Fig 1: (A) Cellular uptake of [64Cu]Cu-Monomer_L, [64Cu]Cu-Monomer_D, and [64Cu]Cu-Dimer_D in CD38-positive MOLP2 cells at 4 °C for 30 min. Uptake was significantly higher for the d-amino-acid analog and further increased for the dimeric construct. Uptake was markedly reduced in the presence of a 250-fold excess (25 μM) of the corresponding unlabeled peptide, daratumumab (DARA), and isatuximab (ISA), confirming CD38-specific binding. (B) Representative saturation binding curves showing specific binding of [64Cu]Cu-Monomer_L, [64Cu]Cu-Monomer_D, and [64Cu]Cu-Dimer_D to MOLP2 cells obtained by nonlinear regression using a one-site binding model (1/Y2 weighting). Replacement of L-with d-amino acids improved affinity (K d = 1043 → ∼740 nM), and the dimeric analog exhibited a comparable K d (∼730 nM) but a markedly higher B max (3024 → 6993 fmol/mg), indicating enhanced avidity due to multivalency. (C) Cellular uptake of the dimeric analog, [64Cu]Cu-Dimer_D, in multiple CD38-expressing cell lines (MOLP2, MOLP8, MM1.S, and U266) following incubation at 4 °C for 30 min. (D) Time-dependent internalization of [64Cu]Cu-Dimer_D in MOLP2 cells at 37 °C. Statistical significance is indicated as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) and P < 0.0001 (****).
Fig 2: Molecular surface representation of human CD38 (PDB:2EF1, orange) highlighting the catalytic pocket (cyan) and the bound poses of the three copper conjugated peptides predicted through AutoDock Vina. (A) Cu-Monomer_ L, (B) Cu-Monomer_D, and (C) Cu-Dimer_D are shown in stick representation. The catalytic region was defined by residues E146, W125, R127, D155, W189, S193, and E226. All peptides docked within a surface groove adjacent to this catalytic pocket. The dimeric analog engaged a broader surface area consistent with its multivalent interaction model. The cyan region serves as a spatial reference indicating proximity of the peptide-binding site to the reported catalytic cavity of CD38.
Fig 3: Functional evaluation of CD38 enzymatic activity. CD38 NADase activity was measured using a fluorescence-based ε-NAD assay in the presence of Monomer_L, Monomer_D, and Dimer_D (0.1–100 μM, 37 °C, 30 min; Ex 300 nm/Em 410 nm). Fluorescence signals were corrected for intrinsic peptide fluorescence and normalized to control reactions without peptide. Enzymatic activity is expressed as percentage of control, and IC50 values were obtained by nonlinear regression. Data are shown as mean ± SD.
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