The absolute structural requirement for a proline in the P3'-position of Bowman-Birk protease inhibitors is surmounted in the minimized SFTI-1 scaffold.

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.

Synthesis and bio-assay of RCM-derived Bowman-Birk inhibitor analogues.

Bowman-Birk inhibitor analogues containing 2, 3 and 4-carbon analogues of the natural disulfide were synthesised via solid phase microwave-assisted RCM and found to have K(i) values against chymotrypsin in the low to sub-micromolar range, the best replacement for the disulfide arising from the linkage by RCM of two l-homoallylglycine residues.

A conserved cis peptide bond is necessary for the activity of Bowman-Birk inhibitor protein.

The Bowman-Birk inhibitor (BBI) family of protease inhibitors has an inhibitory region comprising a disulfide-linked nine-residue loop that adopts the characteristic canonical motif found in many serine protease inhibitors. A unique feature of the BBI loop is the presence of a cis peptide bond at the edge of the inhibitory loop. BBI-related protein fragments that encapsulate this loop retain the structure and inhibitory activity of the parent protein. The most common BBI loop sequence has a proline-proline element with a cis-trans geometry at P3'-P4'. We have examined this element by analysis of the inhibitory activity and structure for a series of synthetic fragments where each of these proline residues has been systematically replaced with alanine. The results show that only when a proline is present at P3' are potent inhibition and a cis peptide bond at that position in the solution structure observed, suggesting that this conformation is required for biological activity. Though a P4' proline is not essential for activity, it effectively stabilizes the cis conformation at P3' by suppressing alternative conformations. This is most evident from the Pro-Ala variant, which comprises a 1:1 mixture of slowly exchanging and structurally different cis and trans isomers. Monitoring the action of trypsin on this mixture by NMR shows that this protease interacts selectively with the cis P3' structure, providing direct evidence for the link between activity and the nativelike structure of the cis isomer. This is, to the best of our knowledge, the first example where cis isomer selectivity can be demonstrated for a proteinase.

Immunoconjugates of soybean Bowman-Birk protease inhibitor as targeted antitumor polymeric agents.

To enhance the antitumor potential of soybean Bowman-Birk inhibitor (BBI), the conjugate of BBI with an antibody via a macromolecular carrier was prepared. Clinical dextran (D) was used as a biocompatible biodegradable carrier for co-immobilization of BBI and antibody. A model immunoglobulin isolated from sheep serum (sIgG), raised against human IgM was utilized to develop the procedure of immunoconjugate synthesis. The molar ratio of the ingredients in the conjugate was the following BBI:D:sIgG=9:1:1. Comparison of the dose response curves for the native sIgG and the BBI-D-sIgG conjugate indicated that sIgG completely retained its specific activity (>90%) after modification with dextran. The determination of the Ki values for chymotrypsin interaction with the native BBI and the BBI-D-sIgG conjugate indicated high anti-chymotrypsin activity. In the next step, the monoclonal antibody (ICO 25 MAb) against the mucin-like human epithelial membrane antigen was used for conjugation as it is the most universal vector for targeting different agents to human tumors of epithelial origin. The influence of conjugation on the specificity of the Mab reaction with its antigen was studied. The conjugated MAb reacted with tumor cells of different epithelial genesis (breast, lung, gastric, ovarian and uterus tumors), but did not react with tumor cells of non-epithelial origin. It was shown that BBI-D-ICO 25 MAb conjugate has almost the same immunohistochemical activity as non-conjugated MAb. These results demonstrated the feasibility of exploiting the activities of covalently bound BBI and ICO 25 MAb for anticarcinogenic agent targeting.

Development and in vitro evaluation of a drug delivery system based on chitosan-EDTA BBI conjugate.

In this study a (poly)peptide drug delivery system providing a protective effect towards serine pancreatic proteases was generated. Tablets containing insulin (3.3%), chitosan-EDTA (56.7%), chitosan-EDTA Bowman Birk Inhibitor (= BBI) conjugate (10%) and mannitol (30%) were homogenised in a mortar and compressed to tablets. The protective effect of this dosage form for the incorporated model drug was evaluated in vitro. Tablets were therefore incubated with an artificial intestinal fluid containing trypsin (1350 spectrophotometric BAEE units/ml), chymotrypsin (3.6 BTEE units/ml) and elastase (0.14 succinyl-Ala-Ala-Ala-p-nitroanilide units/ml) for 4.5 h at 37 degrees C. Following analysis of the dosage form demonstrated that 58.6+/-26.8% (mean +/- SD; n = 3) insulin in lateral parts and 44.4+/-12.4% (mean +/- SD: n = 3) insulin in inner parts of the swollen carrier-matrix were degraded, whereas insulin was completely metabolised in lateral parts and by 90.3+/-12.5% (mean +/- SD: n = 3) in inner parts of tablets without the chitosan-EDTA BBI conjugate. As chitosan-EDTA also provides a protective effect towards zinc-dependent proteases, the delivery system described in this study should therefore guarantee a protection towards the most abundant intestinal proteases. It might be a promising formulation for the peroral administration of peptide and protein drugs.

Synthesis and biodistribution of Bowman-Birk soybean protease inhibitor conjugate with amphiphilic polyester.

The modification of Bowman-Birk soybean protease inhibitor (BBI) with the monoaldehyde derivative of block copolymer of ethylene oxide and propylene oxide (PE), M(r) 2,000 is described. The conjugate contains five covalently bound polymer chains per protein molecule, and retains the ability to inhibit trypsin and chymotrypsin-like proteinases. The distribution of native BBI and the BBI-PE conjugate was examined in mice. After i.v. injection of [125I]BBI and [125I]BBI-PE, both inhibitors distributed very rapidly to the liver, kidney, and lungs, and more slowly to the brain. At the same time-points (up to 24 h), radioactivity in the blood and organs of mice injected with modified inhibitor was higher than that of the native inhibitor. The blood concentration time profile following i.v. administration of two BBI preparations at a dose 3 mg/kg was reasonable well described by a two-compartment open model with first-order elimination kinetics. The total clearance of BBI-PE decreased by a factor of 8, body mean residence time increased by a factor of 5 in comparison with BBI. A physiological pharmacokinetic model was developed to describe the tissue-to-blood distribution of two inhibitors. One-compartment physiological organ model (flow limited) was used to describe of time-course profiles of BBI concentration in organs. A two-compartment physiological organ model (membrane limited) was used to predict tissue-to-blood distribution of conjugated BBI in some organs of mice (liver, lungs). The predicted concentration curves of BBI and BBI-PE in blood and organs in mice (with the exception of kidney) showed good agreement with the observed values.

Design and inhibitory properties of synthetic Bowman-Birk loops.

A cyclic tridecapeptide based on the sequence of an anti-tryptic loop of a Bowman-Birk inhibitor was synthesized, and demonstrated to be active as an inhibitor of trypsin. Molecular modeling of this sequence suggested an improved sequence which demonstrated an order of magnitude improvement in the inhibitory constant.

Studies on the synthesis of proteinase inhibitors. II. Synthesis of cyclic nonapeptide fragments and analogs related to the reactive sites of soybean Bowman-Birk inhibitor.

Several heterodetic cyclic nonapeptides modeled after the disulfide loops of Bowman-Birk inhibitor which involve either the anti-tryptic or anti-chymotryptic region have been synthesized by the conventional method. The inhibitory properties and the stabilities of these peptides toward trypsin and chymotrypsin, as well as the properties of dimers of the above nonapeptides, were examined.

Studies on the synthesis of proteinase inhibitors. I. Synthesis and activity of nonapeptide fragments of soybean Bowman-Birk inhibitor.

Two heterodetic cyclic nonapeptides, X-Cys-Thr-Lys-Ser-Asn-Pro-Pro-Gln-Cys-Y (Ia: X = Ac, Y = NH2; Ib: X = H, Y = OH), which correspond to residues 14-22 in the sequence of Bowman-Birk inhibitor, have been synthesized by Merrifield's solid-phase method. Inhibitory activities of Ia and Ib on tryptic hydrolysis of amide and ester substrates were examined. When Gly2-Lys-Gly3 and Tos-Arg-OMe were used as substrates, the values of I50 for the peptide Ia were calculated to be 3.6 micron and 40 micron, respectively. When Gly2-Lys-Gly3 was used as a substrate, the value of Ki was calculated to be 1.5 micron. Ia was hydrolyzed slowly by trypsin, losing the inhibitory activity. When the Lys-Ser bond of Ia was cleved with trypsin, the modified Ia could not be regenerated by trypsin. The linear peptide S, S'-dicarboxamidomethyl-Ia also was inactive and appeared to be a good substrate. Optical rotatory dispersion studies showed that the active fragments have characteristic conformations which were lost upon modification to inactive derivatives.