Recruitment of FBXO22 for targeted degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

Distinct antithrombin activation modes for fondaparinux and natural heparins detected using millisecond hydrogen deuterium exchange and collision induced unfolding.

Low molecular weight heparin and synthetic mimetics such as fondaparinux show different binding kinetics, protease specificity, and clinical effects. A combination of allosteric and template-mediated bridging mechanisms have been proposed to explain the differences in rate acceleration and specificity. The difficulty in working with heterogeneous heparin species has rendered a crystallographic interpretation of the differences in antithrombin activation between mimetics and natural heparin inaccessible. In this study, we examine the allosteric changes in antithrombin caused by binding fondaparinux, enoxaparin and depolymerized natural heparins using millisecond hydrogen deuterium exchange mass spectrometry (TRESI-HDX MS) and relate these conformational changes to complex stability in the gas phase using collision induced unfolding (CIU). This exploration reveals that in addition to the dynamic changes caused by fondaparinux, long chain heparins reduce structural flexibility proximal to Arg393, the cleavable residue in the reactive centre loop of the protein. These local changes in protein dynamics are associated with an increase in overall complex stability that increases with heparin chain length. Ultimately, these results shed light on the molecular mechanisms underlying differences in activity and specificity between heparin mimetics and natural heparins.

Diindoles produced from commensal microbiota metabolites function as endogenous CAR/Nr1i3 ligands.

Numerous studies have demonstrated the correlation between human gut bacteria and host physiology, mediated primarily via nuclear receptors (NRs). Despite this body of work, the systematic identification and characterization of microbe-derived ligands that regulate NRs remain a considerable challenge. In this study, we discover a series of diindole molecules produced from commensal bacteria metabolites that act as specific agonists for the orphan constitutive androstane receptor (CAR). Using various biophysical analyses we show that their nanomolar affinities are comparable to those of synthetic CAR agonists, and that they can activate both rodent and human CAR orthologues, which established synthetic agonists cannot. We also find that the diindoles, diindolylmethane (DIM) and diindolylethane (DIE) selectively up-regulate bona fide CAR target genes in primary human hepatocytes and mouse liver without causing significant side effects. These findings provide new insights into the complex interplay between the gut microbiome and host physiology, as well as new tools for disease treatment.

Small Molecule Screen Identifies Non-catalytic USP3 Chemical Handle.

Zinc-finger ubiquitin-binding domains (ZnF-UBDs) are noncatalytic domains mostly found in deubiquitylases (DUBs) such as USP3. They represent an underexplored opportunity for the development of deubiquitylase-targeting chimeras (DUBTACs) to pharmacologically induce the deubiquitylation of target proteins. We previously showed that ZnF-UBDs are ligandable domains. Here, a focused small molecule library screen against a panel of 11 ZnF-UBDs led to the identification of compound 59, a ligand engaging the ZnF-UBD of USP3 with a KD of 14 µM. The compound binds the expected C-terminal ubiquitin binding pocket of USP3 as shown by hydrogen-deuterium exchange mass spectrometry experiments and does not inhibit the cleavage of K48-linked diubiquitin by USP3. As such, this molecule is a chemical starting point toward chemical tools that could be used to interrogate the function of the USP3 Znf-UBD and the consequences of recruiting USP3 to ubiquitylated proteins.

In vitro safety signals for potential clinical development of the anti-inflammatory pregnane X receptor agonist FKK6.

Based on the mimicry of microbial metabolites, functionalized indoles were demonstrated as the ligands and agonists of the pregnane X receptor (PXR). The lead indole, FKK6, displayed PXR-dependent protective effects in DSS-induced colitis in mice and in vitro cytokine-treated intestinal organoid cultures. Here, we report on the initial in vitro pharmacological profiling of FKK6. FKK6-PXR interactions were characterized by hydrogen-deuterium exchange mass spectrometry. Screening FKK6 against potential cellular off-targets (G protein-coupled receptors, steroid and nuclear receptors, ion channels, and xenobiotic membrane transporters) revealed high PXR selectivity. FKK6 has poor aqueous solubility but was highly soluble in simulated gastric and intestinal fluids. A large fraction of FKK6 was bound to plasma proteins and chemically stable in plasma. The partition coefficient of FKK6 was 2.70, and FKK6 moderately partitioned into red blood cells. In Caco2 cells, FKK6 displayed high permeability (A-B: 22.8 × 10-6 cm.s-1) and no active efflux. These data are indicative of essentially complete in vivo absorption of FKK6. The data from human liver microsomes indicated that FKK6 is rapidly metabolized by cytochromes P450 (t1/2 5 min), notably by CYP3A4. Two oxidized FKK6 derivatives, including DC73 (N6-oxide) and DC97 (C19-phenol), were detected, and these metabolites had 5-7 × lower potency as PXR agonists than FKK6. This implies that despite high intestinal absorption, FKK6 is rapidly eliminated by the liver, and its PXR effects are predicted to be predominantly in the intestines. In conclusion, the PXR ligand and agonist FKK6 has a suitable pharmacological profile supporting its potential preclinical development.

Recruitment of FBXO22 for Targeted Degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

Broadly neutralizing humanized SARS-CoV-2 antibody binds to a conserved epitope on Spike and provides antiviral protection through inhalation-based delivery in non-human primates.

The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses.

Innate Conformational Dynamics Drive Binding Specificity in Anti-Apoptotic Proteins Mcl-1 and Bcl-2.

The structurally conserved B-cell lymphoma 2 (Bcl-2) family of protein function to promote or inhibit apoptosis through an exceedingly complex web of specific, intrafamilial protein-protein interactions. The critical role of these proteins in lymphomas and other cancers has motivated a widespread interest in understanding the molecular mechanisms that drive specificity in Bcl-2 family interactions. However, the high degree of structural similarity among Bcl-2 homologues has made it difficult to rationalize the highly specific (and often divergent) binding behavior exhibited by these proteins using conventional structural arguments. In this work, we use time-resolved hydrogen deuterium exchange mass spectrometry to explore shifts in conformational dynamics associated with binding partner engagement in the Bcl-2 family proteins Bcl-2 and Mcl-1. Using this approach combined with homology modeling, we reveal that Mcl-1 binding is driven by a large-scale shift in conformational dynamics, while Bcl-2 complexation occurs primarily through a classical charge compensation mechanism. This work has implications for understanding the evolution of internally regulated biological systems composed of structurally similar proteins and for the development of drugs targeting Bcl-2 family proteins for promotion of apoptosis in cancer.

Apparatus for Automated Continuous Hydrogen Deuterium Exchange Mass Spectrometry Measurements from Milliseconds to Hours.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a rapidly growing technique for protein characterization in industry and academia, complementing the "static" picture provided by classical structural biology with information about the dynamic structural changes that accompany biological function. Conventional hydrogen deuterium exchange experiments, carried out on commercially available systems, typically collect 4-5 exchange timepoints on a timescale ranging from tens of seconds to hours using a workflow that can require 24 h or more of continuous data collection for triplicate measurements. A small number of groups have developed setups for millisecond timescale HDX, allowing for the characterization of dynamic shifts in weakly structured or disordered regions of proteins. This capability is particularly important given the central role that weakly ordered protein regions often play in protein function and pathogenesis. In this work, we introduce a new continuous flow injection setup for time-resolved HDX-MS (CFI-TRESI-HDX) that allows automated, continuous or discrete labeling time measurements from milliseconds to hours. The device is composed almost entirely of "off-the-shelf" LC components and can acquire an essentially unlimited number of timepoints with substantially reduced runtimes compared to conventional systems.

Multifunctional metallochaperone modifications for targeting subsite cavities in mutant p53-Y220C.

The p53 protein, known as the 'guardian of the genome', plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.

Developments in rapid hydrogen-deuterium exchange methods.

Biological macromolecules, such as proteins, nucleic acids, and carbohydrates, contain heteroatom-bonded hydrogens that undergo exchange with solvent hydrogens on timescales ranging from microseconds to hours. In hydrogen-deuterium exchange mass spectrometry (HDX-MS), this exchange process is used to extract information about biomolecular structure and dynamics. This minireview focuses on millisecond timescale HDX-MS measurements, which, while less common than 'conventional' timescale (seconds to hours) HDX-MS, provide a unique window into weakly structured species, weak (or fast cycling) binding interactions, and subtle shifts in conformational dynamics. This includes intrinsically disordered proteins and regions (IDPs/IDRs) that are associated with cancer and amyloidotic neurodegenerative disease. For nucleic acids and carbohydrates, structures such as isomers, stems, and loops, can be elucidated and overall structural rigidity can be assessed. We will provide a brief overview of technical developments in rapid HDX followed by highlights of various applications, emphasising the importance of broadening the HDX timescale to improve throughput and to capture a wider range of function-relevant dynamic and structural shifts.

The protein dynamics of bovine and caprine ß-lactoglobulin differ as a function of pH.

The properties of milk proteins differ between mammalian species. ß-Lactoglobulin (ßlg) proteins from caprine and bovine milk are sequentially and structurally highly similar, yet their physicochemical properties differ, particularly in response to pH. To resolve this conundrum, we compared the dynamics of both the monomeric and dimeric states for each homologue at pH 6.9 and 7.5 using hydrogen/deuterium exchange experiments. At pH 7.5, the rate of exchange is similar across both homologues, but at pH 6.9 the dimeric states of the bovine ßlg B variant homologue have significantly more conformational flexibility compared with caprine ßlg. Molecular dynamics simulations provide a mechanistic rationale for the experimental observations, revealing that variant-specific substitutions encode different conformational ensembles with different dynamic properties consistent with the hydrogen/deuterium exchange experiments. Understanding the dynamic differences across ßlg homologues is essential to understand the different responses of these milks to processing, human digestion, and differences in immunogenicity.

Role of Half-of-Sites Reactivity and Inter-Subunit Communications in DAHP Synthase Catalysis and Regulation.

α-Carboxyketose synthases, including 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS), are long-standing targets for inhibition. They are challenging targets to create tight-binding inhibitors against, and inhibitors often display half-of-sites binding and partial inhibition. Half-of-sites inhibition demonstrates the existence of inter-subunit communication in DAHPS. We used X-ray crystallography and spatially resolved hydrogen-deuterium exchange (HDX) to reveal the structural and dynamic bases for inter-subunit communication in Escherichia coli DAHPS(Phe), the isozyme that is feedback-inhibited by phenylalanine. Crystal structures of this homotetrameric (dimer-of-dimers) enzyme are invariant over 91% of its sequence. Three variable loops make up 8% of the sequence and are all involved in inter-subunit contacts across the tight-dimer interface. The structures have pseudo-twofold symmetry indicative of inter-subunit communication across the loose-dimer interface, with the diagonal subunits B and C always having the same conformation as each other, while subunits A and D are variable. Spatially resolved HDX reveals contrasting responses to ligand binding, which, in turn, affect binding of the second substrate, erythrose-4-phosphate (E4P). The N-terminal peptide, M1-E12, and the active site loop that binds E4P, F95-K105, are key parts of the communication network. Inter-subunit communication appears to have a catalytic role in all α-carboxyketose synthase families and a regulatory role in some members.

Addressing Sugar-Sweetened Beverage Consumption in North Carolina.

Consuming sugar-sweetened beverages is one of the main concerns in addressing obesity in North Carolina. Many interventions have been developed to limit their consumptionwhile promoting drinking more water. Major sugarsweetened beverage producers take no accountability for the harm they cause to communities.

Exploring student perceptions of the learning environment in four health professions education programs.

The desire to support student learning and professional development, in combination with accreditation requirements, necessitates the need to evaluate the learning environment of educational programs. The Health Education Learning Environment Survey (HELES) is a recently-developed global measure of the learning environment for health professions programs. This paper provides evidence of the applicability of the HELES for evaluating the learning environment across four health professions programs: medicine, nursing, occupational therapy and pharmaceutical sciences. Two consecutive years of HELES data were collected from each program at a single university (year 1 = 552 students; year 2 = 745 students) using an anonymous online survey. Reliability analyses across programs and administration years supported the reliability of the tool. Two-way factorial ANOVAs with program and administration year as the independent variables indicated statistically- and practically-significant differences across programs for four of the seven scales. Overall, these results support the use of the HELES to evaluate student perceptions of the learning environment multiple of health professions programs.

Epitope screening using Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): An accelerated workflow for evaluation of lead monoclonal antibodies.

BACKGROUND: Epitope mapping is an increasingly important aspect of biotherapeutic and vaccine development. Recent advances in therapeutic antibody design and production have enabled candidate mAbs to be identified at a rapidly increasing rate, resulting in a significant bottleneck in the characterization of "structural" epitopes, that are challenging to determine using existing high throughput epitope mapping tools. Here, a Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) epitope screening workflow was introduced that is well suited for accelerated characterization of epitopes with a common antigen. MAIN METHODS AND MAJOR RESULTS: The method is demonstrated on set of six candidate mAbs targeting Pertactin (PRN). Using this approach, five of the six epitopes were unambiguously determined using two HDX mixing timepoints in 24 h total run time, which is equivalent to the instrument time required to map a single epitope using the conventional workflow. CONCLUSION: An accelerated HDX-MS epitope screening workflow was developed. The "screening" workflow successfully characterized five (out of six attempted) novel epitopes on the PRN antigen; information that can be used to support vaccine antigenicity assays.

Subsecond Time-Resolved Mass Spectrometry in Dynamic Structural Biology.

Life at the molecular level is a dynamic world, where the key players─proteins, oligonucleotides, lipids, and carbohydrates─are in a perpetual state of structural flux, shifting rapidly between local minima on their conformational free energy landscapes. The techniques of classical structural biology, X-ray crystallography, structural NMR, and cryo-electron microscopy (cryo-EM), while capable of extraordinary structural resolution, are innately ill-suited to characterize biomolecules in their dynamically active states. Subsecond time-resolved mass spectrometry (MS) provides a unique window into the dynamic world of biological macromolecules, offering the capacity to directly monitor biochemical processes and conformational shifts with a structural dimension provided by the electrospray charge-state distribution, ion mobility, covalent labeling, or hydrogen-deuterium exchange. Over the past two decades, this suite of techniques has provided important insights into the inherently dynamic processes that drive function and pathogenesis in biological macromolecules, including (mis)folding, complexation, aggregation, ligand binding, and enzyme catalysis, among others. This Review provides a comprehensive account of subsecond time-resolved MS and the advances it has enabled in dynamic structural biology, with an emphasis on insights into the dynamic drivers of protein function.

Population genomics of Drosophila suzukii reveal longitudinal population structure and signals of migrations in and out of the continental United States.

Drosophila suzukii, or spotted-wing drosophila, is now an established pest in many parts of the world, causing significant damage to numerous fruit crop industries. Native to East Asia, D. suzukii infestations started in the United States a decade ago, occupying a wide range of climates. To better understand invasion ecology of this pest, knowledge of past migration events, population structure, and genetic diversity is needed. In this study, we sequenced whole genomes of 237 individual flies collected across the continental United States, as well as several sites in Europe, Brazil, and Asia, to identify and analyze hundreds of thousands of genetic markers. We observed strong population structure between Western and Eastern US populations, but no evidence of any population structure between different latitudes within the continental United States, suggesting that there are no broad-scale adaptations occurring in response to differences in winter climates. We detect admixture from Hawaii to the Western United States and from the Eastern United States to Europe, in agreement with previously identified introduction routes inferred from microsatellite analysis. We also detect potential signals of admixture from the Western United States back to Asia, which could have important implications for shipping and quarantine policies for exported agriculture. We anticipate this large genomic dataset will spur future research into the genomic adaptations underlying D. suzukii pest activity and development of novel control methods for this agricultural pest.

Immunocartography: Charting vaccine-driven immunity by applying single cell proteomics to an in vitro human model.

The ability to measure immunomodulatory effects of a vaccine is crucial for novel vaccine design. While traditional animal models have been effective, a better understanding of the response in humans to new vaccines in pre-clinical development is critical for advancement to clinical trials. A translational methodology that can capture the complexity of a vaccine-driven response in a human model, which does not require human exposure, is needed. Here we have designed a platform that uses fresh human whole blood as a key component to study the adaptive immune memory response to vaccine formulations. The response is monitored by high-parameter single cell analysis using mass cytometry (Helios, CyTOF System), allowing for a rapid, in-depth characterization of antigen specific proliferation and expansion of preexisting memory T cells in concert with an innate adjuvant-driven response. In this work we demonstrate the capability of this platform to characterize biologically relevant changes in the cellular response across memory T-cells, B cells, monocytes, and NK cells, at an unprecedented level of detail. This approach that we call Immunocartography has the potential to transform the way new vaccines can be assessed before and throughout clinical development.

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization "Hotspots" in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction.

The novel severe respiratory syndrome-like coronavirus (SARS-CoV-2) causes COVID-19 in humans and is responsible for one of the most destructive pandemics of the last century. At the root of SARS-CoV infection is the interaction between the viral spike protein and the human angiotensin converting enzyme 2 protein, which allows the virus to gain entry into host cells through endocytosis. In this work, we apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a detailed view of the functional footprint and conformational dynamics associated with this interaction. Our results broadly agree with the binding interface derived from high resolution X-ray crystal structure data but also provide insights into shifts in structure and dynamics that accompany complexation, including some that occur immediately outside of the core binding interface. We propose that dampening of these "binding-site adjacent" dynamic shifts could represent a mechanism for neutralizing activity in a multitude of spike protein-targeted mAbs that have been found to specifically bind these "peripheral" sites. Our results highlight the unique capacity of HDX-MS to detect potential neutralization "hotspots" outside of the core binding interfaces defined by high resolution structural data.