Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid with 2,4-dinitrophenyl groups at various positions

Abstract

The development of renin inhibitors for the treatment of hypertension requires highly sensitive substrates to evaluate potency and to characterize the mechanism of tight-binding inhibitors. A series of intramolecularly quenched fluorogenic renin substrates, based on the N-terminal tetradecapeptide sequence of human angiotensinogen (hTDP), was synthesized using a solid-phase technique. Incorporation of the fluorescent amino acid L-Amp [L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid] and the DNP (2,4-dinitrophenyl) group at various positions resulted in >90% quenching efficiency and strong product fluorescence. Shortening the hTDP sequence to an octapeptide from histidine in P5 to histidine in P3' (substrate 3) resulted in an acceptable k(cat)/K(m) (41000 M(-1).s(-1)) and further systematic variation gave substrate 9, DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-L-Amp, with a k(cat)/K(m) value of 350000 M(-1).s(-1) and 94% quenching efficiency. The free side chain of lysine, replacing the isoleucine residue at P6 position in the angiotensinogen sequence, contributed to the increased value for k(cat). The pH dependence of k(cat)/K(m) for renin and substrate 9 showed that the optimal pH is at pH 6-7. It also showed two titrating groups on the acidic side of the pH optimum, and one titrating group with a pK(a) of 7.8 on the alkaline side. The combination of good kinetic and spectroscopic properties resulted in a >20-fold improvement in the sensitivity of renin assay, compared with the commercial substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg [where EDANS is 5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid and DABCYL is 4-(4-dimethylaminophenylazo)benzoic acid] (k(cat)/K(m)=268000 M(-1) x s(-1), quenching efficiency <80%). The detection limit in a microplate renin assay was 60 pM, making substrate 9 well suited for the evaluation of inhibitors at picomolar concentrations.

Figure 1. Determination of Michaelis–Menten parameters for the renin-catalysed hydrolysis of substrate 9 by the initial-rate method

The initial-rate assay was performed at the following conditions: renin concentration, 0.27 nM; five substrate concentrations over the K_m range 0.5–3; assay buffer, 50 mM Mops/NaOH, pH 7.0, 2 mM EDTA, 0.5% BSA, 0.1% NaN₃ and 1% DMSO; excitation/emission wavelengths 328/388 nm; and temperature, 37 °C. Product concentration was determined by HPLC. Data were fitted to the Michaelis–Menten equation.

Figure 2. Renin-catalysed hydrolysis of substrate 9 (▪) and substrate 20 (○)

Progress curves under pseudo-first-order conditions: renin concentration, 8.2 nM; substrate concentration 0.2 μM; assay buffer, 50 mM Mops/NaOH, pH 7.0, 2 mM EDTA, 0.5% BSA, 0.1% NaN₃ and 1% DMSO; excitation/emission wavelengths, 328/388 nm for substrate 9 and 340/485 nm for substrate 20; and temperature, 37 °C. The inset shows hydrolysis of substrate 20 using a 7-fold-increased sensitivity of the spectrofluorimeter.

Figure 3. Determination of renin activity using substrates 9 (▪) and 20 (○) in a 96-well microplate

Conditions: renin concentration, 0.03–2 nM; substrate concentration, 5 μM; assay buffer, 50 mM Mops/NaOH, pH 7.0, 2 mM EDTA, 0.5% BSA, 0.1% NaN₃ and 1% DMSO; excitation/emission wavelengths, 328/388 nm for substrate 9 and 340/485 nm for substrate 20; and temperature, 37 °C. Assay time was 10 min at 37 °C, the plate was read at 30 s intervals and slopes were calculated. Results are means±S.D. for three wells. RFU, relative fluorescence units.

Figure 4. pH-dependence of k_cat/K_m of renin-catalysed hydrolysis of substrate 9

Buffers (50 mM) containing 0.5% BSA, 2 mM EDTA and 0.1% NaN₃ were prepared from sodium acetate (pH 4.5, 4.9 and 5.4), Mes (pH 5.5, 5.8 and 6.2), Mops (pH 6.7, 7.1, 7.7 and 8.1) and Taps (8.2, 8.7, 9.1 and 9.6) using correction factors for the change of pK_a values with temperature. The final renin concentration was 8.2–41 nM. Values for k_cat/K_m were determined by the progress-curve method at 0.2 μM substrate concentrations and fitted to eqns (5) (continuous line) and (6) (dotted line). Results from two experiments are shown.