David R Adams

Publications (41)157.27 Total impact

• Article: Chemical informatics uncovers a new role for moexipril as a novel inhibitor of cAMP phosphodiesterase-4 (PDE4).

  Ryan T Cameron, Ryan G Coleman, Jon P Day, Krishna C Yalla, Miles D Houslay, David R Adams, Brian K Shoichet, George S Baillie
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  ABSTRACT: PDE4 is one of eleven known cyclic nucleotide phosphodiesterase families and plays a pivotal role in mediating hydrolytic degradation of the important cyclic nucleotide second messenger, cyclic 3'5' adenosine monophosphate (cAMP). PDE4 inhibitors are known to have anti-inflammatory properties, but their use in the clinic has been hampered by mechanism-associated side effects that limit maximally tolerated doses. In an attempt to initiate the development of better-tolerated PDE4 inhibitors we have surveyed existing approved drugs for PDE4-inhibitory activity. With this objective, we utilised a high-throughput computational approach that identified moexipril, a well tolerated and safe angiotensin-converting enzyme (ACE) inhibitor, as a PDE4 inhibitor. Experimentally we showed that moexipril and two structurally related analogues acted in the micro molar range to inhibit PDE4 activity. Employing a FRET-based biosensor constructed from the nucleotide binding domain of the type 1 exchange protein activated by cAMP, EPAC1, we demonstrated that moexipril markedly potentiated the ability of forskolin to increase intracellular cAMP levels. Finally, we demonstrated that the PDE4 inhibitory effect of moexipril is functionally able to induce phosphorylation of the Hsp20 by cAMP dependent protein kinase A. Our data suggest that moexipril is a bona fide PDE4 inhibitor that may provide the starting point for development of novel PDE4 inhibitors with an improved therapeutic window.
  Biochemical pharmacology 03/2013; · 4.25 Impact Factor
• Article: Phosphodiesterase inhibitors. Part 5: Hybrid PDE3/4 inhibitors as dual bronchorelaxant/anti-inflammatory agents for inhaled administration.

  Koji Ochiai, Satoshi Takita, Akihiko Kojima, Tomohiko Eiraku, Kazuhiko Iwase, Tetsuya Kishi, Akira Ohinata, Yuichi Yageta, Tokutaro Yasue, David R Adams, Yasushi Kohno
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  ABSTRACT: (-)-6-(7-Methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (KCA-1490) exhibits moderate dual PDE3/4-inhibitory activity and promises as a combined bronchodilatory/anti-inflammatory agent. N-alkylation of the pyridazinone ring markedly enhances potency against PDE4 but suppresses PDE3 inhibition. Addition of a 6-aryl-4,5-dihydropyridazin-3(2H)-one extension to the N-alkyl group facilitates both enhancement of PDE4-inhibitory activity and restoration of potent PDE3 inhibition. Both dihydropyridazinone rings, in the core and extension, can be replaced by achiral 4,4-dimethylpyrazolone subunits and the core pyrazolopyridine by isosteric bicyclic heteroaromatics. In combination, these modifications afford potent dual PDE3/4 inhibitors that suppress histamine-induced bronchoconstriction in vivo and exhibit promising anti-inflammatory activity via intratracheal administration.
  Bioorganic & medicinal chemistry letters 11/2012; · 2.65 Impact Factor
• Article: Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs.

  Brian D Hudson, Elisabeth Christiansen, Irina G Tikhonova, Manuel Grundmann, Evi Kostenis, David R Adams, Trond Ulven, Graeme Milligan
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  ABSTRACT: When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC(50) values for inhibition of cAMP, 5.83 ± 0.11; Ca(2+) mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.-Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs.
  The FASEB Journal 08/2012; · 5.71 Impact Factor
• Article: Phosphodiesterase inhibitors. Part 4: design, synthesis and structure-activity relationships of dual PDE3/4-inhibitory fused bicyclic heteroaromatic-4,4-dimethylpyrazolones.

  Koji Ochiai, Satoshi Takita, Akihiko Kojima, Tomohiko Eiraku, Naoki Ando, Kazuhiko Iwase, Tetsuya Kishi, Akira Ohinata, Yuichi Yageta, Tokutaro Yasue, David R Adams, Yasushi Kohno
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  ABSTRACT: (-)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and anti-inflammatory activity. Here we show that a 4,4-dimethylpyrazolone subunit serves as an effective surrogate for the 5-methyl-4,5-dihydropyridazin-3(2H)-one ring of KCA-1490 whilst lacking a stereogenic centre. The 2- and 7-substituents in the pyrazolo[1,5-a]pyridine subunit markedly influence the PDE-inhibitory profile and can be adjusted to afford either potent PDE4-selective inhibitors or dual PDE3/4 inhibitors. A survey of bicyclic heteroaromatic replacements for the pyrazolo[1,5-a]pyridine allowed further refinement of the inhibitory profile and identified 3-(8-methoxy-2-(trifluoromethyl)imidazo[1,2-a]pyridin-5-yl)-4,4-dimethyl-1H-pyrazol-5(4H)-one as an orally active, achiral KCA-1490 analog with well-balanced dual PDE3/4-inhibitory activity.
  Bioorganic & medicinal chemistry letters 07/2012; 22(18):5833-8. · 2.65 Impact Factor
• Article: Serine phosphorylation of the insulin-like growth factor I (IGF-1) receptor C-terminal tail restrains kinase activity and cell growth.

  Geraldine M Kelly, Deirdre A Buckley, Patrick A Kiely, David R Adams, Rosemary O'Connor
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  ABSTRACT: Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the (1248)SFYYS(1252) motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling.
  Journal of Biological Chemistry 06/2012; 287(33):28180-94. · 4.77 Impact Factor
• Article: Phosphodiesterase inhibitors. Part 3: Design, synthesis and structure-activity relationships of dual PDE3/4-inhibitory fused bicyclic heteroaromatic-dihydropyridazinones with anti-inflammatory and bronchodilatory activity.

  Koji Ochiai, Satoshi Takita, Tomohiko Eiraku, Akihiko Kojima, Kazuhiko Iwase, Tetsuya Kishi, Kazunori Fukuchi, Tokutaro Yasue, David R Adams, Robert W Allcock, Zhong Jiang, Yasushi Kohno
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  ABSTRACT: (-)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and anti-inflammatory activity. A survey of potential bicyclic heteroaromatic replacement subunits for the pyrazolo[1,5-a]pyridine core of KCA-1490 has identified the 4-methoxy-2-(trifluoromethyl)benzo[d]thiazol-7-yl and 8-methoxy-2-(trifluoromethyl)quinolin-5-yl analogues as dual PDE3/4-inhibitory compounds that potently suppress histamine-induced bronchoconstriction and exhibit anti-inflammatory activity in vivo.
  Bioorganic & medicinal chemistry 03/2012; 20(5):1644-58. · 2.82 Impact Factor
• Article: An analysis of exome sequencing for diagnostic testing of the genes associated with muscle disease and spastic paraplegia

  Cristina Dias, Murat Sincan, Praveen F. Cherukuri, Rosemarie Rupps, Yan Huang, Hannah Briemberg, Kathryn Selby, James C. Mullikin, Thomas C. Markello, David R. Adams, William A. Gahl, Cornelius F. Boerkoel
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  ABSTRACT: In this study, we assess exome sequencing (ES) as a diagnostic alternative for genetically heterogeneous disorders. Because ES readily identified a previously reported homozygous mutation in the CAPN3 gene for an individual with an undiagnosed limb girdle muscular dystrophy, we evaluated ES as a generalizable clinical diagnostic tool by assessing the targeting efficiency and sequencing coverage of 88 genes associated with muscle disease (MD) and spastic paraplegia (SPG). We used three exome-capture kits on 125 individuals. Exons constituting each gene were defined using the UCSC and CCDS databases. The three exome-capture kits targeted 47–92% of bases within the UCSC-defined exons and 97–99% of bases within the CCDS-defined exons. An average of 61.2–99.5% and 19.1–99.5% of targeted bases per gene were sequenced to 20X coverage within the CCDS-defined MD and SPG coding exons, respectively. Greater than 95–99% of targeted known mutation positions were sequenced to ≥1X coverage and 55–87% to ≥20X coverage in every exome. We conclude, therefore, that ES is a rapid and efficient first-tier method to screen for mutations, particularly within the CCDS annotated exons, although its application requires disclosure of the extent of coverage for each targeted gene and supplementation with second-tier Sanger sequencing for full coverage. Hum Mutat 33:614–626, 2012. © 2012 Wiley Periodicals, Inc.†
  Human Mutation 02/2012; 33(4):614 - 626. · 5.69 Impact Factor
• Article: Analysis of DNA sequence variants detected by high-throughput sequencing.

  David R Adams, Murat Sincan, Karin Fuentes Fajardo, James C Mullikin, Tyler M Pierson, Camilo Toro, Cornelius F Boerkoel, Cynthia J Tifft, William A Gahl, Tom C Markello
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  ABSTRACT: The Undiagnosed Diseases Program at the National Institutes of Health uses high-throughput sequencing (HTS) to diagnose rare and novel diseases. HTS techniques generate large numbers of DNA sequence variants, which must be analyzed and filtered to find candidates for disease causation. Despite the publication of an increasing number of successful exome-based projects, there has been little formal discussion of the analytic steps applied to HTS variant lists. We present the results of our experience with over 30 families for whom HTS sequencing was used in an attempt to find clinical diagnoses. For each family, exome sequence was augmented with high-density SNP-array data. We present a discussion of the theory and practical application of each analytic step and provide example data to illustrate our approach. The article is designed to provide an analytic roadmap for variant analysis, thereby enabling a wide range of researchers and clinical genetics practitioners to perform direct analysis of HTS data for their patients and projects.
  Human Mutation 01/2012; 33(4):599-608. · 5.69 Impact Factor
• Article: Recombination mapping using Boolean logic and high-density SNP genotyping for exome sequence filtering.

  Thomas C Markello, Ted Han, Hannah Carlson-Donohoe, Chidi Ahaghotu, Ursula Harper, MaryPat Jones, Settara Chandrasekharappa, Yair Anikster, David R Adams, William A Gahl, Cornelius F Boerkoel
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  ABSTRACT: Whole genome sequence data for small pedigrees has been shown to provide sufficient information to resolve detailed haplotypes in small pedigrees. Using such information, recombinations can be mapped onto chromosomes, compared with the segregation of a disease of interest and used to filter genome sequence variants. We now show that relatively inexpensive SNP array data from small pedigrees can be used in a similar manner to provide a means of identifying regions of interest in exome sequencing projects. We demonstrate that in those situations where one can assume complete penetrance and parental DNA is available, SNP recombination mapping using Boolean logic identifies chromosomal regions identical to those detected by multipoint linkage using microsatellites but with much less computation. We further show that this approach is successful because the probability of a double crossover between informative SNP loci is negligible. Our observations provide a rationale for using SNP arrays and recombination mapping as a rapid and cost-effective means of incorporating chromosome segregation information into exome sequencing projects intended for disease-gene identification.
  Molecular Genetics and Metabolism 12/2011; 105(3):382-9. · 3.19 Impact Factor
• Article: Direct interaction between scaffolding proteins RACK1 and 14-3-3ζ regulates brain-derived neurotrophic factor (BDNF) transcription.

  Jérémie Neasta, Patrick A Kiely, Dao-Yao He, David R Adams, Rosemary O'Connor, Dorit Ron
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  ABSTRACT: RACK1 is a scaffolding protein that spatially and temporally regulates numerous signaling cascades. We previously found that activation of the cAMP signaling pathway induces the translocation of RACK1 to the nucleus. We further showed that nuclear RACK1 is required to promote the transcription of the brain-derived neurotrophic factor (BDNF). Here, we set out to elucidate the mechanism underlying cAMP-dependent RACK1 nuclear translocation and BDNF transcription. We identified the scaffolding protein 14-3-3ζ as a direct binding partner of RACK1. Moreover, we found that 14-3-3ζ was necessary for the cAMP-dependent translocation of RACK1 to the nucleus. We further observed that the disruption of RACK1/14-3-3ζ interaction with a peptide derived from the RACK1/14-3-3ζ binding site or shRNA-mediated 14-3-3ζ knockdown inhibited cAMP induction of BDNF transcription. Together, these data reveal that the function of nuclear RACK1 is mediated through its interaction with 14-3-3ζ. As RACK1 and 14-3-3ζ are two multifunctional scaffolding proteins that coordinate a wide variety of signaling events, their interaction is likely to regulate other essential cellular functions.
  Journal of Biological Chemistry 11/2011; 287(1):322-36. · 4.77 Impact Factor
• Article: Phosphodiesterase inhibitors. Part 2: design, synthesis, and structure-activity relationships of dual PDE3/4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory and bronchodilatory activity.

  Koji Ochiai, Naoki Ando, Kazuhiko Iwase, Tetsuya Kishi, Kazunori Fukuchi, Akira Ohinata, Hitomi Zushi, Tokutaro Yasue, David R Adams, Yasushi Kohno
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  ABSTRACT: A structural survey of pyrazolopyridine-pyridazinone phosphodiesterase (PDE) inhibitors was made with a view to optimization of their dual PDE3/4-inhibitory activity for respiratory disease applications. These studies identified (-)-6-(7-methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridine-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490, compound 2ac) as a compound with potent combined bronchodilatory and anti-inflammatory activity and an improved therapeutic window over roflumilast.
  Bioorganic & medicinal chemistry letters 09/2011; 21(18):5451-6. · 2.65 Impact Factor
• Article: Extracellular loop 2 of the free fatty acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric modulator.

  Nicola J Smith, Richard J Ward, Leigh A Stoddart, Brian D Hudson, Evi Kostenis, Trond Ulven, Joanne C Morris, Christian Tränkle, Irina G Tikhonova, David R Adams, Graeme Milligan
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  ABSTRACT: Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu(173) or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB.
  Molecular pharmacology 07/2011; 80(1):163-73. · 4.53 Impact Factor
• Article: Phosphodiesterase inhibitors. Part 1: Synthesis and structure-activity relationships of pyrazolopyridine-pyridazinone PDE inhibitors developed from ibudilast.

  Robert W Allcock, Haakon Blakli, Zhong Jiang, Karen A Johnston, Keith M Morgan, Georgina M Rosair, Kazuhiko Iwase, Yasushi Kohno, David R Adams
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  ABSTRACT: Ibudilast [1-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-2-methylpropan-1-one] is a nonselective phosphodiesterase inhibitor used clinically to treat asthma. Efforts to selectively develop the PDE3- and PDE4-inhibitory activity of ibudilast led to replacement of the isopropyl ketone by a pyridazinone heterocycle. Structure-activity relationship exploration in the resulting 6-(pyrazolo[1,5-a]pyridin-3-yl)pyridazin-3(2H)-ones revealed that the pyridazinone lactam functionality is a critical determinant for PDE3-inhibitory activity, with the nitrogen preferably unsubstituted. PDE4 inhibition is strongly promoted by introduction of a hydrophobic substituent at the pyridazinone N(2) centre and a methoxy group at C-7' in the pyrazolopyridine. Migration of the pyridazinone ring connection from the pyrazolopyridine 3'-centre to C-4' strongly enhances PDE4 inhibition. These studies establish a basis for development of potent PDE4-selective and dual PDE3/4-selective inhibitors derived from ibudilast.
  Bioorganic & medicinal chemistry letters 06/2011; 21(11):3307-12. · 2.65 Impact Factor
• Source

  Available from: PubMed Central

  Article: RACK1, A multifaceted scaffolding protein: Structure and function.

  David R Adams, Dorit Ron, Patrick A Kiely
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  ABSTRACT: ABSTRACT: The Receptor for Activated C Kinase 1 (RACK1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and shares significant homology to the β subunit of G-proteins (Gβ). RACK1 adopts a seven-bladed β-propeller structure which facilitates protein binding. RACK1 has a significant role to play in shuttling proteins around the cell, anchoring proteins at particular locations and in stabilising protein activity. It interacts with the ribosomal machinery, with several cell surface receptors and with proteins in the nucleus. As a result, RACK1 is a key mediator of various pathways and contributes to numerous aspects of cellular function. Here, we discuss RACK1 gene and structure and its role in specific signaling pathways, and address how posttranslational modifications facilitate subcellular location and translocation of RACK1. This review condenses several recent studies suggesting a role for RACK1 in physiological processes such as development, cell migration, central nervous system (CN) function and circadian rhythm as well as reviewing the role of RACK1 in disease.
  Cell Communication and Signaling 01/2011; 9:22. · 5.50 Impact Factor
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  Available from: Graeme Milligan

  Article: High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules.

  Paul D Andrews, Melissa Becroft, Anders Aspegren, Jane Gilmour, Martyn J James, Scott McRae, Robert Kime, Robert W Allcock, Achamma Abraham, Zhong Jiang, Raimund Strehl, Joanne C Mountford, Graeme Milligan, Miles D Houslay, David R Adams, Julie A Frearson
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  ABSTRACT: The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of 'lead-like' small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N⁶,N⁶-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by 'off-target' inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.
  Biochemical Journal 11/2010; 432(1):21-33. · 4.90 Impact Factor
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  Available from: Graeme Milligan

  Article: Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

  Peter Burton, David R Adams, Achamma Abraham, Robert W Allcock, Zhong Jiang, Angela McCahill, Jane Gilmour, John McAbney, Alexandra Kaupisch, Nicole M Kane, George S Baillie, Andrew H Baker, Graeme Milligan, Miles D Houslay, Joanne C Mountford
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  ABSTRACT: hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.
  Biochemical Journal 10/2010; 432(3):575-84. · 4.90 Impact Factor
• Article: p62 (SQSTM1) and cyclic AMP phosphodiesterase-4A4 (PDE4A4) locate to a novel, reversible protein aggregate with links to autophagy and proteasome degradation pathways.

  Frank Christian, Diana F Anthony, Surakiran Vadrevu, Tracy Riddell, Jonathan P Day, Ruth McLeod, David R Adams, George S Baillie, Miles D Houslay
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  ABSTRACT: Chronic challenge of cyclic AMP phosphodiesterase-4A4 (PDE4A4) with certain PDE4 selective inhibitors causes it to reversibly form intracellular aggregates that are not membrane-encapsulated. These aggregates are neither stress granules (SGs) nor processing bodies (PBs) as they contain neither PABP-1 nor Dcp1a, respectively. However, the PDE4 inhibitor rolipram decreases arsenite-induced SGs and increases the amount of PBs, while arsenite challenge ablates rolipram-induced PDE4A4 aggregates. PDE4A4 aggregates are neither autophagic vesicles (autophagosomes) nor aggresomes, although microtubule disruptors ablate PDE4A4 aggregate formation. PDE4A4 constitutively co-immunoprecipitates with p62 protein (sequestosome1, SQSTM1), which locates to both PDE4A4 aggregates and autophagosomes in cells constitutively challenged with rolipram. The mTor inhibitor, rapamycin, activates autophagy, prevents PDE4A4 from forming intracellular aggregates and triggers the loss of bound p62 from PDE4A4. siRNA-mediated knockdown of p62 attenuates PDE4A4 aggregate formation. The p62-binding protein, light chain 3 (LC3), is not found in PDE4A4 aggregates. Blockade of proteasome activity and activation of autophagy with MG132 both increases the level of ubiquitinated proteins found associated with PDE4A4 and inhibits PDE4A4 aggregate formation. Activation of autophagy with either thapsigargin or ionomycin inhibits PDE4A4 aggregate formation. Inhibition of autophagy with either wortmannin or LY294002 activates PDE4A4 aggregate formation. The protein kinase C inhibitors, RO 320432 and GO 6983, and the ERK inhibitors UO 126 and PD 98059 all activated PDE4A4 aggregate formation, whilst roscovitine, thalidomide and the tyrosine kinase inhibitors, genistein and AG17, all inhibited this process. We suggest that the fate of p62-containing protein aggregates need not necessarily be terminal, through delivery to autophagic vesicles and aggresomes. Instead, we propose a novel regulatory mechanism where a sub-population of p62-containing protein aggregates would form in a rapid, reversible manner so as to sequester specific cargo away from their normal, functionally important site(s) within the cell. Thus an appropriate conformational change in the target protein would confer reversible recruitment into a sub-population of p62-containing protein aggregates and so provide a regulatory function by removing these cargo proteins from their functionally important site(s) in a cell.
  Cellular signalling 10/2010; 22(10):1576-96. · 4.09 Impact Factor
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  Available from: Graeme Milligan

  Article: Identification and characterization of small-molecule ligands that maintain pluripotency of human embryonic stem cells.

  Peter Burton, David R Adams, Achamma Abraham, Robert W Allcock, Zhong Jiang, Angela McCahill, Jane Gilmour, John McAbney, Nicole M Kane, George S Baillie, Fergus R McKenzie, Andrew H Baker, Miles D Houslay, Joanne C Mountford, Graeme Milligan
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  ABSTRACT: hESCs (human embryonic stem cells) offer great potential for pharmaceutical research and development and, potentially, for therapeutic use. However, improvements in cell culture are urgently required to allow the scalable production of large numbers of cells that maintain pluripotency. Supplementing feeder-free conditions with either EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine] or readily synthesized analogues of this compound maintains hESC pluripotency in the absence of exogenous cytokines. When the hESC lines SA121 or SA461 were maintained in feeder-free conditions with EHNA they displayed no reduction in stem-cell-associated markers such as Nanog, Oct4 (octamer-binding protein 4) and SSEA4 (stage-specific embryonic antigen 4) when compared with cells maintained in full feeder-free conditions that included exogenously added bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but EHNA did not limit efficient spontaneous or directed differentiation following its removal. We conclude that EHNA or related compounds offers a viable alternative to exogenous cytokine addition in maintaining hESC cultures in a pluripotent state and might be a particularly useful replacement for bFGF for large-scale or GMP (good manufacturing practice)-compliant processes.
  Biochemical Society Transactions 08/2010; 38(4):1058-61. · 3.71 Impact Factor
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  Available from: tcd.ie

  Article: Selective SUMO modification of cAMP-specific phosphodiesterase-4D5 (PDE4D5) regulates the functional consequences of phosphorylation by PKA and ERK.

  Xiang Li, Suryakiran Vadrevu, Allan Dunlop, Jon Day, Noopur Advant, Jessica Troeger, Enno Klussmann, Ellis Jaffrey, Ron T Hay, David R Adams, Miles D Houslay, George S Baillie
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  ABSTRACT: Enzymes from the PDE (phosphodiesterase) 4 cAMP-specific PDE family are crucial for the maintenance of compartmentalized cAMP responses in many cell types. Regulation of PDE activity can be achieved via post-translational modification such as phosphorylation by ERK (extracellular-signal-regulated kinase) MAPKs (mitogen-activated protein kinases) and PKA (protein kinase A). In the present paper, we report for the first time that PDE4 isoforms from the PDE4A and PDE4D subfamilies can be selectively modified by SUMO (small ubiquitin-related modifier). We have identified a single SUMO site within a consensus tetrapeptide motif, PsiKXE (where Psi represents a hydrophobic residue), which lies in the catalytic unit of these enzymes. SUMO modification of PDE4 at this site was observed upon overexpression of the SUMO E3 ligase PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y] in HEK (human embryonic kidney)-293 cells and we identify PIASy as a novel binding partner for long PDE4 isoforms. Site-directed mutagenesis of the acceptor lysine residue ablated conjugation of PDE4 with SUMO, suggesting the presence of a single SUMO site in the first subdomain of the conserved PDE4 catalytic unit. This observation was supported by both cell-free in vitro SUMOylation assays and analysis of SUMOylated spot-immobilized peptide arrays. SUMO modification of long PDE4 isoforms serves to augment their activation by PKA phosphorylation and repress their inhibition by ERK phosphorylation. Following ligation of beta-adrenergic receptors, SUMOylation of PDE4 isoforms sufficiently amplified PKA-stimulated PDE4 activity to reduce markedly the PKA phosphorylation status of the beta2-adrenergic receptor. These results highlight a new means whereby cells might achieve the selective regulation of the activity of cAMP-specific PDE4 enyzmes.
  Biochemical Journal 03/2010; 428(1):55-65. · 4.90 Impact Factor
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  Available from: PubMed Central

  Article: Novel anti-infective compounds from marine bacteria.

  Hafizur Rahman, Brian Austin, Wilfrid J Mitchell, Peter C Morris, Derek J Jamieson, David R Adams, Andrew Mearns Spragg, Michael Schweizer
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  ABSTRACT: As a result of the continuous evolution of microbial pathogens towards antibiotic-resistance, there have been demands for the development of new and effective antimicrobial compounds. Since the 1960s, the scientific literature has accumulated many publications about novel pharmaceutical compounds produced by a diverse range of marine bacteria. Indeed, marine micro-organisms continue to be a productive and successful focus for natural products research, with many newly isolated compounds possessing potentially valuable pharmacological activities. In this regard, the marine environment will undoubtedly prove to be an increasingly important source of novel antimicrobial metabolites, and selective or targeted approaches are already enabling the recovery of a significant number of antibiotic-producing micro-organisms. The aim of this review is to consider advances made in the discovery of new secondary metabolites derived from marine bacteria, and in particular those effective against the so called "superbugs", including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), which are largely responsible for the increase in numbers of hospital acquired, i.e., nosocomial, infections.
  Marine Drugs 01/2010; 8(3):498-518. · 3.85 Impact Factor