Fluorescent probes for cytochrome P450 structural characterization and inhibitor screening

Article abstract of DOI:10.1021/ja0271678

We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Foerster energy transfer upon binding (K_d = 0.83 μM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (K_d ≈ 0.02 μM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 A resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B-, F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors. Copyright

Full text of DOI:10.1021/ja0271678

Published on Web 08/10/2002
Fluorescent Probes for Cytochrome P450 Structural Characterization and
Inhibitor Screening
Alexander R. Dunn,1a Anna-Maria A. Hays,1b David B. Goodin,1b C. David Stout,1b Richard Chiu,1a
Jay R. Winkler,*,1a and Harry B. Gray*,1a
Beckman Institute, California Institute of Technology, Pasadena, California 91125, and Department of Molecular
Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received June 4, 2002
Substrate-specific cytochromes P450 play major roles in steroid
and eicosanoid biosynthesis and thus constitute important drug
design targets.2 In contrast, P450 isozymes expressed in the liver
take part in the metabolism of nearly all drugs.3 Adverse drug
reactions, for instance to Prozac,4 result from individual variations
in hepatic P450s.5 It is thus important to predict which P450s
interact with a potential drug candidate and to understand the nature
of these interactions.
We have developed fluorescent probe molecules for P450cam
(the prototypical P450) that consist of an R,ω-diaminoalkane chain
connecting a dansyl fluorophore to the P450cam substrate ada-
mantane:
Figure 1. (A) Absorption spectra showing the binding of camphor to
P450cam (4.9 µM) in the presence of 1 equiv of D-4-Ad. The initial addition
of D-4-Ad to P450cam results in a shift in the Soret absorption from 416
to 414 nm. A fit of the data to a competitive binding model gives a
dissociation constant of 0.83 µM. (B) The camphor-induced shift from low-
to high-spin P450cam (5.7 µM) in the presence of 1 equiv of D-8-Ad. Black,
P450cam; purple, P450cam + 1 equiv of dansyl probe; blue to red, 0.5, 1,
2, 4, 8, 16, 32, and 64 equiv of camphor.
A shift in Soret absorption (Figure 1) and greatly diminished dansyl
luminescence attributable to Fo¨rster energy transfer to the heme6
(Figure 2) accompany probe binding. When D-4-Ad is displaced
from the active site by camphor, fluorescence is restored (Figure
2a).7 Because a bright signal stands out against a dark background,
substrate or inhibitor binding is readily detected. This assay, which
is both simple and sensitive, can be employed to screen combina-
torial chemical libraries.8
Both fluorescence and absorption spectra show that D-4-Ad
binding to P450cam is competitive with camphor. The Soret shift
(416-414 nm) induced by D-4-Ad indicates that it binds in the
active site. With a Kd of 0.83 µM, D-4-Ad binds twice as strongly
as the natural substrate.9 D-8-Ad also induces a shift in the Soret
Figure 2. Fluorescence spectra of D-4-Ad (A) and D-8-Ad (B). Black, 2
µM D-8-Ad or D-4-Ad; purple, 2 µM dansyl probe + 1 equiv of P450cam;
blue to red, 2 µM P450cam and dansyl probe + 0.5, 1, 2, 4, 8, 16, 32, and
maximum from 416 to 414 nm: from the integrated D-8-Ad
fluorescence in the presence and in the absence of P450cam, we
estimate an upper limit Kd ≈ 0.02 µM for this probe.10
64 equiv of camphor (Kd ) 1.6 µM).9
The crystal structure of the P450cam:D-8-Ad complex shows
that the probe binds in the same channel as RuII(bpy)3-linker-Ad
The structure reveals a hydrogen bond between the amide
carbonyl of the probe and Tyr96 in P450cam:D-8-Ad, mimicking
the hydrogen bond between camphor and Tyr96 in the P450cam:
(bpy ) 2,2′-bipyridine) analogues (Figure 3).11 The eight-carbon
chain is nearly fully extended, allowing the dansyl moiety to bind
at the surface of the protein. The good fit is attributable to
substrate complex.12 In addition, there are a great many hydrophobic
conformational flexibility; that is, the F and G helices open just
interactions between the probe molecule and the enzyme; analysis
enough to allow the probe to enter and bind. The observed
of these contacts shows that much of the solvent-accessible surface
conformation is midway between the “closed” (camphor)12 and
area is buried. The estimated Kd of 0.02 µM corresponds to a
“open” (Ru-linker-Ad)11d structures.
binding energy of ∼11 kcal/mol, or ∼11 cal/(mol Å2) (Figure 4).
2+
The Ru(bpy)3 analogues (Ru-9-Ad and Ru-F8bp-Ad) do not
* Address correspondence to these authors. E-mail: hbgray@caltech.edu;
winklerj@caltech.edu.
bind as tightly to P450cam, but the free energy changes per buried
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J. AM. CHEM. SOC. 2002, 124, 10254-10255
10.1021/ja0271678 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Acknowledgment. This work was supported by the Fannie and
John Hertz Foundation (A.R.D.), the National Science Foundation,
and the National Institutes of Health (Metalloprotein Program
Project Grant P01 GM48495; NRSA fellowship GM20703 to A.-
M.A.H.).
Supporting Information Available: Syntheses of compounds;
spectroscopic data; and crystallographic experimental details (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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Figure 3. The 2.2 Å resolution structure of the D-8-Ad:P450cam cocrystal,
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Figure 4. (Top) Ruthenium tris-bipyridyl photosensitizers known to bind
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Figure 2b). These data are consistent with simultaneous binding of
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The two probes described herein illustrate the usefulness of our
methodology. D-4-Ad can be employed to screen potential P450
inhibitors, as it is easily displaced by other molecules with
comparable or lower dissociation constants. In contrast, D-8-Ad
binds extremely tightly: the conformational flexibility of the P450
fold allows the enzyme to close around the probe, thereby making
a great many productive hydrophobic contacts. The insight gained
from the D-8-Ad:P450cam structure could potentially lead to a more
rational design strategy for P450 inhibitors.
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