The Parasol Protocol for computational mutagenesis.

To aid in the discovery and development of peptides and proteins as therapeutic agents, a virtual screen can be used to predict trends and direct workflow. We have developed the Parasol Protocol, a dynamic method implemented using the AMBER MD package, for computational site-directed mutagenesis. This tool can mutate between any pair of amino acids in a computationally expedient, automated manner. To demonstrate the potential of this methodology, we have employed the protocol to investigate a test case involving stapled peptides, and have demonstrated good agreement with experiment.

Potent and specific inhibition of the biological activity of the type-II transmembrane serine protease matriptase by the cyclic microprotein MCoTI-II.

Matriptase is a type-II transmembrane serine protease involved in epithelial homeostasis in both health and disease, and is implicated in the development and progression of a variety of cancers. Matriptase mediates its biological effects both via as yet undefined substrates and pathways, and also by proteolytic cleavage of a variety of well-defined protein substrates, several of which it shares with the closely-related protease hepsin. Development of targeted therapeutic strategies will require discrimination between these proteases. Here we have investigated cyclic microproteins of the squash Momordica cochinchinensis trypsin-inhibitor family (generated by total chemical synthesis) and found MCoTI-II to be a high-affinity (Ki 9 nM) and highly selective (> 1,000-fold) inhibitor of matriptase. MCoTI-II efficiently inhibited the proteolytic activation of pro-hepatocyte growth factor (HGF) by matriptase but not by hepsin, in both purified and cell-based systems, and inhibited HGF-dependent cell scattering. MCoTI-II also selectively inhibited the invasion of matriptase-expressing prostate cancer cells. Using a model of epithelial cell tight junction assembly, we also found that MCoTI-II could effectively inhibit the re-establishment of tight junctions and epithelial barrier function in MDCK-I cells after disruption, consistent with the role of matriptase in regulating epithelial integrity. Surprisingly, MCoTI-II was unable to inhibit matriptase-dependent proteolytic activation of prostasin, a GPI-anchored serine protease also implicated in epithelial homeostasis. These observations suggest that the unusually high selectivity afforded by MCoTI-II and its biological effectiveness might represent a useful starting point for the development of therapeutic inhibitors, and further highlight the role of matriptase in epithelial maintenance.

Structural analysis of foot-and-mouth disease virus 3C protease: a viable target for antiviral drugs?

Foot-and-mouth disease virus causes a major global agricultural problem that is difficult to control with existing vaccines. Structural analyses of the viral 3C protease not only have provided fresh insights into the catalytic mechanism of an unusual class of chymotrypsin-like cysteine proteases, but also are generating valuable information to drive the quest for effective antiviral therapies.

Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides.

Four 11-residue peptides based on the Bowman-Birk inhibitor (BBI) structure were synthesized. These were tested for their ability to inhibit human beta-tryptase. Peptides with a basic residue at P1 inhibited tryptase even though the intact BBI protein is inactive. This result is interpreted in terms of the unique structural arrangement of active sites in tryptase which prevent access by large protein inhibitors.

Introduction of the new dipeptide isostere 7-endo-BtA as reverse turn inducer in a Bowman-Birk proteinase inhibitor: synthesis and conformational analysis.

Two dipeptide isosteres 7-exo-BTG (1) and 7-endo-BtA (2), belonging to the new class of gamma/delta-bicyclic amino acid BTAa, were inserted into an 11-residue peptide deriving from the Bowman Birk Inhibitor (BBI) class of serine protease inhibitors, and the conformational properties of these modified peptides have been studied by NMR and molecular modelling. The dipeptide isostere 7-endo-BtA [(1R,4S,5R,7R)-4-endo-methyl-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-endo-carboxylic acid] (2), derived from L-alanine and meso tartaric acid, gave rise to the modified BBI peptide 5 whose structure was very similar to that of the original peptide 3, suggesting a possible reverse turn inducing property for this dipeptide isostere.

Synthetic peptide mimics of the Bowman-Birk inhibitor protein.

Proteins of the Bowman-Birk inhibitor family of serine proteinase inhibitors interact with the enzymes they inhibit via an exposed surface loop that adopts the canonical proteinase inhibitory conformation. The resulting non-covalent complex renders the proteinase inactive. This inhibition mechanism is common for the majority of serine proteinase inhibitor proteins and many analogous examples are known. A particular feature of the Bowman-Birk inhibitor protein, however, is that the interacting loop is a particularly well-defined disulfide-linked short beta-sheet region. Moreover, synthetic peptides based on this region keep the same structure as the corresponding part of the full sized protein and also retain inhibitory activity. This review describes the background to inhibition by Bowman-Birk inhibitor proteins (and derived peptides) and shows how peptides based on the reactive site can be manipulated in order to generate potent proteinase inhibitors with redirected specificities.

The Bowman-Birk inhibitor reactive site loop sequence represents an independent structural beta-hairpin motif.

We have determined the NMR structure in aqueous solution of a disulphide-cyclised 11-residue peptide that forms a stable beta-hairpin, incorporating a type VIb beta-turn. The structure is found to be extremely well ordered for a short peptide, with the 30 lowest energy simulated annealing structures having an average pairwise r.m.s. deviation of only 0.36 A over the backbone. All but three side-chains adopt distinct conformations, allowing a detailed analysis of their involvement in cross-strand interactions. The peptide sequence analysed originates from a previously reported study, which identified potent inhibitors of human leukocyte elastase from screening a combinatorial peptide library based on the short protein beta-sheet segment that forms the reactive site loop of Bowman-Birk inhibitors. A detailed comparison of the peptide's solution structure with the corresponding region in the whole protein structure reveals a very good correspondence not only for the backbone (r.m.s. deviation approximately 0.7 A) but also for the side-chains. This isolated beta-hairpin retains the biologically active "canonical conformation" typical of small serine proteinase inhibitor proteins, which explains why it retains inhibitory activity. Since the structural integrity is sequence-inherent and does not depend upon the presence of the remaining protein, this beta-hairpin represents an independent structural motif and so provides a useful model of this type of protein architecture and its relation to biological function. The relationship between the conformation of this beta-hairpin and its biological activity is discussed.

Identification of chymotrypsin inhibitors from a second-generation template assisted combinatorial peptide library.

In an earlier study (McBride JD, Freeman N, Domingo GJ, Leatherbarrow RJ. Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library. J. Mol. Biol. 1996; 259: 819-827) we described a resin-bound cyclic peptide library, constructed based on the sequence of the anti-tryptic reactive site loop of Bowman Birk Inhibitor (BBI), a proteinase inhibitor protein. This library was used to identify re-directed chymotrypsin inhibitors with Ki values as low as 17 nM. We have now extended this work by constructing an enhanced library in which a further position, at the P4 site of the inhibitor, has been randomized. This new library has variation at three target locations (P4, P1 and P2) within the inhibitory loop region, producing 8,000 variants. Screening this library allowed selection of new inhibitor sequences with Ki values as low as 3.4 nM. The success of this approach is reflected by the fact that the inhibition constant given by the selected peptide sequence is slightly lower than that reported against chymotrypsin for the most studied full length BBI protein, Soybean BBI 2-IV.

Selection of human elastase inhibitors from a conformationally constrained combinatorial peptide library.

A resin-bound cyclic peptide library was constructed based on the sequence of the reactive-site loop of Bowman-Birk inhibitor, a proteinase inhibitor protein. The constrained loop sequence, which incorporates the minimal proteinase-binding motif, was retained throughout the library, but selected residues known to be important for inhibitor specificity were randomised. The approach was used to create a 'one bead, one peptide' library with 8000 variants resulting from randomization at three target locations in the sequence (P4, P1 and P2'). This library allows us to examine the degree to which variations in this proteinase-binding motif can redirect activity, as well as providing information about the binding specificity of a proteinase target. Screening this library for binding to human leucocyte elastase identified sequences with a strong consensus, and on resynthesis all were found to act as inhibitors, with Ki values as low as 65 nM. Human leucocyte elastase is known to have a substrate preference for small alkyl chains at the P1 locus, with valine being preferred. However, alanine and not the expected valine was found in 21 out of 23 identified sequences. The remaining two sequences had threonine at P1, a finding that would be hard to predict based on substrate specificity alone. Further analysis of resynthesized peptides demonstrated that valine substitution results in an analogue that is hydrolysed far more rapidly than ones having library-selected P1 residues. Testing of the human leucocyte elastase-selected sequences as inhibitors of porcine pancreatic elastase demonstrates a significant difference in the specificity of the P4 locus between these two proteinases.

Analysis of molecular recognition using optical biosensors.

Optical biosensors have made the analysis of molecular interactions rapid and convenient. The field is still young, with commercial instrumentation having been available for less than ten years. For this reason instrument development is still an important factor and the past year has seen continuing advances in instrumentation and particularly in novel sensor surfaces.

The role of the P2' position of Bowman-Birk proteinase inhibitor in the inhibition of trypsin. Studies on P2' variation in cyclic peptides encompassing the reactive site loop.

The role of the P2' residue in proteinase inhibitors of the Bowman-Birk family was investigated using synthetic cyclic peptides based on the reactive site loop of the inhibitor. A series of 21 variants having different P2' residues was tested for inhibition of trypsin, and the rate at which they were hydrolysed by this enzyme was also measured. Variation at P2' was found to result in marked differences in inhibitory potency, with the best sequence (Ile) having a Ki value of 9 nM. Peptides with P2' Gly, Pro or Glu failed to demonstrate any measurable inhibition (Ki>1 mM). The peptides also displayed significant differences in the rates at which they were hydrolysed, which varied by over three orders of magnitude between the difference sequences. There was found to be overall correlation between the Ki value and the rate of hydrolysis, with peptides that inhibited best also being hydrolysed more slowly. The results are discussed in light of the sequence information for Bowman-Birk inhibitor proteins.

Second-order kinetic analysis of IAsys biosensor data: its use and applicability.

The kinetic analysis of IAsys biosensor association data usually relies upon the assumption of constant ligate concentration. In certain circumstances this assumption may no longer be valid. In a similar vein, the analysis of the dissociation phase assumes the concentration of ligate to be negligible in the liquid phase-an assumption that may not be sustainable for high-affinity interactions. In this paper we derive analytical solutions of the second-order differential kinetic equations for the association and dissociation phases, together with a binding isotherm that also allows for changes in concentration of both the ligand and the ligate. Using these equations it is possible to determine the conditions under which the pseudo-first-order assumption ceases to be valid. It is found that the effect of ligate depletion on the association rate constant becomes significant only when binding low ligate concentrations to ligand on surfaces with high binding capacities or high affinities. Similarly, the rebinding in the dissociation phase is dependent upon the affinity and the ligand capacity together with the starting dissociation response compared to the capacity. Finally, depletion also affects the form of the binding isotherm, particularly in situations involving high matrix capacities for ligate and high-affinity interactions.

The role of threonine in the P2 position of Bowman-Birk proteinase inhibitors: studies on P2 variation in cyclic peptides encompassing the reactive site loop.

Previously, we have described a template-assisted combinatorial peptide library based on the anti-tryptic reactive site loop of a Bowman-Birk inhibitor (BBI). Sequences that displayed inhibitory activity re-directed towards chymotrypsin were found to have a consensus binding motif, with their most striking feature being that exclusively threonine was found at the P2 position. The present study investigates the reason for this surprising specificity by maintaining the binding motif but systematically varying the P2 residue. From analysis of 26 variants, it is found that the requirements for inhibitory activity at P2 are finely tuned, and in agreement with the library work, threonine at P2 provides optimal inhibition. In addition, peptides with threonine at P2 are significantly less susceptible to hydrolysis. Examination of all available BBI sequences shows that threonine is very highly conserved at P2, which implies that the functional requirement extends to the full-length BBI protein. Our results are consistent with a dual requirement for hydrophobic recognition within the S2 pocket and maintenance of an inhibitory conformation via hydrogen bonding within the reactive-site loop. As the isolated peptide loop reproduces the active region of full-length BBI, these results explain why threonine is well conserved at P2 in this class of inhibitor. Furthermore, they illustrate that proteinase inhibitor specificity can have characteristics that are not easily predicted from information on the substrate preferences of a proteinase.

A study of the specificity of barley chymotrypsin inhibitor 2 by cysteine engineering of the P1 residue.

A combination of oligonucleotide-directed mutagenesis and chemical modification was used to produce reactive site (P1) variants of chymotrypsin inhibitor II (CI2) in an attempt to create more potent inhibitors and examine inhibitory specificity. Mutagenesis to introduce a unique cysteine (CI2M59C) followed by modification to S-carboxamidocysteine with iodoacetamide produced a 7-fold more potent inhibitor of subtilisin BPN' than the wild type inhibitor. Modification with iodoacetic acid, which gives a negatively charged P1 residue (S-carboxymethylcysteine), generates a weaker inhibitor of subtilisin BPN' and chymotrypsin. Further chemical modification experiments of CI2M59C with a series of iodoalkanes of increasing chain lengths was used to determine the optimal P1 side chain length required for potent inhibition of porcine pancreatic elastase. A trend was observed which implies that for CI2 the original methionine residue or its isostere S-ethylcysteine are most effective residues at this position and not alanine as might have been expected from the substrate specificity. The mutant CI2M59C did not inhibit human neutrophil elastase. The iodoalkane modifications not only resulted in recovery of inhibitory activity but also proved to be substantially more potent inhibitors of human neutrophil elastase than wild-type CI2.

Effect of osmolytes on the exchange rates of backbone amide protons in proteins.

Osmolytes are small organic solutes produced by the cells of all organisms (except halobacteria) in high stress situations (e.g. extremes of salt concentration, high temperature, etc.) to stabilize their macromolecules and so conserve biological activity. They do not interact with the macromolecule directly but act by altering the solvent properties in the cellular environment, and so their presence indirectly modifies the stability of proteins. In this paper we examine the effect of a model osmolyte, glycine, on the stabilization of two proteins, chymotrypsin inhibitor 2 and horse heart cytochrome c. We have used NMR to monitor the effect of this osmolyte on amide hydrogen exchange rates, which allows a probe at discrete points within the protein structure. Hydrogen exchange rates of specific backbone amide protons provide information about the localized structural fluctuations that expose these amides to solvent and allow exchange to take place. We find that the presence of a high concentration of glycine osmolyte has a profound stabilizing effect on the proteins studied, which is accompanied by a large reduction of the exchange rate constants of most slowly exchanging amide protons. The spectra indicate that this arises without significant changes in the three-dimensional structure. However, the effects on individual amide protons within a single protein were not uniform, and a wide variation in the magnitude of the effects was observed. This ranged from no apparent change in the exchange rate, to decreases in the exchange rate constant by over 2 orders of magnitude. The osmolyte appears to alter a number of different processes that contribute to the observed exchange rates, and no simple generalization allows prediction of the extent of stabilization at any individual location. The results are discussed in light of the available structures of the proteins studied.

Stability of protease inhibitors based on the Bowman-Birk reactive site loop to hydrolysis by proteases.

Bowman-Birk proteinase inhibitor proteins contain two inhibitory regions, each of which is encapsulated within nine-residue disulfide-linked loops. It is known that short cyclic peptides that retain the nine-residue disulfide-bridged motif have inhibitory activity, and can be used as models of the natural inhibitor protein. Two factors are important in determining the effectiveness of such inhibitor peptides: the value of the inhibition constant, and rate at which the inhibitor peptide is hydrolyzed by the proteinase. In this paper we report a study of the inhibitory properties and stability towards proteolytic hydrolysis of a family of synthetic peptides derived from the trypsin reactive site loop of the Bowman-Birk inhibitors. The addition of a single amino acid residue to each end of the nine-residue disulfide-linked loop is found to reduce the rate at which the peptide is hydrolyzed. In addition, changing the P2' residue from Asn-->Ile gives inhibitors with considerably enhanced stability to proteolysis, as well as reduced values of Ki. The implications of these factors for the design of inhibitors based on this loop motif is discussed.

Ligand loading at the surface of an optical biosensor and its effect upon the kinetics of protein-protein interactions.

Optical biosensors are finding increasing use in the determination of kinetic and equilibrium constants for a variety of biomolecular interactions. Usually these biosensors require one biomolecule, the ligand, to be covalently attached to a hydrogel matrix which itself is bonded to the sensing surface. The ligands partner, the ligate, then binds from solution resulting in a measurable change in response which the instrument records as a function of time. Although in many cases, optical biosensors are used in order to obtain parameters that relate to interactions in solution, it is becoming clear that measurements involving the interaction of ligate with immobilized ligands on surfaces require careful experimental design. Here we report on how the density of ligand loading within the hydogel matrix affects the measured interaction kinetics. It is found that crowding of ligand within this matrix results in a significant reduction in the measured association rate constant, with a corresponding effect in the calculated overall affinity. However, measurements at low ligand loadings show association rate constants that are comparable to those measured in solution. Clearly, where this comparison is required, it is important to perform measurements under such conditions.

Determination of association rate constants by an optical biosensor using initial rate analysis.

We show that initial rate analysis can be successfully applied to analyze experimental binding data generated by an optical biosensor. The initial rates of binding obtained from linear regression are concentration dependent, and plots of initial rate against ligate concentration yield a straight line that passes through the origin. The slope of this graph is the product of the association constant times the maximal binding capacity of the immobilized ligand. This latter parameter is easily obtained from a single binding curve at high ligate concentration, allowing rapid determination of the association rate constant. The association rate constant obtained in this manner is found to be in good agreement with that obtained by the more customary method of nonlinear regression analysis of the entire binding profile. Initial rate analysis is more simple than fitting the full association profile and needs less data collection time. It also requires fewer assumptions about the functional form of the association profile. This can be advantageous when fitting biosensor-derived data, which often show complex association kinetics. Furthermore, it avoids the potential complication of second-order kinetics which may be found at low ligate concentrations with high-affinity interactions.

Analysis of kinetic data of antibody-antigen interaction from an optical biosensor by exponential curve fitting.

An optical biosensor system employing a resonant mirror (RM), with a stirred cuvette has been used to follow the interaction of a recombinant antibody fragment with its antigen, hen egg lysozyme. The data generated by the biosensor were analysed in order to determine the kinetic constants for the interaction using a linear transform (derivative analysis). For comparison the data were also analysed using an exponential curve fitting routine. It was demonstrated that the exponential curve fitting method produced results which were in agreement with the existing linear transform method. It was also shown that early fitting of the association phase response, using the exponential curve fitting routine between 0 and 70 s after sample addition, yielded sufficient information to provide a prediction of Kon. The potential use of the optical biosensor for the rapid monitoring of protein production and purification is discussed.

Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library.

A synthetic library of cyclic peptides was constructed utilizing the anti-tryptic loop region of the Bowman-Birk inhibitor, D4 from Macrotyloma axillare, as a template. The loop region of this proteinase inhibitor was reproduced by an 11 residue sequence, conformationally constrained by the presence of a disulfide bridge, to act as a mimetic of the functional reactive site region of this protein. This sequence, plus a pentaglycine spacer arm, was used to create a "one bead, one peptide" combinatorial library after on-resin deprotection and cyclization. Randomization at three positions considered to be important for proteinase specificity (P2, P1 and P'2) with the genetically coded amino acids (minus cysteine) plus norleucine generated 8000 permutations. Screening this library with biotinylated alpha-chymotrypsin under appropriate conditions revealed a small number (<0.05%) of beads that selectively bound the labeled proteinase. The sequences present on these active beads were determined, and found to have a well-defined consensus. Analysis of chymotrypsin inhibition in solution using re-synthesized peptides reveals that the sequences identified are potent inhibitors with Ki values in the nanomolar range. These results show that directed randomization of the canonical loop is a powerful way of generating proteinase inhibitors with targeted specificities. Incorporation of selective random changes within a defined structural framework is found to be an effective means of generating variation in large synthetic systems. The functional basis for inhibition by the identified sequences is discussed.