Absence Of Compound Research Articles

Targeted protein degradation via intramolecular bivalent glues

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)—bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1–4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change ‘glues’ BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target–ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4–IBG1–DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

Alteration in Mechanical Compliance via Mast Cell Activation in Juvenile and Adult Urinary Bladder

Balance between bladder wall stiffness and mechanical compliance is essential to maintain normal bladder function. These biomechanical properties are directly influenced by the integrity of the extracellular matrix (ECM) and its interaction with urinary bladder smooth muscle (UBSM). While it is clear that normal storage and voiding functions differ between juveniles and adults, it is unclear if these differences are due solely to increases in bladder size. Differences in the mechanical properties of the wall may also exist and are unexplored. Changes in mechanical compliance are implicated in the pathophysiology of many lower urinary tract symptoms (LUTS) such as overactivity and fibrosis due to inflammation. Interestingly, juvenile bladders are particularly susceptible to inflammatory LUTS by various factors (e.g., early life stress) compared to their adult counterparts. We were interested in how bladders from juvenile and adult mice respond to inflammation, specifically driven by mast cell degranulation. We hypothesized that: (1) juvenile bladders are inherently more compliant than adult bladders and (2) mast cell degranulation increases mechanical compliance in both juvenile and adult mouse bladders.Whole mouse bladders from juvenile (4–5 weeks old) and adult (12–14 weeks old) C57Bl/6 male mice were dissected and mounted in a custom designed pentaplanar reflected image macroscopy (PRIM) system for simultaneous measurement of transient pressure events, intravesical pressure, bladder volume, wall stress, and wall stretch during ex vivo bladder filling. Mechanical properties of the bladder wall were measured as the relationship between mechanical stress versus stretch. Properties of the transient contractions were calculated using acquired pressure-volume curves. Measurements were performed in the absence or presence of the mast cell activator Compound 48/80 (10 μg/mL).In the absence of Compound 48/80, the average stress-stretch curves had no significant shift for adult mice compared to young mice, implying no changes in mechanical compliance. However, the mechanical stress in adult mouse bladders was higher compared to juvenile bladders, implying that adult bladders were stiffer. In presence of Compound 48/80, both age groups show similar effects. Mechanical compliance increased with a paradoxical increase in amplitude of transient contractions. Together, these data suggest that while juvenile mouse bladders are less stiff, they respond to mast cell activation in a similar manner as adult bladders. Consequently, rapid degranulation of mast cells in juvenile bladders leads to increased mechanical compliance and contractility. Thus, preventing mast cell degranulation could represent a viable target for treating LUTS in children as well as adults. R01-DK119615 (NRT) and P20-DK127554 (SR and NRT) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Investigation of Secondary Metabolites from Marine‐Derived FungiAspergillus

AbstractMarine fungiAspergillus flavipesandA. terreuswere investigated for secondary metabolites using electrospray ionization‐quadrupole time‐of‐flight mass spectrometry (ESI‐QTOF MS) analysis. The ESI‐MS analysis showed thatA. flavipesshowed molecular ion peaks for compounds, terrein(1), butyrolactone I(2), butyrolactone I‐carboxylic acid(3), butyrolactone III(4), while,A. terreushad additional presence of new compound aspernolide Q(5)with absence of compound(3). By using tandem mass spectrometry data, the prominent fragment ions helped in the establishing the structure of compounds(2‐5). Further, chemical transformation of butyrolactone I to butyrolactone III and aspernolide Q were carried out followed by NMR analysis which strongly suggested their structures. The evaluation of salt stress experiment ofA. terreusculture medium affected the production of major bioactive metabolites, terrein and butyrolactone I(2). The HPLC analysis showed that culture produces high amount of terrein at 35 ‰, while, maximum production of butyrolactone I was observed in fresh water i. e. 0‰. The study highlighted an importance of salinity in producing these metabolites where, terrein and butyrolactone I could be produced in equal ratio at 18‰. Importantly, we have demonstrated that using this fungal strain and by growing in medium at specific salinity could be way forward for commercial production of these bioactive compounds.

Foldscope Embedded Pedagogy in Stem Education: A Case Study of SDG4 Promotion in India

Most government schools and colleges run on low budgets in India, yet they cater to a large population of students. In government settings, biology labs lack basic equipment such as simple microscopes or compound microscopes or sometimes possess just one instrument. In the absence of compound or simple microscopes, the students lose interest in science. To solve this issue, the Department of Biotechnology, Ministry of Science and Technology, Government of India, introduced the origami microscope, the foldscope, into the Indian educational system. In this article, we describe the design of a sustainable, inclusive, and equitable curricula for teaching biological concepts using the foldscope. We describe the implementation of foldscope-adapted curricula in post-secondary settings to teach natural selection, developmental biology, parasitology, and economic Zoology via individual, small-group, and large-group field trips, and project-based learning that involves experiential learning. We found that these culturally sensitive pedagogies involving translingual instructions had the potential to retain students and make science education accessible for the linguistically and culturally diverse population of India. We could successfully implement our project as per the five priority action areas outlined by UNESCO. Therefore, we propose the adoption of the foldscope-adapted curricula under SDG4 to make STEM education accessible in budget-frugal settings.

Crataegus aronia prevents high-fat diet-induced hepatic steatosis in rats by activating AMPK-induced suppression of SREBP1 and activation of PPARα.

This study examined if the aqueous extract of Crataegus aronia (C. aronia) can prevent high-fat diet (HFD)-induced hepatic steatosis in rats by activating AMPK. Adult male Wistar rats were fed either a control diet or HFD for 12weeks and treated either with vehicle (normal saline) or C. aronia extract (200mg/kg/orally), daily. Also, hepatocytes were treated with increasing concentrations of the extract in the presence or absence of compound C (CC), an AMPK inhibitor. C. aronia prevented the increase in serum and hepatic lipids, reduced hepatic levels of reactive oxygen species, and increased hepatic glutathione and superoxide dismutase levels. It also downregulated the hepatic expression of SREBP1/2, fatty acid synthase, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase but stimulated the activity of AMPK and levels of peroxisome proliferator-activated receptor-alpha. Similar effects were reported in the cultured cells, in a dose-dependent manner but were prevented by CC. In conclusion, C. aronia ameliorates HFD-induced hepatic steatosis and oxidative stress by activating AMPK. PRACTICAL APPLICATIONS: The use of the aqueous extract of Crataegus aronia has been extensively used during the last years in traditional medicine to treat chronic disorders including nonalcoholic fatty liver disease. The findings of this study support these findings and suggest that oral administration of C. aronia aqueous extract has potent hypoglycemic effect and demonstrate the mechanism of action mimics such drugs such as metformin and involves activation of AMPK and peroxisome proliferator-activated receptor-alpha. These findings are very encouraging for further biochemical analysis and isolation of active ingredients responsible for these effects to be used in more clinical trials.

Effect of Ginsenoside-Rh2 and Curcurbitacin-B on Cryptosporidium parvum in vitro

Ginsenoside-Rh2 and cucurbitacin-B (CuB) are secondary metabolites of Ginseng (Panax ginseng) and Cucurbitaceae plants respectively. We assessed the anticryptosporidial activity of these two functional compounds in a cell culture model of cryptosporidiosis. The highest concentration of each compound that was not toxic to the host cells was used to assess the activity against C. parvum during infection/invasion and growth in HCT-8 cell monolayers. Monolayers were infected with pre-excysted C. parvum oocysts. Infected monolayers were incubated at 37 °C for 24 h and 48 h in the presence of different concentrations of each test compound. A growth resumption assay was performed by incubating infected monolayers in the presence of compounds for 24 h followed by a second 24-h incubation in the absence of compound. To screen for invasion inhibiting activity, freshly excysted C. parvum sporozoites were pre-treated with different concentrations of compounds prior to adding them to the cell monolayers. Paromomycin, a known inhibitor of C. parvum, and DMSO were used as positive and negative control, respectively. The level of infection was initially assessed using an immunofluorescent assay and quantified by real-time PCR. Both compounds were found to strongly inhibit C. parvum intracellular development in a dose-dependent manner. IC50 values of 25 μM for a 24 h development period and 5.52 μM after 48 h development were measured for Rh2, whereas for CuB an IC50 value of 0.169 μg/ml and 0.118 μg/ml were obtained for the same incubation periods. CuB also effectively inhibited resumption of growth, an activity that was not observed with Rh2. CuB was more effective at inhibiting excystation and/or host cell invasion, indicating that this compound also targets extracellular stages of the parasite.

Naringenin improves mitochondrial function and reduces cardiac damage following ischemia-reperfusion injury: the role of the AMPK-SIRT3 signaling pathway.

Mitochondrial dysfunction contributed greatly to myocardial ischemia-reperfusion (MI/R)-induced cardiomyocyte apoptosis. Naringenin is a flavonoid exhibiting potential protective effects on myocardial mitochondria under stress conditions. However, the detailed down-stream signaling pathway involved remains uncovered. This study was designed to elucidate naringenin's mitochondrial protective actions during MI/R with a focus on AMPK-SIRT3 signaling. Sprague-Dawley rats were administered with naringenin (50 mg kg-1 d-1) and subjected to MI/R surgery in the presence or absence of compound C (0.25 mg kg-1, Com.C, an AMPK inhibitor) co-treatment. An in vitro study was performed on H9c2 cardiomyoblasts subjected to simulated ischemia-reperfusion treatment. Before the treatment, the cells were administered with naringenin (80 μmol L-1) with or without SIRT3 siRNA/AMPK1α siRNA transfection. Naringenin improved post-reperfusion left ventricular systolic pressure and the instantaneous first derivative of left ventricular pressure, and reduced the infarction size and myocardial apoptosis index by suppressing mitochondrial oxidative stress damage (as evidenced by decreased mitochondrial cytochrome c release and oxidative markers) and enhancing mitochondrial biogenesis [as evidenced by increased NRF1, TFAM and oxidative phosphorylation subunit complexes (II, III and IV)]. These protective actions were abolished by Com.C (in vivo) or SIRT3 siRNA (in vitro) administration. Further investigation revealed that Com.C (in vivo) or AMPK1α siRNA (in vitro) markedly suppressed PGC-1α and SIRT3 levels while SIRT3 siRNA (in vitro) inhibited SIRT3 expression without significantly changing AMPK phosphorylation and PGC-1α levels. Taken together, we found that naringenin directly inhibits mitochondrial oxidative stress damage and preserves mitochondrial biogenesis, thus attenuating MI/R injury. Importantly, AMPK-SIRT3 signaling played a key role in this process.

Use of Molecular Weight and Elemental Composition as an Additional Constraint in Library Search

Identification of organic compounds based only on searching against electron ionization mass spectral libraries is tentative. To increase the reliability of the identification, additional information should be used. Gas chromatographic retention indices, in addition to mass spectra, are most often used. Molecular weight and elemental composition is less frequently used, because additional experiments are required to determine these characteristics. In the present paper, we examined the influence of the molecular weight, elements present in a molecule and molecular formula on the results of the library search. It is shown that, determination of the nominal mass or molecular formula slightly increases efficiency of the library search (for example, probability that the correct compound occupies the first position in the list of possible candidates increases by only 2.8 and 3.0% respectively). On the other hand, use of additional constraints in the library search can significantly reduce the number of possible candidates and simplify predicting the absence of compound in the database.

Beta estradiol and norepinephrine treatment of differentiated SH-SY5Y cells enhances tau phosphorylation at (Ser396) and (Ser262) via AMPK but not mTOR signaling pathway

Hyperphosphorylation of tau is one of the main hallmarks for Alzheimer's disease (AD) and many other tauopathies. Norepinephrine (NE), a stress-related hormone and 17-β-estradiol (E2) thought to influence tau phosphorylation (p-tau) and AD pathology. The controversy around the impact of NE and E2 requires further clarification. Moreover, the combination effect of physiological and psychological stress and estrogen alteration during menopause, which affect p-tau, has not been addressed. Exposure to E2 is believed to reduce NE release, however, the link between these two hormones and AD at cellular level was also remained unknown. Here, we examined whether NE and E2 treatment of differentiated SH-SY5Y cells affected tau phosphorylation. The involvement of adenosine monophosphate kinase protein kinase (AMPK) and target of Rapamycin (mTOR) as the possible mechanisms, underlying this effect was also investigated. Subsequent to SH-SY5Y differentiation to mature neurons, we treated the cells with NE, E2 and NE plus E2 in presence and absence of Compound C and Rapamycin. Cell viability was not affected by our treatment while our Western blot and immunofluorescent findings showed that exposure to NE and E2 separately, and in combination enhanced p-tau (Ser396) and (Ser262)/tau but not (Ser202/Thr205)/tau. Blocking AMPK by Compound C reduced p-tau (Ser396) and (Ser262), while GSK-3β and PP2A activities were remained unchanged. We also found that blocking mTOR by Rapamycin did not change increased p-tau (Ser396) and (Ser262) due to NE + E2 treatment. Collectively, our results suggested that tau hyperphosphorylation due to exposure to NE/E2 was mediated by AMPK, the main energy regulator of cells during stress with no significant involvement of mTOR, GSK-3β and PP2A.

Diallyl trisulfide exerts cardioprotection against myocardial ischemia-reperfusion injury in diabetic state, role of AMPK-mediated AKT/GSK-3β/HIF-1α activation.

Diallyl trisulfide (DATS), the major active ingredient in garlic, has been reported to confer cardioprotective effects. However, its effect on myocardial ischemia-reperfusion (MI/R) injury in diabetic state and the underlying mechanism are still unknown. We hypothesize that DATS reduces MI/R injury in diabetic state via AMPK-mediated AKT/GSK-3β/HIF-1α activation. Streptozotocin-induced diabetic rats received MI/R surgery with or without DATS (20mg/kg) treatment in the presence or absence of Compound C (Com.C, an AMPK inhibitor, 0.25mg/kg) or LY294002 (a PI3K inhibitor, 5mg/kg). We found that DATS significantly improved heart function and reduced myocardial apoptosis. Additionally, in cultured H9c2 cells, DATS (10μM) also attenuated simulated ischemia-reperfusion injury. We found that AMPK and AKT/GSK-3β/HIF-1α signaling were down-regulated under diabetic condition, while DATS markedly increased the phosphorylation of AMPK, ACC, AKT and GSK-3β as well as HIF-1α expression in MI/R-injured myocardium. However, these protective actions were all blunted by Com.C administration. Additionally, LY294002 abolished the stimulatory effect of DATS on AKT/GSK-3β/HIF-1α signaling without affecting AMPK signaling. While 2-methoxyestradiol (a HIF-1α inhibitor) reduced HIF-1α expression without affecting AKT/GSK-3β signaling. Taken together, these data showed that DATS protected against MI/R injury in diabetic state by attenuating cellular apoptosis via AMPK-mediated AKT/GSK-3β/HIF-1α signaling. Its cardioprotective effect deserves further study.

Abstract 3037: Tumor cell death triggered by sustained IRES inhibition is a population-wide event involving a quorum-sensing mechanism in which putative tumor stem cells are eliminated along with their progeny

Abstract The internal ribosome entry site (IRES) is a feature contained within the 5’-untranslated region of the mRNA which allows the efficiency with which that mRNA is translated to be regulated independently of the general rate of protein synthesis in the cell. It appears that tumor cells rely on IRES-mediated translation to promote their own survival under adverse microenvironmental conditions, ultimately contributing to chemoresistance, relapse, and metastasis. Our lab has identified and characterized a series of compounds (prototype IRES inhibitors) which selectively modulate IRES function. Here we examine the phenotypic consequences of sustained IRES inhibition in triple-negative breast carcinoma and glioblastoma. 72 hours continuous exposure to the IRES inhibitor results in sudden loss of viability affecting >99.9% of the tumor cell population. Cell death is a consequence of the inhibition of synthesis of critical IRES-driven proteins, and not a typical cytotoxic response. The treated cells exhibit prominent features of terminal differentiation, with marked gains in cytoskeletal organization and planar polarity, extensive intercellular networking, and formation of tight junctions or neuronal processes. In addition to depletion of Myc protein, specific changes in IGF1R, connexin 43, BiP, and CHOP also correlate with phenotypic outcome. The highly undifferentiated triple-negative breast tumor and glioblastoma cells apparently cannot tolerate the transition to a fully differentiated state, or the restoration of normal homeostatic mechanisms allows the abnormalities that cause these cells to be malignant to be recognized. Once triggered, cell death is rapid and comprehensive. PARP cleavage is not observed, caspase 3/7 activation is limited, and no protection afforded by Z-VAD-fmk. The cells show no evidence of recovery even following prolonged incubation in the absence of compound. In contrast, normal human mammary epithelial cells (which are already differentiated) are resistant to IRES inhibition. The synchronized cell death response to IRES inhibition is a highly orchestrated event, with the population of malignant cells becoming physically linked together and responding as a unit, in contrast to typical stochastic processes such as apoptosis, programmed necrosis, and autophagy, where cell death decisions are made at the level of the individual cell. Importantly, tumor stem cells, which typically are resilient to conventional cytotoxic or targeted therapeutic regimens, appear to be vulnerable to IRES inhibition. Together, these findings support the concept that IRES-mediated translation is critical for maintenance of the undifferentiated phenotype. IRES inhibition potentially represents a new strategy for treatment of undifferentiated tumor types such as triple-negative breast cancer and glioblastoma. Citation Format: Christos Vaklavas, William E. Grizzle, Hyoungsoo Choi, Zheng Meng, Kurt R. Zinn, Scott W. Blume. Tumor cell death triggered by sustained IRES inhibition is a population-wide event involving a quorum-sensing mechanism in which putative tumor stem cells are eliminated along with their progeny. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3037.

Resveratrol ameliorates high glucose-induced oxidative stress injury in human umbilical vein endothelial cells by activating AMPK.

To investigate the effects of resveratrol on high glucose (HG)-induced vascular injury, and to establish the mechanism(s) underlying these effects. Human umbilical vein endothelial cells (HUVECs) were treated with glucose, and then incubated with resveratrol in the presence or absence of Compound C, an AMP-activated protein kinase (AMPK) inhibitor. Cell viability was determined using the Cell Counting Kit-8 (CCK-8) method. Reactive oxygen species, malondialdehyde, and superoxide dismutase were detected by flow cytometry, thiobarbituric acid reaction, and the nitroblue tetrazolium method, respectively. Protein levels of total and phosphorylated AMPKα and acetyl-CoA carboxylase were detected by immunoblotting. Resveratrol significantly ameliorated HG-induced decreases in cell viability and superoxide dismutase levels and increases in reactive oxygen species and MDA levels. Moreover, resveratrol significantly reversed HG-induced dephosphorylation of AMPKα and acetyl-CoA carboxylase. However, treatment with Compound C curtailed the beneficial effects of resveratrol on HG-treated HUVECs. Resveratrol ameliorates HG-induced injury in HUVECs by activation of AMPKα, leading to increased cellular reductive reactions and decreased oxidative stress. These results provide further evidence for resveratrol-mediated activation of AMPKα.

AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa

Spermatozoa undergo energy- and metabolism-dependent processes to successfully fertilize the oocyte. AMP-activated protein kinase, AMPK, is a sensor of cell energy. We recently showed that AMPK controls spermatozoa motility. Our aims are i) to investigate the intracellular localization of AMPK in boar spermatozoa by immunofluorescence, ii) to study whether AMPK plays a role in other relevant processes of spermatozoa: mitochondrial membrane potential (∆Ψm), plasma membrane lipid disorganization, outward phosphatidylserine (PS) exposure, acrosome integrity and induced-acrosome reaction by flow cytometry and iii) to investigate intracellular AMPK pathways by western blot. Spermatozoa were incubated under different conditions in the presence or absence of compound C (CC, 30μM), an AMPK inhibitor and/or cAMP analog 8Br-cAMP. AMPKα protein is expressed at the entire acrosome and at the midpiece of spermatozoa flagellum, whereas phospho-Thr172-AMPK is specifically localized at the apical part of acrosome and at flagellum midpiece. CC treatment rapidly confers head-to-head aggregation-promoting property to spermatozoa. Long term AMPK inhibition in spermatozoa incubated in TCM significantly reduces high ∆Ψm. Moreover, AMPK inhibition significantly induces plasma membrane lipid disorganization and simultaneously reduces outward PS translocation at plasma membrane in a time-dependent manner. Acrosomal integrity in TCM is significantly enhanced when AMPK is inhibited. However, neither acrosome reaction nor membrane lipid disorganization induced by ionophore A23187 are affected by CC. AMPK phosphorylation is potently stimulated upon PKA activation in spermatozoa. This work suggests that AMPK, lying downstream of PKA, regulates at different levels mammalian spermatozoa membrane function.

Abstract 4369: Compound 1, a quinoxaline-based small molecule, regulates the cancer cell proliferantion by the down-regulation of maternal embryonic leucine zipper kinase gene expression in brain tumor cells.

Abstract Maternal embryonic leucine zipper kinase (MELK), a member of the snf1/AMPK family of serine-threonine kinase, was indicated in stem cell renewal, cell cycle progression. Recently, several groups reported that MELK expression level is highly correlated with malignancy of a variety of tumors and its expression in the cancer cells is a negative factor of radiotherapy in cancer patients. Therefore, MELK can be a good target for cancer drug development. Previously, we tested the neuronal cell differentiation by the compound-1, a quinoxaline-based small molecule, with rat mesenchymal stem cells, and we found that the gene expression level of MELK is dramatically decreased in the differentiated cells in comparison with the parental stem cells. Based on this phenomenon, we checked the MELK gene expression in the several central nerve system cancer cell lines such as U87MG, XL498 and SNB19, and examined the change of the gene expression level by treatment of compound 1. All the cancer cells tested in this study expressed the MELK gene and its expression level is significantly decreased by the treatment of 20 uM compound 1. To confirm the relationship between the MELK gene expression and cell proliferation in brain tumor cells, we transfected the MELK gene to the U87MG cells and tested the cell growth rate in the presence or absence of compound 1 in comparison with parental cells. The cell growth rate of MELK gene-transfected cancer cells increased in comparison with the non-transfected control cells, and its overexpression can recover the down-regulation of the cell proliferation by treatment of compound 1. Meanwhile, the treatment of MELK gene siRNA decreased the cell proliferation rate of the U87MG cells in time dependent manner. In the cell cycle analysis, compound 1 induced both the increase of apoptotic cell rate from less than 5% to 22% and the decrease of G0/G1 phase U87MG cells in comparison with control. Meanwhile, the S and G2/M phase is not changed by the treatment of compound 1 in the U87MG cells. These results support MELK has an effect on regulation of cancer cell proliferation, and it may be a good target for cancer drug development. Citation Format: Sang-Un Choi, Chi Hoon Park, Mansoor M. Ahmed, Stephen Yoo, Shin-Young Park, Kwangrok Kim. Compound 1, a quinoxaline-based small molecule, regulates the cancer cell proliferantion by the down-regulation of maternal embryonic leucine zipper kinase gene expression in brain tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4369. doi:10.1158/1538-7445.AM2013-4369

English

The active compounds, cis-3-(2',4',5'-trimethoxyphenyl)-4-{(E)-2''',4''',5'''-trimethoxystyryl}cyclohex-1-ene (Compound C) and (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol (Compound D), have been identified in hexane fraction of Zingiber cassumunar Roxb., the medicinal plant which has been used for pain relief in arthritis including osteoarthritis (OA) and rheumatoid arthritis (RA). It was therefore interesting to investigate the chondroprotective activity of these compounds in vitro. Articular cartilage explants were cultured in the culture media containing 7 ng/ml of interleukin-1β (IL-1β), in the presence or absence of Compound C or Compound D at the concentration range of 1 to 100 μM. It was found that these compounds at concentrations of 10 and 100 μM significantly inhibited the IL-1β-induced cartilage degeneration by conserving the content of the cartilage matrix biomolecules such as collagen and uronic acid (UA) within the cartilage explants; and also resulted in the decline of releasing sulfated glycosaminoglycans and hyaluronic acid (HA) into the culture media. The increase in activities of matrix metalloproteinase-2 (MMP-2) and MMP-13 caused by IL-1β was significantly diminished by Compound C and Compound D. Additionally, the results showed no significant difference between the two active constituents and diacerein, an anti-arthritic agent used in OA, in those activities at a concentration of 100 μM. This is a pioneering evidence that indicate the potential chondroprotective property of the Z. cassumunar active compounds.   Key words: Zingiber cassumunar, chondroprotective activity, cartilage explant, rheumatoid arthritis, osteoarthritis, cis-3-(2',4',5'-trimethoxyphenyl)-4-{(E)-2''',4''',5'''-trimethoxystyryl}cyclohex-1-ene, (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol.

Active Compound of <em>Zingiber Cassumunar</em> Roxb. Down-Regulates the Expression of Genes Involved in Joint Erosion in a Human Synovial Fibroblast Cell Line

Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovium. It is involved in up-regulation of pro-inflammatory cytokines and matrix metalloproteinases (MMPs), resulting in joint inflammation and erosion. Zingiber cassumunar Roxb. has long been used to reduce joint pain and inflammation. This study aimed to investigate the inhibitory activities of an active compound of Z. cassumunar, (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol (compound D), against cytokine-induced up-regulation of catabolic genes involved in cartilage degradation in RA. Synovial fibroblast cell line, SW982, was cultured in media containing interleukin-1β (IL-1β), in the presence or absence of compound D at the concentration range of 1 to 100 µM. After 24 hours, the cells were analyzed for the expressions of MMPs, IL-1β and interleukin-1β-converting enzyme (ICE) by RT-PCR. MMPs activities in the culture media were analyzed by zymographic techniques. Dexamethasone was used as the positive control. It was found that compound D at the concentration of 10 - 100 µM significantly decreased the mRNA expressions of MMP-1, -2, -3, and -13 which was induced by IL-1β (P<0.05) concomitantly with a decrease in activities of these MMPs in the culture media. An increase in the mRNA expression of IL-1β and ICE was also suppressed by compound D. The results suggest that the potent activities of this compound may be involved in the reduction of IL-1β protein synthesis in both pro-form and active form which played an important role in up-regulation of MMPs. This study first revealed the chondroprotective activity of Z. cassumunar in the transcriptional level by suppressing cytokine-induced catabolic genes which caused cartilage erosion in RA.

Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3β by Tideglusib

Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [(35)S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.

SRT1720, SRT2183, SRT1460, and Resveratrol Are Not Direct Activators of SIRT1

Sirtuins catalyze NAD(+)-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1-7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.

Vaccinia virus p37 interacts with host proteins associated with LE-derived transport vesicle biogenesis

BackgroundProteins associated with the late endosome (LE) appear to play a central role in the envelopment of a number of taxonomically diverse viruses. How viral proteins interact with LE-associated proteins to facilitate envelopment is not well understood. LE-derived transport vesicles form through the interaction of Rab9 GTPase with cargo proteins, and TIP47, a Rab9-specific effector protein. Vaccinia virus (VV) induces a wrapping complex derived from intracellular host membranes to envelope intracellular mature virus particles producing egress-competent forms of virus.ResultsWe show that VV p37 protein associates with TIP47-, Rab9-, and CI-MPR-containing membranes. Mutation of a di-aromatic motif in p37 blocks association with TIP47 and inhibits plaque formation. ST-246, a specific inhibitor of p37 function, inhibits these interactions and also blocks wrapped virus particle formation. Vaccinia virus expressing p37 variants with reduced ST-246 susceptibility associates with Rab9 and co-localizes with CI-MPR in the presence and absence of compound.ConclusionThese results suggest that p37 localizes to the LE and interacts with proteins associated with LE-derived transport vesicle biogenesis to facilitate assembly of extracellular forms of virus.

Impact of Human Immunodeficiency Virus Type 1 Resistance to Protease Inhibitors on Evolution of Resistance to the Maturation Inhibitor Bevirimat (PA-457)

The maturation inhibitor bevirimat [3-O-(3',3'dimethysuccinyl)betulinic acid; BVM; also known as PA-457 or DSB] potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking protease (PR)-mediated cleavage at the junction between capsid (CA) and spacer peptide 1 (SP1) in Gag. We previously isolated a panel of single-amino-acid substitutions that confer resistance to BVM in vitro (C. S. Adamson, S. D. Ablan, I. Boeras, R. Goila-Gaur, F. Soheilian, K. Nagashima, F. Li, K. Salzwedel, M. Sakalian, C. T. Wild, and E. O. Freed, J. Virol. 80:10957-10971, 2006). The BVM resistance mutations cluster at or near the CA-SP1 cleavage site. Because BVM likely will be used clinically in patients harboring viruses resistant to PR inhibitors (PIs), in this study we evaluated the interplay between a PI-resistant (PIR) PR and the BVM resistance mutations in Gag. As expected, the PIR mutations had no effect on inhibition by BVM; however, we observed general processing defects and a slight delay in viral replication in Jurkat T cells associated with the PIR mutations, even in the absence of compound. When combined, most BVM resistance and PIR mutations acted additively to impair viral replication, particularly in the presence of BVM. The BVM-resistant mutant SP1-A1V was an exception, as it supported robust replication in the context of either wild-type (WT) or PIR PR, even at high BVM concentrations. Significantly, the emergence of BVM resistance was delayed in the context of the PIR PR, and the SP1-A1V mutation was acquired most frequently with either WT or PIR PR. These results suggest that resistance to BVM is less likely to emerge in patients who have failed PIs than in patients who are PI naive. We predict that the SP1-A1V substitution is the most likely to emerge in vivo, as this mutant replicates robustly independently of PR mutations or BVM. These findings offer insights into the effect of PIR mutations on the evolution of BVM resistance in PI-experienced patients.