Inhibition of β-lactamase function by de novo designed peptide

Abstract

Antimicrobial resistance is a great public health concern that is now described as a "silent pandemic". The global burden of antimicrobial resistance requires new antibacterial treatments, especially for the most challenging multidrug-resistant bacteria. There are various mechanisms by which bacteria develop antimicrobial resistance including expression of β-lactamase enzymes, overexpression of efflux pumps, reduced cell permeability through downregulation of porins required for β-lactam entry, or modifications in penicillin-binding proteins. Inactivation of the β-lactam antibiotics by β-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Although several effective small-molecule inhibitors of β-lactamases such as clavulanic acid and avibactam are clinically available, they act only on selected class A, C, and some class D enzymes. Currently, none of the clinically approved inhibitors can effectively inhibit Class B metallo-β-lactamases. Additionally, there is increased resistance to these inhibitors reported in several bacteria. The objective of this study is to use the Resonant Recognition Model (RRM), as a novel strategy to inhibit/modulate specific antimicrobial resistance targets. The RRM is a bio-physical approach that analyzes the distribution of energies of free electrons and posits that there is a significant correlation between the spectra of this energy distribution and related protein biological activity. In this study, we have used the RRM concept to evaluate the structure-function properties of a group of 22 β-lactamase proteins and designed 30-mer peptides with the desired RRM spectral periodicities (frequencies) to function as β-lactamase inhibitors. In contrast to the controls, our results indicate 100% inhibition of the class A β-lactamases from Escherichia coli and Enterobacter cloacae. Taken together, the RRM model can likely be utilized as a promising approach to design β-lactamase inhibitors for any specific class. This may open a new direction to combat antimicrobial resistance.

Fig 1. Phase circle with phases at frequency f1 = 0.0352 chosen for the design of peptides.

Fig 2. RRM cross-spectrum of β-lactamase protein sequences with common RRM characteristic frequency at f1 = 0.0352±0.0041.

Fig 3. β-lactamase activity assay of E. coli TEM-1 using 200–500 μg of pep3 peptide.

(A) The assay was performed in 96-well plate in a 100 μl total reaction volume containing assay buffer, TEM-1 β-lactamase and nitrocefin in absence or presence of pep3 peptide. The reactions were followed by measuring absorbance at 490 nm for 10 minutes with 1-minute interval. The results represent the means of three independent experiments. Error bars represent the standard deviations from the means. (B) Brown color in well with no peptide is due to hydrolyzed nitrocefin which rapidly changes color from yellow to brown when degraded due to hydrolysis. Statistical analysis was performed using two-tailed paired Student’s t-test (*, p < 0.2; **, p < 0.01; ***, p ≤ 0.001 vs. no peptide control).

Fig 4. β-lactamase activity assay of E. coli TEM-1 using 500 μg of pep1, pep2 and pep3 peptides.

(A) The assay was performed in 96-well plate in a 100 μl total reaction volume containing assay buffer, TEM-1 β-lactamase and nitrocefin in absence or presence of peptides pep1, pep2 and pep3. The reactions were followed by measuring absorbance at 490 nm for 10 minutes with 1-minute interval. The results represent the means of three independent experiments. Error bars represent the standard deviations from the means. (B) Brown color in wells with no peptide, 500 μg of pep1 and 500 μg of pep2 is due to hydrolyzed nitrocefin which rapidly changes color from yellow to brown when degraded due to hydrolysis. Statistical analysis was performed using two-tailed paired Student’s t-test (*, p < 0.5; **, p < 0.005 vs. no peptide control).

Fig 5. β-lactamase activity assay of E. cloacae using 100–400 μg of pep3 peptide.

(A) The assay was performed in 96-well plate in a 100 μl total reaction volume containing assay buffer, E. cloacae β-lactamase and nitrocefin in absence or presence of pep3 peptide. The reactions were followed by measuring absorbance at 490 nm for 10 minutes with 1-minute interval. The results represent the means of three independent experiments. Error bars represent the standard deviations from the means. (B) Brown color in well with no peptide is due to hydrolyzed nitrocefin which rapidly changes color from yellow to brown when degraded due to hydrolysis. Statistical analysis was performed using two-tailed paired Student’s t-test (*, p = 0.5; **, p < 0.05; ***, p ≤ 0.005 vs. no peptide control).

Fig 6. β-lactamase activity assay of E. cloacae using 400 μg of pep1, pep2 and pep3 peptides.

(A) The assay was performed in 96-well plate in a 100 μl total reaction volume containing assay buffer, E. cloacae β-lactamase and nitrocefin in absence or presence of peptides pep1, pep2 and pep3. The reactions were followed by measuring absorbance at 490 nm for 10 minutes with 1-minute interval. The results represent the means of three independent experiments. Error bars represent the standard deviations from the means. (B) Brown color in wells with no peptide, 400 μg of pep1 and 400 μg of pep2 is due to hydrolyzed nitrocefin which rapidly changes color from yellow to brown when degraded due to hydrolysis. Statistical analysis was performed using two-tailed paired Student’s t-test (*, p < 0.5; **, p < 0.05; ***, p ≤ 0.005 vs. no peptide control).