Therapeutic target-site variability in α1-antitrypsin characterized at high resolution

The intrinsic propensity of [alpha]1-antitrypsin to undergo conformational transitions from its metastable native state to hyperstable forms provides a motive force for its antiprotease function. However, aberrant conformational change can also occur via an intermolecular linkage that results in polymerization. This has both loss-of-function and gain-of-function effects that lead to deficiency of the protein in human circulation, emphysema and hepatic cirrhosis. One of the most promising therapeutic strategies being developed to treat this disease targets small molecules to an allosteric site in the [alpha]1-antitrypsin molecule. Partial filling of this site impedes polymerization without abolishing function. Drug development can be improved by optimizing data on the structure and dynamics of this site. A new 1.8 Å resolution structure of [alpha]1-antitrypsin demonstrates structural variability within this site, with associated fluctuations in its upper and lower entrance grooves and ligand-binding characteristics around the innermost stable enclosed hydrophobic recess. These data will allow a broader selection of chemotypes and derivatives to be tested in silico and in vitro when screening and developing compounds to modulate conformational change to block the pathological mechanism while preserving function

Polymers and inflammation: disease mechanisms of the serpinopathies

Members of the serpin (serine proteinase inhibitor) superfamily play a central role in the control of inflammatory, coagulation, and fibrinolytic cascades. Point mutations that cause abnormal conformational transitions in these proteins can trigger disease. Recent work has defined three pathways by which these conformers cause tissue damage. Here, we describe how these three mechanisms can be integrated into a new model of the pathogenesis of emphysema caused by mutations in the serpin α1-antitrypsin

Non-invasive testing for liver pathology in alpha-1 antitrypsin deficiency

BACKGROUND: Many patients with alpha-1 antitrypsin deficiency (A1ATD) receive care in respiratory clinics without access to specialist hepatology expertise. Liver disease can develop asymptomatically, and non-invasive markers of fibrosis may help identify patients who require definitive assessment with liver biopsy. We evaluated the utility of non-invasive markers of liver fibrosis in A1ATD to guide testing in settings without ready access to hepatology expertise. METHODS: Patients attending the London A1ATD service undergo assessment using blood tests to calculate the 'APRI' and 'FIB-4' score, liver ultrasound and Fibroscan. Liver biopsy is offered to patients who have abnormal liver function tests with abnormal liver ultrasound and/or liver stiffness >6 kPa on Fibroscan. Liver biopsies were assessed for the presence of A1AT, steatosis, fibrosis and inflammation. RESULTS: 75 patients with A1ATD had results for analysis, 56% were female, age 16-82 years. 75% of patients had Fibroscan 8 kPa. There was a significant correlation between FIB-4 and Fibroscan (r=0.244, p=0.035). Fibroscan >6 kPa corresponded to a FIB-4 score of >1.26. However, FIB-4 >1.26 had poor sensitivity (47%), specificity (32%) and positive-predictive value (PPV; 36%) to identify Fibroscan >6 kPa. The negative-predictive value (NPV) was stronger at 81%. APRI data were similar. Twelve patients underwent liver biopsy, with 11 reports available for analysis. Six had FIB-4 scores<1.26 and five had Fibroscan of <6 kPa. A1AT was present in 64% of biopsies, steatosis in 82%, mild fibrosis in 36%, moderate fibrosis in 9% and severe fibrosis in 9%. CONCLUSION: A combination of liver ultrasound and non-invasive fibrosis tests can help identify patients with A1ATD liver injury. However, APRI and FIB-4 scores alone had poor sensitivity and specificity to justify use as an independent tool for liver pathology in A1ATD

In silico assessment of potential druggable pockets on the surface of α1-Antitrypsin conformers

The search for druggable pockets on the surface of a protein is often performed on a single conformer, treated as a rigid body. Transient druggable pockets may be missed in this approach. Here, we describe a methodology for systematic in silico analysis of surface clefts across multiple conformers of the metastable protein α1-antitrypsin (A1AT). Pathological mutations disturb the conformational landscape of A1AT, triggering polymerisation that leads to emphysema and hepatic cirrhosis. Computational screens for small molecule inhibitors of polymerisation have generally focused on one major druggable site visible in all crystal structures of native A1AT. In an alternative approach, we scan all surface clefts observed in crystal structures of A1AT and in 100 computationally produced conformers, mimicking the native solution ensemble. We assess the persistence, variability and druggability of these pockets. Finally, we employ molecular docking using publicly available libraries of small molecules to explore scaffold preferences for each site. Our approach identifies a number of novel target sites for drug design. In particular one transient site shows favourable characteristics for druggability due to high enclosure and hydrophobicity. Hits against this and other druggable sites achieve docking scores corresponding to a Kd in the µM–nM range, comparing favourably with a recently identified promising lead. Preliminary ThermoFluor studies support the docking predictions. In conclusion, our strategy shows considerable promise compared with the conventional single pocket/single conformer approach to in silico screening. Our best-scoring ligands warrant further experimental investigation

An in vitro investigation of the inflammatory response to the strain amplitudes which occur during high frequency oscillation ventilation and conventional mechanical ventilation

The research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College Londo

Deconvolution of ion mobility mass spectrometry arrival time distributions using a genetic algorithm approach: application to α1-antitrypsin peptide binding

Ion mobility mass spectrometry (IM-MS) is a fast and sample-efficient method for analysing the gas phase conformation of proteins and protein complexes. Subjecting proteins to increased collision energies prior to ion mobility separation can directly probe their unfolding behaviour. Recent work in the field has utilised this approach to evaluate the effect of small ligand binding upon protein stability, and to screen compounds for drug discovery. Its general applicability for high-throughput screening will, however, depend upon new analytical methods to make the approach scalable. Here we describe a fully automated program, called Benthesikyme, for summarising the ion mobility results from such experiments. The program automatically creates collision induced unfolding (CIU) fingerprints and summary plots that capture the increase in collision cross section and the increase in conformational flexibility of proteins during unfolding. We also describe a program, based on a genetic algorithm, for the deconvolution of arrival time distributions from the \{CIU\} data. This multicomponent analysis method was developed to require as little user input as possible. Aside from the IM-MS data, the only input required is an estimate of the number of conformational families to be fitted to the data. In cases where the appropriate number of conformational families is unclear, the automated procedure means it is straightforward to repeat the analysis for several values and optimize the quality of the fit. We have employed our new methodology to study the effects of peptide binding to α1-antitrypsin, an abundant human plasma protein whose misfolding exemplifies a group of conformational diseases termed the serpinopathies. Our analysis shows that interaction with the peptide stabilises the protein and reduces its conformational flexibility. The previously unresolved patterns of unfolding detected by the deconvolution algorithm will allow us to set up a fully automated screen for new ligand molecules with similar properties

A multicentre retrospective cohort comparison of aetiology and survival in patients with chronic hypersensitivity pneumonitis versus idiopathic pulmonary fibrosis

This is the author accepted manuscript. The final version is available from BMJ Publishing Group via the DOI in this recordWinter Meeting of the British Thoracic Society, 5-7 December 2018, London, U

Interactions between N-linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum.

The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site-directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early-onset dementia. Our findings indicate that N401 glycosylation reports lability of the C-terminal end of neuroserpin in its native state. This C-terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant-specific N-linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin

The structural basis for Z α1-antitrypsin polymerization in the liver

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α1-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α1-antitrypsin as “polymer” chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α1-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α1-antitrypsin

Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

The common severe Z mutation (E342K) of α1-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular 'snapshots', the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α1-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P14P12 and P4, but not P10P8 or P2P1', resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P14P12 region self-inserts early during activation, while the interaction between P6P4 and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P6P4, but not P14P12, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol-1