Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death.

Parkinson's disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z' factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.

Identification of a small molecule SIRT2 inhibitor with selective tumor cytotoxicity.

As a member of the class III histone deacetylases, Sirtuin-2 (SIRT2) is critical in cell cycle regulation which makes it a potential target for cancer therapeutics. In this study, we identified a novel SIRT2 inhibitor, AC-93253, with IC(50) of 6 microM in vitro. The compound is selective, inhibiting SIRT2 7.5- and 4-fold more potently than the closely related SIRT1 and SIRT3, respectively. AC-93253 significantly enhanced acetylation of tubulin, p53, and histone H4, confirming SIRT2 and SIRT1 as its cellular targets. AC-93253 as a single agent exhibited submicromolar selective cytotoxicity towards all four tumor cell lines tested with a therapeutic window up to 200-fold, comparing to any of the three normal cell types tested. Results from high content analysis suggested that AC-93253 significantly triggered apoptosis. Taken together, SIRT2 selective inhibitor AC-93253 may serve as a novel chemical scaffold for structure-activity relationship study and future lead development.

Identification of small-molecule HSF1 amplifiers by high content screening in protection of cells from stress induced injury.

Small molecule amplifiers of heat shock response have shown promising results in rescuing stress related injury through chaperone amplification. Herein, we report the results of a high content target-based primary screening of several known bioactive libraries. Screening resulted in the identification of three potent gedunin derivatives and a sappanone A derivative. Western blot results confirmed compound-induced activation of HSF1 and increased expression level of HSP70. These compounds rescued cells from cell death caused by proteasome inhibitor MG-132 and RNAi knockdown of HSF1 significantly reversed the cytoprotective effects, confirming an HSF1-dependent mechanism of action. These HSF1 amplifiers were tested in two mammalian cell based models of Huntington's disease (HD) and found to improve survival. Therefore, these screening hits may have therapeutic potential for HD and possibly other protein conformational disorders.

Pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione derivatives as novel small molecule chaperone amplifiers.

Pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione derivatives were investigated as novel small molecule amplifiers of heat shock factor 1 transcriptional activity. Lead optimization led to the discovery of compound 4A-13, which displayed potent HSF1 activity under mild heat stress (EC(50)=2.5microM) and significant cytoprotection in both rotenone (EC(50)=0.23microM) and oxygen-glucose deprivation cell toxicity models (80% protection at 2.5microM).

Pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione derivatives: their cytoprotection effect from rotenone toxicity and preliminary DMPK properties.

Pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione derivatives exhibited potent cytoprotective effect from rotenone toxicity. Lead optimization focused on the CC50/EC50 ratio and DMPK properties led to the overall improvement of the compound profile of this series with high CC50/EC50 ratio (92 for 1f), good metabolic stability in rat microsomes and medium to high aqueous solubility.

Chloro-oxime derivatives as novel small molecule chaperone amplifiers.

Chloro-oxime derivatives were investigated as novel small molecule chaperone amplifiers. Lead optimization led to the discovery of compounds that displayed potent HSF1 activation activity, significant cytoprotection in MG-132 stress, ER stress and PolyQ stress cell models (EC(50)<10 microM).

High-content image-based screening for small-molecule chaperone amplifiers in heat shock.

Heat shock proteins represent the major elements of the cellular stress response that protects cells from diseases caused by protein misfolding. Small-molecule amplifiers of heat shock proteins have shown promising results in several animal models, demonstrating the potential importance of such compounds for therapeutics. The expression of many heat shock proteins is controlled by HSF1, which forms stress granules in the nucleus when transcriptionally activated. Activation of the cellular stress also correlates with the translocation of HSP70 into nucleoli. The authors have developed an image-based, multiparametric assay to simultaneously monitor the effects of compounds on HSF1/HSP70 stress granule formation in heat-shocked Hela cells. High-content screening of the compound library was performed with a Z' of 0.62, demonstrating a highly robust assay for large-scale screening. The resulting hits showed prolonged amplification of HSP70 induction in heat-stressed cells but no effects in cells without stress. Treatment of cells with selected hits exhibited significant cytoprotection from both oxygen glucose deprivation and rotenone-induced stresses. Thus, high-content screening of HSF1/HSP70 amplifiers provides a practical opportunity for clinical therapeutics targeting protein misfolding diseases.

Ribozyme to proliferating cell nuclear antigen to treat proliferative vitreoretinopathy.

PURPOSE: A DNA-RNA chimeric ribozyme was developed that targets the mRNA of a cell cycle regulatory protein, proliferating cell nuclear antigen (PCNA). The hypothesis was that inhibition of PCNA, essential in DNA replication, would decrease the proliferation of cells that are involved in formation of granuloma after surgical procedures in the eye. The ability of intravitreous injection of this ribozyme to prevent or inhibit development of proliferative vitreoretinopathy (PVR) was tested in a dispase-induced rabbit PVR model. METHODS: Rabbit genomic DNA encoding PCNA was cloned and sequenced. The cleavage of rabbit PCNA by the chimeric ribozyme was tested in vitro. Delivery of the ribozyme to rabbit retinal pigment epithelial (RPE) or fibroblast cells and its effects on proliferation of fibroblasts were examined. The stability of the ribozyme in vitreous fluid and serum was studied as well. In the dispase-induced rabbit model of PVR, the ability of the PCNA ribozyme to prevent or inhibit development of PVR and retinal detachment (RD) was tested. Experimental groups receiving intravitreous PCNA ribozyme, with or without a lipid vehicle, were compared with sham-treated control groups. Progression of PVR in rabbit eyes was followed by indirect ophthalmic examination and observations documented by fundoscopic photography, gross pathology, and histopathology. RESULTS: The chimeric ribozyme targeted a specific sequence in the rabbit PCNA that was identical with that in the human. In vitro cleavage assays confirmed the ability of the ribozyme to cleave the mRNA of PCNA. The catalytic efficiency in vitro, calculated as k(2)/K(m)(app), was 0.26 microM(-1) x min(-1). In vitro studies with fluoresceinated ribozyme indicated that lipid vehicles facilitated delivery of the ribozyme into cells causative of PVR (RPE and fibroblasts); however, the PCNA ribozyme decreased the proliferation of fibroblasts, with or without lipid vehicle. The ribozyme displayed good stability in vitreous fluid, whereas, it degraded quite rapidly in serum. In animal experiments, rabbits in sham-treated groups usually exhibited development of severe PVR characterized by focal traction or RD. Animals in the PCNA ribozyme-treated groups usually did not exhibit an RD. If they did have RD, it was small and localized, or focal tractions developed that did not progress to the degree that the sham-treated animal eyes did over the follow-up period. The in vivo use of a lipid delivery vehicle resulted in a precipitate; however, an effective naked ribozyme dose was identified that did not cause this side effect. CONCLUSIONS: In addition to validating the newly developed dispase PVR rabbit model, the results indicate that ribozyme targeted against the cell cycle agent PCNA is efficacious in the treatment or prevention of PVR in the rabbit eye. These experiments suggest that chimeric ribozyme targeted against PCNA may have a therapeutic or preventative role in humans.

Identification of chaulmoogric acid as a small molecule activator of protein phosphatase 5.

Protein phosphatase 5 (PP5) is an important protein phosphatase that is abundantly expressed in the central nervous system. Recent studies showed that PP5 activity in the neocortex from patients with Alzheimer's disease (AD) is decreased significantly, suggesting that small molecule PP5 activator may have therapeutic potential for AD. We performed a biochemical screening for PP5 activators with the microsource compound library. Chaulmoogric acid was identified to be an effective activator with EC(50) value of 134.5 microM. Importantly, results from circular dichroism (CD) and limited proteolysis study showed that chaulmoogric acid binds to a region of tetratricopeptide repeat (TPR) domain of PP5 resulting in complete loss of helical contents. These results demonstrate a different mechanism of action from that of arachidonic acid, a known activator for PP5 dephosphorylation activity. Synergistic activation of PP5 enzymatic activity was also observed with combined application of both compounds at relatively low concentrations. Therefore, further structure activity relationship study of chaulmoogric acid may facilitate the discovery of small molecules that can synergize with endogenous arachidonic acid for PP5 activation.

Identification of inhibitors of HSF1 functional activity by high-content target-based screening.

Cancer cells are known to experience a high level of stress and may require constant repair for survival and proliferation. Recent studies showed that inhibition of heat shock factor 1 (HSF1), the key regulator for the stress-activated transcription of heat shock protein (HSP), can reduce the tumorigenic potential of cancer cells. Such a "nononcogene addiction" phenomenon makes HSF1 an attractive cancer drug target. Here, the authors report an image-based high-content screening (HCS) assay for HSF1 functional inhibitors. A heat shock-based methodology was used to stimulate the stress response followed by quantitative measurement of HSF1/HSP70 granules for compound-induced inhibitory effects. The authors discovered a small molecule from a compound library that inhibits HSF1 granule formation substantially in heat-shocked HeLa cells with IC(50) at 80 nM. Electorphoretic mobility shift of HSF1 by this compound suggested significant inhibition of HSF1 phosphorylation, accompanied by reduced expression levels of HSP70 and HSP90 after heat induction. Importantly, HeLa cells stably transfected with HSF1 shRNA were more resistant to the compound treatment under lethal temperature than cells containing HSF1, further validating an HSF1-dependent mechanism of action. The HCS assay the authors developed was robust with a Z' factor of 0.65 in a 384-well plate format, providing a valuable method for identifying small-molecule functional inhibitors of HSF1 for potential cancer treatment.

Arimoclomol at dosages up to 300 mg/day is well tolerated and safe in amyotrophic lateral sclerosis.

Arimoclomol is an investigational drug for amyotrophic lateral sclerosis (ALS) that amplifies heat shock protein gene expression during cell stress. The objectives of the present study were to assess the safety, tolerability, and pharmacokinetics of arimoclomol in ALS. Eighty-four participants with ALS received arimoclomol at one of three oral doses (25, 50, or 100 mg three times daily) or placebo. The primary outcome measure was safety and tolerability. A subset of 44 participants provided serum and cerebrospinal fluid (CSF) samples for pharmacokinetic analysis. Participants who completed 12 weeks of treatment could enroll in a 6-month open-label study. Arimoclomol at doses up to 300 mg/day was well tolerated and safe. Arimoclomol resulted in dose-linear pharmacologic exposures and the half-life did not change with continued treatment. Arimoclomol CSF levels increased with dose. Arimoclomol was shown to be safe, and it crosses the blood-brain barrier. Serum pharmacokinetic profiles support dosing of three times per day. An efficacy study in ALS is planned.