Publications by authors named "Ian G Jennings"

22 Publications

  • Page 1 of 1

Disrupting the platelet internal membrane via PI3KC2α inhibition impairs thrombosis independently of canonical platelet activation.

Sci Transl Med 2020 07;12(553)

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC 3004, Australia.

Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2α) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2α is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2α in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window.
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http://dx.doi.org/10.1126/scitranslmed.aar8430DOI Listing
July 2020

Synthesis of linear and angular aryl-morpholino-naphth-oxazines, their DNA-PK, PI3K, PDE3A and antiplatelet activity.

Bioorg Med Chem Lett 2016 11 5;26(22):5534-5538. Epub 2016 Oct 5.

Pharmacy and Applied Science, La Trobe Institute for Molecular Science, La Trobe University, PO Box 199, Bendigo, VIC 3552, Australia. Electronic address:

To continue our study of 2-morpholino-benzoxazine based compounds, which show useful activity against PI3K family enzymes or antiplatelet activity, we designed and synthesized a series of linear 6.7-fused, 5,6-angular fused and 7,8-angular fused-aryl-morpholino-naphth-oxazines. The compounds were prepared from substituted 2-hydroxynaphthoic acid to give the corresponding thioxo analogues 8, 9, 15 and 19. The thioxo products were then converted to the morpholino substituted analogue. The aryl group was introduced by Suzuki coupling of bromo precursors. The products were evaluated for activity at PI3K family enzymes and as platelet aggregation inhibitors and compared to reported unsubstituted analogues. The linear 6.7-fused product 13a and 13b were moderated potent but selective PI3Kδ isoform inhibitors (IC=7.7 and 5.61μM). Good antiplatelet activity was noticed for the angular 7,8-fused compounds 22a, b, k and l with IC=3.0,14.0, 2.0 and 5.0μM respectively. The antiplatelet activity is independent of PDE3.
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http://dx.doi.org/10.1016/j.bmcl.2016.10.003DOI Listing
November 2016

Development of single and mixed isoform selectivity PI3Kδ inhibitors by targeting Asn836 of PI3Kδ.

Bioorg Med Chem Lett 2016 10 11;26(19):4790-4794. Epub 2016 Aug 11.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

A series of PI3Kδ inhibitors derived from the pan-PI3K inhibitor ZSTK474 was prepared that target a non-conserved region of the catalytic site. Dependent upon the substituents present, these analogues show different levels of isoform selectivity and sensitivity to the mutation N836D in PI3Kδ. As a marker of 'on-target' activity and permeability, a selection of the most potent PI3Kδ inhibitors were shown to inhibit pAkt production in the Nawalma Burkitt lymphoma cell line.
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http://dx.doi.org/10.1016/j.bmcl.2016.08.028DOI Listing
October 2016

Discovery and antiplatelet activity of a selective PI3Kβ inhibitor (MIPS-9922).

Eur J Med Chem 2016 Oct 10;122:339-351. Epub 2016 Jun 10.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia. Electronic address:

A series of amino-substituted triazines were developed and examined for PI3Kβ inhibition and anti-platelet function. Structural adaptations of a morpholine ring of the prototype pan-PI3K inhibitor ZSTK474 yielded PI3Kβ selective compounds, where the selectivity largely derives from an interaction with the non-conserved Asp862 residue, as shown by site directed mutagenesis. The most PI3Kβ selective inhibitor from the series was studied in detail through a series of in vitro and in vivo functional studies. MIPS-9922, 10 potently inhibited ADP-induced washed platelet aggregation. It also inhibited integrin αIIbβ3 activation and αIIbβ3 dependent platelet adhesion to immobilized vWF under high shear. It prevented arterial thrombus formation in the in vivo electrolytic mouse model of thrombosis without inducing prolonged bleeding or excess blood loss.
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http://dx.doi.org/10.1016/j.ejmech.2016.06.010DOI Listing
October 2016

Synthesis, structure elucidation, DNA-PK and PI3K and anti-cancer activity of 8- and 6-aryl-substituted-1-3-benzoxazines.

Eur J Med Chem 2016 Mar 27;110:326-39. Epub 2016 Jan 27.

Pharmacy and Applied Science, La Trobe Institute for Molecular Science, La Trobe University, P.O. Box 199, Bendigo, VIC 3552, Australia. Electronic address:

The synthesis of 6-aryl, 8- aryl, and 8-aryl-6-chloro-2-morpholino-1,3-benzoxazines with potent activity against PI3K and DNA-PK is described. Synthesis of thirty one analogues was facilitated by an improved synthesis of 3-bromo-2-hydroxybenzoic acid 13 by de-sulphonation of 3-bromo-2-hydroxy-5-sulfobenzoic acid 12 en route to 2-methylthio-substituted-benzoxazine intermediates 17-19. From this series, compound 20k (LTURM34) (dibenzo[b,d]thiophen-4-yl) (IC50 = 0.034 μM) was identified as a specific DNA-PK inhibitor, 170 fold more selective for DNA-PK activity compared to PI3K activity. Other compounds of the series show markedly altered selectivity for various PI3K isoforms including compound 20i (8-(naphthalen-1-yl) a potent and quite selective PI3Kδ inhibitor (IC50 = 0.64 μM). Finally, nine compounds were evaluated and showed antiproliferative activity against an NCI panel of cancer cell lines. Compound 20i (8-(naphthalen-1-yl) showed strong anti-proliferative activity against A498 renal cancer cells that warrants further investigation.
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http://dx.doi.org/10.1016/j.ejmech.2016.01.042DOI Listing
March 2016

Class II but Not Second Class-Prospects for the Development of Class II PI3K Inhibitors.

ACS Med Chem Lett 2015 Jan 24;6(1):3-6. Epub 2014 Sep 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus) , 381 Royal Parade, Parkville, Victoria 3052, Australia.

The Class II PI3 kinases are emerging from the shadows of their Class I cousins. The data emerging from PIK3C2 genetic modification studies and from siRNA knockdown suggest important roles in physiology and pathology. With some well-studied Class I isoform inhibitors showing strong Class II activity and a wealth of crystallographic information available, the structural similarity of these isoforms to Class I provides both the opportunity and the challenge in design of selective pharmacological inhibitors.
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http://dx.doi.org/10.1021/ml500354eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291705PMC
January 2015

Identification of potent phosphodiesterase inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites.

ACS Chem Biol 2015 Apr 4;10(4):1145-54. Epub 2015 Feb 4.

†Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis, respectively, undergo several critical developmental transitions during their lifecycle. Most important for human pathogenesis is the asexual cycle, in which parasites undergo rounds of host cell invasion, replication, and egress (exit), destroying host cell tissue in the process. Previous work has identified important roles for Protein Kinase G (PKG) and Protein Kinase A (PKA) in parasite egress and invasion, yet little is understood about the regulation of cyclic nucleotides, cGMP and cAMP, that activate these enzymes. To address this, we have focused upon the development of inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) to block the breakdown of cyclic nucleotides. This was done by repurposing human PDE inhibitors noting various similarities of the human and apicomplexan PDE binding sites. The most potent inhibitors blocked the in vitro proliferation of P. falciparum and T. gondii more potently than the benchmark compound zaprinast. 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO) was found to be a potent inhibitor of recombinant P. falciparum PfPDEα and activated PKG-dependent egress of T. gondii and P. falciparum, likely by promoting the exocytosis of micronemes, an activity that was reversed by a specific Protein Kinase G inhibitor. BIPPO also promotes cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion, suggesting that the compound inhibits single or multiple PDE isoforms that regulate both cGMP and cAMP levels. BIPPO is therefore a useful tool for the dissection of signal transduction pathways in apicomplexan parasites.
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http://dx.doi.org/10.1021/cb501004qDOI Listing
April 2015

Structural basis of nSH2 regulation and lipid binding in PI3Kα.

Oncotarget 2014 Jul;5(14):5198-208

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP₂, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP₂ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4170646PMC
http://dx.doi.org/10.18632/oncotarget.2263DOI Listing
July 2014

L-Aminoacyl-triazine derivatives are isoform-selective PI3Kβ inhibitors that target non-conserved Asp862 of PI3Kβ

ACS Med Chem Lett 2013 Feb;4(2):206-210

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052 Australia.

A series of aminoacyl-triazine derivatives based upon the pan-PI3K inhibitor ZSTK474 were identified as potent and isoform selective inhibitors of PI3Kβ. The compounds showed selectivity based upon stereochemistry with L-amino acyl derivatives preferring PI3Kβ while their D-congeners favoured PI3Kδ. The mechanistic basis of this inhibition was studied using site-directed mutants. One Asp residue, D862 was identified as a critical participant in binding to the PI3Kβ-selective inhibitors distinguishing this class from other reported PI3Kβ-selective inhibitors. The compounds show strong inhibition of cellular Akt phosphorylation and growth of PTEN-deficient MD-MBA-468 cells.
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http://dx.doi.org/10.1021/ml300336jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3688631PMC
February 2013

Potent inhibitors of phosphatidylinositol 3 (PI3) kinase that have antiproliferative activity only when delivered as prodrug forms.

ChemMedChem 2013 Jun 8;8(6):914-8. Epub 2013 Apr 8.

Department of Chemistry, La Trobe University Institute for Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia.

Prodrugs for PI3K: A series of substituted analogues of the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002 were prepared and found to potently inhibit the isolated enzyme but not MCF7 cell proliferation. Two tetrazolyl-substituted analogues were further derivatized as prodrugs resulting in restoration of cell-based activity. These data provide a conceptual model for development of tumor-targeting prodrug forms of cell-impermeable PI3K inhibitors.
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http://dx.doi.org/10.1002/cmdc.201200583DOI Listing
June 2013

Mechanisms of PI3Kβ-selective inhibition revealed by reciprocal mutagenesis.

ACS Chem Biol 2013 Apr 6;8(4):679-83. Epub 2013 Feb 6.

The p110β isoform of PI3 kinase (PI3Kβ) has been implicated in pathological disorders such as thrombosis and cancer and a number of PI3Kβ-selective inhibitors have recently progressed into clinical studies. Although crystallography studies identify a binding site conformation favored by the inhibitors, no specific interaction explains the observed selectivity. Using site-directed mutagenesis we have identified a specific tyrosine residue of the binding site Y778 that dictates the ability of the PI3Kβ isoform to bind these inhibitors. When mutated to isoleucine, PI3Kβ has reduced ability to present a specific cryptic binding site into which a range of reported PI3Kβ inhibitors can bind, and conversely when tyrosine is introduced into the same position in PI3Kα, the same inhibitors gain potency. The results provide a cogent explanation for the selectivity profiles displayed by these PI3K inhibitors and maybe others as well.
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http://dx.doi.org/10.1021/cb300666sDOI Listing
April 2013

Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines as inhibitors of phosphoinositide 3-kinase.

Bioorg Med Chem Lett 2013 Feb 1;23(3):802-5. Epub 2012 Dec 1.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, 381 Royal Pde, Parkville, Victoria 3052, Australia.

Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treatment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benzimidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by the traditional route results in a mixture of the two regioisomers. We have developed a straightforward regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be probed.
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http://dx.doi.org/10.1016/j.bmcl.2012.11.076DOI Listing
February 2013

Definition of the binding mode of a new class of phosphoinositide 3-kinase α-selective inhibitors using in vitro mutagenesis of non-conserved amino acids and kinetic analysis.

Biochem J 2012 Jun;444(3):529-35

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052, Australia.

The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110α and p110β isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor's p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser(773), A-66S bound the region 1 non-conserved amino acid p110α Gln(859), and J-32 binding had an indirect interaction with Lys(776) and Ile(771). The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.
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http://dx.doi.org/10.1042/BJ20120499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3474370PMC
June 2012

Synthesis and Pharmacological Evaluation of 4-Iminothiazolidinones for Inhibition of PI3 Kinase.

Aust J Chem 2012 Jan;65(10):1396-1404

Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), Parkville, Vic. 3052, Australia.

The thiazolidinedione, compound , has previously shown pan-inhibition of the phosphoinositide 3-kinase (PI3K) class I isoforms. We hypothesized the derivatization of the thiazolidinedione core of compound could introduce isoform selectivity. We report the synthesis, characterization, and inhibitory activity of a novel series of 4-iminothiazolidin-2-ones for inhibition of the class I PI3K isoforms. Their synthesis was successfully achieved by multiple pathways described in this paper. Initial in vitro data of 28 analogues demonstrated poor inhibition of all class I PI3K isoforms. However, we identified an alternate target, the phosphodiesterases, and present preliminary screening results showing improved inhibitory activity.
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http://dx.doi.org/10.1071/CH12140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3755381PMC
January 2012

Thiophene inhibitors of PDE4: crystal structures show a second binding mode at the catalytic domain of PDE4D2.

Bioorg Med Chem Lett 2011 Dec 5;21(23):7089-93. Epub 2011 Oct 5.

Medicinal Chemistry & Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville campus), Parkville, Victoria, Australia.

PDE4 inhibitors have been identified as therapeutic targets for a variety of conditions, particularly inflammatory diseases. We have serendipitously identified a novel class of phosphodiesterase 4 (PDE4) inhibitor during a study to discover antagonists of the parathyroid hormone receptor. X-ray crystallographic studies of PDE4D2 complexed to four potent inhibitors reveal the atomic details of how they inhibit the enzyme and a notable contrast to another recently reported thiophene-based inhibitor.
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http://dx.doi.org/10.1016/j.bmcl.2011.09.109DOI Listing
December 2011

Isoform-selective inhibition of phosphoinositide 3-kinase: identification of a new region of nonconserved amino acids critical for p110α inhibition.

Mol Pharmacol 2011 Oct 21;80(4):657-64. Epub 2011 Jul 21.

Medicinal Chemistry & Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus),Victoria, Australia.

The combination of molecular modeling and X-ray crystallography has failed to yield a consensus model of the mechanism for selective binding of inhibitors to the phosphoinositide 3-kinase (PI3K) p110 α-isoform. Here we have used kinetic analysis to determine that the p110α-selective inhibitor 2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-α]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid (PIK-75) is a competitive inhibitor with respect to a substrate, phosphatidylinositol (PI) in contrast to most other PI3K inhibitors, which bind at or near the ATP site. Using sequence analysis and the existing crystal structures of inhibitor complexes with the p110γ and -δ isoforms, we have identified a new region of nonconserved amino acids (region 2) that was postulated to be involved in PIK-75 p110α selectivity. Analysis of region 2, using in vitro mutation of identified nonconserved amino acids to alanine, showed that Ser773 was a critical amino acid involved in PIK-75 binding, with an 8-fold-increase in the IC(50) compared with wild-type. Kinetic analysis showed that, with respect to PI, the PIK-75 K(i) for the isoform mutant S773D increased 64-fold compared with wild-type enzyme. In addition, a nonconserved amino acid, His855, from the previously identified region 1 of nonconserved amino acids, was found to be involved in PIK-75 binding. These results show that these two regions of nonconserved amino acids that are close to the substrate binding site could be targeted to produce p110α isoform-selective inhibitors.
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http://dx.doi.org/10.1124/mol.111.072546DOI Listing
October 2011

Thiazolidinedione-based PI3Kα inhibitors: an analysis of biochemical and virtual screening methods.

ChemMedChem 2011 Mar 4;6(3):514-22. Epub 2011 Jan 4.

Medicinal Chemistry & Drug Action, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052, Australia.

A series of synthesized and commercially available compounds were assessed against PI3Kα for in vitro inhibitory activity and the results compared to binding calculated in silico. Using published crystal structures of PI3Kγ and PI3Kδ co-crystallized with inhibitors as a template, docking was able to identify the majority of potent inhibitors from a decoy set of 1000 compounds. On the other hand, PI3Kα in the apo-form, modeled by induced fit docking, or built as a homology model gave only poor results. A PI3Kα homology model derived from a ligand-bound PI3Kδ crystal structure was developed that has a good ability to identify active compounds. The docking results identified binding poses for active compounds that differ from those identified to date and can contribute to our understanding of structure-activity relationships for PI3K inhibitors.
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http://dx.doi.org/10.1002/cmdc.201000467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187668PMC
March 2011

Inflammatory twins from PI3K gang brought to justice?

Chem Biol 2010 Feb;17(2):101-2

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Australia.

PI3 kinase inhibitors are hot property. In this issue of Chemistry & Biology, Williams et al. add a dual PI3Kdelta/gamma inhibitor to the collection and show that its anti-inflammatory profile in vitro is quite different from pan-PI3K inhibitors, but bears an uncanny resemblance to that of the glucocorticoid drugs.
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http://dx.doi.org/10.1016/j.chembiol.2010.02.001DOI Listing
February 2010

Dissecting isoform selectivity of PI3K inhibitors: the role of non-conserved residues in the catalytic pocket.

Biochem J 2008 Sep;414(3):383-90

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC 3004, Australia.

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 Thr(886) and Lys(890) of the PI3Kgamma 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 beta isoform (Glu(858) and Asp(862)) and those in the alpha isoform (His(855) and Gln(859)) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of beta-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the alpha-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 PI3Kbeta 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 alpha-isoform-selectivity of the compounds studied.
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http://dx.doi.org/10.1042/BJ20080512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820364PMC
September 2008

Src kinase activates endothelial nitric-oxide synthase by phosphorylating Tyr-83.

J Biol Chem 2005 Oct 25;280(43):35943-52. Epub 2005 Aug 25.

Vascular Biology Center and Department of Pediatrics, Medical College of Georgia, Augusta, Georgia 30912-2500, USA.

The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.
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http://dx.doi.org/10.1074/jbc.M504606200DOI Listing
October 2005

AMPK beta subunit targets metabolic stress sensing to glycogen.

Curr Biol 2003 May;13(10):867-71

St. Vincent's Institute of Medical Research, University of Melbourne, 41 Victoria Parade, Fitzroy, Australia.

AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients, as well as hormones, including adiponectin and leptin. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK. Mammalian AMPK is an alphabetagamma heterotrimer with multiple isoforms of each subunit comprising alpha1, alpha2, beta1, beta2, gamma1, gamma2, and gamma3, which have varying tissue and subcellular expression. Mutations in the AMPK gamma subunit cause glycogen storage disease in humans, but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK beta subunit contains a functional glycogen binding domain (beta-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished beta-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK beta1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.
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http://dx.doi.org/10.1016/s0960-9822(03)00292-6DOI Listing
May 2003

Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase.

Protein Sci 2002 Aug;11(8):2041-7

Structural Biology Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, 3065, Australia.

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.
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http://dx.doi.org/10.1110/ps.4560102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373677PMC
August 2002
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