Synthesis and Photophysical Properties of a Deazaflavin‐Bridged Porphyrinatoiron(III) That Mimics the Interaction of a Deazaflavin Inhibitor with the Heme‐Thiolate Cofactor of Cytochrome P450 3A4

Abstract

Cytochrome P450 3A4 metabolizes a majority of administered therapeutic agents in the human liver. We recently reported the synthesis of a new inhibitor, 1, whose binding to and displacement from the active site of CYP 3A4 can be conveniently followed by the associated changes in fluorescence intensity. Here we report the synthesis of a bichromophoric compound, 6, in which deazaflavin was strapped over the distal side of a porphyrinatoiron(III) complex to mimic the envisaged enzyme-inhibitor interaction within the active site. Femtosecond pump-probe and fluorescence spectroscopies were used to study the photophysical processes of 6. Rapid intramolecular energy transfer and enhanced intersystem-crossing processes induced by the high-spin Fe III central ion are responsible for the complete suppression of deazaflavin fluorescence in 6. Fluorescence quenching is less efficient in the iron-free analogue of 6, i.e., in 21.1. Introduction. -Cytochrome P450 3A4 (CYP 3A4), a heme-thiolate protein, is one of the most important P450s in the human liver [1]. The ability of CYP 3A4 to metabolize a majority of administered therapeutic agents accounts for the large number of documented drug-drug interactions associated with CYP 3A4 inhibition [2]. It is therefore of great importance to know the affinity of new drug candidates to CYP 3A4 at an early stage, in order to determine their potential as inhibitors, and to evaluate their influence on the metabolism of co-administered drugs. Some particular features of CYP 3A4 make it difficult to achieve this goal. Though X-ray structures are now available from truncated CYP 3A4 [3], these pictures provide no clues on how the enzyme accepts substrates as different as, e.g., erythromycin and nifedipine, how the active site accommodates more than one substrate [4], and why partial inhibition and even activation is observed when pairs of drugs are co-incubated [5].Our group has recently reported [6] the synthesis of a new inhibitor 1 that displayed favourable IC 50 values in the range of 1 -4 mM for three characteristic substrates of CYP 3A4 such as testosterone (2), midazolam (3), and nifedipine (4) (Fig. 1). The binding of 1 to the active site of CYP 3A4 and its displacement by other substrates can be followed by the associated change in the intensity of the deazaflavin fluorescence [7], such that a screening for drugs that might cause drug-drug interactions requires neither the determination of substrate turnover nor product analyses.From these experiments, it was suggested [6] that the fluorescence of 1 is quenched upon binding to CYP 3A4 due to an interaction such as that shown in structure 5 of the

Fluorescent Probes for Rapid Screening of Potential Drug–Drug Interactions at the CYP3A4 Level

“…Thus the difference in the extent of quenching for the two systems is not due to differences in affinities. It may be due to distinct orientations of 4 relative to the heme within the binding pocket, the fluorescence intensities being influenced by the distance and/or angle of the emitter to the quencher 15. It may also depend on the presence of aggregates in the solubilized enzyme 3A4‐Cal.…”

“… Complex 5 : proposed interaction of 4 with the heme of CYP3A4; the synthetic model system 6 was used for investigation of the photophysical interaction between deazaflavin and the iron(III)‐containing porphyrin 15…”

“…In this construct the putative binding sites of the steroid are maintained, the site of hydroxylation is occupied, and the deazaflavin unit that takes the stacking role of nifedipine permits photophysical interaction with the iron porphyrin. Using a synthetic model system 6 which mimics this interaction we have recently shown that the fluorescence of the deazaflavin moiety is quenched mainly through intersystem crossing (ISC) processes supported by the paramagnetic iron(III) 15. Furthermore, the combination of two different binding modes as in 5 is expected to produce an affinity higher than for each isolated subunit, restricting the flexibility of the CYP3A4 active site and hence preventing the access of other molecules.…”

Competing Pathways in the Photo-Favorskii Rearrangement and Release of Esters: Studies on Fluorinated p-Hydroxyphenacyl-Caged GABA and Glutamate Phototriggers

“…30 The sample was excited with a frequency-quadrupled pulse from a Ti/Sa laser system (775 nm, pulse energy 0.8 mJ, full width at half maximum ( fwhm ) 150 fs, operating frequency 426 Hz) described previously. 31 The output at 540 nm was frequency doubled to 266 nm and after compression provided pump pulses with an energy of 1 μJ and <100 fs pulse width. A probe beam continuum was produced by focusing the 775 nm 1 mm in front of a CaF 2 of 2 mm path length.…”

Mechanistic Organic Photochemistry

“…The interaction with other research groups has repeatedly led us into exciting new areas such as protein folding, [26] abiotic photodegradation in surface waters, [27] photoinitiators [28] and photochromism, [29] and the study of electron transfer in proteins [30] or in a synthetic cytochrome P450 mimic. [31] Photochemistry and photophysics have emerged from a relatively small community of specialists to become an integral part of and powerful tool for all branches of science: chemistry, biochemistry, biophysics, materials science, analytical chemistry, etc . Fluorescence measurements with spatial, temporal and spectral resolution are becoming available at decreasing cost along with continuously rising sensitivity and resolution, ultimately allowing the detection of single molecules and to follow their reactions and movements in space and time.…”