Synthesis and Preclinical Evaluation of PSMA-Targeted 111In-Radioconjugates Containing a Mitochondria-Tropic Triphenylphosphonium Carrier.

Nuclear DNA is the canonical target for biological damage induced by Auger electrons (AE) in the context of targeted radionuclide therapy (TRT) of cancer, but other subcellular components might also be relevant for this purpose, such as the energized mitochondria of tumor cells. Having this in mind, we have synthesized novel DOTA-based chelators carrying a prostate-specific membrane antigen (PSMA) inhibitor and a triphenyl phosphonium (TPP) group that were used to obtain dual-targeted 111In-radioconjugates ([111In]In-TPP-DOTAGA-PSMA and [111In]In-TPP-DOTAGA-G3-PSMA), aiming to promote a selective uptake of an AE-emitter radiometal (111In) by PSMA+ prostate cancer (PCa) cells and an enhanced accumulation in the mitochondria. These dual-targeted 111In-radiocomplexes are highly stable under physiological conditions and in cell culture media. The complexes showed relatively similar binding affinities toward the PSMA compared to the reference tracer [111In]In-PSMA-617, in line with their high cellular uptake and internalization in PSMA+ PCa cells. The complexes compromised cell survival in a dose-dependent manner and in the case of [111In]In-TPP-DOTAGA-G3-PSMA to a higher extent than observed for the single-targeted congener [111In]In-PSMA-617. µSPECT imaging studies in PSMA+ PCa xenografts showed that the TPP pharmacophore did not interfere with the excellent in vivo tumor uptake of the "golden standard" [111In]In-PSMA-617, although it led to a higher kidney retention. Such kidney retention does not necessarily compromise their usefulness as radiotherapeutics due to the short tissue range of the Auger/conversion electrons emitted by 111In. Overall, our results provide valuable insights into the potential use of mitochondrial targeting by PSMA-based radiocomplexes for efficient use of AE-emitting radionuclides in TRT, giving impetus to extend the studies to other AE-emitting trivalent radiometals (e.g., 161Tb or 165Er) and to further optimize the designed dual-targeting constructs.

Stability profile of vimentin rod domain.

Intermediate filaments (IFs) form an essential part of the metazoan cytoskeleton. Despite a long history of research, a proper understanding of their molecular architecture and assembly process is still lacking. IFs self-assemble from elongated dimers, which are defined by their central "rod" domain. This domain forms an α-helical coiled coil consisting of three segments called coil1A, coil1B, and coil2. It has been hypothesized that the structural plasticity of the dimer, including the unraveling of some coiled-coil regions, is essential for the assembly process. To systematically explore this possibility, we have studied six 50-residue fragments covering the entire rod domain of human vimentin, a model IF protein. After creating in silico models of these fragments, their evaluation using molecular dynamics was performed. Large differences were seen across the six fragments with respect to their structural variability during a 100 ns simulation. Next, the fragments were prepared recombinantly, whereby their correct dimerization was promoted by adding short N- or C-terminal capping motifs. The capped fragments were subjected to circular dichroism measurements at varying temperatures. The obtained melting temperatures reveal the relative stabilities of individual fragments, which correlate well with in silico results. We show that the least stable regions of vimentin rod are coil1A and the first third of coil2, while the structures of coil1B and the rest of coil2 are significantly more robust. These observations are in line with the data obtained using other experimental approaches, and contribute to a better understanding of the molecular mechanisms driving IF assembly.