Substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. Analysis of fatty acid analogs containing carbonyl groups, nitrogen heteroatoms, and nitrogen heterocycles in an in vitro enzyme assay and subsequent identification of inhibitors of human immunodeficiency virus I replication.

Covalent attachment of myristic acid (C14:0) to the amino-terminal glycine residue of a variety of eukaryotic cellular and viral proteins can have a profound influence on their biological properties. The enzyme that catalyzes this modification, myristoyl-CoA-protein N-myristoyltransferase (NMT), has been identified as a potential target for antiviral and antifungal therapy. Its reaction mechanism is ordered Bi Bi with myristoyl-CoA binding occurring before binding of peptide and CoA release preceding release of myristoylpeptide. Perturbations in the binding of its acyl-CoA substrate would therefore be expected to have an important influence on catalysis. We have synthesized 56 analogs of myristic acid (C14:0) to further characterize the acyl-CoA binding site of Saccharomyces cerevisiae NMT. The activity of fatty acid analogs was assessed using a coupled in vitro assay system that employed the reportedly nonspecific Pseudomonas acyl-CoA synthetase, purified S. cerevisiae NMT, and octapeptide substrates derived from residues 2-9 of the catalytic subunit of cyclic AMP-dependent protein kinase and the Pr55gag polyprotein precursor of human immunodeficiency virus I (HIV-I). Analysis of ketocarbonyl-, ester-, and amide-containing myristic acid analogs (the latter in two isomeric arrangements, the acylamino acid (-CO-NH-) and the amide (-NH-CO)) indicated that the enzyme's binding site is able to accommodate a dipolar protrusion from C4 through C13. This includes the region of the acyl chain occurring near C5-C6 (numbered from carboxyl) that appears to be bound in a bent conformation of 140-150 degrees. The activities of NMT's acyl-CoA substrates decrease with increasing polarity. This relationship was particularly apparent from an analysis of a series of analogs in which the hydrocarbon chain was terminated by (i) an azido group or (ii) one of three nitrogen heterocycles (imidazole, triazole, and tetrazole) alkylated at either nitrogen or carbon. This inverse relationship between polarity and activity was confirmed after comparison of the activities of the closely related ester- or amide-containing tetradecanoyl-CoA derivatives. Members from all of the analog series were surveyed to determine whether they could inhibit replication of human immunodeficiency virus I (HIV-I), a retrovirus that depends upon N-myristoylation of its Pr55gag for propagation. 12-Azidododecanoic acid was the most active analog tested, producing a 60-90% inhibition of viral production in both acutely and chronically infected T-lymphocyte cell lines at a concentration of 10-50 microM without associated cellular toxicity.     

Development of a pharmacologically improved peptide agonist of the leptin receptor

Leptin, a hormone produced by adipose tissue, regulates energy balance in the hypothalamus and is involved in fertility, immune response and carcinogenesis. The existence of disorders related to leptin deficit and leptin overabundance calls for the development of drugs activating or inhibiting the leptin receptor (ObR). We synthesized four proposed receptor-binding leptin fragments (sites I, IIa and IIb, III), their reportedly antagonist analogs, and a peptide chimera composed of the two discontinuous site II arms. To assess the pharmacological utility of leptin fragments, we studied the peptides' ability to stimulate the growth of ObR-positive and ObR-negative cells. The combined site II construct and site III derivatives selectively reversed leptin-induced growth of ObR-positive cells at mid-nanomolar concentrations. However, these peptides appeared to be partial agonists/antagonists as they activated cell growth in the absence of exogenous leptin. A designer site III analog, featuring non-natural amino acids at terminal positions to decrease proteolysis and a blood-brain barrier (BBB) penetration-enhancing carbohydrate moiety, proved to be full agonist to ObR, i.e., stimulated proliferation of different ObR-positive but not ObR-negative cells in the presence or absence of leptin. This glycopeptide bound to isolated ObR on solid-phase assays and activated ERK-1/2 signaling in ObR-positive MCF-7 cells at 100-500 nM concentrations. The glycopeptide was stable in mouse serum, readily crossed endothelial/astrocyte cell layers in a cellular BBB model, and was distributed into the brain of Balb/c mice after intraperitoneal administration. These characteristics suggest a potential pharmaceutical utility of the designer site III glycopeptide in leptin-deficient diseases.     

Mechanism of auxin interaction with Auxin Binding Protein (ABP1): a molecular dynamics simulation study

Auxin Binding Protein 1 (ABP1) is ubiquitous in green plants. It binds the phytohormone auxin with high specificity and affinity, but its role in auxin-induced processes is unknown. To understand the proposed receptor function of ABP1 we carried out a detailed molecular modeling study. Molecular dynamics simulations showed that ABP1 can adopt two conformations differing primarily in the position of the C-terminus and that one of them is stabilized by auxin binding. This is in agreement with experimental evidence that auxin induces changes at the ABP1 C-terminus. Simulations of ligand egress from ABP1 revealed three main routes by which an auxin molecule can enter or leave the ABP1 binding site. Assuming the previously proposed orientation of ABP1 to plant cell membranes, one of the routes leads to the membrane and the other two to ABP1's aqueous surroundings. A network of hydrogen-bonded water molecules leading from the bulk water to the zinc-coordinated ligands in the ABP1 binding site was formed in all simulations. Water entrance into the zinc coordination sphere occurred simultaneously with auxin egress. These results suggest that the hydrogen-bonded water molecules may assist in protonation and deprotonation of auxin molecules and their egress from the ABP1 binding site.     

Overwintering of the sycamore aphid, Drepanosiphum platanoidis

Spatial and temporal aspects of overwintering in the holocyclic sycamore aphid Drepanosiphum platanoidis (Schrank) (Homoptera: Callaphididae) were investigated. Eggs of D. platanoidis were principally laid on trunks and branches of Acer pseudoplatanusL., at considerable distances from the buds. Trunks with rough bark, followed by branches with moderate roughness supported more overwintering eggs than the relatively exposed smooth surfaces of twigs and terminal buds. Eggs were aggregated on rough bark. Aspect had no effect on the number of eggs laid. Experimental manipulations varying ovipara densities showed intra-specific competition among oviparae for optimal oviposition sites. Overwintering egg mortality was a function of time, with the greatest mortality rate occurring in late winter. Egg mortality varied from 80.4% to 76.9% on trunks and 83.0% to 65.9% on branches in 1996 and 1997.