Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein.

Leukotrienes are proinflammatory products of arachidonic acid oxidation by 5-lipoxygenase that have been shown to be involved in respiratory and cardiovascular diseases. The integral membrane protein FLAP is essential for leukotriene biosynthesis. We describe the x-ray crystal structures of human FLAP in complex with two leukotriene biosynthesis inhibitors at 4.0 and 4.2 angstrom resolution, respectively. The structures show that inhibitors bind in membrane-embedded pockets of FLAP, which suggests how these inhibitors prevent arachidonic acid from binding to FLAP and subsequently being transferred to 5-lipoxygenase, thereby preventing leukotriene biosynthesis. This structural information provides a platform for the development of therapeutics for respiratory and cardiovascular diseases.

The discovery of a potent and selective lethal factor inhibitor for adjunct therapy of anthrax infection.

A potent and selective anthrax LF inhibitor 40, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, was identified through SAR study of a high throughput screen lead. It has an IC50 of 54 nM in the enzyme assay and an IC50 of 210 nM in the macrophage cytotoxicity assay. Compound 40 is also effective in vivo in several animal model studies.

Homogeneous assays for adenosine 5'-monophosphate-activated protein kinase.

Allosteric activation of 5(')-AMP-activated protein kinase (AMPK) is currently of interest as an approach for the treatment of metabolic disorders because AMPK plays multiple roles in glucose and lipid metabolism. The availability of ultrafast, ultrasensitive, and robust assays suited to high-throughput screening (HTS) is key to obtaining small-molecule AMPK activators. In the absence of high-affinity and selective antiphospho Ser/Thr antibodies for AMPK substrates, we have developed two homogeneous AMPK assays with the commercially available antibody Anti-pS(133)-CREB and an engineered peptide ACC-CREBp. Anti-pS(133)-CREB antibody was raised against the phospho-CREB peptide derived from cAMP response element binding protein (CREB). ACC-CREBp was a variant (Arg to Pro) of ACC-CREB, a hybrid peptide consisting of a 9-amino-acid peptide from rat acetyl-CoA carboxylase (ACC), CREB peptide, and the addition of two hydrophobic Leu residues. ACC-CREBp showed increased suitability as a substrate for AMPK, eliminated phosphorylation by PKA, and preserved antibody binding. The homogeneous time-resolved fluorescence and AlphaScreen AMPK assays were developed using both Anti-pS(133)-CREB antibody and ACC-CREBp that are either labeled with a fluorescent probe or linked to a photoactivated bead, respectively. Thus, ACC-CREBp phosphorylation can be measured as a signal change resulting from the formation of antibody-antigen complex. This approach of adapting known antibody and antigenic peptide pairs to monitor alternate Ser/Thr kinases may be of general use.

Identification, localization and receptor characterization of novel mammalian substance P-like peptides.

Hemokinin-1 (HK-1) is a novel substance P (SP)-like peptide that is encoded by the preprotachykinin C (PPT-C) gene recently identified in mouse B cells and shown to be a potentially important regulator of B cell development (Nat. Immunol. 1 (2000) 392). We have now isolated and characterized the human and rat orthologs of PPT-C and examined activities of human and mouse HK-1 on the three tachykinin receptors, neurokinin-1-3 (NK1-3). The rat PPT-C polypeptide is highly homologous to mouse PPT-C and contains the same processing sites to generate predicted HK-1. The human PPT-C polypeptide is also homologous to mouse PPT-C, however, it contains two potential monobasic cleavage sites rather than a single dibasic cleavage site at the amino-terminal end of the predicted HK-1 peptide. Thus, human PPT-C has the potential to generate full length predicted HK-1 as well as a truncated version (HK-1(4-11)). Polymerase chain reaction analysis revealed that both human and mouse PPT-C were expressed in a variety of tissues with strong signals detected in the skin of both species and in the mouse brain. Binding and functional analysis indicated that human and mouse HK-1 peptides were nearly identical to SP in their overall activity profile on the three NK receptors with the most potent affinity for the NK1 receptor. The results indicate that PPT-C encodes another high affinity ligand of the NK1 receptor which may play an important role in mediating some of the physiological roles previously assigned to the NK1 receptor.

A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease.

A fluorescence resonance energy transfer assay has been developed for monitoring Bacillus anthracis lethal factor (LF) protease activity. A fluorogenic 16-mer peptide based on the known LF protease substrate MEK1 was synthesized and found to be cleaved by the enzyme at the anticipated site. Extension of this work to a fluorogenic 19-mer peptide, derived, in part, from a consensus sequence of known LF protease targets, produced a much better substrate, cleaving approximately 100 times more efficiently. This peptide sequence was modified further on resin to incorporate donor/quencher pairs to generate substrates for use in fluorescence resonance energy transfer-based appearance assays. All peptides cleaved at similar rates with signal/background ranging from 9-16 at 100% turnover. One of these substrates, denoted (Cou)Consensus(K(QSY-35)GG)-NH(2), was selected for additional assay optimization. A plate-based assay requiring only low nanomolar levels of enzyme was developed for screening and inhibitor characterization.

Photochemical preparation of a pyridone containing tetracycle: a Jak protein kinase inhibitor.

Jak3 is a protein tyrosine kinase that is associated with the shared gamma chain of receptors for cytokines IL2, IL4, IL7, IL9, and IL13. We have discovered that a pyridone-containing tetracycle (6) may be prepared from trisubstituted imidazole (5) in high yield by irradiation with >350 nm light. Compound 6 inhibits Jak3 with K(I)=5 nM; it also inhibits Jak family members Tyk2 and Jak2 with IC(50)=1 nM and murine Jak1with IC(50)=15 nM. Compound 6 was tested as an inhibitor of 21 other protein kinases; it inhibited these kinases with IC(50)s ranging from 130 nM to >10 microM. Compound 6 also blocks IL2 and IL4 dependent proliferation of CTLL cells and inhibits the phosphorylation of STAT5 (an in vivo substrate of the Jak family) as measured by Western blotting.