Interaction of Follicle-Stimulating Hormone (FSH) Receptor Binding Inhibitor-8: A Novel FSH- Binding Inhibitor, with FSH and its Receptor Swati S. Chitnis 1 , Chellasamy Selvaakumar 2 , Dhanashree D. Jagtap 1 , Ravi P. Barnwal 3 , Kandala V. R. Chary 3 , Smita D. Mahale 1 and Tarala D. Nandedkar 1, * 1 National Institute for Research in Reproductive Health, Indian Council of Medical Research, J.M. Street, Parel, Mumbai 400 012, India 2 Department of Bioinformatics, Padmashree Dr. D.Y. Patil University's Department of Biotechnology and Bioinformatics, CBD Belapur, Navi Mumbai 400614, India 3 Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India *Corresponding author: Tarala D. Nandedkar, cellbioirr@hotmail.com Follicle-stimulating hormone (FSH) receptor bind- ing inhibitor (FRBI-8) is a novel octapeptide puri- fied from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing folli- cles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI-8 and understand its mechanism of inhibiting inter- action of FSH to its receptors. Homology model- ing predicted that the FRBI-8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI- 8 with reported FSH–FSHR hormone binding (FSHR HB ) domain complex using ZDOCK algorithm revealed that the FRBI-8 binds to FSHbL2–FSHR HB binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI-8 analogs were designed by replac- ing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI-8 D6A ) had a higher binding affinity than the native FRBI-8. In vitro radioreceptor assay with FRBI-8 D6A showed 50% lower IC 50 compared with the FRBI- 8, confirming the in silico observations. Thus, the study reveals that both FRBI-8 and FRBI-8 D6A interfered with the binding of FSH to its receptor. Key words: circular dichroism spectra, follicle-stimulating hormone receptor, FSH receptor binding inhibitor-8, NMR spectroscopy, ZDOCK Received 4 February 2009, revised 19 March 2009 and accepted for publication 19 March 2009 Follicle-stimulating hormone (FSH) is a glycoprotein hormone secreted by the anterior pituitary gland and is associated with the major functions of the ovary like follicular development, maturation and ovulation (1,2). Follicle-stimulating hormone is a heterodimer consisting of a common a-subunit which it shares with other glycoprotein hormones namely leutinizing hormone (LH), chorionic gonadotropin and thyroid-stimulating hormone. The b-subunit is hormone-specific for a given species (3). Follicle-stimulating hormone acts via the FSH receptor (FSHR) which belongs to the G protein-coupled receptor family (4). These are present exclusively on granulosa cells of ovarian follicles in females (5). The FSHR is com- posed of 695 amino acid residues (aa) including the 17 aa long hydrophobic signal peptide. The 349 aa form the extracellular hor- mone binding (HB) domain, 264 aa form the transmembrane domain and the remaining 65 aa encode for the short carboxy terminal intracellular domain (6). Follicle-stimulating hormone is known to bind to the concave face of the curved HB domain of FSHR, which comprises the N-terminal Cys cluster along with the extracellular Leu-rich repeats (7). Binding of FSH to FSHR leads to mainly activa- tion of various G proteins resulting in cyclic AMP production (8) thereby regulating steroidogenesis (9). Our group has been working on a peptide purified from human ovarian follicular fluid (hOFF). Human ovarian follicular fluid was ultrafiltered and the filtrate was then passed through Sephadex G- 25 column (collected from patients undergoing in vitro fertilisation in Fertility Clinic, Mumbai, India). Of the fractions obtained by gel exclusion chromatography, the active fraction was identified as that exhibiting the FSH-binding inhibitory activity in a radioreceptor assay (RRA). This fraction was further purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The single frac- tion obtained after analytical RP-HPLC was sequenced. A partial N- terminal eight amino-acid residue long octapeptide (AESNEDGY) thus obtained is referred to as a FSH receptor binding inhibitor-8 (FRBI-8). Synthetic FRBI-8 specifically inhibits the binding of FSH to its receptors on granulosa cells (10) as no binding inhibition was observed in a human chorionic gonadotrophin (hCG) RRA wherein binding of hCG to LH receptors on luteal cells was not inhibited in the presence of FRBI-8 (data not shown). Further, in vitro, it also affects the FSH-induced progesterone (P 4 ) secretion by granulosa cells (11) and induces follicular atresia in mice (12). 637 Chem Biol Drug Des 2009; 73: 637–643 Research Article ª 2009 John Wiley & Sons A/S doi: 10.1111/j.1747-0285.2009.00810.x