John M. Ostresh

Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity

Apoptosis resistance commonly occurs in cancers, preventing activation of Caspase family cell death proteases. XIAP is an endogenous inhibitor of Caspases overexpressed in many cancers. We developed an enzyme derepression assay, based on overcoming XIAP-mediated suppression of Caspase-3, and screened mixture-based combinatorial chemical libraries for compounds that reversed XIAP-mediated inhibition of Caspase-3, identifying a class of polyphenylureas with XIAP-inhibitory activity. These compounds, but not inactive structural analogs, stimulated increases in Caspase activity, directly induced apoptosis of many types of tumor cell lines in culture, and sensitized cancer cells to chemotherapeutic drugs. Active compounds also suppressed growth of established tumors in xenograft models in mice, while displaying little toxicity to normal tissues. These findings validate IAPs as targets for cancer drug discovery.

Induced conformational states of amphipathic peptides in aqueous/lipid environments

Specific conformational effects have been reported for amphipathic model peptides upon binding of defined hydrophobic domains to nonpolar stationary phases during reversed-phase high performance liquid chromatography (RP-HPLC). Such induced conformations are found to be especially pronounced for peptides that are amphipathic in an alpha-helical conformation. Such induced amphipathic conformations resulted in substantially later elution than predicted using amino acid-based retention coefficients. In the present study, the induced conformational behavior of model peptides observed during RP-HPLC was correlated with their secondary structure as determined by circular dichroism (CD) spectroscopy in both aqueous solution and C18-mimetic environments. The experimental retention times of the peptides studied were found to correlate with their CD spectra in the presence of lipids, whereas a poor correlation was observed with their CD spectra in the presence of trifluoroethanol. A new approach was developed to evaluate the induction of secondary structure in peptides due to interactions at aqueous/lipid interfaces, which involves the measurement of the CD ellipticities of peptides bound to a set of C18-coated quartz plates. An excellent correlation was found in this environment between the RP-HPLC retention times and CD ellipticities of the bound peptides.

Combinatorial chemistry: From peptides and peptidomimetics to small organic and heterocyclic compounds

Modified dipeptides have been used successfully for the generation of a variety of small organic and heterocyclic combinatorial libraries, including linear urea, polyamine, hydantoin, thiohydantoin, cyclic urea, cyclic thiourea and bicyclic guanidine. The synthesis and screening results for a number of these libraries are described. The solid phase synthesis of heterocyclic compounds such as diazepine and thiomorpholinone are also described.

Solid phase synthesis of heterocyclic compounds from linear peptides: Cyclic ureas and thioureas

Abstract The solid phase synthesis of cyclic ureas and thioureas is described. The reduction of acylated dipeptides followed by treatment with carbonyldiimidazole or thiocarbonyldiimidazole affords the corresponding cyclic urea or thiourea in good yield and high purity. This is an example of a broader approach to the solid phase synthesis of individual heterocyclic compounds and combinatorial libraries using peptides as starting materials.

The synthesis of peptidomimetic combinatorial libraries through successive amide alkylations

A soluble peptidomimetic combinatorial library of 57,500 compounds was prepared. This library has a dipeptide scaffold with each amide hydrogen replaced with five different alkyl groups (methyl, ethyl, allyl, benzyl, or naphthylmethyl). Solid-phase methodology in combination with N-alkylation were used to synthesize the library, which incorporated 50 different L-, D-, and unnatural amino acids. Repetitive amide alkylations were carried out on the solid support following each amino acid coupling step. Individual model compounds were synthesized in order to optimize the alkylation conditions, to study potential amino acid side chain modifications, to determine the extent of racemization, and to provide analytical controls during the library synthesis.

Solid phase synthesis of 1,3,4,7-Tetrasubstituted Perhydro-1,4-diazepine-2,5-diones

Abstract The solid phase synthesis of 1,3,4,7-Tetrasubstituted Perhydro-1,4-diazepine-2,5-diones is described. Starting from the resin-bound tBu ester of aspartic acid and employing reductive alkylation and amide formation, 40 diazepines have been synthesized in good yield and high purity. The general nature of this approach permits not only large numbers of individual diazepines to be prepared, but also combinatorial libraries.

Distal Recognition Site for Classical Pathway Convertase Located in the C345C/Netrin Module of Complement Component C5

Previous studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the α-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSFRYIYPLD. Alanine substitutions affected activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with <20% of the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the effects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5. These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase. This site lies ∼880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases.

GENERATION AND USE OF NONSUPPORT-BOUND PEPTIDE AND PEPTIDOMIMETIC COMBINATORIAL LIBRARIES

Publisher Summary The practical use of nonsupport-bound combinatorial libraries represents an important breakthrough in all the areas of basic research and drug discovery. The use of a wide variety of chemical transformations permits a range of peptidomimetic libraries to be generated that greatly expands the chemical diversity available. The results described in this chapter demonstrate that an existing peptide positional scanning- synthetic combinatorial libraries (PS-SCL) can be chemically transformed to generate a peptidomimetic SCL, from which highly active individual compounds can be identified. The synthesis and deconvolution methods developed for peptide libraries are easily applied to other types of chemical pharmacophores. The soluble nature of the nonsupport-bound combinatorial libraries is a distinct advantage over the other methods in that membrane-bound and whole cell assays can also be used. In addition, the deconvolution methods used allow the chemical structure of peptidic, peptidomimetic, and organic compounds to be determined based solely on the structural similarities of compounds, within each active pool or sublibrary.

Parallel solid phase synthesis of tetrasubstituted diethylenetriamines via selective amide alkylation and exhaustive reduction of N-acylated dipeptides

Abstract Polyamines are a rapidly developing area of vital importance to biomedical science. Selective N-alkylation followed by N-terminal acylation and the complete reduction of carbonyl amide bonds enables the preparation by parallel solid phase synthesis of a wide range of N 1 ,N 5 ,1,4-tetrasubstituted-1,5-diamino-3-azapentane derivatives.

Solid phase synthesis of 1,4-benzothiazepin-5-one derivatives

Abstract The solid phase synthesis of 1,4-benzothiazepin-5-one derivatives, resulting from the reaction of resin-bound protected cysteine with 2-fluoro-5-nitro-benzoic acid followed by a reductive alkylation and an intra molecular cyclization, is described.