Synthetic linear and cyclic glucagon antagonists

The synthesis and biological activities of seven new glucagon analogues are reported. The design of com- pounds 2-5 is based on potent antagonists recently reported from this laboratory, where we have focused on modifications in the N-terminal region. In this report we have concentrated specifically on modifications to histidine-1. In addition we have prepared two cyclic compounds 7 and 8 , related to a linear in vivo antagonist [Glu⁹]glucagon, reported by Medeld (Unson et al. (1987) Proc. Natl. Acad. Sci. USA 84 , 4083-4087). The N-terminal modifications involved substitution of His¹ by the unnatural conformationally constrained residue (S)-5,6,7,8-tetrahydro-5-oxoimidazo(1,5-c)pyrimidine-7-carboxylic acid (Toc), desaminohistidine (dHis) and 3-(4-nitrobenzyl)histidine. The structures of the new compounds are as follows. [Toc¹,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17,18,Glu²¹]glucagon ( 2 ); [Toc¹,d Phe⁴,Tyr⁵,Arg¹²,Lys^17,18,Glu²¹]glucagon amide ( 3 ); [3-(4-nitrobenzyl)His¹,d Phe⁴,Tyr⁵,Arg¹²,Lys^17,18,Glu²¹]glucagon ( 4 ); [dHis¹,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17,18,Glu²¹]glucagon ( 5 ); [dHis¹,Glu⁹]glucagon ( 6 ); (desHis¹)glucagon amide ( 7 ); (desHis¹)-glucagon amide ( 8 ). The binding potencies of the linear analogues, as expressed a percentage of glucagon binding, are 2.6 ( 2 ), 0.13 ( 3 ), 0.8 ( 4 ), 0.8 ( 5 ), 2.2 ( 6 ). Both cyclic analogues 7 and 8 show biphasic binding curves. The IC₅₀ values for 7 at the high and low finity sites are 1.5 and 167 nm , respectively (IC₅₀ of glucagon = 1.3 nm ). The IC₅₀ values for ₅₀ at the high and low affinity sites are 4.7 and 3451 nm , respectively. The cyclic analogues are characterized by fast atom bombardment mass spectrometry of endoproteinase ASP-N digests. The specificity of the enzyme used in these studies enables differentiation of isomers of the cyclic glucagon analogues which differ only in the position of cyclic amide bond. Analogues 2,3 and 5–8 are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase (AC) system. Analogue 4 is a partial agonist (5.7% compared to glucagon) of AC. Introduction of unusual amino acids which do not contain a primary α-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.     

MOLECULAR MAKEUP OF HIV-1 ENVELOPE PROTEIN

A broad range of structural, functional, and immunological similarities between HIV-1 gp120 and human proteins, especially those participating in immune responses, highlight gp120 as a pleiotropic protein that can in different ways affect many important functions of the human immune system. Here we described some of these properties of HIV-1 gp120 that represent the main obstacle in the development of effective and safe AIDS vaccine.     

RECOMBINATION PROPERTY OF THE HIV-1 gp120 GENE

Using a chimeric primer consisting of the nucleotide sequence derived from the HIV-1 envelope gene coding for the second conserved region of gp120, and the highly conserved sequence derived from the human immunoglobulin gene coding for the V_HIII domain, it has been identified in sera of AIDS patients HIV-1 field isolates carrying the complete and active Chi recombination hot spot (GCTGGTGG). The recombination between the HIV-1 gene coding for the central portion of gp120 and the bacterial gene coding for the clp protease was also demonstrated in vivo. These results point out serious concern that vectored AIDS vaccine candidates carrying the HIV-1 env gene on viral and bacterial vectors could become the source of potentially new infectious diseases rather than an effective instrument for AIDS prevention.