GRPR inhibitors(Alpha-9 Theranostics)

The gastrin-releasing peptide receptor (GRPR) is overexpressed in a variety of cancers and represents a promising target for diagnosis and therapy. However, the extremely high accumulation in the pancreas observed for most of the clinically evaluated GRPR-targeted radiopharmaceuticals could limit their applications. In this study, we synthesized one GRPR antagonist (ProBOMB5) and two GRPR agonists (LW02056 and LW02057) by replacing the 4-thiazolidinecarboxylic acid (Thz¹⁴) residue in our previously reported GRPR-targeted tracers with Pro¹⁴. The ⁶⁸Ga and ¹⁷⁷Lu labeling were conducted in HEPES (2 M, pH 5.0) buffer and acetate (0.1 M, pH 4.5) buffer, respectively, and the radiolabeled products were obtained in a 24-57% decay-corrected radiochemical yield and >92% radiochemical purity. The binding affinities (K_i) of Ga-ProBOMB5, Ga-LW02056, Ga-LW02057, and Lu-ProBOMB5 were measured via in vitro competition binding assays and were 12.2 ± 1.89, 14.7 ± 4.81, 13.8 ± 2.24, and 13.6 ± 0.25 nM, respectively. The PET imaging and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice at 1 h post injection. [⁶⁸Ga]Ga-ProBOMB5, [⁶⁸Ga]Ga-LW02056, and [⁶⁸Ga]Ga-LW02057 enabled clear tumor visualization in PET images. The tumor uptake values of [⁶⁸Ga]Ga-ProBOMB5, [⁶⁸Ga]Ga-LW02056, and [⁶⁸Ga]Ga-LW02057 were 12.4 ± 1.35, 8.93 ± 1.96, and 7.64 ± 0.55%ID/g, respectively, and their average pancreas uptake values were minimal (0.60-1.37%ID/g). Longitudinal SPECT imaging and ex vivo biodistribution studies were also conducted for [¹⁷⁷Lu]Lu-ProBOMB5 and clinically validated [¹⁷⁷Lu]Lu-RM2. Despite comparable tumor uptake at 1 h post injection ([¹⁷⁷Lu]Lu-ProBOMB5:8.09 ± 1.70%ID/g; [¹⁷⁷Lu]Lu-RM2:7.73 ± 0.96%ID/g), a faster clearance from PC-3 tumor xenografts was observed for [¹⁷⁷Lu]Lu-ProBOMB5, leading to a lower radiation-absorbed dose delivered to tumors. Our data demonstrate that [⁶⁸Ga]Ga-ProBOMB5 is a promising tracer for clinical translation for detecting GRPR-expressing tumor lesions. However, further optimizations are needed for [¹⁷⁷Lu]Lu-ProBOMB5 to prolong tumor retention for therapeutic applications.

Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β₂ and α₂ subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-β interface that restricts the ability of β₂ to obtain a position that is capable of radical transfer. ATP breaks the α-β interface, freeing β₂ and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α-β interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.