Discovery and Control of Succinimide Formation and Accumulation at Aspartic Acid Residues in The Complementarity-Determining Region of a Therapeutic Monoclonal Antibody.

PURPOSE: Succinimide formation and isomerization alter the chemical and physical properties of aspartic acid residues in a protein. Modification of aspartic acid residues within complementarity-determining regions (CDRs) of therapeutic monoclonal antibodies (mAbs) can be particularly detrimental to the efficacy of the molecule. The goal of this study was to characterize the site of succinimide accumulation in the CDR of a therapeutic mAb and understand its effects on potency. Furthermore, we aimed to mitigate succinimide accumulation through changes in formulation. METHODS: Accumulation of succinimide was identified through intact and reduced LC-MS mass measurements. A low pH peptide mapping method was used for relative quantitation and localization of succinimide formation in the CDR. Statistical modeling was used to correlate levels of succinimide with basic variants and potency measurements. RESULTS: Succinimide accumulation in Formulation A was accelerated when stored at elevated temperatures. A strong correlation between succinimide accumulation in the CDR, an increase in basic charge variants, and a decrease in potency was observed. Statistical modeling suggest that a combination of ion exchange chromatography and potency measurements can be used to predict succinimide levels in a given sample. Reformulation of the mAb to Formulation B mitigates succinimide accumulation even after extended storage at elevated temperatures. CONCLUSION: Succinimide formation in the CDR of a therapeutic mAb can have a strong negative impact on potency of the molecule. We demonstrate that thorough characterization of the molecule by LC-MS, ion exchange chromatography, and potency measurements can facilitate changes in formulation that mitigate succinimide formation and the corresponding detrimental changes in potency.

A role for Saccharomyces cerevisiae Centrin (Cdc31) in mitochondrial function and biogenesis.

Centrins belong to a family of proteins containing calcium-binding EF-hand motifs that perform well-established roles in centrosome and spindle pole body (SPB) duplication. Yeast encodes a single Centrin protein (Cdc31) that binds components in the SPB. However, further studies revealed a role for Centrins in mRNA export, and interactions with contractile filaments and photoreceptors. In addition, human Centrin-2 can bind the DNA-lesion recognition factor XPC, and improve the efficiency of nucleotide excision repair. Similarly, we reported that yeast Cdc31 binds Rad4, a functional counterpart of the XPC DNA repair protein. We also found that Cdc31 is involved in the ubiquitin/proteasome system, and mutations interfere with intracellular protein turnover. In this report, we describe new findings that indicate a role for Cdc31 in the energy metabolism pathway. Cdc31 and cdc31 mutant proteins showed distinct interactions with proteins in energy metabolism, and mutants showed sensitivity to oxidative stress and poor growth on non-fermentable carbon. Significant alteration in mitochondrial morphology was also detected. Although it is unclear how Cdc31 contributes to so many unrelated mechanisms, we propose that by controlling SPB duplication Centrin proteins might link the cellular responses to DNA damage, oxidative load and proteotoxic stresses to growth control.

Green tea polyphenol EGCG suppresses lung cancer cell growth through upregulating miR-210 expression caused by stabilizing HIF-1α.

(-)-Epigallocatechin-3-gallate (EGCG) has been reported to affect many cellular regulatory pathways. This study aims to determine whether EGCG could target microRNA (miRNA), one of the mechanisms for cells to achieve subtle change in multiple targets. We found that, in both human and mouse lung cancer cells in culture, EGCG specifically upregulated the expression of miR-210, a major miRNA regulated by HIF-1α. Furthermore, we found that overexpression of miR-210 led to reduced cell proliferation rate and anchorage-independent growth as well as reduced sensitivity to EGCG. On the mechanisms of miR-210 regulation by EGCG, we demonstrated that the regulation was mediated through the hypoxia-response element in miR-210 promoter. Consistently, the upregulation of miR-210 was found to be correlated with the stabilized HIF-1α in lung cancer cell lines after EGCG treatment. This EGCG-induced stabilization of HIF-1α was further shown by the stabilization of HA-tagged HIF-1α but not the P402A/P564A-mutated HIF-1α by EGCG, suggesting that EGCG targets the oxygen-dependent degradation (ODD) domain. Direct evidence was obtained by affinity binding assay showing that EGCG specifically binds HIF-1α with a K(d) = 3.47 µM. This result suggests that EGCG binding interferes with the hydroxylation of key Pro residues in the ODD domain, preventing HIF-1α from the Pro hydroxylation-dependent ubiquitination and subsequent proteosome-mediated degradation. In summary, our results demonstrated, for the first time, the elevation of miR-210 by EGCG in lung cancer cell lines and this is mediated by the stabilization of HIF-1α. This event contributes to the anticancer activity of EGCG.

Post-translational modifications of connexin26 revealed by mass spectrometry.

Gap junctions play important roles in auditory function and skin biology; mutations in the Cx26 (connexin26) gene are the predominant cause of inherited non-syndromic deafness and cause disfiguring skin disorders. Mass spectrometry (MS) was used to identify PTMs (post-translational modifications) of Cx26 and to determine whether they occur at sites of disease-causing mutations. Cx26 was isolated from transfected HeLa cells by sequential immunoaffinity and metal chelate chromatography using a tandem C-terminal haemagglutinin epitope and a (His-Asn)6 sequence. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase GluC. Peptides were fractionated using a reversed-phase matrix by stepwise elution with increasing concentrations of organic solvent. To improve detection of low-abundance peptides and to maximize sequence coverage, MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; MS) and MALDI-TOF/TOF-MS/MS (matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry; MS/MS) spectra were acquired from each elution step using an Applied Biosystems 4800 tandem mass spectrometer. Acquisition, processing and interpretation parameters were optimized to improve ionization and fragmentation of hydrophobic peptides. MS and MS/MS coverage of Cx26 was significantly above that reported for other membrane proteins: 71.3% by MS, with 29.9% by MS/MS. MS coverage was 92.6% if peptides resulting from in-source collisions and/or partial enzymatic cleavages were considered. A variety of putative PTMs of Cx26 were identified, including acetylation, hydroxylation, gamma-carboxyglutamation, methylation and phosphorylation, some of which are at sites of deafness-causing mutations. Knowledge of the PTMs of Cx26 will be instrumental in understanding how alterations in the cellular mechanisms of Cx26 channel biogenesis and function lead to losses in auditory function and disfiguring skin disorders.

Altered proteolytic events in experimental autoimmune encephalomyelitis discovered by iTRAQ shotgun proteomics analysis of spinal cord.

BACKGROUND: Abnormal activation of protease activities during experimental autoimmune encephalomyelitis (EAE) in rats, a rodent model of multiple sclerosis, have been implicated in either the direct destruction of myelin components or the intracellular signal transduction pathways that lead to lymphocyte infiltration, oligodendrocyte destruction, neuronal dysfunctions and axonal degeneration. The identification of changes in regulated proteolytic events during EAE is crucial for uncovering activated proteases that may underline the pathological features such as inflammation and demyelination. We searched for either non-tryptic or semi-tryptic peptides from a previous shotgun proteomics study using isobaric tags for relative and absolute quantification (iTRAQ) to compare the proteomes of normal and EAE rat lumbar spinal cords. RESULTS: We discovered that several proteins, such as alpha1-macroglobulin, a protease inhibitor, alpha1B-glycoprotein, beta2-microglobulin, neurofilament light polypeptide and sulfated glycoprotein 1 had non-tryptic peptide iTRAQ ratios that were substantially different from the overall protein iTRAQ ratios, suggesting that such peptides may be markers for the proteolytic products generated by the protease(s) altered during EAE. Indeed, subsequent Western blotting confirmed the dysregulation of specific protein cleavages in EAE tissues. Additional proteolytic changes in alpha2-macroglobulin, another protease inhibitor similar to alpha1-macroglobulin was also observed. CONCLUSION: The results from this study revealed changes among both neuronal protein processing and endogenous proteolysis modulators in EAE animals. This information may provide a rationale for protease inhibitor-based therapeutic interventions for multiple sclerosis.

Purification and mass spectrometric analysis of the kappa opioid receptor.

A clonal human embryonic kidney (HEK) 293 cell line was established that stably expressed the rat kappa-opioid receptor (rKOR) with a FLAG epitope at the amino terminus. The Kd for [3H]diprenorphine was 1.1+/-0.2 nM, and the Bmax was 2.6+/-0.4 pmol/mg. Dynorphin A (1-13), U69,593 and naloxone competitively inhibited [3H]diprenorphine binding with Ki values of 2.0, 18 and 18 nM, respectively, in good agreement with previously reported affinities for the unmodified receptor. U69,593 stimulated [35S]GTPgammaS binding in a concentration-dependent manner and caused phosphorylation of mitogen-activated protein (MAP) kinase, indicating that the activated epitope-tagged receptor triggered appropriate signaling pathways. Immunoblot analysis demonstrated that two immunoreactive receptor species with apparent molecular masses of 42 and 52 kDa were expressed. Previous studies indicated that the 42 kDa protein was localized intracellularly and was a precursor of the 52 kDa receptor, which was present at the cell surface. rKOR was extracted from transfected HEK 293 cell membranes with n-dodecyl-beta-D-maltopyranoside. Sequential use of wheat germ agglutinin chromatography, Sephacryl S300 gel filtration chromatography, anti-FLAG immunoaffinity chromatography and SDS/PAGE permitted purification of the 52 kDa receptor. MALDI-TOF mass spectrometry was used to identify peptides derived from rKOR following sequential in-gel digestion with trypsin and cyanogen bromide. Eighteen rKOR peptides were detected, corresponding to 27.1% coverage of the receptor. Precursor-selective MS/MS confirmed the identity of most of these peptides. In addition, we have identified heat shock protein 70 (HSP70) as a rKOR-interacting protein.

Repeated cocaine administration increases N-methyl-d-aspartate NR1 subunit, extracellular signal-regulated kinase and cyclic AMP response element-binding protein phosphorylation and glutamate release in the rat dorsal striatum.

This study was conducted to determine the phosphorylation state of N-methyl-d-aspartate (NMDA) NR1 subunit on serine residues 896 (Ser896) and 897 (Ser897), the extracellular signal-regulated kinase 1/2 (ERK1/2), and the cyclic AMP response element-binding protein (CREB) after repeated exposure to cocaine (20 mg/kg, once daily for 9 days) in the dorsal striatum of rats. The real-time changes of glutamate concentration evoked by repeated cocaine injections were examined using a glutamate biosensor in order to evaluate the correlation between glutamate concentration and the change in these phosphoproteins. The results of this study showed that the immunoreactivity of phosphorylated (p)NMDA NR1 subunit at Ser896 and Ser897 as well as pERK1/2, but not pCREB, in the dorsal striatum was increased at 30 min and then returned to basal levels 4 h after repeated cocaine injections. Similarly, glutamate responses evoked by repeated cocaine injections were also increased 30 min after repeated cocaine injections for 3 days and were prolonged by the 9th day of treatment. However, the glutamate responses were not detected at 4 h after repeated cocaine injections for 5 days. In addition, the elevated immunoreactivity of the phosphoproteins 2 h after repeated cocaine injections was attenuated by the blockade of dopamine D1 receptors and NMDA receptors with the SCH23390 or MK801 antagonists, respectively. These findings suggest that glutamate release and dopamine D1 and NMDA receptor stimulation after repeated exposure to cocaine are associated with NMDA NR1 subunit, ERK1/2 and CREB phosphorylation in the dorsal striatum.

Air-liquid interface (ALI) culture of human bronchial epithelial cell monolayers as an in vitro model for airway drug transport studies.

Serially passaged normal human bronchial epithelial (NHBE) cell monolayers were established on Transwell inserts via an air-liquid interface (ALI) culture method. NHBE cells were seeded on polyester Transwell inserts, followed by an ALI culture from day 3, which resulted in peak TEER value of 766+/-154 Omegaxcm2 on the 8th day. Morphological characteristics were observed by light microscopy and SEM, while the formation of tight junctions was visualized by actin staining, and confirmed successful formation of a tight monolayer. The transepithelial permeability (Papp) of model drugs significantly increased with the increase of lipophilicity and showed a good linear relationship, which indicated that lipophilicity is an important factor in determining the Papp value. The expression of P-gp transporter in NHBE cell monolayers was confirmed by the significantly higher basolateral to apical permeability of rhodamine123 than that of reverse direction and RT-PCR of MDR1 mRNA. However, the symmetric transport of fexofenadine.HCl in this NHBE cell monolayers study seems to be due to the low expression of P-gp transporter and/or to its saturation with high concentration of fexofenadine.HCl. Thus, the development of tight junction and the expression of P-gp in the NHBE cell monolayers in this study imply that they could be a suitable in vitro model for evaluation of systemic drug absorption via airway delivery, and that they reflect in vivo condition better than P-gp over-expressed cell line models.

Enhancing effect of surfactants on fexofenadine.HCl transport across the human nasal epithelial cell monolayer.

The effect of various surfactants (sodium cholate, sodium taurocholate, Tween 80 and Poloxamer F68) on enhancing the transepithelial permeability of fexofenadine.HCl was evaluated in a human nasal epithelial cell monolayer model. The cytotoxicity of the surfactants on the human nasal epithelial cells was evaluated by the MTT assay. A dose-dependent reduction of cell viability was observed at higher than critical micelle concentration (CMC) of the surfactants, and the IC50 of non-ionic surfactants (Tween 80 and Poloxamer F68) was higher than that of ionic surfactants (sodium cholate and sodium taurocholate). The TEER values significantly decreased after 2 h incubation with the ionic surfactants, but were recovered after 24 h in the fresh culture media. Ionic surfactants significantly increased the transepithelial permeability (P(app)) of fexofenadine.HCl compared to the non-ionic surfactants. The reduction of TEER values upon exposing the cell monolayer to the surfactants for 2 h correlated well with the P(app) of fexofenadine.HCl, which suggests that the permeation-enhancing mechanism of the ionic surfactants is by altering the tight junction property of the paracellular pathway. F-actin staining showed that the effect of ionic surfactants on the tight junction is temporary and reversible, which is consistent with the TEER value recovery within 24 h. These results imply that ionic surfactants are potentially useful permeation enhancers for nasal delivery of hydrophilic compounds, such as fexofenadine.HCl. This study also indicated the usefulness of the human nasal epithelial cell monolayer model not only for evaluating the in vitro nasal drug transport but also for studying the mechanism and toxicity of enhancers.

Evaluation of the physicochemical stability and skin permeation of glucosamine sulfate.

Glucosamine sulfate (GS) is known to stop the degenerative process of osteoarthritis. Because most of the GS formulation on the market is in the oral form, an alternative formulation such as a transdermal delivery system (TDS) is necessary in order to increase patient compliance. As the initial step to develop a TDS of GS, the physicochemical stability and permeation study in rat skin were examined. Evaluation of the stability of GS at different pHs showed the compound to be most stable at pH 5.0. The degradation rate constant at 25 degrees C was estimated to be 5.93 x 10(-6) hr(-1) (t90 approximately 2.03 years) in a pH 5 buffer solution. Due to its hydrophilic characteristic, low skin permeability was expected of GS. However, the skin permeation rate was determined to be 13.27 microg/cm2/hr at 5% concentration. Results of this study suggest the possibility of developing GS into a transdermal delivery system.

In vitro evaluation of patch formulations for topical delivery of gentisic acid in rats.

Gentisic acid (GA) is used in cosmetics as a skin-whitening agent for the treatment of skin pigmentary disorders by influencing the synthesis of melanin through inhibition of melanosomal tyrosinase activity. In order to achieve effective topical delivery of GA to the active site in the skin, a matrix-type transdermal delivery system was developed. The in vitro skin permeation as well as skin deposition of GA was studied in rats. Among the five pressure-sensitive adhesives tested, DuroTak 87-2510 was the most effective to achieve the highest permeation rate of GA. Dodecylamine showed the most potent enhancement among the enhancers tested, and significantly increased the permeation rate of GA up to 112.99 (+/-30.12) microg/cm(2) per h at the concentration of 1%, when 6% GA was incorporated in DuroTak 87-2510. Moreover, a linear relationship was observed between the skin permeation rate of GA and the amount of the skin deposition after 12 h of permeation (r(2)=0.95). Thus, the in vitro skin permeation data may be useful to determine the amount of GA actually deposited in the skin.