Introduction of alpha-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity

In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P1-P1' reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P1 by alpha-hydroxymethylserine (HmSer) and both enantiomers of alpha-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine beta-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine beta-trypsin, whereas [Val5]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser5]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated epsilon-NH2 group of the inhibitor P1 position and beta-carboxylate of trypsin Asp189 is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.     

Introduction of Pro and its analogues in the conserved P1' position of trypsin inhibitor SFTI-1 retains its inhibitory activity

A number of monocyclic SFTI-1 analogues modified in the conserved inhibitor P1' position by Pro, its L-hydroxyproline (Hyp) derivative as well as mimetics with different ring size were synthesized by the solid-phase method. Replacement of Ser6 by Pro, Hyp, and a four-member ring, L-azetidine-2-carboxylic acid (Aze), retained trypsin or chymotrypsin inhibitory activity. The determined association equilibrium constants of these analogues with a cognate enzyme were about two orders of magnitude lower than those obtained for ones with conserved Ser6. In all analogues, with the exception of one, [Phe5,Aze6]SFTI-1, the P1-P1' reactive site remained intact. The results provide first evidence that the conserved Ser in the P1' position of Bowman-Birk inhibitors can be successfully replaced by an amino acid with a secondary amine group.     

Proteinase inhibitors from desert locust, Schistocerca gregaria: engineering of both P(1) and P(1)' residues converts a potent chymotrypsin inhibitor to a potent trypsin inhibitor.

Two peptides, SGCI and SGTI, that inhibited chymotrypsin and trypsin, respectively, were isolated from the haemolymph of Schistocerca gregaria. Their primary structures were found to be identical with SGP-2 and SGP-1, two of a series of peptides isolated from ovaries of the same species (A. Hamdaoui et al., FEBS Lett. 422 (1998) 74-78). All these peptides are composed of 35-36 amino acid residues and contain three homologous disulfide bridges. The residues imparting specificity to SGCI and SGTI were identified as Leu-30 and Arg-29, respectively. The peptides were synthesised by solid-phase peptide synthesis, and the synthetic ones displayed the same inhibition as the natural forms: SGCI is a strong inhibitor of chymotrypsin (K(i) = 6.2 x 10(-12) M), and SGTI is a rather weak inhibitor of trypsin (K(i) = 2.1 x 10(-7) M). The replacement of P(1) then P(1)' residues of SGCI with trypsin-specific residues increased affinity towards trypsin 3600- and 1100-fold, respectively, thus SGCI was converted to a strong trypsin inhibitor (K(i) = 5.0 x 10(-12) M) that retained some inhibitory affinity towards chymotrypsin (K(i) = 3.5 x 10(-8) M). The documented role of both P(1) and P(1)' highlights the importance of S(1)'P(1)' interactions in enzyme-inhibitor complexes.     

Structural consequences of accommodation of four non-cognate amino acid residues in the S1 pocket of bovine trypsin and chymotrypsin

Crystal structures of P1 Gly, Val, Leu and Phe bovine pancreatic trypsin inhibitor (BPTI) variants in complex with two serine proteinases, bovine trypsin and chymotrypsin, have been determined. The association constants for the four mutants with the two enzymes show that the enlargement of the volume of the P1 residue is accompanied by an increase of the binding energy, which is more pronounced for bovine chymotrypsin. Since the conformation of the P1 side-chains in the two S1 pockets is very similar, we suggest that the difference in DeltaG values between the enzymes must arise from the more polar environment of the S1 site of trypsin. This results mainly from the substitutions of Met192 and Ser189 observed in chymotrypsin with Gln192 and Asp189 present in trypsin. The more polar interior of the S1 site of trypsin is reflected by a much higher order of the solvent network in the empty pocket of the enzyme, as is observed in the complexes of the two enzymes with the P1 Gly BPTI variant. The more optimal binding of the large hydrophobic P1 residues by chymotrypsin is also reflected by shrinkage of the S1 pocket upon the accommodation of the cognate residues of this enzyme. Conversely, the S1 pocket of trypsin expands upon binding of such side-chains, possibly to avoid interaction with the polar residues of the walls. Further differentiation between the two enzymes is achieved by small differences in the shape of the S1 sites, resulting in an unequal steric hindrance of some of the side-chains, as observed for the gamma-branched P1 Leu variant of BPTI, which is much more favored by bovine chymotrypsin than trypsin. Analysis of the discrimination of beta-branched residues by trypsin and chymotrypsin is based on the complexes with the P1 Val BPTI variant. Steric repulsion of the P1 Val residue by the walls of the S1 pocket of both enzymes prevents the P1 Val side-chain from adopting the most optimal chi1 value.     

Dipeptidyl aspartyl fluoromethylketones as potent caspase inhibitors: peptidomimetic replacement of the P(2) amino acid by 2-aminoaryl acids and other non-natural amino acids

As a continuation of our SAR studies of dipeptidyl aspartyl-fmk as caspase inhibitors, we explored the replacement of the P(2) amino acid by a 2-aminoaryl acid or other non-natural amino acids. Several of these compounds, such as 6l and 6p, were found to have good activities with inhibition potencies of around 100 nM in a caspase-3 enzyme assay. EP1113, Z-Val-(2-aminobenzoyl)-Asp-fmk (9b), is identified as a potent broad-spectrum caspase inhibitor with IC(50) values of 6-60 nM in different caspases. EP1113 also has good activity in a cell apoptosis protection assay.     

Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by (1)H NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 A, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H(alpha) protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.     

Purification and amino acid analysis of plasma acid stable trypsin inhibitor

Acid stable trypsin inhibitor (ASTI), with a molecular weight of about 85,000 by gel filtration, specific activity of 1,498 U/mg protein and pI of 1.6, from renal failure patient plasma was first purified. The amino acid composition of the purified ASTI was found to be that of a Gly- and Glu-rich protein which lacked His, closely resembling that of urinary trypsin inhibitor. The NH2-terminal amino acid sequence was Ala-Val-Leu-Pro-Gln-Glu- Glu-Glu-Gly-X-Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr-Lys-Lys-Glu- Asp-Ser-Ser-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro.     

Differences between the binding of trypsin and chymotrypsin by alpha 1-proteinase inhibitor

Complexes of alpha 1-proteinase inhibitor with proteases were examined by SDS-PAGE in 7.5% polyacrylamide gel and in a gel gradient. While the inhibitor-chymotrypsin complex was stable under both sets of conditions, the inhibitor-trypsin complex quantitatively dissociated under the second set of conditions, indicating that trypsin, unlike chymotrypsin, is not linked covalently to the inhibitor. Although the inhibitor sustained at least two discrete cleavages by trypsin, its overall recovery after dissociation was 100%. Due to an increased rate of autolytic breakdown in the presence of the inhibitor, the recovery of trypsin after dissociation was appreciably less than 100%. Based on these observations, a new theory of trypsin inhibition by alpha 1-proteinase inhibitor is proposed. This method is suitable for the examination of other inhibition systems as well.     

Implication of the disulfide bridge in trypsin inhibitor SFTI-1 in its interaction with serine proteinases

Fourteen monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds were synthesized by the solid-phase method. The purpose of this work was to establish the role of a disulfide bridge present in inhibitor’s side chains of Cys3 and Cys11 in association with serine proteinases. This cyclic fragment was replaced by the disulfide bridges formed by l-pencillamine (Pen), homo-l-cysteine (Hcy), N-sulfanylethylglycine (Nhcy) or combination of the three with Cys. As in the substrate specificity the P₁ position of the synthesized analogues Lys, Nlys [N-(4-aminobutyl)glycine], Phe or Nphe (N-benzylglycine) were present, and they were checked for trypsin and chymotrypsin inhibitory activity. The results clearly indicated that Pen and Nhcy were not acceptable at the position 3, yielding inactive analogues, whereas another residue (Cys11) could be substituted without any significant impact on the affinity towards proteinase. On the other hand, elongation of the Cys3 side chain by introduction of Hcy did not affect inhibitory activity, and an analogue with the Hcy–Hcy disulfide bridge was more than twice as effective as the reference compound ([Phe⁵] SFTI-1) in inhibition of bovine α-chymotrypsin.     

Hypochlorous acid is a potent inhibitor of GST P1-1.

Glutathione S-transferase is a phase II detoxification enzyme that can be inactivated by H(2)O(2). During oxidative stress various other reactive oxygen species are generated that are more reactive than the relatively stable H(2)O(2). Hypochlorous acid (HOCl) is a powerful oxidant which is highly reactive towards a range of biological substrates. We studied the influence of HOCl on the activity of GST P1-1. HOCl inhibits purified glutathione S-transferase P1-1 in a concentration dependent manner with an IC(50)-value of 0.6 microM, which is more than 1000 times as low as IC(50) reported for H(2)O(2). HOCl lowered the V(max) value, but did not affect the K(m) for CDNB. Our results show that HOCl is a potent, non-competitive inhibitor of GST P1-1. The relevance of this effect is discussed.     

Dipeptidyl aspartyl fluoromethylketones as potent caspase-3 inhibitors: SAR of the P2 amino acid

This article describes the synthesis and biological evaluation of a series of dipeptidyl aspartyl fluoromethylketones as caspase-3 inhibitors. Structure-activity relationship (SAR) studies showed that for caspase-3 inhibition, Val is the best P(2) amino acid. The SAR studies also showed that the Asp free carboxylic acid in P(1) is important for caspase inhibiting activities, as well as for selectivity over other proteases.     

Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI)

Summary The molecular conformation of the basic pancreatic trypsin inhibitor (BPTI) is known in considerable detail from both X-ray studies in single crystals and NMR studies in solution. The NMR experiments showed that the aromatic rings of the phenylalanyl and tyrosyl residues can undergo rapid rotational motions about the C-C bond. The present paper describes a model investigation of the mechanistic aspects of these intramolecular rotational motions. From calculations of the conformational energies for molecular species derived from the X-ray structure by rotations of individual aromatic rings, it was apparent that the rotational motions of the aromatics could only be understood in a flexible structure. Flexibility was simulated by allowing the protein to relax to an energetically favorable conformation for each of the different rotation states of the aromatic rings. It was then of particular interest to investigate how the perturbations caused by different rotation states of the aromatic rings were propagated in the protein structure. It was found that the rotation axes C-C were only slightly affected ( ¹20°). The most sizeable perturbations are caused by through space interactions with nearby atoms, which move away from the ring center and thus release the steric hindrance opposing the rotational motions. The values for the energy barriers obtained from the energy minimization are of the same order of magnitude as those measured by NMR.     

1H assignments and secondary structure determination of the soybean trypsin/chymotrypsin Bowman-Birk inhibitor

The 1H resonance assignments and secondary structure of the trypsin/chymotrypsin Bowman-Birk inhibitor from soybeans were determined by nuclear magnetic resonance spectroscopy (NMR) at 600 MHz in an 18% acetonitrile-d3/aqueous cosolvent. Resonances from 69 of 71 amino acids were assigned sequence specifically. Residues Q11-T15 form an antiparallel beta-sheet with residues Q21-S25 in the tryptic inhibitory domain and an analogous region of antiparallel sheet forms between residues S38-A42 and Q48-V52 in the chymotryptic inhibitory domain. The inhibitory sites of each fragment (K16-S17 for trypsin, L43-S44 for chymotrypsin) are each part of a type VI like turn at one end of their respective region of the antiparallel beta-sheet. These structural elements are compared to those found in other Bowman-Birk inhibitors.     

Solution structures by 1H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant

SFTI-1 is a recently discovered cyclic peptide trypsin inhibitor from sunflower seeds comprising 14 amino acid residues. It is the most potent known Bowman-Birk inhibitor and the only naturally occurring cyclic one. The solution structure of SFTI-1 has been determined by 1H-NMR spectroscopy and compared with a synthetic acyclic permutant. The solution structures of both are remarkably similar. The lowest energy structures from each family of 20 structures of cyclic and acyclic SFTI-1 have an rmsd over the backbone and heavy atoms of 0.29 A and 0.66 A, respectively. The structures consist of two short antiparallel beta-strands joined by an extended loop containing the active site at one end. Cyclic SFTI-1 also has a hairpin turn completing the cycle. Both molecules contain particularly stable arrangements of cross-linking hydrogen bonds between the beta-strands and a single disulfide bridge, making them rigid and well defined in solution. These stable arrangements allow both the cyclic and acyclic variants of SFTI-1 to inhibit trypsin with very high potencies (0.5 nM and 12.1 nM, respectively). The cyclic nature of SFTI-1 appears to have evolved to provide higher trypsin inhibition as well as higher stability. The solution structures are similar to the crystal structure of the cyclic inhibitor in complex with trypsin. The lack of a major conformational change upon binding suggests that the structure of SFTI-1 is rigid and already pre-organized for maximal binding due to minimization of entropic losses compared to a more flexible ligand. These properties make SFTI-1 an ideal platform for the design of small peptidic pharmaceuticals or pesticides.     

Conversion of peanut trypsin-chymotrypsin inhibitor B-III to a chymotrypsin inhibitor by deimination of the P1 arginine residues in two reactive sites

The deimination of the arginine residues in peanut trypsin-chymotrypsin inhibitor B-III caused the disappearance of its trypsin-inhibitory activity. Peanut protease inhibitor B-III was incubated with peptidylarginine deiminase, resulting in the conversion of 2.5 mol of arginine to citrulline and in the loss of its trypsin-inhibitory activity. However, the ability of the deiminated inhibitor to inhibit chymotrypsin was as strong as before. Structural analysis of the deiminated B-III indicated that the P1 arginine residues at both reactive sites, Arg(10) and Arg(38), were completely modified to citrulline by the action of peptidylarginine deiminase, and that the Arg(60) in the C-terminal region of B-III was partially deiminated. These residues seem to be exposed on the surface of the molecule. The P1' arginine residue at the first reactive site, Arg(11), was not deiminated at all.     

Primary structure and properties of the cathepsin G/chymotrypsin inhibitor from the larval hemolymph of Apis mellifera.

A member of the Ascaris inhibitor family exhibiting anti-cathepsin G and anti-chymotrypsin activity was purified from the larval hemolymph of the honey bee (Apis mellifera). Three forms of the inhibitor, designated AMCI 1-3, were isolated using gel filtration and anion-exchange chromatographies followed by reverse-phase HPLC. The amino-acid analyses indicated that AMCI-1 and AMCI-2 have an identical composition whereas AMCI-3 is shorter by two residues (Thr, Arg). All three forms contain as many as 10 cysteine residues and lack tryptophan, tyrosine, and histidine. The sequence of the isoinhibitors showed that the major form (AMCI-1) consisting of 56 amino-acid residues was a single-chain protein of molecular mass 5972 Da, whereas the other two forms were two-chain proteins with a very high residue identity. The AMCI-2 appeared to be derived from AMCI-1, as a result of the Lys24-Thr25 peptide bond splitting, while AMCI-3 was truncated at its N-terminus by the dipeptide Thr25-Arg26. The association constants for the binding of bovine alpha-chymotrypsin to all purified forms of the inhibitor were high and nearly identical, ranging from 4.8 x 10(10) M-1 for AMCI-1 to 2.7 x 10(9) M-1 for AMCI-3. The sensitivity of cathepsin G to inhibition by each inhibitor was different. Only the association constant for the interaction of this enzyme with AMCI-1 was high (2 x 10(8) M-1) whereas those for AMCI-2 and AMCI-3 were significantly lower, and appeared to be 3.7 x 10(7) M-1 and 4.5 x 10(6) M-1, respectively. The reactive site of the inhibitor, as identified by cathepsin G degradation and chemical modification, was found to be at Met30-Gln31. A search in the Protein Sequence Swiss-Prot databank revealed a significant degree of identity (44%) between the primary structure of AMCI and the trypsin isoinhibitor from Ascaris sp (ATI). On the basis of the cysteine residues alignment, the position of the reactive site as well as some sequence homology, the cathepsin G/chymotrypsin inhibitor from larval hemolymph of the honey bee may be considered to be a member of the Ascaris inhibitor family.