Impermeant antitumor sulfonylurea conjugates that inhibit plasma membrane NADH oxidase and growth of HeLa cells in culture. Identification of binding proteins from sera of cancer patients.
The antitumor sulfonylurea LY237868 (N-(4-aminophenyl-sulfonyl)-N'-(4-chlorophenyl)urea) was conjugated through the A ring to alpha-cyclodextrin or agarose bead material (Affigel 10) to prepare impermeant conjugates for activity measurements and affinity isolation of binding proteins from serum. When conjugated to alpha-cyclodextrin, the resulting LY237868 conjugate inhibited both NADH oxidase activity and growth of HeLa cells in culture. The conjugate was at least one order of magnitude more potent as an inhibitor than the parent compound. These findings confirm previous results that demonstrate an antitumor sulfonylurea-binding protein with NADH oxidase activity at the external plasma membrane surface of HeLa cells that is shed into culture media conditioned by growth of HeLa cells. A comparable activity, responsive to sulfonylurea, was present in sera of cancer patients. LY237868 conjugated to agarose beads as the affinity support bound a large number of serum proteins. However, compared to serum from normal patients, the affinity support bound two proteins of M(r) approx. 33.5 and 29.5 not found in sera of normal patients. The 33.5 kDa protein from human sera reacted with antisera to a 33.5 kDa protein from culture media conditioned by growth of HeLa cells that blocked and immunoprecipitated the sulfonylurea-responsive activity from HeLa cell plasma membranes. The results point to the 33.5 kDa protein from cancer patient sera that bound to the sulfonylurea affinity support as representing the circulating equivalent of the previously identified 34 kDa sulfonylurea-binding protein, with NADH oxidase activity at the external cell surface of cultured HeLa cells and a corresponding 33.5 kDa protein shed into culture media conditioned by growth of HeLa cells.
Kim
C
C
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
MacKellar
W C
WC
Cho
N M
NM
Byrn
S R
SR
Morré
D J
DJ
D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000633:toxicity; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D003505:Cyclodextrins; D004791:Enzyme Inhibitors; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D009363:Neoplasm Proteins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D009369:Neoplasms / Q000097:blood; D011485:Protein Binding; D013453:Sulfonylurea Compounds / Q000494:pharmacology* / Q000633:toxicity; D047391:alpha-Cyclodextrins
D000970:Antineoplastic Agents; D001798:Blood Proteins; D003505:Cyclodextrins; D004791:Enzyme Inhibitors; C105269:LY 237868; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D013453:Sulfonylurea Compounds; D047391:alpha-Cyclodextrins; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; C032613:alpha-cyclodextrin