Synthesis and structure-activity studies on novel analogs of human growth hormone releasing hormone (GHRH) with enhanced inhibitory activities on tumor growth.

The syntheses and biological evaluations of new GHRH analogs of Miami (MIA) series with greatly increased anticancer activity are described. In the design and synthesis of these analogs, the following previous substitutions were conserved: D-Arg2, Har9, Abu15, and Nle27. Most new analogs had Ala at position 8. Since replacements of both Lys12 and Lys21 with Orn increased resistance against enzymatic degradation, these modifications were kept. The substitutions of Arg at both positions 11 and 20 by His were also conserved. We kept D-Arg28, Har29 -NH2 at the C-terminus or inserted Agm or 12-amino dodecanoic acid amide at position 30. We incorporated pentafluoro-Phe (Fpa5), instead of Cpa, at position 6 and Tyr(Me) at position 10 and ω-amino acids at N-terminus of some analogs. These GHRH analogs were prepared by solid-phase methodology and purified by HPLC. The evaluation of the activity of the analogs on GH release was carried out in vitro on rat pituitaries and in vivo in male rats. Receptor binding affinities were measured in vitro by the competitive binding analysis. The inhibitory activity of the analogs on tumor proliferation in vitro was tested in several human cancer cell lines such as HEC-1A endometrial adenocarcinoma, HCT-15 colorectal adenocarcinoma, and LNCaP prostatic carcinoma. For in vivo tests, various cell lines including PC-3 prostate cancer, HEC-1A endometrial adenocarcinoma, HT diffuse mixed ß cell lymphoma, and ACHN renal cell carcinoma cell lines were xenografted into nude mice and treated subcutaneously with GHRH antagonists at doses of 1-5µg/day. Analogs MIA-602, MIA-604, MIA-610, and MIA-640 showed the highest binding affinities, 30, 58, 48, and 73 times higher respectively, than GHRH (1-29) NH2. Treatment of LNCaP and HCT-15 cells with 5µM MIA-602 or MIA-690 decreased proliferation by 40%-80%. In accord with previous tests in various human cancer lines, analog MIA-602 showed high inhibitory activity in vivo on growth of PC-3 prostate cancer, HT-mixed ß cell lymphoma, HEC-1A endometrial adenocarcinoma and ACHN renal cell carcinoma. Thus, GHRH analogs of the Miami series powerfully suppress tumor growth, but have only a weak endocrine GH inhibitory activity. The suppression of tumor growth could be induced in part by the downregulation of GHRH receptors levels.

Synthesis and biological evaluation of novel structure-related hGHRH agonistic analogs.

Activity and half-life play key roles in the application of GHRH analogues. The GHRH monomers produced in a solid synthesizer were incubated, respectively, in NH4OH solution and lyophilized to obtain their dimers. The activities, specificities, and receptor affinities of the GHRH dimers were evaluated in rGH release/inhibition, rACTH/LH/PRL release, pituitary homogenate binding, and fluorescent staining. Compared to hGHRH(1-44)NH2 (S), PP-hGHRH(1-44)-GGC-CGG-hGHRH(44-1)-PP (2D), P-hGHRH(1-44)-GGC-CGG-hGHRH(44-1)-P (2E), (1)P-hGHRH(2-44)-GGC-CGG-hGHRH(44-2)-(1)P (2F), or hGHRH(1-44)-GGC-CGG-hGHRH(44-1) (2Y) had potency of 104 ± 16.7%, 94 ± 32.6%, 114 ± 16.6%, or 122 ± 14.5% and similar specificities. The inhibition effect of GHIH on rGH stimulated by GHRH dimer was in dose-/time-dependent manner. The staining of FITC-labeled dimer showed cytomembrane distribution and the binding ranking was 2F>2D>2Y>2E>S. 2F presents the strongest activity and the highest affinity to pituitary cells. The dimer with (1)Pro-GHRH stimulates stronger rGH release than that with (1)Tyr-GHRH and the N-terminal single cyclic amino acid is required for the stimulation.

Synthesis of human growth hormone-releasing hormone via three-fragment serine/threonine ligation (STL).

The synthesis of human growth hormone-releasing hormone (hGH-RH), by the chemoselective serine/threonine ligations (STLs) of three unprotected peptide fragments, is reported. To allow for the multiple-fragment ligation, we chose the Msz (p-(methylsulfinyl)benzyloxycarbonyl) group, which is compatible with the preparation of peptide salicylaldehyde esters via Fmoc-SPPS and readily removed by reductive acidolysis, to protect the serine and threonine residue at the N-terminus.

Site-specific PEGylation for high-yield preparation of Lys(21)-amine PEGylated growth hormone-releasing factor (GRF) (1-29) using a GRF(1-29) derivative FMOC-protected at Tyr(1) and Lys(12).

PEGylation has been viewed as an effective means of overcoming the therapeutic restriction of growth hormone-releasing factor (1-29) (GRF(1-29)) due to its short biological lifetime caused by severe proteolysis and rapid glomerular filtration. Of three isomers according to the PEGylation sites (Tyr1, Lys12, or Lys21), PEGylated GRF(1-29) at Lys21-amine (Lys21-PEG-GRF(1-29)) was shown to have the highest bioactivity. In this report, we propose a unique two-step site-specific PEGylation method capable of producing only Lys21-PEG-GRF(1-29) with a single composition in high yield using a GRF(1-29) derivative protected at Tyr1 and Lys12 and remained available at Lys21 (FMOC1,12-GRF(1-29)). The first step of this reaction involved the PEG attachment to FMOC1,12-GRF(1-29), and the second step involved the removal of FMOC moieties. This PEGylation process was optimized at the following conditions: 0.2-0.3% (v/v) triethylamine concentration, 5.0-6.0-fold molar amount of PEG, reaction temperature of 25-45 degrees C, and reaction time of 30 min. Under these conditions, the maximum yield of Lys21-PEG-GRF(1-29) produced was ca. approximately 95%, 6.3-fold higher than that by nonspecific PEGylation at pH 8.5. Significantly, this site-specific Lys21-PEG-GRF(1-29) was found to have greatly increased resistance to rat plasma, liver, and kidney homogenates, with 7.0-, 25.4-, and 16.4-fold longer half-lives vs GRF(1-29), respectively. Furthermore, 125I-Lys21-PEG-GRF(1-29) displayed significantly reduced liver and kidney distributions and extended blood presence vs 125I-GRF(1-29) in rats. Due to these benefits, Lys21-PEG-GRF(1-29) displayed an enhanced initial growth hormone release in vivo despite having 15% remaining activity in vitro. This devised PEGylation method using an FMOC-protection/deprotection strategy would provide great usefulness for PEGylating bioactive peptides in terms of improved biological potency, elevated production yield, and a uniform composition.

Drug evaluation: tesamorelin, a synthetic human growth hormone releasing factor.

Theratechnologies, under license from Valeant, is developing tesamorelin as a potential vaccine adjuvant and for the potential treatment of wasting, hip fracture recovery, immune disorders, HIV-related lipodystrophy, sleep maintenance insomnia and mild cognitive impairment. Phase III clinical trials for the treatment of HIV-associated lipodystrophy and phase II clinical trials for sleep disorder, chronic obstructive pulmonary disorder, hip fracture and immune system dysfunction are underway. Phase II trials are also assessing the influenza vaccination immune response and cognitive effects of tesamorelin.

NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor, hGRF(1-29)-NH2.

Two novel mono-PEGylated derivatives of hGRF(1-29)-NH(2) [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys(12) or Lys(21) to a monomethoxy-PEG(5000) chain (compounds Lys(12)PEG-GRF and Lys(21)PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys(12)PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys(21)PEG-GRF. To assess whether the differences in the biological activity of the PEGylated analogues could be related to conformational rearrangements induced by the PEG moiety, the structure of these PEGylated derivatives has been worked out (TFE solution) by means of NMR spectroscopy and molecular dynamics. Secondary structure shifts, hydrogen/deuterium exchange kinetics, temperature coefficients of amide protons, and NOE-based molecular models point out that hGRF(1-29)-NH(2), Lys(21)PEG-GRF and Lys(12)PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an alpha-helix conformation which spans the whole length of the molecule, and which becomes increasingly rigid on going from the N-terminus to the C-terminus. Residues Lys(12) and Lys(21) are enclosed in all the compounds considered into well-defined alpha-helical domains, indicating that PEGylation either at Lys(12) or Lys(21) does not alter the tendency of the peptide to adopt a stable alpha-helix conformation, nor does it induce appreciable conformational mobility in the proximity of the PEGylation sites. No significant variation of the amphiphilic organization of the alpha-helix is observed among the three peptides. Therefore, the different biological activities observed for the PEGylated analogues are not due to conformational effects, but are rather due to sterical hindrance effects. The relationship between the biological activitiy of the mono-PEGylated derivatives and sterical hindrance is discussed in terms of the topology of interaction between hGRF(1-29)-NH(2) and its receptor.

Inhibition of PC-3 human prostate cancers by analogs of growth hormone-releasing hormone (GH-RH) endowed with vasoactive intestinal peptide (VIP) antagonistic activity.

Vasoactive intestinal peptide (VIP) stimulates the proliferation and invasiveness of malignant prostatic cells. Receptors for VIP and the closely related growth hormone-releasing hormone (GH-RH) show considerable homology and are found in prostatic and other carcinomas. Among various analogs of GH-RH synthesized, JV-1-52 is a non-selective VIP/GH-RH antagonist, whereas JV-1-53 is a VIP antagonist devoid of GH-RH antagonistic effect. In our study, nude mice bearing PC-3 human androgen-independent prostate carcinomas were treated with JV-1-52 or JV-1-53 (20 microg/day, s.c.) for 28 days. Both antagonists produced a similar reduction in tumor volume (62-67%, p < 0.01) and tumor weight (59-62%; p < 0.05) vs. controls and extended tumor doubling-time from 9.1 to about 16 days (p < 0.05). To investigate the mechanisms involved, in another study we compared the effects of JV-1-53 with those of somatostatin analog RC-160. VIP antagonist JV-1-53 reduced tumor weight by 67% (p < 0.01) and suppressed the expression of mRNA for c-fos and c-jun oncogenes by about 34% (p < 0.05), without affecting serum levels of insulin-like growth factor-I (IGF-I). In contrast, RC-160 (50 microg/day) reduced serum IGF-I by 19% (p < 0.05), but did not significantly decrease tumor weight. mRNA for VIP and high affinity receptors for VIP were detected on PC-3 tumors. Our results suggest that VIP/GH-RH antagonists can inhibit the growth of androgen-independent prostate cancer by abrogating the autocrine/paracrine mitogenic stimuli of VIP. The ability of GH-RH antagonists to block tumoral VIP receptors, in addition to GH-RH receptors, could be potentially beneficial for prostate cancer therapy.

Use of the HIV-1 protease for excision of growth-hormone-releasing factor from synthetic and recombinant peptide precursors.

An autolysis-resistant mutant of the HIV-I protease was employed for removal of metabolically stabilized and highly bioactive analogues of bovine growth-hormone-releasing factor (bGRF) from their larger either synthetic or recombinant precursors. The N-terminal four amino acids in two selected model GRF analogues, Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQVF32-OH (I; GRF32) and Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQ30-OH (IA; GRF30), conform well to the specificity of the HIV-I protease for residues in the P1' to P4' positions of its peptide substrates. A variety of amino acids were tried in the N-terminal extension (positions P4-P1) to fit the protease substrate specificity for the 8 amino acids in positions P4-P4'. A synthetic precursor of I, extended N-terminally with RQVF-, a sequence representing the four C-terminal residues in I, was effectively cleaved by the protease at the Phe-1-Tyr1 bond (... RQVF-decreases-YIDA ...) to release GRF32. However, when several soluble fusion proteins linked to GRF32 by the RQVF sequence were expressed in Escherichia coli, attempts to cleave out the core GRF32 met with variable, and only limited, success. By random mutagenesis in a propeptide segment, [MGQSVAQVF]-decreases-GRF30, (II) was identified as a construct that showed reasonably high-level expression in E. coli and was effectively processed by the HIV-I protease. A yield of 5 mg of pure GRF30 was obtained/litre of culture medium after a single HPLC purification step.

Synthesis and in vitro evaluation of new potent antagonists of growth hormone-releasing hormone (GH-RH).

In the search for more potent antagonists of hGH-RH, 20 new analogs were synthesized, purified and tested in vitro. All the analogs were based on the N-terminal sequence of 28 or 29 amino acid residues of hGH-RH, but contained D-Arg2 and Nle27 modifications. Most analogs had Phe (pCl)6 and Agm29 substituents. The effect of other substitutions such as Abu8 and/or Abu15 and Ala15 and various hydrophobic and hydrophilic D or L amino acids at position 8 were also investigated. All the peptides were acylated at the N-terminus in an attempt to increase the antagonistic activity. In the superfused rat pituitary cell system, most analogs inhibited more powerfully the GH release induced by GH-RH than the standard antagonist [Ac-Tyr1, D-Arg2]hGH-RH (1-29)-NH2. Some antagonists were long acting. Among the peptides synthesized, antagonist PhAc[D-Arg2, Phe(pCl)6, Abu15, Nle27]hGH-RH (1-28) Agm (MZ-5-156) appeared to be the most potent and inhibited GH release in vitro 63-200 times more powerfully than the standard antagonist. MZ-5-156 and other antagonists showed high binding affinities to membrane receptors for GH-RH. Some of these hGH-RH antagonists could be further developed for possible onocological applications.

Synthesis and biological evaluation of superactive agonists of growth hormone-releasing hormone.

Analogs of the 29 amino acid sequence of human growth hormone-releasing hormone (hGH-RH) with agmatine (Agm) in position 29, desaminotyrosine (Dat) in position 1, norleucine (Nle) in position 27, and L-alpha-aminobutyric acid (Abu) in position 15 have been synthesized, and their biological activity was evaluated. Some peptides contained one or two residues of ornithine (Orn) instead of Lys in positions 12 and 21 and additional replacements in positions 8 and 28. All analogs were found to be more potent than hGH-RH-(1-29)-NH2 in the superfused rat pituitary cell system. In tests in vivo in rats after subcutaneous administration, the analogs JI-22, [Dat1, Orn12,21, Abu15, Nle27, Agm29]hGH-RH-(1-29); JI-34, [Dat1, Orn12,21,Abu15,Nle27, Asp28, Agm29]hGH-RH-(1-29); JI-36, [Dat1, Thr8, Orn12,21, Abu15,Nle27,Asp28,Agm29]hGH-RH-(1-29); and JI-38, [Dat1,Gln8, Orn12,21,Abu15,Nle27,Asp28,Agm29]hGH-RH-(1 -29) displayed a potency 44.6,80.9,95.8, and 71.4 times greater, respectively, than that of hGH-RH-(1-29)-NH2 at 15 min and 217.1, 89.7, 87.9, and 116.8 times greater at 30 min. After intravenous administration, JI-22, JI-36, and JI-38 were 3.2-3.8 times more potent than hGH-RH-(1-29)-NH2 at 5 min and 6.1-8.5 times more active at 15 min. All analogs were found to have higher binding affinities for GH-RH receptors on rat pituitary cells than hGH-RH-(1-29)-NH2. Because of high activity and greater stability, these analogs could be considered for therapy of patients with growth hormone deficiency.

Rational design, synthesis, and biological evaluation of novel growth hormone releasing factor analogues.

Since its initial discovery in 1982, growth hormone-releasing factor (GRF) has been the subject of intense investigation. This interest was prompted by the potential application of GRF for stimulating growth in dwarf humans and for performance enhancement in livestock. Substantial research has been focused upon the development of potent, long-acting analogs as therapeutics. Herein is described a summary of the cumulative efforts of various laboratories endeavoring in this quest. The rationale utilized in GRF analog development is discussed: 1) determination of bioactive core, 2) evaluation of secondary structure, and 3) elucidation of degradation pathways (chemical and enzymatic). Using this information, several series of linear (unnatural and natural sequence) and cyclic GRF analogs were designed, synthesized, and evaluated. Stimulated by the constraints of commercial production, innovative, alternative methods of synthesis were explored: solid-phase, solution-phase, enzymatic, and recombinant. To date, the most promising candidate for drug development is [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH. This natural sequence analog, consisting of rodent and human sequences, incorporates the bioactive core, preferred secondary structure, resistance to chemical and enzymatic degradation; with the added benefit of amenability to large-scale recombinant synthesis.

Synthesis and biological activities of highly potent antagonists of growth hormone-releasing hormone.

In the search for antagonists of human growth hormone-releasing hormone (hGHRH) with high activity, 22 analogs were synthesized by solid-phase methods, purified, and tested biologically. Within the N-terminal sequence of 28 or 29 amino acids of hGHRH, all the analogs contained D-Arg2, Phe(4-Cl)6 (para-chlorophenylalanine), Abu15 (alpha-aminobutyric acid), and Nle27 and most of them had Agm29 (agmatine) substituents. All the peptides, except one, were acylated at the N terminus with different hydrophobic acids--e.g., isobutyric acid (Ibu) or 1-naphthylacetic acid (Nac) in order to study the effect of N-terminal acylation on the antagonistic activity. In the superfused rat pituitary cell system, all the analogs inhibited more powerfully the GHRH-induced growth hormone (GH) release than the standard GHRH antagonist [Ac-Tyr1,D-Arg2]hGHRH-(1-29)NH2. Antagonists [Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-28) Agm (MZ-4-71), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-28) Agm (MZ-4-243), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-29) NH2 (MZ-4-169), [Nac0-His1,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]-hGH RH-(1-29)NH2 (MZ-4-181), and [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27,Asp28]hGH RH-(1-28)Agm (MZ-4-209) inhibited GH release at 3 x 10(-9) M. Among these peptides, MZ-4-243, MZ-4-169, and MZ-4-181 were also long acting in vitro. Antagonist MZ-4-243 inhibited GH release 100 times more powerfully than the standard antagonist and was the most potent in vitro among GHRH antagonists synthesized. Analogs with high inhibitory effects in vitro were also found to have high affinities to rat pituitary GHRH receptors. In experiments in vivo, antagonists [Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]-hGHRH-(1-28 )Agm (MZ-4-71), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-29) NH2 (MZ-4-169), and [Nac0-His1,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHR H-(1-29)NH2 (MZ-4-181) induced a significantly greater inhibition of GH release than the standard antagonist. In view of their high antagonistic activity and prolonged duration of action, some of these antagonists of GHRH may find clinical applications, including treatment of certain endocrine disorders and insulin-like growth factor I-dependent tumors.

Peptide design using a genetically patterned binary code: growth hormone-releasing hormone as a model.

This paper reviews a method for the design of peptides and proteins of predefined structure and function and provides an example. Specifically, an analog of rat growth hormone-releasing hormone (GHRH) (residues 1-23) was synthesized by solid-phase methods based on a reversed sequence of the mRNA for GHRH (1-23). The new peptide, designated GHRH 3'-5', had a hydropathic profile similar to that of native GHRH 5'-3' (GHRH) but had only 17% primary sequence homology. GHRH 3'-5' specifically bound to the GHRH receptor on rat pituitary cells and to polyclonal anti-GHRH antibody in ELISA and RIA procedures. Additionally, GHRH 3'-5' blocked the in vitro stimulation of GH RNA synthesis and in vitro and in vivo GH release mediated by GHRH. These data show that 3'-5' GHRH with little sequence homology to native rat GHRH is an antagonist and further supports the importance of the linear pattern of hydropathy to the gross secondary and/or tertiary structure and rudimentary function of peptides and proteins. The impact of these findings on the interaction of complementary peptides is discussed.

New Gaba-containing analogues of human growth hormone-releasing hormone (1-30)-amide: I. Synthesis and in vitro biological activity.

Analogues of human growth hormone-releasing hormone (1-30)-amide have been developed. All analogues have been modified in position 27 with Nle and with Gaba in position 30. Additional D-amino-acids have been inserted in the GHRH(1-30)-NH2 sequence: A-1741: Nle27,Gaba30-GH-RH(1-30)-NH2 A-495: D-Ala2,Nle27,Gaba30-GH-RH(1-30)-NH2 A-515: D Ala2,Leu15,Nle27,Gaba30-GH-RH(1-30)-NH2 A-527: D-Ala2,D-Arg11,Leu15,Nle27,Gaba30-GH-RH(1-30)-NH2. Our analogues were synthesized by solid phase peptide synthesis and were tested is two different in vitro systems and in rat pituitary cell cultures. A-495 and A-1741 were found to be the most active in releasing GH, however they showed different activities in the two different test systems. A-495 exhibited higher potency in the superfusion system (1.63 fold potency of the GHRH (1-29)-amide), while A-1741 evoked higher GH release from cultured pituitary cells (1.5-2.5 times higher than the GH-RH(1-44)-amide). The other analogues (A-515 and A-527) were found to be equipotent to the standard molecule. We can conclude that Nle27 and Gaba30 substitutions appeared to be a good modification in in vitro test systems, and Gaba30 substitution served as a good spacer during the synthesis, since it made the coupling of the C-terminal amino acids easier and produced quantitative coupling. In addition to the advantageous properties in the synthesis these modifications with or without D-Ala at the N-terminus increased the in vitro biological activity to 1.5-2.5 fold of the GHRH molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Semisynthesis of human growth hormone-releasing factor by trypsin catalyzed coupling of leucine amide to a C-terminal acid precursor.

Human growth hormone-releasing factor, GRF(1-44)-NH2, was synthesized by trypsin catalyzed coupling of Leu-NH2 to Arg43 of the precursor, GRF(1-43)-OH, prepared by solid phase peptide synthesis. The semisynthetic GRF(1-44)-NH2 was fully characterized and showed full potency in the rat pituitary in vitro bioassay. Conversion to GRF(1-44)-NH2 was limited to 60-70% in both 75% v:v N,N'-dimethylacetamide and 95% v:v 1,4-butanediol due to competing transpeptidations at Arg41 and Arg38 generating [Leu42]-GRF(1-42)-NH2 and [Leu39]-GRF(1-39)-NH2 side-products, respectively. The rates of formation and yields of GRF(1-44)-NH2 versus pH, Leu-NH2 concentration, and solvent composition were also studied.

Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs.

A series of novel hGRF(1-29)-NH2 analogs were synthesized and biotinylated. The immunological and biological activities of these analogs were then characterized. To distance the biotin moiety from the putative bioactive core, a C-terminal spacer arm consisting of -Gly-Gly-Cys-NH2 (-GGC) was added to hGRF(1-29)-NH2 (hGRF29) and analogs, with subsequent biotinylation performed at the cysteine residue. Neither addition of the C-terminal spacer arm nor biotinylation affected affinity of these analogs for GRF antibody. Relative to hGRF(1-44)-NH2 (hGRF44: potency = 1.0), the biotinylated analogs were equipotent in vitro to their nonbiotinylated, parent compounds: [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2 (4.7) = [Ala15]hGRF29-GGC-(tpBiocytin)-NH2 (3.9) greater than hGRF29-GGC-(tpBiocytin)-NH2 (0.8). Based upon cumulative GH release data in vivo (0-60 min postinjection), [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2, [Ala15]hGRF29-GGC-(tpBiocytin)-NH2, and hGRF29-GGC-(tpBiocytin)-NH2 displayed 8.6, 5.5, and 0.8 times, respectively, the potency of hGRF44. These in vivo potency values were not significantly different from the corresponding parent compounds (i.e., with or without the C-terminal spacer arm). In summary, biotinylated hGRF analogs have been developed that retain full immunoreactivity and potent bioactivity (in vitro and in vivo), thus permitting their use in GRF receptor isolation, ELISA, and histochemical procedures.

Synthesis and biological evaluation of mouse growth hormone-releasing factor.

The recently described mouse growth hormone-releasing factor (mGRF) was synthesized by the solid phase procedure, purified by 2 stages of preparative high performance liquid chromatography and fully characterized. The biologic activity of the 42-amino acid peptide (H-His-Val-Asp-Ala-Ile-Phe- Thr-Thr-Asn-Tyr- Arg-Lys-Leu-Leu-Ser-Gln-Leu-Tyr-Ala-Arg-Lys-Val-Ile-Gln-Asp-Ile-Met-Asn- Lys- Gln-Gly-Glu-Arg-Ile- Gln-Glu-Gln-Arg-Ala-Arg-Leu-Ser-OH) was assessed in primary cultures of both mouse and rat anterior pituitary cells and compared to synthetic rat (rGRF) and human (hGRF) growth hormone-releasing factors. mGRF was equipotent to rGRF in mouse somatotrophs but slightly less potent in rat somatotrophs, while hGRF was 3-5 times less potent in both rodent species.

Comparison of enzymatic semisyntheses of peptide amides: human growth hormone releasing factor and analogs.

Enzymatic semisyntheses of growth hormone releasing factor (GRF), a 44-residue peptide amide hormone, from C-terminal acid precursors, are compared. A recombinant alpha-amidating enzyme was used to convert the glycine-extended precursor, GRF(1-44)-Gly-OH, to GRF(1-44)-NH2 in an essentially quantitative fashion. Trypsin was used to convert the precursors, GRF(1-43)-OH and GRF(1-44)-OH, to GRF(1-44)-NH2 (60 and 15% conversion, respectively) in a 75% v:v N,N'-dimethylacetamide solution containing a large excess of leucine amide. Carboxypeptidase Y catalyzed transpeptidations of the precursors, GRF(1-44)-OH and [Ala44]-GRF(1-44)-OH, to GRF(1-44)-NH2 in aqueous leucine amide solutions were also attempted. The trypsin catalyzed direct amidation of [Ala15]-GRF(1-29)-OH in concentrated ammonium acetate/ammonia buffer (95% 1,4-butanediol cosolvent) to form the superactive analog, [Ala15]-GRF(1-29)-NH2 (ca. 25% conversion at equilibrium), is also described.

Analogues of growth hormone-releasing factor (1-29) amide containing the reduced peptide bond isostere in the N-terminal region.

Previous peptide structure-activity investigations employing the psi[CH2NH] peptide bond isostere have produced antagonists when inserted into various sequences. These include bombesin, in which the incorporation of Leu13 psi[CH2NH]Leu14 produced a potent antagonist, and tetragastrin, with which Boc-Trp-Leu psi[CH2NH]Asp-Phe-NH2 is an antagonist. In this study, we chose to investigate the effect of this isostere on growth hormone-releasing factor (1-29) amide. Analogues were prepared by solid-phase synthesis and the isosteres incorporated by racemization-free reductive alkylation with a preformed protected amino acid aldehyde in the presence of NaBH3CN. The aldehydes were prepared by the reduction of the protected N,O-dimethyl hydroxamates with LiAlH4 at 0 degrees C. The purified analogues were assayed in a 4-day primary culture of male rat anterior pituitary cells for growth hormone (GH) release. Potential antagonists were retested in the presence of GRF(1-29)NH2. The following results were obtained: At position 5-6, a very weak agonist was produced with much less than 0.01% activity. Incorporation of the isostere in positions 1-2, 2-3, and 6-7 gave weak agonists with approximately 0.1% activity. Agonists with 0.39% and 1.6% activity were produced by incorporation at 10-11 and 3-4, respectively. The analogue [Ser9 psi[CH2NH]Tyr10]GRF(1-29)NH2 was found to be an antagonist in the 10 microM range vs 1 nM GRF and had no agonist activity at doses as high as 0.1 mM.