Browsing by Subject "Imidazoles"

Now showing 1 - 7 of 7
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    A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.
    (Sci Signal, 2013-05-07) Kurokawa, Manabu; Kim, Jiyeon; Geradts, Joseph; Matsuura, Kenkyo; Liu, Liu; Ran, Xu; Xia, Wenle; Ribar, Thomas J; Henao, Ricardo; Dewhirst, Mark W; Kim, Wun-Jae; Lucas, Joseph E; Wang, Shaomeng; Spector, Neil L; Kornbluth, Sally
    In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.
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    Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.
    (Nature chemical biology, 2005-07) Degterev, Alexei; Huang, Zhihong; Boyce, Michael; Li, Yaqiao; Jagtap, Prakash; Mizushima, Noboru; Cuny, Gregory D; Mitchison, Timothy J; Moskowitz, Michael A; Yuan, Junying
    The mechanism of apoptosis has been extensively characterized over the past decade, but little is known about alternative forms of regulated cell death. Although stimulation of the Fas/TNFR receptor family triggers a canonical 'extrinsic' apoptosis pathway, we demonstrated that in the absence of intracellular apoptotic signaling it is capable of activating a common nonapoptotic death pathway, which we term necroptosis. We showed that necroptosis is characterized by necrotic cell death morphology and activation of autophagy. We identified a specific and potent small-molecule inhibitor of necroptosis, necrostatin-1, which blocks a critical step in necroptosis. We demonstrated that necroptosis contributes to delayed mouse ischemic brain injury in vivo through a mechanism distinct from that of apoptosis and offers a new therapeutic target for stroke with an extended window for neuroprotection. Our study identifies a previously undescribed basic cell-death pathway with potentially broad relevance to human pathologies.
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    Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.
    (Circ Res, 1999-01-08) Xiao, RP; Avdonin, P; Zhou, YY; Cheng, H; Akhter, SA; Eschenhagen, T; Lefkowitz, RJ; Koch, WJ; Lakatta, EG
    -Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.
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    Identification of BfmR, a response regulator involved in biofilm development, as a target for a 2-Aminoimidazole-based antibiofilm agent.
    (Biochemistry, 2012-12) Thompson, Richele J; Bobay, Benjamin G; Stowe, Sean D; Olson, Andrew L; Peng, Lingling; Su, Zhaoming; Actis, Luis A; Melander, Christian; Cavanagh, John
    2-Aminoimidazoles (2AIs) have been documented to disrupt bacterial protection mechanisms, including biofilm formation and genetically encoded antibiotic resistance traits. Using Acinetobacter baumannii, we provide initial insight into the mechanism of action of a 2AI-based antibiofilm agent. Confocal microscopy confirmed that the 2AI is cell permeable, while pull-down assays identified BfmR, a response regulator that is the master controller of biofilm formation, as a target for this compound. Binding assays demonstrated specificity of the 2AI for response regulators, while computational docking provided models for 2AI-BfmR interactions. The 2AI compound studied here represents a unique small molecule scaffold that targets bacterial response regulators.
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    Structure of the Francisella response regulator QseB receiver domain, and characterization of QseB inhibition by antibiofilm 2-aminoimidazole-based compounds.
    (Molecular microbiology, 2017-10) Milton, Morgan E; Allen, C Leigh; Feldmann, Erik A; Bobay, Benjamin G; Jung, David K; Stephens, Matthew D; Melander, Roberta J; Theisen, Kelly E; Zeng, Daina; Thompson, Richele J; Melander, Christian; Cavanagh, John
    With antibiotic resistance increasing at alarming rates, targets for new antimicrobial therapies must be identified. A particularly promising target is the bacterial two-component system. Two-component systems allow bacteria to detect, evaluate and protect themselves against changes in the environment, such as exposure to antibiotics and also to trigger production of virulence factors. Drugs that target the response regulator portion of two-component systems represent a potent new approach so far unexploited. Here, we focus efforts on the highly virulent bacterium Francisella tularensis tularensis. Francisella contains only three response regulators, making it an ideal system to study. In this study, we initially present the structure of the N-terminal domain of QseB, the response regulator responsible for biofilm formation. Subsequently, using binding assays, computational docking and cellular studies, we show that QseB interacts with2-aminoimidazole based compounds that impede its function. This information will assist in tailoring compounds to act as adjuvants that will enhance the effect of antibiotics.
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    Structure-Guided Synthesis of FK506 and FK520 Analogs with Increased Selectivity Exhibit In Vivo Therapeutic Efficacy against Cryptococcus.
    (mBio, 2022-06) Hoy, Michael J; Park, Eunchong; Lee, Hyunji; Lim, Won Young; Cole, D Christopher; DeBouver, Nicholas D; Bobay, Benjamin G; Pierce, Phillip G; Fox, David; Ciofani, Maria; Juvvadi, Praveen R; Steinbach, William; Hong, Jiyong; Heitman, Joseph
    Calcineurin is an essential virulence factor that is conserved across human fungal pathogens, including Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans. Although an excellent target for antifungal drug development, the serine-threonine phosphatase activity of calcineurin is conserved in mammals, and inhibition of this activity results in immunosuppression. FK506 (tacrolimus) is a naturally produced macrocyclic compound that inhibits calcineurin by binding to the immunophilin FKBP12. Previously, our fungal calcineurin-FK506-FKBP12 structure-based approaches identified a nonconserved region of FKBP12 that can be exploited for fungus-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C-22 position, which was less immunosuppressive yet maintained antifungal activity. We now report high-resolution protein crystal structures of fungal FKBP12 and a human truncated calcineurin-FKBP12 bound to a natural FK506 analog, FK520 (ascomycin). Based on information from these structures and the success of APX879, we synthesized and screened a novel panel of C-22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05, demonstrates broad-spectrum antifungal activity in vitro and is nonimmunosuppressive in vivo. In murine models of pulmonary and disseminated C. neoformans infection, JH-FK-05 treatment significantly reduced fungal burden and extended animal survival alone and in combination with fluconazole. Furthermore, molecular dynamic simulations performed with JH-FK-05 binding to fungal and human FKBP12 identified additional residues outside the C-22 and C-21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity. IMPORTANCE Due to rising rates of antifungal drug resistance and a limited armamentarium of antifungal treatments, there is a paramount need for novel antifungal drugs to treat systemic fungal infections. Calcineurin has been established as an essential and conserved virulence factor in several fungi, making it an attractive antifungal target. However, due to the immunosuppressive action of calcineurin inhibitors, they have not been successfully utilized clinically for antifungal treatment in humans. Recent availability of crystal structures of fungal calcineurin-bound inhibitor complexes has enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound with increased fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and maintained therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.
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    Survivin as a therapeutic target in Sonic hedgehog-driven medulloblastoma.
    (Oncogene, 2015-07) Brun, SN; Markant, SL; Esparza, LA; Garcia, G; Terry, D; Huang, J-M; Pavlyukov, MS; Li, X-N; Grant, GA; Crawford, JR; Levy, ML; Conway, EM; Smith, LH; Nakano, I; Berezov, A; Greene, MI; Wang, Q; Wechsler-Reya, RJ
    Medulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Development of such therapies depends in part on identification of genes that are necessary for growth and survival of tumor cells. Survivin is an inhibitor of apoptosis protein that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB and when expressed at high levels predicts poor clinical outcome. Therefore, we hypothesized that Survivin may have a critical role in growth and survival of MB cells and that targeting it may enhance MB therapy. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Genetic deletion of survivin in Ptch mutant tumor cells significantly inhibits proliferation and causes cell cycle arrest. Treatment with small-molecule antagonists of Survivin impairs proliferation and survival of both murine and human MB cells. Finally, Survivin antagonists impede growth of MB cells in vivo. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.