HIV-1 integrase inhibitor-inspired antibacterials targeting isoprenoid biosynthesis

Article abstract of DOI:10.1021/ml300038t

We report the discovery of antibacterial leads, keto- and diketo-acids, targeting two prenyl transferases: undecaprenyl diphosphate synthase (UPPS) and dehydrosqualene synthase (CrtM). The leads were suggested by the observation that keto- and diketo-acids bind to the active site Mg²⁺/Asp domain in HIV-1 integrase, and similar domains are present in prenyl transferases. We report the X-ray crystallographic structures of one diketo-acid and one keto-acid bound to CrtM, which supports the Mg²⁺ binding hypothesis, together with the X-ray structure of one diketo-acid bound to UPPS. In all cases, the inhibitors bind to a farnesyl diphosphate substrate-binding site. Compound 45 had cell growth inhibition MIC₉₀ values of ～250-500 ng/mL against Staphylococcus aureus, 500 ng/mL against Bacillus anthracis, 4 μg/mL against Listeria monocytogenes and Enterococcus faecium, and 1 μg/mL against Streptococcus pyogenes M1 but very little activity against Escherichia coli (DH5α, K12) or human cell lines.

Full text of DOI:10.1021/ml300038t

Letter
pubs.acs.org/acsmedchemlett
HIV-1 Integrase Inhibitor-Inspired Antibacterials Targeting
Isoprenoid Biosynthesis
Yonghui Zhang,*,†,‡ Fu-Yang Lin,§,○ Kai Li,‡,○ Wei Zhu,§ Yi-Liang Liu,§ Rong Cao,‡ Ran Pang,∥
Eunhae Lee,∥ Jordan Axelson,‡ Mary Hensler,∇ Ke Wang,‡ Katie J. Molohon,⊥ Yang Wang,‡
Douglas A. Mitchell,‡,⊥,# Victor Nizet,∇ and Eric Oldfield*,‡,§
†PrenylX Research Institute, Zhangjiagang, 215600, People's Republic of China
§
∥
‡Department of Chemistry, Center for Biophysics & Computational Biology, School of Molecular and Cellular Biology,
⊥Department of Microbiology, and Institute for Genomic Biology, University of Illinois at Urbana−Champaign, Urbana, Illinois
#
61801, United States
∇Department of Pediatrics and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla,
California 92093, United States
S
* Supporting Information
ABSTRACT: We report the discovery of antibacterial leads, keto-
and diketo-acids, targeting two prenyl transferases: undecaprenyl
diphosphate synthase (UPPS) and dehydrosqualene synthase
(CrtM). The leads were suggested by the observation that keto-
and diketo-acids bind to the active site Mg2+/Asp domain in HIV-1
integrase, and similar domains are present in prenyl transferases. We
report the X-ray crystallographic structures of one diketo-acid and
one keto-acid bound to CrtM, which supports the Mg2+ binding
hypothesis, together with the X-ray structure of one diketo-acid
bound to UPPS. In all cases, the inhibitors bind to a farnesyl
diphosphate substrate-binding site. Compound 45 had cell growth inhibition MIC90 values of ∼250−500 ng/mL against
Staphylococcus aureus, 500 ng/mL against Bacillus anthracis, 4 μg/mL against Listeria monocytogenes and Enterococcus faecium, and
1 μg/mL against Streptococcus pyogenes M1 but very little activity against Escherichia coli (DH5α, K12) or human cell lines.
KEYWORDS: antibacterials, isoprenoid biosynthesis, HIV integrase, undecaprenyl diphosphate synthase, dehydrosqualene synthase
in developing phosphorus-free prenyl transferase inhibitors,
which might have even more druglike properties. For example,
Jahnke et al. reported a series of FPPS inhibitors, dicarboxylic
acids, that bound to a novel, allosteric site.9 In addition, other
species such as tetramic acid UPPS inhibitors have been
described (e.g., 4, Chart 1),10 but to date, their X-ray structures
have not been reported, although an allosteric model has been
proposed.11
A key component of the active site of most prenyl
transferases is a Mg2+/Asp motif that interacts with a substrate's
diphosphate group. We reasoned that HIV-1 integrase (IN)
inhibitors12 might provide clues for new prenyl transferase
inhibitors, since IN contains a similar Asp/Mg2+ motif13 and IN
inhibitors such as 5 (L-708,906, Chart 1)14 and 6 (elvitegravir,
Chart 1),15 diketo-acids and keto-acids, respectively, are
thought to bind at or near the Mg2+/Asp motif in the IN
active site.16,17 In addition, many other IN inhibitors like
raltegravir, dolutegravir, MK2048, etc. (structures not shown)
have been found to bind Mg2+.17−19
here is currently an urgent need for new types of
Tantibacterials exhibiting novel modes of action, due to the
rapid rise in drug resistance,1 and isoprenoid biosynthesis2,3 is
one attractive target. For example, cell wall biosynthesis can be
inhibited by targeting farnesyl diphosphate synthase (FPPS) or
undecaprenyl diphosphate synthase (UPPS), involved in lipid I
biosynthesis (Figure 1). In addition, in Staphylococcus aureus,
the formation of the virulence factor staphyloxanthin4 can be
blocked by inhibiting dehydrosqualene synthase (CrtM),
resulting in a lowering of the antioxidant shield to host derived
reactive oxygen species (ROS)5 (Figure 1). The bisphospho-
nate class of drugs such as zoledronate (1, Chart 1) are potent,
low nanomolar inhibitors of FPPS, but 1 has little antibacterial
activity (presumably due to lack of cell penetration), although
more lipophilic bisphosphonates such as 2 (BPH-210, Chart 1)
have modest activity (IC50 ∼ 30 μM) against Escherichia coli.6
More lipophilic bisphosphonates also potently target UPPS,7 as
well as CrtM,5 but again, they have essentially no activity in
bacteria. Replacing one phosphonate group by a sulfonate to
form a phosphonosulfonate results, however, in potent CrtM
inhibitors (e.g., 3, BPH-652, Chart 1, IC50 ∼ 7.9 μM, Ki ∼ 80
nM) that also blocks carotenoid pigment formation in cells
(IC50 ∼ 110 nM).8 In addition, there has recently been interest
Received: February 12, 2012
Accepted: April 3, 2012
Published: April 3, 2012
© 2012 American Chemical Society
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To see how these inhibitors bound to CrtM, we carried out
cocrystallization and soaking experiments with 7 (class I) and
41 (class II) and obtained crystals (by soaking) that diffracted
to 2.3 and 1.9 Å, respectively. Full X-ray crystallographic data
and structure refinement details are given in the Supporting
Information, Table S1. Electron density results for 7 are shown
in Figure 2a and indicate the presence of 7 in addition to one
molecule of farnesyl monophosphate (FMP) that copurified
with the protein. The identity of FMP was further confirmed by
LC-MS (Supporting Information, Figure S2) and the electron
density results (Figure 2a). The diphenyl ether fragment in 7
(cyan) binds into the CrtM S1 site23 and is shown in Figure 2b
superimposed on one of the S-thiolo-farnesyl diphosphate
(FSPP) inhibitors (in yellow, green) whose structures were
reported previously.5 This binding mode is similar to that seen
with the phosphonosulfonate 3 (Figure 2c), with the diketo-
acid headgroup interacting with two of the three Mg2+
(Mg2+B,C) seen in the CrtM-FSPP structure (Figure 2d). The
farnesyl side chain in FMP bound to the S2 site and had a 0.8 Å
rmsd from the S2 FSPP reported previously.5 With 41, the
ligand electron density is again well-defined (Figure 3a), and
the crystallographic results show that the side chain binds in S2,
similar to the farnesyl side chain in the FSPP structures (Figure
3b), as well as the phosphonoacetamide analogue of 7 (43,
BPH-830,24 Figure 3c). There are three Mg2+ in the X-ray
Figure 1. Biosynthetic reactions catalyzed by CrtM and UPPS, with
the end products of the pathways shown.
We thus made a small screening library (38 compounds) of
IN inhibitor-inspired molecules and their structures, and
inhibition of S. aureus CrtM, E. coli UPPS, and S. aureus
UPPS are shown in Figure S1 in the Supporting Information.
Most compounds were amide-diketo acids (7−40, class I,
Figure S1 in the Supporting Information) and were
conveniently prepared from the synthon (Z)-2,2-dimethyl-5-
carboxymethylene-1,3-dioxolan-4-one20 by amine coupling.
Among these compounds, 7 (Chart 1) inhibited CrtM with
IC50 ∼ 24 μM, Ki ∼ 250 nM (for comparison, Ki of 3 ∼ 70
structure. However, these are not the Mg2+
seen in most
ABC
prenyl transferases25 but rather Mg2+BCD. That is, there is a new
Mg2+ binding site, Mg2+D. The dihydropyridone side chain
interacts with Mg2+
but, surprisingly, via the two ring
CD
oxygens, not the carboxylate (Figure 3d), which interacts with
two water molecules (Supporting Information, Figure S3).
These inhibition and structural results for 7 and 41 clearly
support the Mg2+ binding hypothesis, at least for CrtM.
CrtM is a so-called head-to-head prenyl transferase so we
next sought to see if any of the molecules synthesized might
also inhibit the head-to-tail prenyl transferase FPPS or the cis-
prenyl transferase, UPPS. There was no activity against FPPS
(probably due to the lack of a positively charged feature that
mimics the carbocation involved in FPP biosynthesis), but most
of the amide-diketo acids (class I) were potent UPPS inhibitors
nM8) and blocked staphyloxanthin pigment formation (IC50
=
4 μM). Inhibitors of class II were keto-acids, dihydropyridone-
3-carboxylates, and were based on 6 (Elvitegravir) and
dihydroquinoline-3-carboxylic acid IN inhibitors,21 which
again are thought to bind via their carboxyl and carbonyl
oxygens to Mg2+/Asp.22 We made two analogues, 41 and 42
(Chart 1), with alkoxy-aryl tails to mimic the substrate farnesyl
diphosphate (FPP). The longer chain species 42 had no
activity, but the shorter chain species 41 had a CrtM IC50= 45
μM, Ki = 450 nM, and a loss of pigmentation IC50 = 33 μM.
Chart 1. Chemical Structures of Selected Compounds and the Inhibition of CrtM, E. coli UPPS, and S. aureus UPPS by 7−9, 41,
42, 44, and 45
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Figure 2. CrtM crystallographic structures. (a) Compound 7 (cyan) plus FMP (yellow) electron density, bound to CrtM. (b) Compound 7 (cyan)
bound to CrtM, superimposed on two FSPP molecules (yellow, green; PDB ID code 2ZCP). Also shown is the FMP (magenta) that cocrystallized.
The Mg2+ are from the FSPP structure. (c) Comparison between 7 (cyan) and 3 (magenta, PDB ID code 2ZCQ) bound to CrtM. Both diphenyl
ether side chains bind in S1. (d) Interactions between 7 (cyan), FMP (magenta), and Mg2+ in CrtM.
Figure 3. CrtM crystallographic structures. (a) Electron density of 41 bound to CrtM. (b) Structure of 41 (cyan) bound to CrtM shown
superimposed on two FSPP molecules (yellow, green). (c) Comparison between 41 (cyan) and the phosphonoacetamide analog of 7 (compound
43, BPH-830)24 (magenta) bound to CrtM (PDB ID code 2ZY1). (d) Interactions between 41 (cyan) and Mg2+ in CrtM.
Figure 4. UPPS crystallographic structures. (a) Electron density of 9 bound to UPPS. (b) Structure of 9 (cyan) bound to UPPS, superimposed on
FSPP/Mg2+ (from PDB ID code 1X06) and four bisphosphonate inhibitors (PDB ID code 2E98). (c) Superposition of 9 (cyan) on FSPP (yellow)
in site 1 in UPPS. The Mg2+ is from the FSPP structure. (d) The diketo-acid headgroup of 9 binds into the active site of UPPS and interacts with
D26 and N28.
with the most active one (8) having an IC50 ∼ 240 nM and Ki
∼ 120 nM, comparable to the most active bisphosphonate
UPPS inhibitor BPH-629 (IC50 ∼ 300 nM for E. coli UPPS).7
There are four different ligand-binding sites in UPPS
(designated 1−4 in ref 7) found with bisphosphonate
inhibitors. This is not unexpected since the UPPS product,
undecaprenyl diphosphate (UPP), contains 55-carbon atoms
and is thus much larger than the (C15) FPP substrate. In
principle, then, novel inhibitors might occupy multiple binding
sites.
Cocrystallization of E. coli UPPS with 9 (IC50 = 560 nM)
produced well-formed crystals with E. coli UPPS, and the
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ACS Medicinal Chemistry Letters
Letter
Funding
electron density was well resolved (Figure 4a). As can be seen
in Figure 4b, 9 binds to site 1,7 the FPP binding site, and as can
be seen in Figure 4c, 9 (in cyan) closely maps the FPP
backbone structure (in yellow) with the diketo-acid fragment
being located close to two of the three most essential residues
in UPPS, D26 and N28 (Figure 4d). We found no evidence for
the presence of Mg2+, but this observation is not entirely
unexpected since even with the five E. coli UPPS X-ray
structures with strong Mg2+ chelators, bisphosphonates (PDB
ID codes 2E98, 2E99, 2E9A, 2E9C, and 2E9D),7 Mg2+ was not
observed.
This work was supported by the U.S. Public Health Service
(NIH Grant 5R01AI074233-16 to E.O.) and the NIH
Director's New Innovator Award Program (DP2 OD008463
to D.A.M.). K.J.M. was supported in part by a NIH Cellular and
Molecular Biology Training Grant (T32 GM007283). The
Advanced Photon Source was supported by Department of
Energy Contract DE-AC02-06CH11357. The Life Science
Collaborative Access Team Sector 21 was supported by the
Michigan Economic Development Corporation and Michigan
Technology Tri-Corridor (Grant 085P000817).
The amide-diketo acids were not growth suppressive toward
S. aureus or E. coli, perhaps due to the instability of the amide
bond inside the cells or a lack of cell permeability. However, 44
and 45 (aryldiketo acids, class III) had good activity against S.
aureus UPPS (44, IC50 = 0.73 μM, Ki = 230 nM; 45, IC50 = 2.0
μM, Ki = 670 nM), and both were active against the USA300
(MRSA) strain of S. aureus with MIC90 values of 500 (44) and
250−500 ng/mL (45). There was no appreciable activity
against the Gram-negative E. coli; however, there was promising
activity against other Gram-positives: ∼ 500 ng/mL against
Bacillus anthracis str. Sterne, ∼ 4 μg/mL against Listeria
monocytogenes and Enterococcus faecium U503, and ∼1 μg/mL
for Streptococcus pyogenes M1. While the precise mechanism of
action of these compounds in each cell remains to be
determined, UPPS inhibition is a likely candidate. In addition,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Andrew H.-J. Wang of the Institute of Biological
Chemistry, Academia Sinica (Taipei, Taiwan), for providing E.
coli UPPS plasmids and S. aureus CrtM plasmids.
ABBREVIATIONS
■
CrtM, dehydrosqualene synthase; UPPS, undecaprenyl diphos-
phate synthase; FPPS, farnesyl diphosphate synthase; FPP,
farnesyl diphosphate; FMP, farnesyl monophosphate; FSPP, S-
thiolo-farnesyl diphosphate; IN, HIV-1 integrase
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ASSOCIATED CONTENT
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S
* Supporting Information
X-ray study, synthesis, and characterization of the screening
library compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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Author Contributions
○These authors contributed equally.
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