A fluorescence polarization assay for inhibitors of Hsp90.

Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC50s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors.

The carbenoid approach to peptide synthesis.

A different approach to the synthesis of dipeptides is described based on the formation of the NHCHR1CONH-CHR2CO bond by carbenoid N-H insertion, rather than the formation of the peptide bond itself. Thus decomposition of triethyl diazophosphonoacetate catalysed by rhodium(II) acetate in the presence of N-protected amino acid amides 8 gives the phosphonates 9. Subsequent Wadsworth-Emmons reaction of 9 with aldehydes in the presence of DBU gives dehydro dipeptides 10. The reaction has been extended to a simple two-step procedure, without the isolation of the intermediate phosphonate, for conversion of a range of amino acid amides 11 into dehydro dipeptides 12 and to an N-methylamide 11 h, and for conversion of a dipeptide to tripeptide (13-->14). Direct conversion, by using methyl diazophenylacetate, of amino acid amides to phenylglycine-containing dipeptides 19 proceeds in good chemical yield, but with poor diastereoselectivity.

Inhibition of inducible nitric oxide synthase by acetamidine derivatives of hetero-substituted lysine and homolysine.

The synthesis and in vitro evaluation of the acetamidine derivatives of hetero-substituted lysine and homolysine analogues have identified potent inhibitors of human nitric oxide synthase enzymes, including examples with marked selectivity for the inducible isoform.

Synthesis and SAR of 4-aryl-2-hydroxy-4-oxobut-2-enoic acids and esters and 2-amino-4-aryl-4-oxobut-2-enoic acids and esters: potent inhibitors of kynurenine-3-hydroxylase as potential neuroprotective agents.

The synthesis and structure-activity relationship of a series of 4-aryl-2-hydroxy-4-oxobut-2-enoic acids and esters and 2-amino-4-aryl-4-oxobut-2-enoic acids and esters as potent inhibitors of kynurenine-3-hydroxylase are described. These compounds are the most potent inhibitors of the kynurenine-3-hydroxylase enzyme so far disclosed. Additionally methyl 4-(3-chlorophenyl)-2-hydroxy-4-oxobut-2-enoate (2d), 4-(3-chlorophenyl)-2-hydroxy-4-oxobut-2-enoic acid (3d), methyl 4-(3-fluorophenyl)-2-hydroxy-4-oxobut-2-enoate (2f), and 4-(3-fluorophenyl)-2-hydroxy-4-oxobut-2-enoic acid (3f) prevent the increase in the interferon-gamma-induced synthesis of quinolinic acid in primary cultures of cultured human peripheral blood monocyte-derived macrophages.

S-aryl cysteine S,S-dioxides as inhibitors of mammalian kynureninase.

A series of 2-amino-S-aryl cysteine S,S-dioxides have been synthesised and shown to inhibit kynureninase an important enzyme in the biosynthesis of the known excitotoxic moiety quinolinic acid. The most potent of these, 2-amino-5-methyl-S-phenyl cysteine S,S-dioxide 6d, inhibits interferon-gamma induced synthesis of quinolinic acid in human macrophages.

Rationally designed "dipeptoid" analogues of CCK. Acid mimics of the potent and selective non-peptide CCK-B receptor antagonist CI-988.

This paper outlines the synthesis of selected acid mimics of the non-peptide CCK-B selective antagonist CI-988, 1. CCK-B and CCK-A binding affinities of these analogues are described and their CCK-B affinity and selectivity rationalized by consideration of the pK(a) values, charge distribution, and geometry of the respective acid mimics. Several of the compounds have CCK-B binding affinities similar to the parent carboxylic acid 1 (CCK-B, IC50 = 1.7 nM; pK(a) = 5.6) and span a pK(a) range of less than 1 (sulfonic acid 27) to greater than 9.5 (5-thio-1,2,4-triazole 24). Among the more active compounds synthesized are tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[2-[[2-[[(3-hydroxy-5-isoxazolyl)acetyl]-amino]-2- phenylethyl]amino]-1-(1H-indol-3-ylmethyl)-1-methyl-2-oxoethyl+ ++]carbamate (15), tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[1-(1H-indol-3-ylmethyl)-1-methyl-2-oxo-2-[[2-[(1-oxo- 3-sulfopropyl)amino]-2-phenylethyl]amino]-ethyl]carbamate, monosodium salt (27), and tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[1-(1H-indol-3-ylmethyl)-1- methyl-2-oxo-2-[[2-[[(1H-1,2,4-triazol-5-ylsulfinyl)acetyl]a mino]-2-phenylethyl]amino]ethyl]carbamic acid (34) which have CCK-B binding affinities of IC50 = 2.6, 1.3, and 1.7 nM, CCK-A/-B ratios of 650, 780, and 550 and pK(a) values of 6.5, less than 1, and 7.0, respectively.