Identification of novel binding interactions in the development of potent, selective 2-naphthamidine inhibitors of urokinase. Synthesis, structural analysis, and SAR of N-phenyl amide 6-substitution.
The preparation and assessment of biological activity of 6-substituted 2-naphthamidine inhibitors of the serine protease urokinase plasminogen activator (uPA, or urokinase) is described. 2-Naphthamidine was chosen as a starting point based on synthetic considerations and on modeling of substituent vectors. Phenyl amides at the 6-position were found to improve binding; replacement of the amide with other two-atom linkers proved ineffective. The phenyl group itself is situated near the S1' subsite; substitutions off of the phenyl group accessed S1' and other distant binding regions. Three new points of interaction were defined and explored through ring substitution. A solvent-exposed salt bridge with the Asp60A carboxylate was formed using a 4-alkylamino group, improving affinity to K(i) = 40 nM. Inhibitors also accessed two hydrophobic regions. One interaction is characterized by a tight hydrophobic fit made with a small dimple largely defined by His57 and His99; a weaker, less specific interaction involves alkyl groups reaching into the broad prime-side protein binding region near Val41 and the Cys42-Cys58 disulfide, displacing water molecules and leading to small gains in activity. Many inhibitors accessed two of these three regions. Affinities range as low as K(i) = 6 nM, and many compounds had K(i) < 100 nM, while moderate to excellent selectivity was gained versus four of five members of a panel of relevant serine proteases. Also, some selectivity against trypsin was generated via the interaction with Asp60A. X-ray structures of many of these compounds were used to inform our inhibitor design and to increase our understanding of key interactions. In combination with our exploration of 8-substitution patterns, we have identified a number of novel binding interactions for uPA inhibitors.
Wendt
Michael D
MD
Cancer Research and Structural Biology, Global Pharmaceutical R & D, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6101, USA. mike.d.wendt@abbott.com
Rockway
Todd W
TW
Geyer
Andrew
A
McClellan
William
W
Weitzberg
Moshe
M
Zhao
Xumiao
X
Mantei
Robert
R
Nienaber
Vicki L
VL
Stewart
Kent
K
Klinghofer
Vered
V
Giranda
Vincent L
VL
D000578:Amidines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D009281:Naphthalenes / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D012997:Solvents; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors*
D000578:Amidines; D009281:Naphthalenes; D012997:Solvents; D014568:Urokinase-Type Plasminogen Activator