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79958695999
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note
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Buffer assay: Human recombinant renin (Proteos) at 100 pM was incubated in the presence or absence of renin inhibitors and 6 μM of Q-FRET substrate 9 DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-D,L-Amp in 50 mM MOPS, 100 mM NaCl, pH 7.4, 0.002% Tween. The reactions take place in a Costar 384 well black plate at 37°C for 3 h. Fluorescence was measured at times 0 and 3 h in a SpectraMax Gemini EM reader with excitation and emission filters at 328 and 388 nm, respectively. Plasma assay: Frozen human EDTA-plasma was rapidly thawed in warm water and centrifuged at 2900 g for 15 min at 40°C. The supernatant was collected and recombinant human renin (Proteos) added at 1 nm nominal concentration. The plasma was transferred to Costar black 384 well plates, renin inhibitors added and the mixture pre-incubated at 37°C for 10 min. The renin Q-FRET substrate QXL520-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-Lys-(5-FAM) (Proteos), diluted in 3 M Tris/200 mM EDTA, pH 7.2 was added to the plasma with final concentrations of 342 mM Tris, 23 mM EDTA and 6.8 μM substrate. The plate was incubated at 37°C for 1 h and the plate read in a SpectraMax Gemini EM reader with excitation and emission filters at 490 and 520 nm, respectively, at time 0 and 1 h.
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9
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79958746645
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note
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In fact, the oral bioavailability of renin inhibitors such as aliskiren increases nonlinearly with dose. It has been hypothesized that because aliskiren is a good substrate of endogenous human and rat PGP transporters, this efflux mechanism would become increasingly saturated at higher doses. Drugs that exhibit this behavior could have the potential to be victims of drug-drug interactions when co-dosed with compounds known to inhibit PGP transporters.
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79958703472
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note
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During in silico docking experiment with compound 15, modeling revealed that the tertiary alcohol residue points out into solvent and does not engage in any stabilizing interactions with the renin enzyme.
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12
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0001610101
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