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

Vitamin D (1,25(OH

Studies to identify novel immune-regulatory functions of active vitamin D (1,25(OH) D3) in human CD4 T cells revealed that 1,25(OH) D3 potently induced expression of the gene SERPINA1, encoding the anti-protease α-1-antitrypsin. We confirmed α-1-antitrypsin protein expression by 1,25(OH) D3-treated CD4 T cells, but not in CD8 T cells or monocytes. α-1-Antitrypsin promotes anti-inflammatory IL-10 synthesis in other immune cell populations. We therefore investigated its immune-regulatory effects in CD4 T cells. Plasma-derived α-1-antitrypsin drove IL-10 synthesis by CD4 T cells, which was not dependent on anti-protease activity, but appeared to require a serum-binding factor, since this could not be achieved with recombinant protein. α-1-Antitrypsin is reported to bind complement components, which regulate T cell function. A role for this interaction was therefore probed. Plasma-derived, but not recombinant α-1-antitrypsin contained C3a. Surface Plasmon Resonance and Microscale Thermophoresis demonstrated α-1-antitrypsin binding to C3a. Addition of C3a to CD4 T cells cultured with recombinant α-1-antitrypsin restored induction of IL-10, whereas neutralisation of C3a abrogated IL-10 induced by plasma-derived α-1-antitrypsin. To interrogate an endogenous role for the α-1-antitrypsin-C3a axis in 1,25(OH) D3-driven CD4 T cell IL-10 synthesis, we treated cells from healthy or α-1-antitrypsin-deficient individuals (which transcribe SERPINA1 but do not secrete protein) with 1,25(OH) D3. A significant correlation was identified between SERPINA1 and IL10 gene expression in healthy donor CD4 T cells, which was absent in cells from α-1-antitrypsin-deficient individuals. Therefore, α-1-antitrypsin is required for 1,25(OH) D3-induced IL-10 expression in CD4 T cells, interacting with C3a to drive IL-10 expression. [Abstract copyright: Copyright © 2019. Published by Elsevier Ltd.

Targeting serpins in high-throughput and structure-based drug design

Native, metastable serpins inherently tend to undergo stabilizing conformational transitions in mechanisms of health (e.g., enzyme inhibition) and disease (serpinopathies). This intrinsic tendency is modifiable by ligand binding, thus structure-based drug design is an attractive strategy in the serpinopathies. This can be viewed as a labor-intensive approach, and historically, its intellectual attractiveness has been tempered by relatively limited success in development of drugs reaching clinical practice. However, the increasing availability of a range of powerful experimental systems and higher-throughput techniques is causing academic and early-stage industrial pharmaceutical approaches to converge. In this review, we outline the different systems and techniques that are bridging the gap between what have traditionally been considered distinct disciplines. The individual methods are not serpin-specific. Indeed, many have only recently been applied to serpins, and thus investigators in other fields may have greater experience of their use to date. However, by presenting examples from our work and that of other investigators in the serpin field, we highlight how techniques with potential for automation and scaling can be combined to address a range of context-specific challenges in targeting the serpinopathies

Therapeutic targeting of misfolding and conformational change in α1-antitrypsin deficiency

Misfolding and conformational diseases are increasing in prominence and prevalence. Both misfolding and ‘postfolding’ conformational mechanisms can contribute to pathogenesis and can coexist. The different contexts of folding and native state behavior may have implications for the development of therapeutic strategies. α1-antitrypsin deficiency illustrates how these issues can be addressed with therapeutic approaches to rescue folding, ameliorate downstream consequences of aberrant polymerization and/or maintain physiological function. Small-molecule strategies have successfully targeted structural features of the native conformer. Recent developments include the capability to follow solution behavior of α1-antitrypsin in the context of disease mutations and interactions with drug-like compounds. Moreover, preclinical studies in cells and organisms support the potential of manipulating cellular response repertoires to process misfolded and polymer states

Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen

Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377–588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line

TARGETING SERPINS IN HIGH-THROUGHPUT AND STRUCTURE-BASED DRUG DESIGN

Native, metastable serpins inherently tend to undergo stabilizing conformational transitions in mechanisms of health (e.g., enzyme inhibition) and disease (serpinopathies). This intrinsic tendency is modifiable by ligand binding, thus structure-based drug design is an attractive strategy in the serpinopathies. This can be viewed as a labor-intensive approach, and historically, its intellectual attractiveness has been tempered by relatively limited success in development of drugs reaching clinical practice. However, the increasing availability of a range of powerful experimental systems and higher-throughput techniques is causing academic and early-stage industrial pharmaceutical approaches to converge. In this review, we outline the different systems and techniques that are bridging the gap between what have traditionally been considered distinct disciplines. The individual methods are not serpin-specific. Indeed, many have only recently been applied to serpins, and thus investigators in other fields may have greater experience of their use to date. However, by presenting examples from our work and that of other investigators in the serpin field, we highlight how techniques with potential for automation and scaling can be combined to address a range of context-specific challenges in targeting the serpinopathies

1H, 15N and 13C backbone resonance assignments of the archetypal serpin α1-antitrypsin

Alpha1-antitrypsin is a 45-kDa (394-residue) serine protease inhibitor synthesized by hepatocytes, which is released into the circulatory system and protects the lung from the actions of neutrophil elastase via a conformational transition within a dynamic inhibitory mechanism. Relatively common point mutations subvert this transition, causing polymerisation of α1-antitrypsin and deficiency of the circulating protein, predisposing carriers to severe lung and liver disease. We have assigned the backbone resonances of α1-antitrypsin using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for a detailed solution state characterization of the structural properties of this highly dynamic protein via NMR methods

Structural dynamics associated with intermediate formation in an archetypal conformational disease

In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α1-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α1-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions