Promiscuous aggregate-based inhibitors promote enzyme unfolding
Kristin E D Coan1, David A Maltby, Alma L Burlingame
1Department of Pharmaceutical Chemistry & Graduate Group in Chemistry and Chemical Biology, University of CaliforniaSan Francisco, San Francisco, California 94158-2550, USA
Journal of Medicinal Chemistry
|March 14, 2009
Summary
Small molecule aggregates cause false positives in drug discovery by inhibiting enzymes. This study reveals that aggregate binding leads to enzyme denaturation and increased susceptibility to proteolysis, clarifying the inhibition mechanism.
Area of Science:
- Biochemistry
- Drug Discovery
- Structural Biology
Background:
- Organic small molecule aggregates are a significant source of false positives in early drug discovery.
- Nonspecific enzyme inhibition by aggregates occurs at micromolar concentrations in aqueous solutions.
- The molecular basis for aggregate-induced enzyme inhibition is not well understood.
Purpose of the Study:
- To elucidate the molecular mechanism by which small molecule aggregates inhibit enzymes.
- To investigate changes in enzyme structure and stability upon binding to aggregates.
- To determine if aggregate-bound enzymes exhibit altered susceptibility to proteolysis.
Main Methods:
- Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to assess changes in solvent accessibility upon enzyme-aggregate binding.
- AmpC beta-lactamase was used as a model enzyme.
- Proteolysis assays with trypsin were performed on enzyme-aggregate complexes and uninhibited enzyme.
Main Results:
- Binding of rottlerin aggregates to AmpC beta-lactamase increased deuterium exchange across 41% of the enzyme sequence, indicating global solvent accessibility changes consistent with denaturation.
- Enzyme-aggregate complexes formed with five different aggregators showed significantly increased susceptibility to trypsin degradation compared to the uninhibited enzyme.
- Uninhibited AmpC beta-lactamase remained largely stable to trypsin digestion under the tested conditions.
Conclusions:
- The mechanism of inhibition by small molecule aggregates involves partial protein unfolding (denaturation) of the enzyme.
- Aggregate binding alters enzyme conformation, leading to increased accessibility and susceptibility to proteolytic degradation.
- Understanding this mechanism is crucial for mitigating false positives in early drug discovery.
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