6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis.

Conformational diseases such as amyloidosis, Alzheimer's disease, prion diseases, and the serpinopathies are all caused by structural rearrangements within a protein that transform it into a pathological species. These diseases are typified by the Z variant of alpha(1)-antitrypsin (E342K), which causes the retention of protein within hepatocytes as inclusion bodies that are associated with neonatal hepatitis and cirrhosis. The inclusion bodies result from the Z mutation perturbing the conformation of the protein, which facilitates a sequential interaction between the reactive center loop of one molecule and beta-sheet A of a second. Therapies to prevent liver disease must block this reactive loop-beta-sheet polymerization without interfering with other proteins of similar tertiary structure. We have used reactive loop peptides to explore the differences between the pathogenic Z and normal M alpha(1)-antitrypsin. The results show that the reactive loop is likely to be partially inserted into beta-sheet A in Z alpha(1)-antitrypsin. This conformational difference from M alpha(1)-antitrypsin was exploited with a 6-mer reactive loop peptide (FLEAIG) that selectively and stably bound Z alpha(1)-antitrypsin. The importance of this finding is that the peptide prevented the polymerization of Z alpha(1)-antitrypsin and did not significantly anneal to other proteins (such as antithrombin, alpha(1)-antichymotrypsin, and plasminogen activator inhibitor-1) with a similar tertiary structure. These findings provide a lead compound for the development of small molecule inhibitors that can be used to treat patients with Z alpha(1)-antitrypsin deficiency. Furthermore they demonstrate how a conformational disease process can be selectively inhibited with a small peptide.

Members of the serine proteinase inhibitor or serpin superfamily are characterized by an exposed 14-residue (P 14-1 ) mobile reactive center loop and a dominant five-stranded ␤-sheet A (1, 2) (Fig. 1a). Biochemical and crystallographic studies have defined the marked flexibility of the reactive loop and have demonstrated its role in inhibiting the target proteinase (3)(4)(5)(6). The proteinase binds to the serpin and cleaves the reactive loop at the P 1 -P 1Ј bond. This cleavage initiates a profound conformational change in the serpin in which the reactive loop peptide inserts into ␤-sheet A to form a new central strand termed s4A (6,7). The conformational change inactivates the proteinase by translocating it over 70 Å to the lower pole of the molecule and disrupting the catalytic site.
This reactive loop-␤-sheet A interaction, while being essential for proteinase inhibition, also renders the serpin vulnerable to conformational disturbances that are associated with disease, the serpinopathies (8). Point mutations perturb the relationship between the reactive loop and ␤-sheet A to allow the sequential interaction between the reactive center loop of one molecule and ␤-sheet A of another (Fig. 1a). These loopsheet polymers are inactive as proteinase inhibitors and are retained within the cell of synthesis. This process is best characterized for the deficiency variants of ␣ 1 -antitrypsin (9 -13). ␣ 1 -Antitrypsin is synthesized in hepatocytes and circulates in the plasma. Most Northern Europeans are M homozygotes, but 1 in 25 has the severe Z ␣ 1 -antitrypsin deficiency variant (E342K). This is at residue P 17 (17 residues proximal to the P 1 reactive center) at the head of strand 5 of ␤-sheet A and the base of the mobile reactive loop (Fig. 1). The mutation opens ␤-sheet A thereby favoring the insertion of the reactive loop of another ␣ 1 -antitrypsin molecule to form a dimer. This can then extend to form chains of polymers that accumulate in the endoplasmic reticulum of the liver to form inclusion bodies (9). These inclusions result in neonatal hepatitis, cirrhosis, and hepatocellular carcinoma (14 -16), and the lack of circulating proteinase inhibitor exposes the lungs to uncontrolled proteolytic attack and early onset emphysema (17).
An effective approach to treatment of the liver disease would be to inhibit polymerization of the Z ␣ 1 -antitrypsin and thus prevent the accumulation of the protein within hepatocytes. Previous studies have shown that synthetic peptides with homology to the reactive loop of ␣ 1 -antitrypsin and the related serpin antithrombin can anneal to ␤-sheet A of both M ␣ 1antitrypsin and antithrombin (18 -21). Furthermore, binding of these 11-13-mer peptides prevents the polymerization of Z ␣ 1 -antitrypsin (9) (Fig. 1a). However, such peptides are promiscuous and can efficiently anneal to, and inactivate, both M and Z ␣ 1 -antitrypsin and other members of the serpin superfamily (20). Their size and lack of specificity precludes the use of these peptides as therapeutic agents or as lead compounds from which to develop mimetic drugs. Consequently other strategies are being developed to prevent polymerization using chemical chaperones (22,23) and by targeting a hydrophobic pocket (24,25) that is filled as polymers form (26).
We report here the use of reactive loop peptides to explore the structural differences between the pathogenic Z and normal M ␣ 1 -antitrypsin. This has allowed us to first define the pathogenic conformation of Z ␣ 1 -antitrypsin and then exploit this difference from M ␣ 1 -antitrypsin to target a 6-mer peptide specifically to Z ␣ 1 -antitrypsin to prevent polymerization.

EXPERIMENTAL PROCEDURES
Purification of M and Z ␣ 1 -Antitrypsin-M and Z ␣ 1 -antitrypsin were purified from the plasma of known homozygotes by 50 and 75% ammonium sulfate fractionation followed by glutathione and anion exchange chromatography as described previously (27). The proteins migrated as a single band on SDS-PAGE and had a normal unfolding profile on transverse urea gradient gel electrophoresis. Both M and Z ␣ 1 -antitrypsin were functional as inhibitors of bovine ␣-chymotrypsin.
Assessment of Synthetic Reactive Loop Peptide Annealing to ␣ 1 -Antitrypsin by PAGE-A synthetic N ␣ -acetyl peptide corresponding to the P 14-3 sequence of the reactive loop of antithrombin (Ac-Ser-Glu-Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-Ile-Ala-OH) was synthesized and purified by Genosys Biotechnologies Inc. (Cambridge, UK) and dissolved in 50 mM Tris, 50 mM NaCl, pH 7.4. The P 7-2 sequence of ␣ 1 -antitrypsin (Ac-Phe-Leu-Glu-Ala-Ile-Gly-OH) was synthesized and purified by MWB (Cambridge, UK) and dissolved in water. The peptides were annealed to M or Z ␣ 1 -antitrypsin, or other serpins, by incubating at a final concentration of 0.1-0.5 mg/ml in 50 mM Tris, 50 mM KCl, pH 7.4 at 37°C. The binary complex between ␣ 1 -antitrypsin, and other serpins, and the reactive loop peptides was assessed on a 7.5% (w/v) nondenaturing polyacrylamide gel containing 8 M urea. 1 The formation of reactive loop-␤-sheet A polymers was assessed on the same gel without urea (28).
Assessment of Synthetic Reactive Loop Peptide Annealing to ␣ 1 -Antitrypsin by Intrinsic Tryptophan Fluorescence-The kinetics of reactive loop peptide annealing to ␣ 1 -antitrypsin were assessed by following the intrinsic tryptophan fluorescence of ␣ 1 -antitrypsin in a PerkinElmer LS 50B spectrophotometer. Intrinsic tryptophan fluorescence of ␣ 1 -antitrypsin was measured in 50 mM Tris, 50 mM KCl, pH 7.4 using an excitation wavelength of 295 nm and an emission wavelength of 340 nm. The excitation and emission slit widths were controlled to give the optimal emission signal as described previously (10). Each experiment was performed with a native molecule control, and the final data were obtained by subtraction of ␣ 1 -antitrypsin incubated with peptide from native ␣ 1 -antitrypsin alone. The data were fitted to a single exponential function using Grafit (Version 3.00, 1992, Erithracus Software Ltd).

RESULTS AND DISCUSSION
The 12-mer P 14-3 peptide, corresponding to the reactive loop of antithrombin, was incubated in 100-fold molar excess with 0.1 mg/ml M and Z ␣ 1 -antitrypsin at 37°C. The binding of the reactive loop peptide to ␣ 1 -antitrypsin was monitored on acrylamide/8 M urea gels and by intrinsic tryptophan fluorescence. Native ␣ 1 -antitrypsin unfolded in the urea and was retarded by the gel. The binary complex of ␣ 1 -antitrypsin with peptide was stable in 8 M urea and hence migrated further into the acrylamide. The peptide annealed at a much slower rate to Z ␣ 1antitrypsin than it did to M ␣ 1 -antitrypsin (Fig. 2, top). Analysis of intrinsic tryptophan fluorescence allowed a more detailed assessment of the rate of peptide annealing. The addition of peptide to M ␣ 1 -antitrypsin resulted in a significant increase in fluorescence as the peptide annealed to ␤-sheet A, reflecting incorporation of the reactive loop peptide into ␣ 1antitrypsin (Fig. 2, bottom). However, the Z variant accepted the 12-mer reactive loop peptide at less than half of the rate of M ␣ 1 -antitrypsin (2.6 ϫ 10 Ϫ5 and 6.0 ϫ 10 Ϫ5 s Ϫ1 for Z and M ␣ 1 -antitrypsin, respectively). There was no change in fluorescence signal from ␣ 1 -antitrypsin cleaved at the reactive loop with Staphylococcus aureus V8 proteinase when incubated under the same conditions (data not shown).
Both reactive loop peptide annealing to serpins and the formation of loop-sheet polymers occurs by intramolecular addi-1 A. Zhou, personal communication.
FIG. 1. Schematic representation of the polymerization of Z ␣ 1 -antitrypsin and the design of a selective inhibitor. a, the Z mutation (E342K) perturbs the structure of ␣ 1 -antitrypsin to allow opening of ␤-sheet A (green). This ␤-sheet then accepts the reactive center loop of another molecule (yellow) to form a dimer that extends as chains of polymers (left). A 12-mer peptide (yellow) can anneal to ␤-sheet A thereby preventing polymer formation (right). b, our data indicate that the Z mutation (ringed in blue) allows partial insertion of the reactive center loop (red). This opens the lower part of ␤-sheet A thereby favoring polymerization (left). Understanding the configuration of the reactive loop prompted the hypothesis that a 6-mer peptide with homology to P 7-2 of the reactive center loop would specifically bind to Z ␣ 1 -antitrypsin and so prevent polymerization (right). 6-mer Peptide Selectively Anneals to Z ␣ 1 -Antitrypsin 6772 tion of a strand to ␤-sheet A (12,18,21,29) (Fig. 1a). The Z mutation of ␣ 1 -antitrypsin readily favors loop-sheet polymerization and therefore must make this region more receptive to the reactive center loop of another molecule (Fig. 1a). However, our data show that the 12-mer reactive loop peptide anneals to Z ␣ 1 -antitrypsin at a slower rate than to the nonpolymerogenic M ␣ 1 -antitrypsin. This can be explained by examining the crystal structure of ␣ 1 -antitrypsin (26), which shows that the Z mutation lies at the head of strand 5 of ␤-sheet A and at the base of the reactive center loop. The positively charged lysine residue in Z ␣ 1 -antitrypsin must destabilize the upper part of ␤-sheet A to allow partial insertion of the reactive center loop (Fig. 1b). This partial loop insertion would explain the slower rate of annealing of full-length reactive loop peptides. It would also explain the rapid conversion to polymers as these are formed by annealing of P 8-3 of the reactive loop to the lower part of ␤-sheet A (12, 30 -32). The conformation adopted by Z ␣ 1 -antitrypsin is therefore similar to that produced by annealing a P 14-8 7-mer reactive loop peptide to antithrombin, which opens the top of ␤-sheet A to favor polymerization (33). It also approximates the conformation seen in our crystal structure of a naturally occurring mutant of ␣ 1 -antichymotrypsin that also readily forms polymers in vitro and in vivo (34).
The hypothesis that the pathogenic conformation adopted by Z ␣ 1 -antitrypsin is associated with partial insertion of the reactive loop was tested using a peptide targeted to the lower part of ␤-sheet A. A 6-mer peptide that was homologous to P 7-2 of the reactive loop of ␣ 1 -antitrypsin was obtained from Dr A. Zhou and colleagues (Department of Hematology, University of Cambridge) who were undertaking a separate study on the structural requirements for peptide-␤-sheet A blockage (Fig.  1b). This peptide was able to anneal to Z, but not to M ␣ 1antitrypsin (Fig. 3, top), and resulted in a 60% reduction in its inhibitory activity against bovine ␣-chymotrypsin (data not shown). Furthermore, the rate of fluorescence increase when the peptide annealed was over 30-fold more rapid with Z ␣ 1antitrypsin than with M ␣ 1 -antitrypsin: 2.2 ϫ 10 Ϫ5 and 0.07 ϫ 10 Ϫ5 s Ϫ1 for Z and M ␣ 1 -antitrypsin, respectively (Fig. 3, middle). The importance of this interaction was highlighted by co-incubation of the 6-mer peptide with Z ␣ 1 -antitrypsin, which resulted in a complete inhibition of polymerization of Z ␣ 1antitrypsin when incubated at 37°C (Fig. 3, bottom) and 41°C (data not shown). It is likely that the peptide also prevented polymerization of Z ␣ 1 -antitrypsin at peptide:protein ratios of less than 25:1, although accurate values were precluded by limited peptide solubility.
Peptide annealing was specific for Z ␣ 1 -antitrypsin as coincubation of the 6-mer peptide with M ␣ 1 -antitrypsin for 3 days resulted in no significant binary complex formation (Fig.  3, top). Moreover it had no effect on inhibitory activity of M ␣ 1 -antitrypsin against bovine ␣-chymotrypsin or the ability of M ␣ 1 -antitrypsin to form SDS-stable complexes with trypsin (data not shown). The specificity of the interaction of the 6-mer peptide with Z ␣ 1 -antitrypsin was underscored by the demonstration that it would not anneal to other members of the serpin superfamily that have the same tertiary structure (␣ 1antichymotrypsin, plasminogen activator inhibitor-1, or ␣-antithrombin) when incubated under physiological conditions (data not shown).
The implications of these findings are 2-fold. First, an effective approach to treat the liver disease associated with Z ␣ 1antitrypsin would be to inhibit polymerization of the Z protein and thus prevent the accumulation of the protein within hepatocytes. However, this approach may result in the release of inactive Z ␣ 1 -antitrypsin and would require intravenous replacement therapy with normal ␣ 1 -antitrypsin to replenish plasma levels to prevent emphysema. This makes it essential to specifically block ␤-sheet A of Z ␣ 1 -antitrypsin but not M ␣ 1 -antitrypsin or other proteins with a similar tertiary structure. We have shown that this is achievable in vitro. Second, these findings extend to other diseases that result from polymer formation. Loop-sheet polymerization is also recognized to underlie the deficiency of other members of the serpin superfamily: antithrombin (35), C1-inhibitor (36,37), ␣ 1 -antichymotrypsin (34), and neuroserpin (38), which are associated with thrombosis, angioedema, emphysema, and an inclusion body dementia, respectively. All the mutations that favor these disease processes have been shown, or been predicted, to open ␤-sheet A and facilitate polymer formation (8,38,39). It is likely that peptides or synthetic mimetics can be created that FIG. 3. Top, 7.5% (w/v) acrylamide, 8 M urea gel showing the insertion of the 6-mer peptide into Z but not M ␣ 1 -antitrypsin. M and Z ␣ 1antitrypsin were incubated at 0.5 mg/ml with 100-fold molar excess of peptide at 37°C for 3 days. Each lane contains 4 g of ␣ 1 -antitrypsin. Middle, intrinsic tryptophan fluorescence of M ␣ 1 -antitrypsin and Z ␣ 1 -antitrypsin (0.1 mg/ml) with 100-fold molar excess of the 6-mer peptide at 37°C for 24 h. Bottom, 7.5% (w/v) nondenaturing PAGE to assess the effect of the 6-mer peptide on the polymerization of Z ␣ 1antitrypsin. Z ␣ 1 -antitrypsin (0.5 mg/ml) was incubated with (right side) and without (left side) 100-fold molar excess of the 6-mer peptide at 37°C for 12 days. Each lane contains 10 g of ␣ 1 -antitrypsin.
6-mer Peptide Selectively Anneals to Z ␣ 1 -Antitrypsin 6773 will bind specifically to these mutant serpins, prevent polymer formation, and so attenuate disease. In summary, these findings offer the real prospect of selectively targeting Z ␣ 1 -antitrypsin to prevent polymerization and so ameliorate the associated liver disease. The challenge for the future is to reconcile the requirements of a small molecule for specific inhibition of Z ␣ 1 -antitrypsin with those properties needed for drug design and targeting to the endoplasmic reticulum.