key: cord-0007434-476gla8b authors: Wang, Suming; Han, Jinbo; Wang, Yanfang; Lu, Wuyuan; Chi, Chengwu title: Design of peptide inhibitors for furin based on the C‐terminal fragment of histone H1.2 date: 2008-10-17 journal: Acta Biochim Biophys Sin (Shanghai) DOI: 10.1111/j.1745-7270.2008.00470.x sha: 17e9cff82de9104419fa213e79fce234133f1b23 doc_id: 7434 cord_uid: 476gla8b The mammalian proprotein convertase furin has been found to play an important role in diverse physiological and pathological events, such as the activation of viral glycoproteins and bacterial exotoxins. Small, non‐toxic and highly active, furin inhibitors are considered to be attractive drug candidates for diseases caused by virus and bacteria. In this study, a series of peptide inhibitors were designed and synthesized based on the C‐terminal fragment of histone H1.2, which has an inhibitory effect on furin. Replacing the reactive site of inhibitors with the consensus substrate recognition sequence of furin has been found to increase inhibitory activity greatly. The most potent inhibitor, I(4), with 14 amino acid residues has a K(i) value of 17 nM for furin. Although most of the synthesized peptides were temporary inhibitors, the inhibitor I(5), with nine amino acids, retained its full potency, even after a 3 h incubation period with furin at 37 °C. These inhibitors may potentially lead to the development of anti‐viral and antibacterial drug compounds. The mammalian proprotein convertase furin has been found to play an important role in diverse physiological and pathological events, such as the activation of viral glycoproteins and bacterial exotoxins. Small, non-toxic and highly active, furin inhibitors are considered to be attractive drug candidates for diseases caused by virus and bacteria. In this study, a series of peptide inhibitors were designed and synthesized based on the C-terminal fragment of histone H1.2, which has an inhibitory effect on furin. Replacing the reactive site of inhibitors with the consensus substrate recognition sequence of furin has been found to increase inhibitory activity greatly. The most potent inhibitor , I 4 , with 14 amino acid residues has a K i value of 17 nM for furin. Although most of the synthesized peptides were temporary inhibitors, the inhibitor I 5 , with nine amino acids, retained its full potency, even after a 3 h incubation period with furin at 37 ºC. These inhibitors may potentially lead to the development of anti-viral and antibacterial drug compounds. furin; inhibitor; histone H1.2; peptide synthesis In the secretory pathway, proproteins are limited and cleaved by a family of proteolytic enzymes called proprotein convertases (PCs). PCs are calcium-dependent serine proteases whose catalytic domain shares some sequence similarities with that of the bacterial subtilisin [1] . This cleavage is an important process widely used to regulate the activation of peptides and proteins that play significant roles in various biological events that are implicated in both homeostasis and various diseases [2] . Furin, a mammalian PC, was the first to be identified, and it has been extensively studied. Furin has been shown to have effects on different substrates, such as blood-clotting factors, growth factors, hormone receptors, matrix metalloproteinases and ion channels [3−5] . Bacterial exotoxins, such as diphtheria toxin, anthrax toxin, and viral envelope glycoproteins of HIV and the SARS virus, are also processed by furin [2,6−9] . Furthermore, many studies have indicated that increased furin activity is closely related to the malignancy of various tumors [10] . Thus, furin is an attractive target for therapeutic drugs. Many furin inhibitors have been studied, including small molecular PC inhibitors and protein-based inhibitors [11] . Each small molecular PC inhibitor is categorized as a peptide inhibitor, peptidomimetic inhibitor or non-peptide inhibitor [12−14] , while protein-based inhibitors include polypeptides derived from the prodomain of PC [15−17] , bioengineered proteins [18−21], and some endogenous proteins [22−26]. Among them, α 1 -antitrypsin Portland and polyarginine have been used to prevent the activation of bacterial toxin, the processing of envelope glycoprotein in viral replication and the metastasis of cancer [10, 27, 28] . By comparison, small peptide inhibitors are more attractive furin inhibitors, since they are more potent but have low toxicity. Many peptide inhibitors have been investigated; for example, some of them were designed based on the sequence of PC prodomain or PC partner proteins and the lysine active domain of the mung bean trypsin inhibitor (MBTI) [29, 30] . Other peptide inhibitors include the consensus substrate recognition sequence of furin, the C-terminals of which are modified by an active group (−CMK, −CHO, =NOH or −CH=NNHCONH 2 ) [17, 31] . Meanwhile, the stability of small peptide inhibitors has been improved by cyclic peptide inhibitors, such as chymotrypsin inhibitor 2 from the barley serine proteinase inhibitor-2 [32] , sunflower trypsin inhibitor-1 [33] , and the Lys fragment of mung bean trypsin inhibitor [34] . In our previous study, three highly active inhibitors against furin were purified from porcine liver and identified as C-terminal truncated fragments with different sizes of histone H1.2. The inhibitory activities of these fragments were greater than that of the full-length histone H1.2, and it has been suggested that inhibitory activity against furin relies upon the C-terminal domain [35] . In the same study, a synthesized 36 amino acid peptide of the C-terminal fragment retained inhibitory activity against furin; however, this 36 amino acid peptide with a K i value of 5.1×10 −7 M is too long for wide application and lacks the ability to inhibit the activity of furin efficiently. In this study, we used this small amino acid peptide as a template to design a shorter but more potent and stable furin inhibitor. Seven peptide inhibitors derived from the 36 amino acid peptide were synthesized, and their potency and stability against furin were characterized. Of them, we found a nonapeptide with high stability and a K i value of 2.7×10 −8 M, which may serve as a leading compound for the development of therapeutic drugs for furin-mediated diseases, such as HIV. The fluorogenic substrate pyrArg-Thr-Lys-Arg-7-amino-4-methylcoumarin (MCA) was purchased from Bachem Bioscience (San Diego, USA). All Fmoc amino acids and Fmoc resins were obtained from Applied Biosystems (Foster City, USA). All the linear peptides were synthesized using the standard Fmoc chemistry. The protected peptide was independently grown on a Wang-resin, using the HBTU (O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate)/ HOBT (N-Hydroxybenzotriazole) amino acid activation method. Solid phase peptide synthesis was performed on a 433A peptide synthesizer (Applied Biosystems). The protected amino acids were Fmoc-Ser (tBu), Fmoc-Lys (tBoc), Fmoc-Thr (tBu), Fmoc-Arg (Pbf) and Fmoc-Asp (otBu). The resin was incubated in TFA containing 5% pcresol and a few drops of triethylsilane and thioanisole for 1.5 h at room temperature for cleavage. The crude peptides were precipitated by cool anhydrous diethyl ether and purified by reverse phase HPLC. All the cyclic peptides were synthesized through their corresponding linear peptide thioester precursors by intramolecular native chemical ligation [36] . The linear peptide thioester precursors were prepared using the Boc solid phase method with in situ neutralization [37] . Typically, S-trityl-β-mercaptopropionic acid was preactivated with HBTU/DIEA (N,N-diisopropylethylamine) and introduced to Leu-Pam resin. After deprotection with neat TFA, the first amino acid from the C-terminal was coupled to the resin with a free thiol group using HBTU/DIEA as coupling reagent. After the chain elongation was finished, all the protection groups were removed and peptides were cleaved from resin by HF (Hydrogen Fluoride)/p-cresol (90:10) at 0 ºC. The peptides were precipitated and washed with cold diethyl ether and purified by reverse phase HPLC. The cyclization of the linear peptides was performed on 0.25 M phosphate buffer containing 6 M guanidine hydrochloride, pH 7.4, overnight. The reaction was monitored by RP-HPLC and the cyclic product was purified by HPLC and identified by electrospray ionization-mass spectrometry. The synthetic peptides were desalted on a Sephadex G15 column (Amersham Biosciences, Piscataway, USA), washed with buffer A (0.1% TFA in water), lyophilized, dissolved in buffer A and then purified on a Zorbax C18 column (9.4×250 mm) (Agilent, Palo Alto, USA) by HPLC. The peptides were equilibrated with buffer A at a flow rate of 2 ml/min and eluted in a gradient of 0% buffer B (0.1% TFA in acetonitrile) for 5 min and 0%−30% buffer B for 25 min. The molecular masses of all synthetic peptides were determined with an ABI API2000 Q-trap mass spectroscope (Applied Biosystems). The fluorogenic MCA substrate (pyrArg-Thr-Lys-Arg-MCA) was used for the furin activity assay. To determine the inhibitory activity, different amounts of the inhibitors were first incubated with a fixed amount of enzyme (1.7 µM) at 37 ºC for 3 min in a final volume of 1 ml HEPES buffer (100 mM HEPES, pH7.5, 1 mM CaCl 2 , 0.5% Triton X-100, and 1 mM β-mercaptoethanol), and the residual enzyme activity was then measured with an F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). For stability assay, the inhibitors were incubated with furin for different periods (0, 30, 60, 90, 120, 150 and 180 min), and then the inhibitory activity was measured. Enzymes incubated without inhibitors were measured as control. The excitation and emission wavelengths were 370 nm (slit width, 10 nm) and 460 nm (slit width, 10 nm), respectively. The K i values of inhibitors against furin were determined by Dixon's plot (1/V against I) using two different concentrations of substrate (1.0 µM, 1.5 µM). The substrate concentration for stability assay was 1.0 µM. Data from three measurements were averaged and graphically analyzed with an equation to obtain the equilibrium inhibition constant K i . HPLC was used to study the stability of peptide inhibitors; 20 µg peptide inhibitors with or without incubation with furin (1.7 µM) in HEPES buffer at 37 ºC for 3 h were placed into 300 µl buffer A and centrifugated. The supernate was then loaded to a PepMap C18 column (4.6×250 mm) (Applied Biosystems). I 5 was equilibrated with buffer A at a flow rate of 0.8 ml/min and eluted in a gradient of 0% buffer B for 5 min and 0%−50% buffer B for 25 min. I 4 was equilibrated with 10% buffer B at a flow rate of 0.8 ml/min and eluted in a gradient of 10% buffer B for 5 min and 10%−50% buffer B for 20 min. The molecular masses of all peaks were determined with an ABI API2000 Q-trap mass spectroscope. As reported in our previous work, a peptide, bearing a K i value of 5.1×10 −7 M, with 36 amino acid residues (PAAATVTKKVAKSPKKAKAAKPKKAAKSAAKAVKPK) derived from the C-terminal fragment of histone H1.2 possesses a potent inhibitory activity against furin [35] . Based on this 36 amino acid peptide template (termed I 1 ), a series of shorter peptides was designed and synthesized (Table 1) (Fig, 1) . The first step in optimization was to shorten the 36 amino acid peptide from both the N-and C-terminals. The resulting peptide, termed I 2 , with 14 amino acid residues exhibited a 10-fold lower inhibitory activity than I 1 . To improve the potency of I 2 , the second step introduced the consensus substrate recognition sequence of furin into the reactive site. Furin recognizes a specific RXRAKR↓ site. The peptide I 3 was then designed by replacing the P 2 residue with Lys and P 1 , P 4 and P 6 residues with Arg. These replacements led to a decrease in the K i value of I 3 by approximately 5.8×10 −8 M, suggesting that the consensus substrate sequence of furin is essential for the inhibitor. When two alanine residues at the P 1 ' and P 2 ' positions of I 3 were replaced with Asp (P 1 ') and Leu (P 2 '), respectively, to achieve I 4 , the K i value for furin further decreased three-fold, indicating that a negatively charged residue at the P 1 ' site is favorable. Though the 14 amino acid peptides I 3 and I 4 have appropriate inhibitory activities, their relatively large sizes restrain their application. The third step was to remove the N-terminal Thr-Lys-Lys-Val and C-terminal Ala residues flanking the reactive site of the inhibitor I 3 to obtain I 5 . The truncation at both termini had no apparent impact on the inhibitory activity of I 5 , resulting in a nonapeptide inhibitor with a K i value of 2.7×10 −8 M. To protect the peptides from possible in vivo degradation by exopeptidase, three cyclic peptide inhibitors with 10, 12 and 14 amino acid residues were also synthesized in the thioester formation, between the N-terminal cysteine and the Cterminal Leu. Unexpectedly, the inhibitory potencies of the peptides I 6 , I 7 and I 8 decreased by 160, 35 and 5 folds, respectively, compared to I 4 ( Table 2 ). Stability assays were carried out to measure the stability of the inhibitors over several hours. To measure the stability of these inhibitors, the IC 50 concentrations of the inhibitors were used based on their K i values. The substrate concentration for the stability assay was 1.0 µM. Enzymes incubated without inhibitors were used as a control to confirm that furin activity would not change during incubation in buffer at 37 ºC. The initial inhibitory activity of each inhibitor was marked as 100%; their inhibitory activities at indicated time points were then compared with initial activity and normalized as percentage values. Inhibitory activities were measured three times. When the synthesized peptides (I 1 −I 8 ) were incubated with furin for an indicated time, their inhibitory activities gradually decreased in a time-dependent manner, with the exception of I 5 . The most stable inhibitor, I 5 , retained 100% potency against furin, even after a 3 h incubation period. In contrast, the inhibitor I 6 was the least stable with a 50% activity loss during the same time period. Among inhibitors I 2 , I 3 and I 4 , the activity of the one with the highest inhibitory activity (K i -I 4