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Promiscuous aggregate-based inhibitors promote enzyme unfolding.

Coan KE, Maltby DA, Burlingame AL, Shoichet BK - J. Med. Chem. (2009)

Bottom Line: This suggested a global increase in proton accessibility upon aggregate binding, consistent with denaturation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry & Graduate Group in Chemistry and Chemical Biology, University of CaliforniaSan Francisco, San Francisco, California 94158-2550, USA

ABSTRACT
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.

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Mass envelopes and corresponding deuterium incorporation plots for two fragments from the mass spectrum of β-lactamase in the presence (dotted line) or absence (solid line) of the aggregator rottlerin. The spectra have been expanded to show the isotopic distribution of the ions of interest: (A) the peptide containing residues 132−146 (monoisotopic m/z = 908.4, +2 charge state) and (B) the peptide containing residues 291−322 (monoisotopic m/z = 813.7, +4 charge state). For each peptide, the isotopic distributions are shown for (i) the undeuterated sample, (ii) the sample that was deuterated for 4 h in the absence of rottlerin, and (iii) the sample that was deuterated for 4 h in the presence of rottlerin.
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fig3: Mass envelopes and corresponding deuterium incorporation plots for two fragments from the mass spectrum of β-lactamase in the presence (dotted line) or absence (solid line) of the aggregator rottlerin. The spectra have been expanded to show the isotopic distribution of the ions of interest: (A) the peptide containing residues 132−146 (monoisotopic m/z = 908.4, +2 charge state) and (B) the peptide containing residues 291−322 (monoisotopic m/z = 813.7, +4 charge state). For each peptide, the isotopic distributions are shown for (i) the undeuterated sample, (ii) the sample that was deuterated for 4 h in the absence of rottlerin, and (iii) the sample that was deuterated for 4 h in the presence of rottlerin.

Mentions: The peptides that we reproducibly observed covered 41% of the β-lactamase sequence (Figure 2), representing several regions of the enzyme spanning both buried and exposed regions. Although our results are not strong enough to determine whether specific areas experienced more exchange than others, it was also not our goal to do so. Given that aggregates are nonspecific inhibitors, we were searching for a global effect, a mechanism that could explain inhibition of many enzymes and that was not restricted to specific residues or peptide sequences. The results suggested such a trend. Across all of the peptides that we measured, enzyme−aggregate samples showed deuterium incorporation greater than or equal to the deuterium incorporation of the enzyme alone (Table 1). Levels of deuteration were very low in both samples with and without inhibitor; however, the trend of higher deuterium content in the aggregate-containing samples is consistent across all of the peptides. We suspect the low levels of incorporation are due to increased back exchange occurring during the protein concentration and chromatography steps. Many peptides incorporated levels of deuterium that were not, in themselves, entirely convincing; however, even here, each showed more deuterium exchange in the presence of aggregates than for the free enzyme. We never observed a peptide that had reduced deuterium exchange in the presence of aggregates. Two peptides that did show significant exchange are shown in Figure 3 (a complete list of peptides is available in the Supporting Information). Again, we observed very low deuterium incorporation, but the time points repeatedly indicated a significant difference between samples with and without aggregates. The higher deuterium incorporation of aggregate-bound enzyme across all 10 peptides suggested that the enzyme may be unfolded when bound to the aggregate.


Promiscuous aggregate-based inhibitors promote enzyme unfolding.

Coan KE, Maltby DA, Burlingame AL, Shoichet BK - J. Med. Chem. (2009)

Mass envelopes and corresponding deuterium incorporation plots for two fragments from the mass spectrum of β-lactamase in the presence (dotted line) or absence (solid line) of the aggregator rottlerin. The spectra have been expanded to show the isotopic distribution of the ions of interest: (A) the peptide containing residues 132−146 (monoisotopic m/z = 908.4, +2 charge state) and (B) the peptide containing residues 291−322 (monoisotopic m/z = 813.7, +4 charge state). For each peptide, the isotopic distributions are shown for (i) the undeuterated sample, (ii) the sample that was deuterated for 4 h in the absence of rottlerin, and (iii) the sample that was deuterated for 4 h in the presence of rottlerin.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664636&req=5

fig3: Mass envelopes and corresponding deuterium incorporation plots for two fragments from the mass spectrum of β-lactamase in the presence (dotted line) or absence (solid line) of the aggregator rottlerin. The spectra have been expanded to show the isotopic distribution of the ions of interest: (A) the peptide containing residues 132−146 (monoisotopic m/z = 908.4, +2 charge state) and (B) the peptide containing residues 291−322 (monoisotopic m/z = 813.7, +4 charge state). For each peptide, the isotopic distributions are shown for (i) the undeuterated sample, (ii) the sample that was deuterated for 4 h in the absence of rottlerin, and (iii) the sample that was deuterated for 4 h in the presence of rottlerin.
Mentions: The peptides that we reproducibly observed covered 41% of the β-lactamase sequence (Figure 2), representing several regions of the enzyme spanning both buried and exposed regions. Although our results are not strong enough to determine whether specific areas experienced more exchange than others, it was also not our goal to do so. Given that aggregates are nonspecific inhibitors, we were searching for a global effect, a mechanism that could explain inhibition of many enzymes and that was not restricted to specific residues or peptide sequences. The results suggested such a trend. Across all of the peptides that we measured, enzyme−aggregate samples showed deuterium incorporation greater than or equal to the deuterium incorporation of the enzyme alone (Table 1). Levels of deuteration were very low in both samples with and without inhibitor; however, the trend of higher deuterium content in the aggregate-containing samples is consistent across all of the peptides. We suspect the low levels of incorporation are due to increased back exchange occurring during the protein concentration and chromatography steps. Many peptides incorporated levels of deuterium that were not, in themselves, entirely convincing; however, even here, each showed more deuterium exchange in the presence of aggregates than for the free enzyme. We never observed a peptide that had reduced deuterium exchange in the presence of aggregates. Two peptides that did show significant exchange are shown in Figure 3 (a complete list of peptides is available in the Supporting Information). Again, we observed very low deuterium incorporation, but the time points repeatedly indicated a significant difference between samples with and without aggregates. The higher deuterium incorporation of aggregate-bound enzyme across all 10 peptides suggested that the enzyme may be unfolded when bound to the aggregate.

Bottom Line: This suggested a global increase in proton accessibility upon aggregate binding, consistent with denaturation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry & Graduate Group in Chemistry and Chemical Biology, University of CaliforniaSan Francisco, San Francisco, California 94158-2550, USA

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus