Impact of ligand and protein desolvation on ligand binding to the S1 pocket of thrombin.

In the present study, we investigate the impact of a tightly bound water molecule on ligand binding in the S1 pocket of thrombin. The S1 pocket contains a deeply buried deprotonated aspartate residue (Asp189) that is, due to its charged state, well hydrated in the uncomplexed state. We systematically studied the importance of this water molecule by evaluating a series of ligands that contains pyridine-type P1 side chains that could potentially alter the binding properties of this water molecule. All of the pyridine derivatives retain the original hydration state albeit sometimes with a slight perturbance. In order to prevent a direct H-bond formation with Asp189, and to create a permanent positive charge on the P1 side chain that is positioned adjacent to the Asp189 carboxylate anion, we methylated the pyridine nitrogen. This methylation resulted in displacement of water but was accompanied by a loss in binding affinity. Quantum chemical calculations of the ligand solvation free energy showed that the positively charged methylpyridinium derivatives suffer a large penalty of desolvation upon binding. Consequently, they have a substantially less favorable enthalpy of binding. In addition to the ligand desolvation penalty, the hydration shell around Asp189 has to be overcome, which is achieved in nearly all pyridinium derivatives. Only for the ortho derivative is a partial population of a water next to Asp189 found. Possibly, the gain of electrostatic interactions between the charged P1 side chain and Asp189 helps to compensate for the desolvation penalty. In all uncharged pyridine derivatives, the solvation shell remains next to Asp189, partly mediating interactions between ligand and protein. In the case of the para-pyridine derivative, a strongly disordered cluster of water sites is observed between ligand and Asp189.

Displacement of disordered water molecules from hydrophobic pocket creates enthalpic signature: binding of phosphonamidate to the S1'-pocket of thermolysin.

BACKGROUND: Prerequisite for the design of tight binding protein inhibitors and prediction of their properties is an in-depth understanding of the structural and thermodynamic details of the binding process. A series of closely related phosphonamidates was studied to elucidate the forces underlying their binding affinity to thermolysin. The investigated inhibitors are identical except for the parts penetrating into the hydrophobic S1'-pocket. METHODS: A correlation of structural, kinetic and thermodynamic data was carried out by X-ray crystallography, kinetic inhibition assay and isothermal titration calorimetry. RESULTS AND CONCLUSIONS: Binding affinity increases with larger ligand hydrophobic P1'-moieties accommodating the S1'-pocket. Surprisingly, larger P1'-side chain modifications are accompanied by an increase in the enthalpic contribution to binding. In agreement with other studies, it is suggested that the release of largely disordered waters from an imperfectly hydrated pocket results in an enthalpically favourable integration of these water molecules into bulk water upon inhibitor binding. This enthalpically favourable process contributes more strongly to the binding energetics than the entropy increase resulting from the release of water molecules from the S1'-pocket or the formation of apolar interactions between protein and inhibitor. GENERAL SIGNIFICANCE: Displacement of highly disordered water molecules from a rather imperfectly hydrated and hydrophobic specificity pocket can reveal an enthalpic signature of inhibitor binding.

The design, synthesis and activity of non-ATP competitive inhibitors of pp60(c-src) tyrosine kinase. Part 1: hydroxynaphthalene derivatives.

A series of hydroxynaphthalene pp60(c-src) non-peptide inhibitors was designed, using the crystal structure of the insulin receptor tyrosine kinase as a qualitative model, to target the peptide substrate binding site. Representative inhibitors were shown to bind non-competitively with respect to ATP.

The design, synthesis and activity of non-ATP competitive inhibitors of pp60(c-src) tyrosine kinase. Part 2: hydroxyindole derivatives.

As part of a continuing effort to identify novel scaffolds that inhibit the pp60(c-src) protein tyrosine kinase, a series of hydroxyindole amides was rationally designed and synthesized. The most potent derivative was found to bind non-competitively with respect to ATP.

The design, synthesis and activity of pentapeptide pp60c-src inhibitors containing L-phosphotyrosine mimics.

Efficient syntheses of 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine and 4-carboxy-L-phenylalanine within the context of the pentapeptide Ac-Ile-X-Gly-Glu-Phe-NH2 (wherein X = the unnatural amino acid) illustrate the use of a divergent synthetic strategy from an advanced common peptide intermediate to more readily access peptide-based tyrosine kinase inhibitors. The key intermediate, Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2, was synthesized by a facile palladium-catalyzed carbonylation of Ac-Ile-Phe(4-iodo)-Gly-Glu(O-tBu)-Phe-NH2. Oxidation of Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2 with tetrabutylammonium permanganate or addition of di-t-butylphosphite, both followed by trifluoroacetic acid deprotection, gave the target pentapeptide inhibitors wherein X = 4-carboxy-L-phenylalanine or 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine, respectively. These two peptides gave somewhat more potent inhibition of the tyrosine kinase pp60c-src than the corresponding pentapeptide wherein X = L-phenylalanine, demonstrating that appended functionalities at the 4-position are accepted and can enhance binding through added interactions within the catalytic region of the active site.

The design and synthesis of substituted biphenyl libraries.

A novel scaffold system for the generation of diversity libraries has been designed which allows for rapid modification not only of functional groups, but their spatial arrangements as well. The biphenyl scaffold allows for display of three or four diverse functional groups in a wide variety of spatial arrangements depending on the substitution pattern selected. The libraries are generated by a combination of solution and solid-phase chemistries and are cleaved off the solid-support for screening.

Identification of efficient pentapeptide substrates for the tyrosine kinase pp60c-src.

The development of inhibitors of protein tyrosine kinases (PTKs) is a promising approach to obtaining new therapeutic agents for a variety of diseases, particularly cancer. However, the discovery of peptide-based inhibitors has been hindered by the lack of small peptide substrate sequences (i.e. five residues or less) with which a variety of inhibitor designs could be readily evaluated by replacing the Tyr with natural and unnatural amino acids. These prototypical small peptide inhibitors could then form the basis for designing analogous conformationally constrained, peptide-mimetic or non-peptide inhibitors with improved therapeutic potential. In this study we have identified the best known small peptide substrate for the PTK pp60c-src, which is the parent of the src family of nonreceptor PTKs. This pentapeptide substrate, Ac-Ile-Tyr-Gly-Glu-Phe-NH2, has a Km of 368 microM and Vmax of 1.02 mumol/min/mg when tested utilizing the assay methodology of Budde et al. (Anal. Biochem. 1992, 200, 347-351) after a series of modifications were made to more closely simulate the conditions inside a typical mammalian cell. This substrate was designed from information obtained by Songyang et al. (Nature 1995, 373, 536-539) with a 2.5 billion member combinatorial library of peptide substrates for pp60c-src. A second pentapeptide substrate, Ac-Glu-Asp-Ala-Ile-Tyr-NH2, with a weaker binding affinity (Km = 880 microM) but improved Vmax (1.86 mumol/min/mg), was also identified. This peptide was designed from the pp60c-src autophosphorylation sequence and information obtained by Songyang et al. (Ibid.) and Till et al. (J. Biol. Chem. 1994, 269, 7423-7428) with combinatorial libraries of peptide substrates. These new substrates provide sufficient binding affinities and rates of phosphorylation to be utilized for evaluating the relative effectiveness of various reversible and mechanism-based irreversible inhibitor designs for pp60c-src while appended to easily prepared small peptides.

Tetrapeptide tyrosine kinase inhibitors. Enantioselective synthesis of p-hydroxymethyl-L-phenylalanine, incorporation into a tetrapeptide, and subsequent elaboration into p-(R,S-hydroxyphosphonomethyl)-L-phenylalanine.

A convenient enantioselective synthesis of p-hydroxymethyl-L-phenylalanine was developed which produces a 4/1 ratio of L/D enantiomers resulting from a chiral phase-transfer-catalyzed alkylation. This amino acid was coupled into the p56(1)ck tyrosine kinase substrate Ac-Leu-Pro-Tyr-Ala-NHCH3 as a replacement for Tyr and can subsequently be elaborated into a variety of potential tyrosine kinase inhibitor designs of general structure Ac-Leu-Pro-AA-Ala-NHCH3, wherein AA is an unnatural amino acid. The contaminating D enantiomer was readily removed after coupling to L-Ala-NHCH3 of this sequence. The utility of the p-hydroxymethyl functionality in an efficient divergent synthetic strategy leading to various inhibitor designs is illustrated with the synthesis of Ac-Leu-Pro-AA-Ala-NHCH3, wherein AA is p-(R,S-hydroxyphosphonomethyl)-L-phenylalanine.

Synthesis and use of 3-amino-4-phenyl-2-piperidones and 4-amino-2-benzazepin-3-ones as conformationally restricted phenylalanine isosteres in renin inhibitors.

The design of P2-P3 conformational restrictions in renin inhibitors by the use of a renin computer graphic model led to the synthesis of inhibitors containing N-Boc, N-acetyl, and N-phthalyl derivatives of 3(S)-amino-4(R,S)-2-piperidones and 4(S)-amino-2-benzazepinones in place of phenylalanine in the control compound N-acetyl-L-phenylalanyl-N-[4(S)-[(butylamino)carbonyl]-1(S)- (cyclohexylmethyl)-2(S)-hydroxy-5-methylhexyl]-L-norleuci namide (32). The piperidone inhibitors were prepared by utilization of the Evans chiral auxilliary to introduce the amino group with enantioselectivity and also to act as a leaving group in an intramolecular cyclization to the piperidone. The most potent inhibitor, 3(S)-(acetylamino)-alpha(S)-butyl-N-[4(S)- [(butylamino)carbonyl]-1(S)-(cyclohexylmethyl)-2(S)-hydroxy-5- methylhexyl]-2-oxo-4(R)-phenyl-1-piperidineacetamide (18, IC50 = 21 nM), was 25-fold less potent than the acyclic control 32. Considerable dependence of potency with the size of the P4 derivative was observed as had been expected based on the presynthetic modeling studies. Attempts to rationalize the observed potencies on the basis of further molecular modeling studies suggested that the loss in inhibitor potency was due to the conformational restrictions distorting the 3S center from the geometry present in the putative extended conformation present when the inhibitor is bound within the renin active site.

Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells.

An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-naphthalenyl-methyl) phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa beta-subunit of the insulin receptor (IC50 = 200 microM). The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 microM, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa beta-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. Our data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases.

An interactive computer graphics study of thermolysin-catalyzed peptide cleavage and inhibition by N-carboxymethyl dipeptides.

Interactive computer graphics was used as a tool in studying the cleavage mechanism of the model substrate Z-Phe-Phe-Leu-Trp by the zinc endopeptidase thermolysin. Two Michaelis complexes and three binding orientations of the tetrahedral intermediate to the crystal structure of thermolysin were investigated. Our results indicate that a Michaelis complex, which does not involve coordination of the scissile peptide to the zinc, is consistent with available experimental data and the most plausible of the two complexes. A tetrahedral intermediate complex wherein the two oxygens of the hydrated scissile peptide straddle the zinc in a bidentate fashion results in the most favorable interactions with the active site. The preferred tetrahedral intermediate and Michaelis complex provide a rationalization for the published substrate data. A trajectory for proceeding from the Michaelis complex to the tetrahedral intermediate is proposed. This trajectory involves a simultaneous activation of the zinc-bound water molecule concurrent with attack on the scissile peptide. A detailed ordered product release mechanism is also presented. These studies suggest some modifications and a number of extensions to the mechanism proposed earlier [Kester, W. R., & Matthews, B. W. (1977) Biochemistry 16, 2506; Holmes, M. A., & Matthews, B. W. (1981) Biochemistry 20, 6912]. The binding mode of the thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan [Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry (preceding paper in this issue)] is compared with that of the preferred tetrahedral intermediate, providing insight into this inhibitor design.