Design and synthesis of a series of (2R)-N(4)-hydroxy-2-(3-hydroxybenzyl)-N(1)- [(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]butanediamide derivatives as potent, selective, and orally bioavailable aggrecanase inhibitors.

A pharmacophore model of the P1' site, specific for aggrecanase, was defined using the specificity studies of the matrix metalloproteinases and the similar biological activity of aggrecanase and MMP-8. Incorporation of the side chain of a tyrosine residue into compound 1 as the P1' group provided modest selectivity for aggrecanase over MMP-1, -2, and -9. A cis-(1S)(2R)-amino-2-indanol scaffold was incorporated as a tyrosine mimic (P2') to conformationally constrain 2. Further optimization resulted in compound 11, a potent, selective, and orally bioavailable inhibitor of aggrecanase.

Discovery of macrocyclic hydroxamic acids containing biphenylmethyl derivatives at P1', a series of selective TNF-alpha converting enzyme inhibitors with potent cellular activity in the inhibition of TNF-alpha release.

SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.

P1, P2'-linked macrocyclic amine derivatives as matrix metalloproteinase inhibitors.

A novel series of 13- and 14-membered macrocyclic amines was developed by linking the P1 and P2' groups. The synthesis entails stereoselective Frater alkylation to install the anti-succinate configuration and macrocyclic amination via nucleophilic displacement. This strategy resulted in a new class of conformationally constrained inhibitors that are potent and selective for MMP-8 and 9 over MMP-1 and 3.

Macrocyclic hydroxamate inhibitors of matrix metalloproteinases and TNF-alpha production.

Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.

Corticotropin-releasing hormone receptor antagonists: framework design and synthesis guided by ligand conformational studies.

As described in the preceding paper (Arvanitis et al. J. Med. Chem. 1999, 42), anilinopyrimidines I were identified as potent antagonists of corticotropin-releasing hormone-1 receptor (CRH1-R, also referred to as corticotropin-releasing factor, CRF1-R). Our next goal was to understand the receptor-bound conformation of the antagonists and to use this information to help guide preclinical optimization of the series and to develop new leads. Since receptor structural information was not available, we assumed that these small, high-affinity antagonists would tend to bind in conformations at or energetically close to their global minima and that rigid analogues that maintained the important stereoelectronic features of the bound anilinopyrimidine would also bind tightly. Conformational preferences and barriers to rotation of the anilinopyrimidines were determined by semiempirical methods, and X-ray and variable-temperature NMR spectroscopy provided experimental results that correlated well with calculated structures. Using these data, a key dihedral angle was constrained to design fused-ring analogues, substituted N-arylpyrrolopyridines II, synthesis of which provided CRH1 receptor antagonists with potency equal to that of the initial congeneric leads (Ki = 1 nM) and which closely matched the conformation held by the original compound, as determined by crystallography. In addition to providing a useful template for further analogue synthesis, the study unequivocally determined the active conformation of the anilinopyrimidines. Theoretical and spectroscopic studies, synthesis, and receptor binding data are presented.

Synthesis, corticotropin-releasing factor receptor binding affinity, and pharmacokinetic properties of triazolo-, imidazo-, and pyrrolopyrimidines and -pyridines.

The synthesis and CRF receptor binding affinities of several new series of N-aryltriazolo- and -imidazopyrimidines and -pyridines are described. These cyclized systems were prepared from appropriately substituted diaminopyrimidines or -pyridines by nitrous acid, orthoester, or acyl halide treatment. Variations of amino (ether) pendants and aromatic substituents have defined the structure-activity relationships of these series and resulted in the identification of a variety of high-affinity agents (Ki's < 10 nM). On the basis of this property and lipophilicity differences, six of these compounds (4d,i,n,x, 8k, 9a) were initially chosen for rat pharmacokinetic (PK) studies. Good oral bioavailability, high plasma levels, and duration of four of these compounds (4d,i,n,x) prompted further PK studies in the dog following both iv and oral routes of administration. Results from this work indicated 4i,x had properties we believe necessary for a potential therapeutic agent, and 4i1 has been selected for further pharmacological studies that will be reported in due course.

Structure-based design and synthesis of a series of hydroxamic acids with a quaternary-hydroxy group in P1 as inhibitors of matrix metalloproteinases.

Examination of the S1 area of the active site of pro-stromelysin has led us to the design of novel and potent inhibitors of matrix metalloproteinases containing constrained quaternary-hydroxy group at P1. The synthesis and biological activity of these compounds with variations at P1', P2', and P3' will be described.

Fitting an inhibitor into the active site of thermolysin: a molecular dynamics case study.

We used molecular dynamics computer simulation to "fly" a small flexible ligand, L-leucine hydroxamic acid, into the active site of thermolysin. The potential, which imposed no constraints between protein and ligand, produced conformations close to the crystallographically determined one. The calculations made use of the combined molecular mechanics/grid method of Luty et al. (J. Comp. Chem. 16:454-464, 1995), in which atoms of the active site are free to move whereas the rest of the protein, assumed to be rigid, is represented as points of a grid, and which also includes an implicit solvation model. The method is sufficiently fast that large sets of simulations could be carried out, enabling statistical sampling and exploration of the effect of initial position and conformation of the ligand on the probability of successful docking. In a charged catalytic Glu/uncharged ligand regime, when the initial position of the ligand was determined by random translations and rotations that kept the center of mass within 8.0 angstroms of the crystal one, none of the 20 runs placed the ligand correctly. In a second set with uncharged Glu and zwitterionic ligand, 3 of 24 similarly placed random structures flew the ligand in successfully. In a third set with the same protonation scheme as the second the starting positions had randomly determined conformations but kept the hydroxamate oxygens within 4.0 angstroms of the zinc; in this case 22 of 25 runs reoriented correctly. A diverse set of energetic, structural, and dynamic criteria was used for evaluation of the calculations. The results indicate the method to be a promising tool for the rational drug design process.

Molecular characterization of helix-loop-helix peptides.

A class of regulators of eukaryotic gene expression contains a conserved amino acid sequence responsible for protein oligomerization and binding to DNA. This structure consists of an arginine- and lysine-rich basic region followed by a helix-loop-helix motif, which together mediate specific binding to DNA. Peptides were prepared that span this motif in the MyoD protein; in solution, they formed alpha-helical dimers and tetramers. They bound to DNA as dimers and their alpha-helical content increased on binding. Parallel and antiparallel four-helix models of the DNA-bound dimer were constructed. Peptides containing disulfide bonds were engineered to test the correctness of the two models. A disulfide that is compatible with the parallel model promotes specific interaction with DNA, whereas a disulfide compatible with the antiparallel model abolishes specific binding. Electron paramagnetic resonance (EPR) measurements of nitroxide-labeled peptides provided intersubunit distance measurements that also supported the parallel model.

A molecular dynamics investigation of the elastomeric restoring force in elastin.

A repetitive polypentapeptide organized as a connected chain of beta-bends is believed to be an important structural element of elastin, the major elastomer in biological systems. Molecular dynamics simulations were carried out on hydrated polymers of (Val-Pro-Gly-Val- Gly)18 at various extensions. Analysis of the fluctuations of backbone angles in relaxed elastin showed that particularly large-amplitude torsional motions occur in phi and psi angles of residues connecting sequentially adjacent hairpin bends. Many such motions reflect peptide plane librations that result from anticorrelated crankshaft rotations of psi i and phi i+1. These effects were much reduced in stretched polymer models. The conformational entropy of relaxed and stretched elastin models was estimated using a treatment due to Meirovitch, and gave a calculated decrease in entropy of about 1 cal/mol deg when the polymer was stretched to 1.75 times its original length. There are large changes in solvent-accessible surface area during the initial stages of elastin stretching. Collectively these results suggest that hydrophobic interactions make contributions to elastin entropy at low extensions, but that librational mechanisms make larger contributions to the elastic restoring force at longer extensions.

Protein design, a minimalist approach.

The question of how the amino acid sequence of a protein specifies its three-dimensional structure remains to be answered. Proteins are so large and complex that it is difficult to discern the features in their sequences that contribute to their structural stability and function. One approach to this problem is de novo design of model proteins, much simpler than their natural counterparts, yet containing sufficient information in their sequences to specify a given function (for example, folding in aqueous solution, folding in membranes, or formation of ion channels). Designed proteins provide simple model systems for understanding protein structure and function.

Synthetic amphiphilic peptide models for protein ion channels.

Ion channel proteins are important for the conduction of ions across biological membranes. Recent analyses of their sequences have suggested that they are composed of bundles of alpha-helices that associate to form ion-conducting channels. To gain insight into the mechanisms by which alpha-helices can aggregate and conduct ions, three model peptides containing only leucine and serine residues were synthesized and characterized. A 21-residue peptide, H2N-(Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2, which was designed to be a membrane-spanning amphiphilic alpha-helix, formed well-defined ion channels with ion permeability and lifetime characteristics resembling the acetylcholine receptor. In contrast, a 14-residue version of this peptide, which was too short to span the phospolipid bilayer as an alpha-helix, failed to form discrete, stable channels. A third peptide, H2N-(Leu-Ser-Leu-Leu-Leu-Ser-Leu)3-CONH2, in which one serine per heptad repeat was replaced by leucine, produced proton-selective channels. Computer graphics and energy minimization were used to create molecular models that were consistent with the observed properties of the channels.

Computer simulation of biological interactions and reactivity.

Computer simulations of molecular motion provide a useful tool for analyzing dynamic aspects of macromolecular structure and function. In many cases, simulations can be compared to experimental results that provide an average estimate of molecular flexibility. For example, variations in computed molecular motions in different regions of a protein structure can be compared to refined B-values obtained from X-ray crystallographic refinement. Such comparisons both provide a detailed view of the motions responsible for crystalline disorder, and allow an evaluation of how crystal packing affects mobility of groups on the protein surface. In these applications, dynamics simulations provide a means of regenerating the temporal dimension of a structure whose average behavior is experimentally well defined in the crystal lattice. An additional benefit of the detailed and instantaneous view of molecular flexibility offered by simulation methods lies in its potential for exploring infrequent structural fluctuations or dynamic states of molecular association that cannot be examined in detail by X-ray methods, but are suggested on the basis of alternative structural information. For example, studies of the effects of surface chemical modification on interacting proteins can produce information concerning the sites, if not the exact details, of the intermolecular interactions. The present work describes some applications of molecular dynamics methods to the study of large molecular aggregates whose dynamic properties thus far have precluded detailed structural descriptions. These include simulations of an electrostatically associated electron transfer complex between cytochromes c and b5, some model systems for trans-membrane ion channels, and a phospholipid micelle.