Enzymatic Characterization of ER Stress-Dependent Kinase, PERK, and Development of a High-Throughput Assay for Identification of PERK Inhibitors

PERK is serine/threonine kinase localized to the endoplasmic reticulum (ER) membrane. PERK is activated and contributes to cell survival in response to a variety of physiological stresses that affect protein quality control in the ER, such as hypoxia, glucose depravation, increased lipid biosynthesis, and increased protein translation. Pro-survival functions of PERK are triggered by such stresses, suggesting that development of small-molecule inhibitors of PERK may be efficacious in a variety of disease scenarios. Hence, we have conducted a detailed enzymatic characterization of the PERK kinase to develop a high-throughput-screening assay (HTS) that will permit the identification of small-molecule PERK inhibitors. In addition to establishing the Km of PERK for both its primary substrate, eIF2?, and for adenosine triphosphate, further mechanistic studies revealed that PERK targets its substrate via either a random/steady-state ordered mechanism. For HTS, we developed a time-resolved fluorescence resonance energy transfer–based assay that yielded a robust Z? factor and percent coefficient of variation value, enabling the successful screening of 79,552 compounds. This approach yielded one compound that exhibited good in vitro and cellular activity. These results demonstrate the validity of this screen and represent starting points for drug discovery efforts

Identification of novel small molecules that elevate Klotho expression

The absence of Klotho (KL) from mice causes the development of disorders associated with human aging and decreased longevity, whereas increased expression prolongs lifespan. With age, KL protein levels decrease, and keeping levels consistent may promote healthier aging and be disease-modifying. Using the KL promoter to drive expression of luciferase, we conducted a high-throughput screen to identify compounds that activate KL transcription. Hits were identified as compounds that elevated luciferase expression at least 30%. Following validation for dose-dependent activation and lack of cytotoxicity, hit compounds were evaluated further in vitro by incubation with opossum kidney and Z310 rat choroid plexus cells, which express KL endogenously. All compounds elevated KL protein compared with control. To determine whether increased protein resulted in an in vitro functional change, we assayed FGF23 (fibroblast growth factor 23) signalling. Compounds G–I augmented ERK (extracellular-signal-regulated kinase) phosphorylation in FGFR (fibroblast growth factor receptor)-transfected cells, whereas co-transfection with KL siRNA (small interfering RNA) blocked the effect. These compounds will be useful tools to allow insight into the mechanisms of KL regulation. Further optimization will provide pharmacological tools for in vivo studies of KL

Lowering of amyloid beta peptide production with a small molecule inhibitor of amyloid-? precursor protein dimerization

The amyloid ? precursor protein (APP) is a single-pass transmembrane glycoprotein that is ubiquitously expressed in many cell types, including neurons. Amyloidogenic processing of APP by ?- and ?-secretases leads to the production of amyloid-? (A?) peptides that can oligomerize and aggregate into amyloid plaques, a characteristic hallmark of Alzheimer’s disease (AD) brains. Multiple reports suggest that dimerization of APP may play a role in A? production; however, it is not yet clear whether APP dimers increase or decrease A? and the mechanism is not fully understood. To better understand the relationship between APP dimerization and production of A?, a high throughput screen for small molecule modulators of APP dimerization was conducted using APP-Firefly luciferase enzyme complementation to detect APP dimerization. Selected modulators identified from a compound library of 77,440 compounds were tested for their effects on A? generation. Two molecules that inhibited APP dimerization produced a reduction in A? levels as measured by ELISA. The inhibitors did not change sAPP? or ?-CTF levels, but lowered sAPP? levels, suggesting that blocking the dimerization is preventing the cleavage by ?-secretase in the amyloidogenic processing of APP. To our knowledge, this is the first High Throughput Screen (HTS) effort to identify small molecule modulators of APP dimerization. Inhibition of APP dimerization has previously been suggested as a therapeutic target in AD. The findings reported here further support that modulation of APP dimerization may be a viable means of reducing the production of A?

A high-throughput screen to identify inhibitors of SOD1 transcription

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. Approximately 20% of familial ALS cases are caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Rodents expressing mutant SOD1 transgenes develop progressive, fatal motor neuron disease and disease onset and progression is dependent on the level of SOD1. We investigated the possibility that a reduction in SOD1 protein may be of therapeutic benefit in ALS and screened 30,000 compounds for inhibition of SOD1 transcription. The most effective inhibitor identified was N-{4-[4-(4-methylbenzoyl)-1-piperazinyl]phenyl}-2-thiophenecarboxamide (Compound ID 7687685), which in PC12 cells showed an EC50 of 10.6 microM for inhibition of SOD1 expression and an LD50 >30 microM. This compound was subsequently shown to reduce endogenous SOD1 levels in HeLa cells and to exhibit a modest reduction of SOD1 protein levels in mouse spinal cord tissue. These data suggest that the efficacy of compound 7687685 as an inhibitor of SOD1 gene expression is not likely to be clinically useful, although the strategy reported could be applied broadly to screening for small molecule inhibitors of gene expression

Pim1 promotes human prostate cancer cell tumorigenicity and c-MYC transcriptional activity

<p>Abstract</p> <p>Background</p> <p>The serine/threonine kinase PIM1 has been implicated as an oncogene in various human cancers including lymphomas, gastric, colorectal and prostate carcinomas. In mouse models, Pim1 is known to cooperate with c-Myc to promote tumorigenicity. However, there has been limited analysis of the tumorigenic potential of Pim1 overexpression in benign and malignant human prostate cancer cells <it>in vivo</it>.</p> <p>Methods</p> <p>We overexpressed Pim1 in three human prostate cell lines representing different disease stages including benign (RWPE1), androgen-dependent cancer (LNCaP) and androgen-independent cancer (DU145). We then analyzed <it>in vitro </it>and <it>in vivo </it>tumorigenicity as well as the effect of Pim1 overexpression on c-MYC transcriptional activity by reporter assays and gene expression profiling using an inducible MYC-ER system. To validate that Pim1 induces tumorigenicity and target gene expression by modulating c-MYC transcriptional activity, we inhibited c-MYC using a small molecule inhibitor (10058-F4) or RNA interference.</p> <p>Results</p> <p>Overexpression of Pim1 alone was not sufficient to convert the benign RWPE1 cell to malignancy although it enhanced their proliferation rates when grown as xenografts <it>in vivo</it>. However, Pim1 expression enhanced the <it>in vitro </it>and <it>in vivo </it>tumorigenic potentials of the human prostate cancer cell lines LNCaP and DU145. Reporter assays revealed increased c-MYC transcriptional activity in Pim1-expressing cells and mRNA expression profiling demonstrated that a large fraction of c-MYC target genes were also regulated by Pim1 expression. The c-MYC inhibitor 10058-F4 suppressed the tumorigenicity of Pim1-expressing prostate cancer cells. Interestingly, 10058-F4 treatment also led to a reduction of Pim1 protein but not mRNA. Knocking-down c-MYC using short hairpin RNA reversed the effects of Pim1 on Pim1/MYC target genes.</p> <p>Conclusion</p> <p>Our results suggest an <it>in vivo </it>role of Pim1 in promoting prostate tumorigenesis although it displayed distinct oncogenic activities depending on the disease stage of the cell line. Pim1 promotes tumorigenicity at least in part by enhancing c-MYC transcriptional activity. We also made the novel discovery that treatment of cells with the c-MYC inhibitor 10058-F4 leads to a reduction in Pim1 protein levels.</p

Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

The ubiquitin-proteasome system is the primary proteolysis machine for controlling protein stability of the majority of regulatory proteins including those that are critical for cancer development. The forkhead box transcription factor FOXO3 plays a key role in regulating tumor suppression; however, the control of FOXO3 protein stability remains to be established. It is crucial to elucidate the molecular mechanisms underlying the ubiquitin-mediated degradation of FOXO3 tumor suppressor.Here we show that betaTrCP1 oncogenic ubiquitin E3-ligase interacts with FOXO3 and induces its ubiquitin-dependent degradation in an IkappaB kinase-beta phosphorylation dependent manner. Silencing betaTrCP1 augments FOXO3 protein level, resulting in promoting cellular apoptosis in cancer cells. In animal models, increasing FOXO3 protein level by silencing betaTrCP1 suppresses tumorigenesis, whereas decreasing FOXO3 by over-expressing betaTrCP1 promotes tumorigenesis and tumor growth in vivo.This is a unique demonstration that the betaTrCP1-mediated FOXO3 degradation plays a crucial role in tumorigenesis. These findings significantly contribute to understanding of the control of FOXO3 stability in cancer cells and may provide opportunities for developing innovative anticancer therapeutic modalities

A Collage of Small Planets from the Lick-Carnegie Exoplanet Survey: Exploring the Super-Earth and Sub-Neptune Mass Regime

Analysis of new precision radial velocity (RV) measurements from the Lick Automated Planet Finder and Keck HIRES has yielded the discovery of three new exoplanet candidates orbiting the nearby stars HD 190007 and HD 216520. We also report new velocities from the APF and the Planet Finder Spectrograph and updated orbital fits for the known exoplanet host stars GJ 686 and HD 180617. Of the newly discovered planets, HD 190007 b has a period of P = 11.72 days, an RV semiamplitude of K = 5.64 ± 0.55 m s-1, a minimum mass of M pl = 16.46 ± 1.66 M ⊕, and orbits the slightly metal-rich, active K4V star HD 190007. For HD 216520 b, we find P = 35.45 days, K = 2.28 ± 0.20 m s-1, and M pl = 10.26 ± 0.99 M ⊕, while for HD 216520 c, P = 154.43 days, K = 1.29 ± 0.22 m s-1, and M pl = 9.44 ± 1.63 M ⊕. Both planets orbit the slightly metal-poor, inactive K0V star HD 216520. Our updated best-fit models for HD 180617 b and GJ 686 b are in good agreement with the published results. For HD 180617 b, we obtain P = 105.91 days and M pl = 12.214 ± 1.05 M ⊕. For GJ 686 b, we find P = 15.53 days and M pl = 6.624 ± 0.432 M ⊕. Using an injection-recovery exercise, we find that HD 190007 b and HD 216520 b are unlikely to have additional planets with masses and orbital periods within a factor of 2, in marked contrast to ∼85% of planets in this mass and period range discovered by Kepler

A TNF-JNK-Axl-ERK signaling axis mediates primary resistance to EGFR inhibition in glioblastoma.

Aberrant epidermal growth factor receptor (EGFR) signaling is widespread in cancer, making the EGFR an important target for therapy. EGFR gene amplification and mutation are common in glioblastoma (GBM), but EGFR inhibition has not been effective in treating this tumor. Here we propose that primary resistance to EGFR inhibition in glioma cells results from a rapid compensatory response to EGFR inhibition that mediates cell survival. We show that in glioma cells expressing either EGFR wild type or the mutant EGFRvIII, EGFR inhibition triggers a rapid adaptive response driven by increased tumor necrosis factor (TNF) secretion, which leads to activation in turn of c-Jun N-terminal kinase (JNK), the Axl receptor tyrosine kinase and extracellular signal-regulated kinases (ERK). Inhibition of this adaptive axis at multiple nodes rendered glioma cells with primary resistance sensitive to EGFR inhibition. Our findings provide a possible explanation for the failures of anti-EGFR therapy in GBM and suggest a new approach to the treatment of EGFR-expressing GBM using a combination of EGFR and TNF inhibition

Developing a clinically relevant radiosensitizer for temozolomide-resistant gliomas.

The prognosis for patients with glioblastoma (GB) remains grim. Concurrent temozolomide (TMZ) radiation-the cornerstone of glioma control-extends the overall median survival of GB patients by only a few months over radiotherapy alone. While these survival gains could be partly attributed to radiosensitization, this benefit is greatly minimized in tumors expressing O6-methylguanine DNA methyltransferase (MGMT), which specifically reverses O6-methylguanine lesions. Theoretically, non-O6-methylguanine lesions (i.e., the N-methylpurine adducts), which represent up to 90% of TMZ-generated DNA adducts, could also contribute to radiosensitization. Unfortunately, at concentrations attainable in clinical practice, the alkylation capacity of TMZ cannot overwhelm the repair of N-methylpurine adducts to efficiently exploit these lesions. The current therapeutic application of TMZ therefore faces two main obstacles: (i) the stochastic presence of MGMT and (ii) a blunted radiosensitization potential at physiologic concentrations. To circumvent these limitations, we are developing a novel molecule called NEO212-a derivatization of TMZ generated by coupling TMZ to perillyl alcohol. Based on gas chromatography/mass spectrometry and high-performance liquid chromatography analyses, we determined that NEO212 had greater tumor cell uptake than TMZ. In mouse models, NEO212 was more efficient than TMZ at crossing the blood-brain barrier, preferentially accumulating in tumoral over normal brain tissue. Moreover, in vitro analyses with GB cell lines, including TMZ-resistant isogenic variants, revealed more potent cytotoxic and radiosensitizing activities for NEO212 at physiologic concentrations. Mechanistically, these advantages of NEO212 over TMZ could be attributed to its enhanced tumor uptake presumably leading to more extensive DNA alkylation at equivalent dosages which, ultimately, allows for N-methylpurine lesions to be better exploited for radiosensitization. This effect cannot be achieved with TMZ at clinically relevant concentrations and is independent of MGMT. Our findings establish NEO212 as a superior radiosensitizer and a potentially better alternative to TMZ for newly diagnosed GB patients, irrespective of their MGMT status