Title: Development of a small molecule that corrects misfolding and increases secretion of Z α <sub>1</sub> ‐antitrypsin
Abstract: Article29 January 2021Open Access Transparent process Development of a small molecule that corrects misfolding and increases secretion of Z α1-antitrypsin David A Lomas Corresponding Author David A Lomas [email protected] orcid.org/0000-0003-2339-6979 UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author James A Irving James A Irving orcid.org/0000-0003-3204-6356 UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author Christopher Arico-Muendel Christopher Arico-Muendel GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Svetlana Belyanskaya Svetlana Belyanskaya GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Andrew Brewster Andrew Brewster GlaxoSmithKline, Stevenage, UK Search for more papers by this author Murray Brown Murray Brown GlaxoSmithKline, Stevenage, UK Search for more papers by this author Chun-wa Chung Chun-wa Chung GlaxoSmithKline, Stevenage, UK Search for more papers by this author Hitesh Dave Hitesh Dave GlaxoSmithKline, Stevenage, UK Search for more papers by this author Alexis Denis Alexis Denis GlaxoSmithKline, Paris, France Search for more papers by this author Nerina Dodic Nerina Dodic GlaxoSmithKline, Paris, France Search for more papers by this author Anthony Dossang Anthony Dossang GlaxoSmithKline, Stevenage, UK Search for more papers by this author Peter Eddershaw Peter Eddershaw GlaxoSmithKline, Stevenage, UK Search for more papers by this author Diana Klimaszewska Diana Klimaszewska GlaxoSmithKline, Stevenage, UK Search for more papers by this author Imran Haq Imran Haq UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author Duncan S Holmes Duncan S Holmes GlaxoSmithKline, Stevenage, UK Search for more papers by this author Jonathan P Hutchinson Jonathan P Hutchinson GlaxoSmithKline, Stevenage, UK Search for more papers by this author Alistair M Jagger Alistair M Jagger UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author Toral Jakhria Toral Jakhria GlaxoSmithKline, Stevenage, UK Search for more papers by this author Emilie Jigorel Emilie Jigorel GlaxoSmithKline, Paris, France Search for more papers by this author John Liddle John Liddle GlaxoSmithKline, Stevenage, UK Search for more papers by this author Ken Lind Ken Lind GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Stefan J Marciniak Stefan J Marciniak Cambridge Institute for Medical Research, Cambridgem, UK Search for more papers by this author Jeff Messer Jeff Messer GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Margaret Neu Margaret Neu GlaxoSmithKline, Stevenage, UK Search for more papers by this author Allison Olszewski Allison Olszewski GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Adriana Ordonez Adriana Ordonez Cambridge Institute for Medical Research, Cambridgem, UK Search for more papers by this author Riccardo Ronzoni Riccardo Ronzoni orcid.org/0000-0002-3981-8104 UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author James Rowedder James Rowedder GlaxoSmithKline, Stevenage, UK Search for more papers by this author Martin Rüdiger Martin Rüdiger GlaxoSmithKline, Stevenage, UK Search for more papers by this author Steve Skinner Steve Skinner GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Kathrine J Smith Kathrine J Smith GlaxoSmithKline, Stevenage, UK Search for more papers by this author Rebecca Terry Rebecca Terry GlaxoSmithKline, Stevenage, UK Search for more papers by this author Lionel Trottet Lionel Trottet GlaxoSmithKline, Paris, France Search for more papers by this author Iain Uings Iain Uings GlaxoSmithKline, Stevenage, UK Search for more papers by this author Steve Wilson Steve Wilson GlaxoSmithKline, Stevenage, UK Search for more papers by this author Zhengrong Zhu Zhengrong Zhu GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Andrew C Pearce Corresponding Author Andrew C Pearce [email protected] orcid.org/0000-0002-4698-037X GlaxoSmithKline, Stevenage, UK Search for more papers by this author David A Lomas Corresponding Author David A Lomas [email protected] orcid.org/0000-0003-2339-6979 UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author James A Irving James A Irving orcid.org/0000-0003-3204-6356 UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author Christopher Arico-Muendel Christopher Arico-Muendel GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Svetlana Belyanskaya Svetlana Belyanskaya GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Andrew Brewster Andrew Brewster GlaxoSmithKline, Stevenage, UK Search for more papers by this author Murray Brown Murray Brown GlaxoSmithKline, Stevenage, UK Search for more papers by this author Chun-wa Chung Chun-wa Chung GlaxoSmithKline, Stevenage, UK Search for more papers by this author Hitesh Dave Hitesh Dave GlaxoSmithKline, Stevenage, UK Search for more papers by this author Alexis Denis Alexis Denis GlaxoSmithKline, Paris, France Search for more papers by this author Nerina Dodic Nerina Dodic GlaxoSmithKline, Paris, France Search for more papers by this author Anthony Dossang Anthony Dossang GlaxoSmithKline, Stevenage, UK Search for more papers by this author Peter Eddershaw Peter Eddershaw GlaxoSmithKline, Stevenage, UK Search for more papers by this author Diana Klimaszewska Diana Klimaszewska GlaxoSmithKline, Stevenage, UK Search for more papers by this author Imran Haq Imran Haq UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author Duncan S Holmes Duncan S Holmes GlaxoSmithKline, Stevenage, UK Search for more papers by this author Jonathan P Hutchinson Jonathan P Hutchinson GlaxoSmithKline, Stevenage, UK Search for more papers by this author Alistair M Jagger Alistair M Jagger UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author Toral Jakhria Toral Jakhria GlaxoSmithKline, Stevenage, UK Search for more papers by this author Emilie Jigorel Emilie Jigorel GlaxoSmithKline, Paris, France Search for more papers by this author John Liddle John Liddle GlaxoSmithKline, Stevenage, UK Search for more papers by this author Ken Lind Ken Lind GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Stefan J Marciniak Stefan J Marciniak Cambridge Institute for Medical Research, Cambridgem, UK Search for more papers by this author Jeff Messer Jeff Messer GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Margaret Neu Margaret Neu GlaxoSmithKline, Stevenage, UK Search for more papers by this author Allison Olszewski Allison Olszewski GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Adriana Ordonez Adriana Ordonez Cambridge Institute for Medical Research, Cambridgem, UK Search for more papers by this author Riccardo Ronzoni Riccardo Ronzoni orcid.org/0000-0002-3981-8104 UCL Respiratory, Rayne Institute, University College London, London, UK Search for more papers by this author James Rowedder James Rowedder GlaxoSmithKline, Stevenage, UK Search for more papers by this author Martin Rüdiger Martin Rüdiger GlaxoSmithKline, Stevenage, UK Search for more papers by this author Steve Skinner Steve Skinner GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Kathrine J Smith Kathrine J Smith GlaxoSmithKline, Stevenage, UK Search for more papers by this author Rebecca Terry Rebecca Terry GlaxoSmithKline, Stevenage, UK Search for more papers by this author Lionel Trottet Lionel Trottet GlaxoSmithKline, Paris, France Search for more papers by this author Iain Uings Iain Uings GlaxoSmithKline, Stevenage, UK Search for more papers by this author Steve Wilson Steve Wilson GlaxoSmithKline, Stevenage, UK Search for more papers by this author Zhengrong Zhu Zhengrong Zhu GlaxoSmithKline, Cambridge, MA, USA Search for more papers by this author Andrew C Pearce Corresponding Author Andrew C Pearce [email protected] orcid.org/0000-0002-4698-037X GlaxoSmithKline, Stevenage, UK Search for more papers by this author Author Information David A Lomas *,1, James A Irving1, Christopher Arico-Muendel2, Svetlana Belyanskaya2, Andrew Brewster3, Murray Brown3, Chun-wa Chung3, Hitesh Dave3, Alexis Denis4, Nerina Dodic4, Anthony Dossang3, Peter Eddershaw3, Diana Klimaszewska3, Imran Haq1, Duncan S Holmes3, Jonathan P Hutchinson3, Alistair M Jagger1, Toral Jakhria3, Emilie Jigorel4, John Liddle3, Ken Lind2, Stefan J Marciniak5, Jeff Messer2, Margaret Neu3, Allison Olszewski2, Adriana Ordonez5, Riccardo Ronzoni1, James Rowedder3, Martin Rüdiger3, Steve Skinner2, Kathrine J Smith3, Rebecca Terry3, Lionel Trottet4, Iain Uings3, Steve Wilson3, Zhengrong Zhu2 and Andrew C Pearce *,3 1UCL Respiratory, Rayne Institute, University College London, London, UK 2GlaxoSmithKline, Cambridge, MA, USA 3GlaxoSmithKline, Stevenage, UK 4GlaxoSmithKline, Paris, France 5Cambridge Institute for Medical Research, Cambridgem, UK *Corresponding author. Tel: +44 20 3108 7929; E-mail: [email protected] *Corresponding author. Tel: +44 1438 551923; E-mail: [email protected] EMBO Mol Med (2021)13:e13167https://doi.org/10.15252/emmm.202013167 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Severe α1-antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1-antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α1-antitrypsin. The lead compound blocks Z α1-antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1-antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1-antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α1-antitrypsin deficiency. Synopsis A chemistry campaign has developed a small molecule that stabilises the severe Z deficiency mutant of α1-antitrypsin. The lead compound binds to a cryptic pocket and blocks the conformational change and pathological polymerisation that underlie α1-antitrypsin deficiency. A small molecule has been developed that blocks the pathological polymerisation of the Z mutant of α1-antitrypsin. Crystallography shows that it binds to a cryptic site and negates the local effects of the Z mutation. The lead compound has good selectivity in off-target screening. The lead compound completely blocks polymerisation and increases the secretion of Z α1-antitrypsin 3 fold in a cell model of disease and 7 fold in a transgenic mouse model of disease. There was no effect on hepatic inclusions following 20 days of treatment. The paper explained Problem Intracellular protein aggregation can result in "gain-of-function" cell toxicity. It has proved challenging to develop small molecules that can stabilise intracellular mutant proteins, prevent self-aggregation and so ameliorate disease. Severe α1-antitrypsin deficiency results largely from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1-antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. Results We have undertaken a medicinal chemistry campaign to develop an orally bioavailable small molecule that binds to intra-endoplasmic reticulum mutant Z α1-antitrypsin, corrects the folding defect and increases secretion in a transgenic model of disease. Impact This study reports the successful targeting of an aggregation-prone mutant in order to prevent the intracellular polymerisation and accumulation of α1-antitrypsin that underlies α1-antitrypsin deficiency. It demonstrates that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α1-antitrypsin deficiency. Introduction Alpha-1 antitrypsin deficiency affects 1 in 2,000 people of Northern European descent, leading to liver and lung diseases (Lomas et al, 2016). Ninety-five per cent of severe deficiency results from the "Z" allele (Glu342Lys) that perturbs the folding of α1-antitrypsin resulting in the secretion of only 15% of the mature protein. The remaining protein is retained within the cell by persistent binding to molecular chaperones (Wu et al, 2003) and then either degraded via the ERAD-proteasome pathway (Le et al, 1992; Qu et al, 1996; Teckman et al, 2001) or folded into ordered polymers that may be cleared by autophagy (Teckman et al, 2004) or accumulate within the endoplasmic reticulum (ER) of hepatocytes (Lomas et al, 1992). The accumulation of polymers causes neonatal hepatitis, cirrhosis and hepatocellular carcinoma, and can sensitise the liver to damage from environmental insults such as alcohol, fat or viral hepatitis (Ordóñez et al, 2013; Strnad et al, 2019). The consequent deficiency of α1-antitrypsin within the circulation results in insufficient protection of the lungs from neutrophil elastase, leading to early-onset emphysema (Lomas et al, 2016). The Z mutation lies at the head of strand 5 of β-sheet A of α1-antitrypsin. It perturbs the local environment, allowing population of an unstable intermediate that we have termed M* (Dafforn et al, 1999) in which β-sheet A opens and the upper part of helix F unwinds (Gooptu et al, 2000; Nyon et al, 2012). Polymerisation from this state involves insertion of the RCL into β-sheet A with a domain-swap of the C-terminal region providing the inter-subunit linkage (Huang et al, 2016; Faull et al, 2020; Laffranchi et al, 2020). The resulting polymer is deposited within hepatocytes. The aim of our work was to develop a small molecule corrector of Z α1-antitrypsin folding that was able to block the formation of polymers within the ER of hepatocytes and that was suitable for oral dosing as a potential treatment for α1-antitrypsin deficiency. To achieve this, we needed to overcome a number of challenges: (i) the drug target is a highly mobile folding intermediate located in the ER; (ii) disparity in the size of the interface between a small molecule and the large protein–protein interaction that it is designed to block; (iii) oral dosing greatly restricts suitable chemical space; (iv) as a non-classical drug target, small molecule binders may well not be well-represented in compound screening libraries; and (v) the relatively high concentration of circulating monomeric Z α1-antitrypsin (~ 5 μM), even in individuals with severe plasma deficiency, represents a high-affinity sink for compound, restricting its access to the target in the hepatocyte and requiring high total blood concentrations of drug to achieve sufficient free drug concentration and target engagement in the liver. Results Identification of GSK716 through encoded library technology screening, structure-guided drug design and cellular profiling Z α1-antitrypsin is a conformationally dynamic molecule (Lomas et al, 1992; Knaupp et al, 2010) that represents a non-classical target for drug discovery. A cell-free assay approach to hit finding was undertaken so as not to miss compounds that bind α1-antitrypsin and block polymerisation but lack the molecular properties to cross cell membranes. This comprised the following: (i) an encoded library technology (ELT) screen (Goodnow et al, 2017) of a library with a nominal diversity of 2 × 1012 unique components to identify binders to Z α1-antitrypsin and (ii) a high-throughput screen (HTS) of the GSK compound collection (~ 1.7 million compounds) for small molecules that could block polymerisation of Z α1-antitrypsin. In both screening approaches, glycosylated Z α1-antitrypsin, purified from the plasma of Z α1-antitrypsin homozygotes (Lomas et al, 1993), was used since this represents the disease-relevant human pathophysiological drug target that populates an intermediate on the polymerisation pathway (Knaupp et al, 2010; Irving et al, 2015). ELT selections were performed by incubating Z α1-antitrypsin with DNA-encoded compound libraries for 1 h at 4 and 37°C for three rounds of selection with subsequent capture of Z α1-antitrypsin using α1-antitrypsin select resin (GE Healthcare). A variation on this protocol using pre-immobilised Z α1-antitrypsin was also used for library selections. In the HTS assay, polymerisation of purified Z α1-antitrypsin was induced by incubation at 37°C for 72 h in the presence of test compounds, with end-point quantification of polymers performed using the polymer-specific monoclonal antibody, 2C1 (Miranda et al, 2010) in a TR-FRET-based immunoassay. A number of small molecules that could block polymerisation of Z α1-antitrypsin were obtained through the HTS but none progressed beyond the early lead optimisation stage. However, a single lead series of chiral hydroxy-carboxamides (GSK425) was identified from the ELT screen that also demonstrated functional activity at blocking polymerisation in the TR-FRET immunoassay (pIC50 6.5; Fig 1A and B). Figure 1. Characteristics of the lead series of chiral hydroxy-carboxamides identified from the ELT screen The structure of GSK425, identified from the ELT screen, and the derived compound GSK716 obtained through a structure-based design pipeline. The degree of polymerisation of Z α1-antitrypsin after 72 h at 37°C, as determined by an end-point immunoassay using the 2C1 monoclonal antibody, in varying concentrations of compound (shown in panel A). Modification of the phenyl and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~ 100-fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716). GSK716 binds to Z α1-antitrypsin with a high-affinity mean pKD of 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative. There was a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin, with a mean pKD of 6.8 ± 0.18 (n = 10). Data presented as mean ± SD. The compound bound to monomeric but not polymeric Z α1-antitrypsin (Z α1-AT) as reported by fluorescence polarisation of an Alexa-488-labelled variant of GSK716. Representative curves reporting the interaction of different concentrations of GSK716 with Z α1-antitrypsin based on changes in intrinsic tryptophan fluorescence. Based on the concentration dependence (inset), the second-order rate constant of association was found to be 4.1 × 104 M−1 s−1. The association of GSK716 with M α1-antitrypsin, giving a second-order rate constant of 2.1 × 102 M−1 s−1. Download figure Download PowerPoint Optimisation of this initial hit followed a structure-based design approach, exploiting knowledge from iterative crystal structures of small molecule ligands complexed with α1-antitrypsin. The central hydroxy carboxamide and propyl chain were found to be critical for binding to Z α1-antitrypsin and hence further medicinal chemistry development focussed on modification of the phenyl and indole heterocycle. This resulted in an ~ 100-fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3; Fig 1A and B). GSK716 is a potent inhibitor of polymerisation in vitro and in cell models of disease GSK716 binds to Z α1-antitrypsin with a high-affinity mean pKD 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative (Fig 1C). The binding demonstrates selectivity with a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin at mean pKD 6.8 ± 0.18 (n = 10; Fig 1C). The shape of the curves and native mass spectrometry (not shown) are consistent with a single high-affinity compound binding site. No binding of the fluorescent derivative to polymers of Z α1-antitrypsin was observed, indicating conformational selectivity for the monomeric protein (Fig 1D). The rate of interaction of the compound with the target was monitored through changes in intrinsic tryptophan fluorescence (Dafforn et al, 1999); this property was used to determine the second-order association rate constants for GSK716 binding to Z (4.1 × 104 M−1 s−1) and M α1-antitrypsin (2.1 × 102 M−1 s−1; Fig 1E and F). From the association rate constants and the affinity values, first-order dissociation rate constants were calculated and found to be of the same order of magnitude for Z (6.1 × 10−5 s−1) and M α1-antitrypsin (1.6 × 10−5 s−1). Therefore, the selectivity of the compound for Z over M α1-antitrypsin is dominated by the difference in the rate of association rather than dissociation. The ability of GSK716 to block Z α1-antitrypsin polymerisation in the ER during folding was assessed by adding GSK716 to CHO-TET-ON-Z-A1AT cells (Ordóñez et al, 2013) with simultaneous induction of Z α1-antitrypsin expression using doxycycline. In comparison with controls, GSK716 completely blocked the intracellular formation of Z α1-antitrypsin polymers, as measured by staining with the 2C1 anti-Z α1-antitrypsin polymer monoclonal antibody (mean pIC50 = 6.3 ± 0.23; n = 71; Fig 2A and B). It also increased the secretion of Z α1-antitrypsin (mean pEC50 6.2 ± 0.23; n = 74; Fig 2B). Similar potency between the effects on secretion and polymerisation was observed throughout members of the lead series supporting the hypothesis that these effects are caused by the same pharmacological mode of action. GSK716 had a similar effect on the secretion and polymerisation of constitutively expressed Z α1-antitrypsin in iPSC-derived human hepatocytes with the ZZ α1-antitrypsin genotype (Yusa et al, 2011). It inhibited polymerisation and increased secretion with a mean pIC50 of 6.4 ± 0.45 (n = 16) and mean pEC50 of 6.5 ± 0.37 (n = 14), respectively, inducing an approximately threefold increase in secreted levels of Z α1-antitrypsin (Fig 2C and D). GSK716 treatment reduced the levels of intracellular Z α1-antitrypsin polymer compared with cells assessed before compound addition (Fig 2C), demonstrating that polymers can be cleared over the time course of the experiment, and that accumulation of polymers is reversible in ZZ-iPSC hepatocytes. Figure 2. GSK716 inhibits polymerisation of Z α1-antitrypsin in cell models of disease A. GSK716 was added to CHO-TET-ON-Z-A1AT cells (Ordóñez et al, 2013) with simultaneous induction of Z α1-antitrypsin expression using doxycycline, and polymer load was quantified with the 2C1 monoclonal antibody that is specific to pathological polymers of α1-antitrypsin (Miranda et al, 2010). The parent cell line that did not express Z α1-antitrypsin provided a negative control. GSK716 completely prevented intracellular polymer formation. Scale bar: 50 µm. B. Quantification of immunostained CHO-TET-ON-Z-A1AT cells showed that GSK716 reduced intracellular polymer formation and increased the secretion of Z α1-antitrypsin in a dose-dependent manner with similar potencies. Data were normalised to vehicle and a control compound from the GSK716 series at saturating concentration. Data presented as mean ± SD, n = 61 (secretion) and n = 67 (polymer inhibition). C, D. GSK716 was (C) administered to iPSC-derived-hepatocytes and (D) inhibited polymerisation and increased secretion with a similar potency. It induced an approximately threefold increase in secreted levels of Z α1-antitrypsin compared with vehicle control. This was apparent even after polymers had been allowed to form. Scale bar: 100 µm. Data presented as mean ± SD, n = 8 (secretion) and n = 6 (polymer inhibition). E. CHO tetracycline-inducible cells expressing Z α1-antitrypsin were induced with 0.5 μg/ml doxycycline for 48 h and treated with 10 μM GSK716. Cells were then lysed in 1% v/v NP-40 buffer at different time points (0, 12, 24, 36, 48, 60, 72, 84, 96 and 108 h). For every time point, NP-40-soluble and NP-40-insoluble fractions were separated and immunoprecipitated with the 2C1 mAb and resolved by 4–12% w/v SDS–PAGE and α1-antitrypsin detected by immunoblotting. PRE indicates cells pre-treated for 48 h with 10 μM GSK716 and induced for the same time with 0.5 μg/ml doxycycline. The rate of clearance of soluble and insoluble polymer is shown. F. CHO-inducible cells expressing either wild-type M or Z α1-antitrypsin were induced with 0.5 μg/ml doxycycline and treated with 10 μM GSK716 or with 0.1% DMSO vehicle (NT, not treated). After induction for 48 h, cells were treated with various doses of tunicamycin for 36 h. Cell viability was measured by Cell Counting Kit-8. The results are shown as mean ± SEM, n = 4. G. CHO-K1 Tet-On cells expressing Z α1-antitrypsin were induced with doxycycline (0.5 μg/ml) for 48 h. Cells were incubated with 10 μM GSK716 (or 0.1% v/v DMSO for the control) during the induction. Culture media containing either the experimental compound or DMSO were changed every 24 h. After the induction, cells were labelled for 10 min with 35S Met/Cys and chased at the indicated times. Culture media were collected and cells lysed in 1% v/v NP-40 buffer. Intracellular fractions and culture media from cells expressing Z α1-antitrypsin were immunoprecipitated either with a mAb against total α1-antitrypsin (3C11) or with a polymer-specific mAb (2C1). Samples were resolved by 4–12% w/v acrylamide SDS–PAGE and detected by autoradiography. H. The graphs show the effect of GSK716 on intracellular and extracellular Z α1-antitrypsin (mean ± SEM, n = 2). Download figure Download PowerPoint The pre-treatment of CHO cells induced to express Z α1-antitrypsin with GSK716 significantly reduced the formation of soluble and insoluble polymers (Fig 2E; compare PRE with time 0). To investigate the ability of GSK716 to protect cells from sensitisation to a secondary insult, Z α1-antitrypsin expression was induced in CHO-TET-ON-Z-A1AT cells in the presence or absence of 10 μM GSK716 before exposure to increasing concentrations of the ER stressor tunicamycin (Ordóñez et al, 2013). Cells expressing wild-type M α1-antitrypsin were less susceptible to tunicamycin toxicity than cells expressing Z α1-antitrypsin in a cell viability assay (Fig 2F). GSK716 restored sensitivity of Z α1-antitrypsin expressing cells to that of the wild-type control cells. The effect of GSK716 on Z α1-antitrypsin was confirmed in pulse-chase experiments (Fig 2G and H). These data collectively show that the small molecule completely blocks the intracellular polymerisation of Z α1-antitrypsin and increases secretion of the monomeric protein. GSK716 binds to a novel cryptic binding site A high-resolution crystal structure of α1-antitrypsin complexed with the lead compound GSK716 was generated by soaking compound into apo α1-antitrypsin crystals (Table 1). The structure reveals that interaction with the compound induces the formation of a cryptic binding site that is not evident in apo structures, at the top of β-sheet A behind strand 5. This region is referred to as the "breach" as it is the point at which the reactive centre loop first inserts during protease inhibition (Whisstock et al, 2000), and includes the site of the Z (Glu342Lys) mutation (Fig 3A). The structure reveals that the 2-oxindole ring of GSK716 stacks with the side chain of Trp194 whilst the carbonyl group forms a hydrogen bond with the mainchain Trp194 (Fig 3B). Trp194 adopts a new position due to rearrangement of residues Gly192 to Thr203 consistent with the change in intrinsic tryptophan fluorescence induced by binding (Fig 3C). The phenyl ring and the propyl chain occupy two highly hydrophobic pockets (Fig 3D–G). Hydrogen bonds are formed between the GSK716 hydroxyl group and the Leu291 backbone, the amide nitrogen hydrogen and the backbone carbonyl oxygen of Pro289, and between the amide carbonyl and the Tyr 244 hydroxyl group (Fig 3B). This causes displacement of residues Thr339 to Ser 359 of strand 5A relative to the apoprotein. Few changes are seen outside of these regions. Table 1. Data collection and refinement statistics. 7AEL Temperature 100K Wavelength 0.9763 Resolution range 55.05–1.76 (1.823–1.76) Space group C 1 2 1 Unit cell 113.95 39.59 90.52 90 104.96 90 Total reflections 127,818 (12,581) Unique reflections 38,772 (3,853) Multiplicity 3.3 (3.3) Completeness (%) 99.2 (99.2) Mean I/sigma (I) 19.61 (2.19) Wilson B-factor 32.95 R-merge 0.02941 (0.5007) R-meas 0.03523 (0.5991) CC1/2 0.999 (0.805) CC* 1 (0.944) Reflections used in refinement 38,771 (3,853) Reflections used for R-free 1,908 (178) R-work 0.1969 (0.3065) R-free 0.2259 (0.3204) CC(work) 0.958 (0.739) CC(free) 0.943 (0.699) Number of non-hydroge