Small molecules block the polymerization of Z alpha1-antitrypsin and increase the clearance of intracellular aggregates.

The Z mutant of alpha1-antitrypsin (Glu342Lys) causes a domain swap and the formation of intrahepatic polymers that aggregate as inclusions and predispose the homozygote to cirrhosis. We have identified an allosteric cavity that is distinct from the interface involved in polymerization for rational structure-based drug design to block polymer formation. Virtual ligand screening was performed on 1.2 million small molecules and 6 compounds were identified that reduced polymer formation in vitro. Modeling the effects of ligand binding on the cavity and re-screening the library identified an additional 10 compounds that completely blocked polymerization. The best antagonists were effective at ratios of compound to Z alpha1-antitrypsin of 2.5:1 and reduced the intracellular accumulation of Z alpha1-antitrypsin by 70% in a cell model of disease. Identifying small molecules provides a novel therapy for the treatment of liver disease associated with the Z allele of alpha1-antitrypsin.

[1]  J. Teckman,et al.  Quantitative isolation of α1AT mutant Z protein polymers from human and mouse livers and the effect of heat , 2005, Hepatology.

[2]  R. Abagyan,et al.  Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins. , 1994, Journal of molecular biology.

[3]  D. Lomas,et al.  The mechanism of Z alpha 1-antitrypsin accumulation in the liver. , 1992, Nature.

[4]  B. Ghetti,et al.  Association between conformational mutations in neuroserpin and onset and severity of dementia , 2002, The Lancet.

[5]  A. Shukla,et al.  Identification of a 4-mer peptide inhibitor that effectively blocks the polymerization of pathogenic Z alpha1-antitrypsin. , 2006, American journal of respiratory cell and molecular biology.

[6]  D. Lomas.  The Selective Advantage of 1-Antitrypsin Deficiency , 2006 .

[7]  Yan Liu,et al.  Organizational diversity among distinct glycoprotein endoplasmic reticulum-associated degradation programs. , 2002, Molecular biology of the cell.

[8]  D. Perlmutter.  Liver injury in alpha1-antitrypsin deficiency: an aggregated protein induces mitochondrial injury. , 2002, The Journal of clinical investigation.

[9]  D. Lomas,et al.  Topography of a 2.0 Å structure of α1‐antitrypsin reveals targets for rational drug design to prevent conformational disease , 2000, Protein science : a publication of the Protein Society.

[10]  D. Lomas,et al.  Mutations Which Impede Loop/Sheet Polymerization Enhance the Secretion of Human α1-Antitrypsin Deficiency Variants (*) , 1995, The Journal of Biological Chemistry.

[11]  P Argos,et al.  Optimal protocol and trajectory visualization for conformational searches of peptides and proteins. , 1992, Journal of molecular biology.

[12]  Neil A. Ravenhill,et al.  Targeting a Surface Cavity of α1-Antitrypsin to Prevent Conformational Disease* , 2003, Journal of Biological Chemistry.

[13]  D. Lomas,et al.  A Kinetic Mechanism for the Polymerization of α1-Antitrypsin* , 1999, The Journal of Biological Chemistry.

[14]  B. Villoutreix,et al.  Antithrombin Phe229Leu: a new homozygous variant leading to spontaneous antithrombin polymerization in vivo associated with severe childhood thrombosis. , 2003, Blood.

[15]  D. Lomas,et al.  Nucleation of α1-antichymotrypsin polymerization , 2003 .

[16]  F. Callea,et al.  Differential detection of PAS‐positive inclusions formed by the Z, Siiyama, and Mmalton variants of α1‐antitrypsin , 2004, Hepatology.

[17]  J. Carlson,et al.  Risk of cirrhosis and primary liver cancer in alpha 1-antitrypsin deficiency. , 1986, The New England journal of medicine.

[18]  J. Teckman,et al.  The Proteasome Participates in Degradation of Mutant α1-Antitrypsin Z in the Endoplasmic Reticulum of Hepatoma-derived Hepatocytes* , 2001, The Journal of Biological Chemistry.

[19]  R. Abagyan,et al.  Pocketome via Comprehensive Identification and Classification of Ligand Binding Envelopes* , 2005, Molecular & Cellular Proteomics.

[20]  T Sveger,et al.  Liver disease in alpha1-antitrypsin deficiency detected by screening of 200,000 infants. , 1976, The New England journal of medicine.

[21]  Neil A. Ravenhill,et al.  Targeting a Surface Cavity of α1-Antitrypsin to Prevent Conformational Disease , 2003 .

[22]  R. Read,et al.  Inactive conformation of the serpin alpha(1)-antichymotrypsin indicates two-stage insertion of the reactive loop: implications for inhibitory function and conformational disease. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Teckman.  Lack of Effect of Oral 4-Phenylbutyrate on Serum Alpha-1-Antitrypsin in Patients with α-1-Antitrypsin Deficiency: A Preliminary Study , 2004, Journal of pediatric gastroenterology and nutrition.

[24]  R. Carrell,et al.  Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 Asn-->Asp) , 1994, The Journal of clinical investigation.

[25]  S. Ōmura,et al.  Degradation of a Mutant Secretory Protein, α1-Antitrypsin Z, in the Endoplasmic Reticulum Requires Proteasome Activity* , 1996, The Journal of Biological Chemistry.

[26]  D. Lomas,et al.  Effect of the Z mutation on the physical and inhibitory properties of alpha 1-antitrypsin. , 1993, Biochemistry.

[27]  D. Lomas.  The selective advantage of alpha1-antitrypsin deficiency. , 2006, American journal of respiratory and critical care medicine.

[28]  D. Lomas,et al.  Mutant Neuroserpin (S49P) That Causes Familial Encephalopathy with Neuroserpin Inclusion Bodies Is a Poor Proteinase Inhibitor and Readily Forms Polymers in Vitro * , 2002, The Journal of Biological Chemistry.

[29]  D. Perlmutter,et al.  Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Hack,et al.  A hinge region mutation in C1-inhibitor (Ala436-->Thr) results in nonsubstrate-like behavior and in polymerization of the molecule. , 1993, The Journal of biological chemistry.

[31]  R. Read,et al.  Structure of a serpin–protease complex shows inhibition by deformation , 2000, Nature.

[32]  E. Verpy,et al.  COOH-terminal Substitutions in the Serpin C1 Inhibitor That Cause Loop Overinsertion and Subsequent Multimerization (*) , 1995, The Journal of Biological Chemistry.

[33]  K. Moremen,et al.  Elucidation of the molecular logic by which misfolded α1-antitrypsin is preferentially selected for degradation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Lomas,et al.  Familial dementia caused by polymerization of mutant neuroserpin , 1999, Nature.

[35]  Glyn L. Devlin,et al.  Prevention of polymerization of M and Z alpha1-Antitrypsin (alpha1-AT) with trimethylamine N-oxide. Implications for the treatment of alpha1-at deficiency. , 2001, American journal of respiratory cell and molecular biology.

[36]  Takahiro Kamimoto,et al.  Intracellular Inclusions Containing Mutant α1-Antitrypsin Z Are Propagated in the Absence of Autophagic Activity* , 2006, Journal of Biological Chemistry.

[37]  J. Teckman,et al.  Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response. , 2000, American journal of physiology. Gastrointestinal and liver physiology.

[38]  D. Lomas,et al.  Alpha1-antitrypsin polymerization and the serpinopathies: pathobiology and prospects for therapy. , 2002, The Journal of clinical investigation.

[39]  Yan Liu,et al.  Processing by Endoplasmic Reticulum Mannosidases Partitions a Secretion-impaired Glycoprotein into Distinct Disposal Pathways* , 2000, The Journal of Biological Chemistry.

[40]  N. Novoradovskaya,et al.  Inhibition of intracellular degradation increases secretion of a mutant form of alpha1-antitrypsin associated with profound deficiency. , 1998, The Journal of clinical investigation.

[41]  J. Kocher,et al.  Cavities of α1‐antitrypsin that play structural and functional roles , 2001 .

[42]  D. Lomas,et al.  6-mer Peptide Selectively Anneals to a Pathogenic Serpin Conformation and Blocks Polymerization , 2002, The Journal of Biological Chemistry.

[43]  B. Cooperman,et al.  Alpha 1-antitrypsin polymerization: a fluorescence correlation spectroscopic study. , 2005, Biochemistry.