Inhibition Of ATP Binding Research Articles

Abstract 3196: Antitumor effect of a Compound A, a derivative of a naturally occurring rotenoid deguelin, by inhibition of ATP binding to heat shock protein 90

Abstract Despite several decades of intensive efforts to develop anti-cancer drugs and therapeutic strategies, lung cancer is still one of the main causes of cancer-related deaths worldwide. Deguelin, a natural plant product, has shown anti-cancer activities in skin, colon, breast and non-small cell lung (NSCLC) cancers. However, the potential side effects of deguelin could be an obstacle to its use in clinic. In the current study, we aimed to develop a derivative of deguelin with reduced potential side effects. We have synthesized a novel deguelin analog, compound A, and evaluated its effects on cancer and normal bronchial epithelial cell proliferation, survival, and colony formation by the MTT, FACS, and/or anchorage-dependent and -independent colony forming assays. Mechanisms underlying anti-tumor effects of compound A were investigated by Western blot, and RT-PCR. Treatment with compound A inhibited cancer cell proliferation and colony formation, promoted cell cycle arrest and induced apoptosis. Compound A also suppressed the expression of HIF-1α protein and its target gene, VEGF, in NSCLC cells. Administration of compound A to NSCLC xenograft-bearing mice significantly decreased tumor growth by inhibiting tumor angiogenesis and by inducing apoptosis. In its action mechanism, compound A was involved in binding to the ATP-binding pocket of HSP90, leading to the suppression of HSP90 function. These collective results suggest that a novel deguelin derivative, compound A, has a strong potential as an anti-cancer drug. Citation Format: Su-Chan Lee, Ju-Sung Lee, Seung-Yeob Hyun, Hoon Choi, Hongchan An, Kyu-Won Kim, Young-Ger Suh, Ho-Young Lee. Antitumor effect of a Compound A, a derivative of a naturally occurring rotenoid deguelin, by inhibition of ATP binding to heat shock protein 90. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3196. doi:10.1158/1538-7445.AM2014-3196

Atovaquone and quinine anti-malarials inhibit ATP binding cassette transporter activity.

BackgroundTherapeutic blood plasma concentrations of anti-malarial drugs are essential for successful treatment. Pharmacokinetics of pharmaceutical compounds are dependent of adsorption, distribution, metabolism, and excretion. ATP binding cassette (ABC) transport proteins are particularly involved in drug deposition, as they are located at membranes of many uptake and excretory organs and at protective barriers, where they export endogenous and xenobiotic compounds, including pharmaceuticals. In this study, a panel of well-established anti-malarial drugs which may affect drug plasma concentrations was tested for interactions with human ABC transport proteins.MethodsThe interaction of chloroquine, quinine, artemisinin, mefloquine, lumefantrine, atovaquone, dihydroartemisinin and proguanil, with transport activity of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), bile salt export pump (BSEP) and multidrug resistance-associated proteins (MRP) 1–4 were analysed. The effect of the anti-malarials on the ATP-dependent uptake of radio-labelled substrates was measured in membrane vesicles isolated from HEK293 cells overexpressing the ABC transport proteins.ResultsA strong and previously undescribed inhibition of BCRP-mediated transport by atovaquone with a 50% inhibitory concentration (IC50) of 0.23 μM (95% CI 0.17-0.29 μM) and inhibition of P-gp-mediated transport by quinine with an IC50 of 6.8 μM (95% CI 5.9-7.8 μM) was observed. Furthermore, chloroquine and mefloquine were found to significantly inhibit P-gp-mediated transport. BCRP transport activity was significantly inhibited by all anti-malarials tested, whereas BSEP-mediated transport was not inhibited by any of the compounds. Both MRP1- and MRP3-mediated transport were significantly inhibited by mefloquine.ConclusionsAtovaquone and quinine significantly inhibit BCRP- and P-gp- mediated transport at concentrations within the clinically relevant prophylactic and therapeutic range. Co-administration of these established anti-malarials with drugs that are BCRP or P-gp substrates may potentially lead to drug-drug interactions.

Cowpox virus protein CPXV012 eludes CTLs by blocking ATP binding to TAP.

CD8(+) CTLs detect virus-infected cells through recognition of virus-derived peptides presented at the cell surface by MHC class I molecules. The cowpox virus protein CPXV012 deprives the endoplasmic reticulum (ER) lumen of peptides for loading onto newly synthesized MHC class I molecules by inhibiting the transporter associated with Ag processing (TAP). This evasion strategy allows the virus to avoid detection by the immune system. In this article, we show that CPXV012, a 9-kDa type II transmembrane protein, prevents peptide transport by inhibiting ATP binding to TAP. We identified a segment within the ER-luminal domain of CPXV012 that imposes the block in peptide transport by TAP. Biophysical studies show that this domain has a strong affinity for phospholipids that are also abundant in the ER membrane. We discuss these findings in an evolutionary context and show that a frameshift deletion in the CPXV012 gene in an ancestral cowpox virus created the current form of CPXV012 that is capable of inhibiting TAP. In conclusion, our findings indicate that the ER-luminal domain of CPXV012 inserts into the ER membrane, where it interacts with TAP. CPXV012 presumably induces a conformational arrest that precludes ATP binding to TAP and, thus, activity of TAP, thereby preventing the presentation of viral peptides to CTLs.

QSAR-based models for designing quinazoline/imidazothiazoles/pyrazolopyrimidines based inhibitors against wild and mutant EGFR.

Overexpression of EGFR is responsible for causing a number of cancers, including lung cancer as it activates various downstream signaling pathways. Thus, it is important to control EGFR function in order to treat the cancer patients. It is well established that inhibiting ATP binding within the EGFR kinase domain regulates its function. The existing quinazoline derivative based drugs used for treating lung cancer that inhibits the wild type of EGFR. In this study, we have made a systematic attempt to develop QSAR models for designing quinazoline derivatives that could inhibit wild EGFR and imidazothiazoles/pyrazolopyrimidines derivatives against mutant EGFR. In this study, three types of prediction methods have been developed to design inhibitors against EGFR (wild, mutant and both). First, we developed models for predicting inhibitors against wild type EGFR by training and testing on dataset containing 128 quinazoline based inhibitors. This dataset was divided into two subsets called wild_train and wild_valid containing 103 and 25 inhibitors respectively. The models were trained and tested on wild_train dataset while performance was evaluated on the wild_valid called validation dataset. We achieved a maximum correlation between predicted and experimentally determined inhibition (IC50) of 0.90 on validation dataset. Secondly, we developed models for predicting inhibitors against mutant EGFR (L858R) on mutant_train, and mutant_valid dataset and achieved a maximum correlation between 0.834 to 0.850 on these datasets. Finally, an integrated hybrid model has been developed on a dataset containing wild and mutant inhibitors and got maximum correlation between 0.761 to 0.850 on different datasets. In order to promote open source drug discovery, we developed a webserver for designing inhibitors against wild and mutant EGFR along with providing standalone (http://osddlinux.osdd.net/) and Galaxy (http://osddlinux.osdd.net:8001) version of software. We hope our webserver (http://crdd.osdd.net/oscadd/ntegfr/) will play a vital role in designing new anticancer drugs.

Anti‐arthritis effects of (E)‐2,4‐bis(p‐hydroxyphenyl)‐2‐butenal are mediated by inhibition of the STAT3 pathway

Products of Maillard reactions between aminoacids and reducing sugars are known to have anti-inflammatory properties. Here we have assessed the anti-arthritis effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal and its possible mechanisms of action. We used cultures of LPS-activated macrophages (RAW264.7 cells) and human synoviocytes from patients with rheumatoid arthritis for in vitro assays and the collagen-induced arthritis model in mice. NO generation, iNOS and COX2 expression, and NF-κB/IKK and STAT3 activities were measured in vitro and in joint tissues of arthritic mice, along with clinical scores and histopathological assessments. Binding of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal to STAT3 was evaluated by a pull-down assay and its binding site was predicted using molecular docking studies with Autodock VINA. (E)-2,4-bis(p-hydroxyphenyl)-2-butenal (2.5-10 μg·mL(-1) ) inhibited LPS-inducedNO generation, iNOS and COX2 expression, and NF-κB/IKK and STAT3 activities in macrophage and human synoviocytes. This compound also suppressedcollagen-induced arthritic responses in mice by inhibiting expression of iNOS and COX2, and NF-κB/IKK and STAT3 activities; it also reduced bone destruction and fibrosis in joint tissues. A pull-down assay showed that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal interfered with binding of ATP to STAT3. Docking studies suggested that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal bound to the DNA-binding interface of STAT3 possibly inhibiting ATP binding to STAT3 in an allosteric manner. (E)-2,4-bis(p-hydroxyphenyl)-2-butenal exerted anti-inflammatory and anti-arthritic effects through inhibition of the NF-κB/STAT3 pathway by direct binding to STAT3. This compound could be a useful agent for the treatment of arthritic disease.

Designed Hsp90 Heterodimers Reveal an Asymmetric ATPase-Driven Mechanism In Vivo

Hsp90 is a homodimeric ATPase that is essential in eukaryotes for the maturation of client proteins frequently involved in signal transduction, including many kinases and nuclear steroid hormone receptors. Competitive inhibitors of ATP binding to Hsp90 prevent client maturation and show promise as anticancer agents in clinical trials. However, the role of ATP binding and hydrolysis in each subunit of the Hsp90 dimer has been difficult to investigate because of an inability to assemble and study dimers of defined composition. We used protein engineering to generate functional Hsp90 subunits that preferentially assemble as heterodimers. We analyzed dimers wherein one subunit harbors a disruptive mutation and observed that ATP binding by both subunits is essential for function in yeast, whereas ATP hydrolysis is only required in one subunit. These findings demonstrate important functional contributions from both symmetric and asymmetric Hsp90 dimers and provide valuable reagents for future investigations of Hsp90 mechanism.

P90 RSK2 Mediates Antianoikis Signals by both Transcription-Dependent and -Independent Mechanisms

How invasive and metastatic tumor cells evade anoikis induction remains unclear. We found that knockdown of RSK2 sensitizes diverse cancer cells to anoikis induction, which is mediated through phosphorylation targets including apoptosis signal-regulating kinase 1 (ASK1) and cyclic AMP (cAMP) response element-binding protein (CREB). We provide evidence to show that RSK2 inhibits ASK1 by phosphorylating S83, T1109, and T1326 through a novel mechanism in which phospho-T1109/T1326 inhibits ATP binding to ASK1, while phospho-S83 attenuates ASK1 substrate MKK6 binding. Moreover, the RSK2→CREB signaling pathway provides antianoikis protection by regulating gene expression of protein effectors that are involved in cell death regulation, including the antiapoptotic factor protein tyrosine kinase 6 (PTK6) and the proapoptotic factor inhibitor-of-growth protein 3 (ING3). PTK6 overexpression or ING3 knockdown in addition to ASK1 knockdown further rescued the increased sensitivity to anoikis induction in RSK2 knockdown cells. These data together suggest that RSK2 functions as a signal integrator to provide antianoikis protection to cancer cells in both transcription-independent and -dependent manners, in part by signaling through ASK1 and CREB, and contributes to cancer cell invasion and tumor metastasis.

Abstract 5673: Novel inhibitors of the interferon-inducible, dsRNA-activated kinase PKR for myelodysplastic syndrome.

Abstract In hematopoietic stem/progenitor cells, the interferon-inducible, dsRNA-activated protein kinase, PKR, is a central mediator for the anti-proliferative effects of a wide variety of hematopoietic, inflammatory or cytotoxic stresses such as deprivation of IL-3, IL-5 or GM-CSF from hematopoietic factor-dependent cells. Once activated, PKR inhibits protein synthesis by phosphorylation of eIF2alpha, inhibits mitochondrial function by activating PP2A-dependent Bcl2 dephosphorylation, and activates signaling pathways such as NF-kappaB, p53 and STAT1 to regulate proliferation and apoptosis in response to cellular stress. Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematologic malignancies characterized by peripheral cytopenias, a hypercellular marrow with ineffective hematopoiesis and risk of transformation to acute myeloid leukemia (AML). Significantly, PKR is highly activated in myeloid progenitor cells from MDS patients and inhibition of PKR activity or siRNA-knockdown partially reverses the suppressive effects of IFN-gamma and TNF-alpha on hematopoietic colony formation using normal or MDS-derived CD34+ cells. Thus, increased PKR activity in hematopoietic stem cells may cooperate with driver/founder mutations to promote bone marrow failure and apoptosis characteristic of MDS, and inhibitors of PKR may be therapeutically useful to delay the onset and/or reduce the severity of MDS and its evolution to AML. Since there is only one commercially available pharmacologic PKR inhibitor that targets ATP binding and has been reported to have off target effects, we performed studies to identify novel small molecule compounds that specifically inhibit PKR-substrate association. Using the crystal structure of the PKR kinase domain-eIF2alpha complex, we employed a molecular docking strategy to screen in silico the NIH Developmental Therapeutic Program's repository of over 300,000 small molecules for compounds that interact with and inhibit PKR. The top 195 scoring small molecule compounds identified were obtained and validated in vitro using a LanthaScreen PKR kinase assay and 32P PKR assay. In addition, we screened the NIH/DTP Diversity and Challenge Compound Sets for inhibitors of PKR (2047 compounds total). Using these methods, we have identified 27 novel PKR inhibitors with an IC50 < 100 μM. Significantly, five are competitive inhibitors for eIF2alpha binding to PKR while the remaining inhibit ATP binding. These results identify a novel class of PKR inhibitors that specifically disrupt association of PKR with its substrate, eIF2alpha, and lay the groundwork for future studies that test whether these PKR inhibitors may be therapeutically useful to delay the onset of cytopenias and bone marrow failure associated with MDS and MDS evolution to AML. Citation Format: Richard L. Bennett, David A. Ostrov, W. Stratford May. Novel inhibitors of the interferon-inducible, dsRNA-activated kinase PKR for myelodysplastic syndrome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5673. doi:10.1158/1538-7445.AM2013-5673

Abstract 2925: p90RSK2 coordinates pro-apoptotic and anti-apoptotic signaling pathways to protect cancer cells from anoikis.

Abstract How invasive and metastatic tumor cells evade from anoikis induction remains unclear. We found that knockdown of RSK2 sensitizes diverse cancer cells to anoikis induction, which is mediated through phosphorylation targets, including apoptosis signal-regulating kinase 1 (ASK1) and cAMP response element-binding (CREB). We provide evidence to show that RSK2 inhibits ASK1 by phosphorylating S83, T1109 and T1326 through novel mechanism in which phospho-T1109/T1326 inhibits ATP binding to ASK1, while phospho-S83 attenuates ASK1 substrate MKK6 binding. Moreover, RSK2-CREB signaling pathway provides anti-anoikis protection by regulating gene expression of protein effectors that are involved in cell death regulation, including anti-apoptotic protein tyrosine kinase 6 (PTK6) and pro-apoptotic inhibitor of growth protein 3 (ING3). PTK6 overexpression or ING3 knockdown in addition to ASK1 knockdown further rescued the increased sensitivity to anoikis induction in RSK2 knockdown cells. These data together suggest that RSK2 functions as a signal integrator to provide anti-anoikis protection to cancer cells in both transcription-independent and -dependent manners, in part by signaling through ASK1 and CREB, and contributes to cancer cell invasion and tumor metastasis. Citation Format: Lingtao Jin, Dan Li, Jongseok Lee, Gina N. Alesi, Jun Fan, Hee-Bum Kang, Dongsheng Wang, Haian Fu, Jack Taunton, Titus J. Boggon, Meghan Tucker, Ting-Lei Gu, Zhuo G. Chen, Dong M. Shin, Fadlo R. Khuri, Sumin Kang. p90RSK2 coordinates pro-apoptotic and anti-apoptotic signaling pathways to protect cancer cells from anoikis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2925. doi:10.1158/1538-7445.AM2013-2925

Functional deficit associated with a missense Werner syndrome mutation

Werner syndrome (WS) is a rare autosomal recessive disorder caused by mutations in the WRN gene. WRN helicase, a member of the RecQ helicase family, is involved in various DNA metabolic pathways including DNA replication, recombination, DNA repair and telomere maintenance. In this study, we have characterized the G574R missense mutation, which was recently identified in a WS patient. Our biochemical experiments with purified mutant recombinant WRN protein showed that the G574R mutation inhibits ATP binding, and thereby leads to significant decrease in helicase activity. Exonuclease activity of the mutant protein was not significantly affected, whereas its single strand DNA annealing activity was higher than that of wild type. Deficiency in the helicase activity of the mutant may cause defects in replication and other DNA metabolic processes, which in turn could be responsible for the Werner syndrome phenotype in the patient. In contrast to the usual appearance of WS, the G574R patient has normal stature. Thus the short stature normally associated with WS may not be due to helicase deficiency.

Allosteric inhibition of the DNA-dependent ATPase activity of Escherichia coli DNA gyrase by a representative of a novel class of inhibitors

A novel class of bacterial DNA gyrase inhibitors has been shown previously to form a ternary complex with DNA and gyrase in a site distinct from the fluoroquinolone and ATP binding sites and does not cause double-strand-cleaved complex stabilization like fluoroquinolones. We show that, unlike fluoroquinolones, a representative compound inhibits DNA-dependent ATP hydrolysis by Escherichia coli gyrase and also blocks cleaved complex stabilization by ciprofloxacin. Conversely, ciprofloxacin blocks ATPase inhibition by the novel compound. We conclude that the compound acts allosterically to inhibit ATP binding or hydrolysis and interferes with the gyrase catalytic cycle at a different point than ciprofloxacin.

Abstract 902: Effective and selective targeting of glioblastoma multiforme using an active-site inhibitor of mTORC1/mTORC2 kinase

Abstract The PI3K-AKT-mTOR signaling axis is central to the transformed phenotype of Glioblastoma multiforme (GBM) cells, due to frequent loss of tumor suppressor PTEN. The mammalian target of rapamycin (mTOR) kinase is present in two cellular multiprotein complexes, mTORC1 and mTORC2, which have distinct subunit composition, substrates and mechanisms of activation. Targeting the mTOR protein is a promising strategy for cancer therapy. However, neither of these complexes are fully inhibited by the allosteric inhibitor rapamycin or its analogs. The aim of this study is to provide evidence that a combined inhibition of mTORC1/2 using an ATP-competitive binding inhibitor, PP242, would effectively suppress GBM growth and dissemination as compared to an allosteric binding inhibitor of mTOR. In addition, we utilized another inhibitor, PI-103, a less selective TORC1/2 inhibitor that also targets PI3K, which is more immunosuppressive than PP242, to deter mTOR pathway. We observed a significant number of GBM tumors showed increased expression of pAKTSer473 and pmTORSer2448, as assessed by immunohistochemistry. GBM cells treated with PP242 significantly reduced the activation of mTORC1 and mTORC2, as shown by reduction in phosphorylation of their substrate levels, p-S6KSer235/236 /4E binding protein-1 (4E-BP1) and Akt, respectively, in a dose dependent manner. Furthermore, insulin induced activation of these kinases was abrogated by pre-treatment with PP242 as compared with rapamycin. The cell cycle analysis using the incorporation of 5-ethynyl-2-deoxyuridine (EdU) into the DNA demonstrated that PP242, but not rapamycin, completely blocked the EdU inclusion. To assess TORC2 function, we studied the phosphorylation forkhead box O (FOXO) transcription factors, which are substrates of both Akt and serum downstream of TORC2. PP242, but not rapamycin, reduced FOXO phosphorylation on Akt consensus sites of FOXO1 correlating with greater inhibition of cell cycling. A combined inhibitor of PI3K and mTOR, p103, an ATP binding inhibitor, caused no change in another substrate of mTORC1, suppressed p-S6KSer235/236 levels. In addition, GBM cell proliferation was significantly suppressed by PP242 which was more pronounced compared to rapamycin. Lastly, migration of GBM cells after treatment with PP242 was significantly suppressed, while analysis of cytoskeleton showed that this migration was associated with cellular behavior rather than cytoskeleton loss. In conclusion, these results suggest a novel combined active-site inhibitor of mTORC1/2 kinase; PP242 suppresses both GBM growth and dissemination, which underscores its potential therapeutic use in treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 902. doi:1538-7445.AM2012-902

Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems

PhoP and PhoQ comprise a two-component system in the bacterium Salmonella enterica. PhoQ is the sensor kinase/phosphatase that modifies the phosphorylation state of the regulator PhoP in response to stimuli. The amount of phosphorylated PhoP surges after activation, then declines to reach a steady-state level. We now recapitulate this surge invitro by incubating PhoP and PhoQ with ATP and ADP. Mathematical modeling identified PhoQ's affinity for ADP as the key parameter dictating phosphorylated PhoP levels, as ADP promotes PhoQ's phosphatase activity toward phosphorylated PhoP. The lid covering the nucleotide-binding pocket of PhoQ governs the kinase to phosphatase switch because a lid mutation that decreased ADP binding compromised PhoQ's phosphatase activity invitro and resulted in sustained expression of PhoP-dependent mRNAs invivo. This feedback mechanism may curtail futile ATP consumption because ADP not only stimulates PhoQ's phosphatase activity but also inhibits ATP binding necessary for the kinase reaction.

Geldanamycin and its derivatives as Hsp90 inhibitors

The Hsp90 molecule, one of the most abundant heat shock proteins in mammalian cells, maintains homeostasis and prevents stress-induced cellular damage. Hsp90 is expressed under normal conditions at a level of about 1-2 Percent of total proteins, while its expression increases 2-10 fold in cancer cells. The two main constitutively expressed isoforms of Hsp90 are known as Hsp90-alpha and Hsp90-beta, and their upregulation is associated with tumor progression, invasion and formation of metastases, as well as development of drug resistance. The Hsp90 is a key target for many newly established, potent anticancer agents containing Hsp90 N-terminal ATP binding inhibitors, such as geldanamycin, and its analogues 17AAG and 17DMAG. The therapeutic usage of geldanamycin has been limited due to its poor water solubility and severe hepatotoxicity. Therefore, its analogues, including 17AAG, 17DMAG, Tanespimycin and Retaspimycin hydrochloride, with improved pharmacokinetic profiles, have been developed.

Sulforaphane Potentiates the Efficacy of 17-Allylamino 17-Demethoxygeldanamycin Against Pancreatic Cancer Through Enhanced Abrogation of Hsp90 Chaperone Function

Heat shock protein 90 (Hsp90), an essential molecular chaperone that regulates the stability of a wide range of oncogenic proteins, is a promising target for cancer therapeutics. We investigated the combination efficacy and potential mechanisms of sulforaphane, a dietary component from broccoli and broccoli sprouts, and 17-allylamino 17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, in pancreatic cancer. MTS assay demonstrated that sulforaphane sensitized pancreatic cancer cells to 17-AAG in vitro. Caspase-3 was activated to 6.4-fold in response to simultaneous treatment with sulforaphane and 17-AAG, whereas 17-AAG alone induced caspase-3 activity to 2-fold compared to control. ATP binding assay and coimmunoprecipitation revealed that sulforaphane disrupted Hsp90-p50Cdc37 interaction, whereas 17-AAG inhibited ATP binding to Hsp90. Concomitant use of sulforaphane and 17-AAG synergistically downregulated Hsp90 client proteins in Mia Paca-2 cells. Co-administration of sulforaphane and 17-AAG in pancreatic cancer xenograft model led to more than 70% inhibition of the tumor growth, whereas 17-AAG alone only suppressed the tumor growth by 50%. Our data suggest that sulforaphane potentiates the efficacy of 17-AAG against pancreatic cancer through enhanced abrogation of Hsp90 function. These findings provide a rationale for further evaluation of broccoli/broccoli sprout preparations combined with 17-AAG for better efficacy and lower dose-limiting toxicity in pancreatic cancer.

Liver X Receptor β (LXRβ) Interacts Directly with ATP-binding Cassette A1 (ABCA1) to Promote High Density Lipoprotein Formation during Acute Cholesterol Accumulation

Cells have evolved multiple mechanisms for maintaining cholesterol homeostasis, and, among these, ATP-binding cassette protein A1 (ABCA1)-mediated cholesterol efflux is highly regulated at the transcriptional level through the activity of the nuclear receptor liver X receptor (LXR). Here, we show that in addition to its well defined role in transcription, LXRβ directly binds to the C-terminal region ((2247)LTSFL(2251)) of ABCA1 to mediate its post-translational regulation. In the absence of cholesterol accumulation in the macrophage-like cell line THP-1, the ABCA1-LXRβ complex stably localizes to the plasma membrane, but apolipoprotein A-I (apoA-I) binding or cholesterol efflux does not occur. Exogenously added LXR ligands, which mimic cholesterol accumulation, cause LXRβ to dissociate from ABCA1, thus freeing ABCA1 for apoA-I binding and subsequent cholesterol efflux. Photoaffinity labeling experiments with 8-azido-[α-(32)P]ATP showed that the interaction of LXRβ with ABCA1 inhibits ATP binding by ABCA1. This is the first study to show that a protein-protein interaction with the endogenous protein suppresses the function of ABC proteins by inhibiting ATP binding. LXRβ can cause a post-translational response by binding directly to ABCA1, as well as a transcriptional response, to maintain cholesterol homeostasis.

Genetic Screen to Isolate “Lariat” Peptides for Characterizing ABL Kinase Activity and Conformation

AbstractAbstract 438Type II Abl kinase inhibitors such as imatinib achieve high selectivity by specifically targeting the inactive Abl/c-Kit conformation. However, these inhibitors are prone to inactivation by resistance mutations that cause conformational changes. A better understanding of mutant Abl conformations would provide information to design inhibitors that recognize multiple states of Abl with lower rates of resistance. To more easily examine Abl kinase conformations, conformer specific reagents are needed that are (i) easily generated against a given Abl conformation, (ii) compatible with genetic selections, in vivo and in vitro assays, and structural studies, and (iii) amenable to chemical synthesis. Recently, we developed a novel genetic screen for isolating “lariat” peptide inhibitors of protein function. Lariats are lactone-cyclized peptides that possess the above-mentioned characteristics and can be used to evaluate the function and therapeutic potential of proteins. Previously, we isolated specific lariat inhibitors against the inactive conformation of the bacterial repressor LexA. To obtain conformer specific lariats against Abl, we screened a combinatorial seven amino acid lariat library for interactions with the Abl SH1 domain using the yeast two-hybrid (Y2H) assay. We obtained two lariat peptides named A1 (SGWQRLPFEY) and A2 (SGWHRLSEEY) that interacted with the Abl SH1 domain. In vitro studies with purified Abl kinase demonstrated that the A1 lariat competitively inhibited ATP binding with an inhibitory constant of 5.95 μM. By performing site-saturation mutagenesis, we defined acceptable and tolerable substitutions at each position of the A1 lariat. To obtain tighter binding variants of the A1 lariat, we rationally designed mutations and constructed a second-generation lariat (GWQTLDWNY) with | 10 times higher affinity for Abl. We compared the affinity of the lariats for imatinib resistant Abl kinase mutants using the lariat Y2H binding assay. Mutations that (i) promote conformational dynamics of the kinase T315I), (ii) destabilize the Abl/c-Kit like inactive state (H396R), (iii) disrupt the flexibility or hydrophobicity of the lariat-binding pocket (E355G), and (iv) distort the ATP phosphate binding loop (Y253F), have a marked decrease in lariat affinity. Together, these results show that the lariat preferentially bound to the Abl/c-Kit like inactive conformation and act similarly to type II kinase inhibitors. A specificity screen against a panel of related and distant kinases also demonstrated the conformer specific nature of the lariats. We used lariats that recognized the Abl/c-Kit like inactive state to probe various conformations of Abl His396 mutants. His396 is located in the activation loop and does not alter the Abl/c-Kit like inactive form. His396Pro and His396Arg substitutions are clinically relevant imatinib resistant mutations that alter the conformation of Abl. Previous structural studies showed that the His396Pro mutation causes Abl to adopt the active conformation. It is hypothesized that the His396Arg mutation favors the inactive Src/Cdk like state. Using inactive conformation specific Abl lariats as probes, we showed that these lariats interact weakly with the His396Arg relative to His396, suggesting that this mutation destabilized the Abl/c-Kit like inactive state. We also showed that the lariats interacted stronger with a previously unreported His396Ile mutation than His396, indicating that His396Ile shifts Abl to a highly inactive conformation. Preliminary studies suggest that this residue plays a key role in determining the flexibility of the activation loop and hence the conformational state of Abl. These results provide information on the structure and sequence requirements for maintaining an auto-inhibited activation loop conformation. In summary, we isolated lariat peptide inhibitors against the Abl SH1 domain, characterized their mechanism of action, and improved their affinity for wild type and mutant Abl SH1domains. Additionally, we demonstrated that lariat peptides can be used as affinity reagents to probe conformational states of Abl. Disclosures:No relevant conflicts of interest to declare.

Oxidation-induced intramolecular disulfide bond inactivates mitogen-activated protein kinase kinase 6 by inhibiting ATP binding

Mitogen-activated protein kinase kinase 6 (MKK6) is a member of the mitogen-activated protein kinase (MAPK) kinase (MAP2K) subfamily that specifically phosphorylates and activates the p38 MAPKs. Based on both biochemical and cellular assays, we found that MKK6 was extremely sensitive to oxidation: It was inactivated by oxidation and its kinase activity was fully restored upon treatment with a reducing agent. Detailed mechanistic studies showed that cysteines 109 and 196, two of the six cysteines in MKK6, formed an intramolecular disulfide bond upon oxidation that inactivated MKK6 by inhibiting its ATP binding. This mechanism is distinct from that seen in other redox-sensitive kinases. The two cysteines involved in intramolecular disulfide formation are conserved in all seven members of the MAP2K family. Consistently, we confirmed that other MAP2Ks were also sensitive to oxidation. Our work reveals that MKK6 and other MAP2Ks are a distinct class of cellular redox sensors.

Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Members of the transforming growth factor-beta superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-beta pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.

Retaspimycin hydrochloride (IPI-504): a novel heat shock protein inhibitor as an anticancer agent

Heat shock proteins are vital to cell survival under conditions of stress. They bind client proteins to assist in protein stabilization, translocation of polypeptides across cell membranes and recovery of proteins from aggregates. Heat shock protein inhibitors are a diverse group of novel agents that have been demonstrated to have pro-apoptotic effects on malignant cells through inhibition of ATP binding on the ATP/ADP-binding pocket of the heat shock protein. Initial development of heat shock protein 90 inhibitors, geldanamycin and 17-AAG, were limited by hepatotoxicity and the need for solvent carrying agents. In contrast, retaspimycin, or IPI-504, a derivative of geldanamycin and 17-AAG, is highly soluble in water and generally well tolerated. In Phase I/II trials, retaspimycin has shown activity in NSCLC and gastrointestinal stromal tumor. The most promising activity was observed in gastrointestinal stromal tumors. Phase I/II trials are currently underway to evaluate the dosing schedules and activity of IPI-504 in breast cancer. Given the in vitro activity in diffuse large B-cell lymphoma, mantle cell lymphoma, melanoma, leukemia and pancreatic cancer, current and future trials are of clinical interest. This article reviews IPI-504 and its utility in a wide variety of cancer phenotypes.