Positive Allosteric Modulators of SERCA Pump Restore Dendritic Spines and Rescue Long-Term Potentiation Defects in Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects memory formation and storage processes. Dysregulated neuronal calcium (Ca2+) has been identified as one of the key pathogenic events in AD, and it has been suggested that pharmacological agents that stabilize Ca2+ neuronal signaling can act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+-stabilizing agents and exhibit neuroprotective properties. In the present study, we evaluated effects of a set of novel SERCA PAM agents on the rate of Ca2+ extraction from the cytoplasm of the HEK293T cell line, on morphometric parameters of dendritic spines of primary hippocampal neurons in normal conditions and in conditions of amyloid toxicity, and on long-term potentiation in slices derived from 5xFAD transgenic mice modeling AD. Several SERCA PAM compounds demonstrated neuroprotective properties, and the compound NDC-9009 showed the best results. The findings in this study support the hypothesis that the SERCA pump is a potential therapeutic target for AD treatment and that NDC-9009 is a promising lead molecule to be used in the development of disease-modifying agents for AD.

Positive Allosteric Modulator of SERCA Pump NDC-1173 Exerts Beneficial Effects in Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease that affects millions of people worldwide. AD does not have a cure and most drug development efforts in the AD field have been focused on targeting the amyloid pathway based on the "amyloid cascade hypothesis". However, in addition to the amyloid pathway, substantial evidence also points to dysregulated neuronal calcium (Ca2+) signaling as one of the key pathogenic events in AD, and it has been proposed that pharmacological agents that stabilize neuronal Ca2+ signaling may act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+ stabilizing agents. In the present study, we report the development of a novel SERCA PAM agent, compound NDC-1173. To test the effectiveness of this compound, we performed behavioral studies with the APP/PS1 transgenic AD mouse model. We also evaluated effects of this compound on expression of endoplasmic reticulum (ER) stress genes in the hippocampus of APP/PS1 mice. The results of this study support the hypothesis that the SERCA pump is a potential novel therapeutic drug target and that NDC-1173 is a promising lead molecule for developing disease-modifying agents in AD.

A new target for Alzheimer's disease: A small molecule SERCA activator is neuroprotective in vitro and improves memory and cognition in APP/PS1 mice.

Amongst the cellular cacophony of altered signals in Alzheimer's disease (AD), disrupted Ca2+ homeostasis and consequential endoplasmic reticulum (ER) stress signals have been recognized as key determinants of neuron fate. This altered Ca2+ state is accompanied by a failing sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, which has been recognized as a causal feature of the underlying disease state. Repair of the Ca2+ dyshomeostasis represents a putative drug target via alleviation of ER stress and rescue of injured neurons, effectively modifying the AD state. Herein, we report a small molecule SERCA activator that rescues brain cells and raises ER Ca2+ in vitro, and shows efficacy in the APP/PS1 double transgenic mouse model of Alzheimer's disease. These results support SERCA activation as a therapeutic target for AD.

Discovery of 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent and orally bioavailable tissue-nonspecific alkaline phosphatase (TNAP) inhibitor.

Tissue-nonspecific alkaline phosphatase (TNAP) is an ectoenzyme crucial for bone matrix mineralization via its ability to hydrolyze extracellular inorganic pyrophosphate (ePPi), a potent mineralization inhibitor, to phosphate (Pi). By the controlled hydrolysis of ePPi, TNAP maintains the correct ratio of Pi to ePPi and therefore enables normal skeletal and dental calcification. In other areas of the body low ePPi levels lead to the development of pathological soft-tissue calcification, which can progress to a number of disorders. TNAP inhibitors have been shown to prevent these processes via an increase of ePPi. Herein we describe the use of a whole blood assay to optimize a previously described series of TNAP inhibitors resulting in 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent, selective and oral bioavailable compound that robustly inhibits TNAP in vivo.

Small molecule SUMOylation activators are novel neuroprotective agents.

Neuronal loss characterizes many of the most intractable nervous system diseases that deprive our ageing population of their quality of life. Neuroprotective pharmacological modalities are urgently needed to address this burgeoning population. Small ubiquitin-like modifier (SUMO) conjugation has been established as an endogenous neuroprotective response, and we have discovered several classes of small molecules that enhance SUMO conjugation. Herein we describe the hit to lead campaign that enabled the discovery of 3 diverse classes of drug-like SUMOylation activators. Optimized compounds were ultimately validated in cell-based models of neuronal loss and provide a foundation for establishing systemically active SUMO activators to treat degenerative diseases such as Parkinson's disease, Alzheimer's disease, and stroke.

A new target for Parkinson's disease: Small molecule SERCA activator CDN1163 ameliorates dyskinesia in 6-OHDA-lesioned rats.

Endoplasmic reticulum (ER) stress is intimately linked to Parkinson's disease (PD) pathophysiology. Disrupted intracellular calcium homeostasis is a major cause of the ER stress seen in dopaminergic neurons, leading to the cell death and subsequent loss of movement and coordination in patients. Dysfunctional calcium handling proteins play a major role in the promulgation of ER stress in PD. Specifically, compromised sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) has been identified as a major cause of ER stress and neuron loss in PD. We have identified a small molecule activator of SERCA that increases ER calcium content, rescues neurons from ER stress-induced cell death in vitro, and shows significant efficacy in the rat 6-hydroxydopamine (6-OHDA) model of PD. Together, these results support targeting SERCA activation as a viable strategy to develop disease-modifying therapeutics for PD.

Cytoprotective small molecule modulators of endoplasmic reticulum stress.

Cellular health depends on the normal function of the endoplasmic reticulum (ER) to fold, assemble, and modify critical proteins to maintain viability. When the ER cannot process proteins effectively, a condition known as ER stress ensues. When this stress is excessive or prolonged, cell death via apoptotic pathways is triggered. Interestingly, most major diseases have been shown to be intimately linked to ER stress, including diabetes, stroke, neurodegeneration, and many cancers. Thus, controlling ER stress presents a significant strategy for drug development for these diseases. The goal of this review is to present various small molecules that alleviate ER stress with the intention that they may serve as useful starting points for therapeutic agent development.

Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders.

Dysregulation of endoplasmic reticulum (ER) Ca(2+) homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) has emerged as a major contributor to ER stress. We pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction.

Small-molecule activation of SERCA2a SUMOylation for the treatment of heart failure.

Decreased activity and expression of the cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a), a critical pump regulating calcium cycling in cardiomyocyte, are hallmarks of heart failure. We have previously described a role for the small ubiquitin-like modifier type 1 (SUMO-1) as a regulator of SERCA2a and have shown that gene transfer of SUMO-1 in rodents and large animal models of heart failure restores cardiac function. Here, we identify and characterize a small molecule, N106, which increases SUMOylation of SERCA2a. This compound directly activates the SUMO-activating enzyme, E1 ligase, and triggers intrinsic SUMOylation of SERCA2a. We identify a pocket on SUMO E1 likely to be responsible for N106's effect. N106 treatment increases contractile properties of cultured rat cardiomyocytes and significantly improves ventricular function in mice with heart failure. This first-in-class small-molecule activator targeting SERCA2a SUMOylation may serve as a potential therapeutic strategy for treatment of heart failure.

Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

BACKGROUND: A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. METHODS AND RESULTS: This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. CONCLUSIONS: DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug.

Design, synthesis and evaluation of benzoisothiazolones as selective inhibitors of PHOSPHO1.

We report the discovery and characterization of a series of benzoisothiazolone inhibitors of PHOSPHO1, a newly identified soluble phosphatase implicated in skeletal mineralization and soft tissue ossification abnormalities. High-throughput screening (HTS) of a small molecule library led to the identification of benzoisothiazolones as potent and selective inhibitors of PHOSPHO1. Critical structural requirements for activity were determined, and the compounds were subsequently derivatized and measured for in vitro activity and ADME parameters including metabolic stability and permeability. On the basis of its overall profile the benzoisothiazolone analogue 2q was selected as MLPCN probe ML086.

Alamar blue reagent interacts with cell-culture media giving different fluorescence over time: potential for false positives.

INTRODUCTION: The cell viability assay by alamar blue is based on the principle of reduction of the non-fluorescent reagent (resazurin) to a fluorescent compound (resarufin) by the intracellular reducing environment of living cells over time. In the present study, we have for the first time shown that even in the absence of cells, there occurs significant interaction between alamar blue and cell-culture media causing an increase in fluorescence. METHODS: We have used Opti-MEM, DMEM and 1:1 DMEM:Opti-MEM as three different media and determined the changes in their relative fluorescence units (RFUs) over time after the addition of 10% (v/v) alamar blue using two-way repeated measures analysis of variance (RM-ANOVA) followed by Tukey's post-hoc test. RESULTS: Our results show that upon the addition of alamar blue, there occurs a significant increase in RFUs in all the three media over time along with a significantly higher RFU for the Opti-MEM overall (p<0.05). We also show that the time-dependent change in RFU of 1:1 DMEM:Opti-MEM was more gradual compared to that of the other two media. DISCUSSION: These findings indicate that the reagent can itself interact with the media causing significantly different fluorescence over time in a manner independent from the effect of intracellular reducing environment of living cells on alamar blue. In addition our results indicate that fluorescence varies as a function of incubation time with the reagent. These findings signify the need for routine subtraction of the background fluorescence of media-only with alamar blue reagent during measurement of cell viability by this method in order to determine an accurate measurement of cell viability.

Design and synthesis of systemically active metabotropic glutamate subtype-2 and -3 (mGlu2/3) receptor positive allosteric modulators (PAMs): pharmacological characterization and assessment in a rat model of cocaine dependence.

As part of our ongoing small-molecule metabotropic glutamate (mGlu) receptor positive allosteric modulator (PAM) research, we performed structure-activity relationship (SAR) studies around a series of group II mGlu PAMs. Initial analogues exhibited weak activity as mGlu2 receptor PAMs and no activity at mGlu3. Compound optimization led to the identification of potent mGlu2/3 selective PAMs with no in vitro activity at mGlu1,4-8 or 45 other CNS receptors. In vitro pharmacological characterization of representative compound 44 indicated agonist-PAM activity toward mGlu2 and PAM activity at mGlu3. The most potent mGlu2/3 PAMs were characterized in assays predictive of ADME/T and pharmacokinetic (PK) properties, allowing the discovery of systemically active mGlu2/3 PAMs. On the basis of its overall profile, compound 74 was selected for behavioral studies and was shown to dose-dependently decrease cocaine self-administration in rats after intraperitoneal administration. These mGlu2/3 receptor PAMs have significant potential as small molecule tools for investigating group II mGlu pharmacology.

Design, synthesis and bioevaluation of an EphA2 receptor-based targeted delivery system.

Because of its overexpression in a range of solid tumors, the EphA2 receptor is a validated target for cancer therapeutics. We recently described a new targeted delivery system based on specific EphA2-targeting peptides conjugated with the chemotherapeutic agent paclitaxel. Here, we investigate the chemical determinants responsible for the stability and degradation of these agents in plasma. Introducing modifications in both the peptide and the linker between the peptide and paclitaxel resulted in drug conjugates that are both long-lived in rat plasma and that markedly decrease tumor size in a prostate cancer xenograft model compared with paclitaxel alone treatment. These studies identify critical rate-limiting degradation sites on the peptide-drug conjugates, enabling the design of agents with increased stability and efficacy. These results provide support for our central hypothesis that peptide-drug conjugates targeting EphA2 represent an innovative and potentially effective strategy to selectively deliver cytotoxic drugs to cancer cells.

Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.

We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.

Identification of a selective inhibitor of murine intestinal alkaline phosphatase (ML260) by concurrent ultra-high throughput screening against human and mouse isozymes.

Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified-one tissue-nonspecific (TNAP) and three tissue-specific-named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.

Pharmacological inhibition of PHOSPHO1 suppresses vascular smooth muscle cell calcification.

Medial vascular calcification (MVC) is common in patients with chronic kidney disease, obesity, and aging. MVC is an actively regulated process that resembles skeletal mineralization, resulting from chondro-osteogenic transformation of vascular smooth muscle cells (VSMCs). Here, we used mineralizing murine VSMCs to study the expression of PHOSPHO1, a phosphatase that participates in the first step of matrix vesicles-mediated initiation of mineralization during endochondral ossification. Wild-type (WT) VSMCs cultured under calcifying conditions exhibited increased Phospho1 gene expression and Phospho1(-/-) VSMCs failed to mineralize in vitro. Using natural PHOSPHO1 substrates, potent and specific inhibitors of PHOSPHO1 were identified via high-throughput screening and mechanistic analysis and two of these inhibitors, designated MLS-0390838 and MLS-0263839, were selected for further analysis. Their effectiveness in preventing VSMC calcification by targeting PHOSPHO1 function was assessed, alone and in combination with a potent tissue-nonspecific alkaline phosphatase (TNAP) inhibitor MLS-0038949. PHOSPHO1 inhibition by MLS-0263839 in mineralizing WT cells (cultured with added inorganic phosphate) reduced calcification in culture to 41.8% ± 2.0% of control. Combined inhibition of PHOSPHO1 by MLS-0263839 and TNAP by MLS-0038949 significantly reduced calcification to 20.9% ± 0.74% of control. Furthermore, the dual inhibition strategy affected the expression of several mineralization-related enzymes while increasing expression of the smooth muscle cell marker Acta2. We conclude that PHOSPHO1 plays a critical role in VSMC mineralization and that "phosphatase inhibition" may be a useful therapeutic strategy to reduce MVC.

High-throughput FRET assay yields allosteric SERCA activators.

Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca(2+) regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS.

Orally active metabotropic glutamate subtype 2 receptor positive allosteric modulators: structure-activity relationships and assessment in a rat model of nicotine dependence.

Compounds that modulate metabotropic glutamate subtype 2 (mGlu(2)) receptors have the potential to treat several disorders of the central nervous system (CNS) including drug dependence. Herein we describe the synthesis and structure-activity relationship (SAR) studies around a series of mGlu(2) receptor positive allosteric modulators (PAMs). The effects of N-substitution (R(1)) and substitutions on the aryl ring (R(2)) were identified as key areas for SAR exploration (Figure 3). Investigation of the effects of varying substituents in both the isoindolinone (2) and benzisothiazolone (3) series led to compounds with improved in vitro potency and/or efficacy. In addition, several analogues exhibited promising pharmacokinetic (PK) properties. Furthermore, compound 2 was shown to dose-dependently decrease nicotine self-administration in rats following oral administration. Our data, showing for the first time efficacy of an mGlu(2) receptor PAM in this in vivo model, suggest potential utility for the treatment of nicotine dependence in humans.

A gatekeeper residue for NEDD8-activating enzyme inhibition by MLN4924.

Inhibition of NEDD8-activating enzyme (NAE) has emerged as a highly promising approach to treat cancer through the adenosine sulfamate analog MLN4924. Here, we show that selective pressure results in HCT116 colorectal carcinoma cells with decreased MLN4924 sensitivity and identify a single-nucleotide transition that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3. This reduces the enzyme's affinity for MLN4924 and ATP while increasing NEDD8 activation at physiological ATP concentrations. Expression of UBA3 A171T is sufficient to decrease MLN4924 sensitivity of naive HCT116 cells, indicating that it is a dominant suppressor of MLN4924-mediated cell death. Our data suggest that the on-target potency of MLN4924 selects for a point mutation in NAE that overcomes the molecule's inhibitory effects, allowing cancer cell survival.