N-alkyl urea hydroxamic acids as a new class of peptide deformylase inhibitors with antibacterial activity

Article abstract of DOI:10.1128/AAC.46.9.2752-2764.2002

Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the struct

Full text of DOI:10.1128/AAC.46.9.2752-2764.2002

N-Alkyl Urea Hydroxamic Acids as a New
Class of Peptide Deformylase Inhibitors
with Antibacterial Activity
Corinne J. Hackbarth, Dawn Z. Chen, Jason G. Lewis, Kirk
Clark, James B. Mangold, Jeffrey A. Cramer, Peter S.
Margolis, Wen Wang, Jim Koehn, Charlotte Wu, S. Lopez,
George Withers III, Helen Gu, Elina Dunn, R. Kulathila,
Shi-Hao Pan, Wilma L. Porter, Jeff Jacobs, Joaquim Trias,
Dinesh V. Patel, Beat Weidmann, Richard J. White and
Zhengyu Yuan
Antimicrob. Agents Chemother. 2002, 46(9):2752. DOI:
10.1128/AAC.46.9.2752-2764.2002.
Updated information and services can be found at:
http://aac.asm.org/content/46/9/2752
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2002, p. 2752–2764
0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.9.2752–2764.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 9
N-Alkyl Urea Hydroxamic Acids as a New Class of Peptide
Deformylase Inhibitors with Antibacterial Activity
Corinne J. Hackbarth,1 Dawn Z. Chen,1 Jason G. Lewis,1 Kirk Clark,2 James B. Mangold,3
Jeffrey A. Cramer,3 Peter S. Margolis,1 Wen Wang,1 Jim Koehn,2 Charlotte Wu,1
S. Lopez,1 George Withers III,1 Helen Gu,3 Elina Dunn,3 R. Kulathila,2
Shi-Hao Pan,2 Wilma L. Porter,3 Jeff Jacobs,1 Joaquim Trias,1
Dinesh V. Patel,1 Beat Weidmann,2 Richard J. White,1
and Zhengyu Yuan1*
Versicor, Inc., Fremont, California 945551; Novartis Pharmaceuticals Corporation, Summit, New Jersey
079012; and Novartis Pharmaceuticals Corporation, East Hanover,
New Jersey 079363
Received 17 December 2001/Returned for modification 16 March 2002/Accepted 5 June 2002
Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new
target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient
antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P1؅ site. Using a combi-
nation of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF
inhibitors with N-alkyl urea at the P1؅ site. Compounds with MICs of <4 ␮g/ml against gram-positive and
gram-negative pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influen-
zae, have been identified. The concentrations needed to inhibit 50% of enzyme activity (IC50s) for Escherichia
coli Ni-PDF were <0.1 ␮M, demonstrating the specificity of the inhibitors. In addition, these compounds were
very selective for PDF, with IC50s of consistently >200 ␮M for matrilysin and other mammalian metallopro-
teases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency
and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-
˚
inhibitor structure was determined at a resolution of 1.7 A. This structural information indicated that the urea
compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two
compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective
doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a
starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.
A largely empirical search for new antibiotics, carried out
since the 1940s, has led to an impressive armamentarium of
clinically useful antibacterial drugs. However, the actual num-
ber of targets interfered with by these agents is very limited.
Cross-resistance for antibiotics, resulting from a sharing of the
same target, plays an important role in the worsening problem
of resistance. It is not surprising that antibacterial drug discov-
ery efforts are now focusing on identifying molecules that block
different targets and thus can inhibit the growth of resistant
bacteria. Analysis of microbial genomes has revealed an abun-
dance of novel and potentially useful targets (26), but little has
so far resulted from this much heralded effort. On the other
hand, many bacterial enzymes that have been well character-
ized hold promise for the discovery of new antibacterial drugs.
One such target that has recently received a great deal of
attention is peptide deformylase (PDF; EC 3.5.1.31).
mammalian cells exhibit overall similarity, there are sufficient
differences to allow for selective blocking of the process in
bacteria. One significant difference is the transformylation and
subsequent deformylation of the initiating methionine in bac-
terial translation (35, 36). Unlike cytosolic protein synthesis in
mammalian cells, protein synthesis in bacteria is initiated with
N-formylmethionine (2, 31), which is generated by enzymatic
transformylation of methionyl-tRNA. In prokaryotes, the N-
formylmethionine of the nascent protein is removed by the
sequential action of PDF and a methionine aminopeptidase to
afford the mature protein (2, 37). This bacterium-specific re-
quirement for PDF in protein synthesis provides a rational
basis for selectivity, making it an attractive drug discovery
target. The possible use of PDF as an antimicrobial target was
recently reviewed elsewhere (17, 57).
PDF activity was first reported by Adams in 1968 (1). How-
ever, further attempts to purify the enzyme from cell lysates
failed because the activity was not stable. The enzyme was not
characterized further until the early 1990s, when the deformy-
lase gene, def, was cloned (42) and PDF was subsequently
overexpressed in Escherichia coli (39, 42).
Protein synthesis has proven to be a rich source of targets for
antibacterial drugs (30). Many of the known antibiotics target
one or more steps of this complex process, including the ami-
noglycosides, macrolides, tetracyclines, and oxazolidinones.
Although the protein-synthesizing machineries of bacterial and
Bacterial PDF belongs to a new class of metallohydrolases
that utilize an Fe2ϩ ion as the catalytic metal ion (20, 51, 52).
The ferrous ion in PDF is very unstable and can be quickly and
irreversibly oxidized to the ferric ion, resulting in an inactive
* Corresponding author. Mailing address: Versicor, Inc., 34790 Ar-
dentech Ct., Fremont, CA 94555. Phone: (510) 739-3026. Fax: (510)
739-3086. E-mail: zyuan@versicor.com.
2752

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N-ALKYL UREA HYDROXAMIC ACIDS INHIBIT DEFORMYLASE
2753
FIG. 1. Generic PDF inhibitor structure derived from the transition state of the deformylation reaction (57).
MATERIALS AND METHODS
enzyme (53). Interestingly, the ferrous ion can be replaced with
a nickel ion in vitro, resulting in much greater enzyme stability
with little loss of enzyme activity (20).
Materials. Actinonin, formate dehydrogenase (FDH), catalase, and NADϩ
were obtained from Sigma; E. coli Ni-PDF and Streptococcus pneumoniae Zn-
PDF were overproduced and purified as previously described (11, 33). N-formyl-
methionine-alanine-serine (fMAS) and thio ester peptide Ac-ProLeuGly-S-
LeuLeuGly-OC2H5 were obtained from Bachem. Matrilysin (MMP-7) and
angiotensin-converting enzyme (ACE) were obtained from Calbiochem. Hy-
droxypropyl-␤-cyclodextrin was purchased from Aldrich. All other chemicals
were of the highest commercial grade.
Preparation of N-alkyl urea hydroxamic acids. The method of preparation of
the urea compounds was recently reported (J. Lewis, J. Jacobs, C. Wu, C.
Hackbarth, W. Wang, S. Lopez, R. White, J. Trias, Z. Yuan, and D. Patel, Abstr.
41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 358, 2001) and will be
published elsewhere. Briefly, the urea hydroxamic acids (compound 4) were
prepared in eight steps from commercially available amino acid precursors as
outlined in Fig. 2. The synthetic sequence used Fukuyama-Mitsunobu chemistry
followed by thiolytic deprotection to provide N-mono-alkyl-glycine methyl esters
(compound 1). Acylation of N-alkyl amino esters with excess phosgene under
aqueous reaction conditions provided N-alkyl-N-chlorocarbamoyl-glycine methyl
esters (compound 2), which were subsequently acylated with the N-alkyl-N-
chlorocarbamoyl-glycine methyl esters in pyridine to produce tetrasubstituted
ureas (compound 3). Trifluoroacetic acid (TFA) deprotection followed by cou-
pling of the R2 amine with PyBop peptide coupling reagent and treatment with
hydroxylamine provided the final product, urea hydroxamic acid PDF inhibitors
(compound 4).
The three-dimensional structures of various PDF molecules,
including structures of enzyme-inhibitor complexes, have been
solved and published (6, 10, 13, 14, 20, 21, 38). Although PDF
is a ferrous aminopeptidase with a primary sequence very dif-
ferent from those of other metalloproteases, it has been noted
that the environment surrounding the catalytic metal ion of
PDF appears to be very similar to the active sites of thermo-
lysin and the matrix metalloproteases (MMPs) (10). The cat-
alytic metal ion of PDF is tetrahedrally coordinated with two
histidines from the conserved zinc hydrolase sequence,
HEXXH, and a conserved cysteine from an EGCLS motif. A
water molecule that presumably hydrolyzes the amide bond
occupies the fourth position in the tetrahedron.
The fact that PDF is a metalloprotease makes the enzyme a
more attractive target for drug discovery. Metalloproteases are
among the best studied of the enzyme classes (29), and there
are excellent precedents for the mechanism-based design of
their inhibitors. In the last few years, several classes of PDF
inhibitors have been reported (3, 11–13, 15, 19, 24, 26, 43, 55).
While all of these compounds inhibit PDF activity, most of
them do not have antibacterial activity, presumably due to
weak potency against PDF and/or an inability to penetrate the
bacterial cell. It is interesting that among these compounds,
those for which the concentrations needed to inhibit 50% of
enzyme activity (IC50s) (or Kis) were greater than 1 ␮M had no
antibacterial activity.
Based on mechanistic and structural information, together
with an understanding of the general principles of inhibiting
metalloproteases, a generic PDF inhibitor structure (Fig. 1)
(57) was proposed. In this structure, X represents a chelating
pharmacophore that will be the major component to provide
binding energy; the n-butyl group mimics the methionine side
chain; and P2Ј and P3Ј are regions of the inhibitor that can
provide additional binding energy, selectivity, and favorable
pharmacokinetic properties. Recently, actinonin, a naturally
occurring antibiotic that was first isolated in 1962 from an
actinomycete (18), was shown to be a PDF inhibitor (11). In
this study, we report that N-alkyl urea hydroxamic acids, which
also fit this generic structure, represent a new class of PDF
inhibitors. The structural characterization and biological eval-
uation of this class of inhibitors are presented here.
Enzyme assays. All absorption measurements were obtained with a Spectra-
Max plate reader (Molecular Devices).
Deformylase activity was assayed by a PDF-FDH coupled assay as previously
described (11, 28). Briefly, the assay was carried out at room temperature with 5
nM E. coli Ni-PDF or 10 nM S. pneumoniae Zn-PDF (33) in a buffer consisting
of 50 mM HEPES (pH 7.2), 10 mM NaCl, and 0.2 mg of bovine serum albu-
min/ml in half-area 96-well microtiter plates (Corning). The reaction was initi-
ated by the addition of a reaction mixture of 0.5 U of FDH/ml, 1 mM NADϩ, and
4 mM fMAS at the desired concentration. To determine the IC50s of the desired
compounds, PDF was preincubated for 10 min with various concentrations of test
compounds prior to the addition of the reaction mixture. The initial reaction
velocity was measured as the initial rate of increase in the absorption at 340 nm.
Matrilysin (MMP-7) activity was assayed as reported previously (56) by using
a thio ester peptide as a substrate, with some modifications. Briefly, 0.12 ␮g of
MMP-7/ml was preincubated at room temperature for 10 min with test com-
pounds at various concentrations in a buffer containing 50 mM Tricine (pH 7.5),
0.2 M NaCl, 10 mM CaCl2, and 0.05% Brij. The reaction was initiated by the
addition of 0.05 mM thio ester peptide substrate (Ac-ProLeuGly-S-LeuLeuGly-
OC2H5) and 0.1 mM 5,5Ј-dithio-bis(2-nitrobenzoic acid). Reaction progress was
monitored by recording the increase in the absorption at 405 nm.
ACE activity was determined with a 96-well format according to the procedure
reported by Maclean et al. (32). The hydrolysis product of the enzyme reaction
was detected by derivatization with o-phthaldialdehyde reagent (Pierce) by fol-
lowing the manufacturer’s protocol. Briefly, 0.03 U of ACE/ml was incubated
with test compounds at different concentrations at room temperature for 10 min
in a buffer consisting of 25 mM HEPES (pH 8.2) and 0.3 M NaCl. The reaction
was initiated when 2 mM substrate (hippuryl-HisLeu) was added to the mixture.
The enzyme reaction was carried out at 35°C for 1 h. An equal volume of

2754
HACKBARTH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. Synthesis of N-alkyl urea hydroxamic acids. The letters in the scheme represent the following solvents: a, NsCl, aqueous NaHCO3,
DCM; b, R1OH, DIAD, PPh3; c, PhSH, TBDE-PS, DMF; d, COCl2, aqueous NaHCO3, Et2O; e, L-Pro-OtBu, pyridine, DCM; f, TFA, DCM; g,
R2NH2, PyBop, DIEA, DMF; and h, aqueous NH2OH, dioxane.
o-phthaldialdehyde reagent was added, and the reaction signal was detected by
recording the change in fluorescence at excitation and emission wavelengths of
360 and 465 nm, respectively.
The E. coli def gene was cloned for overexpression in E. coli BL21(DE3)/pLysS
as described previously (11). Fermentation was performed by using a Bioflo 2000
fermentor (New Brunswick Scientific) with an on-demand fed-batch strategy and
minimal medium. A 4-liter fermentation was maintained at 37°C until the culture
reached an OD600 of ϳ8.9. The temperature was then lowered to 18°C, and the
culture was induced overnight with isopropyl-␤-D-galactopyranoside (IPTG) at a
final concentration of 0.4 mM. The bacterial cells (final OD600, ϳ40) were
harvested by centrifugation and yielded ϳ240 g of paste (wet weight). The cells
were frozen as pellets and stored at Ϫ80°C.
The IC50 was calculated with the following equation:
y ϭ yo/͕͑1 ϩ IC50͒/͓In͔͖
(1)
In this equation, yo is enzyme activity in the absence of inhibitor, and [In] is the
inhibitor concentration.
All data fitting was carried out by using nonlinear least-squares regression with
the commercial software package DeltaGraph 4.0 (Deltapoint, Inc).
Cytotoxicity assays. The cytotoxicities of the test compounds were assessed by
using human K562 (ATCC CCL-243) and murine P388D1 (ATCC CCL-46)
leukemia cell lines. The human cell line K562 was maintained in RPMI 1640
medium supplemented with 10% fetal bovine serum (Gibco BRL) and 1 mM
sodium pyruvate. P388D1 cells were grown in Dulbecco’s modified Eagle’s me-
dium supplemented with 10% bovine calf serum (Gibco BRL). The assays were
conducted with 96-well microtiter plates (Corning), and test compounds were
serially diluted in 10% dimethyl sulfoxide. A volume of 10 ␮l of each dilution was
added to wells 1 to 11 in each row; well 12, used as a control, contained 10 ␮l of
10% dimethyl sulfoxide solution without drug; and well 12H contained 0.25 ␮g
of puromycin/ml as a no-growth control. Ninety microliters of log-phase cells (5.5
ϫ 104 cells/ml) was suspended in the appropriate assay medium and added to
each well. The cells were exposed to the drug for 3 days at 37°C in the presence
of 5% CO2. On day 4, an indicator solution containing 1 mg of XTT/ml and 7.7
␮g of phenazine methosulfate (Sigma)/ml in phosphate-buffered saline was
added to each well, and the suspension was reincubated for 4 h under the same
conditions. The XTT cleavage product was detected by recording the change in
the absorption at 450 nm, and cell growth as a percentage of that in the corre-
sponding control well was used to calculate the IC50 with equation 1.
Susceptibility studies and killing curves. Bacteria used in these studies were
stored at Ϫ80°C and grown at 35°C in accordance with NCCLS recommenda-
tions (44).
For susceptibility tests, MICs were determined by the broth microdilution
method in accordance with NCCLS guidelines (44) but with the following mod-
ification. Because of the high frequency of resistance previously reported with
other PDF inhibitors (4, 13, 34), a modified bacterial inoculum size of 0.5 ϫ 105
to 1.0 ϫ 105 CFU/ml was used to determine the relative activities of the com-
pounds. The organisms used are all part of the Versicor strain collection (11).
The MIC was defined as the lowest concentration that yielded no visible growth
after 24 h of incubation at 35°C; end points were obtained by measuring the
optical density at 600 nm (OD600). Minimum bactericidal concentrations
(MBCs) were determined for Haemophilus influenzae (ATCC 31517;
VHIN1004) and S. pneumoniae (ATCC 49219; VSPN1001) (45). The MBC was
defined as the lowest concentration of antibiotic that resulted in a Ն3-log10
decrease in the bacterial titer.
All purification steps were performed at 4°C unless noted otherwise. Aliquots
of cells (ϳ50 g) were thawed briefly at 37°C and resuspended in 250 ml of lysis
buffer (20 mM morpholineethanesulfonic acid [MES]-KOH [pH 6.5], 100 mM
KCl, 5 mM NiSO4, 5 mM MgSO4, 10 ␮g of catalase/ml, 100 ␮M phenylmethyl-
sulfonyl fluoride, 10.0 ␮g of DNase I/ml, five tablets of complete protease
inhibitor cocktail without EDTA [Boehringer Mannheim]). The cells were lysed
with four freeze-thaw cycles and then sonicated (four 10-s bursts, full power) with
a Virsonic 60 apparatus (Virtus) equipped with a titanium horn. The pellets were
collected at 35,000 ϫ g and washed by resuspension in 120 ml of lysis buffer. The
supernatants were combined and clarified by centrifugation at 100,000 ϫ g for 30
min. DNA was removed by centrifugation at 35,000 ϫ g following a 15-min
treatment with 0.1% polyethyleneimine (final concentration). The supernatant
was buffer exchanged with diafiltration against a 5-kDa-cutoff membrane in 20
mM morpholinepropanesulfonic acid (MOPS)-KOH (pH 7.7)–50 mM KCl–1
mM NiSO4 and filtered through a 0.2-␮m-pore-size filter. The sample was then
loaded at a flow rate of 30 ml/min onto a POROS 20HQ (perfusion chromatog-
raphy medium; PE Biosystems) column (2 by 16 cm) equilibrated with 20 mM
MOPS-KOH (pH 7.7)–1 mM NiSO4 at room temperature. A linear salt gradient,
0 to 300 mM KCl in 20 column volumes, was used to elute bound proteins, and
the fractions were immediately placed at 4°C. Fractions containing Ni-PDF were
identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis,
pooled, and further purified by size exclusion chromatography on a Superdex 75
HiLoad (16/60) column (Amersham Pharmacia Biotech) equilibrated with 20
mM MOPS-KOH (pH 7.7)–100 mM KCl at a flow rate of 1 ml/min. Proteins
were pooled and concentrated to ϳ5 to 10 mg/ml. After the addition of glycerol
to 10% and 1 mM Tris-(2-carboxyethyl)-phosphine (TCEP) hydrochloride, the
enzyme was stored at Ϫ80°C. The protein concentration was determined with
Bradford reagent (Bio-Rad) and bovine serum albumin as a standard. This highly
purified Ni-PDF was used directly for crystallization.
Crystallization and structure determination. An enzyme-inhibitor complex
was formed by incubation of a twofold molar excess of PDF inhibitor VRC4307
with 1.5 mg of PDF/ml in binding buffer (20 mM HEPES [pH 7.5], 100 mM
NaCl, 0.5 mM TCEP) for 30 min at room temperature. The complex was buffer
exchanged four times in binding buffer with 50 ␮M compound VRC4307 by using
a Millipore concentrator (5,000-molecular-weight cutoff) and finally concen-
trated to 36 mg/ml. Crystals were grown by the hanging-drop vapor diffusion
method by mixing equal volumes of protein solution and well buffer [100 mM
HEPES (pH 7.0), 2.0 M (NH4)2SO4] at 17°C. Crystals were cryoprotected at
room temperature by a 15-min, 10-step transfer to cryobuffer solution [100 mM
HEPES (pH 7.0), 2.4 M (NH4)2SO4, 25% glycerol] with 5 ␮M compound
VRC4307 and immediately flash-frozen in a 100 K nitrogen gas cold stream
(model 700 apparatus; Oxford Cryosystems) for data collection. Diffraction data
were collected by using an R-AXIS-IIc image-plate system (Molecular Structures
Time-kill curves were determined by using log-phase cultures of H. influenzae
(ATCC 31517), S. pneumoniae (ATCC 49219), and Staphylococcus aureus
(ATCC 25923; VSAU1003). The bacteria were exposed to 5 or 10 times the MIC
of each compound tested. Cultures were incubated at 35°C, and titers were
quantitatively determined at 0, 2, 6, 8, and 24 h of incubation. Results were
expressed as log10 CFU per milliliter versus time.
Expression and purification of E. coli PDF used for crystallographic studies.

VOL. 46, 2002
N-ALKYL UREA HYDROXAMIC ACIDS INHIBIT DEFORMYLASE
2755
Corp. [MSC]) with Osmic Confocal Max-Flux optics (Blue-3 configuration;
Rigaku/MSC) and an RU-2HR generator (MSC) operating at 100 mA and 50
kV. A complete data set was collected from a single crystal (0.2 by 0.18 by 0.06
mm) maintained at 100 K. The images were indexed, and integration was per-
formed by using Denzo/Scalepack (50).
The structure of the enzyme or enzyme complex was determined by molecular
replacement with AMoRe (46) as implemented in the CCP4 (9) package with a
structure of E. coli PDF (e.g., 1ICJ) (6) as a search model. The structure was
refined with the CNX 2000 (8) package (Accelrys Inc.).
Isolation of resistant mutants of H. influenzae and S. pneumoniae. Spontaneous
resistant mutants were selected as previously described (33, 34). Briefly, 1010
CFU of H. influenzae (ATCC 31517) were plated on chocolate agar plates
containing 30 ␮g of antibiotic/ml. In a second experiment, approximately 1010
CFU of log-phase S. pneumoniae (ATCC 49219) were spread on tryptic soy agar
(TSA)–5% sheep blood agar plates containing 30 ␮g of test compound/ml. The
plates were incubated for 48 h at 35°C in a 10% CO2 atmosphere. Colonies were
picked and transferred to drug-free plates; stable mutants that survived this
passage were subjected to further characterization.
MS to identify additional metabolites in the serum. In a separate experiment, test
compounds (10 ␮M) were incubated with mouse, rat, and human liver micro-
somes as described above at 37 °C for 20 min, and the resulting samples were
extracted before being analyzed for metabolites.
Protection from infection in the mouse septicemia model (S. aureus strain
Smith). A standard peritonitis model of infection was used as an initial screen to
determine the relative efficacies of the PDF inhibitors (MDS Pharma Services,
Bothell, Wash.). Briefly, outbred ICR-derived mice (20 to 25 g each) were
inoculated intraperitoneally with a 90 to 100% lethal dose (9.0 ϫ 105 CFU/
mouse) of S. aureus strain Smith (ATCC 19636) in 0.5 ml of brain heart infusion
broth containing 5% mucin. Compounds were dissolved in 20% cyclodextrin and
administered subcutaneously (s.c.) and/or p.o. at 1 and 5 h after infection.
Vancomycin was included as a control antibiotic, and animals in this antibiotic
regimen were dosed s.c. Groups of five mice were used for each dose level. Mice
were monitored daily for 6 days, and cumulative mortality was used to determine
the 50% effective dose (ED50) by nonlinear regression with Graph-Pad Prism
(Graph Pad Software).
Molecular techniques and sequence analysis. Primary sequences for the def
and fmt homologs of H. influenzae and S. pneumoniae were obtained from public
sequence databases and previously published work (16, 34). The def-fmt operon
and flanking DNA (230 bp upstream; 60 bp downstream) from wild-type H.
influenzae VHIN1004 and a single PDF inhibitor-resistant derivative were am-
plified by PCR with respective upstream and downstream primers oPV743 (5Ј-
CGCCAGGTTACCAAATACTTATCTAAG-3Ј) and oPV378 (5Ј-TTTTCCGC
TATAAATTTACCG-3Ј) for def and oPV177 (5Ј-GCCAGCGCATTAAAGAA
AAGTTG-3Ј) and oPV179 (5Ј-CGAAAGTGCGGTCATTTTTACTG-3Ј) for
fmt.
The defA, defB, and fmt genes and flanking DNAs (approximately 120, 350,
and 60 bp upstream, respectively; 60 bp downstream for all three) from wild-type
S. pneumoniae ATCC 49619 and two PDF inhibitor-resistant derivatives were
amplified by PCR with the respective upstream and downstream primers oPV369
(5Ј-CGTGGCTTGGAAGTAACT-3Ј) and oPV370 (5Ј-GATATACCAGGTCG
TTGC-3Ј) for defA, oPV385 (5Ј-TCCCTTATTGCTCCACTTG-3Ј) and oPV368
(5Ј-TATCATTTGTTTTCATGCCTC-3Ј) for defB, and oPV383 (5Ј-CCACAAC
CTCTATCATTACCAG-3Ј) and oPV384 (5Ј-ACCGTTCCATAGACCAGC-3Ј)
for fmt.
RESULTS
Previously, the alkyl succinate hydroxamic acid VRC3324,
(2R)-butyl-N4-hydroxy-N1-[2-methyl-(1S)-(naphthalen-2-
ylcarbamoyl)-propyl]-succinamide, was identified as a potent
PDF inhibitor with excellent antibacterial activity (C. Hack-
barth, S. Lopez, M. Gomez, W. Wang, J. Jacobs, R. Jain, Z.-J.
Ni, J. Trias, D. Chen, G. Withers, K. Clark, J. Koehn, B.
Weidmann, D. Patel, and Z. Yuan, Abstr. 40th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. 2173, 2000). Similar
compounds of this chemical class exhibit broad-spectrum an-
tibacterial activity and in vivo activity in a mouse septicemia
model (D. Chen, C. Hackbarth, Z.-J. Ni, W. Wang, C. Wu, D.
Young, R. White, J. Trias, D. Patel, and Z. Yuan, Abstr. 40th
Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2175,
2000). In our efforts to discover new pharmacophores that
would improve the biological properties of this class of inhib-
itors, N-alkyl urea hydroxamic acids were identified. VRC3852
(Table 1) represents an early lead in this new class of PDF
inhibitors (Lewis et al., 41st ICAAC). Analogs of VRC3852,
prepared in both library format and as individual compounds,
were screened to identify those with optimized activity and
structure.
In vitro activity tests. From more than 200 analogs tested, 21
compounds (Table 1) were purified by high-pressure liquid
chromatography and subjected to additional biological charac-
terization. All of these compounds were tested for the inhibi-
tion of deformylase activity with the E. coli and S. pneumonia
enzymes overproduced and purified from E. coli. In addition,
whole-cell activity was assessed with broth microdilution MICs
against a panel of 26 organisms, including both gram-positive
and -negative bacteria and Candida albicans.
All PCR products were purified with QiaQuick DNA purification columns
(Qiagen), and DNA sequences were determined by the dideoxy chain termina-
tion method (Sequetech). In all instances, mutations were confirmed by analysis
of a second independent PCR product.
Pharmacokinetic studies. CD-1 female outbred mice (Charles River Labora-
tories) were used for pharmacokinetic analyses of selected PDF inhibitors. The
compounds were formulated in 20% cyclodextrin (Aldrich) and filter sterilized.
After 1 week of acclimation, mice (20 to 25 g each) were dosed either intrave-
nously (i.v.) or orally (p.o.) with antibiotic; at 0.083, 0.25, 0.5, 1, 2, and 4 h after
dosing, blood samples were collected from anesthetized mice via cardiac punc-
ture. Groups of four mice were used for each time point. The blood was allowed
to clot, and the serum samples were stored immediately at Ϫ80°C. Serum was
extracted with a mixture of acetonitrile, ethanol, and acetic acid and analyzed by
liquid chromatography with triple quadropole mass spectrometry (LC-MS/MS)
MS (Phenomenex LUNA-C8, 50 by 2 mm, 3 ␮m, HP1100, Micromass Quattro
LC) with an internal standard. The pharmacokinetic parameters, including Tmax
(time to maximum concentration), Cmax (maximum concentration measured),
t1/2 (terminal half-life), and AUC (area under the curve), were calculated. The
p.o. bioavailability was calculated as the ratio of the AUC for p.o. administration
to the AUC for i.v. administration.
Metabolic stability in liver microsomes. Preparations of hepatic microsomes
from male CD-1 mice, male Sprague-Dawley rats, and human donors were
obtained from Xenotech LLC. To assess their metabolic stability, test com-
pounds (1 ␮M) in phosphate buffer (100 mM, pH 7.4) were incubated at 37°C
with liver microsomal proteins (0.2 mg/ml) supplemented with NADPH (1 mM),
UDP-glucuronic acid (4 mM), and alamethecin (60 ␮g/mg of protein) for 0, 10,
20, and 30 min. Aliquots were removed at various times, and the reaction was
terminated by the addition of acetonitrile. Loss of the parent compound was
monitored by LC-MS/MS (PE-SCIEX API3000; triple quadrupole) with reverse-
phase high-pressure liquid chromatography separation (Phenomenex LUNA-
C18, 2.1 by 30 mm, 3 ␮m). The viability and activity of the microsomal prepara-
tions were separately verified by using a positive metabolic control. Intrinsic
clearance was calculated from the slope of the curve of log10 linear concentration
versus incubation time.
Twenty of these 21 compounds have IC50s of Յ100 nM for
E. coli deformylase. Eighteen of these 20 compounds also have
excellent activity (IC50s, Յ75 nM) against S. pneumoniae PDF.
These IC50s corresponded to improved activity against S. pneu-
moniae. In fact, the two compounds with poor IC50s for the S.
pneumoniae enzyme, VRC4347 and VRC4390, also lacked
whole-cell activity against this organism, providing further ev-
idence that this class of compounds targets deformylase for the
inhibition of bacterial growth.
Compared with the MICs of the initial lead in this series,
VRC3852, the MICs against the three gram-positive pathogens
tested (S. aureus, S. pneumoniae, and Enterococcus faecium)
improved for the majority of the compounds listed. One of the
Metabolite identification. Mouse serum samples from the pharmacokinetic
studies described above were extracted with acetonitrile and analyzed by LC-MS/

2756
HACKBARTH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Structure and activity of N-alkyl urea hydroxamic acidsa
a S. pn., S. pneumoniae; S. aur, S. aureus; E. faec, E. faecium; H. influ, H. influenzae; M. cat, M. catarrhalis; E. coli acr, efflux pump mutant of E. coli;
C. alb, C. albicans. The number of strains tested is indicated in parentheses. P388, P388D1.

VOL. 46, 2002
N-ALKYL UREA HYDROXAMIC ACIDS INHIBIT DEFORMYLASE
2757
TABLE 2. Summary of diffraction data and refinement data for the
crystal structure of the E. coli PDF complex
compounds that had poor gram-positive activity, VRC4390,
nonetheless maintained its activity against H. influenzae. On
the other hand, VRC4234 and VRC4305 were more active
against S. aureus and S. pneumoniae but less potent against the
gram-negative pathogen H. influenzae. Apparently, these com-
pounds readily undergo efflux in E. coli, since none was active
against wild-type E. coli but all possessed low MICs against an
E. coli acr mutant which lacks the Acr efflux pump (49). None
of these compounds was active against C. albicans.
To determine whether this class of compounds inhibited
bacterial growth through the inhibition of deformylase, each of
the compounds was tested for its ability to inhibit the growth of
an actinonin-resistant S. aureus strain, VSAU6011. This strain
harbors a loss-of-function mutation in the formyltransferase-
encoding gene, bypassing the need for deformylase activity and
permitting growth even when PDF activity is inhibited (34). S.
aureus VSAU6011 was highly resistant to all of the compounds
tested, demonstrating that N-alkyl urea hydroxamic acids, like
alkyl succinates (11, 13), inhibit bacterial growth through the
inhibition of deformylase.
These compounds were also tested for their potential cyto-
toxicity and inhibition of matrilysin, a member of the mamma-
lian MMP superfamily which closely resembles PDF (5, 10,
38). Matrilysin activity was not inhibited in the presence of up
to and in most instances exceeding 100 ␮M PDF inhibitor. In
addition, the IC50s of VRC4232 and VRC4307 against human
ACE, another metalloprotease, were higher than 100 ␮M.
Thus, these N-alkyl urea hydroxamic acids are over 1,000 times
more potent against bacterial deformylase than the eukaryotic
metalloenzymes tested.
When these compounds were tested against two different
mammalian cell lines, the IC50s, determined after 72 h of
continuous exposure, were at least 10-fold higher than the
MICs for the major bacterial pathogens in the screening panel.
A closer examination of the structure-activity relationships of
these compounds (see Discussion) suggested that the cytotox-
icity of these compounds generally correlated with their hydro-
phobicity. Given their overall in vitro profiles, VRC4232 and
VRC4307 were selected as new leads for additional in vitro
and in vivo characterizations.
Parameter
Description or value
Space group..............................................................C2
Cell
a, b, c (Å).............................................................138.85, 163.37, 85.96
␣, ␤, ␥ (°)..............................................................90, 121.39, 90
Asymmetric unit ......................................................Three complexes
Resolution (Å).........................................................1.80 (1.86–1.80)
No. of unique reflections........................................56,820 (5,097)
Redundancy .............................................................4.3 (2.7)
Rmerge (%)................................................................5.2 (23.1)
Intensity (SE)...........................................................13.7 (2.7)
No. of atoms
Protein ..................................................................3,977
Solvent ..................................................................480
Inhibitor................................................................93
R (%) ........................................................................18.6 (22.5)
Rfree (%) (test set of 5%).......................................21.9 (25.3)
Completeness (F Ͼ 1.0␴) (%)...............................90.5 (69.4)
RMSD from ideal
Bond lengths (Å).................................................0.01
Bond angles (°)....................................................1.4
Dihedral angle .....................................................22.2
Mean B value (Å2)..............................................24.9
14, 40). When the PDF structure with bound VRC4307 is
examined, the hydroxamic acid is observed to coordinate the
active-site Ni2ϩ, as expected; the cyclopentyl ring system of
VRC4307 lies in the S1Ј pocket of the enzyme (Fig. 3A). The
carbonyl oxygen of the proline group of VRC4307 forms a
hydrogen bond with the backbone nitrogen of Ile44, maintain-
ing the peptidic interaction observed for the PDF structure
with the bound product MAS (6). Details of the metal coor-
dination, hydrogen bonding, and Van der Waals interactions
are shown in Fig. 4A. The electron density for most of the
compound is clear and unambiguous. The electron density for
the amide-dimethylthiazole (Fig. 4A, green atoms) portion of
the inhibitor is weak and broken. Apparently, the inhibitor is
able to adopt multiple conformations when bound; this hy-
pothesis is illustrated by superimposition of the three com-
plexes (Fig. 3B). Surprisingly, the peptidic interaction with the
backbone carbonyl of Glu42 is seen in only one of the three
complexes (Fig. 4A).
X-ray analysis of binding interactions. Key parameters and
summary statistics for the refined PDF model are provided in
Table 2. After independent rigid-body refinement of the three
PDF proteins in the asymmetric unit, the electron density for
the three inhibitors was clearly seen in each of the active sites.
Several rounds of iterative model building (including the ad-
dition of water molecules and correcting loops involved in
crystal contacts) with the program O (27) and refinement were
conducted before the three inhibitor molecules were added to
the model. In complex A, the inhibitor could be positioned
unambiguously in the electron density. For complexes B and C,
there was a weak, broken electron density for the 4,5-dimeth-
ylthiazole ring of the inhibitor. Rfree was monitored while ad-
ditional water molecules were added. The final model con-
tained three PDF-compound VRC4307 complexes as well as
435 water, 3 sulfate, and 5 glycerol molecules, resulting in an R
factor of 18.6% (Rfree ϭ 21.6%) in the resolution range of 30.0
The hydroxamic acid-metal coordination in the PDF-
VRC4307 complex maintains a tetrahedral configuration (Fig.
4B). Although the overall arrangement of the ligand atoms
approximates a distorted trigonal bipyrimidal geometry, the
carbonyl oxygen of the inhibitor is more distant from the metal
than is typically observed in metal-ligand complexes. In all
three complexes, the Ni2ϩ distances are reasonable for the N
˚
˚
and O atoms (2.0 to 2.1 A) and for the S atoms (2.3 A);
˚
however, the carbonyl O atom of VRC4307 is 2.7 to 3.0 A from
the metal. This suboptimal interaction may be offset, in part, by
an ideal hydrogen bonding interaction distance between the
carbonyl oxygen and the backbone nitrogen of Leu91. The
unusual metal-ligand geometry observed appears to be a direct
consequence of the relatively planar N compared to an alpha-
carbon in substrate/product. The VRC4307 cyclopentyl analog
of the methionine side chain is packed on top of His132 of
PDF, meaning that the relatively planar configuration of the
urea N prevents the hydroxamic acid from fully entering the
˚
to 1.8 A.
The structure of E. coli PDF has already been extensively
characterized and will not be elaborated on further here (6, 10,

2758
HACKBARTH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 3. Surface representation of VRC4307 binding to E. coli PDF. PDF is represented as a smooth surface created by GRASP; the catalytic
Ni2ϩ is shown as a magenta sphere. (A) The cyclopentyl ring system is positioned in the S1Ј pocket. (B) The inhibitors of three complexes have
been superimposed to illustrate relative disorder in the dimethylthiazole rings of the inhibitors.
binding pocket for an optimal interaction with the metal. The
favorable hydrogen bond interaction between a carbonyl of
VRC4307 and Ile44 is accompanied by a steric clash between
the carbonyl and Gly43 (Fig. 4A).
Killing curves and MBCs. It was previously shown that ac-
tinonin, a succinate hydroxamic acid, is bacteriostatic against S.
aureus (11). In this study, killing curves for the urea com-
pounds VRC4232 and VRC4307 were determined at 10 times
the MICs for S. aureus, S. pneumoniae, and H. influenzae (Fig.
5). Like actinonin, the urea-based compounds are also bacte-
riostatic against S. aureus, but a killing effect was observed
against the other two organisms. For S. pneumoniae, viable
counts dropped by greater than 2 log10 units over the initial
12 h of exposure; for H. influenzae, a drop of up to 5 log10 units
in viable organisms was observed after 24 h. Both compounds
were bacteriostatic over the initial 8 h of exposure; increasing
the drug concentration to 20 times the MIC (80 ␮g/ml) did not
improve the initial killing rate (data not shown). These obser-
vations were consistent with the MBC (Table 3) determina-
tions for H. influenzae and S. pneumoniae. Unlike S. aureus
(data not shown), these two fastidious pathogens are killed by
PDF inhibitors, although the reason for the late killing effect is
unclear.
Resistance and its mechanism. The in vitro selection of
spontaneously resistant H. influenzae and S. pneumoniae mu-
tants occurred at a frequency of 1 in 109 for both VRC4232 and
FIG. 4. Interactions between E. coli PDF and VRC4307. (A) Schematic representation of key interactions between PDF and VRC4307.
(B) Interactions of the hydroxamic acid with the active-site metal. Although the ligand coordination geometry is a distorted trigonal bipyramid,
the fifth ligand, the carbonyl oxygen, is positioned too far from the Ni2ϩ atom for optimal interactions with three complexes (2.7, 2.7, and 3.0 A).
˚
Solid lines indicate proposed interactions; the potential fifth ligand interaction is shown as broken lines.

VOL. 46, 2002
N-ALKYL UREA HYDROXAMIC ACIDS INHIBIT DEFORMYLASE
2759
FIG. 5. Bacterial killing curves. Log-phase organisms were exposed to either no drug (■), 10 times the MIC of VRC4232 (ࡗ), or 10 times the
MIC of VRC4307 (F). Results are expressed as log10 CFU per milliliter versus time.
VRC4307 (Table 4). These resistance frequencies are similar
to those observed previously for actinonin against S. pneu-
moniae (33).
original 1-bp deletion in fmt, a T residue inserted following bp
299 of the fmt ORF. This compensatory frameshift mutation
restores the fmt reading frame in mutant VHIN6529, such that
the revertants express a full-length transformylase with a three-
amino-acid substitution at codons 99 through 101 (Fig. 6).
Two independent S. pneumoniae mutants, VSPN6521 and
VSPN6522, were further characterized. Morphologically, the
resistant mutant strains resemble the parent strain. The dou-
bling times of the mutant strains (74 to 82 min) are slightly
longer than that of the corresponding wild type (65 min), a
finding that is very similar to what was found with mutants of
S. pneumoniae selected with actinonin (33). Like that of the H.
influenzae mutants, the overall antibiotic profile of the pneu-
mococcal mutants does not vary from that of the parent strain
(Table 4). The defB (PDF), defA (PDF paralog), and fmt
(transformylase) genes were amplified from these mutants by
PCR, and the DNA sequences were compared to the sequence
of the wild-type parent strain. No changes were observed in
defA or fmt, but for both mutants, a G-to-T transversion was
observed at bp 211 of the defB ORF. This change is predicted
to generate a Val (GTT)-to-Phe (TTT) substitution at amino
acid 71 of the PDF product, a substitution that is distinct from
those observed previously for S. pneumoniae mutants selected
with actinonin (33).
One major difference can be observed between the resistant
mutants from S. pneumoniae and those from H. influenzae:
while H. influenzae mutants selected with urea are cross resis-
tant to both succinate and N-alkyl urea PDF inhibitor classes,
mutants from S. pneumoniae are cross resistant to other N-
alkyl urea compounds but display increased susceptibility to
actinonin, a succinate hydroxamic acid.
Pharmacokinetic studies with mice. The clearance of
VRC4232 and VRC4307 in mouse serum was measured after
the compounds were administered i.v. and p.o. The concentra-
tions in serum at different times were used to calculate the
corresponding pharmacokinetic parameters presented in Table
5. VRC4232 has a longer half-life (1.1 h) than VRC4307 (0.1
h) after i.v. administration. Both compounds have poor p.o.
bioavailability (3.2 and 0.1%, respectively), suggesting that
these compounds will not have significant p.o. efficacy.
In vitro metabolic stability. The metabolic stability of the
compounds was determined in vitro: the compounds were in-
cubated with mouse, rat, and human liver microsomes and
Two independent H. influenzae mutants, VHIN6529 and
VHIN6536, were selected for further characterization. Both
mutants were highly resistant to all PDF inhibitors tested: the
MICs of all such compounds, except actinonin, were Ͼ64 ␮g/
ml; the MIC of actinonin was 32 ␮g/ml (Table 4). The antibi-
otic profile of the mutants is comparable to that of the parent
strain, suggesting that there is no cross-resistance with other
classes of antibiotics and that this resistance is not due to a
defect in a transport mechanism that is shared with other
antibiotics. The resistant mutants appear on solid medium as
small colonies; they grow slowly both in broth and on agar.
When these small colonies are streaked on solid agar in the
absence of a drug, large, normally sized colonies frequently
appear on the plate. These revertants are again as susceptible
to deformylase inhibitors as the parent strain, VHIN1004 (data
not shown).
To better define the mechanism of resistance to compounds
of the urea series, the def and fmt homologs from H. influenzae
mutant VHIN6529 and from three independent revertants
were sequenced and compared to those of the wild type. In
comparison with the sequences of the genes from parent strain
VHIN1004, no change was observed in the def homolog. How-
ever, the fmt gene from mutant VHIN6529 (and each rever-
tant) contained a frameshift mutation: the deletion of a C
residue following bp 307 of the open reading frame (ORF).
The resulting protein is predicted to diverge from the wild-type
protein at amino acid 103, terminating four amino acids later
(Fig. 6). The def sequences from the three independent rever-
tants were not changed compared to that from the parent
strain. However, all three revertants carried, in addition to the
TABLE 3. MICs and MBCs of PDF inhibitors against H. influenzae
VHIN1004 and S. pneumoniae VSPN1001a
VRC4232
VRC4307
Strain
MIC
MBC
MIC
MBC
VHIN1004
VSPN1001
4
4
32
Ͼ64
4
1
32
32
a MICs and MBCs are given in micrograms per milliliter.

2760
HACKBARTH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
then analyzed for loss of the parent compound over time. As
summarized in Table 6, both compounds were rapidly metab-
olized by mouse and rat liver microsomes (t1/2, Ͻ3 min) but
were much more stable in human liver microsomes (t1/2, Ͼ19
min).
Identification of major metabolites for VRC4232. The serum
samples collected from mice after dosing with VRC4232 were
analyzed for major metabolites by LC-MS/MS. Similar analy-
ses were also carried out for samples collected after incubation
of VRC4232 with liver microsomes. The major metabolites
identified from these samples are summarized in Fig. 7. Since
the authentic compounds were not available, quantitative anal-
ysis was not performed. Although these metabolites were iden-
tified in all samples, their relative intensities in the LC-MS
spectrum varied. The three major peaks found by LC-MS for
these metabolites (in order of decreasing relative peak areas)
were E, A, and B for in vivo mouse serum; A, E, and F for
mouse liver microsomes; D, E, and A for rat liver microsomes;
and C, A, and E for human liver microsomes.
The majority of the major metabolites correspond to mod-
ifications of the hydroxamic acid, including hydrolysis (metab-
olites A, B, and C in Fig. 7) and glucuronic acid conjugation
(metabolites E and F in Fig. 7). All of these modifications
would result in loss of the chelating activity of the parent
compound, an effect which should render the compounds de-
void of PDF inhibitory activity. Other major modifications
identified include hydroxylation of the proline group (metab-
olite C in Fig. 7) and oxidation of dimethylthiazole (metabo-
lites B, D, and F in Fig. 7).
Mouse S. aureus septicemia model. VRC4232 and VRC4307
both have MICs of 0.015 ␮g/ml against S. aureus strain Smith,
used in the septicemia model. After s.c. administration of
VRC4232, four of five mice survived at 32 mg/kg of body
weight and none of five survived at 16 mg/kg, resulting in an
ED50 of 29.7 mg/kg. VRC4307 was more efficacious in this
model. The survival of three, three, one, and none of five mice
was observed after s.c. doses of 32, 16, 8, and 4 mg/kg, respec-
tively, resulting in an ED50 of 17.9 mg/kg. VRC4307 was also
tested for its efficacy after p.o. administration; no protection
was observed at the highest tested dose of 30 mg/kg (i.e., none
of five mice survived). The ED50 for the control antibiotic,
vancomycin, was 2.4 mg/kg after s.c. dosing.
DISCUSSION
N-alkyl urea hydroxamic acids represent a new class of PDF
inhibitors. All previously published PDF inhibitors with rea-
sonable antibacterial activity share the structural feature of a
2-substituted alkanoyl, particularly 2-substituted hexanoyl, at
the P1Ј site (57). In this study, it was shown that bacterial PDF
can accommodate N-alkyl urea at the P1Ј site. The tested
compounds also can effectively inhibit the growth of both
gram-positive and gram-negative bacteria. The N-alkyl urea
structure allows easy preparation of analogs with different P1Ј
and P3Ј substitutions to explore the combination of moieties at
either site that might provide the best antibacterial activity.
Various alkyl groups at the P1Ј site were explored in a
combinatorial library format: branched and cyclic aliphatic
groups such as N-cyclopentylethyl and N-isopentyl showed the
best antibacterial activity (J. Lewis, unpublished data). Com-

VOL. 46, 2002
N-ALKYL UREA HYDROXAMIC ACIDS INHIBIT DEFORMYLASE
2761
FIG. 6. Internal DNA sequence and predicted protein sequence (codons 94 to 109) of the H. influenzae fmt ORF from wild-type strain
VHIN1004 (top), resistant mutant VHIN6529 (middle), and a susceptible revertant (bottom). Note that the insertion of T at codon 99 restores
the fmt reading frame in the revertant. Underlined amino acids indicate divergence from the wild-type formyltransferase protein sequence.
pounds combining these two P1Ј substitutions with different P3Ј
groups were prepared as purified compounds and tested in a
panel of in vitro assays (Table 1). The data in Table 1 demon-
strate that the N-alkyl urea compounds can very effectively
inhibit bacterial PDFs with IC50s well below 1 ␮M. In general,
compounds with substituted thiazoles have excellent activity
against S. aureus and S. pneumoniae. Overall, all of the N-alkyl
urea compounds prepared resulted in moderate inhibition of
H. influenzae growth.
the active-site metal. The result is a single metal-ligand inter-
action with the hydroxamate, while the remaining oxygen atom
forms a hydrogen bond interaction with the backbone amide of
Leu91. In order to achieve these interactions and maintain the
P1Ј interactions, the peptidic interaction between the carbonyl
of the inhibitor and the backbone amide of Ile44 is also dis-
torted relative to the actinonin complex. A good hydrogen
bonding distance is achieved, and close contact with the car-
˚
bonyl and the alpha-carbon of Gly43 results (2.8 A).
A comparison of VRC4390, VRC4156, VRC5157, and
VRC4234, all of which share the P1Ј N-isopentyl substitution,
suggests that increasing hydrophobicity at the P3Ј site improves
enzymatic and whole-cell activities for this class of compounds.
The trend is particularly obvious when a methyl group is in-
corporated in the P3Ј site, a substitution which results in a
dramatic loss of inhibition of S. pneumoniae PDF but which
has only a slight effect on E. coli PDF inhibition. The methyl
substitution also renders the compound inactive against S. au-
reus and S. pneumoniae in whole-cell assays, while activity
against H. influenzae and efflux-deficient E. coli is maintained.
These differences suggest that VRC4390 lacks activity against
S. aureus and S. pneumoniae because of a species-specific loss
of activity against the corresponding PDF. This observation
further suggests that deformylases from different bacterial spe-
cies may have different preferences at their S3Ј sites.
The crystal structure of the complex of VRC4307 with E. coli
PDF revealed the interactions between the urea hydroxamate
and the enzyme. Surprisingly, the hydroxamate did not adopt
the expected bidentate interaction that is common with MMP
inhibitors (7) and that was observed in the recently published
PDF-actinonin structure (13). The relatively planar N (relative
to an alpha-C atom of a substrate or actinonin) coupled with
the P2Ј proline limits the ability of the inhibitor to fully reach
Due to the structural resemblance of the key catalytic resi-
dues between PDFs and MMPs, the 2-substituted alkanoyl can
fit very well into the S1Ј site of the active center of both
enzymes (10, 41). This similarity could result in the inhibition
of MMPs by many deformylase inhibitors. In this study, we
show that bacterial PDF can accommodate N-alkyl urea in the
P1Ј site. In addition to structural novelty, this alternative P1Ј
substitution also provides excellent selectivity, as demonstrated
by the absence of MMP inhibition. The data in Table 1 indicate
that none of the compounds exhibits inhibitory activity against
MMP-7 at 200 ␮M; analogous compounds with a 2-substituted
alkanoyl substitution at the P1Ј site usually display complete
inhibition of MMP-7 at this concentration (data not shown).
Based on the crystal structure, it appears that the selectivity
may require both the N-alkyl urea hydroxamate and a P2Ј
proline group. The conformation adopted by the urea hydrox-
amate places its P2Ј proline ring at a sterically unfavorable
distance from the MMP carbonyl of Pro238 (matrilysin num-
bering). This proline is conserved in the MMP family, and the
orientation of its carbonyl is quite different from that of Gly89,
which occupies the equivalent position in E. coli PDF. This
structural property of the N-alkyl urea hydroxamic acids
should avoid the potential toxicity of hydroxamic acids that is
due to the inhibition of MMPs.
TABLE 5. Mouse pharmacokinetic parameters for VRC4232 and VRC4307a
Compound
VRC4232
Route
Dose (mg/kg)
Tmax (h)
Cmax (ng/ml)
t1/2 (h)
AUC (ng · h/ml)
% Absolute bioavailability
3.2b
i.v.
p.o.
13
13
0.083
0.083
5,580
167
1.1
NC
1,120
35.3
VRC4307
i.v.
p.o.
3.7
3.7
0.083
0.25b
1,720
1.5
0.1
NC
413
0.3
0.1b
a NC, not calculated.
b Value was not accurate due to an extremely low concentrations of the compound.

2762
HACKBARTH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
both H. influenzae and S. pneumoniae is approximately 10Ϫ9, as
was previously reported for actinonin, a PDF inhibitor of the
succinate hydroxamic acid series. This value is comparable to
what was found for a peptide thiol PDF inhibitor against B.
subtilis (25) and is 2 to 3 orders of magnitude smaller that what
has been reported for the spontaneous selection of resistance
to other deformylase inhibitors in S. aureus (13, 34) and E. coli
(4, 13). Two mechanisms of resistance to succinyl hydroxam-
ate-based inhibitors of bacterial PDF have been reported. In E.
coli, S. aureus, Moraxella catarrhalis, and H. influenzae, loss-of-
function mutations in fmt have been identified (4, 13, 33; P.
Margolis, C. Hackbarth, M. Maniar, S. Lopez, W. Wang, R.
White, Z. Yuan, and J. Trias, Abstr. 40th Intersci. Conf. An-
timicrob. Agents Chemother., abstr. 2174, 2000). The data
presented here indicate that resistance to urea-based PDF
inhibitors in H. influenzae occurs through a mechanism similar
to that seen for succinyl hydroxamate-based compounds in the
same species. As was observed previously for actinonin resis-
tance mutations, resistance to VRC4232 and VRC4307 in H.
influenzae apparently is derived from a frameshift mutation
that results in a truncated (and presumably nonfunctional)
formyltransferase protein. Consistent with previous observa-
tions made for E. coli, S. aureus, and H. influenzae, H. influen-
zae fmt mutant strain VHIN6529 grew more slowly than the
wild-type parent strain. Revertants of strain VHIN6529 simul-
taneously displayed an improved growth phenotype, increased
susceptibility to PDF inhibitors, and a compensatory frame-
shift mutation that restored the fmt reading frame. This cor-
relation strongly supports a model whereby resistance to urea-
based PDF inhibitors can occur through bypassing of the
formylation cycle.
TABLE 6. Comparison of metabolic stability of VRC4232 and
VRC4307, as determined in vitro by incubation with liver
microsomes from mice, rats, and humans
t1/2 (min) in:
Clearance (␮l/min/mg) in:
Compound
Mice
Rats
Humans
Mice
Rats
Humans
VRC4232
VRC4307
3.1
2.2
2.6
2.0
26.8
19.9
1,100
1,594
1,309
1,700
129
174
The urea hydroxamates appear to be bacteriostatic against S.
aureus, as was observed previously for other PDF inhibitors
against both S. aureus and E. coli (4, 13, 34). Huntington et al.
(25) reported the lysis of Bacillus subtilis by a peptide thiol
PDF inhibitor, although the compound appeared to be bacte-
riostatic when tested with log-phase organisms. In contrast to
the situation with S. aureus, VRC4232 and VRC4307 both had
a killing effect on S. pneumoniae and H. influenzae, two patho-
gens that cause upper respiratory tract infections. Notably,
neither compound displayed a significant degree of killing of
H. influenzae over the initial hours of the experiments, but
there was significant death of this species by 24 h of drug
exposure. The basis for this delayed killing effect is not clear,
but these data and the characterization of the resistant mutants
indicate that the inability to remove the N-formyl group from
proteins is toxic in H. influenzae. Hypothetically, several cycles
of growth in the presence of deformylase inhibitors would
deplete the pool of normally processed polypeptides, while
N-formylated proteins would accumulate. If, at that late stage,
one or more enzymes are required but are nonfunctional due
to N formylation (e.g., improper folding), then cell death will
ensue.
The frequency of resistance to the urea-based inhibitors in
It is not evident why the loss-of-function mutations found in
FIG. 7. Modification of VRC4232 in vivo (mice) and in vitro (liver microsomes) and identification of its major metabolites (A to F).

VOL. 46, 2002
N-ALKYL UREA HYDROXAMIC ACIDS INHIBIT DEFORMYLASE
2763
H. influenzae fmt occur at a lower frequency (10Ϫ8) than those
found with succinate inhibitors in E. coli or S. aureus (10Ϫ6).
One hypothesis is that the difference in resistance rates may be
due to the bactericidal effects of these compounds on H. in-
fluenzae, which may lead to a decreased number of viable
mutants able to survive the selection process. Alternatively,
rather than a bypass mechanism, perhaps the truncated Fmt
created by the frameshift mutation remains partly functional,
and fmt is essential in H. influenzae. In S. pneumoniae, a second
mechanism has been identified; resistance to actinonin results
from missense mutations in the defB gene. The distinction in
resistance mechanisms observed in different species presum-
ably reflects the fact that fmt is essential in pneumococci (33).
Resistance to the urea-based compounds VRC4232 and
VRC4307 in S. pneumoniae also appears to be mediated by an
alteration in the target, but the location of the missense mu-
tation in defB (V71F) is distinct from those of two other pneu-
mococcal defB mutations, A123D and Q172K, that were iden-
tified previously (33). The V71F mutation seen in strains
VSPN6521 and VSPN6522 is located in the highly conserved
box 1 (70GVGLAAPQ77) of S. pneumoniae PDF. As a com-
parison with the PDF consensus sequence shows, Val71 does
not correspond to a strictly conserved residue; an Ile-to-Ala
substitution of the equivalent residue in the E. coli enzyme is
well tolerated (41). However, this residue does lie between Gly
residues known to be critical for enzyme activity in the E. coli
enzyme.
It also appears that the defB (V71F) mutants VSPN6521 and
VSPN6522 are able to distinguish between inhibitors of the
urea and succinate hydroxamate series. These mutants are
more susceptible to actinonin (Table 4) as well as to other
succinate inhibitors (data not shown), although they are highly
resistant to all urea-series PDF inhibitors tested. In contrast,
mutants selected with actinonin contain distinct defB muta-
tions (33) and are resistant to both classes of PDF inhibitors
(data not shown). A crystal structure of the enzyme containing
this V71F mutation is not available; however, the crystal struc-
ture of E. coli PDF complexed with VRC4307 can be used to
infer the effect of this mutation. The two most common rota-
mers for Phe71 would place it in either the S1Ј or the S3Ј
pocket. Given that the V71F mutant is still active, the more
likely position would be in the large S3Ј pocket, which could
accommodate this large side chain with only minor perturba-
tions. The urea hydroxamate inhibitors are less likely to toler-
ate any movement of Phe71 (and the adjacent Gly70) into the
active site due to the close contact between Gly70 and the
inhibitor. These observations may reflect a species-specific
structural aspect of the pneumococcal PDF enzyme.
both instances, and the sites of modification were also compa-
rable.
VRC4232 and VRC4307 were selected for further in vivo
studies. In an S. aureus septicemia model, both compounds
showed moderate protective activity after s.c. administration,
establishing N-alkyl urea hydroxamic acids as potential anti-
bacterial agents. However, when the more potent compound
VRC4307 was tested after p.o. administration in the same
model, no protective effect was observed up to 30 mg/kg. This
lack of p.o. efficacy presumably reflects the poor p.o. bioavail-
ability of VRC4307, as demonstrated by the in vivo pharma-
cokinetic analysis (absolute p.o. bioavailability, 0.1%). These
data suggest the need to incorporate bioavailability tests at
early stages of compound screening by using Caco-2 cell in
vitro testing or by using cassette dosing.
In summary, we have identified N-alkyl urea hydroxamic
acids as a new class of PDF inhibitors. This class of compounds
has potent whole-cell activity against both gram-positive and
gram-negative bacteria but is devoid of MMP inhibition. The
potential use of this class of compounds as antibacterial agents
is supported by their protective activity in an in vivo infection
model.
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