ABSTRACT
To evaluate N-hydroxyurea as zinc binding group in the design of MMP inhibitors, two peptidyl 1-hydroxyureas were prepared by N-hydroxycarbamoylation of the diastereomeric dipeptides H-Leu-Phe-NHMe and H-D-Leu-Phe-NHMe. Peptidyl 1-hydroxyureas were more potent than the parent peptides, but dramatically weaker (4-5 orders of magnitude) than the isosteric (R)-succinylhydroxamate analogue, which displays IC(50) in the range of nM vs MMP-1, -3, -7 and sub-nM vs MMP-2, -8, and -9. The peptidyl 1-hydroxyurea 1a attained an IC(50) of 20 microM vs MMP-9, and substantially approaches inhibition of known N-hydroxyureas based on aminoacids or peptides against other zinc metalloenzymes and non-peptidic N-hydroxyureas against MMPs. Strong preference of the O-N1-C=O unit for the antiperiplanar amide bond conformation seems to be the major limit for more effective zinc chelation. Methylation of a peptidyl 1-hydroxyurea at N3, to promote the synperiplanar O-N1-C=O conformation required for zinc chelation and improve affinity, resulted in release of a methylimidazolidine-2,4-dione through an undesired intramolecular reaction reminiscent of the Edman peptide degradation.
Subject(s)
Dipeptides/chemical synthesis , Hydroxamic Acids/chemistry , Hydroxyurea/analogs & derivatives , Hydroxyurea/chemical synthesis , Matrix Metalloproteinase Inhibitors , Succinates/chemistry , Animals , Dipeptides/chemistry , Humans , Hydroxyurea/chemistry , Matrix Metalloproteinases/chemistry , Mice , Stereoisomerism , Structure-Activity RelationshipABSTRACT
(R)-alpha-Biphenylsulfonylamino 2-methylpropyl phosphonates attain nM potency against several MMPs and are the most effective inhibitors based on phosphonate as zinc binding group. Since their preparation by direct N-acylation of expensive, enantiopure, alpha-aminophosphonic acids proceeds in low yields, we devised and evaluated a stereoselective and straightforward method of synthesis that avoids the unfavourable step of N-acylation. The key intermediate (R)-4-bromophenylsulfonylamino 2-methylpropyl phosphonate 9 was obtained by highly stereoselective addition of dibenzylphosphite to the enantiopure (S)-N-isobutylidene-p-bromobenzenesulfinamide 3, followed by oxidation with m-CPBA. Suzuki coupling of 9 with the desired arylboronic acids, gave the expected biphenylsulfonylamino derivatives in satisfactory yields. Liberation of the phosphonic group by hydrogenolysis led to the desired (R)-alpha-biphenylsulfonylamino 2-methylpropyl phosphonates 14a-i. Screening of the new compounds on MMP-1, -2, -3, -7, -8, -9, -13 and -14 showed IC(50) in the range of nM in most cases.
Subject(s)
Biphenyl Compounds/chemical synthesis , Biphenyl Compounds/pharmacology , Matrix Metalloproteinase Inhibitors , Organophosphonates/chemical synthesis , Organophosphonates/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Humans , Indicators and Reagents , Isoenzymes/antagonists & inhibitors , Magnetic Resonance Spectroscopy , Models, Molecular , Recombinant Proteins , Spectrophotometry, Ultraviolet , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The first crystallographic structure of an N-hydroxyurea inhibitor bound into the active site of a matrix metalloproteinase is reported. The ligand and three other analogues were prepared and studied as inhibitors of MMP-2, MMP-3, and MMP-8. The crystal structure of the complex with MMP-8 shows that the N-hydroxyurea, contrary to the analogous hydroxamate, binds the catalytic zinc ion in a monodentate rather than bidentate mode and with high out-of-plane distortion of the amide bonds.
Subject(s)
Enzyme Inhibitors/pharmacology , Hydroxyurea/chemistry , Matrix Metalloproteinase 8/chemistry , Matrix Metalloproteinase Inhibitors , Zinc/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Escherichia coli/metabolism , Humans , Hydrogen-Ion Concentration , Inhibitory Concentration 50 , Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase 3/chemistry , Matrix Metalloproteinase 8/metabolism , Models, Chemical , Models, Molecular , Oxygen/chemistry , Phenylalanine/analogs & derivatives , Phenylalanine/chemistry , Protein Binding , Protein Conformation , Thiophenes/chemistryABSTRACT
Three novel peptidomimetic phosphinate inhibitors have been synthesized and evaluated as inhibitors of matrix metalloproteinases MMP-2 and MMP-8. Their IC50 values are in the micromolar range, and one of them showed to be the most effective inhibitor of MMP-2. The differences in binding affinities for MMP-2 and MMP-8 of the three phosphinates have been rationalized by means of modelling studies and molecular dynamics simulations.
Subject(s)
Drug Design , Matrix Metalloproteinase Inhibitors , Models, Chemical , Phosphorous Acids/chemistry , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Inhibitory Concentration 50 , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 8/metabolism , Molecular Structure , Phosphorous Acids/chemical synthesis , Phosphorous Acids/pharmacology , Protease Inhibitors/chemistryABSTRACT
The phosphotryptophan derivative l-Pro-l-Leu-l-(P)Trp(OH)(2) (2b) was reported as the first example of left-hand-sideLeft-hand-side inhibitors: inhibitors that bind in the unprime region of the enzyme active site, in reference to the convention of drawing the unprimed residues of a peptide substrate on the left side. [R.P. Beckett et al., Drug Discov. Today 1 (1996) 16-26]. The opposite applies to right-hand-side inhibitors. phosphonate inhibitor of MMP-8. Its uncommon mode of binding to MMP-8 was mainly ascribed to the presence of the proline residue in P(3). Ten new analogues of 2b were obtained by replacement of the aminoterminal l-Pro with aminoacid residues bearing small side chains. Most of the new analogues show an increase of affinity for MMP-2 and MMP-8, and different profiles of selectivity. Computer simulations were performed to explain the effects of substitutions on the preferred mode of binding. They reveal that most of the new analogues are probably accommodated in the right, rather than left-hand side of MMP-8 active site.
