Potential Rheumatoid Arthritis-Associated Interstitial Lung Disease Treatment and Computational Approach for Future Drug Development.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by swelling in at least one joint. Owing to an overactive immune response, extra-articular manifestations are observed in certain cases, with interstitial lung disease (ILD) being the most common. Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is characterized by chronic inflammation of the interstitial space, which causes fibrosis and the scarring of lung tissue. Controlling inflammation and pulmonary fibrosis in RA-ILD is important because they are associated with high morbidity and mortality. Pirfenidone and nintedanib are specific drugs against idiopathic pulmonary fibrosis and showed efficacy against RA-ILD in several clinical trials. Immunosuppressants and disease-modifying antirheumatic drugs (DMARDs) with anti-fibrotic effects have also been used to treat RA-ILD. Immunosuppressants moderate the overexpression of cytokines and immune cells to reduce pulmonary damage and slow the progression of fibrosis. DMARDs with mild anti-fibrotic effects target specific fibrotic pathways to regulate fibrogenic cellular activity, extracellular matrix homeostasis, and oxidative stress levels. Therefore, specific medications are required to effectively treat RA-ILD. In this review, the commonly used RA-ILD treatments are discussed based on their molecular mechanisms and clinical trial results. In addition, a computational approach is proposed to develop specific drugs for RA-ILD.

Discovery of an Orally Bioavailable Benzofuran Analogue That Serves as a ß-Amyloid Aggregation Inhibitor for the Potential Treatment of Alzheimer's Disease.

We developed an orally active and blood-brain-barrier-permeable benzofuran analogue (8, MDR-1339) with potent antiaggregation activity. Compound 8 restored cellular viability from Aß-induced cytotoxicity but also improved the learning and memory function of AD model mice by reducing the Aß aggregates in the brains. Given the high bioavailability and brain permeability demonstrated in our pharmacokinetic studies, 8 will provide a novel scaffold for an Aß-aggregation inhibitor that may offer an alternative treatment for AD.

Discovery of Potent Human Glutaminyl Cyclase Inhibitors as Anti-Alzheimer's Agents Based on Rational Design.

Glutaminyl cyclase (QC) has been implicated in the formation of toxic amyloid plaques by generating the N-terminal pyroglutamate of ß-amyloid peptides (pGlu-Aß) and thus may participate in the pathogenesis of Alzheimer's disease (AD). We designed a library of glutamyl cyclase (QC) inhibitors based on the proposed binding mode of the preferred substrate, Aß3E-42. An in vitro structure-activity relationship study identified several excellent QC inhibitors demonstrating 5- to 40-fold increases in potency compared to a known QC inhibitor. When tested in mouse models of AD, compound 212 significantly reduced the brain concentrations of pyroform Aß and total Aß and restored cognitive functions. This potent Aß-lowering effect was achieved by incorporating an additional binding region into our previously established pharmacophoric model, resulting in strong interactions with the carboxylate group of Glu327 in the QC binding site. Our study offers useful insights in designing novel QC inhibitors as a potential treatment option for AD.

3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis.

Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.

Tomoregulin (TMEFF2) Binds Alzheimer's Disease Amyloid-ß (Aß) Oligomer and AßPP and Protects Neurons from Aß-Induced Toxicity.

Amyloid-ß (Aß) protein causes neurotoxicity and its abnormal aggregation into amyloid is a pathological hallmark of Alzheimer's disease (AD). Cellular proteins able to interact with Aß or its precursor, AßPP (amyloid-ß protein precursor), may regulate Aß production and neurotoxicity. We identified a brain-enriched type I transmembrane protein, tomoregulin (TR), that directly binds Aß and Aß oligomers (AßO). TR co-immunoprecipitated with Aß and AßO in cultured cells and co-localized with amyloid plaques and intraneuronal Aß in the 5xFAD AD mouse model. TR was also enriched in astrocytic processes reactive to amyloid plaques. Surface plasmon resonance spectroscopy studies showed that the extracellular domain of TR binds to AßO with a high affinity (KD = 76.8 nM). Electron paramagnetic resonance spectroscopy also demonstrated a physical interaction between spin-labeled Aß and the TR extracellular domain in solution. Furthermore, TR also interacted with AßPP and enhanced its cleavage by α-secretase. Both cellular expression of TR and application of recombinant TR extracellular domain protected N2a neurons from AßO-induced neuronal death. These data provide first evidence that neuronal and astrocytic expression of TR is intimately related to Aß metabolism and toxicity, and could be neuroprotective through its direct interaction with Aß and AßPP.

6-Phenoxy-2-phenylbenzoxazoles, novel inhibitors of receptor for advanced glycation end products (RAGE).

Receptor for advanced glycation end products (RAGE) is known to be involved in the transportation of amyloid ß (Aß) peptides and causes the accumulation of Aß in the brain. Moreover, recent studies suggest that the interactions between RAGE and Aß peptides may be the culprit behind Alzheimer's disease (AD). Inhibitors of the RAGE-Aß interactions would not only prevent the accumulation of toxic Aß in the brain, and but also block the progress of AD, therefore, have the potential to provide a 'disease-modifying therapy'. In this study, we have developed a series of 6-phenoxy-2-phenylbenzoxazole analogs as novel inhibitors of RAGE. Among these derivatives, we found several effective inhibitors that block the RAGE-Aß interactions without causing significant cellular toxicity. Further testing showed that compound 48 suppressed Aß induced toxicity in mouse hippocampal neuronal cells and reduced Aß levels in the brains of a transgenic mouse model of AD after oral administration.

Synthesis and Evaluation of a Novel Deguelin Derivative, L80, which Disrupts ATP Binding to the C-terminal Domain of Heat Shock Protein 90.

The clinical benefit of current anticancer regimens for lung cancer therapy is still limited due to moderate efficacy, drug resistance, and recurrence. Therefore, the development of effective anticancer drugs for first-line therapy and for optimal second-line treatment is necessary. Because the 90-kDa molecular chaperone heat shock protein (Hsp90) contributes to the maturation of numerous mutated or overexpressed oncogenic proteins, targeting Hsp90 may offer an effective anticancer therapy. Here, we investigated antitumor activities and toxicity of a novel deguelin-derived C-terminal Hsp90 inhibitor, designated L80. L80 displayed significant inhibitory effects on the viability, colony formation, angiogenesis-stimulating activity, migration, and invasion of a panel of non-small cell lung cancer cell lines and their sublines with acquired resistance to paclitaxel with minimal toxicity to normal lung epithelial cells, hippocampal cells, vascular endothelial cells, and ocular cells. Biochemical analyses and molecular docking simulation revealed that L80 disrupted Hsp90 function by binding to the C-terminal ATP-binding pocket of Hsp90, leading to the disruption of the interaction between hypoxia-inducible factor (HIF)-1α and Hsp90, downregulation of HIF-1α and its target genes, including vascular endothelial growth factor (VEGF) and insulin-like growth factor 2 (IGF2), and decreased the expression of various Hsp90 client proteins. Consistent with these in vitro findings, L80 exhibited significant antitumor and antiangiogenic activities in H1299 xenograft tumors. These results suggest that L80 represents a novel C-terminal Hsp90 inhibitor with effective anticancer activities with minimal toxicities.

Acute ER stress regulates amyloid precursor protein processing through ubiquitin-dependent degradation.

Beta-amyloid (Aß), a major pathological hallmark of Alzheimer's disease (AD), is derived from amyloid precursor protein (APP) through sequential cleavage by ß-secretase and γ-secretase enzymes. APP is an integral membrane protein, and plays a key role in the pathogenesis of AD; however, the biological function of APP is still unclear. The present study shows that APP is rapidly degraded by the ubiquitin-proteasome system (UPS) in the CHO cell line in response to endoplasmic reticulum (ER) stress, such as calcium ionophore, A23187, induced calcium influx. Increased levels of intracellular calcium by A23187 induces polyubiquitination of APP, causing its degradation. A23187-induced reduction of APP is prevented by the proteasome inhibitor MG132. Furthermore, an increase in levels of the endoplasmic reticulum-associated degradation (ERAD) marker, E3 ubiquitin ligase HRD1, proteasome activity, and decreased levels of the deubiquitinating enzyme USP25 were observed during ER stress. In addition, we found that APP interacts with USP25. These findings suggest that acute ER stress induces degradation of full-length APP via the ubiquitin-proteasome proteolytic pathway.

Two ß-strands of RAGE participate in the recognition and transport of amyloid-ß peptide across the blood brain barrier.

Amyloid-ß (Aß) peptide is central to the development of brain pathology in Alzheimer disease (AD) patients. Association with receptors for advanced glycation end-products (RAGE) enables the transport of Aß peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two ß-strands of RAGE participate in the interaction with Aß peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth ß-strands are required for interaction with Aß peptide. Site-directed mutagenesis of amino acids located in the third and eighth ß-strands abolish the interaction of RAGE with Aß peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aß peptide treatment, while a RAGE mutant defective in Aß binding does not. Furthermore, use of peptide for the third ß-strand or a RAGE monoclonal antibody that targets the RAGE-Aß interaction interface inhibited transport of the Aß peptide across the blood brain barrier in a mice model. These results provide information crucial to the development of RAGE-derived therapeutic reagents for Alzheimer disease.

Ligand-based design, synthesis, and biological evaluation of 2-aminopyrimidines, a novel series of receptor for advanced glycation end products (RAGE) inhibitors.

Using the approach of ligand-based drug design, we discovered a novel series of 4,6-disubstituted 2-aminopyrimidines as RAGE inhibitors. In transgenic mouse models of AD, one of the 4,6-bis(4-chlorophenyl)pyrimidine analogs, 59, significantly lowered the concentration of toxic soluble Aß in the brain and improved cognitive function. SPR analysis confirmed the direct binding of 59 with RAGE, which should contribute to its biological activities via inhibition of the RAGE-Aß interaction. We also predicted the binding mode of the 4,6-bis(4-chlorophenyl)pyrimidine analogs to the RAGE V-domain through flexible docking study.

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aß peptide.

BACKGROUND: The deposition and oligomerization of amyloid ß (Aß) peptide plays a key role in the pathogenesis of Alzheimer's disease (AD). Aß peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by ß and γ secretases. Several lines of evidence point to the soluble Aß oligomer (AßO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AßO species. METHODOLOGY/PRINCIPAL FINDINGS: To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aß peptide to investigate the binding and influence of fluorene compounds on AßO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AßO toxicity and a route to directly observe the binding of the fluorene to the AßO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AßO toxicity, scavenge free radicals and diminish the formation of intracellular AßO species. CONCLUSIONS: Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aß peptide toxicity, because they possess both antioxidant properties and the ability to disrupt AßO species.

Mitochondria-specific accumulation of amyloid ß induces mitochondrial dysfunction leading to apoptotic cell death.

Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid ß (Aß) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aß impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aß accumulation has been detected in the AD brain. However, the underlying mechanism of how Aß affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aß accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aß(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aß(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aß(1-42) in HT22 cells using Aß(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aß(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aß(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aß(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aß(1-42) accumulation, which mimics the apoptosis process in exogenous Aß(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aß(1-42) accumulation is the necessary and sufficient condition for Aß-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aß-mediated signaling alterations.

Intracellular amyloid-ß accumulation in calcium-binding protein-deficient neurons leads to amyloid-ß plaque formation in animal model of Alzheimer's disease.

One of the major hallmarks of Alzheimer's disease (AD) is the extracellular deposition of amyloid-ß (Aß) as senile plaques in specific brain regions. Clearly, an understanding of the cellular processes underlying Aß deposition is a crucial issue in the field of AD research. Recent studies have found that accumulation of intraneuronal Aß (iAß) is associated with synaptic deficits, neuronal death, and cognitive dysfunction in AD patients. In this study, we found that Aß deposits had several shapes and sizes, and that iAß occurred before the formation of extracellular amyloid plaques in the subiculum of 5XFAD mice, an animal model of AD. We also observed pyroglutamate-modified Aß (N3pE-Aß), which has been suggested to be a seeding molecule for senile plaques, inside the Aß plaques only after iAß accumulation, which argues against its seeding role. In addition, we found that iAß accumulates in calcium-binding protein (CBP)-free neurons, induces neuronal death, and then develops into senile plaques in 2-4-month-old 5XFAD mice. These findings suggest that N3pE-Aß-independent accumulation of Aß in CBP-free neurons might be an early process that triggers neuronal damage and senile plaque formation in AD patients. Our results provide new insights into several long-standing gaps in AD research, namely how Aß plaques are formed, what happens to iAß and how Aß causes selective neuronal loss in AD patients.

Ghrelin ameliorates cognitive dysfunction and neurodegeneration in intrahippocampal amyloid-ß1-42 oligomer-injected mice.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by cognitive deficits, neuroinflammation, and loss of neurons. Recently, it has been shown that ghrelin, a 28 amino acid peptide hormone produced from the stomach and hypothalamus, has been reported as a potential therapeutic agent for several neurological disorders, including Parkinson's disease (PD), stroke, epilepsy, multiple sclerosis, and spinal cord injury. Here we determined the effects of ghrelin on memory impairments and neuropathological changes in an AD mouse model induced by intrahippocampal injection of amyloid-ß oligomers (AßO). We report that ghrelin: 1) rescues memory deficits in mice injected with AßO in the hippocampus; 2) decreases AßO-induced microgliosis in hippocampus; 3) attenuates hippocampal neuronal loss mediated by AßO; 4) prevents AßO-associated synaptic degeneration including cholinergic fiber loss. Taken together, our findings demonstrate that ghrelin can ameliorate AßO-induced cognitive impairment associated with neuroinflammation and neuronal loss. These results suggest that ghrelin may be a promising therapeutic agent for the treatment of AD.

A differential association of Apolipoprotein E isoforms with the amyloid-ß oligomer in solution.

The molecular pathogenesis of disorders arising from protein misfolding and aggregation is difficult to elucidate, involving a complex ensemble of intermediates, whose toxicity depends upon their state of progression along distinct processing pathways. To address the complex misfolding and aggregation that initiates the toxic cascade resulting in Alzheimer's disease (AD), we have developed a 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin-labeled amyloid-ß (Aß) peptide to observe its isoform-dependent interaction with the apoE protein. Although most individuals carry the E3 isoform of apoE, ∼15% of humans carry the E4 isoform, which is recognized as the most significant genetic determinant for Alzheimer's. ApoE is consistently associated with the amyloid plaque marker for AD. A vital question centers on the influence of the two predominant isoforms, E3 and E4, on Aß peptide processing and hence Aß toxicity. We used electron paramagnetic resonance (EPR) spectroscopy of incorporated spin labels to investigate the interaction of apoE with the toxic oligomeric species of Aß in solution. EPR spectra of the spin-labeled side chain report on side chain and backbone dynamics as well as the spatial proximity of spins in an assembly. Our results indicate oligomer binding involves the C-terminal domain of apoE, with apoE3 reporting a much greater response through this conformational marker. Coupled with SPR binding measurements, apoE3 displays a higher affinity and capacity for the toxic Aß oligomer. These findings support the hypothesis that apoE polymorphism and Alzheimer's risk can largely be attributed to the reduced ability of apoE4 to function as a clearance vehicle for the toxic form of Aß.

FK506 reduces amyloid plaque burden and induces MMP-9 in AßPP/PS1 double transgenic mice.

Deposition of amyloid-ß peptide (Aß) and neurofibrillary tangles are pathological hallmarks of Alzheimer's disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. Recently, calcineurin (CaN) has been reported as a potential modulator of memory function, synaptic plasticity, and neural degeneration in brains of AD animal models. In the present study, we examined the relationship between Aß accumulations and CaN activity in brains of the AßPP/PS1 double transgenic mice. Treatment with FK506, a CaN inhibitor, significantly reduces Aß burden and restores synaptic proteins (synaptophysin and postsynaptic density protein-95; PSD-95) while inducing matrix metallopeptidase-9 (MMP-9) expression in GFAP-positive astrocytes in the brain. These results suggest a role of FK506 and control of CaN activity in neuroprotection associated with Aß deposition in AD.

Single-cell mechanics provides a sensitive and quantitative means for probing amyloid-beta peptide and neuronal cell interactions.

By using a highly sensitive technique of atomic force microscopy-based single-cell compression, the rigidity of cultured N2a and HT22 neuronal cells was measured as a function of amyloid-beta42 (Abeta42) protein treatment. Abeta42 oligomers led to significant cellular stiffening; for example, 90-360% higher force was required to reach 80% deformation for N2a cells. Disaggregated or fibrillar forms of Abeta42 showed much less change. These observations were explained by a combination of two factors: (i) incorporation of oligomer into cellular membrane, which resulted in an increase in the Young's modulus of the membrane from 0.9+/-0.4 to 1.85+/-0.75 MPa for N2a cells and from 1.73+/-0.90 to 5.5+/-1.4 MPa for HT22 cells, and (ii) an increase in intracellular osmotic pressure (e.g., from 7 to 40 Pa for N2a cells) through unregulated ion influx. These findings and measurements provide a deeper, more characteristic, and quantitative insight into interactions between cells and Abeta42 oligomers, which have been considered the prime suspect for initiating neuronal dysfunction in Alzheimer's disease.

Candidate anti-A beta fluorene compounds selected from analogs of amyloid imaging agents.

Alzheimer's disease (AD) is characterized by depositions of beta-amyloid (A beta) aggregates as amyloid in the brain. To facilitate diagnosis of AD by radioligand imaging, several highly specific small-molecule amyloid ligands have been developed. Because amyloid ligands display excellent pharmacokinetics properties and brain bioavailability, and because we have previously shown that some amyloid ligands bind the highly neurotoxic A beta oligomers (A beta O) with high affinities, they may also be valuable candidates for anti-A beta therapies. Here we identified two fluorene compounds from libraries of amyloid ligands, initially based on their ability to block cell death secondary to intracellular A beta O. We found that the lead fluorenes were able to reduce the amyloid burden including the levels of A beta O in cultured neurons and in 5xFAD mice. To explain these in vitro and in vivo effects, we found that the lead fluorenes bind and destabilize A beta O as shown by electron paramagnetic resonance spectroscopy studies, and block the harmful A beta O-synapse interaction. These fluorenes and future derivatives, therefore, have a potential use in AD therapy and research.

Secondary amyloidosis associated with multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. Secondary amyloidosis can occur as a complication of chronic systemic inflammatory and infectious diseases. Until now there has been no report of secondary amyloidosis associated with MS. We report herein a case of renal biopsy-proven secondary amyloidosis in a patient with MS. CASE REPORT: A 41-year-old woman with MS was hospitalized due to aggravated quadriparesis and edema in both lower extremities. Laboratory findings showed nephrotic-range proteinuria and hypoalbuminemia. A percutaneous renal biopsy procedure was performed, the results of which revealed secondary amyloid-A-type amyloidosis associated with MS. CONCLUSIONS: This is the first report of secondary amyloidosis associated with MS.

Inhibition of Alzheimer's amyloid toxicity with a tricyclic pyrone molecule in vitro and in vivo.

Small beta-amyloid (Abeta) 1-42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer's disease (AD). Methods to reduce the level of Abeta, prevent Abeta aggregation, and eliminate existing Abeta aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Abeta oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Abeta42 oligomer. Circular dichroism spectroscopy reveals monomeric Abeta42 peptide remains as a random coil/alpha-helix structure in the presence of CP2 over 48 h. Atomic force microscopy studies show CP2 exhibits similar ability to inhibit Abeta42 aggregation as that of Congo red and curcumin. Atomic force microscopy closed-fluid cell study demonstrates that CP2 disaggregates Abeta42 oligomers and protofibrils. CP2 also blocks Abeta fibrillations using a protein quantification method. Treatment of 5x familial Alzheimer's disease mice, a robust Abeta42-producing animal model of AD, with a 2-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Abeta species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Abeta aggregation and disaggregating existing Abeta oligomers and protofibrils.