Identification and prediction of promiscuous aggregating inhibitors among known drugs.

Some small molecules, often hits from screening, form aggregates in solution that inhibit many enzymes. In contrast, drugs are thought to act specifically. To investigate this assumption, 50 unrelated drugs were tested for promiscuous inhibition via aggregation. Each drug was tested against three unrelated model enzymes: beta-lactamase, chymotrypsin, and malate dehydrogenase, none of which are considered targets of these drugs. To be judged promiscuous, the drugs had to inhibit all three enzymes, do so in a time-dependent manner, be sensitive to detergent and to enzyme concentration, and form particles detectable by light scattering. Of the 50 drugs tested, 43 were nonpromiscuous by these criteria. Surprisingly, four of the drugs showed promiscuous, aggregation-based inhibition at concentrations below 100 microM: clotrimazole, benzyl benzoate, nicardipine, and delavirdine. Three other drugs also behaved as aggregation-based inhibitors, but only at high concentrations (about 400 microM). To investigate possible structure-activity relationships among promiscuous drugs, five analogues of the antifungal clotrimazole were studied. Three of these, miconazole, econazole, and sulconazole, were promiscuous but the other two, fluconazole and ketoconazole, were not. Using recursive partitioning, these experimental results were used to develop a model for predicting aggregate-based promiscuity. This model correctly classified 94% of 111 compounds-47 aggregators and 64 nonaggregators-that have been studied for this effect. To evaluate the model, it was used to predict the behavior of 75 drugs not previously investigated for aggregation. Several preliminary points emerge. Most drugs are not promiscuous, even at high concentrations. Nevertheless, at high enough concentrations (20-400 microM), some drugs can aggregate and act promiscuously, suggesting that aggregation may be common among small molecules at micromolar concentrations, at least in biochemical buffers.

Heterodimeric GTPase core of the SRP targeting complex.

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.