Exploring the UDP pocket of LpxC through amino acid analogs

Article abstract of DOI:10.1016/j.bmcl.2013.02.055

Lipopolysaccharide (LPS) biosynthesis is an attractive antibacterial target as it is both conserved and essential for the survival of key pathogenic bacteria. Lipid A is the hydrophobic anchor for LPS and a key structural component of the outer membrane of Gram-negative bacteria. Lipid A biosynthesis is performed in part by a unique zinc dependent metalloamidase, LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase), which catalyzes the first non-reversible step in lipid A biosynthesis. The UDP portion of the LpxC substrate-binding pocket has been relatively unexplored. We have designed and evaluated a series of hydroxamate based inhibitors which explore the SAR of substitutions directed into the UDP pocket with a range of substituted α-amino acid based linkers. We also provide the first wild type structure of Pseudomonas aeruginosa LpxC which was utilized in the design of many of these analogs.

Full text of DOI:10.1016/j.bmcl.2013.02.055

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2362–2367
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploring the UDP pocket of LpxC through amino acid analogs
⇑
Michael R. Hale , Pamela Hill, Sushmita Lahiri, Matthew D. Miller, Philip Ross, Richard Alm, Ning Gao,
Amy Kutschke, Michele Johnstone, Bryan Prince, Jason Thresher, Wei Yang
Infection Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipopolysaccharide (LPS) biosynthesis is an attractive antibacterial target as it is both conserved and
essential for the survival of key pathogenic bacteria. Lipid A is the hydrophobic anchor for LPS and a
key structural component of the outer membrane of Gram-negative bacteria. Lipid A biosynthesis is per-
formed in part by a unique zinc dependent metalloamidase, LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-N-
acetylglucosamine deacetylase), which catalyzes the first non-reversible step in lipid A biosynthesis.
The UDP portion of the LpxC substrate-binding pocket has been relatively unexplored. We have designed
and evaluated a series of hydroxamate based inhibitors which explore the SAR of substitutions directed
Received 16 November 2012
Revised 6 February 2013
Accepted 12 February 2013
Available online 1 March 2013
Keywords:
LpxC
Inhibitor
Antibiotic
Hydroxamate
into the UDP pocket with a range of substituted a-amino acid based linkers. We also provide the first wild
type structure of Pseudomonas aeruginosa LpxC which was utilized in the design of many of these analogs.
Ó 2013 Elsevier Ltd. All rights reserved.
There is a critical need for discovery of novel agents targeting
Gram-negative bacteria as there is a rising incidence of resistance
to existing therapies.1 Additionally, new agents are not being
developed fast enough to rebuild our arsenal of treatments. The
composition of the outer membrane includes lipopolysaccharide
(LPS) providing a charged layer to protect the cells from the passive
entry of hydrophobic agents including antibiotics.2 Getting
through this protective outer membrane is one of the challenges
in targeting Gram-negative bacteria. The inner membrane of
Gram-negative bacteria provides a more lipophilic barrier; inhibi-
tors must maintain a balance of physical properties to penetrate
both membranes effectively.
LpxC is an attractive target as it is the first non-reversible step
in the synthesis of lipid A, the hydrophobic anchor for LPS present
in the leaflet of the outer membrane of Gram-negative bacteria.3
The lipid A biochemical pathway has recently become an impor-
tant target for Gram-negative antibacterial drug discovery.4 Lipid
A is made on the inner side of the cytosolic membrane by a cascade
of enzymes including LpxC.5 After synthesis, lipid A is transported
to the extracellular side of the outer membrane. LpxC has been
determined to be essential, as failure of bacteria to produce lipid
A results in inhibition of bacterial growth and cell death.6 Recently
there have been reports of the in vivo validation of LpxC as a drug
target.7 LpxC inhibitors could be utilized in the treatment of clini-
cal indications where Gram-negative pathogens predominate.
Clinical indications such as urinary tract, intra-abdominal infec-
tions as well as hospital acquired pneumonia are predominated
by the following pathogens; Pseudomonas aeruginosa, Escherichia
coli, Klebsiella pneumoniae.
LpxC contains an active site zinc ion which is required for
catalyzing the deacetylation of UDP-3-O-(R-3-hydroxymyristoyl)-
N-acetylglucosamine. There have been numerous reports of
hydroxamate based inhibitors that have been effective at binding
LpxC through coordination to the active site Zn.8 The active site
zinc in both P. aeruginosa and E. coli LpxC has an atypical binding
motif, bound by two histidines and an aspartic acid which may
be beneficial in providing inhibitors with selectivity over other
Zn2+ dependent enzymes. Additionally the secondary structure of
the protein is a typical, a bÀ
a
À
a
Àb sandwich fold where four
a
helices are held between two b sheets, which could contribute to
additional selectivity.9
Design: A series of inhibitors as shown in Figure 1 were designed
and synthesized to evaluate the SAR of analogs with diversity at R1
and to compare activities to CH-12.10 CH-12 is an LpxC inhibitor dis-
closed by Chiron and is used as a comparator in this effort. CH-9011 is
one of the more potent and widely used comparators for LpxC inhib-
itors in which there are now numerous examples. The initial com-
pounds were designed and prioritized based on a P. aeruginosa
LpxC homology model. The goal of the design set was to evaluate
diversity at R1 through a series of amino acid based linkers and study
the impact on enzymatic potency, physical properties and target
accessibility (Fig. 1).
Many Zn dependant enzymes including LpxC have similar phar-
macophores. The catalytic Zn is coordinated to the warhead, a
hydroxamate in this case. The warhead can also bind to active site
residues. The warhead is quite often linked to a core that is
substituted by a hydrophobic tail and in some cases additional
⇑
Corresponding author. Tel.: +1 781 839 4113; fax: +1 781 839 4630.
E-mail address: michael.hale@astrazeneca.com (M.R. Hale).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.055

M. R. Hale et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2362–2367
2363
Figure 1. CH-90, CH-12, generic for R1 library diversity.
Scheme 1. Description of general route. Reagents and conditions: (a) Sonagashira conditions, (b) HATU coupling conditions, (c) 50% aq. Hydroxylamine, MeOH, rt 12 h.
substitution into what is referred to as the UDP pocket in LpxC. The
inhibitors discussed in this Letter focus on diversity in the UDP pock-
et through R1 variations, we then studied how they impact potency.
The differences in R1 sizes, properties and geometry can be used to
design selective inhibitors and enhance potency for LpxC.
The synthesis of the compounds evaluated is outlined generi-
cally in Scheme 1. The phenylacetylene benzoic acid is first assem-
bled through a Sonagashira coupling. This is followed by a HATU
In comparing the relative biochemical potency of the top three
compounds in Table 1, one can note the approximate 300-fold
improvement in potency against the P. aeruginosa LpxC enzyme
when comparing the simple glycine analog (1) to the s,s-threonine
analog (CH-12). The enzymatic potency is modestly enhanced by
the addition of the tethered morpholine tail even though the
extension is outside of the active site and exposed to solvent
(CH-90). On a cellular level the addition of the morpholino tail does
not enhance P. aeruginosa activity but leads to a significant
improvement in E. coli MIC values.
The activity of the glycine analog (1) may be partially restored
by alternate substitutions. The racemic-Ala derivative (2) is four-
fold more potent than the glycine analog although not sufficiently
active to show cellular activity. Addition of a second methyl (gem
dimethyl, 3) reduces the activity of the series. The racemic valine
(4) analog which builds further into the pocket is considerably
more potent than the alanine analog (18-fold). A matched pair of
both serine analogs shows the importance of chirality, which has
previously been noted.15 The more potent enantiomer is similar
in potency to the valine analog in both biochemical and cellular
activity despite the change in polarity. Compound 7, an amine, dis-
plays similar activity to the valine and serine analogs which is
interesting given the change in H-bonding nature of these func-
tional groups.
mediated coupling and the introduction of a series of
a-amino
acids. The final step is conversion to the hydroxamic acid which
is accomplished using 50% aqueous hydroxylamine in methanol.
In some cases the intermediate ester was modified prior to
hydroxamic acid formation.
The plan was to vary the substitution and chirality by incorpo-
rating a series of natural and unnatural
a-amino acids as linkers
and to evaluate the impact of R1 on enzymatic and cellular potency.
The compounds were evaluated for enzymatic inhibition (IC50),
wild type (WT) and efflux pump deficient (PD) MIC valuess in both
P. aeruginosa (WT: Pae545 and PD: Pae546), and E. coli (WT:
Eco523 and PD: Eco524), as well as being evaluated for changes
in physical properties (AZ Log D and plasma protein binding).12,13
AZ Log is a modified algorithm for calculation of lipophilicity
developed at AstraZeneca.14
For an initial evaluation of the series we made the glycine based
analog and compared the properties to the s,s-threonine compound
bearing the same hydrophobic substituent, the diphenylacetylene
A series of unnatural amino acids were then evaluated as linkers,
this included methionine and phenyl glycine based analogs.
Compound 9, the sulfone, showed a marked improvement in bio-
chemical potency over the methione itself, this may be the result
of the formation of an H-bond between the sulfone and Lys238.
(CH-12), see Table 1.
A second important comparison was
the extension of the hydrophobic group where it can interact with
solvent and perhaps improve physical properties, CH-90
(Fig. 1).CH-12 does not probe deeply into the UDP portion of the
active site. The series of compounds described here was designed
to evaluate potential for improved binding through extension of
the R1 into the phosphate and ribose binding region of the UDP
pocket. As shown in Table 1, the length of the substituent affects
the enzyme potency at the target site.
The addition of an additional
a-methyl group to the methionine
analog 8 was detrimental to the biochemical activity (data not
shown), as was observed in comparing compounds 2 and 3.
Addition of an aryl substituent improves the biochemical potency
70-fold when compared to the glycine based compound; the substi-
tution provides good cellular activity despite both the size and the

2364
M. R. Hale et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2362–2367
Table 1
Amino acid based R1
Compd. #
R1
AZ Log D
2.3
PaeIc50 (nM)
0.6
Pae546 (
l
M)
Pae545 (
l
M)
EcoIc50 (nM)
0.2
Eco524 (
l
M)
Eco523 (
lM)
CH-90
<0.2
1.6
<0.2
<0.2
CH-12
2.5
1.6
<0.2
1.6
1
<0.2
1.6
1
2
3
2.1
2.8
3.3
516
128
12
12
>200
>200
>200
148
19
12.5
6.3
50
100
25
2067
100
219
100
4
3.3
7.1
0.8
25
3.1
<0.2
6.3
5
6
2.0
2.2
5.3
0.8
50
12
1.6
0.8
50
6.3
187
>200
206
>200
7
8
1.8
3.1
28
1.6
0.8
12
25
15
1.6
0.8
6.3
6.3
9.8
1.8
9
2.4
3.8
7.3
7.4
0.8
0.8
>200
12
1.8
2
0.8
0.4
6.3
3.2
10
11
12
3.4
2.1
80
35
2.5
1.6
>200
>200
14
3.2
0.8
12.5
6.3
1.6
lipophilic character of the group. The less active phenyl glycine
enantiomer is still more potent than the glycine analog (10 vs
11). Substitution with a more polar aromatic group, imidazole
(12), resulted in similar binding but a reduced AZ Log D, this substi-
tution unfortunately also increased efflux in P. aeruginosa but re-
sulted in no change in E. coli.
In general the MIC’s against the pump deficient strains of
P. aeruginosa are less active than those observed in E. coli, which
may be due to the difference in number, types and expression lev-
els of efflux pumps or cell membrane composition for the two
pathogens.
other (Fig. 2).17 Domain II contains insert regions (Insert I and In-
sert II) that form the active site pocket. Of these, Insert I is a loop
forming a part of the UDP-binding region, while Insert II, which
is critical to the inhibitor binding, forms a hydrophobic tunnel with
a bÀ Àb structure that stabilizes the substrate. The active site, in
a
the bottom of Insert II, is comprised of zinc coordinated with
His78, His237 and Asp241. A second, non-catalytic zinc atom
was identified in the crystal structure (not shown) which is likely
a result of the crystallization conditions. The residues from Insert
I (residues 160–169) were not defined in the electron density
hence are not included in the final structural model.
Glutamine analogs: A series of amides which can access further
into the pocket and maintain flexibility was designed and evalu-
ated. It was noted in an early survey of amino acid substitutions
that the Gln analog (13) had good biochemical potency, the phys-
ical properties of this compound were also attractive: protein bind-
ing, solubility, and calculated Log D. Having established the role of
amide in binding with Lys238, additional substitutions were ex-
plored to further enhance binding affinity. Amide substitutions
were evaluated and all of the analogs showed weaker IC50 values
than the initial compound (13), although all compounds in this ser-
ies maintained the attractive physical properties of the lead. A
crystal structure of compound 14 was obtained in the P. aeruginosa
LpxC enzyme which was used in the design of additional analogs
(Table 2).
Compound 14 contains three separate components that contrib-
ute to enzyme potency. The hydroxamate coordinates with the cat-
alytic zinc in trigonal bipyrimidyl geometry. This five-coordinate
complexation with zinc is similar to other known LpxC inhibitors18
,
such as CH-90,17 BB-78485,19 and others.17 In the crystal structure
of compound 14 the hydroxyl amine occupies an axial position and
the carbonyl of the hydroxamate is in the equatorial position
(Fig. 3). The hydroxamate hydroxyl is located 2.4 Å from the
Glu77 acid side chain and the hydroxamate amine is 3.0 Å from
the Met62 backbone oxygen. The hydroxamate carbonyl is 2.7 Å
from the Thr191 side chain. These three interactions in addition
to the metal chelation contribute to the strong potency of the
hydroxamate group.
Compound 14 has a hydroxypropyl substituted by a Gln amide
in the R1 position. This group was designed to form hydrogen
bonds to residues within the binding pocket occupied by the
UDP group of the natural substrate.20 In Figure 4a, the modeled
substrate in LpxC forms two hydrogen bonds between the Lys238
and phosphate groups. To mimic the Lys238 interactions, com-
Structural analysis: The wild-type P. aeruginosa LpxC structure
co-complexed with compound 14 was solved to 2.0 Å resolution.16
Each asymmetric unit contained two nearly identical monomers
(0.27 rsmd) with the previously observed bÀ
a
À Àb sandwich fold
a
formed by two domains of -helix and b-sheet layers against each
a

M. R. Hale et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2362–2367
2365
M)
Table 2
Glutamine based linkers.
Compd #
R1
AZ Log D
1.3
PaeIc50 (nM)
4.4
Pae546 (
4.4
lM)
Pae545 (
>200
lM)
EcoIc50 (nM)
11
Eco524 (
l
M)
Eco523 (
l
13
2.2
35.4
14
15
16
2.1
2.6
2.7
5.3
33
5.3
33
200
50
1.3
3.6
4
3.2
1.6
0.8
50
25
14
14
50
12.5
Figure 3. Residue and ligand interactions with hydroxamate. Hydroxamate forms
two metal chelations and each heteroatom interacts with the protein.
Figure 2. Overall structure of LpxC with compound 14. Shown are Domain I (blue),
Domain II (magenta), and two insert regions (green). Residues 160–169 of Insert I
was unable to be solved, located at the lower portion of the figure.
hydrogen bonding in the Asp196 region of the active site requires
further optimization to improve the potency over compound 13.
The third essential motif contributing to enzyme potency is the
amide/biphenyl acetylene for compound 14. This portion of the
molecule forms both hydrophobic and hydrophilic interactions.
The amide nitrogen is within hydrogen bonding distance to
Thr190 in the crystal structure. In contrast, the amide carbonyl
cannot make any additional H-bonds. The phenyl-acetylene-
phenyl group extends into a hydrophobic tunnel and is surrounded
by many non-polar side chains including Leu18, Gly192, Ile197,
Leu200, Ala206, Gly209, Ala214, and Val216 (Fig. 5). The outer phe-
nyl ring is 15° out of plane with the interior phenyl group to con-
form to the shape of the hydrophobic tunnel.
The last substitution to be evaluated was based on compound 3
in which the amino acid utilized in the design was disubstituted.
Compound 3 was found to be 30-fold less active than the alanine
analog (2). We had noted that the addition of an additional alpha
substitution in the methionine series also reduced enzyme inhibi-
tion (data not shown). Could a cyclic R1 substituent restore activity
and provide some rigidity to the substitution? The carbocyclic
pounds with ethylene linked amides were designed (compound
13–16). Previous docking in a Pae homology model predicted the
formation of a hydrogen bond between Lys238 and the amide car-
bonyl. In the crystal structure, a hydrogen bond occurs between
the Lys side chain and the carbonyl, with a distance of 2.7 Å
(Fig. 4b). Furthermore, the crystal structure shows hydrophobic
interactions between the Phe191 sidechain and ethylene linker
due to the stereochemistry of the compound, likely contributing
to the 5 nM P. aeruginosa IC50 value. The hydroxypropyl group at-
tached to the amide was designed to form hydrogen bonds with
the Asp196 side chain and Phe193 backbone amine. The structure
shows a hydrogen bond between the hydroxyl and Asp196 side
chain (2.6 Å) but not between the hydroxyl and Phe193 backbone
amine. This hydroxyl-Asp196 hydrogen bond found in the com-
pound 14 structure could explain the sixfold improvement in the
P. aeruginosa IC50 over compound 15 which cannot form this
hydrogen bond (Fig. 4c). However, the P. aeruginosa IC50 value for
the unsubstituted amide (compound 13) is 4.4 nM, illustrating that

2366
M. R. Hale et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2362–2367
Figure 4. (a) The modeled substrate has two interactions with Lys238. (b) The crystal structure of compound 14 has hydrogen bonds with Lys238 and Asp196. (c) Compound
21 docked in Pae are predicted to hydrogen bond with Lys238.
amino acids evaluated. The smaller and positively charged
piperidine analog (22) was found to be significantly weaker than
the other compounds in this series perhaps due to the proximity
to Lys238 (Table 3).
We have designed and prepared a series of amino acid linked ana-
logs using the CH-12 scaffold as a template to evaluate the potential
for improved potency at both enzymatic and cellular levels by build-
ing into the UDP pocket. The UDP region of P. aeruginosa LpxC sub-
strate binding site is relatively unexplored with respect to
inhibitor development.21 These efforts were guided by both compu-
tational design and structural input to probe the UDP portion of the
LpxC pocket. Substituents with a variety of stereochemistry and
chemical diversity were utilized to study their impact. The small
substituted aliphatic amino acids; alanine, serine and amine based
linkers were all well tolerated in the base of the UDP pocket. This
study included a series of matched pairs demonstrating strong
preference for stereochemistry at the R1 position. We were able to
replace the threonine with methionine, phenylglycine and gluta-
mine based analogs and maintain enzymatic activity, but not main-
tain cellular activity and physical properties. The glutamine analogs
were among the most potent (IC50) in this series and retained desir-
able physical properties. A co-crystal structure of compound 14
Figure 5. The amide and phenyl-acetylene-phenyl hydrophobic tails of compound
14 form polar and nonpolar interactions with the protein. Seven nonpolar side
bound to the wild-type LpxC P. aeruginosa was obtained and utilized
for the design of many of the analogs presented. The structure re-
vealed a hydrogen bond between Lys238 and provided a foundation
for further potency improvements by extending into the UDP pock-
et. The biochemical potency was maintained but not improved in
comparison to CH-12, with glutamine and sulfone based UDP substi-
tutions offering the most promise for continued exploration. The
whole cell activity of the compounds made did not improve in the
pump deficient or WT cellular activity when compared to CH-12,
likely as a function of reduced biochemical activity and increased ef-
flux. Both series were designed to utilize interactions with Lys238.
Additional compounds which target improvements in potency
through building into the UDP pocket are in progress.
chains surround the hydrophobic tunnel.
series as initially represented by compounds 17 and 18 were quite
similar in potency to compound 3, but when an acceptor hetero-
atom was added on the far side of the cyclic substituents the enzy-
matic potency improved 3 to 10-fold for O, S and an N-benzyl
analogs. The most significant improvement resulted from the con-
version of compound 20 to the sulfone which has the potential for
forming a hydrogen bond with Lys238 as was noted with the Gln
amides. This modification showed an additional 10-fold improve-
ment and has the most potent biochemical potency of the cyclic

M. R. Hale et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2362–2367
2367
M)
Table 3
Cyclic amino acid based linkers.
Compd #
R1
AZ Log D
3.1
PaeIc50 (nM)
8100
Pae546 (
l
M)
Pae545 (
l
M)
EcoIc50 (nM)
178
Eco524 (
50
lM)
Eco523 (
l
17
200
>200
>200
18
19
3.8
2.2
3500
334
100
50
>200
>200
98
12
6.3
6.3
100
50
20
21
3.0
2.1
296
26
12.5
25
>200
100
22
<4
1.6
0.8
25
50
22
23
0.8
3.4
11000
200
25
>200
>200
102
50
>200
208
3434
3.2
12.5
8. (a) Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M.-
H.; Patchett, A. A.; Galloway, S. M.; Hyland, S. A.; Anderson, M. S.; Raetz, C. R. H.
Science 1996, 274, 980; (b) Warmus, J. S.; Quinn, C. L.; Taylor, C.; Murphy, S. T.;
Johnson, T. A.; Limberakis, C.; Ortwine, D.; Bronstein, J.; Pagano, P.; Knafels, J.
D.; Lightle, S.; Mochalkin, I.; Brideau, R.; Podoll, T. Bioorg. Med. Chem. Lett. 2012,
22(7), 2536.
9. (a) Coggins, B. E.; Li, X.; McClerren, A. L.; Hindsgaul, O.; Raetz, C. R. H.; Zhou, P.
Nat. Struct. Biol. 2003, 10, 645; (b) Whittington, D. A.; Rusche, K. M.; Shin, H.;
Fierke, C. A.; Christianson, D. W. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 8146.
10. Anderson, N. H.; Bowman, J.; Erwin, A.; Harwood, E.; Kline, T.; Mdluli, K.; Ng, S.;
Pfister, K. B.; Shawar, R. I.; Wagman, A.; Yabannavar, A. WO2004062601 A2, see
example 12 (CH-12).
Acknowledgements
We would like to thank Maria Uria-Nickelsen for her helpful
suggestions and feedback on the document. We would also like
to acknowledge contributions from Senthilkumar Kuppusamy
and the chemists at Syngene.
Supplementary data
11. Anderson, N. H.; Bowman, J.; Erwin, A.; Harwood, E.; Kline, T.; Mdluli, K.; Ng, S.;
Pfister, K. B.; Shawar, R. I.; Wagman, A.; Yabannavar, A. WO2004062601 A2, see
example 83 (CH-90).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.02.055.
12. For more detailed information on IC 50 assays, cell lines used in microbiology
and AZ log D and PPB methods see supplementary material.
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