  Biased signaling has recently emerged as an interesting mean to modulate the
function of many G protein-coupled receptors (GPCRs). Previous studies reported
two negative allosteric modulators (NAMs) of follicle-stimulating hormone
receptor (FSHR), ADX68692 and ADX68693, with differential effects on
FSHR-mediated steroidogenesis and ovulation. In this study, we attempted to
pharmacologically profile these NAMs on the closely related
luteinizing/chorionic gonadotropin hormone receptor (LH/CGR) with regards to
its canonical Gs/cAMP pathway as well as \beta-arrestin recruitment in HEK293
cells. The NAMs effects on progesterone and testosterone production were also
assessed in murine Leydig tumor cell line (mLTC-1). We found that both NAMs
strongly antagonized LH/CGR signaling in both HEK293 and mLTC-1 cells. ADX68693
appeared more potent than ADX68692 to inhibit hCG-induced cAMP and
\beta-arrestin 2 in HEK293 and mLTC-1 cells whereas no significant difference
in their efficacy on hCG-promoted \beta-arrestin 2 recruitment. Interestingly,
differential antagonism of the two NAMs on hCG-promoted steroidogenesis in
mLTC-1 cells was observed with significant inhibition of testosterone but not
progesterone production. These observations suggest biased effects of the two
NAMs on LH/CGR-dependent pathways controlling steroidogenesis, which appeared
to be different to that previously shown on FSHR. This also illustrates the
complexity of signaling pathways controlling FSHR- and LH/CGR-mediated
steroidogenesis, suggesting differential implication of cAMP and
\beta-arrestins. Together, our data demonstrate that ADX68692 and ADX68693 are
NAMs at the LH/CGR in addition to FSHR. These pharmacological characteristics
are important to consider for potential contraceptive and therapeutic
applications based on such compounds.
