Design of selective and soluble inhibitors of tumor necrosis factor-alpha converting enzyme (TACE).

A program to improve upon the in vitro, in vivo, and physicochemical properties of N-hydroxyformamide TACE inhibitor GW 3333 (1) is described. Using the primary structure of pro-TNF-alpha, along with a homology model of the catalytic domain of TACE based on the X-ray diffraction coordinates of adamalysin, we synthesized N-hydroxyformamide TACE inhibitors containing a P2' arginine side chain. Introduction of nitro and sulfonyl electron-withdrawing groups covalently bound to the P2' guanidine moiety rendered the inhibitors electronically neutral at cellular pH and led to potent inhibition of TNF-alpha release from stimulated macrophages. Inhibitors containing these arginine mimetics were found to have increased solubility in simulated gastric fluid (SGF) relative to 1, allowing for the incorporation of lipophilic P1' side chains which had the effect of retaining potent TACE inhibition, but reducing potency against matrix metalloproteases (MMPs) thus increasing overall selectivity against MMP1, MMP3, and MMP9. Selected compounds showed good to excellent in vivo TNF inhibition when administered via subcutaneous injection. One inhibitor, 28a, with roughly 10x selectivity over MMP1 and MMP3 and high solubility in SGF, was evaluated in the rat zymosan-induced pleuisy model of inflammation and found to inhibit zymosan-stimulated pleural TNF-alpha elevation by 30%.

N-hydroxyformamide peptidomimetics as TACE/matrix metalloprotease inhibitors: oral activity via P1' isobutyl substitution.

N-Hydroxyformamide-class metalloprotease inhibitors were designed and synthesized, which have potent broad-spectrum activity versus matrix metalloproteases and TNF-alpha converting enzyme (TACE). Compound 13c possesses good oral and intravenous pharmacokinetics in the rat and dog.

Phospholipid-transfer activities in cytosols from lung, isolated alveolar type II cells and alveolar type II cell-derived adenomas.

We have examined phospholipid-transfer activities in cytosols from rat and mouse whole lung, isolated rat alveolar type II cells and alveolar type II cell-derived mouse pulmonary adenomas. We report an enrichment in phosphatidylcholine and phosphatidylglycerol (but not phosphatidylinositol) protein-catalysed transfer in the type II cell and adenoma cytosols compared with the whole-lung cytosols. The activities from these cytosols were resolved using column chromatofocusing, which clearly demonstrated the presence of a phosphatidylcholine-specific transfer protein in each of the four tissues. In addition, two proteins (rat) or three proteins (mouse) catalysing both phosphatidylcholine and phosphatidylglycerol transfer were resolved from whole lung, whereas in both the rat isolated alveolar type II cells and the mouse type II cell-derived adenomas one of these less specific proteins is not present.

Spontaneous and protein-catalyzed transfer of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) between phospholipid bilayers.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor or alkylacetyl-GPC), a bioactive phospholipid that possesses hypotensive, platelet-aggregating and inflammatory properties, is known to be secreted by a variety of cell types. The biological activity of alkylacetyl-GPC is related to a precise chemical structure that implicates interaction with proteins. We have studied the spontaneous and protein-catalyzed transfer of alkylacetyl-GPC between phospholipid vesicles and have demonstrated the following: 1. There are at least two transferable pools of alkylacetyl-GPC in sonicated phospholipid vesicles. 2. These two pools differ in the rate at which they dissociate from the vesicles; one pool equilibrates between donor and acceptor vesicles instantaneously while the other pool is transferred much more slowly. 3. Dialysis of alkylacetyl-GPC between phospholipid vesicles through the aqueous phase is slow. 4. A protein fraction derived from rat lung cytosol catalyzes the transfer of the nonequilibrating pool of alkylacetyl-GPC between phospholipid vesicles; this transfer is superimposed on the spontaneous transfer and is unchanged in experiments using vesicles from which the rapidly equilibrating pool has been removed.