Activating transcription factor 4 regulates hypoxia inducible factor 1α in chronic hypoxia in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and difficult to treat cancers with tumors typically exhibiting high levels of chronic hypoxia. Hypoxia activates hypoxia-inducible factors (HIFs) that mediate cellular responses to adapt to low oxygen environments. Hypoxia also causes endoplasmic reticulum (ER) stress, increasing activating transcription factor 4 (ATF4), a master regulator of the unfolded protein response (UPR) pathway that mediates cellular response to ER stress. ATF4 is overexpressed in PDAC and is associated with poor prognoses. While ATF4 promotes cell proliferation and tumorigenesis, most studies have been conducted under normoxia or acute hypoxia. The functions of ATF4 in chronic hypoxia remain largely unexplored. Using siRNA knockdown experiments of healthy skin fibroblast cells WS1 and PDAC cell lines PANC-1 and Mia-PaCa2 to analyze mRNA and protein expression levels, a novel ATF4 function was identified, in which it decreases HIF2α mRNA and increases HIF1α mRNA in chronic hypoxia while having no effect in acute hypoxia. A scratch assay was used to show that ATF4 decreases cell migration in chronic hypoxia as opposed to the increase in cell migration ATF4 imparts in acute hypoxia. Colony formation assay and cell viability assay showed that ATF4 promotes colony formation and cell viability in both chronic and acute hypoxia. In addition to the differential response of ATF4 in chronic hypoxia compared with acute hypoxia, this is the first time ATF4 has been implicated in regulation of response to hypoxia via interaction with HIF proteins in PDAC.

JOTROL, a Novel Formulation of Resveratrol, Shows Beneficial Effects in the 3xTg-AD Mouse Model.

BACKGROUND: Alzheimer's disease (AD) has minimally effective treatments currently. High concentrations of resveratrol, a polyphenol antioxidant found in plants, have been reported to affect several AD-related and neuroprotective genes. To address the low bioavailability of resveratrol, we investigated a novel oral formulation of resveratrol, JOTROL™, that has shown increased pharmacokinetic properties compared to non-formulated resveratrol in animals and in humans. OBJECTIVE: We hypothesized that equivalent doses of JOTROL, compared to non-formulated resveratrol, would result in greater brain exposure to resveratrol, and more efficacious responses on AD biomarkers. METHODS: For sub-chronic reversal studies, 15-month-old male triple transgenic (APPSW/PS1M146V/TauP301L; 3xTg-AD) AD mice were treated orally with vehicle or 50 mg/kg JOTROL for 36 days. For prophylactic studies, male and female 3xTg-AD mice were similarly administered vehicle, 50 mg/kg JOTROL, or 50 mg/kg resveratrol for 9 months starting at 4 months of age. A behavioral battery was run, and mRNA and protein from brain and blood were analyzed for changes in AD-related gene and protein expression. RESULTS: JOTROL displays significantly increased bioavailability over non-formulated resveratrol. Treatment with JOTROL resulted in AD-related gene expression changes (Adam10, Bace1, Bdnf, Psen1) some of which were brain region-dependent and sex-specific, as well as changes in inflammatory gene and cytokine levels. CONCLUSION: JOTROL may be effective as a prophylaxis and/or treatment for AD through increased expression and/or activation of neuroprotective genes, suppression of pro-inflammatory genes, and regulation of central and peripheral cytokine levels.

A novel strategy for combination of clofarabine and pictilisib is synergistic in gastric cancer.

Gastric cancer (GC) is frequently characterized by resistance to standard chemotherapeutic regimens and poor clinical outcomes. We aimed to identify a novel therapeutic approach using drug sensitivity testing (DST) and our computational SynerySeq pipeline. DST of GC cell lines was performed with a library of 215 Federal Drug Administration (FDA) approved compounds and identified clofarabine as a potential therapeutic agent. RNA-sequencing (RNAseq) of clofarabine treated GC cells was analyzed according to our SynergySeq pipeline and identified pictilisib as a potential synergistic agent. Clonogenic survival and Annexin V assays demonstrated increased cell death with clofarabine and pictilisib combination treatment (P<0.01). The combination induced double strand breaks (DSB) as indicated by phosphorylated H2A histone family member X (γH2AX) immunofluorescence and western blot analysis (P<0.01). Pictilisib treatment inhibited the protein kinase B (AKT) cell survival pathway and promoted a pro-apoptotic phenotype as evidenced by quantitative real time polymerase chain reaction (qRT-PCR) analysis of the B-cell lymphoma 2 (BCL2) protein family members (P<0.01). Patient derived xenograft (PDX) data confirmed that the combination is more effective in abrogating tumor growth with prolonged survival than single-agent treatment (P<0.01). The novel combination of clofarabine and pictilisib in GC promotes DNA damage and inhibits key cell survival pathways to induce cell death beyond single-agent treatment.

Dual Screen for Efficacy and Toxicity Identifies HDAC Inhibitor with Distinctive Activity Spectrum for BAP1-Mutant Uveal Melanoma.

Drug screens leading to successful targeted therapies in cancer have been mainly based on cell viability assays identifying inhibitors of dominantly acting oncogenes. In contrast, there has been little success in discovering targeted therapies that reverse the effects of inactivating mutations in tumor-suppressor genes. BAP1 is one such tumor suppressor that is frequently inactivated in a variety of cancers, including uveal melanoma, renal cell carcinoma, and mesothelioma. Because BAP1 is an epigenetic transcriptional regulator of developmental genes, we designed a two-phase drug screen involving a cell-based rescue screen of transcriptional repression caused by BAP1 loss, followed by an in vivo screen of lead compounds for rescue of a BAP1-deficient phenotype with minimal toxicity in Xenopus embryos. The first screen identified 9 compounds, 8 of which were HDAC inhibitors. The second screen eliminated all except one compound due to inefficacy or toxicity. The resulting lead compound, quisinostat, has a distinctive activity spectrum, including high potency against HDAC4, which was recently shown to be a key target of BAP1. Quisinostat was further validated in a mouse model and found to prevent the growth of BAP1-mutant uveal melanomas. This innovative strategy demonstrates the potential for identifying therapeutic compounds that target tumor-suppressor mutations in cancer. IMPLICATIONS: Few drugs have been identified that target mutations in tumor suppressors. Using a novel 2-step screening approach, strategy, we identified quisinostat as a candidate for therapy in BAP1-mutant uveal melanoma. HDAC4 is implicated as a key target in uveal melanoma and perhaps other BAP1-mutant cancers.

Pathogenesis and Treatment of Pancreatic Cancer Related Pain.

Pancreatic cancer is often diagnosed due to the patient seeking medical attention for abdominal pain. It is among the most painful cancers, with pain severity strongly correlating with prognosis. Perineural invasion is a prominent feature of pancreatic cancer and often the first route of metastasis resulting in neuropathic pain. While surgical pain is present, it is generally short-lived; chemo- and radio-therapy associated side effect pain is often longer lasting and more difficult to manage. Treatment-induced mucositis in response to chemotherapy occurs throughout the GI tract resulting in infection-prone ulcers on the lip, buccal mucosa, palate or tongue. Cisplatin treatment is associated with axonal neuropathy in the dorsal root ganglion, although other large sensory fibers can be affected. Opioid-induced hyperalgesia can also emerge in patients. Along with traditional means to address pain, neurolytic celiac plexus block of afferent nociceptive fibers has been reported to be effective in 74% of patients. Moreover, as cancer treatments become more effective and result in improved survival, treatment-related side effects become more prevalent. Here, pancreatic cancer and treatment associated pain are reviewed along with current treatment strategies. Potential future therapeutic strategies to target the pathophysiology underlying pancreatic cancer and pain induction are also presented.

Ovarian Cancer Treatment Stratification Using Ex Vivo Drug Sensitivity Testing.

BACKGROUND: Treatment options for patients with platinum-resistant ovarian cancer are generally palliative in nature and rarely have realistic potential to be curative. Because many patients with recurrent ovarian cancer receive aggressive chemotherapy for prolonged periods, sometimes continuously, therapy-related toxicities are a major factor in treatment decisions. The use of ex vivo drug sensitivity screens has the potential to improve the treatment of patients with platinum-resistant ovarian cancer by providing personalized treatment plans and thus reducing toxicity from unproductive therapy attempts. MATERIALS AND METHODS: We evaluated the treatment responses of a set of six early-passage patient-derived ovarian cancer cell lines towards a set of 30 Food and Drug Administration-approved chemotherapy drugs using drug-sensitivity testing. RESULTS: We observed a wide range of treatment responses of the cell lines. While most compounds displayed vastly different treatment responses between cell lines, we found that some compounds such as docetaxel and cephalomannine reduced cell survival of all cell lines. CONCLUSION: We propose that ex vivo drug-sensitivity screening holds the potential to greatly improve patient outcomes, especially in a population where multiple continuous treatments are not an option due to advanced disease, rapid disease progression, age or poor overall health. This approach may also be useful to identify potential novel therapeutics for patients with ovarian cancer.

Vitamin C supplementation expands the therapeutic window of BETi for triple negative breast cancer.

BACKGROUND: Bromodomain and extra-terminal inhibitors (BETi) have shown efficacy for the treatment of aggressive triple negative breast cancer (TNBC). However, BETi are plagued by a narrow therapeutic window as manifested by severe toxicities at effective doses. Therefore, it is a limitation to their clinical implementation in patient care. METHODS: The impact of vitamin C on the efficacy of small compounds including BETi was assessed by high-throughput screening. Co-treatment of TNBC by BETi especially JQ1 and vitamin C was evaluated in vitro and in vivo. FINDINGS: High-throughput screening revealed that vitamin C improves the efficacy of a number of structurally-unrelated BETi including JQ1, I-BET762, I-BET151, and CPI-203 in treating TNBC cells. The synergy between BETi and vitamin C is due to suppressed histone acetylation (H3ac and H4ac), which is in turn caused by upregulated histone deacetylase 1 (HDAC1) expression upon vitamin C addition. Treatment with JQ1 at lower doses together with vitamin C induces apoptosis and inhibits the clonogenic ability of cultured TNBC cells. Oral vitamin C supplementation renders a sub-therapeutic dose of JQ1 able to inhibit human TNBC xenograft growth and metastasis in mice. INTERPRETATION: Vitamin C expands the therapeutic window of BETi by sensitizing TNBC to BETi. Using vitamin C as a co-treatment, lower doses of BETi could be used to achieve an increased therapeutic index in patients, which will translate to a reduced side effect profile. FUND: University of Miami Sylvester Comprehensive Cancer Center, Bankhead Coley Cancer Research program (7BC10), Flight Attendant Medical Research Institute, and NIH R21CA191668 (to GW) and 1R56AG061911 (to CW and CHV).

mRNA-to-protein translation in hypoxia.

Cells respond to hypoxia by shifting cellular processes from general housekeeping functions to activating specialized hypoxia-response pathways. Oxygen plays an important role in generating ATP to maintain a productive rate of protein synthesis in normoxia. In hypoxia, the rate of the canonical protein synthesis pathway is significantly slowed and impaired due to limited ATP availability, necessitating an alternative mechanism to mediate protein synthesis and facilitate adaptation. Hypoxia adaptation is largely mediated by hypoxia-inducible factors (HIFs). While HIFs are well known for their transcriptional functions, they also play imperative roles in translation to mediate hypoxic protein synthesis. Such adaptations to hypoxia are often hyperactive in solid tumors, contributing to the expression of cancer hallmarks, including treatment resistance. The current literature on protein synthesis in hypoxia is reviewed here, inclusive of hypoxia-specific mRNA selection to translation termination. Current HIF targeting therapies are also discussed as are the opportunities involved with targeting hypoxia specific protein synthesis pathways.

HDAC Inhibitors Induce BDNF Expression and Promote Neurite Outgrowth in Human Neural Progenitor Cells-Derived Neurons.

Besides its key role in neural development, brain-derived neurotrophic factor (BDNF) is important for long-term potentiation and neurogenesis, which makes it a critical factor in learning and memory. Due to the important role of BDNF in synaptic function and plasticity, an in-house epigenetic library was screened against human neural progenitor cells (HNPCs) and WS1 human skin fibroblast cells using Cell-to-Ct assay kit to identify the small compounds capable of modulating the BDNF expression. In addition to two well-known hydroxamic acid-based histone deacetylase inhibitors (hb-HDACis), SAHA and TSA, several structurally similar HDAC inhibitors including SB-939, PCI-24781 and JNJ-26481585 with even higher impact on BDNF expression, were discovered in this study. Furthermore, by using well-developed immunohistochemistry assays, the selected compounds were also proved to have neurogenic potential improving the neurite outgrowth in HNPCs-derived neurons. In conclusion, we proved the neurogenic potential of several hb-HDACis, alongside their ability to enhance BDNF expression, which by modulating the neurogenesis and/or compensating for neuronal loss, could be propitious for treatment of neurological disorders.

Inhibition of HDAC3 reverses Alzheimer's disease-related pathologies in vitro and in the 3xTg-AD mouse model.

Alzheimer's disease (AD) is the leading cause of age-related dementia. Neuropathological hallmarks of AD include brain deposition of ß-amyloid (Aß) plaques and accumulation of both hyperphosphorylated and acetylated tau. RGFP-966, a brain-penetrant and selective HDAC3 inhibitor, or HDAC3 silencing, increases BDNF expression, increases histone H3 and H4 acetylation, decreases tau phosphorylation and tau acetylation at disease-associated sites, reduces ß-secretase cleavage of the amyloid precursor protein (APP), and decreases Aß1-42 accumulation in HEK-293 cells overexpressing APP with the double Swedish mutation (HEK/APPsw). In the triple transgenic AD mouse model (3xTg-AD), repeated administration of 3 and 10 mg/kg of RGFP-966 reverses pathological tau phosphorylation at Thr181, Ser202, and Ser396, increases levels of the Aß degrading enzyme Neprilysin in plasma, decreases Aß1-42 protein levels in the brain and periphery, and improves spatial learning and memory. Finally, we show that RGFP-966 decreases Aß1-42 accumulation and both tau acetylation and phosphorylation at disease residues in neurons derived from induced pluripotent stem cells obtained from APOEε4-carrying AD patients. These data indicate that HDAC3 plays an important regulatory role in the expression and regulation of proteins associated with AD pathophysiology, supporting the notion that HDAC3 may be a disease-modifying therapeutic target.

Naturally occurring compounds as pancreatic cancer therapeutics.

Naturally occurring small molecule compounds have long been in the spotlight of pancreatic cancer research as potential therapeutics to prevent cancer progression and sensitize chemoresistant tumors. The hope is that terminal pancreatic cancer patients receiving aggressive chemotherapy can benefit from an increase in treatment efficacy without adding further toxicity by way of utilizing natural compounds. While preclinical studies on a number of natural compounds, such as resveratrol, curcumin, rapalogs and cannabinoids, show promising preclinical results, little has translated into clinical practice, though a number of other compounds hold clinical potential. Nevertheless, recent advances in compound formulation may increase the clinical utility of these compounds.

Ex vivo drug sensitivity testing as a means for drug repurposing in esophageal adenocarcinoma.

BACKGROUND: Esophageal cancer remains one of the hardest cancers to treat with rising incidence rates, low overall survival and high levels of treatment resistance. The lack of clinically available biomarkers hinder diagnosis and treatment stratification. While large scale sequencing approaches have uncovered a number of molecular makers, little has translated in the routine treatment of esophageal cancer patients. MATERIAL AND METHODS: We evaluate the treatment response towards a panel of 215 FDA-approved and 163 epigenetic compounds of 4 established and 2 patient-derived esophageal cancer cell lines. Cell viability was evaluated after 72h of treatment using cell titer glow. The drug sensitivity testing results for gemcitabine and cisplatin were validated using clonogenic assays. RESULTS: The tested cell lines display different drug sensitivity profiles, although we found compounds that display efficacy in all of the tested established or patient-derived cell lines. Clonogenic assays confirmed the validity of the drug sensitivity testing results. Using the epigenetic library, we observed high sensitivity towards a number of epigenetic modifiers. DISCUSSION: Ex vivo drug sensitivity testing may present a viable option for the treatment stratification of esophageal cancer patients and holds the potential to greatly improve patient outcome while reducing treatment toxicity.

EZH1 is an antipsychotic-sensitive epigenetic modulator of social and motivational behavior that is dysregulated in schizophrenia.

BACKGROUND: With the capacity to modulate gene networks in an environmentally-sensitive manner, the role of epigenetic systems in mental disorders has come under intense investigation. Dysregulation of epigenetic effectors, including microRNAs and histone-modifying enzymes, may better explain the role of environmental risk factors and the observed heritability rate that cannot be fully attributed to known genetic risk alleles. Here, we aimed to identify novel epigenetic targets of the schizophrenia-associated microRNA 132 (miR-132). METHODS: Histone modifications were quantified by immunodetection in response to viral-mediated overexpression of miR-132 while a luminescent reporter system was used to validate targets of miR-132 in vitro. Genome-wide profiling, quantitative PCR and NanoSting were used to quantify gene expression in post-mortem human brains, neuronal cultures and prefrontal cortex (PFC) of mice chronically exposed to antipsychotics. Following viral-mediated depletion of Enhancer of Zeste 1 (EZH1) in the murine PFC, behaviors including sociability and motivation were assessed using a 3-chambered apparatus and forced-swim test, respectively. RESULTS: Overexpression of miR-132 decreased global histone 3 lysine 27 tri-methylation (H3K27me3), a repressive epigenetic mark. Moreover, the polycomb-associated H3K27 methyltransferase, EZH1, is regulated by miR-132 and upregulated in the PFC of schizophrenics. Unlike its homolog EZH2, expression of EZH1 in the murine PFC decreased following chronic exposure to antipsychotics. Viral-mediated depletion of EZH1 in the mouse PFC attenuated sociability, enhanced motivational behaviors, and affected gene expression pathways related to neurotransmission and behavioral phenotypes. CONCLUSIONS: EZH1 is dysregulated in schizophrenia, sensitive to antipsychotic medications, and a brain-enriched miR-132 target that controls neurobehavioral phenotypes.

Precision medicine in the treatment stratification of AML patients: challenges and progress.

Recent advances in high throughput technologies have led to the generation of vast amounts of clinical data and the development of personalized medicine approaches in acute myeloid leukemia (AML). The ability to treat cancer patients based upon their individual molecular characteristics or drug sensitivity profiles is expected to significantly advance cancer treatment and improve the long-term survival of patients with refractory AML, for whom current treatment options are restricted to palliative approaches. The clinical development of omics-based and phenotypic screens, however, is limited by a number of bottlenecks including the generation of cost-effective high-throughput data, data interpretation and integration of multiple approaches, sample availability, clinically relevant timelines, and the development and education of multidisciplinary teams. Recently, a number of small clinical trials have shown survival benefits in patients treated based on personalized medicine approaches. While these preliminary studies are encouraging, larger trials are needed to evaluate the utility of these technologies in routine clinical settings.

BET Bromodomain Inhibitors Which Permit Treg Function Enable a Combinatorial Strategy to Suppress GVHD in Pre-clinical Allogeneic HSCT.

A recent approach for limiting production of pro-inflammatory cytokines has been to target bromodomain and extra-terminal (BET) proteins. These epigenetic readers of histone acetylation regulate transcription of genes involved in inflammation, cardiovascular disease, and cancer. Development of BET inhibitors (BETi) has generated enormous interest for their therapeutic potential. Because inflammatory signals and donor T cells promote graft-versus-host disease (GVHD), regulating both pathways could be effective to abrogate this disorder. The objective of the present study was to identify a BETi which did not interfere in vivo with CD4+FoxP3+ regulatory T cell (Treg) expansion and function to utilize together with Tregs following allogeneic hematopoietic stem cell transplantation (aHSCT) to ameliorate GVHD. We have reported that Tregs can be markedly expanded and selectively activated with increased functional capacity by targeting TNFRSF25 and CD25 with TL1A-Ig and low dose IL-2, respectively. Here, mice were treated over 7 days (TL1A-Ig + IL-2) together with BETi. We found that the BETi EP11313 did not decrease frequency/numbers or phenotype of expanded Tregs as well as effector molecules, such as IL-10 and TGF-ß. However, BETi JQ1 interfered with Treg expansion and altered subset distribution and phenotype. Notably, in Treg expanded mice, EP11313 diminished tnfa and ifng but not il-2 RNA levels. Remarkably, Treg pSTAT5 expression was not affected by EP11313 supporting the notion that Treg IL-2 signaling remained intact. MHC-mismatched aHSCT (B6 → BALB/c) was performed using in vivo expanded donor Tregs with or without EP11313 short-term treatment in the recipient. Early post-transplant, improvement in the splenic and LN CD4/CD8 ratio along with fewer effector cells and high Treg levels in aHSCT recipients treated with expanded Tregs + EP11313 was detected. Interestingly, this group exhibited a significant diminution of GVHD clinical score with less skin and ocular involvement. Finally, using low numbers of highly purified expanded Tregs, improved clinical GVHD scores were observed in EP11313 treated recipients. In total, we conclude that use of this novel combinatorial strategy can suppress pre-clinical GVHD and posit, in vivo EP11313 treatment might be useful combined with Treg expansion therapy for treatment of diseases involving inflammatory responses.

Ex-vivo sensitivity profiling to guide clinical decision making in acute myeloid leukemia: A pilot study.

A precision medicine approach is appealing for use in AML due to ease of access to tumor samples and the significant variability in the patients' response to treatment. Attempts to establish a precision medicine platform for AML, however, have been unsuccessful, at least in part due to the use of small compound panels and having relatively slow turn over rates, which restricts the scope of treatment and delays its onset. For this pilot study, we evaluated a cohort of 12 patients with refractory AML using an ex vivo drug sensitivity testing (DST) platform. Purified AML blasts were screened with a panel of 215 FDA-approved compounds and treatment response was evaluated after 72h of exposure. Drug sensitivity scoring was reported to the treating physician, and patients were then treated with either DST- or non-DST guided therapy. We observed survival benefit of DST-guided therapy as compared to the survival of patients treated according to physician recommendation. Three out of four DST-treated patients displayed treatment response, while all of the non-DST-guided patients progressed during treatment. DST rapidly and effectively provides personalized treatment recommendations for patients with refractory AML.

Vitamin C Sensitizes Melanoma to BET Inhibitors.

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in Gulo-/- mice diminished the treatment outcome of JQ1 for melanoma tumorgraft. In contrast, ascorbate supplementation lowered the effective dose of JQ1 needed to successfully inhibit melanoma tumors in mice. On the basis of our findings, future clinical trials with BETi should consider ascorbate levels in patients. Furthermore, ascorbate supplementation might help reduce the severe side effects that arise from BETi therapy by reducing the dosage necessary for treatment.Significance: This study shows that ascorbate can enhance the efficacy of BET inhibitors, providing a possible clinical solution to challenges arising in phase I trials from the dose-dependent side effects of this class of epigenetic therapy. Cancer Res; 78(2); 572-83. ©2017 AACR.

M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer's disease genes and improving memory.

Alzheimer's disease (AD) comprises multifactorial ailments for which current therapeutic strategies remain insufficient to broadly address the underlying pathophysiology. Epigenetic gene regulation relies upon multifactorial processes that regulate multiple gene and protein pathways, including those involved in AD. We therefore took an epigenetic approach where a single drug would simultaneously affect the expression of a number of defined AD-related targets. We show that the small-molecule histone deacetylase inhibitor M344 reduces beta-amyloid (Aß), reduces tau Ser396 phosphorylation, and decreases both ß-secretase (BACE) and APOEε4 gene expression. M344 increases the expression of AD-relevant genes: BDNF, α-secretase (ADAM10), MINT2, FE65, REST, SIRT1, BIN1, and ABCA7, among others. M344 increases sAPPα and CTFα APP metabolite production, both cleavage products of ADAM10, concordant with increased ADAM10 gene expression. M344 also increases levels of immature APP, supporting an effect on APP trafficking, concurrent with the observed increase in MINT2 and FE65, both shown to increase immature APP in the early secretory pathway. Chronic i.p. treatment of the triple transgenic (APPsw/PS1M146V/TauP301L) mice with M344, at doses as low as 3 mg/kg, significantly prevented cognitive decline evaluated by Y-maze spontaneous alternation, novel object recognition, and Barnes maze spatial memory tests. M344 displays short brain exposure, indicating that brief pulses of daily drug treatment may be sufficient for long-term efficacy. Together, these data show that M344 normalizes several disparate pathogenic pathways related to AD. M344 therefore serves as an example of how a multitargeting compound could be used to address the polygenic nature of multifactorial diseases.

Orphan diseases: state of the drug discovery art.

Since 1983 more than 300 drugs have been developed and approved for orphan diseases. However, considering the development of novel diagnosis tools, the number of rare diseases vastly outpaces therapeutic discovery. Academic centers and nonprofit institutes are now at the forefront of rare disease R&D, partnering with pharmaceutical companies when academic researchers discover novel drugs or targets for specific diseases, thus reducing the failure risk and cost for pharmaceutical companies. Considerable progress has occurred in the art of orphan drug discovery, and a symbiotic relationship now exists between pharmaceutical industry, academia, and philanthropists that provides a useful framework for orphan disease therapeutic discovery. Here, the current state-of-the-art of drug discovery for orphan diseases is reviewed. Current technological approaches and challenges for drug discovery are considered, some of which can present somewhat unique challenges and opportunities in orphan diseases, including the potential for personalized medicine, gene therapy, and phenotypic screening.