ABSTRACT
The design, synthesis, and evaluation of dipeptide analogues as ligands for the pp60c-src SH2 domain are described. The critical binding interactions between Ac-Tyr-Glu-N(n-C5H11)2 (2) and the protein are established and form the basis for our structure-based drug design efforts. The effects of changes in both the C-terminal (11-27) and N-terminal (51-69) portions of the dipeptide are explored. Analogues with reduced overall charge (92-95) are also investigated. We demonstrate the feasibility of pairing structurally diverse subunits in a modest dipeptide framework with the goal of increasing the druglike attributes without sacrificing binding affinity.
Subject(s)
Dipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , src Homology Domains , Crystallography, X-Ray , Dipeptides/chemical synthesis , Dipeptides/chemistry , Dipeptides/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Ligands , Models, Molecular , Molecular Conformation , Proto-Oncogene Proteins pp60(c-src)/metabolism , Structure-Activity RelationshipABSTRACT
Thermodynamic measurements, structural determinations, and molecular computations were applied to a series of peptide ligands of the pp60(c-src) SH2 domain in an attempt to understand the critical binding determinants for this class of molecules. Isothermal titration calorimetry (ITC) measurements were combined with structural data derived from X-ray crystallographic studies on 12 peptide-SH2 domain complexes. The peptide ligands studied fall into two general classes: (1) dipeptides of the general framework N-acetylphosphotyrosine (or phosphotyrosine replacement)-Glu or methionine (or S-methylcysteine)-X, where X represents a hydrophobic amine, and (2) tetra- or pentapeptides of the general framework N-acetylphosphotyrosine-Glu-Glu-Ile-X, where X represents either Glu, Gln, or NH2. Dipeptide analogs which featured X as either hexanolamine or heptanolamine were able to pick up new hydrogen bonds involving their hydroxyl groups within a predominantly lipophilic surface cavity. However, due to internal strain as well as the solvent accessibility of the new hydrogen bonds formed, no net increase in binding affinity was observed. Phosphatase-resistant benzylmalonate and alpha,alpha-difluorobenzyl phosphonate analogs of phosphotyrosine retained some binding affinity for the pp60(c-src) SH2 domain but caused local structural perturbations in the phosphotyrosine-binding site. In the case where a reversible covalent thiohemiacetal was formed between a formylated phosphotyrosine analog and the thiol side chain of Cys-188, deltaS was 25.6 cal/(mol K) lower than for the nonformylated phosphotyrosine parent. Normal mode calculations show that the dramatic decrease in entropy observed for the covalent thiohemiacetal complex is due to the inability of the phosphotyrosine moiety to transform lost rotational and translational degrees of freedom into new vibrational modes.
Subject(s)
Peptides/chemistry , Protein Structure, Tertiary , Proto-Oncogene Proteins pp60(c-src)/chemistry , src Homology Domains , Amino Acid Sequence , Crystallography, X-Ray , Dipeptides/chemistry , Dipeptides/metabolism , Kinetics , Ligands , Oligopeptides/chemistry , Oligopeptides/metabolism , Peptides/metabolism , Phosphotyrosine/chemistry , Phosphotyrosine/metabolism , Protein Binding , Proto-Oncogene Proteins pp60(c-src)/metabolism , Structure-Activity Relationship , ThermodynamicsABSTRACT
1-[5-(2-Hydroxy-1,3-dimethyl-4-pentenyl)-2,2,4-trimethyl-1,3- dioxolan-4-yl]-2-methyl-3-pentanone, C19H34O4, Mr = 326.48, monocline, P2i, a = 11.409 (3), b = 7.889 (2), c = 11.690 (3) A, beta = 109.24 (2) degrees, V = 993.4 (4) A3, Z = 2, Dx = 1.09 g cm-3, Mo K alpha, lambda = 0.71069 A, mu = 0.6963 cm-1, F(000) = 360, T = 194 K, R = 0.0595 for 1496 reflections [Fo greater than or equal to 4 sigma(Fo)]. Molecules stack into columns parallel to the screw axis. Molecules are hydrogen bonded within each column. A hydrogen bond involving the hydroxyl group of one molecule and the carbonyl O of a second molecule (related by - x, -1/2 + y, - z) with an O...O distance of 2.859 (7) A, an H...O distance of 2.01 (7) A and an O--H...O angle of 164 (6) degrees is observed.
Subject(s)
Erythromycin/analogs & derivatives , Chemical Phenomena , Chemistry, Physical , Molecular Structure , X-Ray DiffractionABSTRACT
C19H34O4, Mr = 326.48, orthorhombic, P2(1)2(1)2(1), a = 7.949 (2), b = 14.880 (5), c = 17.047 (5) A, V = 2016 (1) A3, Z = 4, Dx = 1.08 g cm-3, mu = 0.6861 cm-1, lambda(Mo K alpha) = 0.71069 A, F(000) = 720, T = 163 K, R = 0.0644 for 1476 reflections [Fo greater than or equal to 4 sigma (Fo)]. There is an intermolecular H bond involving the hydroxyl group and the carbonyl O (related by 1/2 + x, 3/2 - y, 1 - z) with an O...O distance of 2.838 (5) A, an H...O distance of 2.06 (5) A and an O-H...O angle of 172 (6) degrees. There are no such intramolecular contacts.
