A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P

Abstract

Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5' end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5' end fluorescein-labeled pre-tRNAAsp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNAAsp with a Kd value that is comparable to their IC50 value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P.

Figure 1.

A fluorescence polarization/anisotropy assay for measuring RNase P-catalyzed pre-tRNA cleavage. A fluorescein dye (orange dot) is attached to the 5′ end of a Bacillus subtilis pre-tRNA^Asp with a 5-nt leader (Fl-pre-tRNA^Asp). When excited with polarized light, the Fl-pre-tRNA^Asp tumbles slower than the lifetime of the fluorophore so that the emitted light remains polarized (high anisotropy). Upon cleavage of the 5′ end leader catalyzed by RNase P, the Fl-5nt-leader product rotates faster leading to enhanced depolarization of the emitted light (lower anisotropy).

Figure 2.

Measurement of pre-tRNA cleavage catalyzed by RNase P using fluorescence anisotropy (FA). (A) The time-dependent change in FA was measured under single-turnover (STO) conditions in buffer A ( 50 mM Tris/MES pH 5.5, 10 mM MgCl₂, 200 mM KCl, 20 mM DTT) with 25 nM Fl-pre-tRNA^Asp and 500 nM Bacillus subtilis RNase P holoenzyme at 37°C. The solid line is a single exponential fit to the data with k_obs = 0.0250 ± 0.0002 s⁻¹; when Equation (2) was used to adjust for the change in total florescence and Equation (1) was fit to the data, the k_obs = 0.032 ± 0.001 s⁻¹. (B) The time-dependent change in FA was measured under multiple-turnover (MTO) conditions in buffer C (50 mM Tris–HCl pH 8, 10 mM MgCl₂, 100 mM KCl, 20 mM DTT) with 20 nM Fl-pre-tRNA^Asp, 0.4 nM B. subtilis RNase P and 4 nM P protein at 37°C. Total fluorescence does not change for multiple turnover reactions. The steady-state cleavage velocity is measured from the linear initial rate (solid line). (C) Steady-state kinetic parameters were determined from the dependence of the MTO initial rate on substrate concentration. Reaction conditions are the same as in B except for varying RNase P (0.3–1 nM) and Fl-pre-tRNA^Asp (2 nM–1 μM) concentrations. Results are from three independent experiments and the error bars are the standard deviations. The Michaelis–Menten equation was fit to the data yielding: k_cat = 0.124 ± 0.003 s⁻¹, K_M = 40 ± 3 nM, k_cat/K_M = 3100 ± 200 mM⁻¹s⁻¹.

Figure 3.

Neomycin B and kanamycin B inhibit Bacillus subtilis RNase P. Dose-response of RNase P inhibition was measured in buffer B (50 mM Tris–HCl pH 7.2, 10 mM MgCl₂, 100 mM KCl, 20 mM DTT) with 50 nM Fl-pre-tRNA^Asp and 0.4 nM RNase P with 4 nM P protein at 37°C. The concentrations for NeoB (•) and KanB (▪) that inhibit activity by 50% (IC₅₀, Equation 5) are 23 ± 3 μM (n = 1.1 ± 0.1) and 52 ± 6 μM (n = 0.9 ± 0.1), respectively. In the presence of 12 mg/ml yeast tRNA^Mix, the IC₅₀ values for NeoB (○) and KanB (□) are 110 ± 20 μM (n = 0.53 ± 0.05) and 2.2 ± 0.3 mM (n = 1.0 ± 0.2), respectively.

Figure 4.

Neomycin B and kanamycin B bind to Fl-pre-tRNA^Asp. (A) The FA of Fl-pre-tRNA^Asp increases upon titration with NeoB (•) and KanB (□), measured in buffer B as described in legend of Figure 3 at 37°C with 50 nM FI-pre-tRNA^Asp. The K_d values for NeoB and KanB are determined from a fit of Equation (8) to these data as 90 ± 5 μM and 220 ± 20 μM, respectively. (B) Yeast tRNA^Mix competes with Fl-pre-tRNA^Asp for binding neomycin B and kanamycin B using either 200 μM NeoB (•) or 1 mM KanB (□) and varying concentrations of yeast tRNA^Mix in buffer B. Error bars show the standard deviations from five replicates.

Figure 5.

High-throughput FP assay is robust for screening inhibitors of Bacillus subtilis RNase P. (A) Reaction progress curves measured by quenching the reaction with 80 mM CaCl₂ at specified time points in HTS buffer [50 mM Tris–HCl pH 7.2, 5 mM MgCl₂, 100 mM KCl, 20 mM DTT, 12 mg/ml yeast tRNA^Mix, 10 mM spermidine and 0.01% (v/v) NP-40] with 1% DMSO, 20 nM FL-pre-tRNA^Asp and 0.15 nM RNase P with 1.5 nM P protein at 30°C. The reactions contained either no inhibitor (1% DMSO blank, •) as a negative control or 80 mM CaCl₂ (□) as a positive control for inhibition. Error bars are derived from 18–32 replicates in one 384-well plate. (B) The Z′- factor values for the FP HTS assay determined from each of the nine 384-well microplates in primary screen. (C) A scatter plot showing the percent inhibition by plate in the primary screen of the 2880 compound library. Positive controls including 80 mM CaCl₂ in the assay are shown by red squares. Negative controls (1% DMSO) are blue. Compound samples are green. The red solid line indicates percent inhibition at three times the standard deviation of negative controls (3SD). The samples showing negative inhibition contain compounds interfering with fluorescence signal.

Figure 6.

Iriginol hexaacetate inhibits Bacillus subtilis RNase P catalyzed cleavage. (A) Chemical structure of iriginol hexaacetate (Ir6Ac). (B) Dose-response curve of inhibition by Ir6Ac of MTO cleavage activity catalyzed by RNase P measured in HTS buffer with 50 nM Fl-pre-tRNA^Asp and 0.1 nM B. subtilis RNase P with 2 nM P protein. Ir6Ac was pre-incubated with RNase P for 40 min at 37°C prior to initiation of reaction. IC₅₀ is 820 ± 10 nM with a Hill coefficient of 1.0 ± 0.1.

Figure 7.

Mechanism of inhibition of Bacillus subtilis RNase P by Ir6Ac. The assays were carried out at a fixed RNase P concentration of 0.4 nM (2 nM P protein) with varying concentrations of Ir6Ac and Fl-pre-tRNA^Asp in HTS buffer at 37°C. Ir6Ac was pre-incubated with RNase P for 40 min. (A and B) Fit of Equation (6) to the apparent k_cat (A) and k_cat/K_M (B) values as a function of concentration of Ir6Ac. The solid line is a fit with n = 1 (R² = 0.9731 for k_cat/K_M and 0.9899 for k_cat) and the dotted line is a fit where n is a variable: for k_cat/K_M, n = 1.4 ± 0.1 (R² = 0.9969) and for k_cat, n = 0.9 ± 0.1 (R² = 0.9919). (C) Best global fit for inhibition of RNase P in the presence of varying concentrations of substrate (6–600 nM). Equation (7) for a non-cooperative mixed inhibition is fit to the data (R² = 0.9775) with K_i = 130 ± 10 nM and K­_is = 480 ± 30 nM (n_i = n_is = 1); (D) Lineweaver–Burk plot for the dependence of RNase P activity on Ir6Ac and substrate concentrations. A non-cooperative mixed inhibition model is fit to the data (R² = 0.9670). Symbols represent means ± SD determined from two to three independent experiments at each concentration.

Figure 8.

Comparison of the theoretical calculation and experimental results for the FA value of fluorescein labeled pre-tRNA^Asp. The dashed line indicates the molecular weight (26,085 g/mol) of pre-tRNA^Asp with a 5-nt leader calculated from the sequence in Figure 1. The solid line is the theoretical FA value calculated using Equation (10). The solid symbols indicate the experimental FA values measured in Buffer B (50 mM Tris–HCl pH 7.2, 10 mM MgCl₂, 100 mM KCl, 20 mM DTT) except values for Fl-pre-tRNA^Asp bound to RNase P were measured in 10 mM CaCl₂ and 200 mM KCl at pH 6: Fl-5-nt-leader (▪), Fl-pre-tRNA^Asp (), Fl-pre-tRNA^Asp•NeoB (▾) and Fl-pre-tRNA^Asp•RNase P (♦).