Structure guided development of potent reversibly binding penicillin binding protein inhibitors

Article abstract of DOI:10.1021/ml100260x

Following from the evaluation of different types of electrophiles, combined modeling and crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding protein. The results suggest that a structurally informed approach to penicillin binding protein inhibition will be useful for the development of both improved reversibly binding inhibitors, including boronic acids, and acylating inhibitors, such as β-lactams.

Full text of DOI:10.1021/ml100260x

LETTER
pubs.acs.org/acsmedchemlett
Structure Guided Development of Potent Reversibly Binding Penicillin
Binding Protein Inhibitors
†
‡
§
||
^
†
ꢀ
Esther C. Y. Woon, Astrid Zervosen, Eric Sauvage, Katie J. Simmons, Matej Zivec, Steven R. Inglis,
Colin W. G. Fishwick,|| Stanislav Gobec,^ Paulette Charlier,§ Andrꢁe Luxen,‡ and Christopher J. Schofield*,†
†Chemistry Research Laboratory, Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United
Kingdom
‡Centre de Recherches du Cyclotron, B30, Universite de Liꢂege, Sart-Tilman, B-4000 Liꢂege, Belgium
§Centre d’Ingꢁenierie des Protꢁeines, Universitꢁe de Liꢂege, Institut de Physique B5, B-4000 Liꢂege, Belgium
School of Chemistry, University of Leeds, Leeds LS2 9JT, United Kingdom
^Faculty of Pharmacy, University of Ljubljana, Aꢀskerꢀceva 7, 1000 Ljubljana, Slovenia
S Supporting Information
b
ABSTRACT: Following from the evaluation of different types of electrophiles, combined modeling and
crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding
protein. The results suggest that a structurally informed approach to penicillin binding protein inhibition
will be useful for the development of both improved reversibly binding inhibitors, including boronic acids,
and acylating inhibitors, such as β-lactams.
KEYWORDS: Penicillin binding proteins, boronic acids, antibiotics, antibiotic-resistance, β-lactams,
transpeptidase-inhibition
enicillin-binding proteins (PBPs) catalyze steps in the bio-
synthesis of peptidoglycan, which is a major component of
crystallographic analyses. The results demonstrate how a struc-
turally informed approach can be applied to PBP inhibition. An
analogous approach should be applicable to other types of PBP
inhibitors, including β-lactams.
P
the bacterial cell wall (for reviews, see refs 1 and 2), and their
inhibition causes irregularities in cell wall structure, lysis, and
eventual cell death.3 PBPs are inhibited by the β-lactam anti-
biotics (including penicillins, cephalosporins, monobactams, and
carbapenems, Figure 1a). From the 1940s onward, very sub-
stantial synthetic and screening efforts were made to optimize the
side chains of β-lactam antibiotics with a view to improving their
potency, spectrum of activity, and pharmacokinetics (for review,
see ref 4). The majority of this work was carried out in the
absence of detailed structural knowledge on PBPs, which has
begun to emerge over the past decade or so.1,2,5
The continuing and increasing problem of resistance to β-
lactam antibiotics due to β-lactamases has motivated work
toward the identification of non-hydrolyzable PBP inhibitors.6
One approach involves the use of appropriately functionalized
electrophiles (sometimes referred to as transition state ana-
logues) that react reversibly with the nucleophilic active site
serine of the PBPs. Peptide-based inhibitors of PBPs from
Streptomyces (R61),7 Escherichia coli (PBP 5),8 Neisseria gonor-
rheae (PBP 3/4),9 and Actinomadura (R39)10 have been re-
ported. Following these pioneering efforts, it has recently been
reported that aryl boronic acids inhibit R39.11 Here, we report
the identification and structural analysis of potent nonacylating,
reversible transition state inhibitiors of R39. Following from
work with different types of electrophiles, the activity of
acetamido-boronic acids was optimized by computational and
Catalysis by both PBPs and the serine subfamilies of β-
lactmases proceeds via an acyl-enzyme complex; “tetrahedral”
intermediates are involved in the formation and breakdown of
this complex (Figure 1b).7-10 Several classes of electrophiles are
proposed to act as structural analogues of these tetrahedral
intermediates, including trifluomethylketones, phosphonates,
and boronic acids (Figure 1c). Using a crystal structure of the
acyl-enzyme complex formed by reaction of nitrocefin with R39
(PDB ID 1W8Y)12 for guidance (Figure 2a), three types of
electrophile were identified and then synthesized as potential
inhibitors (compounds 1-17, Figure 1d); for synthetic details,
see the Supporting Information. The chosen side chains reflect
those in β-lactam antibiotics, that is, the thiopheneacetyl group of
cefalotin and the 2,6-dimethoxyphenyl group of methicillin (of
interest as the bulk of this side chain confers some β-lactamase
resistance). Screening of these potential inhibitors against R39
revealed little or no activity for the trifluoromethyl ketones and
the phosphonates (Supporting Information Table S1) but iden-
tified alkyl boronic acids 10 and 11 as potential lead structures,
Received: October 29, 2010
Accepted: December 29, 2010
Published: January 11, 2011
r
2011 American Chemical Society
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Figure 1. Irreversible and reversible inhibition of PBPs. (a) Penicillin and cephalosporin antibiotics; (b) reaction of β-lactam antibiotics with a PBP or
β-lactamase; (c) the intermediate analogue approach; (d) structures of intermediate analogs in the initial screen.
Figure 2. Active site views from structures of R39 (green) bound to nitrocefin (magenta, PDB ID 1W8Y12) and 10 (blue, PDB ID 2XLN). (a)
R39-nitrocefin acyl-enzyme complex. The side-chain amide of nitrocefin is positioned to form hydrogen bonds with the side chain of Asn300. The C-3
side chain of nitrocefin is not shown. (b) Formation of a tetrahedral adduct between the boron of 10 and Oγ atom of Ser49. The bond angle values
around boron are 113.5ꢀ (C1-B-OH1), 112.0ꢀ (C1-B-OH2), 103.9ꢀ (OH1-B-OH2), 103.8ꢀ (OH1-B-Oγ), 116.1ꢀ(OH1-B-Oγ), and
107.4ꢀ (C1-B-Oγ). (c) Superimposition of the nitrocefin (magenta) and 10 (blue) complexes showing the active site surface.
with IC50 values of 33 and 75 μM, respectively (Supporting
Information Table S1).
We then determined a crystal structure of 10 in complex with
R39 to investigate its binding mode (PDB ID 2XLN, Figure 2b
and Supporting Information Figure S1a). The structure reveals
reaction of the electrophilic boron atom with the Oγ atom of
active site Ser49 to form a tetrahedral adduct. This complex is
apparently stabilized by hydrogen-bonding interactions between
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LETTER
one of the boronate hydroxyl group and the backbone nitrogen
atoms of Ser49 and Thr413 (which forms an “oxyanion hole”), in
a manner analogous to that observed between the nitrocefin
acyl-enzyme carbonyl oxygen and the oxyanion hole (Figure 2a).
The C-R group of 10 is positioned next to that of Leu349,
possibly explaining why analogues 12-17 with substituents
bulkier than a methyl group are inactive. The side chain amides
of nitrocefin and 10 are positioned to make similar hydrogen-
bonding interactions to the Asn300 side chain and Thr413
backbone carbonyl. Although the overall structures for the
complexes with nitrocefin and 10 are very similar, in the case
of 10 the side chain of Tyr147 is rotated to enable an apparent π-
stacking interaction with the phenyl ring of 10.
Table 1. Activity of Computationally Designed Second-
(18-27) and Third-Generation (28-30) Boronic Acids
against R39a
The structural data suggest that 10 reacts with R39 to form a
reasonable mimic of an intermediate complex; hence, it was
selected as a starting point for further optimization, using the
inhibitor design software SPROUT.13,14 In this strategy, the
protein is first examined for potential ligand interaction sites.
Fragments are then “added” to the lead structure. A scoring
system based on predicted binding affinity, structural complexity,
and synthetic accessibility guides the “identification” of poten-
tially improved inhibitors (Supporting Information Figure S2).
Initial computational analysis of 10 and nitrocefin R39 com-
plexes reveals two regions of relatively high hydrophobicity
which we considered could be utilized for structural modifica-
tion. One region (A) is adjacent to the 4- and 5-positions of the
phenyl ring of 10, and the other (B) is a relatively large area of
space formed by Trp139, Asp142, Tyr147, Arg351, and Met414,
which is occupied by the nitrocefin C-7 side chain (Figure 2).
A series of second-generation boronic acid inhibitors 18-27
(Table 1) was identified by computationally modifying 10 using a
virtual fragment library of mainly hydrophobic elements. Boronic
acids 18-27 were then prepared to explore structure-activity
relationships in regions A and B, respectively.
The synthesis of the boronic acids 18-27 was adapted from
the strategy of Matteson et al.,15 starting from boronate esters
formed from (þ)- or (-)-pinanediol (Scheme 1). Key steps are
the stereoselective chain homologation using dichloromethyl-
lithium and nucleophilic displacement of chloride with bistri-
methylsilylamide. Deprotection of the N-R-acetyl boronate ester
was achieved via the trifluoroborate intermediate, formed by
reaction of the boronate esters with KHF2, followed by acid
hydrolysis.16,17
a Percentage residual activities were determined in triplicate at 100 μM.
Scheme 1. Synthesis of Boronic Acid Inhibitorsa
The computationally designed compounds 18-24, 26, and
27 (IC50 values 0.27-14 μM) are more active against R39 than
10 (IC50 = 33 μM) (Table 1). The benzothiazole ring in 19 (IC50
= 2.2 μM) is predicted to fit snugly within the smaller region A.
Consistent with this proposal, an analogue 18, with a larger
naphthyl ring, was less active (IC50 = 14 μM). The presence of
bulky groups at the ortho-position generally improves potency as
demonstrated by the results for 20-22 (SPROUT score =
-7.04, -7.27, -7.37, respectively; observed IC50 = 1.80, 1.31,
and 0.27 μM, respectively). An exception is the ortho-substituted
isopropyl derivative 25. The most potent compounds in this
series are compounds 22 and 23, with IC50 values of 0.27 and
0.50 μM, respectively.
a Conditions: (a) (-)-pinanediol, THF, RT, 95%; (b) lithium diiso-
propylamide, CH2Cl2, ZnCl2, THF, -78 ꢀC to rt, 50-80%; (c)
LiN(TMS)2, THF, -78 ꢀC to rt, 60-80%; (d) HCl/Et2O, 95%; (e)
R2COCl, 4-dimethylaminopyridine, Et3N, THF, DMF, 0 ꢀC to rt,
50-90%; (f) R2CO2H, (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate), 1-hydroxybenzotriazole, N-methyl-
morpholine, THF, DMF, 0ꢀC to rt, 50-90%; (g) KHF2, MeOH, 70-90%;
(h) chlorotrimethylsilane, MeCN, H2O, 60-90%.
A crystal structure was then obtained with 20 complexed to
R39 (PDB ID 2XK1, Figure 3, Supporting Information Figure
S1b). The mode of binding of the boronic acid with Ser49 is very
similar to that of 10 (Figure 2b), with all apparent hydrogen
bonds being conserved. The structure implies that the increased
potencies of the S-enantiomers (10, 20, and 23) compared to the
R-enantiomers (11, 27, and 26) are due to better fit of the C-R
group within a small pocket adjacent to region A. Interestingly,
and as predicted from the modeling studies, the presence of the
bulkier side chain of 20 (compared to 10) causes it to adopt a
different conformation, wherein the benzyl group occupies a
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previously unexplored region in the PBP active site can be used to
achieve potent inhibition by reversibly binding low molecular
weight boronic acids. This approach may be useful for the
development of both improved β-lactam and reversibly binding,
non-β-lactam inhibitors. Detailed structural information on PBP
and β-lactamases is now available, and a challenge will be to use
this to achieve the breadth of selectivity and potency required for
useful antibiotics.
’ ASSOCIATED CONTENT
S
Supporting Information. Chemical synthesis and com-
b
pound characterizations, inhibition assay methods, protein pur-
ification, crystallization and structure solution methods, and
SPROUT modeling methods. This material is available free of
charge via the Internet at http://pubs.acs.org.
Accession Codes
Coordinates for R39 in complexed with 10 and 20 have been
deposited with the RCSB Protein Data Bank under the codes
2XLN and 2XK1, respectively.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: Christopher.schofield@chem.ox.ac.uk. Telephone: þ44
(0) 1865 275 625 Fax: þ44(0)1865 285 022.
Funding Sources
This work was supported by the European Union (European
Community Sixth Framework Programme) via EUR-INTAFAR,
by the Fonds de la Recherche Scientifique (IISN 4.4505.00,
IISN4.4509.09), and the University of Liꢂege (Fonds spꢁeciaux,
Crꢁedit classique, 2009).
Figure 3. Views from a crystal structure of 20 (gold) bound to R39
(green) (PDB ID 2XK1). (a) The apparent hydrogen bonds for 10
(Figure 2) are conserved. The benzyl group of 20 occupies region C (in
the nitrocefin structure, this is occupied by the dihydrothiazolidine ring).
(b) Superimposition of the structures of 10 (blue) and 20 (gold). The
phenyl group of 20 and the methoxy group of 10 occupy region B. (c)
Superimposition of the nitrocefin (magenta) and 20 (gold) structures.
’ ACKNOWLEDGMENT
We thank the staff of ESRF BM30a for assistance in data
collection, R. Herman for expert work in protein crystallization,
and A. Dessen for discussion.
’ REFERENCES
space defined by residues Ser49, Glu178, Phe297, and Ser298
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(Table 1). These compounds were designed to have restricted
flexibility because of their ortho-substituents and were predicted
to have better binding affinities compared to 20 (Supporting
Information Figure S3). Indeed, following their synthesis, assays
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