Development of a small molecule that corrects misfolding and increases secretion of Z α1‐antitrypsin

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.

7th Sep 2020 1st Editorial Decision 7th Sep 2020 Dear Prof. Lomas, Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the three referees who agreed to evaluate your manuscript. As you will see from the reports below, while referee #1 and #2 are overall supporting publication of your work, referee #3 highlights the interest of the study but also raises a number of concerns that should be addressed in a major revision of the current manuscript. After a cross-commenting exercise it became clear that a control experiment with M alpha1-antitrypsin-expressing cells treated with GSK716 should be included, while no further in vivo experiments are required. However, addressing the reviewers' concerns in full, experimentally or in writing, will be necessary for consideration of your manuscript in our journal. Particularly, you should provide a detailed response to the referee #3 criticism of the mouse model, and in vivo experiments and discuss the limitations of the study in that regard. Also, PDB validation reports should be made available as suggested by the referee #1.
Please be aware that the acceptance of the manuscript will entail a second round of review. Please note that EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision. from the initial proposal of a conformational basis for a common inherited disorder to, here, the selection and design of a pharmaceutically effective counter. In terms of molecular diseases it is an unique achievement: e.g. even after 70 years there is still no effective counter to the sicklehaemoglobin mutation. The paper outlines and clearly describes careful and painstaking studies. The deductions and conclusions are data-based and frank (no hype!). There are some caveats that do not affect the strength of the paper but to which the authors should respond and modify as appropriate. Over the period of thirty years there has inevitably been relevant refinements as to the underlying mechanism of polymerisation. There is now totally persuasive evidence that the fundamental defect is the aberration of the bonding centred on Glu342 that pinions strand 5a in place and hence guides the final incorporation of the C-terminus into the body of the antitrypsin molecule and subsequently hinders its aberrant release. The supporting evidence is summarised in the paper of Huang et al (JBC 2016: 291,15674) on the 'Molecular mechanism of Z alpha-1-antitrypsin deficiency', which includes the crystal structure of Z antitrypsin. It should be cited. The Huang paper in no way pre-empts the current submission and indeed greatly strengthens it, almost as a preliminary study. The two papers are complementary, with satisfying agreements as to the drugbinding site. The very real advance in the submitted paper being the confirmation structurally of what is otherwise a deduced binding-site and in the design of a ligand that preferentially binds to the Z variant rather than the M (normal wild-type). The submitted paper describes new and important findings but it presents these in what is in places a dated context. The findings here precisely fit with the consensus mechanism that is now supported by a whole body of studies in the field. It is important to get this right which could be achieved with some minor rewriting. As it is, the Discussion can be misleading, as in 'The finding that GSK716 mediates its action by binding to a cryptic pocket implies that intrahepatic polymers form a near native or native conformation, rather than a more extended intermediate.' This is not so, and should be removed. We are looking at the stabilisation of equilibria, be it in the partially folded form or the fully-folded but labile aberrant native form. An outstanding finding in the submitted paper is in the determination of the structure of the antitrypsin-GSK17 complex. Although this structure seemed instinctively correct the data was succinct and a backup expert crystallographic opinion was sought, as follows. 'The crystallography is almost certainly fine, judging from the overall statistics and the resolution, but there are a few things that should be explained better. When a structure is deposited at the PDB, the authors receive a validation report that is intended for potential reviewers. This should be provided with the manuscript to allow referees to assess the quality of the structural work. The authors have left some blanks in Table 1. There is a line for Resolution in the Data collection section, but it's blank. Similarly, in the Refinement section the line for Water under "No. atoms" is blank, and both lines for Protein and Water under B-factors are blank. If numbers are given in these two subsections for protein and water, they should also be given for the ligand. In particular, it's important to be able to compare the mean B-factor of the ligand with that of the protein.
Whenever an important part of a structural paper is the binding of a ligand, it is accepted practice to provide a figure showing electron density for the ligand. Ideally, this should be unbiased electron density, computed either from a difference map before the ligand was added or after omitting the ligand and carrying out some refinement. One comment: the paper reports that the structure was solved by molecular replacement, using a model that had originally been obtained from PDB entry 2qug. Since 2qug seems to be isomorphous to this structure, it would have been simpler to carry out rigid-body refinement than to use molecular replacement, which will give a structure with a different choice of origin and symmetry copy; this makes structure comparison less convenient to users of the structures than the rigidbody refinement option.' The authors should respond to these points and the Editor/Journal note the desirability of making PDB validation reports available to reviewing referees.
Referee #2 (Comments on Novelty/Model System for Author): The models are well-suited to this proof of principle analysis.
Referee #2 (Remarks for Author): The paper presents an analysis of a small molecule corrector of AAT folding that interdicts polymerization of Z-type AAT. The candidate molecule was identified by high throughput screening from a large library. The paper from a distinguished group of investigators is well-written and compelling. The curious findings are that there is no reduction in intrahepatic polymers despite a 7fold increase in circulating Z-type AAT and that the lower doses (10 and 30 mg/kg) exerted the same effect as the 100 mg/kg dose despite low free durg levels. The authors do offer conjecture to explain these findings. They also offer an explanation as to how the opposing effects of GSK716 to decrease serine proteinase inhibition of AAT vs. to enhance secretion of Z-type AAT might result in net benefit. Of course, definitively understanding the net effect of GSK716 will require further study. Within the context of a proof-of-principle analysis, including examining the effects in mice, the paper offers an important contribution.
Referee #3 (Comments on Novelty/Model System for Author): Cell line models are used to demonstrate that the lead compound, GSK716, reduces the intracellular accumulation of polymers and increases secretion of Z-AAT. Similar results were obtained using the PiZ mouse model. However, this study did not provide convincing evidence demonstrating the beneficial effects of abolished polymerization and increased secretion of Z-AAT in the cell-or mouse-models. In Fig. 2f, the authors show that the treatment with GSK716 modestly improved the viability of Z-AAT expressing cells following a "second insult" namely, tunicamycin treatment. However, M-AAT expressing cells + GSK716 control was notably missing. Furthermore, it is unclear why 13-15 week old, PiZ hemizygous mice were used in this study. At this age, the authors themselves report that there were no signs of liver fibrosis. Use of mice at an age with early signs of fibrosis may have been more informative in determining whether GSK716 treatment could prevent progression or reverse existing fibrosis.

Referee #3 (Remarks for Author):
It is widely accepted that the Z-mutant of AAT accumulates as polymers within the endoplasmic reticulum of hepatocytes and this is responsible for the liver disease associated with severe AATdeficiency. It is also widely accepted that the accumulation of Z-AAT in the liver, significantly impairs secretion of AAT into the plasma. The reduced circulating AAT is thought to be responsible, as least in part, for the lung disease associated with AAT-deficiency. As such, a compound that abolishes Z-AAT polymer formation and consequently enhances secretion of AAT would have beneficial effects on both the liver and lung diseases associated with AAT-deficiency. In an effort to identify such a compound, the authors conducted a screen and identified a strong lead compound, GSK716, that blocked Z-polymer accumulation and increased secretion of Z-AAT in their cell line models. Further studies performed in mice showed an apparent increase in secretion as determined by an increase in circulating Z-AAT in the plasma. Unfortunately, the compound had no effect on preventing the accumulation of Z-AAT polymers in the mouse hepatocytes.
Although the in vitro data on the lead compound's ability to prevent polymerization is impressive, it is unclear, from this study, whether the decrease in accumulation of Z-AAT is actually beneficial to the cells in anyway. The authors tried to answer this question by generating a tunicamycin survival curve (Fig. 2f). Interestingly, GSK716 treatment modestly improved viability of Z-expressing cells, however, an important control, notably M-expressing cells treated with GSK716 was missing. Further studies are required to address this important question.
In some instances, there is inadequate information, in the figure legends and in the methods, to adequately understand how the experiments were conducted and what the results actually mean. The authors indicated that GSK716 "increased the secretion of Z a1-antitrypsin approximately 3fold compared to vehicle control (mean pEC50 6.2 {plus minus} 0.23; n = 74) (Fig. 2b)," however, it is unclear how they came to that conclusion from the data provided. Fig. 2b show 100% secretion. Fig.  2d shows 3-fold secretion. What does 100% secretion mean? What does 3-fold secretion mean? More information should be included to assist the reader in determining the significance of the data.
The authors then provide data in the PiZ mouse model showing that GSK716 treatment significantly increased plasma levels of Z-AAT. They concluded that this was due to increased secretion of Z-AAT from the liver, however, it is unclear whether this is actually the case since the amount of Z-AAT in the hepatocytes remained unchanged in control-and GSK716-treated mice.
Although, GSK716 failed to alter Z-AAT polymer accumulation in the liver, one could argue that the 7-fold increase in secretion of Z-AAT would be beneficial to the lung disease associated with AATdeficiency. Unfortunately, GSK716 binds to Z-AAT in such a way that it abolishes the antiproteinase activity of Z-AAT. This is a critically important as it implies that increasing secretion of Z-AAT has no apparent physiological benefits.
The authors' conclusion that "This study provides proof-of-principle that 'mutation ameliorating' small molecules are a viable approach to treat protein conformational diseases" Is not supported by the data presented in this manuscript.
Minor points: GSK716 is sometimes referred to as GSK'716A. Is this a different analog or a typo? Fig. 2g, bottom right, should be "extracellular" not intracellular. This paper represents a triumphant progress, led by the first author over a period of nearly 30 years, from the initial proposal of a conformational basis for a common inherited disorder to, here, the selection and design of a pharmaceutically effective counter. In terms of molecular diseases it is an unique achievement: e.g. even after 70 years there is still no effective counter to the sickle-haemoglobin mutation. The paper outlines and clearly describes careful and painstaking studies. The deductions and conclusions are data-based and frank (no hype!). There are some caveats that do not affect the strength of the paper but to which the authors should respond and modify as appropriate.
We are very grateful to the reviewer for his/her supportive comments.
Over the period of thirty years there has inevitably been relevant refinements as to the underlying mechanism of polymerisation. There is now totally persuasive evidence that the fundamental defect is the aberration of the bonding centred on Glu342 that pinions strand 5a in place and hence guides the final incorporation of the C-terminus into the body of the antitrypsin molecule and subsequently hinders its aberrant release. The supporting evidence is summarised in the paper of Huang et al (JBC 2016: 291,15674) on the 'Molecular mechanism of Z alpha-1-antitrypsin deficiency', which includes the crystal structure of Z antitrypsin. It should be cited. The Huang paper in no way pre-empts the current submission and indeed greatly strengthens it, almost as a preliminary study. The two papers are complementary, with satisfying agreements as to the drug-binding site. The very real advance in the submitted paper being the confirmation structurally of what is otherwise a deduced binding-site and in the design of a ligand that preferentially binds to the Z variant rather than the M (normal wild-type).
The submitted paper describes new and important findings but it presents these in what is in places a dated context. The findings here precisely fit with the consensus mechanism that is now supported by a whole body of studies in the field. It is important to get this right which could be achieved with some minor rewriting. As it is, the Discussion can be misleading, as in 'The finding that GSK716 mediates its action by binding to a cryptic pocket implies that intrahepatic polymers form a near native or native conformation, rather than a more extended intermediate.' This is not so, and should be removed. We are looking at the stabilisation of equilibria, be it in the partially folded form or the fully-folded but labile aberrant native form.
We fully agree with the reviewer and have added the reference to Huang et al, 2016. Indeed, our group has recently used cryo-EM to characterise isolated polymers from the liver tissue of Z  1 -AT homozygotes (Glu342Lys) who had undergone liver transplantation. The data show that the inter-subunit linkage of Z  1 -AT is best explained by a C-terminal domain swap between molecules of  1 -AT (Faull et al, 2020). These data are consistent with a head-to-tail arrangement of subunits in heat-induced polymers revealed by a complex with the non-Z-selective antibody 2H2 (Laffranchi et al, 2020). This is in keeping with the view expressed by the reviewer. We have removed 'The finding that GSK716 mediates its action by binding to a cryptic pocket implies that intrahepatic polymers form a near native or native conformation, rather than a more extended intermediate.' We have changed the introduction to read 'Polymerisation from this state involves insertion of the RCL into -sheet A with a domain-swap of the C-terminal region providing the inter-  and therefore' has been deleted from the results section. 'The finding that GSK716 mediates its action by binding to a cryptic pocket implies that intrahepatic polymers form from a near-native or native conformation, rather than a more extended intermediate' has been deleted from the discussion and replaced with 'GSK716 stabilises the partially folded  1 -antitrypsin or the fully-folded but labile aberrant native form.' An outstanding finding in the submitted paper is in the determination of the structure of the antitrypsin-GSK17 complex. Although this structure seemed instinctively correct the data was succinct and a backup expert crystallographic opinion was sought, as follows. 'The crystallography is almost certainly fine, judging from the overall statistics and the resolution, but there are a few things that should be explained better. When a structure is deposited at the PDB, the authors receive a validation report that is intended for potential reviewers. This should be provided with the manuscript to allow referees to assess the quality of the structural work.
We have provided the validation report from the PDB submission (D_1292111290_valreport-full_P1.pdf). The PDB entry is 7EAL. Unfortunately, the submission has been given the wrong title. We have asked for this to be corrected but there is currently a technical issue with the platform. The data in the PDB are correct. We are sorry that this was missed from the paper. The full Table has been included in the paper and is reproduced below. Whenever an important part of a structural paper is the binding of a ligand, it is accepted practice to provide a figure showing electron density for the ligand. Ideally, this should be unbiased electron density, computed either from a difference map before the ligand was added or after omitting the ligand and carrying out some refinement.

Table 1. Data collection and refinement statistics
The OMIT density maps for the ligand is shown below and reproduced in Figs. 3F and G.

±3.0sigma (blue/red) +1.0 sigma (blue)
One comment: the paper reports that the structure was solved by molecular replacement, using a model that had originally been obtained from PDB entry 2qug. Since 2qug seems to be isomorphous to this structure, it would have been simpler to carry out rigid-body refinement than to use molecular replacement, which will give a structure with a different choice of origin and symmetry copy; this makes structure comparison less convenient to users of the structures than the rigid-body refinement option.' We have ensured the deposited structure, 7AEL.pdb, shares the same choice of origin as 2qug.pdb The authors should respond to these points and the Editor/Journal note the desirability of making PDB validation reports available to reviewing referees.
The validation report has been included with the resubmitted manuscript.

Referee #2 (Comments on Novelty/Model System for Author):
The models are well-suited to this proof of principle analysis.

Referee #2 (Remarks for Author):
The paper presents an analysis of a small molecule corrector of AAT folding that interdicts polymerization of Z-type AAT. We are grateful to the reviewer for his/her comments. We have repeated the experiment with M  1 -AT expressing cells + GSK716. The new figure is reproduced below along with the figure legend. Cells expressing M  1 -AT were more resistant to tunicamycin than cells expressing Z  1 -AT. 716 increased the resistance of Z  1 -AT cells to tunicamycin to that of M  1 -AT. It had no effect on the survival of cells expressing M  1 -AT. This is included as revised Fig. 2F Hepatology. 2017; 65:1865-1874). The primary aim of the mouse experiments was to assess target engagement and in particular whether GSK716 could increase the secretion of Z  1 -AT. Having shown this, we then looked to evaluate whether there was an effect on hepatic inclusions. We selected younger, and hemizygous, rather than older mice, as our longevity studies showed that circulating levels of Z  1 -AT increase with age (to levels much higher than seen in patients) and these artificially high levels may act as a high affinity sink sequestering drug and preventing bioavailability at the site of action in the liver. Further, older mice have larger  1 -AT inclusions that may be more difficult to reverse and any changes would be more difficult to detect than in younger animals. As the reviewer comments, the conclusion from our studies is that GSK716 did not reduce PAS positive inclusions, following testing in animals where the compound had the best chance of impacting them, and would also therefore be unlikely to reverse fibrosis.
The following text has been added to the results section 'Younger hemizygous, rather than older mice, were selected as our longevity studies showed that circulating levels of Z  1antitrypsin increase with age (to levels much higher than seen in patients) and these artificially high levels may act as a high affinity sink sequestering drug and preventing bioavailability at the site of action in the liver. Further, older mice have larger  1 -antitrypsin inclusions that may be more difficult to reverse and any changes would be more difficult to detect than in younger animals.'

Referee #3 (Remarks for Author):
It is widely accepted that the Z-mutant of AAT accumulates as polymers within the endoplasmic reticulum of hepatocytes and this is responsible for the liver disease associated with severe AAT-deficiency. It is also widely accepted that the accumulation of Z-AAT in the liver, significantly impairs secretion of AAT into the plasma. The reduced circulating AAT is thought to be responsible, as least in part, for the lung disease associated with AAT- (Fig. 2f). Interestingly, GSK716 treatment modestly improved viability of Z-expressing cells, however, an important control, notably M-expressing cells treated with GSK716 was missing. Further studies are required to address this important question.

deficiency. As such, a compound that abolishes Z-AAT polymer formation and consequently enhances secretion of AAT would have beneficial effects on both the liver and lung diseases associated with AAT-deficiency. In an effort to identify such a compound, the authors conducted a screen and identified a strong lead compound, GSK716, that blocked Zpolymer accumulation and increased secretion of Z-AAT in their cell line models. Further studies performed in mice showed an apparent increase in secretion as determined by an increase in circulating Z-AAT in the plasma. Unfortunately, the compound had no effect on preventing the accumulation of Z-AAT polymers in the mouse hepatocytes. Although the in vitro data on the lead compound's ability to prevent polymerization is impressive, it is unclear, from this study, whether the decrease in accumulation of Z-AAT is actually beneficial to the cells in anyway. The authors tried to answer this question by generating a tunicamycin survival curve
We have repeated the experiment in Fig 2F with M  1 -AT expressing cells + GSK716 as described in the section above In some instances, there is inadequate information, in the figure legends and in the methods, to adequately understand how the experiments were conducted and what the results actually mean. The authors indicated that GSK716 "increased the secretion of Z a1-antitrypsin approximately 3-fold compared to vehicle control (mean pEC50 6.2 {plus minus} 0.23; n = 74) (Fig. 2b)," however, it is unclear how they came to that conclusion from the data provided. Fig. 2b show 100% secretion. Fig. 2d shows 3-fold secretion. What does 100% secretion mean? What does 3-fold secretion mean? More information should be included to assist the reader in determining the significance of the data.
We thank the reviewer for raising this point. We have changed the legend to Fig 2B and 2D to make it clear that data in Fig. 2B (CHO-TET-ON-Z-A1AT cell assay) are normalised, using vehicle and a control compound (from the GSK716 series) at saturating concentration as high/low controls, as is standard practise for screening assays. The actual concentrations of  1 -AT in the supernatants were not determined in the same experiments, and we have removed the comment about 3-fold increase of secretion in this context. However, in the iPSC-hepatocyte assays (Fig 2D), calibration curves were used to determine the levels of  1 -AT in the supernatant in each individual experiment, therefore the fold increase induced by GSK716, compared with vehicle control, could be calculated and is shown in Fig 2D. The statement in the text is consistent with these data.
The text has been changed to read (new text in red). In comparison with controls, GSK716 completely blocked the intracellular formation of Z  1 -antitrypsin polymers, as measured by staining with the 2C1 anti-Z  1 -antitrypsin polymer monoclonal antibody (mean pIC50 = 6.3 ± 0.23; n = 71) ( Figs. 2A, B). It also increased the secretion of Z  1 -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  1 -antitrypsin in iPSC-derived human hepatocytes with the ZZ  1 -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 3-fold increase of secreted levels of Z  1 -antitrypsin (Figs 2C, D). We have also amended the abstract to read 'The lead compound blocks Z  1 -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z  1 -antitrypsin three-fold in an iPSC model of disease' and have corrected the text in the Discussion.
The authors then provide data in the PiZ mouse model showing that GSK716 treatment significantly increased plasma levels of Z-AAT. They concluded that this was due to increased secretion of Z-AAT from the liver, however, it is unclear whether this is actually the case since the amount of Z-AAT in the hepatocytes remained unchanged in control-and GSK716-treated mice.
The conclusion that the increased secretion is from the liver is based on: (i) the pulse chase experiments that showed GSK716 increased the secretion of Z  1 -AT from CHO cells and iPSC derived hepatocytes (Fig. 3). (ii) treatment with GSK716 increased the monomer measured in liver homogenate by approx. 4-fold in keeping with the changes seen in blood and (iii) liver is the only tissue that can produce sufficient  1 -AT to cause a 7 fold increase in circulating protein.
Although, GSK716 failed to alter Z-AAT polymer accumulation in the liver, one could argue that the 7-fold increase in secretion of Z-AAT would be beneficial to the lung disease associated with AAT-deficiency. Unfortunately, GSK716 binds to Z-AAT in such a way that it abolishes the anti-proteinase activity of Z-AAT. This is a critically important as it implies that increasing secretion of Z-AAT has no apparent physiological benefits.
The authors' conclusion that "This study provides proof-of-principle that 'mutation ameliorating' small molecules are a viable approach to treat protein conformational diseases" Is not supported by the data presented in this manuscript.
We are grateful to the reviewer for raising this issue. The statement referred to our finding that GSK716 can bind to a site close to the mutation in Z-AAT and change its polymerisation behaviour to approximate that of M-AAT. We apologise for the confusion and have amended this sentence in the abstract and the impact section to read 'can block the aberrant polymerisation that underlies Z  1 -antitrypsin deficiency'.
Minor points: GSK716 is sometimes referred to as GSK'716A. Is this a different analog or a typo?
GSK716 and GSK'716 are the same. We have amended Fig. 2 to only refer to GSK716.

Fig. 2G, bottom right, should be "extracellular" not intracellular.
This has been corrected in the revised manuscript.
1st Dec 2020 1st Revision -Editorial Decision 1st Dec 2020 Dear Prof. Lomas, Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments:

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