The shortcomings of native peptides as pharmaceuticals have been long known: short duration of action, lack of receptor selectivity, lack of oral bioavailability. However medicinal chemistry offers solutions to the first two limitations and oral bioavailability issues have been addressed with novel routes of administration (e.g. intranasal, inhalation) and injectable depot formulations. The principal issue for peptide drugs has been a short duration of action, widely assumed to be due to proteolysis. While proteolysis is a problem for native peptide structures, modification of the peptide structure by acylation, PEGylation, unnatural amino acids or restricted conformation can largely remove this issue. However rapid clearance from the blood into the urine remains an issue for even proteolytically stable molecules. Medicinal chemistry approaches here have been peptide modifications to slow release from the injection site (hydrophobic, hydrophilic, self-associating depots), PEGylation, fatty acid acylation, and the like. Medicinal chemistry approaches used in successful peptide pharmaceuticals using unnatural amino acids to achieve depot formation are highlighted in this review.

Vaccinia virus encodes VGF, an early protein of relative molecular mass 19,000 (19K) which, from amino-acid residues 45 to 85, is homologous in 19 residues to epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). The conserved sequence includes a region of high homology (6 out of 10 amino acids) from residues 71 to 80, corresponding to the third disulphide loop of both EGF and TGF-alpha. This region has recently been shown to contain a binding region of TGF-alpha for the EGF receptor, and this raises the question of whether vaccinia virus utilizes the EGF receptor in order to bind to and infect cells. We now show that occupancy of the EGF receptor inhibits vaccinia virus infection. Inhibition is observed in a dose-dependent fashion by pre-treatment with either EGF or synthetic decapeptide antagonists of EGF's mitogenic activity which correspond to the sequence of the third disulphide loop of VGF or TGF-alpha. The relative ability of the peptides to inhibit vaccinia virus infection parallels their binding affinity to the EGF receptor.

The role of the pituitary gonadotrophins in controlling luteal function in the stumptailed macaque has been investigated by examining profiles of serum concentrations of LH, FSH, progesterone and oestradiol in daily blood samples from 13 monkeys during the menstrual cycle, and in blood samples taken at hourly intervals between 09.00 and 21.00 h on different days of the luteal phase in 13 cycles. The effects of acute withdrawal of gonadotrophins was investigated by administering a single injection of 300 micrograms LHRH antagonist/kg body weight at different stages of the luteal phase during 28 cycles. Although there were high basal values and marked fluctuations of bioactive LH during the first 4 days after the LH peak, progesterone profiles showed no corresponding short-term changes, there being a slow and steady rise in progesterone concentrations during the sampling periods. After day 5, basal LH secretion decreased, but high amplitude LH pulses were identified which were associated with episodes of progesterone secretion. Administration of the LHRH antagonist caused a suppression of bioactive LH and progesterone concentrations at all stages of the luteal phase, although some basal secretion of progesterone was maintained through the 24-h period of effective antagonist gonadotroph blockade. Luteal function recovered apparently normally in all monkeys treated in the early-mid-luteal phase. Serum concentrations of FSH and oestradiol fluctuated comparatively less during the 12-h sampling periods, and the antagonist had less suppressive effects on the concentrations of these hormones. The LHRH antagonist had no apparent effect on prolactin release.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as '25I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (I opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.To label one type of receptor binding site in the presence of related types, a radioligand should (i) have very high affinity for the site to be labeled, (ii) be highly selective for that site, (iii) have high enough specific radioactivity to be usable at a low concentration to maximize its binding at the preferred site relative to that at the next-preferred site, (iv) for convenience incorporate a radioisotope such as 125I with a high-energy mode of decay.Dynorphin A (Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-ProLys-Leu-Lys-Trp-Asp-Asn-Gln) is a 17-residue endogenous opioid peptide with selectivity for the K opioid receptor (1-3). Dynorphin A-(1-13) is as potent and as selective for the K binding site as is the natural full-length peptide (4), so we surmised that this fragment could be modified safely by extension at the C terminus. For flexibility of functionalization with various reporter groups, we used a primary amino function at the C terminus as a handle. Lysine-11 and lysine-13 (especially the former) play essential roles in determining K affinity and selectivity (5). To abolish the reactive E-amino groups, yet retain the charges at these two positions, we replaced lysine with arginine. Our C-terminal extension consisted of glycine-14, followed by 1,5-diaminopentane. The resulting peptide is called "DAKLI" (Dynor-After synthesis of the peptide by the solid-phase method and its removal from the resin by aminolysis with 1,5-diaminopentane, the free a-amino group of tyrosine-1 remains blocked by the t-butoxycarbonyl (Boc) group and the only reactive amine is at the extended C terminus. This feature allows the facile production, from [Boc] 25 -22°(c 0.4, AcOH). Reversed-phase analytical HPLC (as above but 4 x 250 mm; particle size, 5 ,um; gradient, 10-50% CH3CN; 0.03 M in NH4OAc, pH 4.5; A = 280 nm) showed the product to be 90% pure, and the amino acid analysis after 6 M HCl hydrolysis (18 hr, 110°C) gave ratios in accord with the desired structure: Pro, 1.1 (1); Gly, 2.9 (3); Ile, 0.9 (1); Leu, 2.0 (2); Tyr, 1.3 (1); Phe, 1.1 (1); Arg, 4.9 (5). The expected amino acid sequence was obtained on an Applied Biosystems (Foster City, CA) 470A microsequencer (K. Jarnagin, Syntex Research).Preparation of lodinated Peptides DAKLII and DAKLII**. BH (Sigma) was iodinated by the chloramine-T method as follows: to 5 mg of dry BH was added 50 mg of NaI in 40 ,ul of 50 mM sodium phosphate buffer, followed immediately by 0.5 ml of chloramine T (30 mg/ml in 250 mM sodium phosphate buffer). The reaction was terminate...

A novel class of heterocyclic aromatic amino acids based on the 3-(2-benzimidazolyl)alanine system has been generated by chiral synthesis from D- or L-aspartic acid. The use of variously substituted o-phenylenediamines for condensation with the beta-carboxyl function of alpha-benzyl N-(benzyloxycarbonyl)-D-aspartate has led to a series of amino acids of graded hydrophobicity with a steric bulk similar to that of tryptophan. In a similar fashion, we have prepared 3-(2-benzothiazolyl)-D-alanine from o-aminothiophenol and 3-(2-benzoxazolyl)-D-alanine from o-aminophenol. Incorporation of these amino acids into the 6-position of luteinizing hormone-releasing hormone (LH-RH) led to a series of very potent agonist analogues (up to 160 times LH-RH potency), active in doses ranging from 0.1 to 0.5 microgram by twice daily injection in a rat estrus cyclicity suppression assay designed to show the paradoxical antifertility effects of these compounds.

The effect of increased hydrophobicity at position 6 of luteinizing hormone-releasing hormone (LH-RH) has been investigated by the incorporation of a series of 15 very hydrophobic, unnatural D-amino acids at this position. The unnatural amino acids studied can be considered analogues of phenylalanine with carbocyclic aromatic side chains consisting of substituted phenyl (e.g., 2,4,6-trimethylphenyl, p-biphenyl) or polycyclic aromatic (e.g., naphthalene, anthracene) units. When enzymatic resolution (subtilisin Carlsberg) of the most hydrophobic amino acids failed, the racemic amino acids were incorporated, and the diastereomeric LH-RH analogues were resolved by preparative high-performance liquid chromatography. The analogues were synthesized by the solid-phase technique. All of the synthetic compounds were very potent LH-RH superagonists, but [6-(3-(2-naphthyl)-D-alanine)]LH-RH, [6-(3-(2-naphthyl)-D-alanine), 7-(N alpha-methylleucine)]LH-RH and [6-(3-(2,4,6-trimethylphenyl)-D-alanine)]LH-RH appear to be among the most potent LH-RH agonist analogues yet reported when tested in a rat estrus cyclicity suppression assay designed to show the paradoxical antifertility effects of these compounds [ED50 approximately 7 x 10(-8) g; twice daily in saline]. These analogues are twice as potent as [D-Trp6,ProNHEt9]LH-RH in this assay system (i.e., approximately 200 times the potency of LH-RH).

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