blue, oxygen = red), and the metals are shown as colored spheres (Ac = magenta, La = gray). The protein binding pockets are noted in yellow text and one of the amino acid residues (K125) that defines Pocket #1 is labeled. Red arrows highlight the positions of the HOPO groups wherein the rings are geometrically different between the La and Ac structures. b Representation of the interactions about the Ac^III metal center with HOPO rings, identifying the oxygen interactions with either N-oxide or ketone oxygen atoms.