Synthesis and protein kinase inhibitory activity of balanol analogues with modified benzophenone subunits.

A series of analogues of the protein kinase C (PKC) inhibitory natural product balanol which bear modified benzophenone subunits are described. The analogues were designed with the goal of uncovering structure-activity features that could be used in the development of PKC inhibitors with a reduced polar character compared to balanol itself. The results of these studies suggest that most of the benzophenone features found in the natural product are important for obtaining potent PKC inhibitory compounds.

Identification of angiotensin II type 2 (AT2) receptor domains mediating high-affinity CGP 42112A binding and receptor activation.

Chimeric angiotensin II (AngII) receptors constructed of portions of the AT2 receptor substituted into the AT1 receptor revealed the AT2 third extracellular loop and seventh transmembrane-spanning domain as major determinants for the ability to bind and activate in response to the AT2 receptor-selective agonist CGP 42112A. Radioligand binding experiments showed that chimeric AngII receptors possessing the AT2 third extracellular loop and seventh transmembrane-spanning domain bound CGP 42112A with high affinity approaching that of the wild-type AT2 receptor. The presence of the AT2 third extracellular loop appeared sufficient for high-affinity CGP 42112A binding, which was further enhanced by the additional presence of the AT2 seventh transmembrane-spanning domain.

Identification and function of disulfide bridges in the extracellular domains of the angiotensin II type 2 receptor.

The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop.

Mutational analysis of the angiotensin type 2 receptor: contribution of conserved amino acids in the region of the sixth transmembrane domain.

Angiotensin II (AngII) mediates its physiological actions through two receptor subtypes: the Type 1 (AT1) and Type 2 (AT2) receptors. The subtypes have identical affinities for AngII, while sharing only 34% homology. Mutagenesis has focused mainly on the AT1 receptor, identifying residues important for AngII binding.

Role of the amino terminus in ligand binding for the angiotensin II type 2 receptor.

Key amino terminal residues in type 1 (AT1) angiotensin II (AngII) receptors are not conserved within type 2 (AT2) receptors. We therefore characterized amino terminal mutants that are transiently expressed in COS-3 membranes. AT2 amino terminal deletion drastically reduced affinity for AngII, suggesting its importance for this subtype.

Mutational analysis of the angiotensin II type 2 receptor: contribution of conserved extracellular amino acids.

While much work has been done examining the ligand-binding characteristics of the AT1 receptor, very little attention has been focused on the AT2 receptor. Both receptors bind angiotensin II (AngII) with identical affinities, but share only 34% homology. Although it is tempting to assume that conserved residues between the two subtypes are responsible for the binding of AngII, there is little data to support this view.

Mutation of a conserved fifth transmembrane domain lysine residue (Lys215) attenuates ligand binding in the angiotensin II type 2 receptor.

A fifth transmembrane domain lysine residue is conserved in both the type 1 (AT1) and type 2 (AT2) angiotensin II (AngII) receptors. This lysine (Lys199) is believed to play a critical role in peptide binding for the AT1 receptor. To evaluate its possible role in the AT2 receptor, the analogous AT2 residue (Lys199) was changed to glutamine.

Synthesis and protein kinase C inhibitory activities of balanol analogs with replacement of the perhydroazepine moiety.

Balanol is a potent protein kinase C (PKC) inhibitor that is structurally composed of a benzophenone diacid, a 4-hydroxybenzamide, and a perhydroazepine ring. A number of balanol analogs in which the perhydroazepine moiety is replaced have been synthesized and their biological activities evaluated against both PKC and cAMP-dependent kinase (PKA). The results suggested that the activity and the isozyme/kinase selectivity of these compounds are largely related to the conformation about this nonaromatic structural element of the molecules.

Synthesis and protein kinase C inhibitory activities of acyclic balanol analogs that are highly selective for protein kinase C over protein kinase A.

A series of balanol analogs in which the perhydroazepine ring and the p-hydroxybenzamide moiety were combined into an acyclic linked unit have been prepared and evaluated for their inhibitory properties against the serine/threonine kinase PKC. Several low-micromolar to low-nanomolar inhibitors of the alpha, beta I, beta II, gamma, delta, epsilon and eta PKC isozymes were prepared. In general, these acyclic balanol analogs were found to be highly selective for PKC over the serine/threonine kinase PKA.

NMR and molecular modeling study of the conformations of taxol 2'-acetate in chloroform and aqueous dimethyl sulfoxide solutions.

Taxol 2'-acetate, an analog of the antitumor drug taxol, displays no significant in vitro microtubule polymerization activity, thus underscoring the importance of a free 2'-OH group to the biological activity of taxol. Previous work had suggested that the inactivity of taxol 2'-acetate is not due to steric interference by the acetyl group. The present study examined the conformations of taxol 2'-acetate in deuteriochloroform and (2)H2O-deuteriodimethyl sulfoxide solutions and found them to be essentially the same as the respective conformations adopted by taxol itself.

Interaction of somatostatin receptors with G proteins and cellular effector systems.

Somatostatin induces its multiple biological actions by interacting with a family of receptors, referred to as sstr1-sstr5. To determine the molecular mechanisms of action of somatostatin, we have investigated the interaction of the different cloned receptors with G proteins and cellular effector systems. sstr2, sstr3 and sstr5 associate with pertussis toxin-sensitive G proteins and are able to mediate the inhibition of adenylyl cyclase activity by somatostatin.

Characterization of two taxol photoaffinity analogues bearing azide and benzophenone-related photoreactive substituents in the A-ring side chain.

Taxol is a structurally novel and clinically effective antitumor drug, which, unlike other antimitotic agents, induces the assembly of tubulin into microtubules. To characterize the binding site(s) of taxol on the microtubule, taxol-based photoaffinity reagents 1 and 2 bearing photoreactive groups on the A-ring side chain were prepared and evaluated. Taxol analogue 1 exhibits better microtubule assembly activity, greater cytotoxicity toward J774.

3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin.

Taxol possesses an unusual chemical structure, a unique mechanism of action, and demonstrated activity in human malignancies. It is the only antitumor agent that has a binding site on the microtubule polymer. The interaction of Taxol with the microtubule polymer results in the formation of stable bundles of cellular microtubules that are resistant to depolymerization.