Selection of a suitable formulation that provides adequate product stability is an important aspect of the development of biopharmaceutical products. Stability of proteins includes not only ...resistance to chemical modifications but also conformational and colloidal stabilities. While chemical degradation of antibodies is relatively easy to detect and control, propensity for conformational changes and/or aggregation during manufacturing or long-term storage is difficult to predict. In many cases, the formulation factors that increase one type of stability may significantly decrease another type under the same or different conditions. Often compromise is necessary to minimize the adverse effects of an antibody formulation by careful optimization of multiple factors responsible for overall stability. In this study, high-throughput stress and characterization techniques were applied to 96 formulations of anti-streptavidin antibodies (an IgG1 and an IgG2) to choose optimal formulations. Stress and analytical methods applied in this study were 96-well plate based using an automated liquid handling system to prepare the different formulations and sample plates. Aggregation and clipping propensity were evaluated by temperature and mechanical stresses. Multivariate regression analysis of high-throughput data was performed to find statistically significant formulation factors that alter measured parameters such as monomer percentage or unfolding temperature. The results of the regression models were used to maximize the stabilities of antibodies under different formulations and to find the optimal formulation space for each molecule. Comparison of the IgG1 and IgG2 data indicated an overall greater stability of the IgG1 molecule under the conditions studied. The described method can easily be applied to both initial preformulation screening and late-stage formulation development of biopharmaceutical products.
We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of
N-acetylglucosamine (GlcNAc), inhibits the enzyme
O-GlcNAc-selective
N-acetyl-β-
d-glucosaminidase (
...O-GlcNAcase) which is responsible for the removal of
O-GlcNAc from proteins. Alloxan, another β-cell toxin is a uracil analog. Since the
O-GlcNAc transferase (OGT) uses UDP–GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in β-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, suggesting that alloxan indeed was inhibiting (OGT). In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0–10 mM alloxan, and OGT activity was measured directly by quantitating UDP–
3
H
-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions, OGT activity was completely inhibited by 1 mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT, and as such, is the first OGT inhibitor described.
Mass spectrometric techniques for identification of proteins by “mass fingerprinting” (matching the masses of tryptic peptides from a protein digest to the theoretical peptides in a database) such as ...matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) are rapidly growing in popularity as the demand for high throughput analysis of the proteome increases. This is due, in part, to the ability to automate the technique and the rapid rate with which mass spectra may be acquired. An important factor in the accuracy of the technique is the number of tryptic peptides that are identified in the various searching algorithms that exist. The greater sequence coverage of the parent protein that is obtained, the higher the level of confidence in the identification that is determined. One impediment to high levels of sequence coverage is the bias of MALDI-TOF mass spectrometry to arginine-containing peptides. Increasing the sensitivity to lysine-containing peptides should increase the sequence coverage obtained. In order to achieve this result we have developed conditions to modify the ϵ-amine group of lysine in tryptic peptides with O-methylisourea. The conditions utilized result in the conversion of lysine to homoarginine with no modification of the amine terminus of the peptides. The sensitivity of MALDI-TOF mass spectrometry detection of peptides was increased dramatically following modification. The modification chemistry may be applied to tryptic peptide mixtures prior to desalting and spotting onto MALDI-TOF plates. This technique will be particularly useful for identifying proteins with a high lysine/arginine ratio.
A homology derived molecular model of prostate specific antigen (PSA) was created and refined. The active site region was investigated for specific interacting functionality and a binding model ...postulated for the novel 2-azetidinone acyl enzyme inhibitor 1 (IC50 = 8.98 ± 0.90 μM) which was used as a lead compound in this study. A single low energy conformation structure II (Figure ) was adopted as most likely to represent binding after minimization and dynamics calculations. Systematic analysis of the binding importance of all three side chains appended to the 2-azetidinone was conducted by the synthesis of several analogues. A proposed salt bridge to Lys-145 with 4 (IC50 = 5.84 ± 0.92 μM) gave improved inhibition, but generally the binding of the N-1 side chain in a specific secondary aromatic binding site did not tolerate much structural alteration. A hydrophobic interaction of the C-4 side chain afforded inhibitor 6 (IC50 = 1.43 ± 0.19 μM), and polar functionality could also be added in a proposed interaction with Gln-166 in 5 (IC50 = 1.34 ± 0.05 μM). Reversal of the C-4 ester connectivity furnished inhibitors 7 (IC50 = 1.59 ± 0.15 μM), 11 (IC50 = 3.08 ± 0.41 μM), and 13 (IC50 = 2.19 ± 0.36 μM) which were perceived to bind to PSA by a rotation of 180° relative to the C-4 ester of normal connectivity. Incorporation of hydroxyl functionality into the C-3 side chain provided 16 (IC50 = 348 ± 50 nM) with the greatest increase in PSA inhibition by a single modification. Multiple copy simultaneous search (MCSS) analysis of the PSA active site further supported our model and suggested that 18 would bind strongly. Asymmetric synthesis yielded 18 (IC50 = 226 ± 10 nM) as the most potent inhibitor of PSA reported to date. It is concluded that our design approach has been successful in developing PSA inhibitors and could also be applied to the inhibition of other enzymes, especially in the absence of crystallographic information.
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Background: BRAF V600E-mutant metastatic colorectal cancer (mCRC) is associated with a poor prognosis and limited clinical data. Based on results from the BEACON CRC trial, targeted treatment with ...encorafenib plus cetuximab (E+C) is available as a standard of care for these patients (pts) after prior systemic therapy. Since data from controlled clinical trials are based on a selected patient population, the non-interventional study (NIS) BERING CRC observes a broader patient population in clinical practice. Methods: BERING CRC is the first NIS investigating the use of E+C in clinical practice of BRAF V600E-mutant mCRC treatment after prior systemic therapy in Germany, Austria, and Switzerland. For the present analysis, disease characteristics and treatment data of the initial 81 pts were documented in 44 sites across Germany and Austria between 09/2020 and 04/2022. BERING-CRC is ongoing and aims to enroll up to 300 pts from 126 German, Austrian, and Swiss sites. The study observes pts treated according to the approved respective Summary of Product Characteristics (SmPC). The primary endpoint is the 1-year overall survival rate. Secondary endpoints include efficacy, QoL, and tolerability of E+C. Results: 81 pts were included in this baseline analysis. Median age was 67 years (range 34-88) and 48% were female. 48 pts (59%) were documented with right-sided tumors and for 62% stage IV disease was noted at initial diagnosis. In the metastatic setting, main sites of metastasis were liver, peritoneum, and lung (52%, 32%, and 22% of pts, respectively), with 16% of pts having ≥ 3 sites documented. For 30% of pts, an ECOG performance status (PS) of 0 was documented at baseline assessment (57% ECOG PS 1 or 2). Adjuvant treatment was reported for 22 pts while relapse ≤ 6 months was documented for 10 of them. Consistent with BEACON CRC, adjuvant systemic therapy with relapse within ≤6 months was counted as metastatic 1st line treatment. 91% of pts with adjuvant treatment received chemotherapy alone in this setting. 4 pts (5%) were documented with E+C treatment directly following adjuvant therapy and 55% of all pts with documented first-line treatment (69 pts) received chemotherapy alone (CT), for 35% chemotherapy was combined with targeted therapy (CT+TT), 3% received immunotherapy, and 1% received TT alone. In second-line, 71% of pts with documented treatment received E+C (7% CT, 16% CT+TT). An initial bi-weekly cetuximab dosing was reported for 10% of pts treated with E+C. Conclusions: While the high number of pts with right-sided tumors was in line with previous findings on BRAF V600E-mutant mCRC pts, synchronous metastasis was reported considerably more often in this real-world cohort. As complementation to previous controlled clinical trial data, pts enrolled in BERING-CRC were notably older and presented with higher ECOG PS compared to the pivotal study. Clinical trial information: NCT04673955 .
Fas ligand (FasL) and Fas receptor are members of the tumor necrosis factor (TNF) receptor and ligand family that play an important role in regulating apoptosis in normal physiology. Decoy receptor 3 ...(DcR3) is a novel member of the TNF receptor superfamily, which binds to and blocks the activities of the ligands FasL and LIGHT. We have demonstrated that DcR3 was degraded rapidly to a major circulating metabolic fragment after subcutaneous administration in primates and mice. This fragment was also generated in subcutaneous tissue homogenate
in
vitro. Mass spectrometry and N-terminal sequencing indicated that DcR3 was proteolytically cleaved between R218 and A219 in the primary sequence to yield the fragment DcR3(1-218). While retaining its ability to bind LIGHT and inhibit LIGHT-mediated activities, DcR3(1-218) no longer bound FasL and did not inhibit FasL-mediated apoptosis
in
vitro. The primary sequence of DcR3 was molecularly engineered, changing the arginine residue at position 218 to glutamine to generate an analog, DcR3(R218Q), which we termed FLINT (LY498919). We demonstrated that FLINT was more stable to proteolytic degradation
in vitro and
in vivo and maintained its activity against both soluble FasL and soluble LIGHT
in vitro. As a result, the modification in the sequence of DcR3 to produce FLINT (LY498919) should result in a pharmacologically superior molecule in the therapeutic intervention of diseases in which the pathogenesis is linked to FasL-mediated apoptotic or inflammatory events.
Little is known about leptin's interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125I-leptin-serum ...complex demonstrated 125I-leptin eluting in significant proportion associated with macromolecules. The 125I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED50: 0.73 +/- 0.09 nM, mean +/- SEM, n = 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80- or 100-kD proteins could be the soluble leptin receptor as approximately 10% of the bound 125I-leptin was immunoprecipitable with leptin receptor antibodies. Significantly higher (P < 0.001) proportions of total leptin circulate in the bound form in lean (46.5 +/- 6.6%) compared with obese (21.4 +/- 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean (P < 0.0005) and three obese (P < 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 +/- 1.9 vs. 1.3 +/- 0.4 ng/ml) compared with those in obese subjects (28.3 +/- 9.8 vs. 14.7 +/- 5.3). No significant (P > 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.
Straightforward methods for the introduction of stable isotopes into proteins with subsequent isolation and purification of the proteins will greatly aid the field of quantitative proteomics. ...Proteins containing amino acids with one or more of the stable isotopes of deuterium, 13C, 15N or 18O can be used as internal standards by addition at an early stage of analysis of a complex protein sample and subsequent measurement using mass spectrometry. There are two approaches for introducing a stable isotope into a protein without chemically modifying that protein, metabolic labeling using whole cells grown in culture, or cell-free labeling using a lysate of either Escherichia coli or wheat germ. Each approach has its advantages and disadvantages which will be discussed. Particular attention will be paid to the cell-free method using an E. coli lysate.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from part-rough, part-smooth transitional elements of the ...endoplasmic reticulum (TER). Vesicle budding from the TER is an ATP-dependent process both in vivo and in vitro. An ATPase with a monomer molecular weight of 100 kD by SDS-PAGE has been isolated from TER and designated as TER ATPase. The native TER ATPase has been characterized as a hexamer of six 100-kD subunits by gel filtration. The protein catalyzes the hydrolysis of γ 32- P ATP and is phosphorylated in the presence of Mg2+. It is distinct from the classical transport ATPases based on pH optima, ion effects, and inhibitor specificity. Electron microscopy of negatively stained preparations revealed the TER ATPase to be a ring-shaped structure with six-fold rotational symmetry. A 19-amino acid sequence of TER ATPase having 84% identity with valosin-containing protein and 64% identity with a yeast cell-cycle control protein CDC48p was obtained. Anti-synthetic peptide antisera to a 15-amino acid portion of the sequence of TER ATPase recognized a 100-kD protein from TER. These antisera reduced the ATP-dependent cell-free formation of transition vesicles from isolated TER of rat liver. In a reconstituted membrane transfer system, TER ATPase antisera inhibited transfer of radiolabeled material from endoplasmic reticulum to Golgi apparatus, while preimmune sera did not. The results suggest that the TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase were isolated by immunoscreening a rat liver cDNA library with the affinity-purified TER ATPase antibody. A computer search of deduced amino acid sequences revealed the cloned TER ATPase to be the rat equivalent of porcine valosin-containing protein, a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide-sensitive fusion protein.
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