Structure-based design of caspase-1 inhibitor containing a diphenyl ether sulfonamide.

A series of compounds was designed and prepared as inhibitors of interleukin-1beta converting enzyme (ICE), also known as caspase-1. These inhibitors, which employ a diphenyl ether sulfonamide, were designed to improve potency by forming favorable interactions between the diphenyl ether rings and the prime side hydrophobic region. An X-ray crystal structure of a representative member of the diphenyl ether sulfonamide series bound to the active site of caspase-1 was obtained.

Development and validation of an ion-pair reversed-phase high-performance liquid chromatographic method for the separation of phosphonodipeptides.

The objective of this research was to develop a rapid, sensitive and reliable method for the separation of phosphonodipeptide prodrugs and parent compounds to facilitate the evaluation of cell permeation using in vitro cell culture models. Separation was accomplished isocratically within 10.0 min using a C18 (150x4.6 mm I.D., 3 microm) reversed-phase column. The mobile phase consisted of 5 mM tetrahexyl ammonium (ion-pair reagent) in 0.02 M phosphate buffer pH 6.5-acetonitrile (48.5:51.5, v/v). The flow-rate was 1.1 ml/min with detection at 221 nm. The standard curves were linear (r2>0.999) over the concentration range 1-100 microM. The method was reliable and reproducible, with the limit of quantitation being 1 microM (25 ng on column).

Hydrophobic D-amino acids in the design of peptide ligands for the pp60src SH2 domain.

Src homology-2 (SH2) domains, containing approximately 100 amino acid residues, are noncatalytic motifs involved with intracellular signal transduction. These domains can be found on nonreceptor kinases, phosphatases, and in regulatory adapter proteins among others. SH2 domains bind proteins containing phosphotyrosine (pTyr) residues in a sequence specific manner. Our efforts have focused on designing peptide mimetic ligands for the SH2 domain of the nonreceptor tyrosine kinase pp60src. We employed the cocrystal structure of the 11mer Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-IIe-Tyr-Leu IC50 = 800 nM as a starting point for our design efforts. These efforts have resulted in the discovery of tripeptide ligands containing D-amino acids that are only 2-fold less potent than the 11mer.

"Diversomers": an approach to nonpeptide, nonoligomeric chemical diversity.

Solid-phase chemistry, organic synthesis, and an apparatus for multiple, simultaneous synthesis have been combined to generate libraries of organic compounds ("diversomers"). Arrays of compounds were synthesized over two to three steps incorporating chemically diverse building blocks on a polystyrene-based solid support in a multiple, simultaneous manner. The generality of this approach is illustrated by the syntheses of dipeptides, hydantoins, and benzodiazepines.

Photo-modulated ion channels based on covalently linked gramicidins.

The covalent coupling of two gramicidin A monomers proved to be a useful tool for the rational design of ion channels with predictable electrophysiological properties (Stankovic, C.J., Heinemann, S.H., Delfino, J.M., Sigworth, F.J. and Schreiber, S.L. (1989) Science 244, 813-817; Stankovic, C.J., Heinemann, S.H. and Schreiber, S.L. (1990) J. Am. Chem. Soc. 112, 3702-3704). Herein we report on our first efforts to equip such channels with an artificial gating mechanism. Gramicidin monomers were covalently linked with 3,3'-azobis(benzeneacetic acid). Based on computer modeling of the beta-helix channel motif, this linker in its dark-adapted (trans) form does not allow for the formation of unimolecular ion channels, while the photo-activated (cis) form was expected to provide this possibility. The electrophysiological assays showed that (A) the trans-isomer does form characteristic ion channels, and (B) irradiation transforms these channels into a new distinct, flickering channel type in a reversible manner. The results are discussed in the framework of intermolecular gramicidin aggregates.

Purification of gramicidin A.

A simple chromatographic purification of the naturally occurring ion channel-forming pentadecapeptide gramicidin A (gA) is presented. This procedure allows gA to be isolated in gram quantities from the commercially available mixture of isomers after chromatography on silica gel. The gramicidin A obtained in this manner is greater than 95% pure as determined by 1HNMR, HPLC, and amino acid analysis.

Transmembrane channels based on tartaric acid-gramicidin A hybrids.

The gramicidin A transmembrane channel is believed to consist of two head-to-head beta helices. Computer-generated models were used to formulate the structure of new single-chain channel molecules based on the gramicidin motif. The chemical synthesis of two tartaric acid-gramicidin A hybrids and single-channel analyses of their conducting properties are reported. These studies illustrate the rational design and synthesis of long-lived channels with tunable conductance properties and provide support for current molecular models of the natural (dimeric) gramicidin channel.