Specific in vitro binding of a new (99m)Tc-radiolabeled derivative of the C-terminal decapeptide of prothymosin alpha on human neutrophils.

Prothymosin alpha (ProTα) is a conserved mammalian polypeptide with intracellular functions associated with cell proliferation and apoptosis and an extracellular role associated with immunopotentiation. The N-terminal fragment [1–28], which is identical with the immunostimulating peptide thymosin α1 (Tα1), was earlier considered as the immunoactive region of the polypeptide; however, recent data suggest that ProTα may exert a discrete immunomodulating action through its central or C-terminal region, via targeting Toll-like receptor- 4 (TLR4). In this work, a derivative of the C-terminal fragment ProTα[100-109] (ProTα-D1) that can be radiolabeled with 99mTc was developed. The biological activity of the non-radioactive 185/187rhenium-complex of this derivative ([185/187Re]ProTα-D1, structurally similar with [99mTc]ProTα-D1) was verified through suitable in vitro bioassays on human neutrophils. Subsequent cell-binding studies revealed specific, time-dependent and saturable binding of [99mTc]ProTα-D1 on neutrophils, which was inhibited by intact ProTα and ProTα[100-109], as well as by a “prototype” TLR4-ligand (lipopolysaccharide from Escherichia coli). Overall, our results support the existence of ProTα-binding sites on human neutrophils, recognizing [99mTc]ProTα-D1, which might involve TLR4. [99mTc]ProTα-D1 may be a useful tool for conducting further in vitro and in vivo studies, aiming to elucidate the extracellular mode of action of ProTα and, eventually, develop ProTα-based immunotherapeutics.