Derek J. Wilson

Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia

Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen–deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.

A versatile microfluidic chip for millisecond time-scale kinetic studies by electrospray mass spectrometry

An electrospray coupled microfluidic reactor for the measurement of millisecond time-scale, solution phase kinetics is introduced. The device incorporates a simple two-channel design that is etched into polymethyl methacrylate (PMMA) by laser ablation. The outlet of the device is laser cut to a sharp tip, facilitating low dead volume ‘on chip’ electrospray. Fabrication is fast, straightforward and highly reproducible, supporting rapid prototyping and large-scale reproduction. Device performance is characterized using a cytochrome c unfolding reaction. Unfolding processes with rates in excess of 30 s−1 are easily measured, including the appearance of a ‘native-like’ intermediate that is maximally populated 180 ms post reaction initiation. To extract reliable rates from the data, a theoretical framework for the analysis of kinetics acquired under square-channel laminar flow is introduced.

Conformer Selection and Intensified Dynamics During Catalytic Turnover in Chymotrypsin

During catalytic turnover, enzymes undergo thermally driven conformational fluctuations (dynamics) that are directly linked to catalytic efficiency. In broad terms, this link exists because enzymes must sample specific dynamic modes in order to access high-energy structures along the catalytic reaction coordinate. Mass spectrometry-based approaches for probing enzyme dynamics are developing rapidly, but have been limited thus far by the application of H/D exchange (HDX) under steady-state conditions, which results in averaging of the data over multiple catalytic states. 6] Models that attempt to define the specific nature of the enzyme dynamics/activity relationship are therefore drawn overwhelmingly from Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion NMR experiments on “active” enzymes. 7,8] This approach is extremely powerful, but is confined to a small set of highly reversible reactions in order to circumvent the issue of substrate depletion during the experiment. 3, 9–12] From the limited set of “CPMG accessible” reactions, two models for catalysis-linked dynamics have been formulated. The “induced fit” model is characterized by a substrate-free (resting) state that samples a different set of dynamic modes compared to the substrate-bound, catalytically active state. This would imply that the dynamics observed in the resting state should be substantially different from those observed during catalysis. A number of studies have reported evidence supporting this model. 14] In the “conformer selection” model, the conformational space sampled by the enzyme is independent of catalysis (i.e., the resting and active-state conformational dynamics are identical). Productive enzyme– substrate interactions occur when incoming substrate “selects” the appropriate conformer for binding. Conformer selection is supported by a substantial number of studies showing no difference in dynamics between free and substrate-bound enzyme. 3, 5,9–11] “Hybrid” models have also been proposed in which substrate binding occurs through conformer selection followed by “induced fit-like” substratedirected conformational sampling during catalysis. These models provide crucial insights into virtually all aspects of enzyme function including substrate binding, specificity, allostery and rate-limiting catalytic processes. However, their formulation from a relatively small pool of similar enzyme systems suggests that they may describe only a fraction of possible catalysis-linked dynamic modes. In this work, we probe conformational dynamics in an active, “CPMG inaccessible” enzyme system using an alternative approach that combines time-resolved electrospray mass spectrometry (TRESI-MS) and sub-second H/D exchange (HDX) labeling to monitor dynamics in the pre-steady state. By this approach, catalytic processes are detected as time-dependent intensity changes in mass-tocharge (m/z) peaks corresponding to the accumulation and/or depletion of enzyme intermediates. For each species that becomes populated during the measurement, dynamics are probed simultaneously, by the rate and magnitude of deuterium uptake. In contrast to CPMG NMR spectroscopy, these measurements are not “site specific”, however, they represent a straightforward and broadly applicable method for characterizing dynamics in active enzyme systems. A schematic illustration of the experimental setup is provided in Figure 1.

Hyperphosphorylation of intrinsically disordered tau protein induces an amyloidogenic shift in its conformational ensemble.

Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabilize microtubules in neuronal axons at all stages of development. In Alzheimers and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphorylation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed picture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to define specific conformational biases that can easily be rationalized as enhancing amyloidogenic propensity. Representative structures for the native and hyperphosphorylated tau ensembles were generated by refinement of a broad sample of conformations generated by low-computational complexity modeling, based on agreement with the TRESI-HDX profiles.

Characterizing rapid, activity-linked conformational transitions in proteins via sub-second hydrogen deuterium exchange mass spectrometry.

This review outlines the application of time‐resolved electrospray ionization mass spectrometry (TRESI‐MS) and hydrogen‐deuterium exchange (HDX) to study rapid, activity‐linked conformational transitions in proteins. The method is implemented on a microfluidic chip which incorporates all sample‐handling steps required for a ‘bottom‐up’ HDX workflow: a capillary mixer for sub‐second HDX labeling, a static mixer for HDX quenching, a microreactor for rapid protein digestion, and on‐chip electrospray. By combining short HDX labeling pulses with rapid digestion, this approach provides a detailed characterization of the structural transitions that occur during protein folding, ligand binding, post‐translational modification and catalytic turnover in enzymes. This broad spectrum of applications in areas largely inaccessible to conventional techniques means that microfluidics‐enabled TRESI‐MS/HDX is a unique and powerful approach for investigating the dynamic basis of protein function.

Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development - A review.

Protein therapeutics have emerged as a major class of biopharmaceuticals over the past several decades, a trend that has motivated the advancement of bioanalytical technologies for protein therapeutic characterization. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful and sensitive technique that can probe the higher order structure of proteins and has been used in the assessment and development of monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs) and biosimilar antibodies. It has also been used to quantify protein-ligand, protein-receptor and other protein-protein interactions involved in signaling pathways. In manufacturing and development, HDX-MS can validate storage formulations and manufacturing processes for various biotherapeutics. Currently, HDX-MS is being refined to provide additional coverage, sensitivity and structural specificity and implemented on the millisecond timescale to reveal residual structure and dynamics in disordered domains and intrinsically disordered proteins.

Changes in Signal Transducer and Activator of Transcription 3 (STAT3) Dynamics Induced by Complexation with Pharmacological Inhibitors of Src Homology 2 (SH2) Domain Dimerization

Background: Salicylic acid-based inhibitors of signal transducer and activator of transcription 3 (STAT3) are predicted to interact with the Src homology 2 (SH2) domain, inhibiting dimerization. Results: STAT3-inhibitor interactions were interrogated by hydrogen-deuterium exchange (HDX) mass spectrometry (MS). Conclusion: HDX MS revealed local and global perturbations in STAT3 dynamics. Significance: Understanding STAT3 inhibitor interactions with the SH2 domain is critical to inhibitor development. The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization.

Time-Resolved Mass Spectrometry for Monitoring Millisecond Time-Scale Solution-Phase Processes

Many chemical and biochemical reactions equilibrate within a few seconds of initiation under “native” conditions. To understand the microscopic processes underlying these reactions, the most direct approach is to monitor the reaction as equilibrium is established (i.e. the reaction kinetics). However, this requires the ability to characterize the reaction mixture on the millisecond time-scale. In this review, we survey the contributions of time-resolved mass spectrometry (TR-MS) to the characterization of millisecond time-scale (bio)chemical processes, with a focus on biochemical applications. Compared to conventional time-resolved techniques, which use optical detection, the primary advantage of TR-MS is the ability to detect virtually all reactive species simultaneously. This provides a singularly high detail account of the reaction without the need for a chromophoric change on turnover or artificial chromophoric probes. To provide millisecond time-resolution, TR-MS set-ups usually employ continuous-flow rapid mixers, corresponding either to a fixed “tee” that provides a single reaction time-point or an adjustable position mixer that allows continuous reaction monitoring. TR-MS has been used to monitor processes with rates up to 500 s−1 and to provide a detailed account of complex reactions involving 10 co-populated species. This corresponds to significantly lower time-resolution than optical methods, which can measure rates in excess of 900 s−1 under ideal conditions, but substantially more detail (optical studies are typically limited to one or two analytes). TR-MS has been implemented on a number of platforms, including capillary and microfluidic set-ups. Capillary-based implementations are straightforward to fabricate and provide the most efficient rapid mixing. Microfluidic implementations have also been devised to enable multi-step experimental workflows that incorporate TR-MS. As a general method for time-resolved measurements, the applications for TR-MS are wide ranging. TR-MS has been used to identify intermediates in organic reactions, reveal protein (un)folding mechanisms, monitor enzyme catalysis in the pre-steady-state and, in conjunction with hydrogen–deuterium exchange, characterize protein conformational dynamics. While not without limitations, TR-MS represents a powerful alternative for measuring solution phase processes on the millisecond time-scale and a new, promising approach for revealing mechanistic details in (bio)chemical reactions.

Kinetic Capillary Electrophoresis with Mass-Spectrometry Detection (KCE- MS) Facilitates Label-Free Solution-Based Kinetic Analysis of Protein-Small Molecule Binding

Tandem tracker: Here we introduce a method for studying the kinetics of protein-small-molecule interactions based on kinetic capillary electrophoresis (KCE) separation and MS detection. Due to the variety of KCE methods and MS modes available, the KCE-MS tandem is a highly versatile platform for label-free, solution-based kinetic studies of affinity interactions.

Understanding and optimizing electrospray ionization techniques for proteomic analysis

Electrospray ionization (ESI) mass spectrometry is a powerful and versatile tool for proteomic analysis. By understanding how proteins and peptides behave during ESI, it is possible to predict source conditions that will maximize ionization efficiency, ultimately leading to lower detection limits for protein identification and more accurate quantitation. In this article, we provide an overview of a variety of electrospray-based ionization methods, including nanospray, liquid chromatography and capillary electrophoresis-coupled sources, and how they are optimized for proteomic samples. We will touch upon analyte characteristics, solvent/eluent conditions as well as optimization of ESI for top-down, bottom-up and quantitative experiments.