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The Absolute Structural Requirement for a Proline in the P3′-position of Bowman-Birk Protease Inhibitors Is Surmounted in the Minimized SFTI-1 Scaffold

2006; Elsevier BV; Volume: 281; Issue: 33 Linguagem: Inglês

10.1074/jbc.m601426200

1083-351X

Norelle L. Daly, Yi-Kuang Chen, Fiona Foley, Paramjit S. Bansal, R Bharathi, Richard J. Clark, Christian P. Sommerhoff, David J. Craik,

Carbohydrate Chemistry and Synthesis

SFTI-1 is a small cyclic peptide from sunflower seeds that is one of the most potent trypsin inhibitors of any naturally occurring peptide and is related to the Bowman-Birk family of inhibitors (BBIs). BBIs are involved in the defense mechanisms of plants and also have potential as cancer chemopreventive agents. At only 14 amino acids in size, SFTI-1 is thought to be a highly optimized scaffold of the BBI active site region, and thus it is of interest to examine its important structural and functional features. In this study, a suite of 12 alanine mutants of SFTI-1 has been synthesized, and their structures and activities have been determined. SFTI-1 incorporates a binding loop that is clasped together with a disulfide bond and a secondary peptide loop making up the circular backbone. We show here that the secondary loop stabilizes the binding loop to the consequences of sequence variations. In particular, full-length BBIs have a conserved cis-proline that has been shown previously to be required for well defined structure and potent activity, but we show here that the SFTI-1 scaffold can accommodate mutation of this residue and still have a well defined native-like conformation and nanomolar activity in inhibiting trypsin. Among the Ala mutants, the most significant structural perturbation occurred when Asp14 was mutated, and it appears that this residue is important in stabilizing the trans peptide bond preceding Pro13 and is thus a key residue in maintaining the highly constrained structure of SFTI-1. This aspartic acid residue is thought to be involved in the cyclization mechanism associated with excision of SFTI-1 from its 58-amino acid precursor. Overall, this mutational analysis of SFTI-1 clearly defines the optimized nature of the SFTI-1 scaffold and demonstrates the importance of the secondary loop in maintaining the active conformation of the binding loop. SFTI-1 is a small cyclic peptide from sunflower seeds that is one of the most potent trypsin inhibitors of any naturally occurring peptide and is related to the Bowman-Birk family of inhibitors (BBIs). BBIs are involved in the defense mechanisms of plants and also have potential as cancer chemopreventive agents. At only 14 amino acids in size, SFTI-1 is thought to be a highly optimized scaffold of the BBI active site region, and thus it is of interest to examine its important structural and functional features. In this study, a suite of 12 alanine mutants of SFTI-1 has been synthesized, and their structures and activities have been determined. SFTI-1 incorporates a binding loop that is clasped together with a disulfide bond and a secondary peptide loop making up the circular backbone. We show here that the secondary loop stabilizes the binding loop to the consequences of sequence variations. In particular, full-length BBIs have a conserved cis-proline that has been shown previously to be required for well defined structure and potent activity, but we show here that the SFTI-1 scaffold can accommodate mutation of this residue and still have a well defined native-like conformation and nanomolar activity in inhibiting trypsin. Among the Ala mutants, the most significant structural perturbation occurred when Asp14 was mutated, and it appears that this residue is important in stabilizing the trans peptide bond preceding Pro13 and is thus a key residue in maintaining the highly constrained structure of SFTI-1. This aspartic acid residue is thought to be involved in the cyclization mechanism associated with excision of SFTI-1 from its 58-amino acid precursor. Overall, this mutational analysis of SFTI-1 clearly defines the optimized nature of the SFTI-1 scaffold and demonstrates the importance of the secondary loop in maintaining the active conformation of the binding loop. Protease inhibitors are of significant interest because they play key roles in an extremely wide range of physiological function, including peptide hormone release, blood coagulation, and complement fixation. They have also been implicated in the treatment of various diseases, including some cancers and inflammatory processes (1Grasberger B.L. Clore G.M. Gronenborn A.M. Structure (Lond.). 1994; 2: 669-678Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Bowman-Birk inhibitors (BBIs), 3The abbreviations used are: BBI, Bowman-Birk inhibitor; RP-HPLC, reverse phase-high performance liquid chromatography; SFTI, sunflower trypsin inhibitor; TCEP, triscarboxylethylphosphine; TOCSY, total correlation spectroscopy; Boc, t-butoxycarbonyl. 3The abbreviations used are: BBI, Bowman-Birk inhibitor; RP-HPLC, reverse phase-high performance liquid chromatography; SFTI, sunflower trypsin inhibitor; TCEP, triscarboxylethylphosphine; TOCSY, total correlation spectroscopy; Boc, t-butoxycarbonyl. one of at least 18 different families of serine protease inhibitors, are involved in plant defense and have been implicated as cancer chemopreventive agents (2Kennedy A.R. Am. J. Clin. Nutr. 1997; 68: S1406-S1412Crossref Scopus (252) Google Scholar). The focus of this paper is SFTI-1 (sunflower trypsin inhibitor 1), a 14-residue cyclic peptide isolated from sunflower seeds that is related to the Bowman-Birk inhibitors and is one of the most potent inhibitors of trypsin of any naturally occurring peptide (3Luckett S. Garcia R.S. Barker J.J. Konarev A.V. Shewry P.R. Clarke A.R. Brady R.L. J. Mol. Biol. 1999; 290: 525-533Crossref PubMed Scopus (317) Google Scholar). SFTI-1 forms a tightly folded scaffold, either when complexed with trypsin or free in solution (3Luckett S. Garcia R.S. Barker J.J. Konarev A.V. Shewry P.R. Clarke A.R. Brady R.L. J. Mol. Biol. 1999; 290: 525-533Crossref PubMed Scopus (317) Google Scholar, 4Korsinczky M.L. Schirra H.J. Rosengren K.J. West J. Condie B.A. Otvos L. Anderson M.A. Craik D.J. J. Mol. Biol. 2001; 311: 579-591Crossref PubMed Scopus (180) Google Scholar). Its compact structure (shown in Fig. 1) and high potency have led to suggestions that it may serve as a scaffold for the design of novel peptide-based drug leads (5Korsinczky M.L. Schirra H.J. Craik D.J. Curr. Protein Pept. Sci. 2004; 5: 351-364Crossref PubMed Scopus (74) Google Scholar).The structure of SFTI-1 consists of two β-strands connected at each end by turns and is braced by a single disulfide bond that creates two distinct loops (3Luckett S. Garcia R.S. Barker J.J. Konarev A.V. Shewry P.R. Clarke A.R. Brady R.L. J. Mol. Biol. 1999; 290: 525-533Crossref PubMed Scopus (317) Google Scholar, 4Korsinczky M.L. Schirra H.J. Rosengren K.J. West J. Condie B.A. Otvos L. Anderson M.A. Craik D.J. J. Mol. Biol. 2001; 311: 579-591Crossref PubMed Scopus (180) Google Scholar). One loop, known as the binding loop, contains the reactive site sequence Lys5–Ser6, although the "secondary" loop contains a β-hairpin turn that essentially cyclizes the binding loop. SFTI-1 inhibits trypsin with a Ki of 0.1 nm (3Luckett S. Garcia R.S. Barker J.J. Konarev A.V. Shewry P.R. Clarke A.R. Brady R.L. J. Mol. Biol. 1999; 290: 525-533Crossref PubMed Scopus (317) Google Scholar) and inhibits cathepsin G with a comparable Ki. SFTI-1 is highly selective as it is 74-fold less inhibitory for chymotrypsin, and 3 orders of magnitude less inhibitory for elastase and thrombin. It has no detectable inhibitory activity against factor Xa (3Luckett S. Garcia R.S. Barker J.J. Konarev A.V. Shewry P.R. Clarke A.R. Brady R.L. J. Mol. Biol. 1999; 290: 525-533Crossref PubMed Scopus (317) Google Scholar).Although SFTI-1 is only 14 residues in size, it has remarkable similarity to the active site sequence of the Bowman-Birk trypsin inhibitors that are typically 60–90 amino acids in length. Three key features have been identified that are conserved between BBIs and SFTI-1, namely covalent cyclization of a hairpin loop via a disulfide bridge, a cis-Pro at the P3′-site (using the protease active site nomenclature of Schechter and Berger (6Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4727) Google Scholar)), and an extensive network of hydrogen bonds. The active site sequences of selected BBIs are given in Fig. 2 to highlight some of these features. A range of synthetic peptides based on the BBIs have been synthesized and their activities determined, as reviewed by Korsinczky et al. (7Korsinczky M.L. Clark R.J. Craik D.J. Biochemistry. 2005; 44: 1145-1153Crossref PubMed Scopus (43) Google Scholar). Essentially all of the important features identified from these studies appear to have been incorporated naturally into the SFTI framework, demonstrating that native SFTI is a highly optimized trypsin inhibitory scaffold.FIGURE 2A comparison of the active site sequences of Bowman-Birk inhibitors with SFTI-1. The conserved cis-proline residue at the P3′-position is highlighted in italics, and the two cysteine residues that are linked in a disulfide bond to form the binding loop are highlighted in boldface. The standard Schechter and Berger nomenclature (6Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4727) Google Scholar) for protease active site positions is illustrated below the BBI sequences.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The precursor protein of SFTI-1 was recently discovered, and the sequence is given in Fig. 1. Interestingly, the sequence of the precursor does not show any similarities with the Bowman-Birk inhibitors, apart from the mature peptide domain, posing questions about the evolution of SFTI-1 (8Mulvenna J.P. Foley F.M. Craik D.J. J. Biol. Chem. 2005; 280: 32245-32253Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The mechanism of cyclization has yet to be elucidated, and the presence of a GR dipeptide motif at both ends of the mature sequence leaves a degree of ambiguity as to the cleavage sites (8Mulvenna J.P. Foley F.M. Craik D.J. J. Biol. Chem. 2005; 280: 32245-32253Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), although it is likely that cyclization occurs between an Asp and a Gly residue (see Fig. 1), which opens up the possibility of a role for an asparaginyl endoprotease in the process (8Mulvenna J.P. Foley F.M. Craik D.J. J. Biol. Chem. 2005; 280: 32245-32253Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The mechanism of cyclization of the largest family of backbone cyclic proteins, the plant cyclotides (9Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (631) Google Scholar), has also been suggested to involve an Asp/Asn-Gly and may also involve an asparaginyl endoprotease, although this has yet to be experimentally verified (10Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (398) Google Scholar). Circular proteins have also been reported in bacteria and mammals, and the mechanism of cyclization is similarly unknown (11Trabi M. Craik D.J. Trends Biochem. Sci. 2002; 27: 132-138Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar).As well as having potential applications as a protein engineering scaffold, SFTI-1 also displays potent inhibitory activity against matriptase, an enzyme implicated in prostate cancer, suggesting that it may also have direct therapeutic applications (12Lin C.Y. Anders J. Johnson M. Sang Q.A. Dickson R.B. J. Biol. Chem. 1999; 274: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 13Friedrich R. Fuentes-Prior P. Ong E. Coombs G. Hunter M. Oehler R. Pierson D. Gonzalez R. Huber R. Bode W. Madison E.L. J. Biol. Chem. 2002; 277: 2160-2168Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 14Long Y.Q. Lee S.L. Lin C.Y. Enyedy I.J. Wang S. Li P. Dickson R.B. Roller P.P. Bioorg. Med. Chem. Lett. 2001; 11: 2515-2519Crossref PubMed Scopus (78) Google Scholar). Matriptase was first isolated as a novel proteinase expressed by human breast cancer cells and is also highly expressed in prostate, breast, and colorectal cancers in vitro and in vivo (12Lin C.Y. Anders J. Johnson M. Sang Q.A. Dickson R.B. J. Biol. Chem. 1999; 274: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 13Friedrich R. Fuentes-Prior P. Ong E. Coombs G. Hunter M. Oehler R. Pierson D. Gonzalez R. Huber R. Bode W. Madison E.L. J. Biol. Chem. 2002; 277: 2160-2168Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Inhibition of matriptase suppresses both primary tumor growth and metastasis in a rat model of prostate cancer.Understanding the structure-activity relationships of SFTI-1 will facilitate potential protein engineering and therapeutic applications of this peptide. Mutagenesis studies involving the systematic replacement of individual residues also have the potential to provide insights into their role in the cyclization mechanism. In this study we have synthesized a complete suite of alanine mutants of SFTI-1 and characterized them structurally and functionally. Lys5 is the only residue that resulted in a significant loss of trypsin inhibitory activity, and most surprisingly, the cis-Pro that is highly conserved across the Bowman-Birk inhibitors is not critical for activity.MATERIALS AND METHODSPeptide Synthesis—Boc-based solid phase peptide synthesis was carried out using standard protocols. Peptides were assembled using a thioester linker assembled on resin allowing subsequent cyclization by a thiazip mechanism (15Tam J.P. Lu Y.-A. Tetrahedron Lett. 1997; 38: 5599-5602Crossref Scopus (83) Google Scholar). Hydrogen fluoride cleavage was conducted on the deprotected resins using standard protocols (0 °C, 90 min, 90% HF, 8% p-cresol, 2% p-thiocresol). Crude cleavage products were purified by RP C18 HPLC (1%/minute gradient of 90% acetonitrile, 10% water, 0.05% trifluoroacetic acid against 100% water, 0.05% trifluoroacetic acid) to give linear, reduced peptides. Peptides were cyclized and oxidized in 0.1 m ammonium bicarbonate at pH 8 overnight and purified as above. Purity of fractions was assessed using electrospray ionization-mass spectrometry and analytical HPLC using a 2%/minute gradient of the same solvents used for previous steps.NMR Spectroscopy—Samples for 1H NMR measurements contained ∼1 mm peptide in 90% H2O, 10% D2O (v/v) at pH ∼5. D2O (99.9 and 99.99%) was obtained from Cambridge Isotope Laboratories, Woburn, MA. Spectra were recorded at 290 K on a Bruker Avance-500 or Avance-600 spectrometer equipped with a shielded gradient unit. Two-dimensional NMR spectra were recorded in phase-sensitive mode using time-proportional phase incrementation for quadrature detection in the t1 dimension (16Marion D. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 113: 967-974Crossref PubMed Scopus (3516) Google Scholar). The two-dimensional experiments consisted of a TOCSY (17Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3101) Google Scholar) using an MLEV-17 spin lock sequence (18Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar) with a mixing time of 80 ms and nuclear Overhauser effect spectroscopy (19Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4817) Google Scholar) with mixing times of 100–250 ms. Solvent suppression was achieved using a modified WATERGATE sequence (20Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3500) Google Scholar). Spectra were acquired over 6024 Hz with 4096 complex data points in F2 and 512 increments in the F1 dimension. Spectra were processed on a Silicon Graphics Indigo work station using XWINNMR (Bruker) software. The t1 dimension was zero-filled to 1024 real data points, and 90° phase-shifted sine bell window functions were applied prior to Fourier transformation.Trypsin Inhibition—The concentrations of the inhibitory active peptides and the equilibrium dissociation constants Ki were determined with trypsin. Bovine pancreatic trypsin (N-p-tosyl-l-phenylalanine chloromethyl ketone-treated; Sigma) was standardized by burst titration with p-nitrophenyl p′-guanidinobenzoate (21Chase Jr., T. Shaw E. Perlmann G.E. Lorand L. Methods in Enzymology. Academic Press, New York1970: 20-27Google Scholar). Trypsin (25 nm) was then incubated with serial dilutions of the peptides in 50 mm HEPES, 150 mm NaCl, 0.01% Triton X-100, 0.01% sodium azide, pH 7.4, for 5 min at room temperature. The residual activity was quantified by following the hydrolysis of the substrate carbobenzoxy-l-arginine-7-amino-4-methylcoumarin (125 μm; Sigma) in an HTS 7000 BioAssay Reader (PerkinElmer Life Sciences). The concentrations of the active peptides were calculated by assuming a 1:1 interaction between inhibitor and trypsin; the mutant K5A could not be titrated because of its considerably lower affinity, and HPLC and amino acid analyses were used for quantification. Subsequently similar experiments were performed at a lower enzyme concentration to determine the equilibrium dissociation constant for the complex (22Bieth J.G. Bull. Eur. Physiopathol. Respir. 1980; 16: 183-197PubMed Google Scholar). Thus, the peptides were incubated with trypsin (0.01 nm), and the residual activity was quantified using the substrate N-p-tosyl-glycine-proline-arginine-7-amido-4-methylcoumarin (5 μm; Sigma). The Ki values were calculated by fitting the steady state velocities to the equation for tight binding inhibitors (23Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (712) Google Scholar) using nonlinear regression analysis with the software ProFit (Quantum Soft, Uetikon am See, Switzerland). Mean values ± S.D. of at least four experiments are reported.RESULTSEach non-cysteine residue in SFTI-1 was replaced individually with an alanine residue in a suite of peptides synthesized using Boc-based solid phase peptide synthesis. The native peptide was also synthesized for comparison. For convenience the peptides are referred to here by reference to the mutation, e.g. G1A for [1-Ala]SFTI-1. For a cyclic sequence of n residues, there are in principle n choices of linear precursors for the synthesis. For the mutants of SFTI-1, the peptide sequences were assembled with the flanking residues being a C-terminal thioester and an N-terminal cysteine residue to facilitate cyclization via an adaptation of native peptide ligation chemistry (24Tam J.P. Lu Y.-A. Yu Q. J. Am. Chem. Soc. 1999; 121: 4316-4324Crossref Scopus (125) Google Scholar, 25Daly N.L. Love S. Alewood P.F. Craik D.J. Biochemistry. 1999; 38: 10606-10614Crossref PubMed Scopus (190) Google Scholar). Fig. 3 outlines the chemical strategy involved. Briefly, nucleophilic attack of the N-terminal Cys on the thioester linker at the C terminus results in a cyclic thiolactam that subsequently rearranges via an S- to N-acyl transfer to produce a native peptide bond. As there are two cysteine residues in SFTI-1, there are two possible ligation points. The preferred ligation point was chosen to be between Cys3 and Arg2, or in the case of R2A between Cys3 and Ala2.FIGURE 3A schematic representation of the strategy for synthesizing the SFTI-1 analogues. A thioester linker is attached to the solid support (resin) and the peptide chain assembled using Boc chemistry. The peptide with the linker attached is cleaved from the resin using HF and a single step reaction used for cyclizing the backbone and oxidizing the disulfide bond. The binding and secondary loops of the SFTI-1 sequence are shown in lighter and darker shading, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Formation of the cyclic backbone and disulfide bond was generally performed in a single step in 0.1 m ammonium bicarbonate at pH 8. For D14A the cyclization reaction did not proceed efficiently and a two-step approach was required. The cyclization reaction was performed in the presence of TCEP, and following purification of the cyclic product, the peptide was oxidized in 0.1 m ammonium bicarbonate. The TCEP was used to keep the two cysteine residues in a reduced state as this appears to facilitate the cyclization reaction. All peptides were purified using preparative reverse phase HPLC (RP-HPLC) and characterized using analytical scale RP-HPLC and mass spectrometry.Sufficient quantities of the 12 SFTI-1 Ala mutants were isolated and purified for structural analysis with NMR spectroscopy and functional analysis of the trypsin inhibitory activity. The NMR spectra were recorded in aqueous solution at 290 K, and NMR spectral assignments were made using established techniques (26Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley Interscience, New York1986: 130-161Google Scholar). Chemical shifts, in particular the αH shifts, are extremely sensitive to structural changes and thus offered a convenient method of assessing structural changes that may have occurred upon alanine substitution.Under the conditions examined, one predominant conformation was present in solution for the native peptide. Analysis of TOCSY spectra between pH 2.8 and 5.8 indicated that Asp14 hasapKa of ∼4, and there were no significant conformational changes over this pH range (as assessed via a lack of αH chemical shift changes). The SFTI-1 mutants also displayed only one predominant conformation, with the exception of D14A, for which two isomers were present. The NMR spectra of the SFTI-1 mutants were recorded at both pH 4 and 5, which gave essentially identical spectra, again showing a lack of pH-induced conformational changes. A comparison of the secondary shifts of native SFTI with the mutants (at pH 5) is shown in Fig. 4. The majority of the mutants display chemical shifts very similar to the native peptide, indicating that no major structural changes occur as a result of the mutations. However, I7A, P8A, and P9A display differences for certain residues that indicate local structural perturbations. I7A differs at residue 7, but this is likely to result from a local effect of the alanine substitution. P8A has changes for residues 6–8, whereas P9A differs at residue 8. T4A also displays minor differences from the native peptide.FIGURE 4Secondary shift analysis of SFTI-1 alanine mutants compared with native SFTI-1. The Hα secondary shifts were calculated by subtracting the random coil shift (40Wishart D.S. Bigam C.G. Holm A. Hodges R.S. Sykes B.D. J. Biomol. NMR. 1995; 5: 67-81Crossref PubMed Scopus (1408) Google Scholar) from the Hα shift.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A comparison of the TOCSY spectrum of D14A with native SFTI-1 is given in Fig. 5. It is clear that one major conformer is present in native SFTI-1, whereas D14A has two sets of spin systems from two distinct conformations. For some residues significant differences are observed for the amide chemical shifts of the two conformers, and in some cases no differences, or only minor differences, are evident. Assignment of both conformers showed that Pro13 is present in a trans conformation in one of the conformers and a cis conformation in the other. The discrimination of the trans and cis forms was based on detection of dαi-1δ and dαα nuclear Overhauser effects, respectively, for the X-Pro peptide bonds. The secondary shifts (i.e. the differences between observed and random coil αH chemical shifts) of the non-native cis conformation of D14A differ from the native peptide at residues 12–14, in contrast to the trans conformation, which is very similar to the native peptide (Fig. 4). Prolines 8 and 9 are present as cis and trans forms, respectively, in both conformers. Clearly, mutating Asp14 to an alanine residue has a significant effect on the stability of the turn region involving residues 12–14 and 1. (Note that residues 14 and 1 are sequentially adjacent in the cyclic peptide.)FIGURE 5Comparison of the TOCSY spectra of native SFTI-1 and the D14A mutant. The spin systems are labeled with the single letter amino acid code and residue number. SFTI-1 exists as a single well defined conformation, whereas in D14A the Pro13 is present in both the cis and trans forms. The cis conformer of D14A is highlighted with boxes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although the geometry of Pro13 is affected in D14A, the native trans conformation is maintained in all other Ala mutants containing this Asp residue. When present, Pro8 and Pro9 also maintain their native geometries (i.e. cis for Pro8 and trans for Pro9) in the Ala mutants.The ability of SFTI-1 and the analogues to inhibit trypsin was compared by determining the equilibrium dissociation constants Ki of the complexes (Table 1). The measured Ki value of SFTI-1 (0.03 nm) is in good agreement with that reported for SFTI-1 isolated from sunflower seeds (0.1 nm (3Luckett S. Garcia R.S. Barker J.J. Konarev A.V. Shewry P.R. Clarke A.R. Brady R.L. J. Mol. Biol. 1999; 290: 525-533Crossref PubMed Scopus (317) Google Scholar)). Not surprisingly, the P1 reactive site mutant K5A is a considerably weaker inhibitor (Ki 190 nm). All of the mutants inhibit trypsin at low nanomolar concentration, the Ki values differing less than 50-fold from that of SFTI-1.TABLE 1Equilibrium dissociation constant Ki for the inhibition of bovine trypsin by SFTI-1 and its alanine mutantsPeptideSequenceKiaValues are mean ± S.D., n ≥ 4.nmSFTI-1-GRCTKSIPPICFPD-0.03 ± 0.01G1A-ARCTKSIPPICFPD-0.08 ± 0.02R2A-GACTKSIPPICFPD-0.47 ± 0.05T4A-GRCAKSIPPICFPD-0.26 ± 0.05K5A-GRCTASIPPICFPD-190 ± 39S6A-GRCTKAIPPICFPD-0.35 ± 0.04I7A-GRCTKSAPPICFPD-0.84 ± 0.25P8A-GRCTKSIAPICFPD-1.49 ± 0.19P9A-GRCTKSIPAICFPD-0.08 ± 0.04I10A-GRCTKSIPPACFPD-0.11 ± 0.02F12A-GRCTKSIPPICAPD-0.53 ± 0.06P13A-GRCTKSIPPICFAD-0.05 ± 0.01D14A-GRCTKSIPPICFPA-0.24 ± 0.05a Values are mean ± S.D., n ≥ 4. Open table in a new tab DISCUSSIONSFTI-1 is one of the most potent BBIs known and has exciting potential therapeutic applications based on its inhibitory activity against matriptase, an enzyme implicated in prostate cancer (14Long Y.Q. Lee S.L. Lin C.Y. Enyedy I.J. Wang S. Li P. Dickson R.B. Roller P.P. Bioorg. Med. Chem. Lett. 2001; 11: 2515-2519Crossref PubMed Scopus (78) Google Scholar). Furthermore, because of its tightly constrained structure, it has been suggested that it may serve as a scaffold for peptide-based drug development (5Korsinczky M.L. Schirra H.J. Craik D.J. Curr. Protein Pept. Sci. 2004; 5: 351-364Crossref PubMed Scopus (74) Google Scholar, 27Brauer A.B. Domingo G.J. Cooke R.M. Matthews S.J. Leatherbarrow R.J. Biochemistry. 2002; 41: 10608-10615Crossref PubMed Scopus (49) Google Scholar, 28Craik D.J. Simonsen S. Daly N.L. Curr. Opin. Drug Discovery Dev. 2002; 5: 251-260PubMed Google Scholar, 29Craik D.J. Cemazar M. Daly N.L. Curr. Opin. Drug Discovery Dev. 2006; 9: 251-260PubMed Google Scholar). In this study, a suite of alanine mutants of SFTI-1 has been synthesized to facilitate a determination of structure-activity relationships, as such an analysis is critical if the therapeutic potential of the SFTI-1 framework is to be realized.We employed Boc chemistry and a thioester-based native chemical ligation approach to cyclize the SFTI-1 mutants. Such an approach has been successfully applied to another family of macrocyclic plant proteins, namely the cyclotides, which contain three disulfide bonds and hence are intrinsically more complex (9Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (631) Google Scholar). The method worked efficiently for the suite of SFTI-1 mutants, and in all but one case only a single step reaction was required. The D14A mutant required a two-step procedure to facilitate cyclization and oxidation. Our approach represents a novel and efficient method of synthesizing SFTI-1 analogues. In the past, several methods have been used to synthesize SFTI-1 and analogues, including an on-resin cyclization approach (4Korsinczky M.L. Schirra H.J. Rosengren K.J. West J. Condie B.A. Otvos L. Anderson M.A. Craik D.J. J. Mol. Biol. 2001; 311: 579-591Crossref PubMed Scopus (180) Google Scholar) and Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry followed by cyclization in solution (30Zablotna E. Kazmierczak K. Jaskiewicz A. Stawikowski M. Kupryszewski G. Rolka K. Biochem. Biophys. Res. Commun. 2002; 292: 855-859Crossref PubMed Scopus (62) Google Scholar). All have been successful, thus emphasizing the synthetic accessibility of the framework, but the thioester approach has the advantage of a single step cyclization and oxidation reaction.The similarity of the αH chemical shifts between the mutants and native SFTI-1 suggests that no major structural changes occur in the majority of the mutants. The most significant differences from the native peptide are for mutants I7A, P8A, and P9A, indicating that the Ile7–Pro9 region of the sequence is important for structural integrity. These residues are in a turn region of the binding loop adjacent to the active site Lys5–Ser6 peptide bond, and include the cis-Pro residue conserved in the P3′-position of all BBIs. The other significant mutation from a structural perspective occurs in the sec