Research Article: An in vivo half-life extended prolactin receptor antagonist can prevent STAT5 phosphorylation

Date Published: May 7, 2019

Publisher: Public Library of Science

Author(s): Shengze Yu, Amira Alkharusi, Gunnar Norstedt, Torbjörn Gräslund, Kevin A. Henry.


Increasing evidence suggests that signaling through the prolactin/prolactin receptor axis is important for stimulation the growth of many cancers including glioblastoma multiforme, breast and ovarian carcinoma. Efficient inhibitors of signaling have previously been developed but their applicability as cancer drugs is limited by the short in vivo half-life. In this study, we show that a fusion protein, consisting of the prolactin receptor antagonist PrlRA and an albumin binding domain for half-life extension can be expressed as inclusion bodies in Escherichia coli and efficiently refolded and purified to homogeneity. The fusion protein was found to have strong affinity for the two intended targets: the prolactin receptor (KD = 2.3±0.2 nM) and mouse serum albumin (KD = 0.38±0.01 nM). Further investigation showed that it could efficiently prevent prolactin mediated phosphorylation of STAT5 at 100 nM concentration and above, similar to the PrlRA itself, suggesting a potential as drug for cancer therapy in the future. Complexion with HSA weakened the affinity for the receptor to 21±3 nM, however the ability to prevent phosphorylation of STAT5 was still prominent. Injection into rats showed a 100-fold higher concentration in blood after 24 h compared to PrlRA itself.

Partial Text

Prolactin (Prl) is a hormone that exerts its functions by homo-dimerization and activation of the prolactin receptor (PrlR) [1]. The Prl/PrlR axis is present in most vertebrates and is involved in more than 300 discrete biological functions, such as stimulation of body growth, stimulation of development during gestation, cell proliferation, homeostasis of different electrolytes etc. The major source of prolactin production in the human body is the pituitary gland. In addition, decidua, prostate, mammary and ovarian tissue as well as vascular endothelial cells and immune cells have been found to produce Prl locally [2]. The Prl/PrlR axis acts on several intracellular pathways [3]. One of the major signaling cascades involves activation of Janus kinase 2, which phosphorylates and activates several down-stream proteins including signal transducer and activator of transcription 5 (STAT5) [4]. Activated STAT5 forms a dimer that is translocated to the nucleus where it acts as a transcription factor on specific DNA elements.

Long acting antagonists of signaling through the Prl/PrlRA axis are highly attractive due to their potential as drugs to treat a number of different diseases, including cancer of various kinds. In this study, we have designed and investigated a half-life extended prolactin receptor antagonist (PrlRA) in the form of a fusion protein consisting of the antagonist with an N-terminal albumin binding domain (ABD). Since the proposed mechanism of action of the PrlRA is based on its strong interaction with only one prolactin receptor molecule, an important consideration was where to attach the ABD so that it would not interfere with the PrlRA/PrlR interaction. The ABD has been found to endow fusion proteins with an extended half-life when placed either at the N- or C-terminus or in the middle of a fusion protein [24–26], so the placement would likely not be constrained by the ABD. The crystal structure between N-terminally truncated G129R-Prl and the PrlR has been determined [27] and clearly shows that the C-terminus of G129R-Prl is directly engaged in the interaction with PrlR, but the N-terminus is more distant from the interface. Based on the crystal structure, the ABD was therefore placed in the N-terminus of the PrlRA.