Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.

3,6-diazabicyclo[3.3.1]heptanes Induces Apoptosis and Arrests Cell Cycle in Prostate Cancer Cells.

BACKGROUND Prostate cancer, non-cutaneous malignant tumor, is the second common cause of cancer related mortalities in American men and is responsible for 13% of deaths related to cancer. The present study investigated the anti-cancer effects of 3,6-diazabicyclo[3.3.1]heptane on LNCaP and PC3 prostate cancer cells in vitro and on tumor growth in vivo in BALB/C nude mice. MATERIAL AND METHODS Reduction of cell viability by 3,6-diazabicyclo[3.3.1]heptane was evaluated by sulphorhodamine-B staining and apoptosis onset using annexin V and propidium iodide (PI) staining. The 2',7'-dichlorofluorescein-diacetate stain was used for assessment of reactive oxygen species (ROS) formation while as western blotting for analysis of protein expression. RESULTS The viability of LNCaP and PC3 cells was reduced significantly (P<0.05) by 3,6-diazabicyclo[3.3.1]heptane in dose-based manner. At 30 µM of 3,6-diazabicyclo[3.3.1]heptane the viability of LNCaP and PC3 cells was reduced to 32 and 28%, respectively. The 3,6-diazabicyclo[3.3.1]heptane treatment increased apoptosis in LNCaP cells to 43.31% at 30 µM. The cell cycle in LNCaP cells was arrested in G1 phase on treatment with 3,6-diazabicyclo[3.3.1]heptane. The expression of cyclin D1 and p21 proteins was significantly increased by 3,6-diazabicyclo[3.3.1]heptane in LNCaP and PC3 cells. The growth of prostate tumor was also suppressed in vivo in mice by 3,6-diazabicyclo[3.3.1]heptane treatment. CONCLUSIONS In summary, the study demonstrated that LNCaP and PC3 prostate cancer cell viability is suppressed by 3,6-diazabicyclo[3.3.1]heptane treatment. The suppression of prostate cancer cell viability by 3,6-diazabicyclo[3.3.1]heptane involves apoptosis induction, cell cycle arrest and upregulation of p21 expression. Therefore, 3,6-diazabicyclo[3.3.1]heptane can be a potential chemotherapeutic agent for prostate cancer.

Design and synthesis of estrogen receptor ligands with a 4-heterocycle-4-phenylheptane skeleton.

The estrogen receptor (ER), a member of the nuclear receptor (NR) family, is involved in the regulation of physiological effects such as reproduction and bone homeostasis. Approximately 70% of human breast cancers are hormone-dependent and ERα-positive, and, thus, ER antagonists are broadly used in breast cancer therapy. We herein designed and synthesized a set of ER antagonists with a 4-heterocycle-4-phenylheptane skeleton.

Synthesis and antibacterial evaluation of macrocyclic diarylheptanoid derivatives.

Bacterial infections, caused by Mycobacterium tuberculosis and other problematic bacterial pathogens, continue to pose a significant threat to global public health. As such, new chemotype antibacterial agents are desperately needed to fuel and strengthen the antibacterial drug discovery and development pipeline. As part of our antibacterial research program to develop natural product-inspired new antibacterial agents, here we report synthesis, antibacterial evaluation, and structure-activity relationship studies of an extended chemical library of macrocyclic diarylheptanoids with diverse amine, amide, urea, and sulfonamide functionalities. Results of this study have produced macrocyclic geranylamine and 4-fluorophenethylamine substituted derivatives, exhibiting moderate to good activity against M. tuberculosis and selected Gram-positive bacterial pathogens.

An analysis on the suppression of NO and PGE2 by diphenylheptane A and its effect on glycerophospholipids of lipopolysaccharide-induced RAW264.7 cells with UPLC/ESI-QTOF-MS.

Diarylheptanoid A, 5-hydroxy-7-(4'-hydroxy-3'-methoxyphenyl)-1-phenyl-3-heptanone, is a naturally occurring phytochemical ingredient isolated from the rhizome of Alpinia officinarum. In order to confirm the anti-inflammatory activity of diphenylheptane A, we investigated its effects on lipopolysaccharide (LPS)-induced pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1ß (IL-1ß), and tumor necrosis factor α (TNF-α), as well as upstream genes, including the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor κB (NF-κB) p65, p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase 1/2 (ERK1/2). Our results have proved the anti-inflammatory property of diphenylheptane A. Based on this finding, an LPS-induced RAW264.7 cell inflammatory model was introduced to evaluate the anti-inflammatory activity associated with glycerophospholipid (GPL) metabolism regulated by diphenylheptane A. We applied ultra-performance liquid chromatography/electrospray ionization-quadruple time of flight-mass spectrometry (UPLC/ESI-QTOF-MS) to the metabolic profiling of GPL synthesis in LPS-stimulated macrophages with the aim of identifying differentially synthesized GPL metabolites. Sixteen GPL metabolites, whose changes were restored to normal level after diphenylheptane A treatment, were further screened to be considered as useful biomarkers of inflammation. Overall, our study revealed for the first time that diphenylheptane A reestablished the production of 16 plasma membrane GPLs to basal level in LPS-activated RAW264.7 cells, suggesting the potential therapeutic property of phytochemical compounds against inflammatory diseases.

7-(4-Hydroxyphenyl)-1-phenyl-4E-hepten-3-one, a Diarylheptanoid from Alpinia officinarum, Protects Neurons against Amyloid-ß Induced Toxicity.

Amyloid-ß (Aß) is one of the major causative agents of Alzheimer's disease (AD), the most common neurodegenerative disorder characterized by progressive cognitive impairment. While effective drugs for AD are currently limited, identifying anti-Aß compounds from natural products has been shown as a promising strategy which may lead to breakthroughs for new drug candidate discovery. We have previously reported that 7-(4-hydroxyphenyl)-1-phenyl-4E-hepten-3-one (AO-1), a diarylheptanoid extracted from the plant Alpinia officinarum, has strong effects on neuronal differentiation and neurite outgrowth in vitro and in vivo. The present study further uncovers that AO-1 exerts neuroprotective effects against the neurotoxicity caused by Aß. Under the damage of Aß oligomers, the major pathological forms of Aß, dendrites of neurons become atrophic and simplified, but such impairments were substantially alleviated by AO-1 treatment. Moreover, AO-1 reduced apoptotic levels and oxidative stress triggered by Aß. Further analysis showed that the anti-caspase and dendrite protective effects of AO-1 were dependent on activation of phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways. These findings collectively identify AO-1 as a beneficial compound to ameliorate the deleterious effects of Aß on dendrite integrity and cell survival, and may provide new insights on drug discovery of AD.

Neuroprotective and Anti-Inflammatory Effects of Diphenylheptanes from the Fruits of Amomum tsaoko, a Chinese Spice.

Two novel diphenylheptanes, 2,3- dihydro-2 - (4' - hydroxy-phenylethyl) - 6 - [(3″,4″ - dihydroxy-5" - methoxy) phenyl] -4 - pyrone (CG-A) and 4 - dihydro-2 - (4' - hydroxy-phenylmethyl) -6 - [(3",4″ - dihydroxy-5″ - methoxyphenyl) methylene]-pyran-3, 5 - dione (CG-B), were isolated from the dried fruits of Amomum tsaoko, a commercially important spice. This study was designed to investigate their protective effects against H2O2-induced nerve injury, using PC-12 cells to determine the cell cytotoxicity and cell viability. The inhibitory effect on (nitric oxide) NO production was also determined in (lipopolysaccharide) LPS-stimulated macrophage RAW 264.7 cells. The results showed that CG-A and CG-B displayed significant neuroprotective effect and exhibited anti-inflammatory activity in a dose-dependent manner. These findings suggest that CG-A and CG-B are very important nutritional ingredients responsible for the neuroprotective and anti-inflammatory health benefits of A. tsaoko.

Transnitrosylation directs TRPA1 selectivity in N-nitrosamine activators.

S-Nitrosylation, the addition of a nitrosyl group to cysteine thiols, regulates various protein functions to mediate nitric oxide (NO) bioactivity. Recent studies have demonstrated that selectivity in protein S-nitrosylation signaling pathways is conferred through transnitrosylation, a transfer of the NO group, between proteins via interaction. We previously demonstrated that sensitivity to activation by synthetic NO-releasing agents via S-nitrosylation is a common feature of members of the transient receptor potential (TRP) family of Ca(2+)-permeable cation channels. However, strategies to confer subtype selectivity to nitrosylating agents targeted to TRP channels are yet to be developed. Here, we show selective activation of TRPA1 channels by novel NO donors derived from the ABBH (7-azabenzobicyclo[2.2.1]heptane) N-nitrosamines, which exhibit transnitrosylation reactivity to thiols without releasing NO. The NNO-ABBH1 (N-nitroso-2-exo,3-exo-ditrifluoromethyl-7-azabenzobicyclo[2.2.1]heptane) elicits S-nitrosylation of TRPA1 proteins, and dose-dependently induces robust Ca(2+) influx via both recombinant and native TRPA1 channels, but not via other NO-activated TRP channels. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, suggesting that cysteine transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by the correlation of N-NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Interestingly, nonelectrophilic derivatives of ABBH also activate TRPA1 selectively, but less potently, compared with NNO-ABBH1. Thus, ABBH N-nitrosamines confer subtype selectivity on S-nitrosylation in TRP channels through synergetic effects of two chemical processes: cysteine transnitrosylation and molecular recognition of the nonelectrophilic moiety.

Potent inhibitors of malarial aspartic proteases, the plasmepsins, by hydroformylation of substituted 7-azanorbornenes.

The increasing prevalence of multidrug-resistant strains of the malarial parasite Plasmodium falciparum requires the urgent development of new therapeutic agents with novel modes of action. The vacuolar malarial aspartic proteases plasmepsin (PM) I, II, and IV are involved in hemoglobin degradation and play a central role in the growth and maturation of the parasite in the human host. We report the structure-based design, synthesis, and in vitro evaluation of a new generation of PM inhibitors featuring a highly decorated 7-azabicyclo[2.2.1]heptane core. While this protonated central core addresses the catalytic Asp dyad, three substituents bind to the flap, the S1/S3, and the S1' pockets of the enzymes. A hydroformylation reaction is the key synthetic step for the introduction of the new vector reaching into the S1' pocket. The configuration of the racemic ligands was confirmed by extensive NMR and X-ray crystallographic analysis. In vitro biological assays revealed high potency of the new inhibitors against the three plasmepsins (IC(50) values down to 6 nM) and good selectivity towards the closely related human cathepsins D and E. The occupancy of the S1' pocket makes an essential contribution to the gain in binding affinity and selectivity, which is particularly large in the case of the PM IV enzyme. Designing non-peptidic ligands for PM II is a valid route to generate compounds that inhibit the entire family of vacuolar plasmepsins.

Synthesis of 2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes via LiAlH4-induced reductive cyclization of 2-(4-chloro-2-cyano-2-phenylbutyl)aziridines and evaluation of their antimalarial activity.

2-(4-Chloro-2-cyano-2-phenylbutyl)aziridines were employed for the one-step stereoselective construction of both endo- and exo-2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes as new azaheterobicyclic scaffolds via a double LiAlH(4)-induced reductive cyclization protocol. Antiplasmodial assessment of these 1-azabicyclo[2.2.1]heptanes revealed moderate to good activities in the micromolar range, with the exo-isomers being the most promising structures. Furthermore, the proposed mode of action was supported by ligand docking studies, pointing to a strong binding interaction with the enzyme plasmepsin II.

7-Azabicyclo[2.2.1]heptane as a scaffold for the development of selective sigma-2 (σ2) receptor ligands.

A series of N-substituted 7-azabicyclo[2.2.1]heptanes (12-17 and 22-25) and similarly substituted pyrrolidines (32-36 and 41-44) were synthesized as sterically-reduced, achiral analogs of adamantane- and trishomocubane-derived σ ligands. In vitro competition binding assays against σ receptors revealed that arylalkyl N-substituents conferred selectivity for the σ(2) subtype, while alicyclic or polycarbocyclic substituents imparted high affinity for both subtypes. The σ(2) binding and subtype selectivities of N-arylalkyl-7-azanorbornanes was generally greater than the analogously-substituted pyrrolidines, indicating that steric bulk and conformational restriction around the nitrogen atom are likely important for subtype discrimination.

First total synthesis of prasinic acid and its anticancer activity.

The first total synthesis of prasinic acid is being reported along with its biological evaluation. The ten step synthesis involved readily available and cheap starting materials and can easily be transposed to large scale manufacturing. The crucial steps of the synthesis included the formation of two different aromatic units (7 and 9) and their coupling reaction. The synthetic prasinic acid exhibited moderate antitumor activity (IC(50) 4.3-9.1 µM) in different lines of cancer cells.

Synthesis of γ-Aminobutyric Acid (GABA) Analogues Conformationally Restricted by Bicyclo[3.1.0]hexane/hexene or [4.1.0]Heptane/heptene Backbones as Potent Betaine/GABA Transporter Inhibitors.

Novel γ-aminobutyric acid (GABA) analogues 3-5, having a bicyclo[3.1.0]hexene, [4.1.0]heptane, or [4.1.0]heptene backbone, respectively, were designed from the bioactive form analysis of the previous inhibitor 2 with a bicyclo[3.1.0]hexane backbone. Compounds 3-5 and 2 were synthesized from a common 1,7-diene intermediate 6 using ring-closing metathesis (RCM) to construct the key bicyclo backbones. Compounds 3-5 strongly inhibit betaine/GABA transporter 1 (BGT1) uptake, but compound 4 stands out with its selective low micromolar potency.

Synthesis and antiviral activity of cyclopropyl-spirocarbocyclic adenosine, (4R,5S,6R,7R)-4-(6-amino-9H-purin-9-yl)-7-(hydroxymethyl)spiro[2.4]heptane-5,6-diol against hepatitis C virus.

An efficient method was developed for the synthesis of 6-exocyclic methylene carbocyclic intermediate 4. The Simmons-Smith cyclopropanation protocol was applied on the 6-exocyclic methylene of intermediate 4 and demonstrated its utility for the synthesis of novel class of a spiro-carbocyclic nucleoside analog 8. The titled compound 8 demonstrated a significant antiviral activity against HCV with EC(50) values of 0.273 and 0.368 µM in genotypes 1A and 1B, respectively.

7-azabicyclo[2.2.1]heptane as a structural motif to block mutagenicity of nitrosamines.

Nitrosamines are potent carcinogens and toxicants in the rat and potential genotoxins in humans. They are metabolically activated by hydroxylation at an α-carbon atom with respect to the nitrosoamino group, catalyzed by cytochrome P450. However, there has been little systematic investigation of the structure-mutagenic activity relationship of N-nitrosamines. Herein, we evaluated the mutagenicity of a series of 7-azabicyclo[2.2.1]heptane N-nitrosamines and related monocyclic nitrosamines by using the Ames assay. Our results show that the N-nitrosamine functionality embedded in the bicyclic 7-azabicylo[2.2.1]heptane structure lacks mutagenicity, that is, it is inert to α-hydroxylation, which is the trigger of mutagenic events. Further, the calculated α-C-H bond dissociation energies of the bicyclic nitrosamines are larger in magnitude than those of the corresponding monocyclic nitrosamines and N-nitrosodimethylamine by as much as 20-30 kcal/mol. These results are consistent with lower α-C-H bond reactivity of the bicyclic nitrosamines. Thus, the 7-azabicyclo[2.2.1]heptane structural motif may be useful for the design of nongenotoxic nitrosamine compounds with potential biological/medicinal applications.

1-Heteroaryl-6-(3,4-dichlorophenyl)-3-azabicyclo[4.1.0]heptane: further insights into a class of triple re-uptake inhibitors.

Further exploration around the recently disclosed potent triple re-uptake inhibitor 6-(3,4-dichlorophenyl)-1-[(methyloxy)methyl]-3-azabicyclo[4.1.0]heptane led to the identification of a new series of potent triple re-uptake inhibitors endowed with good developability characteristics. The insertion of a further aryl moiety into the template allowed the 'titration' of the SERT/NET/DAT ratio leading to the identification of further tools in this important area.

Syntheses and structure-activity relationship (SAR) studies of 2,5-diazabicyclo[2.2.1]heptanes as novel alpha7 neuronal nicotinic receptor (NNR) ligands.

Biaryl substituted 2,5-diazabicyclo[2.2.1]heptanes have been synthesized and tested for their affinity toward alpha7 neuronal nicotinic receptors (NNRs). SAR studies established that 5-N-methyl substituent, heteroaryl linker and the nature of terminal aryl group are critical for the ligand to achieve potent alpha7 NNR agonist activity.

The effect of Tween 80 on eggshell permeabilization in Galleria mellonella (L.) (Lepidoptera, Pyralidae).

The development of a species-specific protocol for dechorionation and permeabilization of insect eggs is a necessary prerequisite to cryopreserve the embryos. Here we tested different procedures based on heptane or the surfactant Tween 80 as an alternative to alkane, evaluating their efficacy and toxicity on the early (24 h post-oviposition) and late (75 h post-oviposition) stage embryos. Heptane efficiently permeabilized the eggs of G. mellonella but the hatching rate ranged from 0.1 to 4.2 percent in the early stage and from 4.3 to 11.2 percent in the late stage. The embryos treated with 1.25 percent NaOCl + 0.08 percent Tween 80 for 2 min showed the same shrinkage and reswelling percentages as eggs exposed to heptane for 10 sec, with a significantly higher hatching percentage in the early (68.2 +/- 1.5 percent) and late stages (22.4 +/- 3.7 percent). Thus, 0.08 percent Tween 80 allows sufficient permeabilization of G. mellonella embryos without the high toxicity of alkane.

Sesquiterpene-like inhibitors of a 9-cis-epoxycarotenoid dioxygenase regulating abscisic acid biosynthesis in higher plants.

Abscisic acid (ABA) is a carotenoid-derived plant hormone known to regulate critical functions in growth, development and responses to environmental stress. The key enzyme which carries out the first committed step in ABA biosynthesis is the carotenoid cleavage 9-cis-epoxycarotenoid dioxygenase (NCED). We have developed a series of sulfur and nitrogen-containing compounds as potential ABA biosynthesis inhibitors of the NCED, based on modification of the sesquiterpenoid segment of the 9-cis-xanthophyll substrates and product. In in vitro assays, three sesquiterpene-like carotenoid cleavage dioxygenase (SLCCD) inhibitor compounds 13, 17 and 18 were found to act as inhibitors of Arabidopsis thaliana NCED 3 (AtNCED3) with K(i)'s of 93, 57 and 87 microM, respectively. Computational docking to a model of AtNCED3 supports a mechanism of inhibition through coordination of the heteroatom with the non-heme iron in the enzyme active site. In pilot studies, pretreatment of osmotically stressed Arabidopsis plants with compound 13 resulted lower levels of ABA and catabolite accumulation compared to levels in mannitol-stressed plant controls. This same inhibitor moderated known ABA-induced gene regulation effects and was only weakly active in inhibition of seed germination. Interestingly, all three inhibitors led to moderation of the stress-induced transcription of AtNCED3 itself, which could further contribute to lowering ABA biosynthesis in planta. Overall, these sesquiterpenoid-like inhibitors present new tools for controlling and investigating ABA biosynthesis and regulation.

Filbertone Ameliorates Adiposity in Mice Fed a High-Fat Diet via Activation of cAMP Signaling.

The aim of this research was to estimate the preventive effects of filbertone, the main flavor compound in hazelnuts, on lipid accumulation in the adipose tissue of mice fed a high-fat diet (HFD) and to reveal the underlying molecular mechanisms. Male C57BL/6N mice were fed chow, a HFD, or a 0.025% filbertone-supplemented HFD for 14 weeks. We found that filbertone supplementation resulted in significant reductions in body weight gain and lipid accumulation in adipose tissue, with parallel improvements in plasma lipid levels (triglycerides, total cholesterol, and free fatty acids) and proinflammatory cytokines (interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α)). Molecular analysis revealed that filbertone treatment led to reprogramming of metabolic signatures in the cyclic adenosine monophosphate (cAMP) pathway. Filbertone supplementation significantly increased the cAMP level and increased downstream protein kinase A catalytic subunit (PKA) signaling in mouse adipose tissue. The mRNA level of adipogenesis-related genes was downregulated in the adipose tissue of filbertone-fed mice compared to control mice fed the HFD alone. Furthermore, filbertone treatment elevated the expression of thermogenic genes in mouse adipose tissue. Filbertone reduced intracellular lipid accumulation and increased the oxygen consumption rate in 3T3-L1 cells and these filbertone-induced changes were abrogated by the adenylate cyclases (ADCY) inhibitor. Taken together, our results suggest that the beneficial effects of filbertone on lipid accumulation may be associated with the activation of cAMP signaling.