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Dive into the research topics where Barry V. L. Potter is active.

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Featured researches published by Barry V. L. Potter.


Neuropharmacology | 1999

Characterisation of the binding of [3H]methyllycaconitine:a new radioligand for labelling α7-type neuronal nicotinic acetylcholine receptors

Andrew R.L Davies; David J. Hardick; Ian S. Blagbrough; Barry V. L. Potter; Adrian J. Wolstenholme; Susan Wonnacott

Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for alpha bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [3H]MLA bound to rat brain membranes with high affinity (Kd = 1.86 +/- 0.31 nM) with a good ratio of specific to non-specific binding. The binding of [3H]MLA was characterised by rapid association (t 1/2 = 2.3 min) and dissociation (t 1/2 = 12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with alpha bungarotoxin-sensitive nAChR. The snake alpha-toxins, alpha bungarotoxin and alpha cobratoxin, displaced [3H]MLA with high affinity (Ki = 1.8 +/- 0.5 and 5.5 +/- 0.9 nM, respectively), whereas nicotine was less potent (Ki = 6.1 +/- 1.1 microM). The distribution of [3H]MLA binding sites in crudely dissected rat brain regions was identical to that of [125I] alpha bungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [3H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake alpha toxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [3H]MLA, therefore, is a novel radiolabel for characterising alpha 7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled alpha bungarotoxin for rapid and accurate equilibrium binding assays.


Nature | 1999

Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose

Andreas H. Guse; C. P. Da Silva; Ingeborg Berg; A. L. Skapenko; Karin Weber; P. Heyer; Martin Hohenegger; Gloria A. Ashamu; H. Schulze-Koops; Barry V. L. Potter; Georg W. Mayr

Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells. Although it can lead to the release of calcium ions in T lymphocytes, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.


Clinical Cancer Research | 2006

Phase I Study of STX 64 (667 Coumate) in Breast Cancer Patients: The First Study of a Steroid Sulfatase Inhibitor

Susannah J. Stanway; Atul Purohit; L. W. Lawrence Woo; Saulat Sufi; David M. Vigushin; Rebecca Ward; Richard Wilson; Frank Z. Stanczyk; Nicola Dobbs; Elena Kulinskaya; Moira A. Elliott; Barry V. L. Potter; Michael J. Reed; R. Charles Coombes

Purpose: Inhibition of steroid sulfatase (STS), the enzyme responsible for the hydrolysis of steroid sulfates, represents a potential novel treatment for postmenopausal women with hormone-dependent breast cancer. Estrone and DHEA are formed by this sulfatase pathway and can be converted to steroids (estradiol and androstenediol, respectively), which have potent estrogenic properties. Experimental Design: STX64 (667 Coumate), a tricylic coumarin-based sulfamate that irreversibly inhibits STS activity, was selected for entry into the first phase I trial of a STS inhibitor in postmenopausal women with breast cancer. STX64 was administered orally (nine patients at 5 mg and five patients at 20 mg) as an initial dose followed 1 week later by 3 × 2 weekly cycles, with each cycle comprising daily dosing for 5 days followed by 9 days off treatment. Blood and tumor tissue samples were collected for the assessment of STS activity and serum was obtained for steroid hormone measurements before and after treatment. Results: The median inhibition of STS activity by STX64 was 98% in peripheral blood lymphocytes (PBL) and 99% in breast tumor tissue at the end of the 5-day dosing period. As expected, serum concentrations of estrone, estradiol, androstenediol, and DHEA all decreased significantly from pretreatment levels. Unexpectedly, androstenedione and testosterone concentrations also decreased. Four patients, all of whom had previously progressed on aromatase inhibitors, showed evidence of stable disease for 2.75 to 7 months. The drug was well tolerated with only minor drug-related adverse events recorded. Conclusion: STX64 is a potent, well-tolerated STS inhibitor. It inhibits STS activity in PBLs and tumor tissues and causes significant decreases in serum concentrations of steroids with estrogenic properties.


Structure | 1999

Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate

Elena Baraldi; Kristina Djinovic Carugo; Marko Hyvönen; Paola Lo Surdo; Andrew M. Riley; Barry V. L. Potter; Ronan O’Brien; John E. Ladbury; Matti Saraste

BACKGROUND The activity of Brutons tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. RESULTS We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D-myo-inositol 1,3,4,5-tetra-kisphosphate (Ins (1,3,4,5)P4). The inositol Ins (1,3,4,5)P4 binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C-delta. A second Ins (1,3,4,5)P4 molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P4 to the wild type (Kd = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. CONCLUSIONS Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a Galpha protein.


Journal of Biological Chemistry | 2003

Identification of Mammalian Vps24p as an Effector of Phosphatidylinositol 3,5-Bisphosphate-dependent Endosome Compartmentalization

Paul Whitley; Barbara J. Reaves; Makoto Hashimoto; Andrew M. Riley; Barry V. L. Potter; Geoffrey D. Holman

Phosphatidylinositol 3,5-bisphosphate is a membrane lipid found in all eukaryotes so far studied but downstream effector proteins of this lipid have yet to be identified. Here we report the use of cDNA phage libraries in conjunction with synthetic biotinylated derivatives of phosphatidylinositol 3,5-bisphosphate in the identification of a mammalian phosphatidylinositol 3,5-bisphosphate-binding protein, mVps24p. This protein is orthologous to the Saccharomyces cerevisiae protein, Vps24p, a class-E vacuolar protein-sorting protein. Using in vitro liposome binding and competition assays, we demonstrate that mVps24p selectively binds to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate in preference to other phosphoinositides tested. When expressed in cultured mammalian cells, full-length mVps24p is cytosolic. However, when cells expressing the full-length mVps24p are co-transfected with a mutated form of mVps4p (which is defective in ATP hydrolysis), or when a N-terminal construct of mVps24p is expressed, the class-E cellular phenotype with swollen vacuoles is induced and mVps24p is membrane-associated. Furthermore, the accumulation of the N-terminal mVps24p construct on the swollen endosomal membranes is abrogated when phosphatidylinositol 3,5-bisphosphate synthesis is blocked with wortmannin. These data provide the first direct link between phosphatidylinositol 3,5-bisphosphate and the protein machinery involved in the production of the class-E cellular phenotype. We hypothesize that accumulation of Vps24 on membranes occurs when membrane association (dependent on interaction of phosphatidylinositol 3,5-bisphosphate with the N-terminal domain of the protein) is uncoupled from membrane disassociation (driven by Vps4p).


Chemistry & Biology | 2000

Potent active site-directed inhibition of steroid sulphatase by tricyclic coumarin-based sulphamates

L. W. Lawrence Woo; Atul Purohit; Bindu Malini; Michael J. Reed; Barry V. L. Potter

BACKGROUND There is now abundant evidence that inhibition of steroid sulphatase alone or in conjunction with inhibition of aromatase may enhance the response of postmenopausal patients with hormone-dependent breast cancer to this type of endocrine therapy. Additionally, sulphatase inhibition has been proposed to be of potential therapeutic benefit in the immune system and for neuro-degenerative diseases. After the finding that our first highly potent active site-directed steroid sulphatase inhibitor, oestrone-3-O-sulphamate (EMATE), was highly oestrogenic, we proposed non-steroidal coumarin sulphamates such as 4-methylcoumarin-7-O-sulphamate (COUMATE) as alternative non-steroidal steroid sulphatase inhibitors. In this work, we describe how tricyclic coumarin-based sulphamates have been developed which are even more potent than COUMATE, are non-oestrogenic and orally active. We also discuss potential mechanisms of action. RESULTS 4-Ethyl- (4), 4-(n-propyl)- (6), 3-ethyl-4-methyl- (8), 4-methyl-3-(n-propyl)coumarin-7-O-sulphamate (11); the tricyclic derivatives 665COUMATE (13), 666COUMATE (15), 667COUMATE (17), 668COUMATE (20) and the tricyclic oxepin sulphamate (22) were synthesised. In a placental microsome preparation, all of these analogues were found to be more active than COUMATE in the inhibition of oestrone sulphatase, with the most potent inhibitor being 667COUMATE which has an IC(50) of 8 nM, some 3-fold lower than that for EMATE (25 nM). In addition, 667COUMATE was also found to inhibit DHEA-sulphatase some 25-fold more potently than EMATE in a placental microsome preparation. Like EMATE, 667COUMATE acts in a time- and concentration-dependent manner, suggesting that it is an active site-directed inhibitor. However, in contrast to EMATE, 667COUMATE has the important advantage of not being oestrogenic. In addition, we propose several diverse mechanisms of action for this active site-directed steroid sulphatase inhibitor in the light of recent publications on the crystal structures of human arylsulphatases A and B and the catalytic site topology for the hydrolysis of a sulphate ester. CONCLUSIONS A highly potent non-steroidal, non-oestrogenic and irreversible steroid sulphatase inhibitor has been developed. Several mechanisms of action for an active site-directed steroid sulphatase inhibitor are proposed. With 667COUMATE now in pre-clinical development for clinical trial, this should allow the biological and/or clinical significance of steroid sulphatase inhibitors in the treatment of postmenopausal women with hormone-dependent breast cancer and other therapeutic indications to be fully evaluated.


European Journal of Neuroscience | 1999

An autoradiographic study of the distribution of binding sites for the novel alpha7-selective nicotinic radioligand [3H]-methyllycaconitine in the mouse brain.

Paul Whiteaker; Andrew R.L Davies; Michael J. Marks; Ian S. Blagbrough; Barry V. L. Potter; Adrian J. Wolstenholme; Allan C. Collins; Susan Wonnacott

[3H]‐Methyllycaconitine ([3H]‐MLA) is a new radioligand with selectivity for α7‐type neuronal nicotinic acetylcholine receptors (nAChRs). In our previous study [Davies, A.R.L., Hardick, D.J., Blagbrough, I.S., Potter, B.V.L., Wolstenholme, A.J. & Wonnacott, S. (1999) Neuropharmacology, 38, 679–690], this radioligand labelled a single class of site in rat brain membranes; its pharmacology and distribution in crudely dissected brain regions closely paralleled that of the well‐established α7‐ligand [125I]‐α‐bungarotoxin. However, a small population of [3H]‐MLA binding sites was apparently insensitive to α‐bungarotoxin. Here we have extended the study to mouse brain, using autoradiography to examine the distribution of [3H]‐MLA and [125I]‐α‐bungarotoxin binding sites. [3H]‐MLA labelled a single class of site in mouse brain membranes with a KD of 2.2 nm and a Bmax of 45.6 fmol/mg protein. Specific binding, defined by unlabelled MLA (Ki = 0.69 nm), was completely inhibited by (–)‐nicotine (Ki = 1.62 μm), whereas α‐bungarotoxin inhibited only 85% of specific binding (Ki = 3.5 nm). The distributions of [125I]‐α‐bungarotoxin and [3H]‐MLA binding sites were compared by autoradiography, and binding was quantitated in 72 brain regions. Binding of both radioligands was highly correlated, with highest densities in the dorsal tegmental nucleus of the pons, colliculi and hippocampus. Serial sections labelled with [3H]‐MLA in the absence or presence of unlabelled MLA or α‐bungarotoxin provided no evidence for any α‐bungarotoxin‐resistant binding. The results are discussed in terms of binding sites that are inaccessible to α‐bungarotoxin in membrane preparations. This study demonstrates the utility of [3H]‐MLA for characterization of α7‐type nicotinic receptors in mammalian brain, and suggests that it labels a population identical to that defined by [125I]‐α‐bungarotoxin.


Cancer Research | 2005

Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway by Inositol Pentakisphosphate Results in Antiangiogenic and Antitumor Effects

Tania Maffucci; Enza Piccolo; Albana Cumashi; Manuela Iezzi; Andrew M. Riley; Adolfo Saiardi; H. Yasmin Godage; Cosmo Rossi; Massimo Broggini; Stefano Iacobelli; Barry V. L. Potter; Paolo Innocenti; Marco Falasca

The purpose of this study was to investigate the antiangiogenic and in vivo properties of the recently identified phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor Inositol(1,3,4,5,6) pentakisphosphate [Ins(1,3,4,5,6)P5]. Because activation of the PI3K/Akt pathway is a crucial step in some of the events leading to angiogenesis, the effect of Ins(1,3,4,5,6)P5 on basic fibroblast growth factor (FGF-2)-induced Akt phosphorylation, cell survival, motility, and tubulogenesis in vitro was tested in human umbilical vein endothelial cells (HUVEC). The effect of Ins(1,3,4,5,6)P5 on FGF-2-induced angiogenesis in vivo was evaluated using s.c. implanted Matrigel in mice. In addition, the effect of Ins(1,3,4,5,6)P5 on growth of ovarian carcinoma SKOV-3 xenograft was tested. Here, we show that FGF-2 induces Akt phosphorylation in HUVEC resulting in antiapoptotic effect in serum-deprived cells and increase in cellular motility. Ins(1,3,4,5,6)P5 blocks FGF-2-mediated Akt phosphorylation and inhibits both survival and migration in HUVEC. Moreover, Ins(1,3,4,5,6)P5 inhibits the FGF-2-mediated capillary tube formation of HUVEC plated on Matrigel and the FGF-2-induced angiogenic reaction in BALB/c mice. Finally, Ins(1,3,4,5,6)P5 blocks the s.c. growth of SKOV-3 xenografted in nude mice to the same extent than cisplatin and it completely inhibits Akt phosphorylation in vivo. These data definitively identify the Akt inhibitor Ins(1,3,4,5,6)P5 as a specific antiangiogenic and antitumor factor. Inappropriate activation of the PI3K/Akt pathway has been linked to the development of several diseases, including cancer, making this pathway an attractive target for therapeutic strategies. In this respect, Ins(1,3,4,5,6)P5, a water-soluble, natural compound with specific proapoptotic and antiangiogenic properties, might result in successful anticancer therapeutic strategies.


The Journal of Steroid Biochemistry and Molecular Biology | 1998

The development of A-ring modified analogues of oestrone-3-O-sulphamate as potent steroid sulphatase inhibitors with reduced oestrogenicity

Atul Purohit; K.A. Vernon; A.E. Wagenaar Hummelinck; L.W.L. Woo; Hatem Hejaz; Barry V. L. Potter; Michael J. Reed

Steroid sulphatases regulate the formation of oestrogenic steroids which can support the growth of endocrine-dependent breast tumours. The development of potent steroid sulphatase inhibitors could therefore have considerable therapeutic potential. Several such inhibitors have now been developed of which the most potent to date is oestrone-3-O-sulphamate (EMATE). Unexpectedly, this inhibitor proved to be a potent oestrogen. In an attempt to reduce the oestrogenicity, whilst retaining the potent sulphatase inhibitory properties associated with this type of molecule, a number of A-ring modified derivatives were designed and synthesized. A-ring modified compounds included the 2-methoxy, 2/4-nitro, 2/4-n-propyl and 2/4-allyl EMATE analogues. The ability of these derivatives to inhibit oestrone sulphatase activity was examined using placental microsomes. The allyl-substituted EMATE derivatives were more potent inhibitors than the propyl analogues but were all considerably less potent than EMATE. In contrast, the 2-methoxy and 2/4-nitro analogues were potent sulphatase inhibitors with 4-nitro EMATE being 5 times more active than EMATE. The 4-nitro, 2-methoxy, 4-n-propyl and 4-allyl derivatives were also tested in vivo for their oestrogenicity and ability to inhibit sulphatase activity. While both 4-nitro and 2-methoxy EMATE were potent inhibitors in vivo, 2-methoxy EMATE had no stimulatory effect on uterine growth in ovariectomized rats. The identification of a potent steroid sulphatase inhibitor lacking any oestrogenicity, such as 2-methoxy EMATE, should be of considerable value in evaluating the potential of steroid sulphatase inhibition for breast cancer therapy.


International Journal of Cancer | 2008

17β‐hydroxysteroid dehydrogenase Type 1, and not Type 12, is a target for endocrine therapy of hormone‐dependent breast cancer

Joanna M. Day; Paul A. Foster; Helena J. Tutill; Michael F.C. Parsons; Simon P. Newman; Surinder K. Chander; Gillian M. Allan; Harshani R. Lawrence; Nigel Vicker; Barry V. L. Potter; Michael J. Reed; Atul Purohit

Oestradiol (E2) stimulates the growth of hormone‐dependent breast cancer. 17β‐hydroxysteroid dehydrogenases (17β‐HSDs) catalyse the pre‐receptor activation/inactivation of hormones and other substrates. 17β‐HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17β‐HSD, 17β‐HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17β‐HSD1 which is important for E2 production and report the inhibition of E1‐stimulated breast tumor growth by STX1040, a non‐oestrogenic selective inhibitor of 17β‐HSD1, using a novel murine model. 17β‐HSD1 and 17β‐HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17β‐HSD12 was highly expressed in breast cancer cell lines, only 17β‐HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1‐stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 μg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1‐stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17β‐HSD, 17β‐HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17β‐HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone‐dependent breast cancer.

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Atul Purohit

Imperial College London

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