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.

Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons.

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.

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 K(m) of PERK for both its primary substrate, eIF2α, 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.

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

The amyloid ß precursor protein (APP) is a single-pass transmembrane glycoprotein that is ubiquitously expressed in many cell types, including neurons. Amyloidogenic processing of APP by ß- and γ-secretases leads to the production of amyloid-ß (Aß) 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 Aß production; however, it is not yet clear whether APP dimers increase or decrease Aß and the mechanism is not fully understood. To better understand the relationship between APP dimerization and production of Aß, 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 Aß generation. Two molecules that inhibited APP dimerization produced a reduction in Aß levels as measured by ELISA. The inhibitors did not change sAPPα or γ-CTF levels, but lowered sAPPß levels, suggesting that blocking the dimerization is preventing the cleavage by ß-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 Aß.

A high-throughput screen to identify inhibitors of SOD1 transcription.

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. Approximately 20 percent 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 more than 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.

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.

A high-throughput screening method for small-molecule inhibitors of the aberrant mutant SOD1 and dynein complex interaction.

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)-compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells.

Screening for inhibitors of the SOD1 gene promoter: pyrimethamine does not reduce SOD1 levels in cell and animal models.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are detected in 20% of familial and 3% of sporadic amyotrophic lateral sclerosis (ALS) cases. Although mutant SOD1 is known to induce motor neuron death via multiple adverse acquired functions, its exact pathogenic mechanism is not well defined. SOD1 toxicity is dose dependent; levels of mutant SOD1 protein in transgenic mice determine disease susceptibility, onset and rate of progression. We therefore sought to identify small molecules that reduce SOD1 levels by inhibiting the SOD1 promoter. We tested pyrimethamine (previously reported to suppress SOD1 expression), several compounds currently in trials in human and murine ALS, and a set of 1040 FDA-approved compounds. In a PC12 cell-based assay, no compounds reduced SOD1 promoter activity without concomitant cytotoxicity. Additionally, pyrimethamine failed to repress levels of SOD1 protein in HeLa cells or homogenates of liver, spinal cord and brain of wild-type mice. Thirty-four compounds (including riluzole, ceftriaxone, minocyclin, PBA, lithium, acetylcysteine) in human and mouse ALS trials and an additional set of 1040 FDA-approved compounds also showed no effect on SOD1 promoter activity. This present study thus failed to identify small molecule inhibitors of SOD1 gene expression.

Further characterization of BC3H1 myogenic cells reveals lack of p53 activity and underexpression of several p53 regulated and extracellular matrix-associated gene products.

To catalog factors that may contribute to the completion of myogenesis, we have been looking for molecular differences between BC3H1 and C2C12 cells. Cells of the BC3H1 tumor line, though myogenic, are nonfusing, and withdraw from the cell cycle only reversibly, whereas cells of the C2C12 line fuse, differentiate terminally, and express several muscle-specific gene products that BC3H1 cells do not. Relative to C2C12 cells, BC3H1 cells underaccumulated cyclin-dependent kinase inhibitor p21 and underaccumulated transcripts for p21, GADD45, CDO, decorin, osteopontin, H19, fibronectin, and thrombospondin-1 (tsp-1). Levels of accumulation of H19, tsp-1, and larger isoforms of fibronectin messenger ribonucleic acid (mRNA) were found to increase in response to expression of myogenic regulatory factors as shown by their accumulation in differentiated myogenically converted 10T1/2 cells but not in 10T1/2 fibroblasts. BC3H1s accumulated a temperature-insensitive, geldanamycin-sensitive, misfolded form of p53 incapable of transactivating a p53 responsive reporter, consistent with underexpression of p21, GADD45, and tsp-1. BC3H1 and C2C12 cells were similar with respect to upregulation of p27 protein, downregulation of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein, upregulation of retinoblastoma (Rb) mRNA, and nuclear localization of hypophosphorylated Rb. Cells of both lines expressed the muscle-specific 1b isoform of MEF2D. Although nonfusing in the short term, after more than 18 d in differentiation medium, some cultures of BC3H1 cells formed viable multinucleated cells in which the nuclei did not reinitiate synthesis of DNA in response to serum. Our findings suggest participation of tsp-1 and specific isoforms of fibronectin in myogenesis and suggest additional avenues of research in myogenesis and oncogenesis.