Undesirable Off-target Effects Research Articles

17β-Estradiol Eye Drops Protect the Retinal Ganglion Cell Layer and Preserve Visual Function in an in Vivo Model of Glaucoma

Neuroprotection in glaucoma as a curative strategy complementary to current therapies to lower intraocular pressure (IOP) is highly desirable. This study was designed to investigate neuroprotection by 17β-estradiol (E2) to prevent retinal ganglion cell (RGC) death in a glaucoma model of surgically elevated IOP in rats. We found that daily treatment with E2-containing eye drops resulted in significant E2 concentration in the retina with concomitant profound neuroprotective therapeutic benefits, even in the presence of continually elevated IOP. The number of apoptotic cells in the RGC layer was significantly decreased in the E2-treated group, when compared to the vehicle-treated controls. Deterioration in visual acuity in these animals was also markedly prevented. Using mass spectrometry-based proteomics, beneficial changes in the expression of several proteins implicated in the maintenance of retinal health were also found in the retina of E2-treated animals. On the other hand, systemic side effects could not be avoided with the eye drops, as confirmed by the measured high circulating estrogen levels and through the assessment of the uterus representing a typical hormone-sensitive peripheral organ. Collectively, the demonstrated significant neuroprotective effect of topical E2 in the selected animal model of glaucoma provides a clear rationale for further studies aiming at targeting E2 into the eye while avoiding systemic E2 exposure to diminish undesirable off-target side effects.

Abstract 5223: Elucidating the complex cross-talk between the MAPK and PIK3 pathways.

Abstract Targeted molecular inhibitors have emerged as a leading anti-cancer strategy; however, despite promising pre-clinical data, many targeted inhibitors induce undesirable off-target effects in the clinic. The large number of off-target effects associated with molecular inhibitors was recently termed the ‘‘whack a mole problem’’ because inhibiting one molecular target often unintentionally activates another molecule. It is increasingly clear that the high incidence of off-target effects associated with targeted inhibitors is related to the complex interactions and emergent behaviors inherent to the highly complex and dysregulated intracellular networks of cancer. Both the mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways are known to be dysregulated in cancer. In previous work, we and others have demonstrated that the MAPK pathway promotes motility, invasion, and angiogenic factors while the PI3K pathway plays an important role in controlling anchorage independent growth. In addition, the PI3K pathway plays an essential role in stimulating glucose metabolism and the Warburg effect. We hypothesize that robust interactions exist between these two pathways that influence efficacy and potentially also acquired resistance to targeted therapies. Using a combination of experimental and theoretical techniques, we developed a predictive network model linking growth factor signaling to the MAPK and PI3K pathways as well as to glucose metabolism. Specifically, we constructed a logic-based network of the cross-talk between MAPK and PI3K signaling that relied on a detailed literature survey to identify known molecular interactions as well as proposed interactions and regulatory feedback connections in the literature. We next performed a set of experiments using a normal-like breast epithelial cell line and a series of pathway specific inhibitors with and without growth factor stimulation to validate our model. Finally, we repeated these experiments using a diverse set of breast cancer cell lines and integrated this data to produce a series of cancer networks representative of different stages of breast cancer progression. Our model was able to recapitulate both our own experimental data and published data in the literature using a smaller subset of regulatory feedback mechanisms than we started with. Together, our results suggest that some proposed interactions and feedback mechanisms attributed to MAPK and PI3K cross-talk in the literature may not be valid. Citation Format: Megan E. Egbert, Michelle L. Wynn, Zhi Fen Wu, Santiago Schnell, Sofia D. Merajver. Elucidating the complex cross-talk between the MAPK and PIK3 pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5223. doi:10.1158/1538-7445.AM2013-5223

RNAi Therapeutics in Autoimmune Disease

Since the discovery of RNA interference (RNAi), excitement has grown over its potential therapeutic uses. Targeting RNAi pathways provides a powerful tool to change biological processes post-transcriptionally in various health conditions such as cancer or autoimmune diseases. Optimum design of shRNA, siRNA, and miRNA enhances stability and specificity of RNAi-based approaches whereas it has to reduce or prevent undesirable immune responses or off-target effects. Recent advances in understanding pathogenesis of autoimmune diseases have allowed application of these tools in vitro as well as in vivo with some degree of success. Further research on the design and delivery of effectors of RNAi pathway and underlying molecular basis of RNAi would warrant practical use of RNAi-based therapeutics in human applications. This review will focus on the approaches used for current therapeutics and their applications in autoimmune diseases, including rheumatoid arthritis and Sjögren’s syndrome.

Milatuzumab-conjugated liposomes as targeted dexamethasone carriers for therapeutic delivery in CD74+ B-cell malignancies.

Corticosteroids are widely used for the treatment of B-cell malignancies, including non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia; however, this class of drug is associated with undesirable off-target effects. Herein, we developed novel milatuzumab-conjugated liposomes as a targeted dexamethasone carrier for therapeutic delivery in CD74(+) B-cell malignancies and explored its effect against the disease. The targeting efficiency of milatuzumab-targeted liposomes to CD74(+) cells was evaluated in vitro. The effect of CD74-targeted liposomal dexamethasone was compared with free dexamethasone in primary CLL cells and cell lines in vitro. The therapeutic efficacy of CD74-targeted liposomal dexamethasone was evaluated in a Raji-severe combined immunodeficient (SCID) xenograft model in vivo. Milatuzumab-targeted liposomes promoted selective incorporation of carrier molecules into transformed CD74-positive B cells as compared with CD74-negative T-cells. The CD74-dexamethasone-targeted liposomes (CD74-IL-DEX) promoted and increased killing in CD74-positive tumor cells and primary CLL cells. Furthermore, the targeted drug liposomes showed enhanced therapeutic efficacy against a CD74-positive B-cell model as compared with free, or non-targeted, liposomal dexamethasone in SCID mice engrafted with Raji cells in vivo. These studies provide evidence and support for a potential use of CD74-targeted liposomal dexamethasone as a new therapy for B-cell malignancies.

Cdk2 Silencing via a DNA/PCL Electrospun Scaffold Suppresses Proliferation and Increases Death of Breast Cancer Cells

RNA interference (RNAi) is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh) RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL) alone (Control) and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i) and EGFP (EGFPi, also served as a control) shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing) and 0.2–3 µm (Control). While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20–60 ng/ml (∼2.5% cumulative release) of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ∼51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ∼40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.

Expression and Role of VEGF-A in the Ciliary Body

The role of VEGF-A in the normal ciliary body is largely unexplored. The ciliary body is similar in many respects to the choroid plexus of the brain, and we demonstrated previously the importance of VEGF-A in maintenance of choroid plexus vasculature and ependymal cells. Therefore, the role of VEGF-A in ciliary body homeostasis was explored. Swiss-Webster mice (VEGF-LacZ) were used to determine VEGF-A expression during ciliary body development and in the adult. VEGFR2 expression was determined in adult wild type C56BL/6J mice. Systemic VEGF-A neutralization in vivo was achieved with adenovirus-mediated overexpression of soluble VEGFR1 (sFlt1). Following VEGF-A neutralization, the ciliary epithelium was analyzed by light microscopy and transmission electron microscopy (TEM). The effect of VEGF-A blockade on ciliary body function also was assessed by measuring intraocular pressure. VEGF-A expression was detected at embryonic day 18.5 (E18.5), the onset of ciliary process formation. In the adult ciliary body, VEGF-A was expressed by the pigmented epithelium, whereas VEGFR2 was localized primarily to the capillary endothelium and nonpigmented epithelium. Systemic VEGF-A neutralization led to a thinning of the nonpigmented epithelium, vacuolization of the pigmented epithelium, loss of capillary fenestrations, and thrombosis. These changes were associated with impaired ciliary body function, as evidenced by decreased intraocular pressure in sFlt1-overexpressing animals (15.31 ± 2.06 mm Hg) relative to controls (18.69 ± 1.49 mm Hg). VEGF-A has an important role in ciliary body homeostasis. Potential for undesired off-target effects should be considered with the chronic use of anti-VEGF-A therapies.

Modification of the siRNA Passenger Strand by 5‐Nitroindole Dramatically Reduces its Off‐Target Effects

During the formation of RNA-induced silencing complex (RISC), the passenger and guide strand of an siRNA duplex separate from each other to generate an active RISC complex. Accumulating evidence shows that an siRNA passenger strand can also assemble into a RISC complex and mediate RNA interference, thereby causing undesired off-target effects. To reduce this effect, the so-called "universal base" 5-nitroindole nucleotides were incorporated into an siRNA passenger strand. Melting temperature and circular dichroism spectrum measurements showed no significant changes compared to the unmodified duplex, thus indicating the formation of normal A-form conformation. Using a dual luciferase reporter assay, we have further shown that 5-nitroindole modification at position 15 of the siRNA passenger strand drastically decreased the RNAi (RNA interfering) potency of this strand, whereas the potency of the RNA guide strand was not much affected. These results could provide a practical approach for reducing off-target effects mediated by the siRNA passenger strand.

Structure Guided Development of Novel Thymidine Mimetics Targeting Pseudomonas aeruginosa Thymidylate Kinase: From Hit to Lead Generation

Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of an essential component, thymidine triphosphate, in DNA replication. All reported TMK inhibitors are thymidine analogues, which might retard their development as potent therapeutics due to cell permeability and off-target activity against human TMK. A small molecule hit (1, IC(50) = 58 μM), which has reasonable inhibition potency against Pseudomonas aeruginosa TMK (PaTMK), was identified by the analysis of the binding mode of thymidine or TP(5)A in a PaTMK homology model. This hit (1) was cocrystallized with PaTMK, and several potent PaTMK inhibitors (leads, 46, 47, 48, and 56, IC(50) = 100-200 nM) were synthesized using computer-aided design approaches including virtual synthesis/screening, which was used to guide the design of inhibitors. The binding mode of the optimized leads in PaTMK overlaps with that of other bacterial TMKs but not with human TMK, which shares few common features with the bacterial enzymes. Therefore, the optimized TMK inhibitors described here should be useful for the development of antibacterial agents targeting TMK without undesired off-target effects. In addition, an inhibition mechanism associated with the LID loop, which mimics the process of phosphate transfer from ATP to dTMP, was proposed based on X-ray cocrystal structures, homology models, and structure-activity relationship results.

Monoclonal Antibodies and Recombinant Immunoglobulins for the Treatment of Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory and degenerative disease leading to demyelination and axonal damage in the CNS. Autoimmunity plays a central role in MS pathogenesis. Per definition, monoclonal antibodies are recombinant biological compounds with a well defined target, thus carrying the promise of targeting pathogenic cells or molecules with high specificity, avoiding undesired off-target effects. Natalizumab was the first monoclonal antibody to be approved for the treatment of MS. Several other monoclonal antibodies are in development and have demonstrated promising efficacy in phase II studies. They can be categorized according to their mode of action into compounds targeting (i) leukocyte migration into the CNS (natalizumab); (ii) cytolytic antibodies (rituximab, ocrelizumab, ofatumumab, alemtuzumab); or (iii) antibodies and recombinant proteins targeting cytokines and chemokines and their receptors (daclizumab, ustekinumab, atacicept, tabalumab [Ly-2127399], secukinumab [AIN457]). In this review, we discuss the specific molecular targets, clinical efficacy and safety of these compounds and discuss criteria to anticipate the position of monoclonal antibodies in the diversifying armamentarium of MS therapy in the coming years.

Expression and Role of VEGF in the Adult Retinal Pigment Epithelium

Despite a lack of active angiogenesis, VEGF is expressed in nearly every adult tissue, and recent evidence suggests that VEGF may serve as a survival factor for both vascular and nonvascular tissues. VEGF blockade is a widely used treatment for neovascular diseases such as wet age-related macular degeneration (AMD). Therefore, it was sought in this study to evaluate the expression and role of endogenous VEGF in RPE. VEGF and VEGFR2 expression in the murine retina were assessed during development. Bevacizumab was used to neutralize VEGF in ARPE-19 cells, and the effects on cell survival and apical microvill were assessed by TUNEL and SEM, respectively. VEGF was systemically neutralized in vivo by adenoviral-mediated overexpression of soluble VEGFR1 (sFlt). RPE and choriocapillaris were analyzed by transmission electron microscopy (TEM). Changes in gene expression were evaluated by quantitative real-time PCR. VEGF expression was detected in the developing RPE as early as embryonic day (E) 9.5, whereas VEGFR2 expression by RPE began nonuniformly between postnatal (P) day 6.5 and P8.5. VEGF neutralization in vitro led to increased apoptosis and reduced microvilli density and length. Systemic VEGF neutralization led to transient degenerative changes; RPE were vacuolated and separated from photoreceptor outer segments, and choriocapillaris fenestrations were decreased. VEGF levels were elevated in RPE of Ad-sFlt1 mice at day 4 postinfection, and there was increased expression of the neurotrophic factor CD59a at day 14. These results indicate that VEGF plays a critical role in survival and maintenance of RPE integrity. Potential undesired off-target effects should be considered with chronic use of anti-VEGF agents.

Kinase inhibitors can produce off-target effects and activate linked pathways by retroactivity

BackgroundIt has been shown in experimental and theoretical work that covalently modified signaling cascades naturally exhibit bidirectional signal propagation via a phenomenon known as retroactivity. An important consequence of retroactivity, which arises due to enzyme sequestration in covalently modified signaling cascades, is that a downstream perturbation can produce a response in a component upstream of the perturbation without the need for explicit feedback connections. Retroactivity may, therefore, play an important role in the cellular response to a targeted therapy. Kinase inhibitors are a class of targeted therapies designed to interfere with a specific kinase molecule in a dysregulated signaling pathway. While extremely promising as anti-cancer agents, kinase inhibitors may produce undesirable off-target effects by non-specific interactions or pathway cross-talk. We hypothesize that targeted therapies such as kinase inhibitors can produce off-target effects as a consequence of retroactivity alone.ResultsWe used a computational model and a series of simple signaling motifs to test the hypothesis. Our results indicate that within physiologically and therapeutically relevant ranges for all parameters, a targeted inhibitor can naturally induce an off-target effect via retroactivity. The kinetics governing covalent modification cycles in a signaling network were more important for propagating an upstream off-target effect in our models than the kinetics governing the targeted therapy itself. Our results also reveal the surprising and crucial result that kinase inhibitors have the capacity to turn "on" an otherwise "off" parallel cascade when two cascades share an upstream activator.ConclusionsA proper and detailed characterization of a pathway's structure is important for identifying the optimal protein to target as well as what concentration of the targeted therapy is required to modulate the pathway in a safe and effective manner. We believe our results support the position that such characterizations should consider retroactivity as a robust potential source of off-target effects induced by kinase inhibitors and other targeted therapies.

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VEGF inhibitor (Iressa) arrests histone deacetylase expression: Single‐cell cotransfection imaging cytometry for multi‐target‐multi‐drug analysis

Multi-target-multi-drug approaches are needed to accelerate the process of drug discovery screening and to design efficient therapeutic strategies against diseases that involve alterations in multiple cellular targets. Herein we report single-cell cotransfection imaging cytometry to quantitatively screen drug-induced off-target effects. Vascular endothelial growth factor (VEGF) and histone deacetylase (HDAC) genes amplified from the genomic DNA were cloned in fluorescently tagged gene constructs (RFP-HDAC/YFP-VEGF). These gene constructs were cotransfected in HEK-293 cells to explore the possibility of off-target effects of 4-phenylbutyrate and Iressa on the expression of VEGF and HDAC through single-cell imaging cytometry. Iressa (10 µM) treatment at the time of cotransfection or 48 h after cotransfection of RFP-HDAC/YFP-VEGF plasmids in HEK-293 cells resulted in off-target effects on HDAC expression. These results suggest possible applications of Iressa in the treatment of diseases in which expression of both HDAC and VEGF should be inhibited. 4-Phenylbutyrate (2.0 mM) did not show any off-target effects on VEGF expression. The developed quantitative multicolor live single-cell cotransfection imaging can be employed to select better drug combinations for faster screening and greater accuracy in multi-target-multi-drug analysis by increasing the on-target/desired off-target effects and eliminating the undesirable off-target effects.

Abstract 4907: Elucidating the role of retroactive signaling and kinase inhibitors on off-target drug effects

Abstract The primary objective of targeted cancer therapies is to modulate cancer progression by perturbing specific molecules involved in aberrant proliferation and invasion. Kinase inhibitors are targeted therapies which are designed to interfere with a specific kinase molecule in a dysregulated oncogenic signaling cascade. While extremely promising as anti-cancer agents, such inhibitors may have undesirable off-target effects, whether by non-specific interactions or by effects from pathway cross-talk. We have shown in published experimental and theoretical work that covalently modified signaling cascades naturally exhibit bidirectional signal propagation. This phenomenon is termed retroactivity and challenges the widespread notion that information in cascades only flows from the cell surface to the nucleus. Previous work has demonstrated that increasing the concentration of a phosphatase in the terminal cycle of a covalently modified cascade may result in a measurable decrease in the concentration of the previous cycle's activated kinase. Thus, a downstream perturbation in a signaling cascade can produce a reverse (or retroactive) response without the need for direct negative feedback connections. This led us to hypothesize that the use of an inhibitory drug in a signaling network may cause an upstream off-target effect simply by inhibiting the activation or deactivation of a downstream kinase. To test the hypothesis that retroactivity contributes to off-target effects, we extended our previous work to a computational model that tested a series of signaling networks. The objective of our approach was two-fold: (1) to probe the effect of retroactivity on a kinase inhibitor in a signaling network and (2) to test whether retroactivity is likely to produce a measurable off-target effect under physiologically realistic conditions. Specifically, our model simulates the targeted inhibition of an activated kinase in a series of multi-cycle networks. The results of our work indicate that at physiologically and therapeutically relevant concentrations, a targeted inhibitor may induce a measurable off-target effect via retroactivity. We also performed local sensitivity analyses to predict the kinetic parameters that most affect the off-target response. Surprisingly, the drug disassociation constant is predicted to have very little effect while parameters such as the enzyme saturation and maximum velocity of some cycles are predicted to be very important. A proper characterization of a pathway's structure is important for identifying which protein in the pathway represents the optimal drug target as well as what concentration of the targeted therapy is likely to modulate the pathway in the manner desired. We believe our results support the position that such characterizations should consider the role of retroactivity as a source of a potential off-target effects by kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4907. doi:10.1158/1538-7445.AM2011-4907

Abstract 2584: eNOS as a therapeutic target in pancreatic cancer

Abstract Pancreatic cancer has a very high mortality rate, and frontline therapy has not changed in over a decade indicating a need for novel therapeutics. In this regard, endothelial Nitric Oxide Synthase (eNOS) has emerged as a new target due to recent studies demonstrating elevated expression in human pancreatic cancers, and inhibition of pancreatic tumor growth with eNOS knockdown or knockout in xenograft and genetically engineered mouse models. By pharmacologically testing eNOS inhibition, we found that a non-selective NOS inhibitor, L-NG-Nitroarginine methyl ester (L-NAME), inhibited tumor growth of human pancreatic cancer cell lines and extended survival in the aggressive Pdx-1-CreTg/+;LSL-KRASG12D/+;LSL-Trp5R172H/+ (KPC) mouse model of pancreatic ductal adenocarcinoma (PDAC) by ∼30 days. This extended survival is particularly significant given that the current frontline therapy, gemcitabine, does not provide these mice with a similar survival advantage. While general inhibitors such as L-NAME are attractive molecules to explore, evidence has shown that targeting all three NOS enzymes can have undesirable off-target effects. As eNOS has been identified as the NOS family member required for pancreatic tumorigenesis, and there currently are no selective eNOS inhibitors, we have created and optimized a cell-based system to identify potent and selective compounds for eNOS inhibition. This assay uses a mammalian baculovirus expression system to distinguish the activity of eNOS, nNOS, and iNOS via measurement of the stable bi-products of nitric oxide synthesis, and has been tested with a variety of published NOS inhibitors including L-NAME, the iNOS specific inhibitor 1400W, and the nNOS specific inhibitor L-NPA. The assay is suitable for high throughput screening of small molecule libraries to identify novel eNOS inhibitors that, when paired with gemcitabine, may represent a novel combination therapy for pancreatic cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2584. doi:10.1158/1538-7445.AM2011-2584

Eating less suppresses microRNA assassins in the brain

The aging brain is perhaps the most fragile of tissues, with a population of structurally elaborate and metabolically demanding neurons that are seldom replenished during adulthood. Neurodegenerative disorders such as Alzheimer's disease (AD) and acute insults such as an ischemic stroke lead to seemingly irreparable damage that impairs cognitive and/or motor function. Considerable evidence suggests that the vulnerability of neurons to aging and associated disorders can be modified by environmental factors including dietary energy intake and exercise [1, 2, 3]. Energy (caloric) restriction (CR) and exercise induce the expression of neurotrophic factors, including brain-derived neurotrophic factor (BDNF), cytoprotective protein chaperones, and proteins that can prevent apoptosis. The mechanisms responsible for the neuro-protective effects of CR and exercise are mediated, in part, by changes in gene transcription [3]. However, new findings reported in this issue of Aging suggest a role for down-regulation of microRNAs (miRNAs) that target mRNAs encoding cell survival proteins in the beneficial effects of CR on the aging brain [4]. miRNAs are short non-coding RNAs that typically bind to scores of transcripts an inhibit translation of the targeted mRNA [5]. Khanna et al found that levels of three miRNAs (miR-34a, miR30e and miR181-a-1*) are significantly lower in brain tissue samples from old mice (24-28 months-old) that had been maintained on a CR diet (40% CR beginning at 4 months of age) compared to mice on the usual ad libitum diet. Interestingly, all three of these miRNAs were predicted to have at least one target site for Bcl2, an anti-apoptotic protein previously been shown to increase with CR. The authors confirmed the repressive action of these miRNAs on Bcl2. These changes in miRNA expression were validated in both cortical and hippocampal tissues, suggesting a more global repression of these miRNAs in the CNS due to CR. Although the authors chose to solely investigate Bcl2 as a target of these three miRNAs in mediating the beneficial effects of CR, eight additional shared mRNA targets were found using multiple target-prediction algorithms. One of these targets, cAMP response element binding protein 1 (CREB1), is an important activator of several immediate response genes that are critical to synaptic plasticity [6]. Repression of CREB1 by these three age-dependent miRNAs could play a role in the cognitive decline observed with aging. Additionally, huntingtin (Htt) is a predicted target of these miRNAs, and this possible interaction may play a role in the beneficial effects of dietary energy restriction in Htt mutant mice, a model of Huntington's disease [7]. Moreover, previous research demonstrated that miR-30a, which has an identical seed region to miR-30e acts to functionally repress BDNF expression in the cortex [8]. Up-regulation of BDNF by CR has been shown to mediate, in part, the increased neurogenesis by CR and is also thought to play an important role in learning and memory [9, 10]. Although not explored by Khanna and colleagues in the context of CR, the regulation of BDNF by the miR-34 family represents yet another potential avenue for miRNAs as mediators of effects of dietary energy intake on neuronal vulnerability in aging and disease. It will be important to determine whether changes in the expression of miRNAs 34a, 30e, and 181a do in fact mediate effects of energy intake on neuronal vulnerability. This might be accomplished by overexpressing or knocking down each of these miRNAs in neurons of interest in animal models of Alzheimer's, Parkinson's and Huntington's diseases. Whether Bcl-2 is a pivotal target of this miRNAs could be determined in experiments with Bcl-2 deficient mice. It seems likely that the findings of Khanna et al. [4] represent the ‘tip of the iceberg’ with regards to miRNAs in brain aging and disease as there are undoubtedly numerous other miRNAs and target mRNAs involved in regulating neuronal survival and plasticity. This arena of research is a promising area for the development of novel therapeutic interventions in neurodegenerative disorders. Indeed, multiple miRNAs appear to be dysregulated in neurological diseases [5]. Initial studies in non-human primates have further emphasized the potential for miRNA-based theraputics [11]. The work by Khanna and colleagues suggest a potential for miRNA-based therapies that utilize the same anti-apoptic mechanisms as CR. However, as a single miRNA can regulate hundreds of transcripts, systemic delivery of a miRNA mimetic or sponge may result in undesirable off-target and tissue specific effects.

Identification of New and Diverse Inducers of Fetal Hemoglobin with High Throughput Screening (HTS)

AbstractAbstract 4277Pharmacologic augmentation of fetal hemoglobin (HbF, γ-globin) production, to replace diminished β-globin chains in the β-thalassemias and to inhibit HbS polymerization in sickle cell disease, is a definitive therapeutic modality. Despite long-term efforts, regulatory approval has been obtained for only one chemotherapeutic agent. Pharmacologic reactivation of high-level HbF expression with non-cytotoxic, tolerable therapeutics is still an unmet medical need for this global health burden. To investigate potential therapeutic libraries for unrecognized HbF inducers, we developed a high-throughput screening (HTS) program to interrogate diverse chemical libraries, including a library of FDA-approved and clinical stage drugs. This program has identified unexpected new and highly potent HbF-inducing drugs, some of which are already in clinical use for other medical indications and have established safety profiles. A human cell-based assay which was previously used in low throughput assays, utilizing a 1.4-kilobase (kb) KpnI-BglII fragment of the HS2 of the locus control region (LCR) linked to the γ-globin gene promoter and the enhanced green fluorescent protein (EGFP) reporter gene, was adapted for high throughput screening and employed as the primary screen. Cytotoxic activity was assayed in a simultaneous counter screen. A number of hits were identified as being more potent than positive controls (such as butyrate). Several hits were immediately eliminated from further development as potential hemoglobinopathy therapeutics because of cytotoxicity (e.g., Idarubicin) or undesirable off-target effects, but nonetheless validated the HTS itself and were validated in secondary confirmatory assays as highly-potent HbF-inducers. The HTS assay identified 8 FDA-approved drugs as potent inducers of γ-globin gene expression, with activity at 1–2 logs lower concentrations (1000-fold higher potency) than prior generation therapeutic candidates. The γ-globin-specificity of hits was determined in a secondary assay employing a stably-transfected dual-luciferase reporter construct containing the LCR and the β-globin promoter linked to renilla luciferase and the Aγ-globin promoter linked to firefly luciferase (μLCRβprRlucAγprFluc cassette). Clinical-stage or clinically-approved agents, including Ambroxol at 1 μM, Desloratadine at 1 μM, Resveratrol at 10 μM, Benserazide at 5 μM, the HDAC inhibitor MS-275 at 5 μM, and an established bioactive, NSC-95397, at 1 μM were all significantly more active in this assay than Butyrate at 2000 μM, with MS-275 and Resveratrol being the most active. These drugs were then assayed for their ability to induce γ-globin mRNA expression in cultured primary human erythroid progenitors, at concentrations which are pharmacologically achievable in humans. Drugs significantly more active in γ -globin mRNA induction than the positive control (2-fold induction) in this system included Ambroxol (3-fold), Desloratadine (up to 6-fold), Resveratrol (up to 3-fold), Benserazide (up to 5-fold), and MS-275 (up to 3.7-fold). Two agents were subsequently studied in anemic baboons, and demonstrated in vivo induction of γ-globin mRNA, HbF, and F-reticulocytes. Unexpectedly, rises in total hemoglobin (>1 gm/dL) also occurred with 2 agents. Thus, a panel of structurally- and functionally-unrelated compounds demonstrate greater HbF-inducing activity, with up to 1000-fold higher potency, than current HbF-inducers which have significant activity in clinical trials. Some of the drugs identified by HTS have entirely benign safety profiles. These candidates could be clinically evaluated rapidly and at significantly less cost than new chemical entities, which require extensive toxicology, manufacturing, and clinical evaluation. These findings demonstrate the utility of a high-throughput screening program based on γ-globin gene promoter induction. Disclosures:No relevant conflicts of interest to declare.

Using cells devoid of RAS proteins as tools for drug discovery

Mutational activation of RAS proteins occurs in nearly 30% of all human tumors. To date direct pharmacological inhibition of RAS oncoproteins has not been possible. As a consequence, current strategies are focusing on the development of inhibitors that target those kinases acting downstream of RAS proteins, including those of the RAF/MEK/ERK and PI3K/AKT pathways. Most of these inhibitors have undesired off-target effects that mask the potential therapeutic effect of blocking their targeted kinases. To facilitate the screening of selective inhibitors, we have generated lines of mouse embryonic fibroblasts that lack endogenous Ras proteins. These cells proliferate due to ectopic expression of either Ras oncoproteins that selectively activate the Raf/Mek/Erk pathway such as H-Ras(G12V/D38E) or constitutively active kinases such as B-Raf and Mek1. These cell lines were exposed to inhibitors against the RAF, MEK, and AKT kinases as well as inhibitors of other kinases known to crosstalk with RAS signaling such as JNK and p38. Amongst all compounds tested, only the MEK inhibitors U0126 and PD0325901, showed the expected specificity pattern. Yet, PD0325901, but not U0126, was able to inhibit a cell line lacking Ras proteins that owed its proliferative properties to loss of p53. Thus, suggesting unexpected off-target activities for this compound. The use of cell lines whose proliferative properties exclusively depend on selective targets provide a novel strategy to analyze the specificity of selective inhibitors designed against molecular targets implicated in human cancer.

RNA interference reduces PmergDNV expression and replication in an in vivo cricket model

RNA interference (RNAi) is an attractive anti-viral preventative because it allows interference with the expression of a viral gene in a highly sequence-specific manner. Thus, essential viral genes can be targeted by design, with little or no risk of undesired off-target effects. To investigate if stealth RNAis can mediate a sequence-specific anti-viral effect against PmergDNV, adult Acheta domesticus were injected with 5 μg of stealth RNAi or control stealth RNAi, targeting the capsid protein. Twenty-four hours post-injection, crickets were challenged with PmergDNV. Mortality was monitored for 14 days and real-time reverse transcriptase PCR was used to enumerate the number of copies of PmergDNV in cricket tissues. Whilst statistically not significant, trends in mortality suggest crickets injected with RNAi targeting PmergDNV had the lowest mortality rate (11.5%) compared to crickets injected with control dsRNAi (33%) and PmergDNV alone (25%). Crickets challenged with specific dsRNAi had statistically significantly reduced PmergDNV titres by one log (3.58 × 10 2) compared to crickets challenged with PmergDNV alone (3.42 × 10 3). Interestingly, even the control dsRNAi was capable of reducing PmergDNV titres by one log (3.95 × 10 2), but did not produce an inhibitory effect quite as strong as the targeted dsRNAi for the capsid protein of PmergDNV. The introduction of dsRNAi corresponding to the capsid protein of PmergDNV, was effective in reducing viral replication in Acheta domesticus. Administration of PmergDNV-specific dsRNAis may provide an efficient counter measure against PmergDNV in prawns.

Predictive Teratology: Teratogenic Risk-Hazard Identification Partnered in the Discovery Process

Unexpected teratogenicity is ranked as one of the most prevalent causes for toxicity-related attrition of drug candidates. Without proactive assessment, the liability tends to be identified relatively late in drug development, following significant investment in compound and engagement in pre clinical and clinical studies. When unexpected teratogenicity occurs in pre-clinical development, three principle questions arise: Can clinical trials that include women of child bearing populations be initiated? Will all compounds in this pharmacological class produce the same liability? Could this effect be related to the chemical structure resulting in undesirable off-target adverse effects? The first question is typically addressed at the time of the unexpected finding and involves considering the nature of the teratogenicity, whether or not maternal toxicity could have had a role in onset, human exposure margins and therapeutic indication. The latter two questions can be addressed proactively, earlier in the discovery process as drug target profiling and lead compound optimization is taking place. Such proactive approaches include thorough assessment of the literature for identification of potential liabilities and follow-up work that can be conducted on the level of target expression and functional characterization using molecular biology and developmental model systems. Developmental model systems can also be applied in the form of in vitro teratogenicity screens, and show potential for effective hazard identification or issue resolution on the level of characterizing teratogenic mechanism. This review discusses approaches that can be applied for proactive assessment of compounds for teratogenic liability.