Theodore Lambros

Enhanced stability and potency of novel growth hormone-releasing factor (GRF) analogues derived from rodent and human GRF sequences

Native human GRF(1-44)-NH2(hGRF44) is subject to biological inactivation by both enzymatic and chemical routes. In plasma, hGRF44 is rapidly degraded via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala2 and Asp3. The hGRF44 is also subject to chemical rearrangement (Asn8-->Asp8, beta-Asp8 via aminosuccinimide formation) and oxidation [Met27-->Met(O)27] in aqueous environments, greatly reducing its bioactivity. It is therefore advantageous to develop long-acting GRF analogues using specific amino acid replacements at the amino-terminus (to prevent enzymatic degradation): residue 8 (to reduce isomerization) and residue 27 (to prevent oxidation). Inclusion of Ala15 substitution (for Gly15), previously demonstrated to enhance receptor binding affinity, would be predicted to improve GRF analogue potency. Substitution of [His1,Val2]-(from the mouse GRF sequence) for [Tyr1,Ala2]-(human sequence) in [Ala15,Leu27]hGRF(1-32)-OH analogues completely inhibited (24-h incubation) DPP-IV cleavage and greatly increased plasma stability in vitro. Additional substitution of Thr8 (mouse GRF sequence), Ser8 (rat GRF sequence), or Gln8 (not naturally occurring) for Asn8 (human GRF sequence) resulted in analogues with enhanced aqueous stability in vitro (i.e., decreased rate of isomerization). These three highly stable and enzymatically resistant hGRF(1-32)-OH analogues, containing His1, Val2, Thr/Gln8, Ala15, and Leu27 replacements, were then bioassayed for growth hormone (GH)-releasing activity in vitro (rat pituitary cell culture) and in vivo (SC injection into pigs). Enhanced bioactivity was observed with all three hGRF(1-32)-OH analogues. In vitro, these analogues were approximately threefold more potent than hGRF44, whereas in vivo they were eleven- to thirteenfold more potent.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of different forms of recombinant human leukocyte interferons and synthetic fragments by high-performance liquid chromatography

Analytical high-performance liquid chromatography (HPLC) conditions are reported for the evaluation of recombinant human interferon monomers: recombinant human leukocyte interferon A (rIFN-alpha A), rIFN-alpha D, and the hybrid rIFN-alpha A1-62/D64-166. The two monomeric forms of rIFN-alpha A (slow-migrating monomer and fast-migrating monomer) were also resolved by HPLC. Conditions are reported for the HPLC separation of oligomers (dimers and trimers) of rIFN-alpha A. The synthesis and analytical HPLC of the carboxy-terminus fragment, corresponding to IFN-alpha D (140-166), and a series of analogues comprising the IFN-alpha A (105-125) region is reported. The syntheses were accomplished by the solid-phase peptide synthesis procedure and the products were purified by preparative HPLC.

Capillary Zone Electrophoresis of Degradative and Cyclic Lactam Derivatives of the Growth Hormone-Releasing Factor Peptide

Abstract Capillary zone electrophoresis (CZE) was used to monitor deamidation of the Asn8 residue in the human growth hormone-releasing factor peptide, GRF(1–44)-NH2. The deamidation proceeds via cyclic imide formation yielding isomeric Asp8 and β-Asp8 containing products. It is demonstrated that GRF peptides differing only by isomerization at a single aspartic acid residue can be separated by CZE at pH 2.5–4.5 as a result of the greater acidity of the β-Asp side-chain carboxylic acid versus that of the normal Asp isomer. The dependence of electrophoretic mobility on the size and charge of GRF peptide fragments was studied by CZE for proteolysis of GRF(1–29)-NH2 by trypsin and endoproteinase Glu-C. CZE was also used to separate cyclic lactam analogs of GRF that all bear approximately the same net charge and differ only by the ring size or orientation of the lactam bridge.

Synthesis of adrenal peptide E and some of its biological activities

A synthesis of peptide E, a highly potent, 25-amino acid adrenal opioid peptide containing both a [Met]enkephalin at the NH2-terminus and [Leu]enkephalin sequence at the COOH-terminus, originally isolated from bovine adrenal medulla [D. L. Kilpatrick, T. Taniguchi, B. N. Jones, A. S. Stern, J. E. Shively, J. Hullihan, S. Kimura, S. Stein, and S. Udenfriend (1981) Proc. Natl. Acad. Sci. USA 78, 3265-3268], is reported. The synthesis was accomplished by the solid-phase method employing the 4-(aminoacyloxymethyl)phenylacetamidomethyl(Pam)-copoly(styrene-1% divinylbenzene) resin. Two synthetic strategies (N-indole formyl protected vs unprotected tryptophan) were followed and results compared and evaluated. It was determined that peptide E prepared with protection of tryptophan (residues 13 and 14) was preferred and gave final product that was readily purified by HPLC. The biological activity of the synthetic material was found to be equivalent to the reported activity of the natural compound.

Capillary Electrophoresis of Multiple Antigenic-Peptide (MAPS) of the Malaria Circumsporozoite Rotein Epitopes

Abstract Capillary electrophoresis (CE) was evaluated as a means of analyzing the homogeneity of synthetic malaria vaccines consisting of multiple-antigenic-peptides (MAPs). The MAPs consist of a branching oligomeric lysine core (Lysn, n = 3, 7, 15) with antigenic peptides coupled to each of the α-amino and ∊-amino arms (n+1 peptides total). In this report we compare CE with reversed-phase and size-exclusion liquid chromatography (RP-HPLC and SE-HPLC) for the analysis of Lys, MAPs corresponding to B-cell and T-cell epitopes of the circumsporozoite protein of the Plasmodium falciparum malaria parasite.

The Synthesis and Physical Properties of Thymosin α1 and Its Analogs Prepared by Fragment Condensation

The isolation of biologically important peptides from the thymus gland has been studied extensively in the last few years. Several thymic peptides have been shown to play certain roles in T-cell maturation (White, 1980; Trainin et al., 1980a, b; Goldstein and Lau, 1980; Bach and Goldstein, 1980). Thymosin α1, a highly acidic Nα-acetyl octacosapeptide, isolated from calf thymus gland (Goldstein et al., 1977) and characterized by sequence analysis (Low and Goldstein, 1979), has been reported to exhibit biological activities involved in the development of thymus-dependent lymphocytes (T cells) (Goldstein et al., 1977). Thymosin a1 has been synthesized by classical procedures in solution (Wang et al., 1979; Birr and Stollenwerk, 1979) and by solid-phase methods (Wong and Merrifield, 1980; Wang et al., 1980; Colombo, 1981).