A panel of cytochrome P450 BM3 variants to produce drug metabolites and diversify lead compounds

Article abstract of DOI:10.1002/chem.200900643

Herein we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply-hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant.

Full text of DOI:10.1002/chem.200900643

FULL PAPER
DOI: 10.1002/chem.200900643
A Panel of Cytochrome P450 BM3 Variants to Produce Drug Metabolites
and Diversify Lead Compounds
Andrew M. Sawayama,[a] Michael M. Y. Chen,[a] Palaniappan Kulanthaivel,[b]
Ming-Shang Kuo,[b] Horst Hemmerle,[b] and Frances H. Arnold*[a]
Abstract: Herein we demonstrate that
a small panel of variants of cytochrome
P450 BM3 from Bacillus megaterium
covers the breadth of reactivity of
human P450s by producing 12 of 13
mammalian metabolites for two mar-
keted drugs, verapamil and astemizole,
and one research compound. The most
active enzymes support preparation of
individual metabolites for preclinical
bioactivity and toxicology evaluations.
Underscoring their potential utility in
drug lead diversification, engineered
P450 BM3 variants also produce novel
metabolites by catalyzing reactions at
carbon centers beyond those targeted
by animal and human P450s. Produc-
tion of a specific metabolite can be im-
proved by directed evolution of the
enzyme catalyst. Some variants are
more active on the more hydrophobic
parent drug than on its metabolites,
which limits production of multiply-hy-
droxylated species, a preference that
appears to depend on the evolutionary
history of the P450 variant.
À
Keywords: C H activation · cyto-
chrome P450 · drug development ·
drug metabolism · oxidation
Introduction
corporating discrete molecular recognition elements into
enzyme catalysts so that they can use specific enzyme–sub-
À
À
Selective C H oxidation represents one of the great chal-
lenges for which synthetic chemists find only very substrate-
strate interactions to impart reactivity to a specific C H
bond.
specific solutions.[1–4] Breakthroughs in selective C H oxida-
Cytochromes P450 (CYPs) are a large superfamily of
heme-containing C H oxidation enzymes. In humans, CYPs
À
À
tion methodology could benefit drug discovery, among other
fields, by enabling rapid and parallel analogue construction
for a specific molecular scaffold. Such improvements in effi-
ciency would greatly increase the number and variety of
compounds that could be produced, and raise the likelihood
of identifying effective therapeutic agents.
play key roles in drug metabolism and clearance. An ability
to predict how potential pharmaceuticals will be metabo-
lized will better equip us to identify derivatives with im-
proved biological activity, solubility, toxicity, stability or bio-
availability.[6] In fact, FDA guidelines indicate that uniquely
human metabolites and metabolites present at dispropor-
tionately higher levels in humans as compared to the animal
species used during standard toxicology testing may require
safety assessment before beginning large-scale clinical
trials.[7]
À
Many synthetic strategies for C H oxidation rely on a re-
À
active intermediate that plays upon subtle differences in C
H bond strength (1–5 kcalmolÀ1) to achieve regioselectivi-
ty.[5] Owing to the large number of C H bonds in most bio-
À
active chemicals, identifying a reagent that can react at one
À
C H bond in preference to all others can be difficult, or
even impossible. Nature solves the selectivity problem by in-
Preparation of these metabolites at sufficient scale for
evaluation is not trivial and often requires a de novo synthe-
sis for each metabolite. Biosynthetic methods that employ
purified human CYPs or crude liver microsomes are not
much better, because human CYPs are poorly stable, mem-
brane-bound, multiprotein systems that exhibit low reaction
rates. As an alternative to using human CYPs as biocatalysts
for metabolite production, we and others have focused on
soluble, bacterial P450 BM3 (also known as CYP102A1)[8]
[a] Dr. A. M. Sawayama, M. M. Y. Chen, Prof. F. H. Arnold
Division of Chemistry and Chemical Engineering 210-41
California Institute of Technology, Pasadena, CA 91125-4100 (USA)
Fax : (+1)626-528-8743
E-mail: frances@cheme.caltech.edu
[b] Dr. P. Kulanthaivel, Dr. M.-S. Kuo, Dr. H. Hemmerle
Eli Lilly & Company, Indianapolis, IN 46285 (USA)
[9–11]
À
as a C H oxidation platform.
Derived from Bacillus
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900643.
megaterium, P450 BM3 has properties that greatly facilitate
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its engineering and use in synthesis: it can be expressed at
high levels in E. coli (~12% dry cell mass), and, unlike
nearly all other CYPs, its hydroxylase, reductase and elec-
tron-transfer domains are all in one contiguous polypeptide
chain. This last feature might contribute to its relatively
high activity (>1000 turnovers per min) on its preferred
fatty acid substrates.[8] Like most CYPs implicated in ana-
bolic pathways, P450 BM3 is substrate specific, and hydroxy-
lates a C-20 fatty acid over a C-12 fatty acid with more than
200-fold higher efficiency.[12]
The variants, their sequences relative to wild type, and the
criteria for their prior selection from mutant libraries are
described in Table S1 in the Supporting Information.
Reactions with human and bacterial CYPs: Verapamil and
astemizole undergo extensive biotransformation by the
major human CYPs to produce demethylated and dealkylat-
ed products[23] by hydroxylation of activated carbon centers
adjacent to heteroatoms (summarized in Tables 1A, 2A).
Experiments conducted with rat liver microsomes in vitro
indicate that LY294002 metabolism is confined to single and
double alkyl hydroxylations of the morpholine ring
(Table 3), a pattern also observed in other drugs possessing
this moiety.
We chose three structurally diverse drug compounds with
known patterns of mammalian CYP-dependent clearance to
evaluate whether P450 BM3 variants can catalyze similar
À
C H oxidations. Verapamil is a calcium channel blocker
used in the treatment of hypertension and arrhythmia.[13]
Astemizole is a potent H1-histamine receptor antagonist
used for treatment of common sinus allergy symptoms.[14]
The third compound, LY294002, is an antiproliferative agent
that inhibits phosphatidylinositol 3-kinase, a target with po-
tential for treatment of malignancies.[15] We report here that
a small collection of P450 BM3 variants can produce nearly
all the known human (or rat) metabolites for each of the
three drugs. Within each set of active enzyme variants, we
identified several that produce selected metabolites in yields
and activities suitable for preparative scale synthesis. We
also identified variants that generate metabolites not pro-
duced by rat liver microsome controls or known to be
human metabolites; the results demonstrate the ability of
Reactions of the 120 P450 BM3 variants with each of the
three drugs were assessed by HPLC. Metabolite products
were evident in the HPLC traces for a significant number of
variants (43 exhibited some activity on verapamil, 42 on as-
temizole and 18 on LY294002); the metabolites and their
distributions were subsequently characterized by using
LCMS and MS/MS (Tables S3, S4 and S5 in the Supporting
Information). Metabolite structure assignments were aided
by comparison of MS and MS/MS spectra with spectra of
known metabolites obtained in our laboratories using rat
microsomal systems.
Verapamil: Table 1 summarizes the performance of selected
enzymes on verapamil (all 43 active enzymes and their prod-
uct distributions are listed in Table S3 in the Supporting In-
formation). For example, entry 4 displays the activity of chi-
mera 22313333 on verapamil. It converts 34% of the start-
ing material into an assortment of products, the distribution
of which is 41% 2, 15% 5, 20% 6, 9% 7 and 15% 10. Var-
iants 2C11 and 9C1 furnished the widest array of products,
with 6 and 8 individual metabolites, respectively. In particu-
lar, 9C1 produced several metabolites that had undergone
two hydroxylation events (1, 4 and 8). Variant 22313333
(entry 4) was the only enzyme capable of producing double
hydroxylation product 5, while 32313233 (entry 6) best pro-
duced norverapamil (3). Interestingly, the addition of reduc-
tase as a fusion to the 32313233 heme domain (entry 7) ren-
dered the enzyme unable to produce new metabolite 7. Var-
iant 7-11D (entry 13) differs by no more than two mutations
from the variants in each of entries 9–12, but its product dis-
tribution more closely parallels chimera 21312332. The 9-
10A family of enzymes was the most active, with conver-
sions exceeding 30% (entries 9–12), and produced metabo-
lite 7 with excellent selectivity.
À
this C H oxidation platform to target a range of carbon
centers.
Results
Selection of P450 variants: From our extensive collections
we selected 120 P450 BM3 variants that had previously
demonstrated activity on substrates not in the wild-type en-
zymeꢁs repertoire. These variants were constructed by using
a variety of commonly implemented genetic diversification
techniques, including error-prone PCR and targeted muta-
genesis of active site residues. We also included variants de-
rived from structure-guided recombination of BM3 and its
homologues, CYP102A2 and CYP102A3, to produce chi-
meric enzymes.[16] These enzymes had been selected based
on their activities towards a variety of substrates, including
straight-chain alkanes, cycloalkanes, alkyl ethers,[17–19] aro-
matic compounds,[20] derivatives of fatty acids,[21] and
drugs.[10,22]
We also distinguished between enzyme variants that func-
tion as monooxygenases and use NADPH to reduce the cat-
alytic FeIII center, and peroxygenases that contain only the
heme domain and use H2O2 in place of O2 and NADPH.
Each has its own catalytic and operational advantages and
disadvantages that have been described elsewhere.[21]
Though we had evidence that the presence of a reductase
domain impacts activity favorably,[20] we were unsure what
effect, if any, this domain would have on regioselectivity.
The enzymes that best mimicked human CYP reactivity
on verapamil were derived from two variant families. Three
had been isolated by directed evolution for activity on pro-
pranolol,[22] while the remaining four were chimeras.[16,24]
The BM3 variants that best produced new metabolites were
derived from the alkane-hydroxylating 9-10A variant.[18,19]
These enzymes catalyzed a new regioselective demethyla-
tion reaction at position R4 and a new benzylic oxidation at
position R3. Even without optimization of the reaction con-
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Directed Evolution of 9-10A F87L for Activity on Astemizole
FULL PAPER
Table 1. Conversion of verapamil to: A) its most abundant[a] human me-
tabolites, and B) new metabolites by different P450 BM3 variants.
Table 2. Conversion of astemizole to: A) its most abundant[a] human me-
tabolites, and B) new metabolites by different P450 BM3 variants.
Variant[b]
% Con-
version
11
12
13
14
15
16
17
Variant[b]
% Con-
version
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
DE10
21313311
22313333
9-10A A78F
9-10A A78T
9-10A A82S
9-10A F87L
41-5B
9
10
9
21
27
30
36
5
11
16
78
15
49
32
31
56
40
56
20
40
22
22
48
37
20
1
2
3
4
5
6
7
8
9
2C11
25
31
78
34
43
24
8
28
29
26
20
6
31
8
8
28
9C1[c]
3
3
8
13
3
10 13
3
38
44
70
88
14
4
3
7
7
3
D6H10
22313333
22313231
32313233
32313233-R1 25
21312332
9-10A A78F 30
24
15
16
17
16
33
41
46
15 20
33
9
5
6
100
50
40
17
21 13
44
33 17
83
2
6
6
9
32313233
45
13
4
27
69
67
80
61
27
9
10
11
12
13
14
15
32313233-R1
32312333-R1
9-10A A78S
9-10A A82L
9-10A A82I
9-10A F87A
6
10
13
24
31
7
6
10 9-10A A82L 51
11 9-10A F87L
12 12-10C
94
94
79
4
49
34
21
63
23
6
28
3
16
45
13 7-11D
29 14
29
[a] Defined as >1% abundance following oral 14C astemizole administra-
tion in humans.[25] [b] See Table 1 for further explanation of variant no-
menclature.
[a] Defined as >1% abundance following oral 14C verapamil administra-
tion in humans.[23] [b] Variants shown in italics were selected for activity
on propranolol,[22] variants in bold text are chimeras,[16] variants in
normal type were selected for activity on alkanes.[18, 19] Chimeras are writ-
ten according to fragment composition: 32313233-R1, for example, repre-
sents a protein that inherits the first fragment from parent CYP102A3,
the second from CYP102A2, the third from CYP102A3, and so on. R1
connotes a fusion to the reductase domain from parent A1. Chimera fu-
sions were used as monooxygenases; chimera heme domains were used
as peroxygenases. [c] Because not all P450 BM3 oxidation products could
be identified, product distribution totals can be less than 100%.
D6H10). By contrast, chimera 22313231 produced metabo-
lite 2, chimera 32313233-R1 made dealkylated compound 6
(entries 5 and 7), and many variants produced new metabo-
lites 7 and 10 (entries 8–13) with sufficient selectivity (ꢀ
30%) for larger scale production without further optimiza-
tion. To demonstrate that useful quantities of metabolites
can be produced, we used purified 9-10A F87L to produce
metabolite 7 (9.4 mg) from verapamil (25 mg) in 39% yield
(1560 TTN, 0.025 mol% catalyst).
ditions, some enzymes were highly active on verapamil;
D6H10 (entry 3), for example, transformed verapamil into
metabolites at 78% conversion and a total turnover number
(TTN) greater than 1500.
The enzymes possess contrasting degrees of regioselectivi-
ty. Some were unselective and produced a spectrum of me-
tabolites (e.g., propranolol-evolved enzymes 2C11, 9C1,
Astemizole: Activity of the enzyme panel on astemizole re-
sulted in the seven metabolites described in Table 2 (all 42
active enzymes and their product distributions are listed in
Table S4 in the Supporting Information). Variants DE10,
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F. H. Arnold et al.
Table 3. Conversion of LY294002 by different P450 BM3 variants into
the most abundant[a] metabolites produced by rat liver microsomes.
Small changes in the substrate channel can affect the re-
À
gioselectivity of aromatic C H oxidation. For instance, the
presence and position of a single methyl group in Thr78 vs.
Ser78 (entry 5 vs. 12) and Ile82 vs. Leu82 (entry 14 vs. 13)
were sufficient to bias oxidation at C6 instead of at another
position on the benzimidazole ring. Product 16 has under-
gone two oxidations. Though this metabolite was observed
in reactions with ten variants, it was produced in greater
than 10% abundance by only one (entry 4). New metabolite
15 is a candidate for scale-up and could be produced at high
conversion (>25% selectivity) by 15 variants of different
lineages (Table S4 in the Supporting Information). Even
without optimization of reaction conditions, several enzymes
showed good activity towards astemizole. Variant 32312333-
R1, for example, converted 78% astemizole into metabolites
(70 TTN).
Variant[b]
% Conversion
18
19
20
LY294002: The most abundant metabolites produced by rat
liver microsomes are detailed in Table 3. Both single hy-
droxylation products 19 and 20 were identified in BM3 var-
iant reactions with very good regioselectivity. Aminoalcohol
18 is the sole metabolite of all three drugs not observed in
the reactions. Derivative 18 requires two oxidations and
might not appear simply due to the low conversion in these
96-well plate reactions.
1
2
3
4
5
9-10A L75I
9-10A A78S
9-10A F87V
9-10A T260S
68-8F
10
7
6
11
9
50
71
100
9
11
[a] Defined as >1% abundance following in vitro reaction of LY294002
with rat liver microsomes. [b] Variants in normal type were selected for
activity on alkanes.[18, 19]
The P450s catalyzing the single hydroxylations were
ACHUTNGERNmNUG onoHCATUNGTRENNoUGN xygenases derived from 9-10A (Table 3). Though sev-
eral variants could produce metabolite 19, only 9-10A F87V
oxidized the other position of the morpholine ring and
made derivative 20 (entry 3). The remaining 12 metabolite-
producing variants were also monooxygenases of both 9-
10A and chimeric origin, and produced a metabolite with
Mw =238; this indicates morpholine loss and addition of
water to the bis-aryl backbone (Table S5 in the Supporting
Information). In all cases, LY294002 conversion was very
low (<15%) and correlates with the low rat liver micro-
some conversion for LY294002 (40%) relative to microsome
activity on verapamil (85%) and astemizole (85%).
LY294002 better evades both mammalian and bacterial
21313311, 22313333, 32313233 (entries 1–3 and 9) produced
dealkylated metabolite 14 in preference to other com-
pounds. The identity of the residue at position 78 had a
strong impact on the product distribution within the 9-10A
backbone; mutations A78F, A78T and A78S (entries 4, 5
and 12) produced metabolites 11, 13 and 15 as the most
abundant respective products. Furthermore, 41-5B (entry 8),
a 9-10A family member that contains the A78F mutation (in
addition to A82G and A328V) also favors demethylated 11.
Variants 9-10A A82S and 9-10A F87L both produced me-
tabolite 13 in good yield and selectivity (entries 6 and 7).
Two chimeric monooxygenases, 32313233-R1 and 32312333-
R1 (entries 10 and 11) were effective in generating new aro-
matic hydroxylation product 15.
À
P450-catalyzed C H oxidation than verapamil and astemi-
zole under the same conditions.
The 9-10A-derived monooxygenases were the most adept
at producing human metabolites of astemizole (entries 4–8).
However, these enzymes were unable to dealkylate astemi-
zole and produce metabolite 14. Oxidation of this more hin-
Activities on singly-hydroxylated metabolites: Of the 12
mammalian metabolites produced by the P450 BM3 var-
iants, eight were made with sufficiently high selectivity to
enable preparative scale production without further optimi-
zation (2, 3, 6, 11, 13, 14, 19 and 20). Of the seven new me-
tabolites to which structures could be assigned, four were
made with sufficiently high regioselectivity to enable prepa-
rative scale production (7, 10, 15 and 17). All 11 of the
highly produced metabolites arose from single oxidations.
Seven of the eight remaining metabolites are the products
of two or more oxidations and are present as minor compo-
nents in mixtures with the singly-hydroxylated products. Of
the 69 individual reactions that underwent appreciable con-
version (ꢀ10% drug consumed) only five generated prod-
À
dered C H bond was best accomplished by the propranolol-
active variants and chimeric peroxygenases (entries 1 and 2,
3, respectively). A new benzylic site not hydroxylated by
human CYPs was targeted by six chimeric BM3 enzymes to
produce 17. A second benzimidazole site was hydroxylated
(as demonstrated by metabolites 15 and 16), which is differ-
ent from that observed in human metabolites 12 and 13.
Discrimination of these sterically and electronically identical
2
À
sp C H bonds is virtually impossible by using traditional
transition metal catalysts and emphasizes the power of mo-
lecular recognition as a regiocontrolling element.
À
ucts of multiple C H oxidations in ꢀ10% abundance.
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Directed Evolution of 9-10A F87L for Activity on Astemizole
FULL PAPER
The remainder contained single-oxidation products, in-
cluding mixtures of singly-oxidized species. The lack of bis-
hydroxylated products could reflect a regiochemical effect
lectivity (Table 2, entry 7); its reaction with astemizole,
which produces metabolite 13 with 88% selectivity, is repre-
sentative of a reaction for which one would like to improve
metabolite production. We speculated that a high-through-
put screen for improved overall aromatic hydroxylation ac-
tivity would yield at least a few variants that retained this
regioselectivity while producing more metabolite. We
screened 2000 variants made by error-prone PCR of the 9-
10A F87L gene using a colorimetric screen for products of
aromatic hydroxylation[29] and identified three new sequen-
ces with improved metabolite production (Table 5). The new
variants improved the conversion to 51–52% while preserv-
ing high regioselectivity (~80% for metabolite 13).
À
in which the second C H site is less reactive. In this case,
one of the two possible single-hydroxylation products would
be favored. Sixteen variants produced only one single-hy-
droxylation product; reaction mixtures from ten additional
variants contained two (metabolites were present in 1:1 to
3:1 ratios in 6/11 reactions). It is also possible that the rela-
tive lack of bis-hydroxylation products reflects discrimina-
tion on the part of the enzymes in which the BM3 binds and
hydroxylates the parent drug in preference to its metabo-
À
lites. Because BM3-catalyzed C H oxidation increases po-
larity by either unmasking heteroatoms or formally substi-
tuting hydrogen with a hydroxyl group, production of poly-
hydroxylated metabolites could be disfavored, for example,
if the enzyme prefers a more hydrophobic substrate.
Table 5. Production of astemizole metabolites by 9-10A F87L variants.
Variant
% Conversion
% Selectivity 13
1
2
3
4
9-10A F87L
E4D D68G
C205R D338G
E4D H92Q
34
51
51
52
88
80
80
75
To assess enzyme activity on the metabolites produced by
a single oxidation, all 120 enzymes were incubated with pu-
rified demethylated metabolites norverapamil (3) and des-
methylastemizole[26] (11). Product distributions and extents
of reaction were determined by HPLC. In every case in
which an enzyme was active on both parent drug and me-
tabolite, regioselectivities were unchanged; this indicates
that the metabolite and parent drug bind in similar orienta-
tions. Comparison of each enzymeꢁs activity on the parent
drug and the associated metabolite showed that enzymes
from the alkane-evolved 9-10A lineage tended to be more
active on astemizole and verapamil than on their metabo-
lites (true of 49 out of 55 active enzymes; Table 4). Because
Discussion
A small, 120-member panel of P450 BM3 variants captured
nearly all of the mammalian P450 scope of reactivity by pro-
ducing 12 of 13 known metabolites. In their ability to mimic
human CYPs, the P450 BM3 variants demonstrated consid-
À
erable versatility, activating C H bonds of varying strength
(90–105 kcalmolÀ1) and steric encumbrance (sp2 vs. sp3 and
18 vs. 28 carbon centers). We were able to assign structures
to seven new metabolites (Tables 1B, 2B) all of which have
undergone oxidation at new carbon centers.
Table 4. Preference for more hydrophobic parent drug over its metabo-
lite depends on evolutionary history of the variant.
Cytochrome P450 enzymes are versatile catalysts the bio-
logical activities of which are encoded in a wide range of
primary sequences. The 26 CYPs for which the crystal struc-
tures have been solved possess as little as 15% sequence
identity,[30] but share a highly conserved fold. Emphasizing
the versatility of the P450 fold, P450cam and P450cin cata-
lyze the oxidation of isosteres camphor and cineole. Al-
though the substrates are nearly identical, the two enzymes
differ not only in sequence (27% identity), but also in the
structure of their active sites, most notably a complete lack
of the B’ helix in P450cin.[31,32] Whereas large changes in se-
quence are possible, only small perturbations are necessary
to produce significant changes in function. For example,
CYP2A4 and CYP2A5 differ by only 11 amino acids, yet
catalyze hydroxylations on structurally dissimilar coumarin
and testosterone substrates.[33]
P450 Variant family
Number of P450s more Number of P450s more
active on parent drug
active on metabolite
Alkane-selected line-
age
Propranolol-selected
and chimera lineages
49/55
6/55
18/30
12/30
neither the N-methyl group of verapamil nor the O-methyl
group of astemizole is the preferred site for C H oxidation
À
by the alkane-evolved variants, this bias could reflect a pref-
erence for the more hydrophobic substrate. In contrast,
when this pairwise reaction matrix is analyzed across the
propranolol-evolved and chimeric P450 BM3 lineages, there
is no statistical difference from random at the 95% confi-
dence interval (0.43ꢁp=0.60ꢁ0.77).
Only 57 human enzymes[34] are responsible for known
CYP-dependent drug metabolism, and a single enzyme,
CYP3A4, accounts for >50% of the burden for xenobiotic
CYP-mediated clearance.[35] Because CYPs can be broadly
or narrowly specific, we and others have speculated that it
should be possible to take advantage of the high native ac-
tivity of P450 BM3 and use mutation to either relax or shift
Directed evolution can improve metabolite production:
None of the 120 members of this catalyst panel had been se-
lected for activity on any of the three drugs. Thus, any initial
activity represents a promiscuous activity; such side activi-
ties are often easy to improve by directed evolution.[27,28] Of
the 103 enzymes that reacted with these substrates, 9-10A
F87L possessed the best combination of activity and regiose-
À
its substrate specificity in order to generate useful C H oxi-
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F. H. Arnold et al.
dation catalysts.[9,36–39] For example, BM3 variant 9-10A,
which is 13 mutations away from the wild type, exhibits
broad activity across short- and medium-chain alkanes—ac-
tivity that is low or completely absent in its wild-type
parent.[18,19] Furthermore, 9-10A could be respecialized to
hydroxylate propane, preferring it over alkanes that differ
by a single methylene group.[40] We also showed previously
that a variant of 9-10A was able to hydroxylate drug-like
compounds efficiently and selectively.[10] Here we wanted to
determine whether variants of 9-10A and other BM3-de-
rived enzymes could cover or even exceed the broad sub-
strate range of mammalian CYPs. Within any catalyst panel,
both extremes of regioselectivity can be useful: enzymes
that already possess the desired selectivity can be used to
produce individual metabolites, whereas less selective en-
zymes can be used to survey metabolite possibilities. Both
can serve as starting sequences for directed evolution to en-
hance activity or tune selectivity. That a systematic and gen-
eral evolutionary algorithm can be used for catalyst im-
provement is a particularly appealing aspect of DNA-en-
coded reagents. Complementary optimization studies used
in traditional synthesis methods usually rely on chemical in-
tuition to improve a catalyst and require a good understand-
ing of the catalytic mechanism.
When we examined conversion of the parent drug versus
its demethylated metabolite, we noted that one enzyme
family consistently converted more of the parent drug than
the demethylated and more polar metabolite. These en-
zymes tend to catalyze single hydroxylations, while enzymes
evolved for activity on propranolol and the chimeric var-
iants often catalyze bis-hydroxylations. Structure–activity re-
lationships of this type should help in the future to select en-
zymes that are most likely to react with as yet untested sub-
strates and could also help predict product profiles.
Experimental Section
General: All chemicals were purchased from Sigma–Aldrich, Inc. or pro-
vided by Eli Lilly directly. Solvents were purchased from EM Sciences.
Lysis enzymes were purchased from Sigma. Absorbance measurements
were conducted by using a SpectraMax 384 Plus plate reader. HPLC sep-
arations were performed by using a Supelco Discovery C18 column (2.1ꢂ
150 mm, 3m) on a Waters 2690 Separation module in conjunction with a
Waters 996 PDA detector. LCMS and MS/MS spectra were obtained by
using the ThermoFinnigan LCQ classic at the shared Caltech MS facility.
Protein expression: LB agar plates supplemented with ampicillin
(100 mgmLÀ1) were streaked with the catalase-deficient strain of E. coli
SN0037[44] (for peroxygenase) or DH5a E. coli (for monooxygenase) con-
taining a desired P450 BM3 variant in the isopropyl b-d-thiogalactopyra-
noside (IPTG)-inducible pCWori vector.[45] These were grown at 378C
for 12 h before single clones were picked and added in quadruplicate to
1 mL 96-well plates containing LB medium (400 mL) supplemented with
ampicillin (100 mgmLÀ1). After being shaken at 80% humidity, 308C for
24 h to grow the precultures to saturation, an aliquot (50 mL) was used to
inoculate 2 mL 96-well plates containing TB medium (900 mL) supple-
mented with ampicillin (100 mgmLÀ1). After being shaken at 80% humid-
ity, 308C for 5 h, P450 expression was induced by addition of IPTG
(500 mm) and the heme precursor d-aminolevulinic acid (d-ALA) to a
final concentration of 1 mm. The cultures were grown for another 24 h
before the cells were centrifuged and stored at À208C.
Activity-based screening: All three drugs and two metabolites were
screened against the cell lysate of the panel of 120 variants. Lysate was
prepared by resuspending cell pellets with a buffer (600 mL) containing
MgCl2 (10 mm), lysozyme (0.5 mgmLÀ1) and DNAseI (8 UmLÀ1). For
the holoenzyme reactions phosphate buffer (0.1m, pH 8) was used and
for the peroxygenase reactions EPPS buffer (0.1m, pH 8.2) was used. The
lysis reactions were incubated at 378C for 1 h and then centrifuged; the
supernatant was used in three assays.
CO binding: Heme proteins absorb light at 450 nm corresponding to the
CO stretch frequency when the Fe-heme is bound to CO. This Soret
band can be used to quantify the amount of folded protein.[46] Lysate
(100 mL) and sodium dithionite (100 mL, 140 mm in 1m buffer, pH 8) were
added to a 96-well flat-bottom screening plate. These were preread by
using a plate reader at 450 and 490 nm before being incubated in a 1 atm
CO chamber for 15 min. The plate was then read again at 450 and
490 nm.
Enzymatic activity: In a 96-well (2 mL) plate, the following mixtures
were prepared for each of the activity-based assays. Holoenzyme: lysate
(60 mL), phosphate (110 mL, 0.1m, pH 8), drug/metabolite (10 mL, 5 mm),
NADPH (20 mL, 20 mm); peroxygenase: lysate (50 mL), EPPS (100 mL,
0.1m, pH 8.2), drug/metabolite (10 mL, 20 mm), hydrogen peroxide
(40 mL, 5 mm).
Conclusion
This panel should enable rapid identification and production
of relevant quantities of the human metabolites of drug can-
didates for pharmacological and toxicological evaluations in
preclinical species.[41,42] Although we have highlighted the
potential of these enzymes to accelerate preparation of me-
tabolites for pharmacological and toxicological testing, this
enzyme panel is likely also to be useful further upstream in
the drug-development process as general reagents for lead
diversification. Reagents that rely on molecular recognition
will always be restricted in their scope of use. However, be-
cause the functionality of small molecules is not evenly dis-
tributed across all possible molecular architectures,[43] it
should be worthwhile to engineer P450-derived reagents
that are active on privileged scaffolds that reside in these
densely functional regions of structure space. The plurality
Upon ultimate addition of NADPH or H2O2, the plates were briefly
shaken and incubated for 2 h. After this time, acetonitrile (200 mL) was
added to quench the reactions. The reactions were centrifuged and the
supernatants were used for subsequent analysis with HPLC and LCMS.
HPLC: Supernatant (25 mL) was analyzed by HPLC. Conditions with sol-
vent A (0.2% formic acid v/v, in H2O) and solvent B (acetonitrile) used
to elute the products of metabolism were: 0–3 min, A/B 90:10; 3–25 min,
linear gradient to A/B 30:70; 25–30 min, linear gradient to A/B 10:90.
LCMS, MS/MS: Identical conditions to the HPLC method detailed
above were used for the LC portion of the analysis. The MS was operat-
ed in positive ESI mode. MS/MS spectra were acquired in a data-depen-
dent manner for the most intense ions.
Preparation of metabolite 7: Glucose-6-phosphate (80 mm), glucose-6-
phosphate
dehydrogenase
(2 UmLÀ1),
superoxide
dismutase
(100 UmLÀ1), 9-10A F87L (250 nm), verapamil (24.6 mg, 1 mm) and
NADP (5 mm) were added to an Erlenmeyer flask (250 mL) containing
potassium phosphate buffer (50 mL, 100 mm, pH 8.0). These reagents
were stirred vigorously at room temperature while metabolite production
was monitored every hour for 4 h by HPLC. The reaction was quenched
by precipitating the enzymes with acetonitrile (50 mL) and by stirring for
À
of C H sites targeted by this small P450 BM3 variant set—
including and extending human P450 metabolism—augurs
well for the development of a truly general panel of C H
À
oxidation catalysts.
11728
www.chemeurj.org
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 11723 – 11729

Directed Evolution of 9-10A F87L for Activity on Astemizole
FULL PAPER
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This work was supported in part by NIH grant GM068664, the Jacobs In-
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Received: March 10, 2009
Revised: August 11, 2009
Published online: September 22, 2009
Chem. Eur. J. 2009, 15, 11723 – 11729
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11729