Measurement and differentiation of ligand-induced calmodulin conformations by dual polarization interferometry.

In early drug discovery, knowledge about ligand-induced conformational changes and their influence on protein activity greatly aids the identification of lead candidates for medicinal chemistry efforts. Efficiently acquiring such information remains a challenge in the initial stages of lead finding. Here we investigated the application of dual polarization interferometry (DPI) as a method for the real-time characterization of low molecular weight (LMW) ligands that induce conformational changes. As a model system we chose calmodulin (CaM), which undergoes large and distinct structural rearrangements in response to calcium ion and small molecule inhibitors such as trifluoperazine (TFP). We measured concentration-dependent mass, thickness, and density responses of an immobilized CaM protein layer, which correlated directly with binding and conformational events. Calcium ion binding to CaM induced an increase in thickness (≤0.05 nm) and decrease in density (≤-0.03 g/cm(3)) whereas TFP induced an increase in both thickness (≤0.05 nm) and density (≤0.01 g/cm(3)). The layer measurements reported here show how DPI can be used to assess and differentiate ligands with distinct structural modes of action.

Promiscuous aggregate-based inhibitors promote enzyme unfolding.

One of the leading sources of false positives in early drug discovery is the formation of organic small molecule aggregates, which inhibit enzymes nonspecifically at micromolar concentrations in aqueous solution. The molecular basis for this widespread problem remains hazy. To investigate the mechanism of inhibition at a molecular level, we determined changes in solvent accessibility that occur when an enzyme binds to an aggregate using hydrogen-deuterium exchange mass spectrometry. For AmpC beta-lactamase, binding to aggregates of the small molecule rottlerin increased the deuterium exchange of all 10 reproducibly detectable peptides, which covered 41% of the sequence of beta-lactamase. This suggested a global increase in proton accessibility upon aggregate binding, consistent with denaturation. We then investigated whether enzyme-aggregate complexes were more susceptible to proteolysis than uninhibited enzyme. For five aggregators, trypsin degradation of beta-lactamase increased substantially when beta-lactamase was inhibited by aggregates, whereas uninhibited enzyme was generally stable to digestion. Combined, these results suggest that the mechanism of action of aggregate-based inhibitors proceeds via partial protein unfolding when bound to an aggregate particle.

Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors.

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) A(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.

Stability and equilibria of promiscuous aggregates in high protein milieus.

At micromolar concentrations, many molecules form aggregates in aqueous solution. In this form, they inhibit enzymes non-specifically leading to false positive "hits" in enzyme assays, especially when screened in high-throughput. This inhibition can be attenuated by bovine serum albumin (BSA); the mechanism of this effect is not understood. Here we present evidence that BSA, lysozyme, and trypsin prevent inhibition when incubated at milligram per millilitre concentrations with aggregates prior to the addition of the monitored enzyme. These solutions still contained aggregates by dynamic light scattering (DLS), suggesting that inhibition is prevented by saturating the aggregate, rather than disrupting it. For most combinations of aggregate and protein, inhibition was not reversed if the competing protein was added after the incubation of aggregates with the monitored enzyme. In the one exception where modest reversal was observed, DLS and flow cytometry indicated that the effect was due to the disruption of aggregates. These results suggest that aggregate-bound enzyme is not in dynamic equilibrium with free enzyme and that bound enzyme cannot be displaced by a competing protein. To further test this hypothesis, we incubated aggregate-bound enzyme with a specific, irreversible inhibitor and then disrupted the aggregates with detergent. Most enzyme activity was restored on aggregate disruption, indicating no modification by the irreversible inhibitor. These results suggest that enzyme is bound to aggregate so tightly as to prevent any noticeable dissociation and that furthermore, aggregates are stable at physiologically relevant concentrations of protein.