A Novel, More Efficient Approach to Generate Bioactive Inhibins.

Gonadal-derived inhibins are essential factors in mammalian reproduction, negatively regulating pituitary production of FSH. Interestingly, declines in inhibin levels across the menopause transition correlate with not only an increase in FSH but also a rapid decrease in bone mass. Therefore, inhibins have been touted as potential therapeutics for osteoporosis in postmenopausal women. However, as heterodimeric proteins of α- and ß- (ßA or ßB)-subunits, inhibins are difficult to produce recombinantly, are poorly processed to their mature bioactive forms, and their expression is always accompanied by production of activins (ß-subunit homodimers), the proteins they antagonize. In this study, we developed the methodology to circumvent most of these issues. Initially, the cleavage sites between the pro- and mature domains of the α- and ßA-subunits were modified to ensure complete processing. These modifications led to a marked increase (9-fold) in the levels of bioactive inhibin A and a striking decrease (12.5-fold) in mature activin A production. Next, a single point mutation (M418A) was incorporated into the ßA-subunit, which reduced residual activin activity approximately 100-fold and, in so doing, increased inhibin bioactivity 8-fold. Finally, we showed that inhibin A noncovalently associated with its prodomain was more potent (∼20-fold) than mature inhibin A in specific in vitro bioassays, indicating an important role of the prodomain in inhibin bioactivity. In conclusion, the production of potent inhibin analogs in the virtual absence of activin activity will greatly facilitate the investigation of the therapeutic potential of these gonadal hormones on bone and other tissues.

[Synthesis and activities of fragments of inhibin alpha subunit].

For further investigation of inhibin which is available only by laborious isolation, four fragments of inhibin alpha subunit (7-28, 37-65, 1-32, Y-1-32) were prepared by solid phase peptide synthesis using the Fmoc strategy. The effects of these fragments on progesterone production of rat corpus luteal (CL) cells in vitro were examined. The results indicate that these four fragments induced significant decrease of basic progesterone production.

[Syntheses and preliminary bioassays of inhibin segments].

Six segments within the subunit beta-A of the follicular inhibin have been synthesized by an improved solid phase procedure on p-methyl-benzhydrylamine (1% divinylbenzene) resin. The courses of the syntheses were monitored by quantitative ninhydrin assays and amino acid analyses. Following the cleavage by HF-p-cresol (9:1 V/V), the peptides were extracted with anhydrous trifluoroacetic acid containing 1% dithiothreitol and precipitated with dry ethyl ether. The purification was achieved uniquely by reverse phase HPLC and final products were characterized by several TLC systems, analytical HPLC and amino acid analyses. Pituitary cell culture bioassays were performed to ascertain their biological activities. However, among these synthetic peptides, the small peptides Ib-beta A (37-39)NH2(I), Ib-beta A(34-39)NH2(II), and Ib-beta A(30-39)NH2(III) showed no significant suppression on the LHRH-induced FSH secretion; the large peptides Ib-beta A(23-39)NH2(IV), Ib-beta A(16-39)NH2(V), and Ib-beta A(14-39)NH2(VI) lack adequate solubility in neutral media, and other methods are to be sought to test their bioactivities.

New segment synthesis of alpha-inhibin-92 by the acyl disulfide method.

The thiocarboxyl group reacts with diaryl disulfides to give an unsymmetrical acyl disulfide in dimethylformamide (DMF) and a symmetrical diacyl disulfide in aqueous DMF. Both acyl disulfides react with the alpha-amino group to form the peptide bond. The method was used in a new segment synthesis of alpha-inhibin-92 (alpha-IB-92) with use of 2,2'-dipyridyl disulfide as activator. Thiocarboxyl peptides were synthesized by the solid-phase method on 4-[alpha-(Boc-Gly-S)benzyl]phenoxyacetamidomethyl-resin. The segments alpha-IB-92-(1-34)SH (I), Msc-alpha-IB-92-(35-65)SH (II), Msc-alpha-IB-92(66-92)OH (III), and Msc-alpha-IB-92-(35-92)OH (VI) were prepared in yields of 33, 36, 41, and 25%, respectively, with use of crystalline symmetrical anhydrides in double and triple coupling protocols. Segments I, II, and III were used in a 3-segment synthesis of alpha-IB-92 with an overall yield based on starting resin of about 8% while a 2-segment synthesis using I and IV gave 11%. An all stepwise synthesis of alpha-IB-92 gave 4.5%.

Synthetic peptide with inhibin-like activity preferentially inhibits follitropin secretion in comparison with lutropin-releasing hormone antagonists.

Biological activity of a synthetic peptide with inhibin-like activity under in vitro and in vivo conditions was compared with three highly potent synthetic lutropin-releasing hormone antagonists. Unlike the synthetic lutropin-releasing hormone antagonists, which effectively inhibited both lutropin and follitropin secretion from the pituitary, the inhibin-like peptide showed a preferential effect by inhibiting follitropin release both in vitro and in vivo. Thus, small peptides such as inhibin-like peptide with a sequence unrelated to lutropin-releasing hormone may provide a basis for design of selective inhibitors of gonadotropin release.

Chemical synthesis of alpha-inhibin-92 by the thiocarboxyl segment coupling method.

The amino acid residue peptide, alpha-inhibin-92 (alpha-IB-92), has been synthesized by the thiocarboxyl segment strategy. Three segments were synthesized by the solid phase method, purified, and characterized: [GlyS34]-alpha-IB-92-(1-34) (I), CF3CO-[GlyS65]-alpha-IB-92-(35-65) (II), and Msc-alpha-IB-92-(66-92) (III). All were reacted with citraconic anhydride followed by removal of the Msc group in III to give Ia, IIa, and IIIa, respectively. Peptide IIIa was coupled to IIa by the silver nitrate/N-hydroxysuccinimide procedure and, after removal of uncoupled segments and the trifluoroacetyl group, Ia was coupled followed again by removal of uncoupled segments. Final deblocking to remove citraconyl groups was accomplished under exceptionally mild conditions in aqueous acetic acid. The synthetic product was identical to natural alpha-IB-92 in amino acid analysis, HPLC, gel electrophoresis, and tryptic mapping. The synthetic peptide was indistinguishable from natural alpha-IB-92 in a radioimmunoassay and in an in vitro mouse pituitary assay for measuring suppression of FSH release in the presence of LHRH.

Peptides of postulated inhibin activity. Lack of in vitro inhibin activity of a 94-residue peptide isolated from human seminal plasma, and of a synthetic replicate of its C-terminal 28-residue segment.

A 94-residue polypeptide isolated from human seminal plasma and its chemically synthesized C-terminal 28-residue segment were studied in an in vitro inhibin bioassay utilizing rat pituitary cell cultures. Both peptides have previously been claimed to have inhibin activities, and the effects on the secretion and cellular content of gonadotrophins (FSH and LH) were now assessed in the in vitro assay. No inhibition was found. After 72 h of culture, both the cellular content and the spontaneous as well as the LHRH-stimulated release of bioactive or immunoactive FSH and LH remained unaffected. Similarly, no effects were found on the storage and/or release of prolactin, growth hormone, or thyrotropin. We conclude that both the native 94-residue peptide and the synthetic replicate of its C-terminal 28-residue segment, do not influence the pituitary FSH secretion when assessed in this in vitro system.