Biology subjects

Seffernick, J. T.

Publications and source records attributed to Seffernick, J. T..

4 recordsLinked to original sources

bioRxiv2025

LCK-targeting molecular glues overcome resistance to inhibitor-based therapy in T-cell acute lymphoblastic leukemia

Drug resistance is a major challenge in cancer therapy, especially in the context of kinase inhibitors. While targeted protein degradation (TPD) was a distinct mode of action compared to inhibition-based therapeutic targeting, the potential value of TPD in drug-resistant cancer remains unclear. Here, we report the discovery of cereblon-recruiting molecular glue degraders (MGDs) targeting LCK, an oncogenic kinase in T-cell acute lymphoblastic leukemia (T-ALL). By high-throughput screening and medicinal chemistry optimization, we developed a series of MGDs that induced CRBN-dependent degradation of LCK as well as potent cytotoxicity in T-ALL in vitro. Structure-activity relationship analysis and ternary complex modeling revealed a non-canonical degron at the LCK-CRBN interface involving the G-loop, whose mutation disrupts this interaction. Unlike inhibitors and inhibitor-based PROTACs, these MGDs engage LCK in regions distal to the ATP binding site and thus their activities in T-ALL are not affected by gate-keeper LCK mutations that drive resistance to inhibitor-based therapeutics. Taken together, our data underscore the potential of LCK-targeting MGDs as a strategy to overcome kinase inhibitor resistance in T-ALL, highlighting a potentially generalizable strategy in cancer therapy.

cancer biology↗

bioRxiv2022

Protein complex prediction using Rosetta, AlphaFold, and mass spectrometry covalent labeling

Structural mass spectrometry offers several techniques for the characterization of protein structures. Covalent labeling (CL) in combination with mass spectrometry can be used as an analytical tool to study and determine structural properties of protein-protein complexes. Degrees of modification obtained from CL experiments for specific labeled residues can be compared between the unbound and bound states of complexes. This analysis can yield insights into structural features of these protein assemblies, specifically the proximity of specific residues to the protein-protein interface. However, this data is sparse and does not unambiguously elucidate protein structure. Thus, computational algorithms are needed to deduce structure from the CL data. In this work we present a novel hybrid method that combines models of protein complex subunits generated with AlphaFold with differential CL data via a CL-guided protein-protein docking in Rosetta. In a benchmark set, the RMSD (root-mean-square deviation) of the best-scoring models was below 3.6 [A] for 5/5 complexes with inclusion of CL data, whereas the same quality was only achieved for 1/5 complexes without CL data. The average improvement in RMSD observed upon inclusion of CL data was 5.2 [A]. This study suggests that our integrated approach can successfully use data obtained from CL experiments to distinguish between nativelike and non-nativelike models. Significance StatementStructural mass spectrometry can be a powerful and versatile approach to characterize the structure of protein complexes. Data obtained from covalent labeling mass spectrometry can provide insights into higher order protein structure (particularly with respect to residue interactions and solvent accessibility) but needs to be supplemented by computational techniques to elucidate accurate, atomic-detail structural information. Here, we present a method to combine bioanalytical data obtained from covalent labeling with models generated using AlphaFold to accurately predict protein-protein complexes in Rosetta. Differential covalent labeling data can be used to determine the proximity of residues to the binding interface of complexes which we utilized to analyze computational models and improve structure prediction algorithms.

biophysics↗

bioRxiv2021

Protein shape sampled by ion mobility mass spectrometry consistently improves protein structure prediction

Among a wide variety of mass spectrometry (MS) methodologies available for structural characterizations of proteins, ion mobility (IM) provides structural information about protein shape and size in the form of an orientationally averaged collision cross-section (CCS). While IM data have been predominantly employed for the structural assessment of protein complexes, CCS data from IM experiments have not yet been used to predict tertiary structure from sequence. Here, we are showing that IM data can significantly improve protein structure determination using the modeling suite Rosetta. The Rosetta Projection Approximation using Rough Circular Shapes (PARCS) algorithm was developed that allows for fast and accurate prediction of CCS from structure. Following successful rigorous testing for accuracy, speed, and convergence of PARCS, an integrative modelling approach was developed in Rosetta to use CCS data from IM experiments. Using this method, we predicted protein structures from sequence for a benchmark set of 23 proteins. When using IM data, the predicted structure improved or remained unchanged for all 23 proteins, compared to the predicted models in the absence of CCS data. For 15/23 proteins, the RMSD (root-mean-square deviation) of the predicted model was less than 5.50 [A], compared to only 10/23 without IM data. We also developed a confidence metric that successfully identified near-native models in the absence of a native structure. These results demonstrate the ability of IM data in de novo structure determination.

biophysics↗

bioRxiv2020

Binding of Calcium and Magnesium to Cardiac Troponin C

Cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) which bind both Ca2+ and Mg2+, and a regulatory site (II) that has been thought to bind only Ca2+. The latter binding initiates a series of conformational changes that culminate in force production. We have quantified the interaction between site II and Ca2+/Mg2+ through Isothermal Titration Calorimetry and Thermodynamic Integration simulations. Direct and competitive binding titrations using wild type and a double mutant that significantly reduces binding to site II demonstrated that physiologically relevant concentrations of both Ca2+/Mg2+ interact with the same locus. Cytosolic free Mg2+ (~1 mM) could occupy a significant population of available site II, as this concentration of Mg2+ decreased the affinity for Ca2+ 1.4-fold. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart at baseline and in diseased states which decrease or increase availability of Mg2+ such as secondary hyperparathyroidism or ischemia, respectively.

cell biology↗