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.

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.

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) ≈50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit “point of no return” model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.

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.

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.

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)-NH2, was selected for additional assay optimization. A plate-based assay requiring only low nano-molar levels of enzyme was developed for screening and inhibitor characterization.

Comparison of SAR for a tetrapeptide inhibitor of MHC class II with existing information endorses a binding mode consistent with naturally loaded full length peptides.

A depsipeptide mimic of the cram-positive muramyl peptide was synthesized on resin using both Boc and Fmoc protection strategies. The depsipeptide unit is chemically and stereochemically compatible with both Boc and Fmoc chemistries and with HF cleavage conditions.

The benzyl‐ester bond linking the C‐terminal δ‐aminovaleric acid residue of a peptide to a polymeric support was cleaved with liquid hydrogen fluoride in the presence of anisole, added as scavenger. Instead of the expected peptide with a free carboxyl group at the C‐terminus, a peptide terminating in a ketone derivative was obtained. The unusual extent of this known side‐reaction was attributed to the effect of the distance between the amino group and the carboxyl group in the C‐terminal residue. The results of model experiments corroborated this view.