Identification of Plasma Biomarkers from Rheumatoid Arthritis Patients Using an Optimized Sequential Window Acquisition of All THeoretical Mass Spectra (SWATH) Proteomics Workflow.

Rheumatoid arthritis (RA) is a systemic autoimmune and inflammatory disease. Plasma biomarkers are critical for understanding disease mechanisms, treatment effects, and diagnosis. Mass spectrometry-based proteomics is a powerful tool for unbiased biomarker discovery. However, plasma proteomics is significantly hampered by signal interference from high-abundance proteins, low overall protein coverage, and high levels of missing data from data-dependent acquisition (DDA). To achieve quantitative proteomics analysis for plasma samples with a balance of throughput, performance, and cost, we developed a workflow incorporating plate-based high abundance protein depletion and sample preparation, comprehensive peptide spectral library building, and data-independent acquisition (DIA) SWATH mass spectrometry-based methodology. In this study, we analyzed plasma samples from both RA patients and healthy donors. The results showed that the new workflow performance exceeded that of the current state-of-the-art depletion-based plasma proteomic platforms in terms of both data quality and proteome coverage. Proteins from biological processes related to the activation of systemic inflammation, suppression of platelet function, and loss of muscle mass were enriched and differentially expressed in RA. Some plasma proteins, particularly acute-phase reactant proteins, showed great power to distinguish between RA patients and healthy donors. Moreover, protein isoforms in the plasma were also analyzed, providing even deeper proteome coverage. This workflow can serve as a basis for further application in discovering plasma biomarkers of other diseases.

Discovery of ABBV-154, an anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody-Drug Conjugate (iADC).

Stable attachment of drug-linkers to the antibody is a critical requirement, and for maleimide conjugation to cysteine, it is achieved by ring hydrolysis of the succinimide ring. During ADC profiling in our in-house property screening funnel, we discovered that the succinimide ring open form is in equilibrium with the ring closed succinimide. Bromoacetamide (BrAc) was identified as the optimal replacement, as it affords stable attachment of the drug-linker to the antibody while completely removing the undesired ring open-closed equilibrium. Additionally, BrAc also offers multiple benefits over maleimide, especially with respect to homogeneity of the ADC structure. In combination with a short, hydrophilic linker and phosphate prodrug on the payload, this afforded a stable ADC (ABBV-154) with the desired properties to enable long-term stability to facilitate subcutaneous self-administration.

Pervilleine F, a new tropane alkaloid aromatic ester that reverses multidrug resistance.

P-glycoprotein (Pgp)-mediated drug efflux can yield a multidrug resistance (MDR) phenotype that is associated with poor response to cancer chemotherapy. Pervilleine F, a new tropane alkaloid aromatic ester obtained from a chloroform extract of the roots of Erythroxylum pervillei as the result of bioactivity-guided fractionation, was found to restore the vinblastine sensitivity of cultured multidrug-resistant KB-V1 cells, with an IC50 value of 0.40 microM. Pervilleine F (8 microM) was also able to partially reverse the cross-resistance of KB-V1 cells to the clinically used or experimental anticancer agents actinomycin D (45.1-fold), baccatin III (> 3.4-fold), daunomycin (> 22.5-fold), ellipticine (1.9-fold), mithramycin A (42.5-fold), podophyllotoxin (1.6-fold), paclitaxel (32.2-fold) and vincristine (73.6-fold). While pervilleine F alone at the concentration of 10 microM had no significant effect on the KB-V1 cell cycle, pervilleine F (at concentrations of 0.2, 1, 2, and 8 microM) combined with vinblastine (1 microgram/ml) induced dose-dependent G2/M phase arrest, ranging from 20.2, 51.0, 63.7, to 79.5%, as an indication of the restoration of vinblastine sensitivity. To confirm this activity with an in vivo animal model, KB-V1 cells were placed in hollow fibers and implanted into NCr nu/nu mice. Cell growth was not significantly inhibited when vinblastine or pervilleine F was administered as single agents, but when these two compounds were used in combination, inhibition of up to 64.1% was observed. Equimolar doses of verapamil were less effective. These data suggest that pervilleine F is an effective inhibitor of Pgp and should be further evaluated for clinical utility.

New bioactive polyphenols from Theobroma grandiflorum ("cupuaçu").

Activity-guided fractionation of Theobroma grandiflorum ("cupuaçu") seeds resulted in the identification of two new sulfated flavonoid glycosides, theograndins I (1) and II (2). In addition, nine known flavonoid antioxidants, (+)-catechin, (-)-epicatechin, isoscutellarein 8-O-beta-d-glucuronide, hypolaetin 8-O-beta-d-glucuronide, quercetin 3-O-beta-d-glucuronide, quercetin 3-O-beta-d-glucuronide 6' '-methyl ester, quercetin, kaempferol, and isoscutellarein 8-O-beta-d-glucuronide 6' '-methyl ester, were identified. Theograndin II (2) displayed antioxidant activity (IC(50) = 120.2 microM) in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical assay, as well as weak cytotoxicity in the HCT-116 and SW-480 human colon cancer cell lines with IC(50) values of 143 and 125 microM, respectively. While 1 was less active as an antioxidant than 2, the known compounds were more potent in the DPPH assay (IC(50) range 39.7-89.7 microM).

Imidazole alkaloids from Lepidium meyenii.

Two new imidazole alkaloids (lepidiline A and lepidiline B) have been isolated from a root extract of Lepidium meyenii with the common name Maca and identified as 1,3-dibenzyl-4,5-dimethylimidazolium chloride (1) and 1,3-dibenzyl-2,4,5-trimethylimidazolium chloride (2), respectively. The structures of these two new compounds were determined by spectroscopic methods, as well as single-crystal X-ray diffraction performed on compound 1.

Characterization of tropane alkaloid aromatic esters that reverse the multidrug-resistance phenotype.

P-Glycoprotein (Pgp) associated multidrug-resistance (MDR) is a significant factor that can lead to the failure of cancer chemotherapy. Several new tropane alkaloid aromatic esters obtained from extracts of Erythroxylum pervillei Baillon (Erythroxylaceae) (1-8) and Erythroxylum rotundifolium Lunan (Erythroxylaceae) (9-12) by means of bioassay-directed fractionation were found to restore vinblastine sensitivity with cultured multidrug-resistant KB-VI cells. With this model, growth was not inhibited by addition of vinblastine (1 microg/ml) to the culture medium, but in combination with tropane alkaloids, inhibition was observed with IC50 values categorized as: low (ranging from 0.17-0.62 microM) for 1 [3alpha-phenylacetoxy-6beta-(3,4,5-trimethaxycinnamoyloxy)-tropane], 3 [3alpha-(3,4,5-trimethoxybenzoyloxy)-6beta-(3,4,5-trimethoxycinnamoyloxy)tropane], 4 [3alpha-(3,4,5-trimethoxybenzoyloxy)-6beta-(3,4,5-trimethoxycinnamoyloxy)-7beta-hydroxytropane], 5 [3alpha,6beta-di-(3,4,5-trimethoxycinnamoyloxy)tropane], 6 [3alpha,6beta-di-(3,4,5-trimethoxycinnamoyloxy)-7beta-hydroxytropane] and 9 [6beta-benzoyloxy-3alpha-(3,4,5-trimethoxycinnamoyloxy)tropane]; medium (2.0-3.7 microM) for 2 [3alpha-(3-hydroxyphenylacetoxy)-6beta-(3,4,5-trimethoxycinnamoyloxy)tropane] and 10 [7beta-acetoxy-6beta-benzoyloxy-3alpha-(3,4,5-trimethoxycinnamoyloxy)tropane]; or high (9.8 microM) for 11 [6beta-benzoyloxy-3alpha-(3,4,5-trimethoxycinnamoyloxy)tropane-7beta-ol]. Compounds 7 (tropane-3alpha,6beta,7beta-triol 3-phenylacetate), 8 (1alphaH, 5alphaH-tropan-3alpha-yl 3,4,5-trimethoxybenzoate) and 12 (6beta-(3,4,5-trimethoxybenzoyloxy)-3alpha-(3,4,5-trimethoxycinnamoyloxy)tropane-7beta-ol) were not active. Among the active compounds, 1 and 3-6 were further tested with drug-resistant CEM/VLB100 cells. In the presence of modulator, sensitivity to vinblastine increased by 50-5,000-fold. Treatment of KB-V1 cells with 1 or 3-6 enhanced the intracellular accumulation of fluorescence dye (rhodamine 123). Visualization by confocal microscopy confirmed the intracellular accumulation of rhodamine 123 in drug-resistant KB-V1 cells was significantly less than drug-sensitive KB-3 cells, while treatment of KB-V1 cells with 10 microM 4 significantly increased intracellular accumulation. The molecular characteristics of the isolates were then determined and compared with their potential to reverse drug resistance. ClogP and molar refractivity were weakly correlated with their potential to reverse MDR

Pervilleines B and C, new tropane alkaloid aromatic esters that reverse the multidrug-resistance in the hollow fiber assay.

P-Glycoprotein (Pgp)-mediated drug efflux can yield a multidrug-resistance phenotype that is associated with poor response to cancer chemotherapy. Pervilleines B and C (PB and PC), two new tropane alkaloid aromatic esters obtained from a chloroform extract of the roots of Erythroxylum pervillei as the result of bioactivity-guided fractionation, were found to restore the vinblastine (VLB) sensitivity of cultured multidrug-resistant KB-V1 cells, with 50% inhibitory concentration values of 0.17 microM in each case. To explore the potential relevance of this response, KB-V1 cells were placed in hollow fibers and implanted into NCr nu/nu mice. Cell growth was not significantly inhibited when VLB or PB or PC were administered as single agents, but when used in combination with vinblastine inhibition of up to 77.7% was observed. Equimolar doses of verapamil were less effective. These data suggest that PB and PC are effective inhibitors of Pgp and should be further evaluated for clinical utility.

Reversal of multidrug resistance by tropane alkaloids from the stems of Erythroxylum rotundifolium.

Six tropane alkaloid esters were isolated from the stems of Erythroxylum rotundifolium. The structures of three new tropane esters, 7beta-hydroxy-6beta-(3,4,5-trimethoxybenzoyloxy)-3alpha-(E)-(3,4,5-trimethoxycinnamoyloxy)tropane (1), 6beta-benzoyloxy-3alpha-(Z)-(3,4,5-trimethoxycinnamoyloxy)tropane (2), and (-)-6beta-benzoyloxy-3alpha-hydroxytropane (3), were established by spectroscopic techniques. When alkaloids 1-6 were evaluated against a panel of human cancer cell lines, the new compound 6beta-benzoyloxy-3alpha-(Z)-(3,4,5-trimethoxycinnamoyloxy)tropane (2) and three known compounds, 6beta-benzoyloxy-3alpha-(3,4,5-trimethoxycinnamoyloxy)tropane (4), 6beta-benzoyloxy-3alpha-(E)-(3,4,5-trimethoxycinnamoyloxy)tropane-7beta-ol (5), and 7beta-acetoxy-6beta-benzoyloxy-3alpha-(E)-(3,4,5-trimethoxycinnamoyloxy)tropane (6), demonstrated greatest activity with multidrug-resistant oral epidermoid carcinoma (KB-V1) cells incubated in the presence of vinblastine. Thus, tropane esters of this type can reverse the multidrug-resistance phenotype, presumably by interacting with P-glycoprotein.