Anti-AIDS agents 81. Design, synthesis, and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors.

In our continuing study of triterpene derivatives as potent anti-HIV agents, different C-3 conformationally restricted betulinic acid (BA, 1) derivatives were designed and synthesized in order to explore the conformational space of the C-3 pharmacophore. 3-O-Monomethylsuccinyl-betulinic acid (MSB) analogues were also designed to better understand the contribution of the C-3' dimethyl group of bevirimat (2), the first-in-class HIV maturation inhibitor, which is currently in phase IIb clinical trials. In addition, another triterpene skeleton, moronic acid (MA, 3), was also employed to study the influence of the backbone and the C-3 modification toward the anti-HIV activity of this compound class. This study enabled us to better understand the structure-activity relationships (SAR) of triterpene-derived anti-HIV agents and led to the design and synthesis of compound 12 (EC(50): 0.0006 microM), which displayed slightly better activity than 2 as a HIV-1 maturation inhibitor.

Emi2-mediated inhibition of E2-substrate ubiquitin transfer by the anaphase-promoting complex/cyclosome through a D-box-independent mechanism.

Vertebrate eggs are arrested at Metaphase II by Emi2, the meiotic anaphase-promoting complex/cyclosome (APC/C) inhibitor. Although the importance of Emi2 during oocyte maturation has been widely recognized and its regulation extensively studied, its mechanism of action remained elusive. Many APC/C inhibitors have been reported to act as pseudosubstrates, inhibiting the APC/C by preventing substrate binding. Here we show that a previously identified zinc-binding region is critical for the function of Emi2, whereas the D-box is largely dispensable. We further demonstrate that instead of acting through a "pseudosubstrate" mechanism as previously hypothesized, Emi2 can inhibit Cdc20-dependent activation of the APC/C substoichiometrically, blocking ubiquitin transfer from the ubiquitin-charged E2 to the substrate. These findings provide a novel mechanism of APC/C inhibition wherein the final step of ubiquitin transfer is targeted and raise the interesting possibility that APC/C is inhibited by Emi2 in a catalytic manner.

A prochelator activated by beta-secretase inhibits Abeta aggregation and suppresses copper-induced reactive oxygen species formation.

The intersection of the amyloid cascade hypothesis and the implication of metal ions in Alzheimer's disease progression has sparked an interest in using metal-binding compounds as potential therapeutic agents. In the present work, we describe a prochelator SWH that is enzymatically activated by beta-secretase to produce a high affinity copper chelator CP. Because beta-secretase is responsible for the amyloidogenic processing of the amyloid precursor protein, this prochelator strategy imparts disease specificity toward copper chelation not possible with general metal chelators. Furthermore, once activated, CP efficiently sequesters copper from amyloid-beta, prevents and disassembles copper-induced amyloid-beta aggregation, and diminishes copper-promoted reactive oxygen species formation.

Tyrosine phosphorylation of G protein alpha subunits by pp60c-src.

A number of lines of evidence suggest that cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp60c-src kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60c-src and G proteins may occur with functional consequences. Preparations of pp60c-src isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein alpha subunits (G alpha) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and G alpha(GDP) uncomplexed by beta gamma subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory G alpha (G alpha s) modestly increases the rate of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gs as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for pp60c-src.

Adrenergic receptors. Models for regulation of signal transduction processes.

Adrenergic receptors are prototypic models for the study of the relations between structure and function of G protein-coupled receptors. Each receptor is encoded by a distinct gene. These receptors are integral membrane proteins with several striking structural features. They consist of a single subunit containing seven stretches of 20-28 hydrophobic amino acids that represent potential membrane-spanning alpha-helixes. Many of these receptors share considerable amino acid sequence homology, particularly in the transmembrane domains. All of these macromolecules share other similarities that include one or more potential sites of extracellular N-linked glycosylation near the amino terminus and several potential sites of regulatory phosphorylation that are located intracellularly. By using a variety of techniques, it has been demonstrated that various regions of the receptor molecules are critical for different receptor functions. The seven transmembrane regions of the receptors appear to form a ligand-binding pocket. Cysteine residues in the extracellular domains may stabilize the ligand-binding pocket by participating in disulfide bonds. The cytoplasmic domains contain regions capable of interacting with G proteins and various kinases and are therefore important in such processes as signal transduction, receptor-G protein coupling, receptor sequestration, and down-regulation. Finally, regions of these macromolecules may undergo posttranslational modifications important in the regulation of receptor function. Our understanding of these complex relations is constantly evolving and much work remains to be done. Greater understanding of the basic mechanisms involved in G protein-coupled, receptor-mediated signal transduction may provide leads into the nature of certain pathophysiological states.

Structural basis of beta-adrenergic receptor subtype specificity studied with chimeric beta 1/beta 2-adrenergic receptors.

The beta 1- and beta 2-adrenergic receptors are two structurally related, but pharmacologically distinguishable, receptor subtypes, both of which activate adenylyl cyclase in a catecholamine-dependent manner through the guanine nucleotide-binding regulatory protein Gs. The receptors are approximately 50% identical in amino acid sequence and each is characterized by the presence of seven putative transmembrane domains. To elucidate the structural basis for the pharmacological distinctions between these two receptor subtypes, we constructed a series of chimeric beta 1/beta 2-adrenergic receptor genes and expressed them by injection of RNA into Xenopus laevis oocytes. The pharmacological properties of the expressed chimeric receptor proteins were assessed by radioligand binding and adenylyl cyclase assays utilizing subtype-selective agonists and antagonists. Our data indicate that transmembrane region IV is largely responsible for determining beta 1 vs. beta 2 properties with respect to agonist binding (relative affinities for epinephrine and norepinephrine). Transmembrane regions VI and VII play an important role in determining binding of beta 1 vs. beta 2 selective antagonists. However, a number of the other transmembrane regions also contribute, to a lesser extent, to the determination of beta-adrenergic receptor subtype specificity for agonists and antagonists. Thus, several of the membrane-spanning regions appear to be involved in the determination of receptor subtype specificity, presumably by formation of a ligand-binding pocket, with determinants for agonist and antagonist binding being distinguishable.

Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.

Molecular engineering of mechanophore activity for stress-responsive polymeric materials

© The Royal Society of Chemistry.Force reactive functional groups, or mechanophores, have emerged as the basis of a potential strategy for sensing and countering stress-induced material failure. The general utility of this strategy is limited, however, because the levels of mechanophore activation in the bulk are typically low and observed only under large, typically irreversible strains. Strategies that enhance activation are therefore quite useful. Molecular-level design principles by which to engineer enhanced mechanophore activity are reviewed, with an emphasis on quantitative structure-activity studies determined for a family of gem-dihalocyclopropane mechanophores. This journal is

Internal Capital, External Knowledge, and Random Draws: Three Drivers of Organizational Structure

This dissertation explores the complex interactions between organizational structure and the environment. In Chapter 1, I investigate the effect of financial development on the formation of European corporate groups. Since cross-country regressions are hard to interpret in a causal sense, we exploit exogenous industry measures to investigate a specific channel through which financial development may affect group affiliation: internal capital markets. Using a comprehensive firm-level dataset on European corporate groups in 15 countries, we find that countries

with less developed financial markets have a higher percentage of group affiliates in more capital intensive industries. This relationship is more pronounced for young and small firms and for affiliates of large and diversified groups. Our findings are consistent with the view that internal capital markets may, under some conditions, be more efficient than prevailing external markets, and that this may drive group affiliation even in developed economies. In Chapter 2, I bridge current streams of innovation research to explore the interplay between R&D, external knowledge, and organizational structure–three elements of a firm’s innovation strategy which we argue should logically be studied together. Using within-firm patent assignment patterns,

we develop a novel measure of structure for a large sample of American firms. We find that centralized firms invest more in research and patent more per R&D dollar than decentralized firms. Both types access technology via mergers and acquisitions, but their acquisitions differ in terms of frequency, size, and i\ntegration. Consistent with our framework, their sources of value creation differ: while centralized firms derive more value from internal R&D, decentralized firms rely more on external knowledge. We discuss how these findings should stimulate more integrative work on theories of innovation. In Chapter 3, I use novel data on 1,265 newly-public firms to show that innovative firms exposed to environments with lower M&A activity just after their initial public offering (IPO) adapt by engaging in fewer technological acquisitions and

more internal research. However, this adaptive response becomes inertial shortly after IPO and persists well into maturity. This study advances our understanding of how the environment shapes heterogeneity and capabilities through its impact on firm structure. I discuss how my results can help bridge inertial versus adaptive perspectives in the study of organizations, by

documenting an instance when the two interact.