Michelle Garrett - Academia.edu (original) (raw)
Papers by Michelle Garrett
Drug Discovery Today, Feb 28, 2010
![Research paper thumbnail of Corrigendum to “Identification and characterisation of 2-aminopyridine inhibitors of checkpoint kinase 2” [Bioorg. Med. Chem. 18 (2010) 707]](https://attachments.academia-assets.com/48889223/thumbnails/1.jpg)
](Bioorganic Medicinal Chemistry, Jun 15, 2010
The authors regret that the residue 'Leu309' was incorrectly labeled in the crystal structures in... more The authors regret that the residue 'Leu309' was incorrectly labeled in the crystal structures in -D, p712, and should correctly be labeled 'Val234'. The accompanying text on p712 should read 'The inhibitor occupied the ATP-binding site of CHK2 and was sandwiched between the hydrophobic side chains of Val234 and Leu354 .' 0968-0896/$ -see front matter Ó
Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yea... more Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79-*L, analogous to the oncogenic mutation of Q-61--->L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-eu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30o of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-ku79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.
Journal of Medicinal Chemistry, Jan 27, 2011
Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kina... more Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might ...
Journal of Medicinal Chemistry, May 1, 2008
Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragmen... more Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3- d]pyrimidine inhibitors of PKBbeta with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.
European Journal of Cancer Supplements, 2004
Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI... more Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI3 kinase. To find novel inhibitors of PKB/AKT kinase activity for progression as anticancer agents, the authors have used a high-throughput screen based on AlphaScreen™ technology. A known kinase inhibitor, the isoquinoline H8, was used as a positive control with mean inhibition in the screen of 43.4% ± 13.1%. The performance of the screen was highly acceptable with Z´ and Z factors of 0.83 ± 0.07 and 0.75 ± 0.04, respectively. A number of confirmed hits (~0.1% hit rate) were identified from 63,500 compounds screened. Five compounds have previously been described as PKB inhibitors, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. Five hits had the potential to interfere with the assay signal and were deemed to be false positives. Two compounds were nonspecific inhibitors of PKB as enzyme inhibition in a filter-based assay was markedly reduced in the presence of 0.01% Triton X100. The authors now include an interference assay during hit confirmation procedures and check compound activity in the presence of Triton X100 in an attempt to eliminate nonspecific aggregators at an early stage. (Journal of Biomolecular Screening 2006:822-827)
Molecular and cellular biology, 1992
Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yea... more Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumu...
European Journal of Cancer Supplements, 2004
Novartis Foundation Symposia, 2007
Sec4 is a Ras-like GTP-binding protein required for exocytosis in yeast. Unlike Ras, it is the ab... more Sec4 is a Ras-like GTP-binding protein required for exocytosis in yeast. Unlike Ras, it is the ability of Sec4 to cycle between the GTP- and GDP-bound forms rather than the absolute levels of the GTP-bound form that is critical for function. This cycle may be coupled to an observed cycle of Sec4 localization within the cell. Sec4 binds to secretory vesicles which then fuse with the plasma membrane in exocytosis. Sec4 can recycle from the plasma membrane through a soluble pool to rebind to a new round of vesicles. We have found an activity in yeast (Saccharomyces cerevisiae) comparable to that of the GDP dissociation inhibitor protein isolated from mammalian cells that releases GDP-bound Sec4 from membranes. DSS4-1, a dominant suppressor of the sec4-8 temperature-sensitive mutation, encodes a nucleotide exchange protein. The cycle of Sec4 may function to allow the assembly and subsequent disassembly of a set of proteins necessary for exocytosis. Candidates for members of this set of proteins are encoded by sec genes which show strong genetic interactions with sec4-8. Two of these (SEC8 and SEC15) encode large proteins which form a complex that is peripherally associated with the plasma membrane.
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 15, 2014
Multiple cancers harbor genetic aberrations that impact AKT signaling. MK-2206 is a potent pan-AK... more Multiple cancers harbor genetic aberrations that impact AKT signaling. MK-2206 is a potent pan-AKT inhibitor with a maximum tolerated dose (MTD) previously established at 60 mg on alternate days (QOD). Due to a long half-life (60-80 hours), a weekly (QW) MK-2206 schedule was pursued to compare intermittent QW and continuous QOD dosing. Patients with advanced cancers were enrolled in a QW dose-escalation phase I study to investigate the safety and pharmacokinetic-pharmacodynamic profiles of tumor and platelet-rich plasma (PRP). The QOD MTD of MK-2206 was also assessed in patients with ovarian and castration-resistant prostate cancers and patients with advanced cancers undergoing multiparametric functional magnetic resonance imaging (MRI) studies, including dynamic contrast-enhanced MRI, diffusion-weighted imaging, magnetic resonance spectroscopy, and intrinsic susceptibility-weighted MRI. A total of 71 patients were enrolled; 38 patients had 60 mg MK-2206 QOD, whereas 33 received MK-...
PLoS ONE, 2012
Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of ... more Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2Acentered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.
The EMBO Journal, 2006
The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. ... more The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serineglutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation. The EMBO Journal VOL 25 | NO 13 | 2006 EMBO THE EMBO JOURNAL THE EMBO JOURNAL
PLoS ONE, 2013
Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DN... more Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragmentbased screening campaign using a combination of a high-concentration AlphaScreen TM kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors.
Molecular Cancer Therapeutics, 2010
The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in ma... more The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in malignant transformation and chemoresistance and is an attractive target for the development of cancer therapeutics. Fragment-based lead discovery, combined with structure-based drug design, has recently identified AT7867 as a novel and potent inhibitor of both AKT and the downstream kinase p70 S6 kinase (p70S6K) and also of protein kinase A. This ATP-competitive small molecule potently inhibits both AKT and p70S6K activity at the cellular level, as measured by inhibition of GSK3β and S6 ribosomal protein phosphorylation, and also causes growth inhibition in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model caused inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also resulted in inhibition of human tumor growth in PTEN-deficient xenograft models. These data suggest that the novel strategy of AKT and p70S6K blockade may have therapeutic value and supports further evaluation of AT7867 as a singleagent anticancer strategy.
Molecular Cancer Therapeutics, 2011
AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT12893... more AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment-and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G 1 arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CAmutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials. Mol Cancer Ther; 10(2); 360-71. Ó2010 AACR.
Journal of Molecular Biology, 2007
Although the crystal structure of the anti-cancer target protein kinase B (PKBβ/Akt-2) has been u... more Although the crystal structure of the anti-cancer target protein kinase B (PKBβ/Akt-2) has been useful in guiding inhibitor design, the closely related kinase PKA has generally been used as a structural mimic due to its facile crystallization with a range of ligands. The use of PKB-inhibitor crystallography would bring important benefits, including a more rigorous understanding of factors dictating PKA/PKB selectivity, and the opportunity to validate the utility of PKA-based surrogates. We present a "backsoaking" method for obtaining PKBβ-ligand crystal structures, and provide a structural comparison of inhibitor binding to PKB, PKA, and PKA-PKB chimera. One inhibitor presented here exhibits no PKB/PKA selectivity, and the compound adopts a similar binding mode in all three systems. By contrast, the PKB-selective inhibitor A-443654 adopts a conformation in PKB and PKA-PKB that differs from that with PKA. We provide a structural explanation for this difference, and highlight the ability of PKA-PKB to mimic the true PKB binding mode in this case.
![Research paper thumbnail of Discovery of 4-Amino-1-(7 H -pyrrolo[2,3- d ]pyrimidin-4-yl)piperidine-4-carboxamides As Selective, Orally Active Inhibitors of Protein Kinase B (Akt)](https://attachments.academia-assets.com/48889232/thumbnails/1.jpg)
](Journal of Medicinal Chemistry, 2010
Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regul... more Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regulating growth and survival. Signaling through PKB is frequently deregulated in cancer, and inhibitors of PKB therefore have potential as antitumor agents. The optimization of lipophilic substitution within a series of 4-benzyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines provided ATP-competitive, nanomolar inhibitors with up to 150-fold selectivity for inhibition of PKB over the closely related kinase PKA. Although active in cellular assays, compounds containing 4-amino-4-benzylpiperidines underwent metabolism in vivo, leading to rapid clearance and low oral bioavailability. Variation of the linker group between the piperidine and the lipophilic substituent identified 4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides as potent and orally bioavailable inhibitors of PKB. Representative compounds modulated biomarkers of signaling through PKB in vivo and strongly inhibited the growth of human tumor xenografts in nude mice at well-tolerated doses. † PDB ID Codes: 2x37 (10-PKB), 2x39 (21-PKB). a Abbreviations: AGC, cAMP-dependent, cGMP-dependent and protein kinase C; ELISA, enzyme-linked immunosorbent assay; mTOR, mammalian target of rapamycin; PH, pleckstrin homology; PKA, protein kinase A; PKB, protein kinase B; PI3K, phosphatidylinositol-3 kinase; PI(3,4,5)P 3 , phosphatidylinositol-3,4,5 triphosphate; PTEN, phosphatase and tensin homologue.
Journal of Medicinal Chemistry, 2008
Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragmen... more Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3- d]pyrimidine inhibitors of PKBbeta with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.
Drug Discovery Today, Feb 28, 2010
Bioorganic Medicinal Chemistry, Jun 15, 2010
The authors regret that the residue 'Leu309' was incorrectly labeled in the crystal structures in... more The authors regret that the residue 'Leu309' was incorrectly labeled in the crystal structures in -D, p712, and should correctly be labeled 'Val234'. The accompanying text on p712 should read 'The inhibitor occupied the ATP-binding site of CHK2 and was sandwiched between the hydrophobic side chains of Val234 and Leu354 .' 0968-0896/$ -see front matter Ó
Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yea... more Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79-*L, analogous to the oncogenic mutation of Q-61--->L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-eu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30o of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-ku79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.
Journal of Medicinal Chemistry, Jan 27, 2011
Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kina... more Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might ...
Journal of Medicinal Chemistry, May 1, 2008
Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragmen... more Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3- d]pyrimidine inhibitors of PKBbeta with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.
European Journal of Cancer Supplements, 2004
Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI... more Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI3 kinase. To find novel inhibitors of PKB/AKT kinase activity for progression as anticancer agents, the authors have used a high-throughput screen based on AlphaScreen™ technology. A known kinase inhibitor, the isoquinoline H8, was used as a positive control with mean inhibition in the screen of 43.4% ± 13.1%. The performance of the screen was highly acceptable with Z´ and Z factors of 0.83 ± 0.07 and 0.75 ± 0.04, respectively. A number of confirmed hits (~0.1% hit rate) were identified from 63,500 compounds screened. Five compounds have previously been described as PKB inhibitors, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. Five hits had the potential to interfere with the assay signal and were deemed to be false positives. Two compounds were nonspecific inhibitors of PKB as enzyme inhibition in a filter-based assay was markedly reduced in the presence of 0.01% Triton X100. The authors now include an interference assay during hit confirmation procedures and check compound activity in the presence of Triton X100 in an attempt to eliminate nonspecific aggregators at an early stage. (Journal of Biomolecular Screening 2006:822-827)
Molecular and cellular biology, 1992
Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yea... more Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumu...
European Journal of Cancer Supplements, 2004
Novartis Foundation Symposia, 2007
Sec4 is a Ras-like GTP-binding protein required for exocytosis in yeast. Unlike Ras, it is the ab... more Sec4 is a Ras-like GTP-binding protein required for exocytosis in yeast. Unlike Ras, it is the ability of Sec4 to cycle between the GTP- and GDP-bound forms rather than the absolute levels of the GTP-bound form that is critical for function. This cycle may be coupled to an observed cycle of Sec4 localization within the cell. Sec4 binds to secretory vesicles which then fuse with the plasma membrane in exocytosis. Sec4 can recycle from the plasma membrane through a soluble pool to rebind to a new round of vesicles. We have found an activity in yeast (Saccharomyces cerevisiae) comparable to that of the GDP dissociation inhibitor protein isolated from mammalian cells that releases GDP-bound Sec4 from membranes. DSS4-1, a dominant suppressor of the sec4-8 temperature-sensitive mutation, encodes a nucleotide exchange protein. The cycle of Sec4 may function to allow the assembly and subsequent disassembly of a set of proteins necessary for exocytosis. Candidates for members of this set of proteins are encoded by sec genes which show strong genetic interactions with sec4-8. Two of these (SEC8 and SEC15) encode large proteins which form a complex that is peripherally associated with the plasma membrane.
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 15, 2014
Multiple cancers harbor genetic aberrations that impact AKT signaling. MK-2206 is a potent pan-AK... more Multiple cancers harbor genetic aberrations that impact AKT signaling. MK-2206 is a potent pan-AKT inhibitor with a maximum tolerated dose (MTD) previously established at 60 mg on alternate days (QOD). Due to a long half-life (60-80 hours), a weekly (QW) MK-2206 schedule was pursued to compare intermittent QW and continuous QOD dosing. Patients with advanced cancers were enrolled in a QW dose-escalation phase I study to investigate the safety and pharmacokinetic-pharmacodynamic profiles of tumor and platelet-rich plasma (PRP). The QOD MTD of MK-2206 was also assessed in patients with ovarian and castration-resistant prostate cancers and patients with advanced cancers undergoing multiparametric functional magnetic resonance imaging (MRI) studies, including dynamic contrast-enhanced MRI, diffusion-weighted imaging, magnetic resonance spectroscopy, and intrinsic susceptibility-weighted MRI. A total of 71 patients were enrolled; 38 patients had 60 mg MK-2206 QOD, whereas 33 received MK-...
PLoS ONE, 2012
Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of ... more Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2Acentered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.
The EMBO Journal, 2006
The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. ... more The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serineglutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation. The EMBO Journal VOL 25 | NO 13 | 2006 EMBO THE EMBO JOURNAL THE EMBO JOURNAL
PLoS ONE, 2013
Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DN... more Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragmentbased screening campaign using a combination of a high-concentration AlphaScreen TM kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors.
Molecular Cancer Therapeutics, 2010
The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in ma... more The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in malignant transformation and chemoresistance and is an attractive target for the development of cancer therapeutics. Fragment-based lead discovery, combined with structure-based drug design, has recently identified AT7867 as a novel and potent inhibitor of both AKT and the downstream kinase p70 S6 kinase (p70S6K) and also of protein kinase A. This ATP-competitive small molecule potently inhibits both AKT and p70S6K activity at the cellular level, as measured by inhibition of GSK3β and S6 ribosomal protein phosphorylation, and also causes growth inhibition in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model caused inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also resulted in inhibition of human tumor growth in PTEN-deficient xenograft models. These data suggest that the novel strategy of AKT and p70S6K blockade may have therapeutic value and supports further evaluation of AT7867 as a singleagent anticancer strategy.
Molecular Cancer Therapeutics, 2011
AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT12893... more AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment-and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G 1 arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CAmutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials. Mol Cancer Ther; 10(2); 360-71. Ó2010 AACR.
Journal of Molecular Biology, 2007
Although the crystal structure of the anti-cancer target protein kinase B (PKBβ/Akt-2) has been u... more Although the crystal structure of the anti-cancer target protein kinase B (PKBβ/Akt-2) has been useful in guiding inhibitor design, the closely related kinase PKA has generally been used as a structural mimic due to its facile crystallization with a range of ligands. The use of PKB-inhibitor crystallography would bring important benefits, including a more rigorous understanding of factors dictating PKA/PKB selectivity, and the opportunity to validate the utility of PKA-based surrogates. We present a "backsoaking" method for obtaining PKBβ-ligand crystal structures, and provide a structural comparison of inhibitor binding to PKB, PKA, and PKA-PKB chimera. One inhibitor presented here exhibits no PKB/PKA selectivity, and the compound adopts a similar binding mode in all three systems. By contrast, the PKB-selective inhibitor A-443654 adopts a conformation in PKB and PKA-PKB that differs from that with PKA. We provide a structural explanation for this difference, and highlight the ability of PKA-PKB to mimic the true PKB binding mode in this case.
Journal of Medicinal Chemistry, 2010
Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regul... more Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regulating growth and survival. Signaling through PKB is frequently deregulated in cancer, and inhibitors of PKB therefore have potential as antitumor agents. The optimization of lipophilic substitution within a series of 4-benzyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines provided ATP-competitive, nanomolar inhibitors with up to 150-fold selectivity for inhibition of PKB over the closely related kinase PKA. Although active in cellular assays, compounds containing 4-amino-4-benzylpiperidines underwent metabolism in vivo, leading to rapid clearance and low oral bioavailability. Variation of the linker group between the piperidine and the lipophilic substituent identified 4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides as potent and orally bioavailable inhibitors of PKB. Representative compounds modulated biomarkers of signaling through PKB in vivo and strongly inhibited the growth of human tumor xenografts in nude mice at well-tolerated doses. † PDB ID Codes: 2x37 (10-PKB), 2x39 (21-PKB). a Abbreviations: AGC, cAMP-dependent, cGMP-dependent and protein kinase C; ELISA, enzyme-linked immunosorbent assay; mTOR, mammalian target of rapamycin; PH, pleckstrin homology; PKA, protein kinase A; PKB, protein kinase B; PI3K, phosphatidylinositol-3 kinase; PI(3,4,5)P 3 , phosphatidylinositol-3,4,5 triphosphate; PTEN, phosphatase and tensin homologue.
Journal of Medicinal Chemistry, 2008
Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragmen... more Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3- d]pyrimidine inhibitors of PKBbeta with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.