Structure-guided microbial targeting of antistaphylococcal prodrugs

Abstract

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue-specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase-specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, ultimately identifying several promoieties likely to be serum esterase-resistant and microbially labile. These studies will enable structure-guided design of antistaphylococcal promoieties and expand the range of molecules to target staphylococcal pathogens.

Keywords: Staphylococcus aureus; antibacterial; biochemistry; chemical biology; drug discovery; esterase; prodrug.

Figure 1.. Prodrug activation model (A) and proposed enzymatic mechanism.

(B) Carboxy ester promoieties highlighted in green.

Figure 2.. Forward and reverse genetics approaches identify FrmB and GloB as candidate POM-prodrug hydrolases in S. aureus.

(A) Reverse genetics identification of candidate prodrug activating enzymes. (B) POM-HEX susceptibility of identified candidate resistance genes from (A) as determined by IC₅₀. Exact values and error reported in Figure 2—source data 1. (C) Forward genetic screen approach, all mutations listed in Figure 2—source data 2. (D) POM-HEX susceptibility of POM-HEX-resistant S. aureus. (E) Nonsynonymous point mutations identified by whole-genome sequencing in frmB and gloB. In all experiments, GloB is colored green and FrmB orange. Displayed are the means of three independent biological experiments.

Figure 2—figure supplement 1.. Conservation of FrmB and GloB within S. aureus.

(A) WhatsGNU analysis of GloB and FrmB. Control proteins: ArgG, argininosuccinate synthase; Fba, fructose-bisphosphate aldolase; MenD, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1carboxylate synthase; and MenC, o-succinylbenzoate synthase. GNU stands for gene novelty unit and is a count of how many protein sequences in the database have an exact match to the queried sequence, with higher counts indicating sequence conservation. Strains across the x-axis are representative strains from the 18 s. aureus colony complexes which were used to query the S. aureus database. (B, C) MAFFT alignment of GloB (B) and FrmB (C) protein sequences across the S. aureus sequence database.

Figure 2—figure supplement 2.. Phylogenetic tree of FrmB and GloB.

Sequences of GloB, FrmB, and RpoB (encoding the beta subunit of RNA polymerase, included for comparison) were retrieved from NCBI using BlastP against each organism. Sequence alignment performed using MUSCLE and alignment visualized using the interactive Tree of Life (iTOL).

Figure 2—figure supplement 3.. Enzymatic characterization of GloB and FrmB.

(A) SDS–PAGE gel of GloB and FrmB protein preparations. Expected molecular weights are 23.3 kDa and 29.5 kDa, respectively. (B) Glyoxalase II activity assay, enzymatic conversion of S-lactoylglutathione releases free glutathione and reacts with DTNB resulting in increased absorbance at 412 nm. (C) 4-Nitrophenyl activation results in increased absorbance at 405 nm. Left to right, activity when supplied 4-nitrophenyl acetate, 4-nitrophenyl butyrate, and 4-nitrophenyl trimethyl acetate. Displayed in points is the mean of two technical replicates for individual experiments, bars indicate mean ± SD of three independent biological experiments performed in technical duplicate.

Figure 2—figure supplement 4.. NMR characterization of POM-HEX activation by GloB and FrmB.

Two-dimensional (2D) ¹H-³¹P HSQC NMR spectra of products following incubation of FrmB, GloB, catalytically inactive (boiled) GloB and FrmB, or buffer alone. Also included are the ¹H-³¹P HSQC NMR spectra of POM-HEX and HEX. Displayed are representative traces of three independent experiments. HEMI-POM HEX peak inferred by predicted shift.

Figure 3.. In vivo activation rates depend on ester promoiety selection.

Time series of activation of various fluorogenic substrates (Figure 3—figure supplement 1). Substrates were added into the microfluidics chamber at t = 10 minutes. On the right, quantification of individual cell or cell cluster fluorescence per area. Faint traces are individual cells and darker traces represent the mean of a given experiment. Each experiment was performed in biological duplicate, and each experiment is displayed in a different color (purple or green). Full movies viewable as Videos 1–4. Error bars denote SD.

Figure 3—figure supplement 1.. Profluorescent substrate library.

Activation of substrates via esterase action results in fluorescence.

Figure 3—figure supplement 2.. Catalytic efficiency of GloB (A) and FrmB (B).

Numbers correspond to the structures displayed in Figure S5, compounds in the carbon series denoted in orange, oxygen series in blue, and sulfur series in green.

Figure 4.. Three-dimensional structure of FrmB.

(A) Overall fold, a-helices colored in orange and β-strands colored in purple. (B) Comparison between SaFrmB (orange) and its closest human ortholog, ESTD (gray). Active site residues denoted in orange spheres. (C, D) Docking of substrate 1O (sticks) in the active site of FrmB. surface view, red indicates highly hydrophobic and white hydrophilic residues. Surface view (C) or stick view with catalytic triad (D).

Figure 4—figure supplement 1.. Structural conservation of FrmB.

(A) Overall structural alignment of FrmB (orange) with S. pneumonia EstA (PDB:2UZ0), B. intestinalis ferulic acid esterase (PDB:5VOL), and deep sea bacteria Est12 (PDB4RGY). (B) Conservation of the serine hydrolase catalytic triad in FrmB and related proteins.

Figure 5.. Three-dimensional structure of GloB.

(A) Overall fold, a-alpha helices colored in green and β-strands colored in purple. (B) Comparison of SaGloB (green) and human GloB (gray). (C) Docking of the substrate 1O (sticks) in the active site of GloB. Left, partial cartoon view; right, surface view. White represents hydrophilic residues, whereas red represents hydrophobic residues. Zn ions indicated as silver spheres; water indicated as blue sphere.

Figure 5—figure supplement 1.. Structural conservation of GloB.

(A) Overall structural alignment of GloB (green) with S. enterica YcbI (PDB:2XF4), T. thermophilus TTHA1623 (PDB:2ZWR), and A. thaliana glyoxalase II (PDB:1XM8). Zinc coordinating residues are colored in green spheres. (B) Positioning of the zinc coordinating residues (green spheres).

Figure 6.. Comparison between microbial esterase and serum esterase catalytic efficiency.

(A–D) Volcano plots of catalytic efficiency. Displayed are the means of three independent experiments. p-values calculated as pairwise t-tests with Holm-Sidak correction for multiple comparisons. (A) Comparison between human sera and GloB, (B) human sera and FrmB, (C) mouse sera and GloB, and (D) mouse sera and FrmB. (E) Structures of ester substrates with 210 enrichment in catalytic efficiency for microbial esterases over human serum (left) or 25 enrichment over mouse serum. Dashed line indicates a p-value of 0.05.

Figure 6—figure supplement 1.. Comparison of esterase activity between fresh and lyophilized human sera.

Points represent individual experiments; bars represent the mean ± SD of the four replicates.

Figure 6—figure supplement 2.. Modified catalytic efficiency (pmol fluorescein produced * min⁻¹*µg⁻¹ protein) of (A) human sera, (B) GloB, (C) FrmB, and (D) mouse sera.

X-axis corresponds to compound identities in Figure S5. Carbon containing compounds indicated in orange, oxygen in blue, and sulfur in green. Displayed are the means ± SD of three independent biological experiments.

Figure 6—figure supplement 3.. Comparison of mouse and human sera.

(A) Modified catalytic efficiency (pmol fluorescein produced * min-1*µg-1 protein) of human and mouse sera. Displayed is a linear regression of the fit between mouse and human sera. (B) Volcano plot of catalytic efficiency. Displayed are the means of three independent experiments. p-values calculated as pairwise t-tests with Holm–Sidak correction for multiple comparisons. Dashed line indicates a p-value of 0.05.

Figure 7.. Model of antistaphylococcal prodrug activation.

Lipophilic carboxy ester prodrugs transit the cell membrane, are first activated by either GloB or FrmB and at least one additional enzyme, before inhibiting the cellular target.