High-resolution structural information of membrane-bound α-synuclein provides insight into the MoA of the anti-Parkinson drug UCB0599.

α-synuclein (αS) is an intrinsically disordered protein whose functional ambivalence and protein structural plasticity are iconic. Coordinated protein recruitment ensures proper vesicle dynamics at the synaptic cleft, while deregulated oligomerization on cellular membranes contributes to cell damage and Parkinson's disease (PD). Despite the protein's pathophysiological relevance, structural knowledge is limited. Here, we employ NMR spectroscopy and chemical cross-link mass spectrometry on 14N/15N-labeled αS mixtures to provide for the first time high-resolution structural information of the membrane-bound oligomeric state of αS and demonstrate that in this state, αS samples a surprisingly small conformational space. Interestingly, the study locates familial Parkinson's disease mutants at the interface between individual αS monomers and reveals different oligomerization processes depending on whether oligomerization occurs on the same membrane surface (cis) or between αS initially attached to different membrane particles (trans). The explanatory power of the obtained high-resolution structural model is used to help determine the mode-of-actionof UCB0599. Here, it is shown that the ligand changes the ensemble of membrane-bound structures, which helps to explain the success this compound, currently being tested in Parkinson's disease patients in a phase 2 trial, has had in animal models of PD.

Common and disparate clinical presentations and mechanisms in different eosinophilic gastrointestinal diseases.

Eosinophilic gastrointestinal diseases (EGIDs) are a group of diseases characterized by selective eosinophil infiltration of the gastrointestinal (GI) tract in the absence of other causes of eosinophilia. These diseases are generally driven by type 2 inflammation, often in response to food allergen exposure. Among all EGIDs, the clinical presentation often includes a history of atopic disease with a variety of GI symptoms. EGIDs are traditionally separated into eosinophilic esophagitis (EoE) and non-EoE EGIDs. EoE is relatively better understood and now associated with clinical guidelines and 2 US Food and Drug Administration-approved treatments, whereas non-EoE EGIDs are rarer and less well-understood diseases without US Food and Drug Administration-approved treatments. Non-EoE EGIDs are further subclassified by the area of the GI tract that is involved; they comprise eosinophilic gastritis, eosinophilic enteritis (including eosinophilic duodenitis), and eosinophilic colitis. As with other GI disorders, the disease presentations and mechanisms differ depending on the involved segment of the GI tract; however, the differences between EoE and non-EoE EGIDs extend beyond which GI tract segment is involved. The aim of this article is to summarize the commonalities and differences between the clinical presentations and disease mechanisms for EoE and non-EoE EGIDs.

Eosinophil Involvement Outside the Esophagus in Eosinophilic Esophagitis.

Eosinophilic gastrointestinal diseases (EGIDs) are chronic, immune-mediated disorders, characterized clinically by gastrointestinal (GI) symptoms and histologically by eosinophil-predominant infiltration in ≥1 GI tract segment.1 A recent, international consensus by 91 experts proposed a new framework for EGID nomenclature to establish updated terms, designations, and conventions.2 Although this framework offers a standardized starting point for the field, debate is ongoing regarding the appropriate terminology for cases involving multiple areas, such as "non-eosinophilic esophagitis (EoE) EGID and EoE" or "non-EoE EGID with esophageal involvement (EI)." Notably, in a survey of these experts, 61% agreed with the later term "non-EoE EGID with EI," because EoE is isolated to the esophagus by current diagnostic criteria.3 However, limited molecular and pathogenic data exist to support the distinction. Furthermore, disease burden of symptoms and comorbidities generally is higher in non-EoE EGIDs than EoE.4 Presently, there is no screen to predict non-EoE EGID concomitance in EoE; therefore, decision-making to further explore other GI segment involvement is clinically challenging. We aimed to answer 2 fundamental questions in the field (Figure 1A): Is there a shared or distinct pathogenesis between patients with isolated EoE and non-EoE EGIDs with EI as assessed by patient characteristics and molecular profiles? Can we predict concomitant non-EoE EGIDs when EoE exists? Herein, we report a similar molecular signature between EoE and EI and a potential predictive model to identify concomitant non-EoE EGIDs in patients with EoE.

A Phase 1/2A trial of idroxioleic acid: first-in-class sphingolipid regulator and glioma cell autophagy inducer with antitumor activity in refractory glioma.

BACKGROUND: The first-in-class brain-penetrating synthetic hydroxylated lipid idroxioleic acid (2-OHOA; sodium 2-hydroxyoleate), activates sphingomyelin synthase expression and regulates membrane-lipid composition and mitochondrial energy production, inducing cancer cell autophagy. We report the findings of a multicentric first-in-human Phase 1/2A trial (NCT01792310) of 2-OHOA, identifying the maximum tolerated dose (MTD) and assessing safety and preliminary efficacy. METHODS: We performed an open-label, non-randomised trial to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics and anti-tumour activity of daily oral treatment with 2-OHOA monotherapy (BID/TID) in 54 patients with glioma and other advanced solid tumours. A dose-escalation phase using a standard 3 + 3 design was performed to determine safety and tolerability. This was followed by two expansion cohorts at the MTD to determine the recommended Phase-2 dose (RP2D). RESULTS: In total, 32 recurrent patients were enrolled in the dose-escalation phase (500-16,000 mg/daily). 2-OHOA was rapidly absorbed with dose-proportional exposure. Treatment was well-tolerated overall, with reversible grade 1-2 nausea, vomiting, and diarrhoea as the most common treatment-related adverse events (AEs). Four patients had gastrointestinal dose-limiting toxicities (DLTs) of nausea, vomiting, diarrhoea (three patients at 16,000 mg and one patient at 12,000 mg), establishing an RP2D at 12,000 mg/daily. Potential activity was seen in patients with recurrent high-grade gliomas (HGG). Of the 21 patients with HGG treated across the dose escalation and expansion, 5 (24%) had the clinical benefit (RANO CR, PR and SD >6 cycles) with one exceptional response lasting >2.5 years. CONCLUSIONS: 2-OHOA demonstrated a good safety profile and encouraging activity in this difficult-to-treat malignant brain-tumour patient population, placing it as an ideal potential candidate for the treatment of glioma and other solid tumour malignancies. CLINICAL TRIAL REGISTRATION: EudraCT registration number: 2012-001527-13; Clinicaltrials.gov registration number: NCT01792310.

Effects of inappropriate cause-of-death certification on mortality from cardiovascular disease and diabetes mellitus in Tonga.

BACKGROUND: Cardiovascular disease (CVD) and diabetes mellitus are major health issues in Tonga and other Pacific countries, although mortality levels and trends are unclear. We assess the impacts of cause-of-death certification on coding of CVD and diabetes as underlying causes of death (UCoD). METHODS: Tongan records containing cause-of-death data (2001-2018), including medical certificates of cause-of-death (MCCD), had UCoD assigned according to International Classification of Diseases 10th revision (ICD-10) coding rules. Deaths without recorded cause were included to ascertain total mortality. Diabetes and hypertension causes were reallocated from Part 1 of the MCCD (direct cause) to Part 2 (contributory cause) if potentially fatal complications were not recorded, and an alternative UCoD was assigned. Proportional mortality by cause based on the alternative UCoD were applied to total deaths then mortality rates calculated by age and sex using census/intercensal population estimates. CVD and diabetes mortality rates for unaltered and alternative UCoD were compared using Poisson regression. RESULTS: Over 2001-18, in ages 35-59 years, alternative CVD mortality was higher than unaltered CVD mortality in men (p = 0.043) and women (p = 0.15); for 2010-18, alternative versus unaltered measures in men were 3.3/103 (95%CI: 3.0-3.7/103) versus 2.9/103 (95%CI: 2.6-3.2/103), and in women were 1.1/103 (95%CI: 0.9-1.3/103) versus 0.9/103 (95%CI: 0.8-1.1/103). Conversely, alternative diabetes mortality rates were significantly lower than the unaltered rates over 2001-18 in men (p < 0.0001) and women (p = 0.013); for 2010-18, these measures in men were 1.3/103 (95%CI: 1.1-1.5/103) versus 1.9/103 (95%CI: 1.6-2.2/103), and in women were 1.4/103 (95%CI: 1.2-1.7/103) versus 1.7/103 (95%CI: 1.5-2.0/103). Diabetes mortality rates increased significantly over 2001-18 in men (unaltered: p < 0.0001; alternative: p = 0.0007) and increased overall in women (unaltered: p = 0.0015; alternative: p = 0.014). CONCLUSIONS: Diabetes reporting in Part 1 of the MCCD, without potentially fatal diabetes complications, has led to over-estimation of diabetes, and under-estimation of CVD, as UCoD in Tonga. This indicates the importance of controlling various modifiable risks for atherosclerotic CVD (including stroke) including hypertension, tobacco use, and saturated fat intake, besides obesity and diabetes. Accurate certification of diabetes as a direct cause of death (Part 1) or contributory factor (Part 2) is needed to ensure that valid UCoD are assigned. Examination of multiple cause-of-death data can improve understanding of the underlying causes of premature mortality to better inform health planning.

The structural basis for high affinity binding of α1-acid glycoprotein to the potent antitumor compound UCN-01.

The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand-binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as antitumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. The recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2-UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. The solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and the supporting NMR data will facilitate rational redesign of small molecules that could evade AGP and therefore improve tissue distribution.

Secondary magnetite in ancient zircon precludes analysis of a Hadean geodynamo.

Zircon crystals from the Jack Hills, Western Australia, are one of the few surviving mineralogical records of Earth's first 500 million years and have been proposed to contain a paleomagnetic record of the Hadean geodynamo. A prerequisite for the preservation of Hadean magnetization is the presence of primary magnetic inclusions within pristine igneous zircon. To date no images of the magnetic recorders within ancient zircon have been presented. Here we use high-resolution transmission electron microscopy to demonstrate that all observed inclusions are secondary features formed via two distinct mechanisms. Magnetite is produced via a pipe-diffusion mechanism whereby iron diffuses into radiation-damaged zircon along the cores of dislocations and is precipitated inside nanopores and also during low-temperature recrystallization of radiation-damaged zircon in the presence of an aqueous fluid. Although these magnetites can be recognized as secondary using transmission electron microscopy, they otherwise occur in regions that are indistinguishable from pristine igneous zircon and carry remanent magnetization that postdates the crystallization age by at least several hundred million years. Without microscopic evidence ruling out secondary magnetite, the paleomagnetic case for a Hadean-Eoarchean geodynamo cannot yet been made.

Conformational dynamics in interleukin 17A and 17F functional complexes is a key determinant of receptor A affinity and specificity.

The proinflammatory cytokines IL-17A and IL-17F have been identified as key drivers of a range of human inflammatory diseases, such as psoriasis, which has led to several therapeutic antibodies targeted at IL-17A. The two cytokines have been shown to tightly associate as functional homo and hetero dimers, which induce signalling via the formation of a cell surface signalling complex with a single copy of both IL-17RA and IL-17RC. Striking differences in affinity have been observed for IL-17RA binding to IL-17AA, IL-17AF and IL-17FF, however, the functional significance and molecular basis for this has remained unclear. We have obtained comprehensive backbone NMR assignments for full length IL-17AA (79%), IL-17AF (93%) and IL-17FF (89%), which show that the dimers adopt almost identical backbone topologies in solution to those observed in reported crystal structures. Analysis of the line widths and intensities of assigned backbone amide NMR signals has revealed striking differences in the conformational plasticity and dynamics of IL-17AA compared to both IL-17AF and IL-17FF. Our NMR data indicate that a number of regions of IL-17AA are interconverting between at least two distinct conformations on a relatively slow timescale. Such conformational heterogeneity has previously been shown to play an important role in the formation of many high affinity protein-protein complexes. The locations of the affected IL-17AA residues essentially coincides with the regions of both IL-17A and IL-17F previously shown to undergo significant structural changes on binding to IL-17RA. Substantially less conformational exchange was revealed by the NMR data for IL-17FF and IL-17AF. We propose that the markedly different conformational dynamic properties of the distinct functional IL-17 dimers plays a key role in determining their affinities for IL-17RA, with the more dynamic and plastic nature of IL-17AA contributing to the significantly tighter affinity observed for binding to IL-17RA. In contrast, the dynamic properties are expected to have little influence on the affinity of IL-17 dimers for IL-17RC, which has recently been shown to induce only small structural changes in IL-17FF upon binding.

Reliability and Validity of Integrated External and Internal Load Ratios as Measures of Fitness in Academy Rugby Union Players.

Taylor, RJ, Sanders, D, Myers, T, and Akubat, I. Reliability and validity of integrated external and internal load ratios as measures of fitness in academy rugby union players. J Strength Cond Res 34(6): 1723-1730, 2020-This study aimed to assess the relationships between integrated ratios of external and internal load measures and aerobic fitness in 3 different training exercise protocols. Twelve academy rugby union players (aged; 17.6 ± 0.44 years, height; 179.4 ± 6.3 cm, body mass 83.3 ± 9.7 kg) performed a lactate threshold/V[Combining Dot Above]O2max test (LT/V[Combining Dot Above]O2max) followed by 3 exercise protocols; 760 m continuous shuttle running (20 m shuttle run at 9 km·h), sprint interval training (SIT: 6 × 6 seconds sprint with 54 seconds recovery), and a small-sided game (SSG: 6 vs. 6, 10 minutes on a 39 × 51 m pitch) on 2 occasions. A LT/V[Combining Dot Above]O2max test was used to determine velocity at lactate threshold (vLT) and at the onset of blood lactate accumulation (vOBLA), maximal oxygen uptake (V[Combining Dot Above]O2max) and the heart rate-blood lactate profile for the calculation of internal load (individualized training impulse or iTRIMP). The total distance (TD), PlayerLoad, metabolic power (MP), high-speed distance >15 km·h (HSD), very high-speed distance >18 km·h (VHSD) and individualized high-speed distance based on each player's vOBLA (iHSD) for the 3 exercise protocols were measured using Micro Electro Mechanical Systems/Global Positioning System technology. Bayesian analysis was used to assess the ratios validity and reliability. Ratios demonstrated large-to-very large associations with vOBLA (Rho = 0.64-0.76), vLT (Rho = 0.63-0.71). Reliability of the ratios ranged from 7.06 to 36.28% (coefficient of variation [CV]%). The results suggest that integrated load ratios from the SIT and SSG protocols provide a measure which is reliable with large-to-very large associations to submaximal aerobic fitness in rugby union.

A Field-Based Cycling Test to Assess Predictors of Endurance Performance and Establishing Training Zones.

Sanders, D, Taylor, RJ, Myers, T, and Akubat, I. A field-based cycling test to assess predictors of endurance performance and establishing training zones. J Strength Cond Res 34(12): 3482-3488, 2020-This study evaluates the relationship between a field-based 8-minute time trial (8MTT) and physiological endurance variables assessed with an incremental laboratory test. Second, lactate thresholds assessed in the laboratory were compared with estimated functional threshold power (FTP) from the 8MTT. Nineteen well-trained road cyclists (aged 22 ± 2 years, height 185.9 ± 4.5 cm, body mass 72.8 ± 4.6 kg, V[Combining Dot Above]O2max 64 ± 4 ml·min·kg) participated. Linear regression revealed that mean 8MTT power output (PO) was strongly to very strongly related to PO at 4 mmol·L, PO at initial rise of 1.00 mmol·L, PO at Dmax and modified (mDmax) (r = 0.61-0.82). Mean 8MTT PO was largely to very largely different compared with PO at fixed blood lactate concentration of 2 mmol·L (ES = 3.20) and 4 mmol·L (ES = 1.90), PO at initial rise 1.00 mmol·L (ES = 2.33), PO at Dmax (ES = 3.47) and mDmax (ES = 1.79) but only trivially different from maximal PO (Wmax) (ES = 0.09). The 8MTT based estimated FTP was moderate to very largely different compared with PO at initial rise of 1 mmol·L (ES = 1.37), PO at Dmax (ES = 2.42), PO at mDmax (ES = 0.77) and PO at 4 mmol·L (ES = 0.83). Therefore, even though the 8MTT can be valuable as a performance test in cycling shown through its relationships with predictors of endurance performance, coaches should be cautious when using FTP and PO at laboratory-based thresholds interchangeably to inform training prescription.

Iridium-Catalyzed Enantioselective Intermolecular Indole C2-Allylation.

The enantioselective intermolecular C2-allylation of 3-substituted indoles is reported for the first time. This directing group-free approach relies on a chiral Ir-(P, olefin) complex and Mg(ClO4 )2 Lewis acid catalyst system to promote allylic substitution, providing the C2-allylated products in typically high yields (40-99 %) and enantioselectivities (83-99 % ee) with excellent regiocontrol. Experimental studies and DFT calculations suggest that the reaction proceeds via direct C2-allylation, rather than C3-allylation followed by in situ migration. Steric congestion at the indole-C3 position and improved π-π stacking interactions have been identified as major contributors to the C2-selectivity.

Structural and functional analysis of Dickkopf 4 (Dkk4): New insights into Dkk evolution and regulation of Wnt signaling by Dkk and Kremen proteins.

Dickkopf (Dkk) family proteins are important regulators of Wnt signaling pathways, which play key roles in many essential biological processes. Here, we report the first detailed structural and dynamics study of a full-length mature Dkk protein (Dkk4, residues 19-224), including determination of the first atomic-resolution structure for the N-terminal cysteine-rich domain (CRD1) conserved among Dkk proteins. We discovered that CRD1 has significant structural homology to the Dkk C-terminal cysteine-rich domain (CRD2), pointing to multiple gene duplication events during Dkk family evolution. We also show that Dkk4 consists of two independent folded domains (CRD1 and CRD2) joined by a highly flexible, nonstructured linker. Similarly, the N-terminal region preceding CRD1 and containing a highly conserved NXI(R/K) sequence motif was shown to be dynamic and highly flexible. We demonstrate that Dkk4 CRD2 mediates high-affinity binding to both the E1E2 region of low-density lipoprotein receptor-related protein 6 (LRP6 E1E2) and the Kremen1 (Krm1) extracellular domain. In contrast, the N-terminal region alone bound with only moderate affinity to LRP6 E1E2, consistent with binding via the conserved NXI(R/K) motif, but did not interact with Krm proteins. We also confirmed that Dkk and Krm family proteins function synergistically to inhibit Wnt signaling. Insights provided by our integrated structural, dynamics, interaction, and functional studies have allowed us to refine the model of synergistic regulation of Wnt signaling by Dkk proteins. Our results indicate the potential for the formation of a diverse range of ternary complexes comprising Dkk, Krm, and LRP5/6 proteins, allowing fine-tuning of Wnt-dependent signaling.

The Aminotriazole Antagonist Cmpd-1 Stabilises a Distinct Inactive State of the Adenosine 2A Receptor.

The widely expressed G-protein coupled receptors (GPCRs) are versatile signal transducer proteins that are attractive drug targets but structurally challenging to study. GPCRs undergo a number of conformational rearrangements when transitioning from the inactive to the active state but have so far been believed to adopt a fairly conserved inactive conformation. Using 19 F NMR spectroscopy and advanced molecular dynamics simulations we describe a novel inactive state of the adenosine 2A receptor which is stabilised by the aminotriazole antagonist Cmpd-1. We demonstrate that the ligand stabilises a unique conformation of helix V and present data on the putative binding mode of the compound involving contacts to the transmembrane bundle as well as the extracellular loop 2.

Nanodisc-Targeted STD NMR Spectroscopy Reveals Atomic Details of Ligand Binding to Lipid Environments.

Saturation transfer difference (STD) NMR spectroscopy is one of the most popular ligand-based NMR techniques for the study of protein-ligand interactions. This is due to its robustness and the fact that it is focused on the signals of the ligand, without any need for NMR information on the macromolecular target. This technique is most commonly applied to systems involving different types of ligands (e.g., small organic molecules, carbohydrates or lipids) and a protein as the target, in which the latter is selectively saturated. However, only a few examples have been reported where membrane mimetics are the macromolecular binding partners. Here, we have employed STD NMR spectroscopy to investigate the interactions of the neurotransmitter dopamine with mimetics of lipid bilayers, such as nanodiscs, by saturation of the latter. In particular, the interactions between dopamine and model lipid nanodiscs formed either from charged or zwitterionic lipids have been resolved at the atomic level. The results, in agreement with previous isothermal titration calorimetry studies, show that dopamine preferentially binds to negatively charged model membranes, but also provide detailed atomic insights into the mode of interaction of dopamine with membrane mimetics. Our findings provide relevant structural information for the design of lipid-based drug carriers of dopamine and its structural analogues and are of general applicability to other systems.

Quantifying Secondary Structure Changes in Calmodulin Using 2D-IR Spectroscopy.

Revealing the details of biomolecular processes in solution needs tools that can monitor structural dynamics over a range of time and length scales. We assess the ability of 2D-IR spectroscopy in combination with multivariate data analysis to quantify changes in secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM) as a function of temperature and Ca2+ concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca2+-free (apo) CaM (reduction in α-helix structure by 13% (CD) and 15% (2D)). 2D-IR also allows accurate differentiation between melting transitions and generic heating effects observed in the more thermally stable Ca2+-bound (holo) CaM. The functionally relevant random-coil-α-helix transition associated with Ca2+ uptake that involves just 7-8 out of a total of 148 amino acid residues was clearly detected. Temperature-dependent Molecular Dynamics (MD) simulations show that apo-CaM exists in dynamic equilibrium with holo-like conformations, while Ca2+ uptake reduces conformational flexibility. The ability to combine quantitative structural insight from 2D-IR with MD simulations thus offers a powerful approach for measuring subtle protein conformational changes in solution.

Low-Temperature, Transition-Metal-Free Cross-Dehydrogenative Coupling Protocol for the Synthesis of 3,3-Disubstituted Oxindoles.

A new low-temperature procedure for the synthesis of 3,3-disubstituted 2-oxindoles via cross-dehydrogenative coupling (CDC) is reported. The use of a strong, nonreversible base in these reactions has been found to effect a dramatic drop in reaction temperature (to room temperature) relative to the current state-of-the-art (>100 °C) procedure. When employing iodine as an "oxidant", new evidence suggests that this transformation may occur via a transiently stable iodinated intermediate rather than by direct single-electron oxidation.

Selective synthesis of three product classes from imine and carboxylic acid precursors via direct imine acylation.

Three divergent Direct Imine Acylation (DIA) procedures are reported that allow the selective generation of δ-lactams, ß-lactams and tetrahydropyrimidinones (via a novel three-component coupling) from imine and carboxylic acid precursors. All operate via initial N-acyliminium ion formation and diverge depending on the reaction conditions and nature of the substrates.

Photoredox-catalyzed procedure for carbamoyl radical generation: 3,4-dihydroquinolin-2-one and quinolin-2-one synthesis.

A reductive approach for carbamoyl radical generation from N-hydroxyphthalimido oxamides under photoredox catalysis is outlined. This strategy was applied to the synthesis of 3,4-dihydroquinolin-2-ones via the intermolecular addition/cyclization of carbamoyl radicals with electron deficient olefins in a mild, redox-neutral manner. Under a general set of reaction conditions, diversely substituted 3,4-dihydroquinolin-2-ones, including spirocyclic systems can be prepared. By using chlorine-substituted olefins, aromatic quinolin-2-ones can also be accessed.

A Cu-Catalysed Radical Cross-Dehydrogenative Coupling Approach to Acridanes and Related Heterocycles.

The synthesis of acridanes and related compounds through a Cu-catalysed radical cross-dehydrogenative coupling of simple 2-[2-(arylamino)aryl]malonates is reported. This method can be further streamlined to a one-pot protocol involving the in situ fomation of the 2-[2-(arylamino)aryl]malonate by α-arylation of diethyl malonate with 2-bromodiarylamines under Pd catalysis, followed by Cu-catalysed cyclisation.

Molecular Insights into the Thermal Stability of mAbs with Variable-Temperature Ion-Mobility Mass Spectrometry.

The aggregation of protein-based therapeutics such as monoclonal antibodies (mAbs) can affect the efficacy of the treatment and can even induce effects that are adverse to the patient. Protein engineering is used to shift the mAb away from an aggregation-prone state by increasing the thermodynamic stability of the native fold, which might in turn alter conformational flexibility. We have probed the thermal stability of three types of intact IgG molecules and two Fc-hinge fragments by using variable-temperature ion-mobility mass spectrometry (VT-IM-MS). We observed changes in the conformations of isolated proteins as a function of temperature (300-550 K). The observed differences in thermal stability between IgG subclasses can be rationalized in terms of changes to higher-order structural organization mitigated by the hinge region. VT-IM-MS provides insights into mAbs structural thermodynamics and is presented as a promising tool for thermal-stability studies for proteins of therapeutic interest.