Dissecting isoform selectivity of P13K inhibitors: the role of non-conserved residues in the catalytic pocket

The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr⁸⁸⁶ and Lys⁸⁹⁰ of the PI3Kγ isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the β isoform (Glu⁸⁵⁸ and Asp⁸⁶²) and those in the α isoform (His⁸⁵⁵ and Gln⁸⁵⁹) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of β-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the α-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kβ activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the α-isoform-selectivity of the compounds studied.

Angiotensin I-converting enzyme transition state stabilization by HIS1089 : evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His¹⁰⁸⁹ as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His¹⁰⁸⁹ to Ala or Leu. At pH 7.5, with Ang I as substrate, k_cat/K_m values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (k_cat) with minor effects on ground state substrate binding (K_m). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His¹⁰⁸⁹ --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His¹⁰⁸⁹ side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His¹⁰⁸⁹ on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.

Phenylalanine-544 plays a key role in substrate and inhibitor binding by providing a hydrophobic packing point at the active site of insulin-regulated aminopeptidase

Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn2+ ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn2+ ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the Ki in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.

Insulin-regulated aminopeptidase : analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine β-naphthalamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.

Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70–86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.

10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin

Objective
Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects.

Design
HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells.

Results
Ten mM glucosamine and 5–10 mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn387 and Asn440, abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells.

Conclusions
Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.

Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.

Properties of an in vitro selected Pb2+ cleavage motif

The addition of Pb 2+ to a small RNA molecule consisting of an asymmetric internal loop of six nucleotides results in site-specific cleavage followed by hydrolysis of the 2′,3′-cyclic phosphate intermediate [Pan, T., & Uhlenbeck, O. C. (1992) Nature 358, 560-563]. Here we show that the reaction is highly specific for Pb 2+ and the cleavage rate increases exponentially with pH from 5.5 to 7.0, both in the presence and in the absence of Mg 2+. This suggests that the reaction mechanism involves Pb 2+ hydroxide acting as a base. Several sequence variants of the RNA are found to be equally active in both steps of the reaction, suggesting that they fold into a similar structure.

Alterations in renal and myocardial adrenoceptors associated with ethynyloestradiol- and levonorgestrel-induced hypertension in the rat

Hypertension is one of many side effects of oral contraceptive use in a small percentage of women. Although the underlying pathology has yet to be fully resolved, alterations in the renin-angiotensin-aldosterone axis, sympathetic nervous system/ renal and cardiac function have been implicated. In the thesis to be presented, the possible involvement of alterations in renal and myocardial adrenoceptor characteristics in the pathogenesis of steroid contraceptive-induced hypertension in rats was examined by radioligand binding techniques. In Chapter 2, a rat model of OC hypertension is described. Chronic low-dose administration of ethynyloestradiol (EE2), levonorgestrel (NG) or a combination of both steroids (EE2/NG) to female Sprague-Dawley rats was shown to significantly increase systolic blood pressure (SBP). Renal and cardiac hypertrophy developed in association with EE2-, EE2/NG- but not NG-induced hypertension. Moreover, whereas administration of NG alone attenuated body weight gain, combined EE2/NG administration increased body weight gain from the second week of treatment onwards. Based on the above observations, it is proposed that EE2 and NG induce hypertension in rats via different mechanisms. Although SBP was elevated to a similar maximum in all steroid-treated groups (+ 20 mmHg compared to controls), only with EE2 administration did SBP remain elevated for the duration of the 17 week treatment regimen. NG may therefore have a protective effect on blood pressure with long-term combined steroid contraceptive treatment. In Chapter 4, renal adrenoceptors were characterized using radioactively labelled adrenocephor antagonists. Under appropriate conditions, binding of [3H]-prazosin and [3H]-rauwolscine to membrane preparations of whole rat kidney displayed the kinetics, saturability and specificity of α1- and α2 -adrenoceptors respectively, which were present in a ratio 3:1. In contrast, [3H]-dihydroergocryptine ([3H]-DHE) apparently bound to both α1 and α2-adrenoceptors. Binding sites identified by [125I] –iodocyanopindolol (ICYP) had the recognition characteristics of β-adrenoceptors. In drug competition studies using the subtype-selective antagonists practolol (β1) and ICI 118,551 (β2)/ the ratio of β1- to β2 -adrenoceptors was found to be approximately 2:1. Subsequently, renal adrenoceptors were investigated at various stages during the development of hypertension with the different steroid contraceptive treatments (Chapters 5 and 6). Preliminary binding studies with [3H]-DHE and [3H]-prazosin suggested that the number of renal α2 - but not α1-adrenoceptors was reduced in rats with established EE2-induced hypertension (17 weeks treatment). This was subsequently confirmed using [3H]-rauwolscine, which in addition showed that the reduction in renal α2 -adrenoceptor number occurred during the developmental stage of EE2/NG~induced hypertension (6 weeks treatment) and established EE2-induced hypertension (12 weeks treatment). NG induced hypertension was unassociated with changes in renal α1- and α2-adrenoceptor characteristics. Renal β-adrenoceptor affinity was reduced in established EE2-, but not NG- or EE2/NG- induced hypertension. Moreover, the β-adrenoceptor agonist (-)-isoprenaline bound to renal β-adrenoceptors with reduced affinity following EE2 administration. Several endogenous and synthetic steroids were found to be ineffective inhibitors of [3H] –prazosin, [3H] –rauwolscine and ICYP binding excluding a direct interaction of these steroids with renal α1-, α2- and β -adrenoceptors. In Chapter 7, myocardial adrenoceptors were characterized and investigated in steroid-treated rats. In membrane preparations of whole myocardium, [3H]-prazosin binding was characteristically to α1- adrenoceptors, whereas there was a notable absence of [3H]-rauwolscine binding. Using ICYP, β-adrenoceptors were also detected, the ratio of β1- to β2~adrenoceptors being 3:1. Steroid contraceptive-induced hypertension was not associated with myocardial α1-adrenoceptor changes. Similarly, myocardial β-adrenoceptors were unchanged in established EE2-, NG- and EE2/NG-induced hypertension (12 weeks treatment). The affinity of (-)-isoprenaline for myocardial β-adrenoceptors was unaffected by EE2 aditiinistration. These studies suggest that established EE2- but not NG-induced hypertension in rats is associated with selective alterations in renal α2- and (β-adrenoceptors. These adrenoceptor changes may help to maintain elevated blood pressure by affecting the control of renal function by the sympathetic nervous system, catecholamines and several hormones which affect renin release and the transport of fluid and electrolytes in the nephron.

Resource distribution influences positive edge effects in a seagrass fish

According to conceptual models, the distribution of resources plays a critical role in determining how organisms distribute themselves near habitat edges. These models are frequently used to achieve a mechanistic understanding of edge effects, but because they are based predominantly on correlative studies, there is need for a demonstration of causality, which is best done through experimentation. Using artificial seagrass habitat as an experimental system, we determined a likely mechanism underpinning edge effects in a seagrass fish. To test for edge effects, we measured fish abundance at edges (0-0.5 m) and interiors (0.5-1 m) of two patch configurations: continuous (single, continuous 9-m2 patches) and patchy (four discrete 1-m2 patches within a 9-m2 area). In continuous configurations, pipefish (Stigmatopora argus) were three times more abundant at edges than interiors (positive edge effect), but in patchy configurations there was no difference. The lack of edge effect in patchy configurations might be because patchy seagrass consisted entirely of edge habitat. We then used two approaches to test whether observed edge effects in continuous configurations were caused by increased availability of food at edges. First, we estimated the abundance of the major prey of pipefish, small crustaceans, across continuous seagrass configurations. Crustacean abundances were highest at seagrass edges, where they were 16% greater than in patch interiors. Second, we supplemented interiors of continuous treatment patches with live crustaceans, while control patches were supplemented with seawater. After five hours of supplementation, numbers of pipefish were similar between edges and interiors of treatment patches, while the strong edge effects were maintained in controls. This indicated that fish were moving from patch edges to interiors in response to food supplementation. These approaches strongly suggest that a numerically dominant fish species is more abundant at seagrass edges due to greater food availability, and provide experimental support for the resource distribution model as an explanation for edge effects.

Economic freedom and economic growth: Does specification make a difference?

In this paper we apply meta-analytic techniques to the literature on the impact of economic freedom on economic growth and find an overall positive direct association between economic freedom and economic growth. A positive indirect effect of economic freedom on economic growth through the stimulation of physical capital is also identified. However, the literature is affected by specification bias with respect to controls for physical capital. The omission of physical capital results in larger estimates of the economic freedom–economic growth association. Further, the use of panel data leads to smaller estimates of the impact of economic freedom on economic growth. The meta-analysis is confirmed by primary cross-sectional and panel data analysis of 82 countries for the period 1970–1999.

Bribery and networking: Is there a difference?

Nature of bribery contrasted with the practice of networking - whether the different moral and legal treatment of the practices is based on principle or simply cultural preference - bribery and the form of networking known as expenditure networking both have the affect of distorting the proper functioning of the market economy by encouraging business decisions to be made on the basis of economically irrelevant considerations - the objective of both practices is to gain a market advantage by providing a 'sweetener' to a person responsible for delegating the provision of goods and services - expenditure networking does have the positive side effect of drawing people together in a social setting.

Removal of VOCs by photocatalysis process using adsorption enhanced TiO2–SiO2 catalyst

Volatile organic compounds (VOCs) exist widely in both the indoor and outdoor environment. The main contributing sources of VOCs are motor vehicle exhaust and solvent utilization. Some VOCs are toxic and carcinogenic to human health, such as benzene. In this study, TiO₂–SiO₂ based photocatalysts were synthesized using the sol–gel method, with high surface areas of 274.1–421.1 m²/g obtained. Two types of pellets were used as catalysts in a fixed-bed reactor installed with a UV black light lamp. Experiments were conducted to compare their efficiencies in degrading the VOCs. Toluene was used as the VOC indicator. When the toluene laden gas stream passed through the photocatalytic reactor, the removal efficiencies were determined using a FTIR multi-gas analyser, which was connected to the outlet of the reactor to analyse the toluene concentrations. As the TiO₂–SiO₂ pellets used have a high adsorption capacity, they had dual functions as a photocatalyst and adsorbent in the hybrid photocatalysis and adsorption system. The experiments demonstrated that the porous photocatalyst with very high adsorptive capacity enhanced the subsequent photocatalysis reactions and lead to a positive synergistic effect. The catalyst can be self-regenerated by photocatalytic oxidation of the adsorbed VOCs. When the UV irradiation and feeding gas is continuous, a destruction efficiency of about 25% was achieved over a period of 20 h. Once the system was designed and operated into adsorption/regeneration mode, a higher removal efficiency of about 55% was maintained. It was found that the catalyst pellets with a higher surface area (421 m²/g) achieved higher conversion efficiency (100%) for a longer period than those with a lower surface area. A full spectrum scan was carried out using a Bio-rad Infrared spectrometer, finding that the main components of the treated gas stream leaving the reactor, along with untreated toluene, were CO₂ and water. The suspected intermediates of aliphatic hydrocarbons and CO were found in minimal amounts or were non detectable. The kinetic rate constants were calculated from the experimental results, it appeared that the stronger adsorption capacity, i.e. larger specific surface area, the higher conversion efficiency would be achieved.

Cortisol disrupts the ability of estradiol-17β to induce the LH surge in ovariectomized ewes

Stress disrupts the preovulatory luteinizing hormone (LH) surge in females, but the mechanisms are unknown. We tested the hypothesis that cortisol compromises the ability of estrogen to induce a preovulatory-like LH surge in ovariectomized ewes in both the breeding and nonbreeding season. Luteinizing hormone surges were induced in ovariectomized ewes by treatment with progesterone followed by a surge-inducing estradiol-17β (E2) stimulus using a crossover design. The experiment was replicated in the breeding and nonbreeding seasons. Cortisol reduced the incidence of LH surges irrespective of season. Cortisol increased the latency from E2 stimulus to the onset of the surge in the breeding season only and suppressed the LH surge amplitude during both seasons (P < 0.01). We conclude that cortisol can interfere with the LH surge in several ways: delay, blunt, and in extreme cases prevent the E2-induced LH surge. Furthermore, the effect of cortisol to delay the E2-induced LH surge is more pronounced in the breeding season. These results show that cortisol disrupts the positive feedback effect of E2 to trigger an LH surge and suggest the involvement of multiple mechanisms.

Effects of strategies to promote children's physical activity on potential mediators

The aim of this paper is to review evidence of the effectiveness of interventions that present physical activity outcomes and potential mediators of behavioural change among 4–12-year-old children. A systematic search of electronic databases for original research articles published in peer-review journals between January 1985 and the end of June 2006 was carried out. A total of 19 studies that reported intervention effects on physical activity and mediators of behavioural change were identified. The most common mediators reported included physical activity knowledge or beliefs (11 studies); self-efficacy (8 studies); and enjoyment or preference for physical activity (6 studies). Less frequently reported mediators included attitudes, behavioural capability, intentions, outcome expectancies, social norms, social support and self-concept. Seven of the 11 interventions that reported intervention effects on knowledge/beliefs stated positive changes in this mediator. Four of the eight studies that reported intervention effects on self-efficacy had significant improvements; however, only two out of six interventions reported significant improvements in physical activity enjoyment or preference. None of the studies reviewed reported whether changes in these constructs mediated changes in children's physical activity behaviours. Although more than half of the studies reviewed reported a positive intervention effect on children's physical activity, no study carried out a mediating analysis to attempt to identify the mechanisms of change. Future research should more clearly identify the mediators of behavioural change that are being targeted and whether this explains intervention effects.