Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors

被引:196
作者
Coan, Kristin E. D.
Shoichet, Brian K. [1 ]
机构
[1] Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA
关键词
D O I
10.1021/ja802977h
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Many false positives in early drug discovery owe to nonspecific inhibition by colloid-like aggregates of organic molecules. Despite their prevalence, little is known about aggregate concentration, structure, or dynamic equilibrium; the binding mechanism, stoichiometry with, and affinity for enzymes remain uncertain. To investigate the elementary question of concentration, we counted aggregate particles using flow cytometry. For seven aggregate-forming molecules, aggregates were not observed until the concentration of monomer crossed a threshold, indicating a "critical aggregation concentration" (CAC). Above the CAC, aggregate count increased linearly with added organic material, while the particles dispersed when diluted below the CAC. The concentration of monomeric organic molecule is constant above the CAC, as is the size of the aggregate particles. For two compounds that form large aggregates, nicardipine and miconazole, we measured particle numbers directly by flow cytometry, determining that the aggregate concentration just above the CAC ranged from 5 to 30 fM. By correlating inhibition of an enzyme with aggregate count for these two drugs, we determined that the stoichiometry of binding is about 10 000 enzyme molecules per aggregate particle. Using measured volumes for nicardipine and miconazole aggregate particles (2.1 x 10(11) and 4.7 x 10(10) angstrom(3), respectively), computed monomer volumes, and the observation that past the CAC all additional monomer forms aggregate particles, we find that aggregates are densely packed particles. Finally, given their size and enzyme stoichiometry, all sequestered enzyme can be comfortably accommodated on the surface of the aggregate.
引用
收藏
页码:9606 / 9612
页数:7
相关论文
共 29 条
[1]  
[Anonymous], NAT STAND REF DATA S
[2]   Stability and equilibria of promiscuous aggregates in high protein milieus [J].
Coan, Kristin E. D. ;
Shoichet, Brian K. .
MOLECULAR BIOSYSTEMS, 2007, 3 (03) :208-213
[3]   Specificity and mechanism of action of some commonly used protein kinase inhibitors [J].
Davies, SP ;
Reddy, H ;
Caivano, M ;
Cohen, P .
BIOCHEMICAL JOURNAL, 2000, 351 (351) :95-105
[4]   Avoiding physicochemical artefacts in early ADME-Tox experiments [J].
DeWitte, Robert S. .
DRUG DISCOVERY TODAY, 2006, 11 (17-18) :855-859
[5]   Small-molecule aggregates inhibit amyloid polymerization [J].
Feng, Brian Y. ;
Toyama, Brandon H. ;
Wille, Holger ;
Colby, David W. ;
Collins, Sean R. ;
May, Barnaby C. H. ;
Prusiner, Stanley B. ;
Weissman, Jonathan ;
Shoichet, Brian K. .
NATURE CHEMICAL BIOLOGY, 2008, 4 (03) :197-199
[6]   A detergent-based assay for the detection of promiscuous inhibitors [J].
Feng, Brian Y. ;
Shoichet, Brian K. .
NATURE PROTOCOLS, 2006, 1 (02) :550-553
[7]   A high-throughput screen for aggregation-based inhibition in a large compound library [J].
Feng, Brian Y. ;
Simeonov, Anton ;
Jadhav, Ajit ;
Babaoglu, Kerim ;
Inglese, James ;
Shoichet, Brian K. ;
Austin, Christopher P. .
JOURNAL OF MEDICINAL CHEMISTRY, 2007, 50 (10) :2385-2390
[8]   High-throughput assays for promiscuous inhibitors [J].
Feng, BY ;
Shelat, A ;
Doman, TN ;
Guy, RK ;
Shoichet, BK .
NATURE CHEMICAL BIOLOGY, 2005, 1 (03) :146-148
[9]   Concentration and pH dependent aggregation of hydrophobic drug molecules and relevance to oral bioavailability [J].
Frenkel, YV ;
Clark, AD ;
Das, K ;
Wang, YH ;
Lewi, PJ ;
Janssen, PAJ ;
Arnold, E .
JOURNAL OF MEDICINAL CHEMISTRY, 2005, 48 (06) :1974-1983
[10]   Surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors [J].
Giannetti, Anthony M. ;
Koch, Bruce D. ;
Browner, Michelle F. .
JOURNAL OF MEDICINAL CHEMISTRY, 2008, 51 (03) :574-580