A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore.

Abstract

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

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A class of selective antibacterials derived from
a protein kinase inhibitor pharmacophore
J. Richard Millera,1,2,3, Steve Dunhama,1,4, Igor Mochalkina,1,2, Craig Banotaia, Matthew Bowmana, Susan Buista,
Bill Dunklea,4, Debra Hannaa,2, H. James Harwoodb, Michael D. Hubanda,2, Alla Karnovskya, Michael Kuhna,2,
Chris Limberakisa,2, Jia Y. Liua, Shawn Mehrensa,2, W. Thomas Muellera,5, Lakshmi Narasimhana,6, Adam Ogdena,2,
Jeff Ohrena,b, J. V. N. Vara Prasada, John A. Shellya,4, Laura Skerlosa, Mark Sulavika, V. Hayden Thomasa,2,
Steve VanderRoesta, LiAnn Wangc, Zhigang Wanga,7, Amy Whittona, Tong Zhua,2, and C. Kendall Stovera,4
aPfizer, Inc., Ann Arbor, MI 48105;bPfizer, Inc., Groton, CT 06340; andcPfizer, Inc., Cambridge, MA 02139
Edited by Michael A. Marletta, University of California, Berkeley, CA, and approved December 5, 2008 (received for review November 10, 2008)
As the need for novel antibiotic classes to combat bacterial drug
resistance increases, the paucity of leads resulting from target-
based antibacterial screening of pharmaceutical compound librar-
ies is of major concern. One explanation for this lack of success is
that antibacterial screening efforts have not leveraged the eukary-
otic bias resulting from more extensive chemistry efforts targeting
eukaryotic gene families such as G protein-coupled receptors and
protein kinases. Consistent with a focus on antibacterial target
space resembling these eukaryotic targets, we used whole-cell
screening to identify a series of antibacterial pyridopyrimidines
derived from a protein kinase inhibitor pharmacophore. In bacte-
ria, the pyridopyrimidines target the ATP-binding site of biotin
carboxylase (BC), which catalyzes the first enzymatic step of fatty
acid biosynthesis. These inhibitors are effective in vitro and in vivo
against fastidious Gram-negative pathogens including Haemophi-
lus influenzae. Although the BC active site has architectural simi-
larity to those of eukaryotic protein kinases, inhibitor binding to
the BC ATP-binding site is distinct from the protein kinase-binding
mode, such that the inhibitors are selective for bacterial BC. In
summary, we have discovered a promising class of potent anti-
bacterials with a previously undescribed mechanism of action. In
consideration of the eukaryotic bias of pharmaceutical libraries,
our findings also suggest that pursuit of a novel inhibitor leads for
antibacterial targets with active-site structural similarity to known
human targets will likely be more fruitful than the traditional focus
on unique bacterial target space, particularly when structure-based
and computational methodologies are applied to ensure bacterial
selectivity.
acetylcoenzyme A carboxylase ? biotin carboxylase ? crystal structure ?
high-throughput screening ? fatty acid biosynthesis
T
antibacterial agents. Literally thousands of new essential bacterial
targets from human pathogens were identified as a result of the
genomics revolution (1–3). This necessitated a means to ‘‘triage’’
these targets to identify the most promising ones to pursue by high
throughput screening of pharmaceutical compound files. In these
target analyses, one desirable attribute was little or no sequence
and/orstructuralhomologytohumangeneproductstoimprovethe
chances of finding bacterial-selective hits. However, this selection
criterion could also have a deleterious effect on the number of
potent hits identified because the medicinal chemistry efforts that
built these compound files were largely focused on human thera-
peutic targets such as kinases, G protein-coupled receptors, pro-
teases, etc. (1–4). Therefore, pursuit of bacterial targets with the
greatest sequence and/or structural relatedness to proven eukary-
otic drug targets could be more fruitful. Consistent with this
hypothesis, we used unbiased whole-bacterial cell screening of the
Pfizer compound library to discover a series of antibacterial pyri-
dopyrimidines (1, 2, and 3; Fig. 1A) that emerged from a structure-
hewell-documentedincreaseinantibacterialresistanceoverthe
past few decades has led to intensive efforts to discover novel
based drug design program targeting eukaryotic tyrosine protein
kinases (5). By using genetic and biochemical tools, the bacterial
target of these compounds was identified as biotin carboxylase
(BC); one portion of the acetyl-CoA carboxylase (ACCase) mul-
tienzyme complex responsible for the first step of fatty acid
biosynthesis (6). BC has an active site with considerable structural
similaritytoeukaryoticproteinkinases,yetitissufficientlydifferent
to allow for selectivity against both human kinases and the eukary-
otic ACCase.
The identification of selective antibacterials containing a
kinase inhibitor pharmacophore has intriguing implications for
antibacterial drug discovery, particularly given that the targets,
biotincarboxylaseandeukaryoticproteinkinases,havestructurally
related ATP-binding sites. It remains to be seen if the huge array
of eukaryotic inhibitors present in pharmaceutical libraries can be
mined for their activity against structurally related bacterial targets
suchasthebacterialhistidinekinasesinvolvedincell–cellsignaling,
lipopolysaccharide sugar kinases involved in Gram-negative cell
wall formation, antibiotic kinases that deactivate specific anti-
bacterial agents, or less obvious targets, such as biotin carboxy-
lase. Our results argue that there may be value in reassessing
antibacterial target space for previously unexplored (or under-
explored) targets amenable to an approach based on repurpos-
ing eukaryotic pharmacophores.
Results
Identification of Antibacterial Pyridopyrimidines. As part of our
antibacterial drug discovery effort, a library of ?1.6 million
Author contributions: J.R.M., S.D., I.M., S.B., D.H., L.N., A.O., J.V.N.V.P., M.S., V.H.T., Z.W.,
T.Z., and C.K.S. designed research; J.R.M., S.D., I.M., C.B., M.B., S.B., B.D., M.D.H., M.K., C.L.,
J.Y.L., S.M., W.T.M., L.N., A.O., J.O., J.V.N.V.P., J.A.S., L.S., S.V., L.W., Z.W., and A.W.
performed research; J.R.M., S.D., I.M., C.B., M.B., B.D., H.J.H., A.K., M.K., C.L., J.Y.L., S.M.,
W.T.M., L.N., J.V.N.V.P., J.A.S., V.H.T., S.V., and Z.W. contributed new reagents/analytic
tools; J.R.M., S.D., I.M., M.B., S.B., D.H., H.J.H., M.D.H., A.K., C.L., J.Y.L., L.N., A.O., J.O.,
J.V.N.V.P., L.S., M.S., L.W., Z.W., A.W., T.Z., and C.K.S. analyzed data; and J.R.M., S.D., I.M.,
J.O., and C.K.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org [PDB IC codes 2V58 (compound 1), 2V59 (compound 2),
and 2V5A (compound 3)].
See Commentary on page 1689.
1J.R.M., S.D., and I.M. contributed equally to this work.
2Present address: Pfizer, Inc., Groton, CT 06340.
3Towhomcorrespondenceshouldbeaddressedatthepresentaddress:Pfizer,Inc.,Eastern
Point Road, Groton, CT 06340. E-mail: richard.miller2@pfizer.com.
4Present address: Pfizer, Inc., Kalamazoo, MI 49007.
5Present address: Pfizer, Inc., Chesterfield, MO 63017.
6Present address: Pfizer, Inc., La Jolla, CA 92121.
7Present address: Pfizer, Inc., Cambridge, MA 02139.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0811275106/DCSupplemental.
© 2009 by The National Academy of Sciences of the USA
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individual compounds was screened for growth inhibition of a
membrane-compromised, efflux pump-deficient strain of Esche-
richia coli (tolC, imp). The pyridopyrimidines, typified by 1, 2, and
3, were among the inhibitors identified in this screen. These
compounds exhibit potent antibacterial activity against the screen-
ing strain and several other human pathogens (Table 1). Most
notable was their exquisite potency against clinical isolates of
fastidious Gram-negative pathogens such as Haemophilus influen-
zae and Moraxella catarrhalis; causative agents of many respiratory
tract infections. Identification of these compounds as antibacterials
was intriguing given their pharmaceutical history. N2-substituted
analogs (typified by 4) are potent inhibitors of the VEGFR2 and
FGFR1 eukaryotic protein kinases (Table 3) (5). In the pretarget-
baseddrugdiscoveryeraofthe1970s–1980s,membersofthisseries
were also investigated as potassium-sparing diuretics with antihy-
pertensive effects in rats (7).
Mechanism of Pyridopyrimidine Antibacterial Activity. To determine
whethertheantibacterialactivityof1and2wascausedbyaspecific
mechanism of action, a biological macromolecular biosynthesis
assay was used. Compounds 1 and 2 specifically inhibit fatty acid
biosynthesis in E. coli (Table 2). Spontaneous mutations conferring
resistance to at least 8 times the minimal inhibitory concentration
(MIC) of 1 were isolated at frequencies of ?1 in 109in E. coli, H.
influenzae, and M. catarrhalis [supporting information (SI) Tables
S1 and S2]. The mutations responsible for resistance were mapped
and confirmed by backcross experiments to the accC gene, which
encodes the BC component of ACCase. ACCase utilizes ATP,
bicarbonate,andacetyl-CoAtocatalyzeformationofmalonyl-CoA
(Fig.S1)andisthefirstcommittedstepinfattyacidbiosynthesis(6).
When the H. influenzae resistance-conferring mutations were
mapped onto the crystal structure of E. coli BC containing bound
ADP (8), the mutations clustered within the ATP-binding pocket
(Fig. 1B and Table S2), indicating that the pyridopyrimidines likely
interact with this portion of the BC active site. This finding is
consistentwithpriorknowledgethatmembersofthischemicalclass
bind the ATP site of FGFR kinase (9) and are competitive with
ATP (5, 10).
Pyridopyrimidines Bind Bacterial BC. Molecular interaction of 1 with
purified BC from E. coli, H. influenzae, Pseudomonas aeruginosa,
and Saccharomyces aureus was confirmed by isothermal titration
calorimetry(ITC)and/orsurfaceplasmonresonance(SPR,Fig.S2,
Table 4, and Table S3). Binding of 1 to E. coli BC is enthalpically
driven, potent (Kd800 ? 200 pM) and exhibits a slow dissociation
rate from the enzyme. The pyridopyrimidine inhibitors are also
potent(Table3)inanenzyme-coupledE.coliACCaseholoenzyme
reaction (Fig. S1). Inhibition in this assay, which requires biotin-
ylated biotin carboxyl carrier protein, carboxytransferase, and BC
for turnover, demonstrates that pyridopyrimidine binding to BC
inhibits the physiologically relevant ACCase holoenzyme reaction.
Purified active BC enzyme from E. coli, H. influenzae, and P.
aeruginosa readily crystallized with 1, 2, and subsequently with
numerous analogs thereof, yielding high-resolution crystal struc-
turestoguidestructure-basedleadoptimization.Theunambiguous
electron density map of inhibitor 1 (Fig. 2A) described a consistent
binding mode of the pyridopyrimidine inhibitors at the BC ATP-
Fig. 1.
conferring mutations in biotin carboxylase. (A) Pyridopyrimidine inhibitors of
BC (1, 2, and 3) and FGFR1 (4). (B) X-ray costructure of ADP and E. coli BC (8).
Residues conferring resistance to 1 upon mutation are highlighted.
Pyridopyrimidine inhibitor structures and sites of resistance-
Table 1. Microbiological data for BC inhibitors: MIC and MBC in
?g mL?1
Strain (genotype)* Compound tested
Additives
Test type
E. coli (tolC, imp?)
E. coli (tolC, imp?), 2% HSA†
E. coli (wild type)
H. influenzae (acrA)
H. influenzae (wild type)
H. influenzae (accC-I437T)
M. catarrhalis (acrA)
M. catarrhalis (wild type)
S. aureus (norA)
S. aureus (MRSA)
S. pneumoniae (wild type)‡
E. faecalis (wild type)§
P. aeruginosa (mexAB oprM)
P. aeruginosa (wild type)
1123 Linezolid
MICMIC
0.125
0.5
16
0.125
0.125
16
0.5
1
16
32
?64
?64
32
?64
MBC
1
MIC
2
4
?64
MIC
88
832
32
?64
?64
0.5
1
64
2
2
32
?64
?64
?64
?64
?64
48
0.5
32
3216
8
8
8
2
2
2
2
64
?64
8
82
64
64
?64
?64
?64
?64
?64
?64
*The following designate targeted knockouts of efflux pumps or subunits of
efflux pumps: tolC, acrA, norA, mexAB, and oprM. The imp gene disruption
furthersensitizesE.colitoinhibitors.TheaccC-I437TgenotypeinHaemophi-
lus results from spontaneously resistant mutants selected in the presence of
compound 1.
†Human serum albumin.
‡Streptococcus pneumoniae.
§Enterococcus faecalis.
Table 2. E. coli (tolC imp) macromolecular synthesis assay IC50
(?g mL?1)
PathwayCompound 1 Triclosan
Cell growth
DNA
RNA
Protein
Fatty acid
0.1250.1
?50
?50
?50
?1
?10
?10
?10
1.3
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binding site with the compound engaged in an extensive ligand-
efficientnetworkofinteractionswithintheadenine-bindingregion,
whereas the bisubstituted phenyl group occupied the hydrophobic
pocket neighboring the ribose-binding site (Fig. 2B and Fig. S3).
These data provide conclusive linkage of pyridopyrimidine anti-
bacterial activity to inhibition of a unique biosynthetic enzyme
target.
Antibacterial Pyridopyrimidines Are Selective for Bacterial BC. The
distinctively different binding modes (Fig. 2 C and D) of pyridopy-
rimidines observed in the BC and FGFR2 kinase domain crystal
structures (9) suggested that BC selectivity could be achieved
despite considerable structural similarity of the ATP-binding sites.
Indeed, 1 and 2 display excellent selectivity for bacterial BC over
FGFR2, VEGFR1, and 28 other eukaryotic protein kinases (Table
3 and Fig. 3). Src was the only eukaryotic protein kinase tested that
wassignificantlyinhibitedby 1(however,theSrcIC50ofcompound
1 is still 70-fold weaker than its IC50against E. coli BC).
Pyridopyrimidines Are Bactericidal. Further efforts focused on as-
sessment of the potential clinical utility of these compounds.
Minimal bactericidal concentrations (MBC) (11, 12) were deter-
mined to be 2- to 4-fold higher than MICs for compounds 1, 2, and
3, indicating that these compounds are bactericidal against E. coli,
H.influenzae,M.catarrhalis,andS.aureus(Table1).Measurements
of the kinetics of bacterial killing confirm that 1 is bactericidal
(defined as a ?99.9% reduction in viable bacteria within 24 h) (12)
againstH.influenzaewithin14hat?2-foldaboveitsMIC(Fig.4A).
Pharmacokinetics of Pyridopyrimidines. The pharmacokinetic prop-
erties of 1 were assessed in vitro and in vivo by using rat and mouse
(Table 5). At low i.v. doses (5 mg/kg), 1 was rapidly cleared. Total
body clearance was greater than liver blood flow in rat and mouse
(clearance ? 100 and 150 mL per min per kg, respectively).
However, at higher oral doses (200 mg/kg), 1 shows ?100%
bioavailability, likely caused by saturation of rodent clearance
mechanisms. The high clearance of 1 observed in rat was predicted
by rat liver microsomes. Because human liver microsomes predict
dramatically lower clearance in humans, scaling from rodent clear-
ance may overestimate human clearance.
Pyridopyrimidine BC Inhibitors Have in Vivo Antibacterial Activity.
Based on the reasonable pharmacokinetic properties of 1, it was
tested in murine models of tissue-localized (thigh) and systemic H.
influenzae infection. In the thigh model, 1 showed a good correla-
tionbetweenplasmaconcentrations24haftertherapy(oraldosing)
and reduction of bacterial levels in thigh tissue (Fig. 4B). Assuming
that 24-h plasma concentrations are representative of total expo-
sures, the 24-h plasma concentration that related to achieving 50%
of the maximum effect was 5.6 ? 1.8 ?g/mL. Similarly, in a H.
influenzae murine systemic infection model, oral dosing of animals
at 200 mg/kg once or twice a day resulted in 63% and 50% survival,
respectively (Table S4). No overt toxicity was observed with 1 at all
doses tested in efficacy studies.
Pyridopyrimidines Show Improved in Vitro Antibacterial Activity in
Combination with Other Antibacterial Agents. Collectively, these
dataindicatethatourpyridopyrimidineBCinhibitorsshowpromise
as clinically useful agents against fastidious Gram-negative organ-
isms. Although a trend toward more selective antibacterial classes
may be desirable to combat unintentional selection for antibiotic
resistance and take advantage of current and future improvements
inmoleculardiagnostics(13),theacceptanceofempirictherapyfor
fastidious Gram-negative or other single-agent infections would be
unlikely in the absence of a rapid, inexpensive, and highly accurate
diagnostic (14). Furthermore, the observation of single-step high-
level resistance to 1 and 2 could be of concern despite the relatively
lowfrequencyofoccurrence.AcceptanceofapyridopyrimidineBC
inhibitor into empiric clinical use might therefore require demon-
stration that it could be used in combination with other antibiotics
possessing a complimentary spectrum and that resistance develop-
ment potential can be minimized by using structural and/or com-
putational tools to guide inhibitor design.
To assess whether the combination of BC inhibitors with other
antibacterials resulted in improved antibacterial spectrum, we
tested 1 along with several other antibacterials in vitro by using a
checkerboard susceptibility approach (15). Compound 1 was not
antagonistic with any of the tested agents or organisms, including
Gram-positivebacteria(Table6).Thisfindingisimportantbecause
compound 1 could be useful in combination with Gram-positive
antibacterials that lack potent activity against H. influenzae and
other fastidious Gram-negative pathogens. Combinations showing
conclusive synergy with compound 1 were triclosan [fractional
inhibitory concentration (FIC) ? 0.37] and ciprofloxacin (FIC ?
0.26) in H. influenzae (Table 6). The observation of triclosan
synergy is consistent with the mechanism of action experiments
because both triclosan and compound 1 target enzymes involved in
fatty acid biosynthesis. This result provides further confirmation of
the validity of the fatty acid biosynthesis pathway as a source of
potential antimicrobial targets (16, 17).
Use of Structural Biology to Ameliorate Pyridopyrimidine Resistance
Development. Low-frequencyspontaneoussingle-stepmutationsin
the accC gene (e.g., I437T in E. coli BC and others described in
TablesS1andS2)resultedinvariablelossofaffinityfor1and2and
corresponding decreases in antibacterial activity (Tables 1 and 3
andTableS2).Themolecularbasisforpyridopyrimidineresistance
in the I437T E. coli mutant was apparent upon examination of the
X-ray crystal structure of 1 bound to the I437T mutant (Fig. S4).
This costructure revealed that the I437T mutation results in a
decrease in shape complementarity between the inhibitor and the
binding pocket as well as a loss of hydrophobic contacts between
I437 and the substituted phenyl ring. Mining the Pfizer compound
file (including compounds not screened in our initial high-
throughput screen) for structurally related pyridopyrimidines less
affected by the I437T mutation identified compound 3 (E. coli BC
IC50?150nM),whichwasonly14-foldlessactiveagainsttheI437T
BC mutant. Based on the crystal structures, the C7-substituted
Table 3. Biochemistry of BC inhibitors
Assay IC50(nM) at 1? Km(ATP)
Compound
1234
Enzyme-coupled E. coli ACCase
Wild-type
I437T mutant
H438P mutant
Rat liver ACCase
FGFR1 kinase
VEGFR2 kinase
?5
560
160
28 150
2,100
1,300
ND
?10,000
?10,000
?10,000
ND*
ND
ND
7,300
1,200
?100,000
5,800
?30,000
?100,000
?30,000
?30,000
17
420
*ND, not determined.
Table 4. Biophysics of inhibitor binding to E. coli BC
ParameterCompound 1 Compound 2
kon, M?1s?1
koff, s?1
Kdfrom SPR, nM
Kdfrom ITC, nM
?H, kcal/mol
?G, kcal/mol
T?S, kcal/mol
(4.90 ? 1.35) ? 107
0.041 ? 0.022
0.82 ? 0.22
?5.0
?18.0 ? 0.1
?(?11.5)
?6.5
(2.36 ? 0.04) ? 107
0.15 ? 0.01
6.53 ? 0.64
18 ? 9
?16.47 ? 0.02
?10.7
?5.8
Miller et al.
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pyrrolidine group of compound 3 utilizes favorable hydrophobic
interactions with the side chain of Ile-287 and the P-loop, partially
compensating for the loss of binding affinity. In this respect, the
ease of obtaining crystal structures of multiple inhibitor analogs
substituted at the C6, C7, or both positions bound to the wild-type
and resistant BC enzymes offers the opportunity to use structure-
baseddrugdesignapproachestocircumventtarget-basedresistance
mechanisms and develop structure activity and structure resistance
relationships. Furthermore, residue 437 (E. coli numbering) rep-
resents a key difference in the Gram-negative and Gram-positive
BC active site. Gram-negatives have isoleucine at this position and
Gram-positives, threonine (Table S5). This suggests that inhibitor
design focusing on this residue difference could enhance the
Gram-positive antibacterial spectrum of the pyridopyrimidines and
alleviate this resistance mechanism in Gram-negative organisms.
Discussion
We report the discovery of a class of potent synthetic antibacterials
that have potential as standalone agents targeting fastidious Gram-
negative pathogens or as broader spectrum agents when used in
combination with existing antibiotics. Although the discovery of a
previously undescribed class of antibacterials is in itself significant,
we believe the pharmaceutical origins of these inhibitors have
far-reaching implications for antibacterial drug discovery in gen-
eral. The BC inhibitors 1, 2, and 3 originated as weakly active
analogsfromstructure-guidedeukaryoticdrugdiscoveryprograms
targeting eukaryotic protein kinases. Despite their origin, or, per-
haps because of their origin, these inhibitors act on the structurally
related ATP-binding site of a previously undescribed bacterial
target.
Discovery of these BC inhibitors by whole-cell screening and
reverse genetics underscores the utility of this screening approach
A
B
CD
Fig. 2.
1duringsimulatedannealing.Mapissuperimposedonthefinalrefinedmodelandcontouredat2.5?level.Inhibitor1isshowninstickswiththefollowingatom
colors: carbon, green; nitrogen, blue; oxygen, red; and bromine, cherry red. Ribbon representation of BC is in green. (B) View of the superimposed ATP-binding
site in complexes with inhibitor 1 and ADP [Protein Data Bank (PDB) ID code 2j9g]. Ribbon representation of the EcBC/inhibitor 1 coordinates is in green. Ribbon
representation of the EcBC/ADP coordinates is in yellow. EcBC residues involved in interactions with ADP are shown in sticks with the following atom colors:
carbon, yellow; nitrogen, blue; oxygen, red. (C) Overlay of compound 1 bound in the ATP-binding site of BC (green carbons) vs. compound 1 docked into the
ATP-binding site of FGFR1 (PDB ID code 2fgi; cyan carbons). The conformation of ATP bound to both kinases is shown in gray as a guide. (D) View of the
distinctively different binding modes of inhibitor 1 and compound 4 in the superimposed ATP-binding sites of BC and FGFR1 (PDB ID code 2fgi). Ribbon
representation of the EcBC/inhibitor 1 coordinates is in green. Ribbon representation of the FGFR/compound 4 coordinates is in pink. Images were prepared by
using PyMOL molecular graphics systems (DeLano Scientific LLC).
Binding modes of pyridopyrimidine inhibitors. (A) View of the unambiguous (Fo? Fc) OMIT electron density map of inhibitor 1 calculated by omitting
Fig. 3.
proteinkinasesbycompounds1and2.(Upper)Scaleshowsrelativepotencies.
The color transition is nonlinear to greater highlight the few kinases that
showed weak inhibition by compound 1. (Lower) Kinase assays were per-
formedbyusingcommerciallyavailableorin-housegeneratedreagentsatthe
Pfizer Kinase Center of Emphasis, Cambridge, MA.
Heat map representation of the inhibition of several eukaryotic
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and demonstrates the need to reassess firmly held notions about
what features constitute the ‘‘best’’ antibacterial drug targets to
interrogate by high-throughput screening of pharmaceutical librar-
ies. We propose that targets with high sequence and/or structural
homology to known human drug targets are more likely to find
potent inhibitors within the chemical space covered by synthetic
pharmaceutical compound libraries and may offer better starting
points for novel antibacterial drug discovery efforts.
Additional evidence supporting this approach comes from a
recent report in which a small set of ATP-competitive eukaryotic
protein kinase inhibitors was screened for inhibition of D-alanine-
D-alanine ligase, an essential bacterial enzyme with active-site
similarity to eukaryotic protein kinases (18). A subset of these
kinase inhibitors exhibited modest inhibition of D-alanine-D-
alanineligase,suggestingthatabroaderscreenofATP-competitive
kinase inhibitor cores might identify additional hits with improved
potency.
Like all other clinically useful antibacterial agents, new agents
emerging from eukaryotic-like targets must penetrate bacterial cell
walls, avoid efflux, and demonstrate selectivity against eukaryotic
homologs and/or paralogs. Our internal experience and that of
others (2) show that, historically, very few target-based screening
hits possess all of these properties, and it is likely that hits identified
from screens of eukaryotic-like targets will be no different. The
advantage of screening eukaryotic-like targets will come from
leveraging the eukaryotic bias of pharmaceutical compound librar-
ies, resulting in more plentiful and potent hits from which to start
lead development efforts. A remaining hurdle is our limited un-
derstanding of the physicochemical requirements needed to design
new inhibitors rationally to penetrate bacterial cell walls and
circumvent efflux efficiently. However, several recent reports and
reviews describe ongoing, knowledge-based and experimental ap-
proaches directed at resolving these fundamental questions (19–
21). Finally, the extensive use of lead optimization tools such as
structure-baseddesignandcomputationalmodelingasdescribedin
this article and elsewhere (4) will likely be key for maintaining the
bacterial selectivity of the initial hits.
Materials and Methods
MacromolecularSynthesisAssay.AsensitizedstrainofE.coli(tolCimp)atmidlog
growthphasewasincubatedwithvariableconcentrationsofcompoundintheE.
coli in the presence of radiolabeled precursors [14C]leucine, [3H]thymidine,
[3H]uracil, [14C]sodium acetate, or [3H]diaminopimelate for 15 min and then
acid-precipitated. The precipitated macromolecules were collected on a filter
plate, and the amount of radioactivity incorporated was compared with control
to determine an IC50. Further details are provided in SI Materials and Methods.
Generation of Mutants Resistant to Pyridopyrimidines. Spontaneously resistant
mutantsto1and2wereisolatedbyplating108,109,or1010cfuofH.influenzae,
E.coli,orM.catarrhalisonplatescontainingcompound1or2in2-foldincrements
from 1 to 128 times the agar MIC. After a 48- to 96-h incubation, resistance was
confirmed by streaking single colonies on medium containing 2 times the agar
MIC of the parent. Selection and propagation of strains are detailed in SI Mate-
rials and Methods.
Fig. 4.
influenzae killing in vitro by compound 1. The number of viable H. influenzae
was determined after treatment with various concentrations of 1 or cipro-
floxacin for various times. Filled squares represent the growth control (no
inhibitor). Open squares represent treatment with ciprofloxacin (4-fold over
MIC). Filled triangles, filled diamonds, open circles, and open squares show
treatment with 1-, 2-, 4-, and 8-fold over the MIC of 1, respectively. The solid
horizontal line denotes a 3-log reduction in bacteria relative to time 0. (B) In
vivo efficacy of compound 1 in a murine H. influenzae thigh infection. Circles
represent the number of recoverable bacteria (y axis) from the infected thigh
of an individual mouse with a given plasma concentration of 1, 24 h after
dosing (x axis). The solid line represents the fit of the data to an inhibitory
sigmoid Emaxmodel.
In vitro and in vivo efficacy of pyridopyrimidines. (A) Kinetics of H.
Table 5. In vivo and in vitro pharmacokinetic properties of
compound 1
Route and parametersRat Mouse
Intravenous
Dose, mg/kg
Clearance, mL/min/kg
Vdss, liters/kg
AUC03?, ?g/h/mL
t1/2, h
Urine recovery, %
Oral
Dose, mg/kg
Cmax, ?g/mL
AUC03?, ?g/h/mL
F, %
In vitro
Human
Microsomal t1/2, min
Microsomal clearanceint,
?L/min/mg protein
Rat
Microsomal t1/2, min
Microsomal clearanceint,
?L/min/mg protein
Plasma protein binding,
fraction unbound
Mouse
Rat
Human
55
100
2.2
0.851
1.9
3.3
150
1.2
0.558
0.22
Not collected
10 200
9.19
131
?100
20200
27.4
601
?100
0.233
0.756
44
0.343
0.531
24
?60
9.63
5.1
171
0.22
0.15
0.14
Table 6. Antibacterial combination studies with compound 1
Combination
agent
Organism
(no. of strains)FIC avg. Outcome
Azithromycin
Linezolid
Ceftazidime
Ciprofloxacin
Triclosan
Linezolid
H. influenzae (4)
H. influenzae (4)
H. influenzae (4)
H. influenzae (1)
H. influenzae (1)
Methicillin-resistant
S. aureus (2)
S. pneumoniae (4)
M. catarrhalis (2)
0.82–1.23
1.05–1.21
0.84–1.19
0.26
0.37
0.94–1.05
Additivity
Additivity
Additivity
Synergy
Synergy
Additivity
Linezolid
Linezolid
0.82–1.54
0.62–0.87
Additivity
Additivity
Miller et al.
PNAS ?
February 10, 2009 ?
vol. 106 ?
no. 6 ?
1741
BIOCHEMISTRY
SEE COMMENTARY

Page 6

Genetic Resistance Mapping in H. influenzae and M. catarrhalis. To identify
region(s) of the H. influenzae chromosome harboring mutations responsible for
resistance, 10-kb PCR amplicons encompassing the entire genome were gener-
ated by using primers described in ref. 22 and a pool of genomic DNA from
resistantmutantsastemplate.TheseampliconswerethenusedasdonorDNAsto
transformHI100in96-wellplates,andcellswereplatedonplatescontaining2,4,
and 8 times the MIC of the parent. These studies identified two overlapping
regionsofthegenome,containingbothHI0971(accB)andHI0972(accC),withthe
abilitytomoveresistancetotheparent.Toidentifythemutationresponsiblefor
resistance, the 5-kb overlapping region was PCR-amplified by using individual
resistantmutantsastemplate,andtheDNAsequencewascomparedwithHI100.
Finally, these individual amplicons were used as donor DNAs to create isogenic
strains containing the mutation of interest. A similar strategy was used in M.
catarrhalis;however,ampliconswerefocusedonaregioncontainingaccCand1
kb flanking either side.
Determination of Antibacterial Activity. Determination of MICs and MBCs were
conducted according to guidelines of the Clinical and Laboratory Standards
Institute [CLSI; formerly National Committee on Clinical Laboratory Standards
(NCCLS) (23, 24) or according to the procedures described in SI Materials and
Methods.
Production of Protein Reagents. Standard molecular biology techniques were
usedtogenerateproteinreagents,andprotocolsareprovidedinSIMaterialsand
Methods.
Assessment of Inhibitor Binding Thermodynamics by ITC and SPR. Binding of
inhibitorstothevariousBCorthologswasexaminedbyusingaVP-ITC(Microcal)
and a Biacore S51 SPR instrument (GE Healthcare). Detailed protocols are pro-
vided in SI Materials and Methods.
ACCase-Coupled Enzyme Assay. A continuous enzyme-coupled phosphate de-
tection assay [based on that of Webb (25) and detailed in Fig. S1] was used to
measure the initial rate of the wild-type and mutant E. coli ACCase holoenzyme
reactions as described in SI Materials and Methods.
Protein Crystallization. Crystals of BC with inhibitors were grown at ambient
temperature by using the hanging-drop vapor diffusion method. Crystallization
dropsconsistedofequalpartsofprotein[12mg/mL,250mMpotassiumchloride,
10mMHepes(pH7.2)]andreservoirsolutionsconsistingof0.1–0.2Mpotassium
chloride and 2–4% wt/vol PEG 8000. Before crystallization, the enzyme was
incubated with the inhibitors (final concentration, 5 mM) at 4 °C for at least 4 h.
The crystals were visible after a few hours and typically grew to dimensions of
0.40?0.40?1.00mm.DetailsofX-raydatacollection,structuredetermination,
and refinement are provided in SI Materials and Methods and Table S6.
In Vitro Checkerboard Assays. In vitro checkerboard testing was conducted by
using 96-well microdilution plates and followed CLSI recommendations for mi-
crobroth growth medium, inoculum preparation, and incubation techniques
(15). A detailed description of test strains and the protocol used is provided in SI
Materials and Methods.
StaticTime–KillAssays.Invitrotime–killstudieswereperformedbyusing10-mL
cultures (initial population of ?5 ? 105cfu/mL) grown in ambient atmosphere
without agitation over a 24-h period. An aliquot of 100 ?L was serial-diluted to
10?7in 0.8% saline solution (Baxter) with 10 ?L of each sample cultured on
chocolate agar (BBL) at selected time intervals according to the method of Miles
and Misera (26). Colony-forming units were read after 24-h incubation and
reconfirmed after 48-h incubation (100 cfu/mL ? limit of detection).
Pharmacokinetic Assessments. Male Sprague–Dawley rats (Charles River Labo-
ratories) were used for rat pharmacokinetic assessments. Animals were housed
and maintained in accordance with Pfizer IACUC, State, and Federal guidelines
for the humane treatment and care of laboratory animals. All animals were
fastedovernightbeforedosingthenextmorning,andfoodwaswithhelduntil4h
after dosing. Water was allowed ad libitum throughout the studies. For each
study, blood samples were collected from a carotid artery cannula into EDTA-
coated tubes. Sampling occurred at time points up to 7 h after the i.v. and oral
dosingofcompounds(dosingformulationdetailedinSIMaterialsandMethods).
Concentrations of compound 1 were determined by a liquid chromatography-
tandemMSmethodaftersamplepreparationbyacetonitrileprecipitation.Phar-
macokinetics calculations were performed by using the noncompartmental ap-
proach (linear trapezoidal rule for AUC calculation) with the aid of Watson 6.4
BioanalyticalLIMS(ThermoElectron).Murinepharmacokineticassessmentswere
similarlyconductedbyusingmaleCD-1mice(CharlesRiverLaboratories),except
that blood samples were collected by tail vein bleeds over 48 h after oral drug
administration.
Murine Infection Models. Animal infection model work was conducted in com-
pliance with National Institutes of Health Guidelines for the Care and Use of
LaboratoryAnimalsunderaprotocolapprovedbythePfizerGlobalResearchand
Development Animal Use Committee. In vivo efficacy of compound 1 was inves-
tigated in a neutropenic murine thigh infection model. Mice (CD-1) were dosed
orally with 150 mg/kg and 100 mg/kg cyclophosphamide 4 days and 1 day,
respectively, before infection. Typically, 40 mice were infected intramuscularly
with 107cfu per animal in 0.1 mL of a 1:1 mixture of Cytodex-1 beads and H.
influenzae strain HI-3543 (a mouse virulent strain) in Haemophilus test medium.
Twohoursafterinfection,groupsoffivemicereceivedoraldosesof400,200,100,
50, 25, 12.5, or 0 mg/kg of compound 1 in nanosuspension (see SI Materials and
Methods). One cohort of mice was killed after inoculation to determine initial
bacterial load. Twenty-four hours after inoculation, thighs were removed asep-
tically and homogenized, and bacterial counts were determined. Efficacy out-
comewasdeterminedbasedonrecoverableorganisms.Plasmasampleswerealso
collected at 24 h for analysis of drug concentrations. Details of a H. influenzae
peritonitis/sepsis model are provided in SI Materials and Methods.
ACKNOWLEDGMENTS. We acknowledge the technical contributions and/or
scientific advice of the following Pfizer colleagues: Loola Al-Kassim, Fred Boyer,
Heather Case, Allison Choy, Phillip Cox, Donna Dunyak, Hongliang Cai, Kelly
Fahnoe, Eric Fauman, Jeffery Gage, Michael Herr, Susan Holley, Denton Hoyer,
KristenKenney,Ji-YoungKim,KarenLeach,AnnetteMeyerRuff,BelindaO’Clair,
Paul Pagano, Joseph Penzien, Stephen Petras, James F. Smith, Fang Sun, Brenda
Vonderwell,DequingXiao,andLupingWu.Wealsoacknowledgetheadviceand
encouragement of Dr. Grover Waldrop.
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