The identification of inhibitory compounds of Rickettsia prowazekii methionine aminopeptidase for antibacterial applications

Article abstract of DOI:10.1016/j.bmcl.2018.03.002

Methionine aminopeptidase (MetAP) is a dinuclear metalloprotease responsible for the cleavage of methionine initiator residues from nascent proteins. MetAP activity is necessary for bacterial proliferation and is therefore a projected novel antibacterial

Full text of DOI:10.1016/j.bmcl.2018.03.002

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The identification of inhibitory compounds of Rickettsia prowazekii
methionine aminopeptidase for antibacterial applications
Travis R. Helgren a, Elif S. Seven a, Congling Chen a, Thomas E. Edwards b,c, Bart L. Staker c,d
,
Jan Abendroth b,c, Peter J. Myler c,d, James R. Horn a, Timothy J. Hagen a,
⇑
a Department of Chemistry and Biochemistry, Northern Illinois University, 1425 W. Lincoln Hwy, DeKalb, IL 60115, USA
b Beryllium Discovery Corp., 7869 NE Day Road West, Bainbridge Island, WA 98110, USA
c Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA, USA
d Center for Infectious Disease Research, Formerly Seattle Biomedical Research Institute, 307 Westlake Avenue N., Seattle, WA 98109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Methionine aminopeptidase (MetAP) is a dinuclear metalloprotease responsible for the cleavage of
methionine initiator residues from nascent proteins. MetAP activity is necessary for bacterial prolifera-
tion and is therefore a projected novel antibacterial target. A compound library consisting of 294 mem-
bers containing metal-binding functional groups was screened against Rickettsia prowazekii MetAP to
determine potential inhibitory motifs. The compounds were first screened against the target at a concen-
tration of 10 mM and potential hits were determined to be those exhibiting greater than 50% inhibition of
enzymatic activity. These hit compounds were then rescreened against the target in 8-point dose–re-
sponse curves and 11 compounds were found to inhibit enzymatic activity with IC50 values of less than
10 mM. Finally, compounds (1–5) were docked against RpMetAP with AutoDock to determine potential
binding mechanisms and the results were compared with crystal structures deposited within the PDB.
Ó 2018 Elsevier Ltd. All rights reserved.
Received 6 October 2017
Revised 28 February 2018
Accepted 1 March 2018
Available online xxxx
The discovery of chemical therapies for the treatment of bacte-
rial infection continues to be the focus of numerous research pro-
the optimized antibacterial compounds may exhibit broad spec-
trum activity against a wide number of distinct bacterial species
in the event a universal pathway is successfully regulated, and sec-
ond, the targeted bacterial species will not have had the opportu-
nity on the evolutionary time scale to develop resistance
mechanisms to these compounds.6 Additionally, if a suitably
potent inhibitory scaffold is discovered, derivatization could afford
potent compounds tailored to target various infective agents.
grams around the world. Epidemic typhus,
a disease often
neglected by research programs, is characterized by a characteris-
tic rash that is often accompanied by sustained fever, muscle pain,
chills, and delirium. The causative agent of epidemic typhus in
humans is Rickettsia prowazekii,1,2 and transmission of R. prowaze-
kii generally occurs in crowded populations with compromised
sanitation and hygienic practices, as the parasite is transmitted
between hosts via the human body louse.3,4 Because R. prowazekii
is an obligate intracellular parasite, there are limited clinically
effective antibiotic treatments to treat rickettsioses. Additionally,
R. prowazekii strains resistant to both tetracycline and chloram-
phenicol antibiotics have been reported,5 and the identification
of novel targets for the development of anti-rickettsial therapeutics
is necessary.
Recently, methionine aminopeptidase (MetAP),
a ubiquitous
enzyme responsible for the cleavage of methionine initiatory resi-
dues from nascent proteins, has been suggested as a potential
broad spectrum antibacterial target.8 MetAP is a dinuclear metallo-
protease, with demonstrated in vitro activity when Co, Mn, Fe, Zn,
and Ni divalent cofactors are utilized.9–11 Additionally, current
inhibitory motifs demonstrate a significant correlation with cofac-
tor identity and are generally only potent against enzymes binding
specific metals.12,13 Regarding MetAP inhibition resulting in
antibacterial outcomes, inactivation of the gene encoding MetAP
in Escherichia coli14 and Salmonella typhimurium15 has demon-
strated MetAP production and function is necessary for bacterial
proliferation. Thus, the inhibition of MetAP is a proposed mecha-
nism of antibacterial activity, and compounds demonstrated to
potently bind MetAP have bactericidal activity against species in
whole cell in vitro assays.16–19 However, MetAP is present in all
To discover effective inhibitory compounds encompassing
novel chemical space and complexity while also possessing the
necessary and desirable antibiotic activity, research programs
should target pathways responsible for necessary functions of bac-
terial life and proliferation.6,7 This will lead to two outcomes: first,
⇑
Corresponding author.
E-mail address: thagen@niu.edu (T.J. Hagen).
https://doi.org/10.1016/j.bmcl.2018.03.002
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Helgren T.R., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.03.002

2
T.R. Helgren et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
eukaryotic life forms, and selective inhibition of bacterial MetAPs is
formidable. Indeed, human and bacterial isoforms have significant
conservation, with Homo sapiens and E. coli isoforms of MetAP type
1 demonstrating 47% sequence identity.20 Additionally, many of
the residues composing the substrate binding pocket are conserved
between human and bacterial MetAPs, resulting in difficulties
associated with isoform selective binding of inhibitors (Fig. 1).20
We therefore set out to determine inhibitory motifs capable
of regulating the activity MetAP from R. prowazekii. Because
R. prowazekii is an obligate intracellular pathogen, the parasite can-
not survive for extended periods outside of a host. Consequently,
screening campaigns targeting R. prowazekii must therefore be per-
formed within host cells,21 affording the bacteria an additional
resistance mechanism; the host cells must first absorb the com-
pounds, which must then be absorbed by the bacteria. For this rea-
son, R. prowazekii is resistant to a wide number of commercially
available antibiotics and few antibiotics are approved to treat this
infection.19 Thus, RpMetAP was chosen as the enzymatic target and
was expressed and purified according to our previously published
methods with slight modifications21–25 (see Supplementary Con-
tent). Additionally, the choice of the metal cofactor to be utilized
in the enzymatic activity assay is important, as inhibitory values
are dependent upon cofactor identity. Here, Mn(II) was used for
our enzyme assays as it is the suggested native cofactor in human
isoforms11 and is the cofactor present in available crystal struc-
tures of RpMetAP.21
Enzymatic activity cleaves the peptide bond of the Met-AMC sub-
strate, resulting in free AMC, which is fluorescent (excitation: 360
nm; emission: 460 nm) (Scheme 1). Because only the product of
enzymatic activity is fluorescent, an increase in fluorescence over
time is directly related to enzyme activity. The addition of inhibi-
tory compounds and the resulting decrease in fluorescence is a
measure of inhibitory activity, where activity is calculated as the
slope of a fluorescence versus time plot as compared to an uninhib-
ited control.26
The members of the compound library screened against the
RpMetAP enzyme were chosen based upon published inhibitory
scaffolds and compounds possessing functional groups capable of
binding to the metal cofactors.27 Typically, MetAP inhibitors con-
tain functional groups capable of potently coordinating the metal
cofactors found within all species of MetAP.20 These functional
groups generally include carboxylic acid, 2,20-bipyridyl, 1,2,4-tria-
zolo, thiazolo, or oxamide derivatives. A commercially available
library was therefore assembled from Otava Chemicals catalog to
include compounds possessing these (or derivatives of these) func-
tional groups. Additionally, reactive functional groups (alkyl
halides, hydrides, oxidizing agents, etc.) were intentionally
excluded from the screening library to minimize the likelihood of
false positive results.
Initially composed of 294 individual chemical species, the inhi-
bition activities were assessed using a single-point screening (10
mM compound). Compounds found to exhibit inhibition greater
than 50% were considered initial hits and were rescreened in 8-
point dose–response curves to determine IC50 values. The final
determinant for hit motifs was IC50 values <10 mM. A total of 11
With the enzyme in hand, enzymatic activity was monitored via
a fluorescence-based activity assay where methionine-amino-
methylcoumarin (Met-AMC) was utilized as the substrate.18,21
Fig. 1. Left: RpMetAP with active site residues displayed; Mn(II) cofactors are shown as purple spheres. Right: Zoom-in of the RpMetAP active site with bound Mn(II) cofactors
(PDB 3MX621). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Scheme 1.
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T.R. Helgren et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
Table 1
the corresponding 5-sulfonamidefuroic acids (1) and 5-phenylisox-
azol-3-carboxylic acids (2) have not yet been reported as possess-
ing MetAP inhibitory activity. Additionally, aryl carboxylic acids
(3–5) have not been discussed in any literature reports to date
regarding MetAP activity. Biaryl chelating inhibitors similar in
structure to (6) have been discovered and often are included in
screening libraries,36–42 although compound (6) has not been
explicitly reported. Finally, numerous 1,2,4-triazole based inhibi-
tors have been discovered,18,19,30,35,43 although essentially all are
derivatives of 5-(benzylthio)-4H-1,2,4-triazol-3-amines. It is there-
fore interesting that a few phenethanone derivatives (7–9) were
found to possess inhibitory activity against RpMetAP.
Experimental and predicted binding affinities of compounds identified as hits against
RpMetAP.
a
Cmpd Structure
IC50
Predicted Kib
(1)
0.27 0.01 0.42c
(2)
(3)
1.4 0.1
3.7 0.2
0.04c
0.07c
Of the compounds screened, the most potent was sulfonamide
(1) with an inhibitory value (IC50) of 0.27 mM. It is noteworthy that
this compound is based upon the 5-aryl-2-furoic acid scaffold that
has been extensively demonstrated to exhibit inhibition of MetAP
isoforms possessing Mn(II) cofactors. Similar aryl carboxylic
acid-based inhibitors demonstrated slightly weaker activity (2–5,
IC50 = 1.4–8.3 mM), although none of these inhibitors have been
previously reported to inhibit any MetAP isoform. We have previ-
ously identified similar 5-aryl-2-furoic acids possessing inhibitory
activity against RpMetAP, notably 5-(2-chlorophenyl)-2-furoic acid
(IC50 = 0.6 mM).21 Interestingly, 2-(pyridin-2-yl)thiazole (6) also
demonstrated moderate activity (IC50 = 1.1 mM), although similar
aryl chelating MetAP inhibitors only have demonstrated activity
against MetAP isoforms utilizing Co(II) cofactors.20 Finally, 1,2,4-
triazole based inhibitors made up half of the hit compounds iden-
tified through this screening campaign. Of these, the most potent
inhibitor was found to be derivative (7), (IC50 = 0.40 mM), and the
other identified inhibitors (8–11) exhibited moderate activity
(IC50 = 1.2–10 mM). These inhibitory activities are noteworthy, as
compounds of this general scaffold typically exhibit the most
potent activity against bacterial MetAP isoforms utilizing Co(II)
cofactors, although these specific compounds have never been
identified as MetAP inhibitors.20
(4)
(5)
3.1 0.3
0.21c
8.3 0.9
1.1 0.1
0.03c
21.8d
(6)
(7)
0.40 0.07 0.37d
(8)
(9)
1.2 0.1
0.06d
4.35d
To gain additional insights into the binding mechanisms and
corresponding activities of the hit compounds, each was docked
into the active site of RpMetAP using the open source docking soft-
ware AutoDock.44 Co-crystal structures of bacterial MetAPs con-
taining bound inhibitors based upon similar motifs (PDB: 1XNZ,29
10.0 1.5
1YVM,45 and 3IU919
) were first examined to determine the
(10)
(11)
9.4 0.7
5.0 0.6
3.98d
1.24d
expected binding mechanism of compounds (1–11). Briefly, furoic
acid based inhibitors are expected to chelate one of the metal
cofactors through the acid O atoms, 1,2,4-triazole based inhibitors
are expected to coordinate to both metal cofactors through the tri-
azole 1 and 2 N atoms, and biaryl chelating inhibitors (such as (6))
are expected to coordinate to a third metal present in the active
site of all crystal structures containing compounds of this class,
even for H. sapiens MetAP isoforms (see PDB: 1YVM,45 2G6P,46
4
a
IC50 values are expressed in units of mM; Mn(II) cofactors, measured in
triplicate.
IU6,41 4HXX,42 4IKR,39 4IKS,39 and 4IKT39).
Ki values represent those predicted by docking software (AutoDock)44 and are
b
With this information, the compounds were docked against the
RpMetAP target (PDB: 3MX621) to determine whether the pre-
dicted binding modes mirrored those observed experimentally.
For compounds similar in structure to furoic acid based inhibitors
(1–5), all docked structures predicted the inhibitors bind to the
metal cofactors through the acid O atoms (Fig. 2). This result was
not unexpected, as published crystal structures (PDB: 1XNZ,29
expressed in units of mM.
Ki value for deprotonated carboxylic acid; protonated species were predicted to
bind in the same manner with only a slight reduction in predicted binding affinity.
No output docking poses correlated well with published crystal structures
including inhibitors of similar docking pose. Structure. The Ki values therefore
represent those for the predicted lowest energy docking pose.
c
d
2EVM,28 2Q92,47 2Q93,47 2Q94,47 2Q95,47 2Q9647 and 3IU719
)
compounds met this cut-off (Table 1). The compounds all possess
metal binding functional groups (carboxylic acids, nitrogenous
heterocycles) and are thus similar to hit motifs already discussed
in the literature20 (ex: furoic acid and 1,2,4-triazoles), although
none have been previously described as inhibitors of MetAP. For
example, 5-phenyl-2-furoic acid-based inhibitors have been exten-
sively described as inhibitors of bacterial MetAPs,13,16–19,28–35 but
demonstrate this binding mechanism. It is noteworthy, however,
that sulfonamide (1) was predicted to coordinate the metal cofac-
tors through the carboxylate, as this compound could conceivably
bind via the sulfonamide functionality. Additionally, no specific
interactions between the inhibitor and the protein target were
predicted to suggest a drastic difference in activity compared to
the other compounds of this series (2–5). This may explain why
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4
T.R. Helgren et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 2. Comparison of the known binding mechanism of furoic acid-based inhibitors against EcMetAP (left, PDB: 1XNZ29) and the predicted docking pose of sulfonamide (1)
against RpMetAP (right). Compound (1) was predicted to bind via chelation to the Mn(II) cofactors through the acid functionality which mirrors the experimentally
determined binding mechanism depicted for EcMetAP containing 5-(2-chlorophenyl)-2-furoic acid.
(1) was predicted to possess the least potent activity against RpMe-
tAP as compared to the other derivatives (2–5), although the com-
pound was the most potent at inhibiting enzymatic activity.
Sulfonamide (1) is a good starting point for further exploration,
as both the acid and sulfonamide functionalities can be easily mod-
ified to determine the effect of both on the inhibitory activity
against RpMetAP. Additionally, 5-sulfonamide-2-furoic acids are
novel inhibitors of MetAP and appear to exhibit superior activity
to the corresponding 5-aryl-2-furoic acid based inhibitors, which
are generally the most potent inhibitors of bacterial MetAPs.
Unfortunately, the docking method was unable to predict docking
poses for compounds (6–11) that closely mirror crystal structures
from MetAP species containing similar inhibitors.
Acknowledgements
This project has been funded in part with Federal funds from the
National Institute of Allergy and Infectious Diseases, National Insti-
tutes of Health, Department of Health and Human Services, under
Contract Nos. HHSN272200700057C and HHSN272201200025C.
Molecular graphics were generated with the UCSF Chimera
package. Chimera is developed by the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San
Francisco (supported by NIGMS P41-GM103311).
A. Supplementary data
Through the testing of a screening library composed of a limited
number of compounds (294 members), we have identified 11 com-
pounds found to inhibit MetAP expressed from R. prowazekii with
IC50 values less than 10 mM. The compounds fit into three distinct
classes of inhibitors for bacterial MetAP species, and none of the
specific compounds identified herein have been previously
reported as inhibitors of this enzyme class. To discern the potential
binding interactions that lead to potent inhibition of MetAP
activity, the compounds were docked with the currently available
crystal structure of RpMetAP (PDB: 3MX621). The docking
output for some compounds (1–5) suggested similar binding
modes to those of currently available crystal structures containing
bound inhibitors of similar composition; however, the remaining
compounds (6–11) were predicted to bind in orientations not
currently revealed by crystal structures of MetAP species contain-
ing similar compounds. With the continual emergence of bacterial
species resistant to currently available therapeutics, the discovery
of a class of antibiotics targeting new pathways is paramount.
The regulation of MetAP is therefore attractive as the enzymatic
activity has been demonstrated as essential for bacterial prolifera-
tion and remains relatively unexplored for this purpose. As novel
inhibitory compounds are identified, MetAP regulation may prove
to be a viable method for the mitigation of bacterial infection, and
future efforts should focus on both the discovery of additional
inhibitory motifs as well as the exploration of the motifs reported
herein.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.03.002.
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