[Show abstract][Hide abstract]ABSTRACT: Background
Clinical failures singularly targeting amyloid-β pathology indicate a critical need for alternative Alzheimer’s disease (AD) therapeutic strategies. The mixed pathology reported in a large population of AD patients demands a multifunctional drug approach. Since activation of cAMP response element binding protein (CREB) plays a crucial role in synaptic strengthening and memory formation, we retooled a clinical drug with known neuroprotective and anti-inflammatory activity to activate CREB, and validated this novel multifunctional drug, NMZ, in 4 different mouse models of AD.
NMZ was tested in three mouse models of familial AD and one model of sporadic AD. In 3 × Tg hippocampal slices, NMZ restored LTP. In vivo, memory was improved with NMZ in all animal models with robust cognitive deficits. NMZ treatment lowered neurotoxic forms of Aβ in both APP/PS1 and 3 × Tg transgenic mice while also restoring neuronal plasticity biomarkers in the 3 × Tg mice. In EFAD mice, incorporation of the major genetic AD risk factor, hAPOE4, did not mute the beneficial drug effects. In a novel sporadic mouse model that manifests AD-like pathology caused by accelerated oxidative stress in the absence of any familial AD mutation, oral administration of NMZ attenuated hallmark AD pathology and restored biomarkers of synaptic and neuronal function.
The multifunctional approach, embodied by NMZ, was successful in mouse models of AD incorporating Aβ pathology (APP/PS1), tau pathology (3xTg), and APOE4, the major human genetic risk factor for AD (EFAD). The efficacy observed in a novel model of sporadic AD (Aldh2−/−) demonstrates that the therapeutic approach is not limited to rare, familial AD genetic mutations. The multifunctional drug, NMZ, was not designed directly to target Aβ and tau pathology; however, the attenuation of this hallmark pathology suggests the approach to be a highly promising, disease-modifying strategy for AD and mixed pathology dementia.
Full-text Article · Dec 2016 · Molecular Neurodegeneration
[Show abstract][Hide abstract]ABSTRACT: Alzheimer disease (AD) is a progressive neurodegenerative disease characterized by amyloid plaques, composed of amyloid-beta peptide (Aβ) and neurofibrillary tangles, composed of aberrantly phosphorylated tau. APOE4 is the greatest genetic risk factor for AD, increasing risk up to 12-fold with a double allele compared to APOE3. In contrast, APOE2 reduces AD risk ~2-fold per allele. Accumulating evidence demonstrates that apolipoprotein E4 (apoE4) plays a multifactorial role in AD pathogenesis, although the exact mechanisms remain unclear. Further data support roles for apoE4 as both a toxic gain of function or loss of positive function in AD, a discrepancy that has significant implications for the future of apoE-directed therapeutics. However, recent evidence repurposing retinoid X receptor (RXR) agonists, or rexinoids, for the treatment of AD demonstrates conflicting, though potentially beneficial effects in familial AD-transgenic (FAD-Tg) mouse models. Of particular note is bexarotene (Targretin®), a selective rexinoid previously utilized in cancer treatment emerging as a viable candidate for AD clinical trials. However, the mechanism of action of bexarotene and similar rexinoids remains controversial, particularly in the context of human APOE. In addition, rexinoids demonstrate distinct adverse event profiles in humans that may have greater detrimental effects in an elderly AD population. Therefore, this special issue review discusses the implications for rexinoid-directed therapeutic strategies in AD, the potential mechanistic targets, and future directions for the improvement of rexinoid-based therapies in AD.
Article · Jun 2016 · Current topics in medicinal chemistry
[Show abstract][Hide abstract]ABSTRACT: Reversible modifications of protein cysteine residues via S-nitrosylation and S-oxidation via disulfide formation are posttranslational modifications (PTM) regulating a broad range of protein activities and cellular signaling. Dysregulated protein nitrosothiol and disulfide formation have been implicated in pathogenesis of neurodegenerative disorders. Under nitrosative or nitroxidative stress, both nitrosylation and oxidation can theoretically occur at redox-sensitive cysteine residues, mediating thiol-regulated stress response. However, few detection strategies address both modifications. Nonquantitative approaches used to observe S-nitrosylation, regardless of unmodified and oxidized thiol forms, may lead to causal conclusions about the importance of protein nitrosothiol in NO-mediated signaling, regulation, and stress response. To observe quantitatively the modification spectrum of the cysteome, we developed a mass spectrometry-based approach, denoted as d-SSwitch, using isotopic labeling and shotgun proteomics to simultaneously identify and quantify different modification states at individual cysteine residues. Both recombinant protein and intact neuroblastoma cells were analyzed by d-SSwitch after treatment with nitrosothiol or NO. In proteins identified to be modified after nitrosothiol treatment, S-oxidation was always observed concomitant with S-nitrosylation and was quantitatively dominant. Herein, we describe the detailed procedures of d-SSwitch and important notes in practice.
[Show abstract][Hide abstract]ABSTRACT: Schistosomiasis affects millions of people in developing countries and is responsible for more than 200,000 deaths annually. Because of toxicity and limited spectrum of activity of alternatives, there is effectively only one drug, praziquantel, available for its treatment. Recent data suggest that drug resistance could soon be a problem. There is therefore the need to identify new drug targets and develop drugs for the treatment of schistosomiasis. Analysis of the Schistosoma mansoni genome sequence for proteins involved in detoxification processes found that it encodes a single cytochrome P450 (CYP450) gene. Here we report that the 1452 bp open reading frame has a characteristic heme-binding region in its catalytic domain with a conserved heme ligating cysteine, a hydrophobic leader sequence present as the membrane interacting region, and overall structural conservation. The highest sequence identity to human CYP450s is 22%. Double stranded RNA (dsRNA) silencing of S. mansoni (Sm)CYP450 in schistosomula results in worm death. Treating larval or adult worms with antifungal azole CYP450 inhibitors results in worm death at low micromolar concentrations. In addition, combinations of SmCYP450-specific dsRNA and miconazole show additive schistosomicidal effects supporting the hypothesis that SmCYP450 is the target of miconazole. Treatment of developing S. mansoni eggs with miconazole results in a dose dependent arrest in embryonic development. Our results indicate that SmCYP450 is essential for worm survival and egg development and validates it as a novel drug target. Preliminary structure-activity relationship suggests that the 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol moiety of miconazole is necessary for activity and that miconazole activity and selectivity could be improved by rational drug design.
Full-text Article · Dec 2015 · PLoS Neglected Tropical Diseases
[Show abstract][Hide abstract]ABSTRACT: In breast tumors, activation of the nuclear factor kappaB (NFkB) pathway promotes survival, migration, invasion, angiogenesis, stem cell-like properties, and resistance to therapy; all phenotypes of aggressive disease where therapy options remain limited. Adding an anti-inflammatory/anti-NFkB agent to breast cancer treatment would be beneficial, but no such drug is approved as either a mono- or adjuvant therapy. To address this need, we examined whether dimethyl fumarate (DMF), an anti-inflammatory drug already in clinical use for multiple sclerosis, can inhibit the NFkB pathway. We find that DMF effectively blocks NFkB activity in multiple breast cancer cell lines and abrogates NFkB-dependent mammosphere formation, indicating that DMF has anti-cancer stem cells properties. In addition, DMF inhibits cell proliferation and significantly impairs xenograft tumor growth. Mechanistically, DMF prevents p65 nuclear translocation and attenuates its DNA binding activity, but has no effect on upstream proteins in the NFkB pathway. Dimethyl succinate (DMS), the inactive analog of DMF that lacks the electrophilic double bond of fumarate, is unable to inhibit NFkB activity. Also, the cell permeable thiol, N-acetyl L-cysteine, reverses DMF inhibition of the NFkB pathway, supporting the notion that the electrophile, DMF, acts via covalent modification. To determine whether DMF directly interacts with p65, we synthesized and used a novel chemical probe of DMF by incorporating an alkyne functionality, and found that DMF covalently modifies p65 with cysteine 38 being essential for DMF activity. These results establish DMF as an NFkB inhibitor with anti-tumor activity that may add therapeutic value in treating aggressive breast cancers.
Full-text Article · Dec 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: Background
Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-β, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property.
The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice.
Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia.
Electronic supplementary material
The online version of this article (doi:10.1186/s12868-015-0208-9) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract]ABSTRACT: Introduction
Activation of cyclooxygenase (COX)/prostaglandin and nuclear factor κB (NFκB) pathways can promote breast tumor initiation, growth, and progression to drug resistance and metastasis. Thus, anti-inflammatory drugs have been widely explored as chemopreventive and antineoplastic agents. Aspirin (ASA), in particular, is associated with reduced breast cancer incidence but gastrointestinal toxicity has limited its usefulness. To improve potency and minimize toxicity, ASA ester prodrugs have been developed, in which the carboxylic acid of ASA is masked and ancillary pharmacophores can be incorporated. To date, the effects of ASA and ASA prodrugs have been largely attributed to COX inhibition and reduced prostaglandin production. However, ASA has also been reported to inhibit the NFκB pathway at very high doses. Whether ASA prodrugs can inhibit NFκB signaling remains relatively unexplored.
A library of ASA prodrugs was synthesized and screened for inhibition of NFκB activity and cancer stem-like cell (CSC) properties, an important PGE2-and NFκB-dependent phenotype of aggressive breast cancers. Inhibition of NFκB activity was determined by dual luciferase assay, RT-QPCR, p65 DNA binding activity and Western blots. Inhibition of CSC properties was determined by mammosphere growth, CD44+CD24−immunophenotype and tumorigenicity at limiting dilution.
While we identified multiple ASA prodrugs that are capable of inhibiting the NFκB pathway, several were associated with cytotoxicity. Of particular interest was GTCpFE, an ASA prodrug with fumarate as the ancillary pharmacophore. This prodrug potently inhibits NFκB activity without innate cytotoxicity. In addition, GTCpFE exhibited selective anti-CSC activity by reducing mammosphere growth and the CD44+CD24−immunophenotype. Moreover, GTCpFE pre-treated cells were less tumorigenic and, when tumors did form, latency was increased and growth rate was reduced. Structure-activity relationships for GTCpFE indicate that fumarate, within the context of an ASA prodrug, is essential for anti-NFκB activity, whereas both the ASA and fumarate moieties contributed to attenuated mammosphere growth.
These results establish GTCpFE as a prototype for novel ASA-and fumarate-based anti-inflammatory drugs that: (i) are capable of targeting CSCs, and (ii) may be developed as chemopreventive or therapeutic agents in breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1868-7) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract]ABSTRACT: Alzheimer's disease, one of the most important brain pathologies associated with neurodegenerative processes, is related to overactivation of calpain-mediated proteolysis. Previous data showed a compelling efficacy of calpain inhibition against abnormal synaptic plasticity and memory produced by the excess of amyloid-β, a distinctive marker of the disease. Moreover, a beneficial effect of calpain inhibitors in Alzheimer's disease is predictable by the occurrence of calpain hyperactivation leading to impairment of memory-related pathways following abnormal calcium influxes that might ensue independently of amyloid-β elevation. However, molecules currently available as effective calpain inhibitors lack adequate selectivity. This work is aimed at characterizing the efficacy of a novel class of epoxide-based inhibitors, synthesized to display improved selectivity and potency towards calpain 1 compared to the prototype epoxide-based generic calpain inhibitor E64. Both functional and preliminary toxicological investigations proved the efficacy, potency, and safety of the novel and selective calpain inhibitors NYC438 and NYC488 as possible therapeutics against the disease.
Article · Oct 2015 · Journal of Alzheimer's disease: JAD
[Show abstract][Hide abstract]ABSTRACT: Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.
[Show abstract][Hide abstract]ABSTRACT: Nitrated and non-nitrated compounds capable of protecting brain tissue from injury and useful as therapeutic agents to treat neurodegenerative diseases and conditions are disclosed. Methods of using the compounds in therapeutic treatments, and methods of preparing the compounds, also are disclosed.
[Show abstract][Hide abstract]ABSTRACT: Previous data demonstrate that Bexarotene (Bex), an RXR agonist, reduces soluble and insoluble amyloid-β (Aβ) in Alzheimer's disease (AD)-transgenic (Tg) mice either via increasing the levels of mouse apolipoprotein E (apoE) or increasing ABCA1/ABCG1-induced apoE lipoprotein-association/lipidation. However, while the mechanism of action of RXR agonists remains unclear, a major concern for their use is human (h)-APOE4, the greatest AD genetic-risk factor. If APOE4 imparts a toxic gain-of-function, then increasing apoE4 may increase soluble Aβ, likely the proximal AD neurotoxin. If APOE4 imparts a loss-of-function, then ABCA1/ABCG1-induced lipidation of apoE4 may be beneficial. In novel EFAD-Tg mice (overexpressing h-Aβ42 with h-APOE), levels of soluble Aβ (Aβ42 and oligomeric Aβ (oAβ)) are highest in E4FAD-hippocampus (HP) > E3FAD-HP > E4FAD-cortex (CX) > E3FAD-CX, while levels of lipoprotein-associated/lipidated apoE have the opposite pattern (6-months, M). In E4FAD-HP, short-term RXR agonist treatment (Bex or LG100268; 5.75-6M) increased ABCA1, apoE4 lipoprotein-association/lipidation and apoE4/Aβ complex, decreased soluble Aβ and increased PSD95. In addition, hydrogel delivery, which mimics low sustained release, was equally effective as gavage for Bex and LG in lowering soluble Aβ with APOE4. RXR agonists induced no beneficial effects in the E4FAD-HP in a prevention protocol (5-6M), and increased soluble Aβ levels in E3FAD-CX and E4FAD-CX (5.75-6M), likely the result of systemic hepatomegaly. Thus, RXR agonists address the loss-of-function associated with APOE4 and exacerbated by Aβ pathology--low levels of apoE4 lipoprotein-association/lipidation. Further studies are vital to address whether RXR agonists are an APOE4-specific AD therapeutic and the systemic side effects that limit translational application.
Full-text Article · Sep 2014 · Journal of Biological Chemistry