Thiadiazolidinone (TDZD) Analogs Inhibit Aggregation-Mediated Pathology in Diverse Neurodegeneration Models, and Extend C. elegans Life- and Healthspan.

Chronic, low-grade inflammation has been implicated in aging and age-dependent conditions, including Alzheimer's disease, cardiomyopathy, and cancer. One of the age-associated processes underlying chronic inflammation is protein aggregation, which is implicated in neuroinflammation and a broad spectrum of neurodegenerative diseases such as Alzheimer's, Huntington's, and Parkinson's diseases. We screened a panel of bioactive thiadiazolidinones (TDZDs) from our in-house library for rescue of protein aggregation in human-cell and C. elegans models of neurodegeneration. Among the tested TDZD analogs, PNR886 and PNR962 were most effective, significantly reducing both the number and intensity of Alzheimer-like tau and amyloid aggregates in human cell-culture models of pathogenic aggregation. A C. elegans strain expressing human Aß1-42 in muscle, leading to AD-like amyloidopathy, developed fewer and smaller aggregates after PNR886 or PNR962 treatment. Moreover, age-progressive paralysis was reduced 90% by PNR886 and 75% by PNR962, and "healthspan" (the median duration of spontaneous motility) was extended 29% and 62%, respectively. These TDZD analogs also extended wild-type C. elegans lifespan by 15-30% (p < 0.001), placing them among the most effective life-extension drugs. Because the lead drug in this family, TDZD-8, inhibits GSK3ß, we used molecular-dynamic tools to assess whether these analogs may also target GSK3ß. In silico modeling predicted that PNR886 or PNR962 would bind to the same allosteric pocket of inactive GSK3ß as TDZD-8, employing the same pharmacophore but attaching with greater avidity. PNR886 and PNR962 are thus compelling candidate drugs for treatment of tau- and amyloid-associated neurodegenerative diseases such as AD, potentially also reducing all-cause mortality.

Biobanked Glioblastoma Patient-Derived Organoids as a Precision Medicine Model to Study Inhibition of Invasion.

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.

Antitumor properties of novel sesquiterpene lactone analogs as NFκB inhibitors that bind to the IKKß ubiquitin-like domain (ULD).

Melampomagnolide B (MMB, 3) is a parthenolide (PTL, 1) based sesquiterpene lactone that has been used as a template for the synthesis of a plethora of lead anticancer agents owing to its reactive C-10 primary hydroxyl group. Such compounds have been shown to inhibit the IKKß subunit, preventing phosphorylation of the cytoplasmic IκB inhibitory complex. The present study focuses on the synthesis and in vitro antitumor properties of novel benzyl and phenethyl carbamates of MMB (7a-7k). Screening of these MMB carbamates identified analogs with potent growth inhibition properties against a panel of 60 human cancer cell lines (71% of the molecules screened had GI50 values < 2 µM). Two analogs, the benzyl carbamate 7b and the phenethyl carbamate7k, were the most active compounds. Lead compound 7b inhibited cell proliferation in M9 ENL AML cells, and in TMD-231, OV-MD-231 and SUM149 breast cancer cell lines. Interestingly, mechanistic studies showed that 7b did not inhibit p65 phosphorylation in M9 ENL AML and OV-MD-231 cells, but did inhibit phophorylation of both p65 and IκBα in SUM149 cells. 7b also reduced NFκB binding to DNA in both OV-MD-231 and SUM149 cells. Molecular docking studies indicated that 7b and 7k are both predicted to interact with the ubiquitin-like domain (ULD) of the IKKß subunit. These data suggest that in SUM149 cells, 7b is likely acting as an allosteric inhibitor of IKKß, whereas in M9 ENL AML and OV-MD-231 cells 7b is able to inhibit an event after IκB/p65/p50 phosphorylation by IKKß that leads to inhibition of NFκB activation and reduction in NFκB-DNA binding. Analog 7b was by far the most potent compound in either carbamate series, and was considered an important lead compound for further optimization and development as an anticancer agent.

Novel hydroxybenzylamine-deoxyvasicinone hybrids as anticholinesterase therapeutics for Alzheimer's disease.

A series of novel 2-hydroxybenzylamine-deoxyvasicinone hybrid analogs (8a-8n) have been synthesized and evaluated as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and as inhibitors of amyloid peptide (Aß1-42) aggregation, for treatment of Alzheimer's disease (AD). These dual acting compounds exhibited good AChE inhibitory activities ranging from 0.34 to 6.35 µM. Analogs8g and 8n were found to be the most potent AChE inhibitors in the series with IC50values of 0.38 µM and 0.34 µM, respectively. All the analogs (8a-8n) exhibited weak BuChE inhibitory activities ranging from 14.60 to 21.65 µM. Analogs8g and 8n exhibited BuChE with IC50values of 15.38 µM and 14.60 µM, respectively, demonstrating that these analogs were greater than 40-fold more selective for inhibition of AChE over BuChE. Additionally, compounds8g and 8n were also found to be the best inhibitors of self-induced Aß1-42 peptide aggregation with IC50values of 3.91 µM and 3.22 µM, respectively; 8g and 8n also inhibited AChE-induced Aß1-42 peptide aggregation by 68.7% and 72.6%, respectively. Kinetic analysis and molecular docking studies indicate that analogs 8g and 8n bind to a new allosteric pocket (site B) on AChE. In addition, the observed inhibition of AChE-induced Aß1-42 peptide aggregation by 8n is likely due to allosteric inhibition of the binding of this peptide at the CAS site on AChE. Overall, these results indicate that 8g and 8n are examples of dual-acting lead compounds for the development of highly effective anti-AD drugs.

Aging-associated skeletal muscle defects in HER2/Neu transgenic mammary tumor model.

BACKGROUND: Loss of skeletal muscle volume and resulting in functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced MyoD levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumor etiology. METHODS: We characterized functional and molecular defects of skeletal muscle in MMTV-Neu (Neu+) mice (n= 6-12), an animal model that represents HER2+ human breast cancer, and compared the results with well-characterized luminal B breast cancer model MMTV-PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle-enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Since NF-κB pathway plays a significant role in skeletal muscle defects, the ability of NF-κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined. RESULTS: Neu+ mice showed skeletal muscle defects similar to accelerated aging. Compared to age and sex-matched wild type mice, Neu+ tumor-bearing mice had lower grip strength (202±6.9 vs. 179±6.8 g grip force, p=0.0069) and impaired rotarod performance (108±12.1 vs. 30±3.9 seconds, P<0.0001), which was consistent with reduced muscle contractibility (p<0.0001). Skeletal muscle of Neu+ mice (n=6) contained lower levels of CD82+ (16.2±2.9 vs 9.0±1.6) and CD54+ (3.8±0.5 vs 2.4±0.4) muscle stem and progenitor cells (p<0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53 and miR-486 expression in muscles (p<0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and Pgc1ß, Neu+ mice displayed accelerated aging-associated changes including muscle fiber shrinkage and increased extracellular matrix deposition. Circulating "aging factor" and cachexia and fibromyalgia-associated chemokine Ccl11 was elevated in Neu+ mice (1439.56±514 vs. 1950±345 pg/ml, p<0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205±6 g force), rotarod performance (74±8.5 seconds), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ±478 pg/ml), and improved animal survival. CONCLUSIONS: These results suggest that breast cancer subtype has a specific impact on the type of molecular and structure changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer-induced skeletal muscle loss and functional limitations.

Evaluation of bone and kidney toxicity of BT2-peg2, a potential carrier for the targeted delivery of antibiotics to bone.

Previous studies have demonstrated that the bone targeting agent BT2-peg2 (BT2-minipeg2, 9), when conjugated to vancomycin and delivered systemically by intravenous (IV) or intraperitoneal (IP) injection accumulates in bone to a greater degree than vancomycin alone, but that this accumulation is associated with severe nephrotoxicity. To determine whether this nephrotoxicity could be attributed to BT2-peg2 itself, we used a rat model to assess the distribution and toxicity of BT2-peg2 after IP injection of 11 mg/kg twice daily for 21 days. The results demonstrated that BT2-peg2 accumulates in bone but there was no evidence of nephrotoxicity or any histopathological abnormalities in the bone. This suggests the nephrotoxicity observed in previous studies is likely due to the altered pharmacokinetics of vancomycin when conjugated to BT2-peg2 rather than to BT2-peg2 itself. Thus, BT2-peg2 may be a safe carrier for the enhanced delivery of antibiotics other than vancomycin to the bone as a means of combating bone infection. However, the data also emphasizes the need to carefully examine the pharmacokinetic characteristics of any BT2-peg2-antibiotic conjugate utilized for treatment of bone infections.

A pharmacokinetic study of morphine-6-O-sulfate in rat plasma and brain.

Morphine-6-O-sulfate (M6S), a polar, zwitterionic sulfate ester of morphine, is a powerful and safe analgesic in several rat models of pain. A sensitive liquid chromatography-tandem mass spectrometry bioanalytical method was developed and validated for the simultaneous determination of M6S and morphine (MOR) in rat plasma and brain after M6S administration. Morphine-d6 was used as internal standard. Multiple reaction monitoring was used for detection and quantitation of M6S, MOR, and morphine-d6 in the turbo ion spray positive mode. The chromatographic separation was carried out on an Alltech Altima C18 column. The analytical method was validated for linearity, precision, accuracy, specificity, and stability over a concentration range of 3-8000 ng/ml in rat plasma and 10-10,000 ng/ml in brain samples for both M6S and MOR. The validated method was applied to determine the PK profile of M6S in plasma after i.v., i.p., and oral dosing in male Sprague-Dawley rats. Rats were administered M6S by i.p. administration (5.6 and 10.0 mg/kg) or orally (10 and 30 mg/kg) and bioavailability compared to an i.v. injection (1 mg/kg) of M6S. The in vivo results indicate that M6S is not a prodrug of morphine, since M6S is not biotransformed into MOR in plasma after either i.p. or oral administration, and MOR was not detected in brain. The bioavailability of M6S was >93% and about 5% after i.p. and oral dosing, respectively. The low oral bioavailability of M6S may be due to poor permeation of the intestinal epithelial membrane. After i.p.-administration, M6S appears to reach brain tissues in low, but significant, concentrations.

Structural modeling of GSK3ß implicates the inactive (DFG-out) conformation as the target bound by TDZD analogs.

Glycogen synthase kinase-3ß (GSK3ß) controls many physiological pathways, and is implicated in many diseases including Alzheimer's and several cancers. GSK3ß-mediated phosphorylation of target residues in microtubule-associated protein tau (MAPTAU) contributes to MAPTAU hyperphosphorylation and subsequent formation of neurofibrillary tangles. Inhibitors of GSK3ß protect against Alzheimer's disease and are therapeutic for several cancers. A thiadiazolidinone drug, TDZD-8, is a non-ATP-competitive inhibitor targeting GSK3ß with demonstrated efficacy against multiple diseases. However, no experimental data or models define the binding mode of TDZD-8 with GSK3ß, which chiefly reflects our lack of an established inactive conformation for this protein. Here, we used metadynamic simulation to predict the three-dimensional structure of the inactive conformation of GSK3ß. Our model predicts that phosphorylation of GSK3ß Serine9 would hasten the DFG-flip to an inactive state. Molecular docking and simulation predict the TDZD-8 binding conformation of GSK3ß to be inactive, and are consistent with biochemical evidence for the TDZD-8-interacting residues of GSK3ß. We also identified the pharmacophore and assessed binding efficacy of second-generation TDZD analogs (TDZD-10 and Tideglusib) that bind GSK3ß as non-ATP-competitive inhibitors. Based on these results, the predicted inactive conformation of GSK3ß can facilitate the identification of novel GSK3ß inhibitors of high potency and specificity.

Binding Modes and Selectivity of Cannabinoid 1 (CB1) and Cannabinoid 2 (CB2) Receptor Ligands.

The cannabinoid (CB) receptors (CB1R and CB2R) represent a promising therapeutic target for several indications such as nociception and obesity. The ligands with nonselectivity can be traced to the high similarity in the binding sites of both cannabinoid receptors. Therefore, the need for selectivity, potency, and G-protein coupling bias has further complicated the design of desired compounds. The bias of currently studied cannabinoid agonists is seldom investigated, and agonists that do exhibit bias are typically nonselective. However, certain long-chain endocannabinoids represent a class of selective and potent CB1R agonists. The binding mode for this class of compounds has remained elusive, limiting the implementation of its binding features to currently studied agonists. Hence, in the present study, the binding poses for these long-chain cannabinoids, along with other interesting ligands, with the receptors have been determined, by using a combination of molecular docking and molecular dynamics (MD) simulations along with molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations. The binding poses for the long-chain cannabinoids implicate that a site surrounded by the transmembrane (TM)2, TM7, and extracellular loop (ECL)2 is vital for providing the long-chain ligands with the selectivity for CB1R, especially I267 of CB1R (corresponding to L182 of CB2R). Based on the obtained binding modes, the calculated relative binding free energies and selectivity are all in good agreement with the corresponding experimental data, suggesting that the determined binding poses are reasonable. The computational strategy used in this study may also prove fruitful in applications with other GPCRs or membrane-bound proteins.

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands.

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure-activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 µM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.

Design and Synthesis of Novel Hybrid 8-Hydroxy Quinoline-Indole Derivatives as Inhibitors of Aß Self-Aggregation and Metal Chelation-Induced Aß Aggregation.

A series of novel hybrid 8-hydroxyquinoline-indole derivatives (7a-7e, 12a-12b and 18a-18h) were synthesized and screened for inhibitory activity against self-induced and metal-ion induced Aß1-42 aggregation as potential treatments for Alzheimer's disease (AD). In vitro studies identified the most inhibitory compounds against self-induced Aß1-42 aggregation as 18c, 18d and 18f (EC50 = 1.72, 1.48 and 1.08 µM, respectively) compared to the known anti-amyloid drug, clioquinol (1, EC50 = 9.95 µM). The fluorescence of thioflavin T-stained amyloid formed by Aß1-42 aggregation in the presence of Cu2+ or Zn2+ ions was also dramatically decreased by treatment with 18c, 18d and 18f. The most potent hybrid compound 18f afforded 82.3% and 88.3% inhibition, respectively, against Cu2+- induced and Zn2+- induced Aß1-42 aggregation. Compounds 18c, 18d and 18f were shown to be effective in reducing protein aggregation in HEK-tau and SY5Y-APPSw cells. Molecular docking studies with the most active compounds performed against Aß1-42 peptide indicated that the potent inhibitory activity of 18d and 18f were predicted to be due to hydrogen bonding interactions, π-π stacking interactions and π-cation interactions with Aß1-42, which may inhibit both self-aggregation as well as metal ion binding to Aß1-42 to favor the inhibition of Aß1-42 aggregation.

Oxone®-Mediated TEMPO-Oxidized Cellulose Nanomaterials form I and form II.

The 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) oxidation of cellulose, when mediated with Oxone® (KHSO5), can be performed simply and under mild conditions. Furthermore, the products of the reaction can be isolated into two major components: Oxone®-mediated TEMPO-oxidized cellulose nanomaterials Form I and Form II (OTO-CNM Form I and Form II). This study focuses on the characterization of the properties of OTO-CNMs. Nanoparticle-sized cellulose fibers of 5 and 16 nm, respectively, were confirmed through electron microscopy. Infrared spectroscopy showed that the most carboxylation presented in Form II. Conductometric titration showed a two-fold increase in carboxylation from Form I (800 mmol/kg) to Form II (1600 mmol/kg). OTO-CNMs showed cellulose crystallinity in the range of 64-68% and crystallite sizes of 1.4-3.3 nm, as shown through XRD. OTO-CNMs show controlled variability in hydrophilicity with contact angles ranging from 16 to 32°, within or below the 26-47° reported in the literature for TEMPO-oxidized CNMs. Newly discovered OTO-CNM Form II shows enhanced hydrophilic properties as well as unique crystallinity and chemical functionalization in the field of bio-sourced material and nanocomposites.

Aggregate Interactome Based on Protein Cross-linking Interfaces Predicts Drug Targets to Limit Aggregation in Neurodegenerative Diseases.

Diagnosis of neurodegenerative diseases hinges on "seed" proteins detected in disease-specific aggregates. These inclusions contain diverse constituents, adhering through aberrant interactions that our prior data indicate are nonrandom. To define preferential protein-protein contacts mediating aggregate coalescence, we created click-chemistry reagents that cross-link neighboring proteins within human, APPSw-driven, neuroblastoma-cell aggregates. These reagents incorporate a biotinyl group to efficiently recover linked tryptic-peptide pairs. Mass-spectroscopy outputs were screened for all possible peptide pairs in the aggregate proteome. These empirical linkages, ranked by abundance, implicate a protein-adherence network termed the "aggregate contactome." Critical hubs and hub-hub interactions were assessed by RNAi-mediated rescue of chemotaxis in aging nematodes, and aggregation-driving properties were inferred by multivariate regression and neural-network approaches. Aspirin, while disrupting aggregation, greatly simplified the aggregate contactome. This approach, and the dynamic model of aggregate accrual it implies, reveals the architecture of insoluble-aggregate networks and may reveal targets susceptible to interventions to ameliorate protein-aggregation diseases.

Inhibition of Human DNA Polymerases Eta and Kappa by Indole-Derived Molecules Occurs through Distinct Mechanisms.

Overexpression of human DNA polymerase kappa (hpol κ) in glioblastoma is associated with shorter survival time and resistance to the alkylating agent temozolomide (TMZ), making it an attractive target for the development of small-molecule inhibitors. We previously reported on the development and characterization of indole barbituric acid-derived (IBA) inhibitors of translesion DNA synthesis polymerases (TLS pols). We have now identified a potent and selective inhibitor of hpol κ based on the indole-aminoguanidine (IAG) chemical scaffold. The most promising IAG analogue, IAG-10, exhibited greater inhibitory action against hpol κ than any other human Y-family member, as well as pols from the A-, B-, and X-families. Inhibition of hpol κ by IAG analogues appears to proceed through a mechanism that is distinct from inhibition of hpol η based on changes in DNA binding affinity and nucleotide insertion kinetics. By way of comparison, both IAG and IBA analogues inhibited binary complex formation by hpol κ and ternary complex formation by hpol η. Decreasing the concentration of enzyme and DNA in the reaction mixture lowered the IC50 value of IAG-10 to submicromolar values, consistent with inhibition of binary complex formation for hpol κ. Chemical footprinting experiments revealed that IAG-10 binds to a cleft between the finger, little finger, and N-clasp domains on hpol κ and that this likely disrupts the interaction between the N-clasp and the TLS pol core. In cell culture, IAG-10 potentiated the antiproliferative activity and DNA damaging effects of TMZ in hpol κ-proficient cells but not in hpol κ-deficient cells, indicative of a target-dependent effect. Mutagenic replication across alkylation damage increased in hpol κ-proficient cells treated with IAG-10, while no change in mutation frequency was observed for hpol κ-deficient cells. In summary, we developed a potent and selective small-molecule inhibitor of hpol κ that takes advantage of structural features unique to this TLS enzyme to potentiate TMZ, a standard-of-care drug used in the treatment of malignant brain tumors. Furthermore, the IAG scaffold represents a new chemical space for the exploration of TLS pol inhibitors, which could prove useful as a strategy for improving patient response to genotoxic drugs.

Reduced Tolerance and Asymmetrical Crosstolerance to Effects of the Indole Quinuclidinone Analog PNR-4-20, a G Protein-Biased Cannabinoid 1 Receptor Agonist in Mice: Comparisons with Δ9-Tetrahydrocannabinol and JWH-018.

Most cannabinoid 1 receptor (CB1R) agonists will signal through both G protein-dependent and -independent pathways in an unbiased manner. Recruitment of ß-arrestin 2 desensitizes and internalizes receptors, producing tolerance that limits therapeutic utility of cannabinoids for chronic conditions. We developed the indole quinuclidinone (IQD) analog (Z)-2-((1-(4-fluorobenzyl)-1H-indol-3-yl)methylene)quinuclidin-3-one (PNR-4-20) as a novel G protein-biased agonist at CB1Rs, and the present studies determine if repeated administration of PNR-4-20 produces lesser tolerance to in vivo effects compared with unbiased CB1R agonists Δ9-tetrahydrocannabinol (Δ9-THC) and 1-pentyl-3-(1-naphthoyl)indole (JWH-018). Adult male National Institutes of Health Swiss mice were administered comparable doses of PNR-4-20 (100 mg/kg), Δ9-THC (30 mg/kg), or JWH-018 (3 mg/kg) once per day for five consecutive days to determine tolerance development to hypothermic, antinociceptive, and cataleptic effects. Persistence of tolerance was then determined after a drug abstinence period. We found that unbiased CB1R agonists Δ9-THC and JWH-018 produced similar tolerance to these effects, but lesser tolerance was observed with PNR-4-20 for hypothermic and cataleptic effects. Tolerance to the effects of PNR-4-20 completely recovered after drug abstinence, while residual tolerance was always observed with unbiased CB1R agonists. Repeated treatment with PNR-4-20 and Δ9-THC produced asymmetric crosstolerance to hypothermic effects. Importantly, binding studies suggest PNR-4-20 produced significantly less downregulation of CB1Rs relative to Δ9-THC in hypothalamus and thalamus of chronically treated mice. These studies suggest that the G protein-biased CB1R agonist PNR-4-20 produces significantly less tolerance than unbiased cannabinoid agonists, and that the IQD analogs should be investigated further as a novel molecular scaffold for development of new therapeutics.

A Novel Microtubule-Binding Drug Attenuates and Reverses Protein Aggregation in Animal Models of Alzheimer's Disease.

Age-progressive neurodegenerative pathologies, including Alzheimer's disease (AD), are distinguished and diagnosed by disease-specific components of intra- or extra-cellular aggregates. Increasing evidence suggests that neuroinflammation promotes protein aggregation, and is involved in the etiology of neurological diseases. We synthesized and tested analogs of the naturally occurring tubulin-binding compound, combretastatin A-4. One such analog, PNR502, markedly reduced the quantity of Alzheimer-associated amyloid aggregates in the BRI-Aß1-42 mouse model of AD, while blunting the ability of the pro-inflammatory cytokine IL-1ß to raise levels of amyloid plaque and its protein precursors in a neuronal cell-culture model. In transgenic Caenorhabditis elegans (C. elegans) strains that express human Aß1-42 in muscle or neurons, PNR502 rescued Aß-induced disruption of motility (3.8-fold, P < 0.0001) or chemotaxis (1.8-fold, P < 0.05), respectively. Moreover, in C. elegans with neuronal expression of Aß1-42, a single day of PNR502 exposure reverses the chemotaxis deficit by 54% (P < 0.01), actually exceeding the protection from longer exposure. Moreover, continuous PNR502 treatment extends nematode lifespan 23% (P ≤ 0.001). Given that PNR502 can slow, prevent, or reverse Alzheimer-like protein aggregation in human-cell-culture and animal models, and that its principal predicted and observed binding targets are proteins previously implicated in Alzheimer's, we propose that PNR502 has therapeutic potential to inhibit cerebral Aß1-42 aggregation and prevent or reverse neurodegeneration.

A novel tetrazole analogue of resveratrol is a potent anticancer agent.

A series of novel tetrazole analogues of resveratrol were synthesized and evaluated for their anti-leukemic activity against an extensive panel of human cancer cell lines and against the MV4-11 AML cell line. These molecules were designed as drug-like derivatives of the resveratrol analogue DMU-212 and its cyano derivatives. Four compounds 8g, 8h, 10a and 10b exhibited LD50 values of 4.60 µM, 0.02 µM, 1.46 µM, and 1.08 µM, respectively, against MV4-11 leukemia cells. The most potent compounds, 8h and 10b, were also found to be active against an extensive panel of human hematological and solid tumor cell lines; compound 8h was the most potent compound with GI50 values <10 nM against more than 90% of the human cancer cell lines in the 60-cell panel. Analogues 8g, 8h, 10a and 10b were also tested for their ability to inhibit the polymerization of tubulin, and compound 8h was found to be the most potent analogue. Molecular modeling studies demonstrated that 8h binds to the colchicine binding site on tubulin. Thus, compound 8h is considered to be a lead druglike molecule from this tetrazole series of compounds.

N-Naphthoyl-substituted indole thio-barbituric acid analogs inhibit the helicase activity of the hepatitis C virus NS3.

The hepatitis C virus (HCV) represents a substantial threat to human health worldwide. The virus expresses a dual-function protein, NS3 having both protease and RNA helicase activities that are essential for productive viral replication and sustained infections. While viral protease and polymerase inhibitors have shown great successes in treating chronic HCV infections, drugs that specifically target the helicase activity have not advanced. A robust and quantitative 96-well plate-based fluorescent DNA unwinding assay was used to screen a class of indole thio-barbituric acid (ITBA) analogs using the full-length, recombinant HCV NS3, and identified three naphthoyl-containing analogs that efficiently inhibited NS3 helicase activity in a dose-dependent manner, with observed IC50 values of 21-24 µM. Standard gel electrophoresis helicase assays using radiolabeled duplex DNA and RNA NS3 substrates confirmed the inhibition of NS3 unwinding activity. Subsequent anisotropy measurements demonstrated that the candidate compounds did not disrupt NS3 binding to nucleic acids. Additionally, the rate of ATP hydrolysis and the protease activity were also not affected by the inhibitors. Thus, these results indicate that the three ITBA analogs containing N-naphthoyl moieties are the foundation of a potential series of small molecules capable of inhibiting NS3 activity via a novel interaction with the helicase domain that prevents the productive unwinding of nucleic acid substrates, and may represent the basis for a new class of therapeutic agents with the potential to aid in the treatment and eradication of hepatitis C virus.

Highly sulphated cellulose: a versatile, reusable and selective desilylating agent for deprotection of alcoholic TBDMS ethers.

A mild, efficient and rapid protocol was developed for the deprotection of alcoholic TBDMS ethers using a recyclable, eco-friendly highly sulphated cellulose sulphate acid catalyst in methanol. This acid catalyst selectively cleaves alcoholic TBDMS ethers in bis-TBDMS ethers containing both alcoholic and phenolic TBDMS ether moieties.

Evaluation of morphine-like effects of the mixed mu/delta agonist morphine-6-O-sulfate in rats: Drug discrimination and physical dependence.

Morphine-6-O-sulfate (M6S) is as a mixed-action mu/delta (µ/δ) opioid receptor agonist with high potency and analgesic efficacy. These studies used assays of drug discrimination and schedule-controlled responding to assess abuse-liability, tolerance, and physical dependence as compared to morphine in rats. Attempts to train 0.3 mg/kg (IP) M6S from saline failed, but all rats rapidly acquired the discrimination when the training dose was changed to 3.0 mg/kg morphine, and substitution tests showed that morphine and fentanyl both fully substituted for the training dose, M6S and M3A6S (3-O-acetyl ester of M6S) only partially substituted, and salvinorin A did not elicit morphine-like effects. Tolerance to response rate-decreasing effects was studied in rats administered either 1.0 or 3.0 mg/kg morphine or M6S before food-reinforced operant sessions. At both unit doses, tolerance to M6S-elicited rate suppression developed more slowly than tolerance to morphine-induced reductions in response rates. To assess dependence, rats were maintained on 1.0 mg/kg morphine or 1.0 mg/kg M6S until food-reinforced response rates were stable for at least 5 days. Rats were then administered saline or increasing doses of the opioid antagonist naltrexone (NTX) (0.3, 1.0, 3.0, or 10.0 mg/kg) in order to determine antagonist-precipitated withdrawal. NTX precipitated withdrawal was similar in both morphine-maintained and M6S-maintained rats. In conclusion, the mixed µ/δ agonist activity of M6S failed to completely protect against the development of physical dependence, but delayed tolerance development to behavioral effects and resulted in decreased morphine-like subjective effects, perhaps implying a decreased abuse liability over µ agonists.