The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.
The dysregulation of Fbxo4-cyclin D1 axis occurs at high frequency in esophageal squamous cell carcinoma (ESCC), where it promotes ESCC development and progression. However, defining a therapeutic vulnerability that results from this dysregulation has remained elusive. Here we demonstrate that Rb and mTORC1 contribute to Gln-addiction upon the dysregulation of the Fbxo4-cyclin D1 axis, which leads to the reprogramming of cellular metabolism. This reprogramming is characterized by reduced energy production and increased sensitivity of ESCC cells to combined treatment with CB-839 (glutaminase 1 inhibitor) plus metformin/phenformin. Of additional importance, this combined treatment has potent efficacy in ESCC cells with acquired resistance to CDK4/6 inhibitors in vitro and in xenograft tumors. Our findings reveal a molecular basis for cancer therapy through targeting glutaminolysis and mitochondrial respiration in ESCC with dysregulated Fbxo4-cyclin D1 axis as well as cancers resistant to CDK4/6 inhibitors.
The su(var)3‐9, enhancer of zeste, trithorax (SET)/inhibitor 2 of protein phosphatase 2A (PP2A) oncoprotein binds and inhibits PP2A, composed of various isoforms of scaffolding, regulatory, and catalytic subunits. Targeting SET with a sphingolipid analog drug fingolimod (FTY720) or ceramide leads to the reactivation of tumor suppressor PP2A. However, molecular details of the SET‐FTY720 or SET‐ceramide, and mechanism of FTY720‐dependent PP2A activation, remain unknown. Here, we report the first in solution examination of the SET‐FTY720 or SET‐ceramide complexes by NMR spectroscopy. FTY720‐ceramide binding resulted in chemical shifts of residues residing at the N terminus of SET, preventing its dimerization or oligomerization. This then released SET from PP2ACα, resulting in PP2A activation, while monomeric SET remained associated with the B56γ. Our data also suggest that the PP2A holoenzyme, composed of PP2A‐Aβ, PP2A‐B56γ, and PP2ACα subunits, is selectively activated in response to the formation of the SET‐FTY720 complex in A549 cells. Various PP2A‐associated downstream effector proteins in the presence or absence of FTY720 were then identified by stable isotope labeling with amino cells in cell culture, including tumor suppressor nonmuscle myosin IIA. Attenuation of FTY720‐SET association by point mutations of residues that are involved in FTY720 binding or dephosphorylation of SET at Serine 171, enhanced SET oligomerization and the formation of the SET‐PP2A inhibitory complex, leading to resistance to FTY720‐dependent PP2A activation.—De Palma, R. M., Parnham, S. R., Li, Y., Oaks, J. J., Peterson, Y. K., Szulc, Z. M., Roth, B. M., Xing, Y., Ogretmen, B. The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction. FASEB J. 33, 7647–7666 (2019). http://www.fasebj.org
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