Macrocyclic Peptides that Selectively Inhibit the Mycobacterium tuberculosis Proteasome

Article abstract of DOI:10.1021/acs.jmedchem.1c00296

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

Full text of DOI:10.1021/acs.jmedchem.1c00296

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Article
Macrocyclic Peptides that Selectively Inhibit the Mycobacterium
tuberculosis Proteasome
Hao Zhang, Hao-Chi Hsu, Shoshanna C. Kahne, Ryoma Hara, Wenhu Zhan, Xiuju Jiang,
Kristin Burns-Huang, Tierra Ouellette, Toshihiro Imaeda, Rei Okamoto, Masanori Kawasaki,
Mayako Michino, Tzu-Tshin Wong, Akinori Toita, Takafumi Yukawa, Francesca Moraca,
Jeremie Vendome, Priya Saha, Kenjiro Sato, Kazuyoshi Aso, John Ginn, Peter T. Meinke, Michael Foley,
Carl F. Nathan, K. Heran Darwin, Huilin Li, and Gang Lin*
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ABSTRACT: Treatment of tuberculosis (TB) currently takes at least
6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically
tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill
nonreplicating (NR) Mtb may shorten treatment when used in
combination with conventional drugs. The Mtb proteasome (Mtb20S)
could be such a target because its pharmacological inhibition kills NR
Mtb and its genetic deletion renders Mtb unable to persist in mice.
Here, we report a series of macrocyclic peptides that potently and
selectively target the Mtb20S over human proteasomes, including
macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S
revealed structural bases for the species selectivity. Inhibition of 20S
within Mtb by 6 dose dependently led to the accumulation of Pup-
tagged GFP that is degradable but resistant to depupylation and death
of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as
anti-TB therapeutics.
chronic phase of disease in mice.2,3,14 We have introduced
several classes of Mtb proteasome inhibitors that kill NO-
stressed or starved Mtb in vitro. These included the first
INTRODUCTION
■
Host immune stresses can drive Mycobacterium tuberculosis
(Mtb) into a state of nonreplication, in which Mtb becomes
inhibitors selective (>1000-fold) for the proteasome of a
phenotypically resistant to many of the drugs used to treat
pathogen and not its host and the first agents to kill bacteria
tuberculosis (TB).1−4 Drugs that kill nonreplicating persisters
by inhibiting protein breakdown rather than protein syn-
are anticipated to shorten the duration of TB treatment.5 Mtb
thesis.2,15,16 We recently discovered a novel class of phenyl-
relies on specific enzymatic pathways to survive the host stresses
that suppress its replication, including the generation of nitric
imidazole-based Mtb20S selective inhibitors.17 However, they
were inactive against Mtb H37Rv under the conditions tested,
oxide (NO) and reactive nitrogen intermediates derived from it
(herein collectively called “NO” for simplicity).6 Among these
which may be attributed to their poor uptake and/or
intracellular biotransformation resulting in insufficient intra-
defenses is the prokaryotic ubiquitin-like protein (Pup)−
proteasome system (Figure 1).2,7−13 Different from human
20S proteasome (hu20S) that contains two copies of seven
distinct α- and seven distinct β-subunits among which only three
β subunits (β1, β2, and β5) are enzymatically active, the Mtb20S
cellular concentrations.
Macrocyclization is a structural optimization strategy that has
been successfully applied in drug research and development.18 It
can potentially improve potency, selectivity, and physicochem-
ical properties through the restriction of conformation.19
proteasome (Mtb20S) has 14 copies each of α and β subunits.
Structures of proteasome are highly conserved in a four-layered,
barrel-shaped complex in the α1−7β1−7β1−7α1−7 fashion.
Although the Mtb20S is dispensable under standard growth
Received: February 15, 2021
Published: May 5, 2021
conditions in vitro, it is indispensable for Mtb to survive in the
host, and it has been validated as a drug target both genetically
and pharmacologically. A null mutant strain lacking prcBA genes
that, respectively, encode the β and α subunits dies in the
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© 2021 American Chemical Society
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Figure 1. Schematic representation of the Pup−proteasome system (PPS) in M. tuberculosis. Pup is expressed with a C-terminal glutamine (Pup-Gln),
which is deamidated to glutamate (Pup-Glu) by Dop. PafA, a Pup ligase, then catalyzes the formation of an isopeptide bond between the C-terminal γ-
carboxylate of Pup-Glu and the lysine ε-amino group of a protein substrate. The pupylated protein is recognized and unfolded by Mpa and degraded by
the Mtb20S proteasome. Alternatively, Dop can rescue the pupylated substrate by cleaving the specific isopeptide bond. Inhibition of the Mtb
proteasome under NO stress (mimicking a host stress that can render Mtb nonreplicating) results in cell death.
Figure 2. Rationale for introducing a tether between P2 and P4 in the DPLG-2 scaffold. (A) X-ray crystal structure of acyclic N,C-capped dipeptide
DPLG-2 in Mtb20SOG (PDB code 5TRG). DPLG-2 is shown in magenta. Hydrogen bonds are indicated by dashed yellow lines. The red arrow
depicts the potential for macrocyclization between the P2 and P4 groups. (B) Our initial macrocyclic strategy for the design of novel Mtb20S selective
inhibitors. The P2 and P4 groups are linked with the conformationally flexible alkyl or ether linkers.
Macrocycle-based inhibitors for eukaryotic proteasomes have
been reported. For example, the natural tripeptide macrocycle
macrocyclic peptide inhibitor of the malaria proteasome was
reported to possess potent antimalarial activity,24,25 and a series
of P1−P3 macrocyclic peptide aldehydes were reported to have
TMZ-95a and its analogs have been extensively studied.20−23
A
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a
Table 1. IC50 Values of Macrocyclic Peptides against Mtb20SOG and β5c and β5i of the Human c-20S and i-20S, Respectively
c
proteasome inhibition IC50 (μM)
SIapp
IC
50−β5c
IC
50−β5i
macrocycle
R
n
X
Mtb20S
β5c
β5i
IC
IC
50−Mtb20S
50−Mtb20S
b
DPLG-2
0.015
70.0
54.7
4670
3650
1
2
3
4
benzyl
H
H
1
1
0
1
CH2
CH2
CH2
O
5.0 0.8
0.15 0.02
0.71 0.07
>100
>100
>100
>100
>100
>100
>100
>100
>200
>674
>141
>984
>200
>674
>141
>984
H
0.10 0.01
a
b
Data shown are the mean of at least three independent experiments. Data quoted from ref 16 for comparison. LLVY: suc-LLVY-AMC, used for
c
all three proteasomes. SIapp: apparent selectivity index. SIapp values were rounded to three or fewer significant figures.
anticancer activity.26 These results suggest the potential to
cyclize either P1−P3 or P2−P4 of peptide-based proteasome
inhibitors to tune them for a specific activity. Here, we describe
our efforts to use this strategy to improve the activity and
stability of a series of selective and potent Mtb proteasome
inhibitors.
Structure−Activity Relationship (SAR) Studies. Our
initial study of macrocyclic Mtb proteasome inhibitors focused
on 15- and 16-membered alkyl- and ether-tethered macrocycles
(Table 1). Considering the synthetic convenience, the 16-
membered macrocycle 1 was synthesized as the racemic form
and showed a significant loss in potency against Mtb20S
compared with acyclic DPLG-2. Truncating the P5 benzyl
substituent of macrocycle 1 gave the 16-membered macrocycle
2, which improved the potency by 33-fold. The 15-membered
macrocycle 3 was 5-fold less potent than macrocycle 2,
suggesting that 16-membered ring is preferable. The potency
of the 16-membered ether-linked macrocycle 4 was slightly
improved over that of the corresponding alkyl-linked macro-
cycle 2.
RESULTS AND DISCUSSION
■
Rationale. The cocrystal structure of the acyclic N,C-capped
dipeptide DPLG-2 and other N,C-capped dipeptides16,27 bound
to the Mtb20S open gate, activated form (Mtb20SOG) shows
that the ligand is occupying the nonprime S1, S2, S3, and S4
binding pockets (Figure 2A). In the case of DPLG-2, the P1
naphthyl moiety inserts into the S1 binding pocket harboring the
proteolytic residue Thr-1. The P2 p-F-benzyl group fills the
shallow S2 pocket and has no interactions with the proteasome.
The P3 N,N-diethyl Asn moiety occupies the S3 pocket, forming
hydrogen bonds with Gln-22 and Ser-27 in addition to binding
to the hydrophobic pocket via van der Waals interaction. The N-
cap 3-phenylpropanoyl group occupies the S4 pocket, forming
additional hydrophobic interactions with residues of neighbor-
ing subunits. Also, the peptide backbone forms several hydrogen
bonds with substrate-binding cleft of Mtb20S. The close spatial
proximity of the P2 and P4 substituents led us to hypothesize
that it may be feasible to link them covalently into a macrocyclic
ring, which may reduce the conformational freedom and
increase the compounds’ potency. Such a macrocyclic strategy
would also allow exploration of the region between P2 and P4
pockets.
Inspired by the recently reported CP124 and our N,C-capped
dipeptide DPLG-2, we next sought to explore biphenyl ether-
linked macrocycles. CP1 was developed to selectively target the
Plasmodium falciparum proteasome (Pf20S) over human
proteasomes. CP1 shares the same backbone with N,C-capped
dipeptides but adds a 3,3′-biphenyl ether tether that links P2 and
P4. We found that CP1 did not inhibit Mtb20S (Table 2), likely
because P3 homo-phenylalanine does not fit well in the shallow
but wide S3 pocket. Our previous study showed that both 2-
fluorobenzyl and naphthyl groups are well tolerated in the S1
pocket of Mtb20S.27 We chose 2-fluorobenzyl as P1 because of
its lower molecular weight28 and better pharmacokinetic
properties than naphthyl. We evaluated several macrocycle 5
analogues with 3,3′-, 3,4′-, or 4,3′-biphenyl ether tethers by
computational docking in the Mtb20SOG (open gate) cocrystal
̈
structure (PDB code 5TS0) using Schrodinger’s Glide
program.29,30 Macrocycle 5 with a 3,4′-biphenyl ether tether
had the highest docking score. The overlay of macrocycle 5 and
the parent N,C-capped dipeptide in the binding pocket
indicated a similar binding pose (Figure 3A). The peptide
backbone of the macrocycle forms four hydrogen bonds with
residues Thr-21, Gly-47, Ala-49, and Asp-124 of the adjacent β-
subunit. The P3 pyrrolidine Asn group forms two hydrogen
bonds with residues Gln-22 and Ser-27. Macrocycle 5 had a sub-
μM IC50 against Mtb20S and little inhibitory activity toward the
human constitutive proteasome (c-20S) or the human
immunoproteasome (i-20S) (Table 2).
As shown in Figure 2B, our initial macrocyclic strategy
involved removing the P2 4-fluorophenyl and connecting the P2
to the α-position of the carboxyl group of P4′s 3-phenyl-
propanoyl group via an alkyl or ether linker of varied length. The
proposed linker would provide conformational flexibility and be
suitable for verifying our strategy. A series of macrocycle 1
analogues ranging from 14- to 17-membered rings were
evaluated with molecular docking using the Mtb20SOG
cocrystal structure (PDB: 5TS0) as the docking template.27
Cyclic peptides with a 15- or 16-membered ring showed good
overlap with the parental N,C-capped dipeptide (Figure S1).
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a
Table 2. IC50 of Macrocyclic Peptides against Mtb20SOG and β5c and β5i of the Human c-20S and i-20S
a
Data are the mean of at least three independent experiments. LLVY: suc-LLVY-AMC, used for all three proteasomes. SIapp: apparent selectivity
b
index. SI values were rounded to three or fewer significant figures.
̈
Figure 3. Molecular docking of macrocycle 5 to the cocrystal structure of Mtb20SOG (PDB 5TS0) executed with Schrodinger’s Glide. (A) Overlay of
macrocycle 5 (yellow) and parent N,C-capped dipeptide PKS-2208 suggests that the two have similar binding modes. Hydrogen bonds are indicated
by dashed yellow lines. (B) Molecular surface representation of the Mtb20SOG in complex with macrocycle 5. The binding pocket is depicted by a
dark gray surface.
We next focused on modifying Asn P3 to further improve
potency against Mtb20S based on macrocycle 5 (IC50 = 0.2
μM). As shown in Figure 3B, molecular docking analysis of
macrocycle 5 revealed that the α-hydrogen atom of P3
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Table 3. Physicochemical Properties and In Vitro Safety Profile of Macrocycles 6, 9, and 10
PAMPA permeability
(nm/s)
solubility pH 6.8
stability in human plasma viability of HepG2 at 30 μM
microsome stability CL
e int
a
b
c
macrocycle
(μg/mL)
(%)
(%)
(μL/min/mg) h/m
6
9
10
227
260
206
1.4
94
86
97
98
91
102
>768/>768
>768/>768
>768/>768
d
33
a
b
c
Kinetic solubility. Plasma stability was determined as the percentage remaining after incubation for 2 h with fresh human plasma. HepG2 cells
d
e
were treated with 30 μM test compounds for 72 h and viability was assessed. Below the level of detection. Stability of the macrocycles in human
(h) and mouse (m) liver microsomes. CLint: intrinsic clearance.
Figure 4. Inhibition of Mtb20S by macrocycle 6. (A) Structure of macrocycle 6. (B) Time-dependent inhibition of Mtb20S by macrocycle 6 at
indicated concentrations. (C) Plot of kobs vs concentration of macrocycle 6 yielded k3 of 5.7 × 104 s−1 M−1 and k4 of 0.7 × 10−3 s−1.
pyrrolidine points to a large S3 subpocket, which is lined by
residues Ala-49 and Val-31. We hypothesized that incorporation
of a hydrophobic substituent to the α-position of pyrrolidine
might provide valuable interactions with the S3 subpocket.
Remarkably, incorporation of a bulky 2-phenyl substituent on
the pyrrolidinyl ring of macrocycle 5 led to macrocycle 6
(TDI5575), which showed a 27-fold increase in potency against
Mtb20S plus about a 5-fold reduced inhibition of human i-20S.
The result was a >13 600-fold selectivity for Mtb20S over both
human c-20S and human i-20S. We then extended the P5 amides
CH3CONH or PhCONH on macrocycle 6, giving macrocycles
7 and 8, which inhibited Mtb20S with IC50 values of 0.17 and
0.84 μM, respectively, which were 23 and 114 times less potent
than macrocycle 6. Structural analysis of the macrocycle 6-
Mtb20SOG complex indicated that the space available for
accommodating the P5 group in the active site was only 3.8 Å
(Figure S2A). This suggested that P5 amides in the context of a
rigid macrocyclic peptide were not well tolerated at the S5
pocket of Mtb20S.
The 16-membered alkyl ether-tethered macrocycle was
identified as a promising scaffold in our initial SAR studies.
We then synthesized a macrocycle 4 analogue, macrocycle 9,
with 2-fluorobenzyl as P1, and 2-phenylpyrrolidinyl-Asn amide
as P3. Macrocycle 9 had improved potency (Mtb20S IC50 = 7.1
nM) and maintained selectivity over human c-20S (hu-β5c IC50
> 100 μM) and human i-20S (hu-β5i IC50 > 100 μM) compared
with macrocycle 4. Despite these favorable characteristics, both
macrocycles 9 and 6 had extremely poor solubility (1.4 μg/mL
and below the level of detection, respectively) (Table 3). To
overcome the solubility issue, we further altered the tether by
replacing the biphenyl ether in macrocycle 6 with a monophenyl
ether, giving macrocycle 10, which showed improved solubility
phenylpyrrolidinyl-Asn. Macrocycle 11 showed comparable
potency to macrocycle 10 against Mtb20S (IC50 = 6.5 nM), but
replacing the phenyl with the cyclopropyl markedly reduced the
selectivity between the Mtb and human proteasomes.
Compared with macrocycle 11, the inhibitory activity of
macrocycle 12 was reduced for both Mtb20S and human
proteasomes by 11−20-fold. These results indicate that the (R)
conformation at the 2-position of pyrrolidinyl is generally
preferred over (S) by proteasomes, and the bulky phenyl group
is responsible for species selectivity for Mtb20S over human c-
20S and i-20S.
We also investigated whether asparagine amide could be
removed without affecting the binding affinity. We synthesized
macrocycle 13 by replacing the CO of P3 asparagine amide in
macrocycle 10 with a CH2. The removal of this H-bond receptor
reduced the potency against Mtb20S by 219-fold, suggesting
that the carbonyl oxygen of the P3 amide is crucial for
proteasome inhibition potency.
We further assessed the inhibitory activity of macrocycles 1−
12 against β1c or β2c subunits of the human c-20S or β1i or β2i
of the human i-20S subunits (Table S1). Except macrocycle 11
exhibiting moderate inhibition against β2i (IC50 44.5 μM), all
compounds showed <50% inhibition even at 100 μM.
Therefore, this series of macrocyclic peptides are highly
species-selective for Mtb20S over all active subunits of both
human c-20S and i-20S.
Inhibition Kinetics. By following the time course of the
peptide LLVY hydrolysis activity of Mtb20S in the presence of
macrocycle 6 (Figure 4A), we found that the macrocyclic
peptides were time-dependent inhibitors (Figure 4B). The plot
of kobs values derived from each time-dependent curve vs
macrocycle 6 yielded a k3 of 5.7 × 104 s−1 M−1 and k4 of 0.7 ×
10−3 s−1, resulting in a ki of 12.1 nM and t1/2 of 16.5 min for the
Mtb20S-inhibitor complex (Figure 4C).
(33 μg/mL) while maintaining high potency (Mtb20S IC50
=
7.0 nM) for Mtb20S and selectivity over human c-20S (>14 400-
fold) and i-20S (>14 400-fold).
To investigate the contributions of P3 side-chain 2-phenyl-
pyrrolidinyl-Asn to potency and selectivity, we synthesized
macrocycles 11 and 12, in which 2-(R)-cyclproylpyrrolidinyl-
Asn or 2-(S)-cyclproylpyrrolidinyl-Asn, respectively, replaced 2-
Crystal Structure of Macrocycle 6 (TDI5575) in
Complex with Mtb20SOG. To understand the binding
modes of macrocyclic dipeptide inhibitors, macrocycle 6 was
soaked into crystals of Mtb20SOG. The inhibitor-soaked
crystals diffracted X-rays to 2.28 Å (Table S2). Molecular
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Figure 5. Binding mode of macrocycle 6 in the Mtb20SOG active site. (A) 2mFo-DFc electron density map is rendered at 1σ threshold and shown as
gray meshes. Macrocycle 6 is in yellow and citrate from the solvent is in magenta sticks. The two neighboring β-subunits are in green and cyan,
respectively, and the catalytic residue Thr-1 is in red. (B, C) Top and a side view of the Mtb20S structure, highlighting 14 macrocycle 6 molecules in the
active sites of the 14 catalytic β-subunits. (D) Surface view of two neighboring β-subunits (green and cyan) as viewed from the interior chamber,
demonstrating that the inhibitor in the substrate pocket of the green subunit also contacts the cyan subunit. (E) Close-up view of the inhibitor-bound
active site. (F) P1 and P3 are inserted into the S1 and S3 binding pockets, respectively. (G) Binding mode of macrocycle 6 in Mtb20SOG. Hydrogen
bonds between macrocycle 6 and the β-subunits are shown as dashed orange lines. The two neighboring β-subunits are in green and cyan; water
molecules are shown as magenta spheres; and citrate is in gray sticks.
replacement was used to solve the inhibitor-bound Mtb20SOG
structure using the available DPLG-2-bound Mtb20S structure
(PDB ID 5TRG) as the search model. After refinement, both the
Fo-Fc and 2Fo-Fc 3D maps had well-defined inhibitor density
for modeling macrocycle 6 (Figure 5A). Each of the 14 β-
subunits of proteasome was occupied by one molecule of
macrocycle 6, consistent with all β-subunits being active in
Mtb20S (Figure 5B,C). Like the reported N,C-capped
dipeptides27 and phenylimidazole-based inhibitors,17 macro-
cycle 6 binds noncovalently to the Mtb20S active site and does
not significantly alter the active site conformation, as compared
with the apo Mtb20S structure (PDB ID 3HFA).
The substrate specificity of a proteasome is mainly
determined by the S1 and S3 binding pockets formed between
two adjacent β-subunits. The P1 2-fluorobenzyl group and P3 2-
phenylpyrrolindyl group are inserted into S1 and S3 pockets,
respectively, with the 3,4′-tethered P2 and P4 phenyl groups
facing the corresponding S2 and S4 sites (Figure 5D−F). No
marked conformational changes were observed in the P1 and P3
pockets upon inhibitor binding, indicating that no conforma-
tional change in the Mtb20S catalytic pocket is required for the
binding of the macrocyclic dipeptide inhibitor. Macrocycle 6
binds to Mtb20S by forming a short antiparallel β-strand with
the β-strands B3 and B6 of the β-subunits. The dipeptide
backbone of the inhibitor forms hydrogen bonds with Thr-21 of
B3, Gly-47, and Ala-49 of B6 and Asp-124 of the adjacent β-
subunit (Figure 5G). The Mtb-specific residue Gln-22, along
with Ser-27 (which is conserved in β5i), contributes an extra
hydrogen bond with the carbonyl group of P3 2-phenyl-
pyrrolidinyl-Asn, explaining the reduced inhibition activity of
macrocycle 13. Macrocycle 6 does not interact directly with the
catalytic Thr-1. However, a citrate molecule from the
crystallization buffer was observed to interact with the OG1 of
Thr-1 through a hydrogen bond. Two water molecules help to
stabilize the inhibitor in the binding site: one water molecule is
coordinated by Thr-21, Ala-180, the citrate, and the C-terminal
carbonyl group of the dipeptide backbone. The other water
molecule is coordinated by Ala-50, Asp-124 of the neighboring
β-subunit, and the first carbonyl group of the dipeptide
backbone. There is no hydrogen bonding between the
connected P2−P4 groups and the neighboring S2 and S4 sites.
To compare the binding modes of the 2-fluorobenzyl and
naphthyl groups, we aligned the β-subunits of macrocycle 6-
Mtb20SOG and DPLG-2-Mtb20SOG. The 2-fluorobenzyl
group in macrocycle 6 and the naphthyl group in DPLG-2
had very similar binding modes in the S1 pockets (Figure S2B).
In the P3 groups, incorporation of a 2-phenyl group (macro-
cycles 6, 9, and 10) or a 2-cyclopropyl group (11 and 12) on the
pyrrolidinyl ring greatly increased the inhibitory potency against
Mtb20S. The aligned macrocycle 6 and DPLG-2 showed similar
binding modes of P3. The phenyl group of P3 in macrocycle 6
points toward the S1 site and the naphthyl group of P1 in
DPLG-2 faces toward the S3 site. Thus, the bulky P3 group 2-
phenylpyrrolindyl cannot coexist with the bulky naphthyl P1
group in the same inhibitor. From the activity and structural
analyses, we observed the preference for bulky hydrophobic
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Figure 6. Macrocyclic inhibitors engage the intracellular target, and macrocycle 6 is mycobactericidal in the presence of nitric oxide. (A) Mtb strain
MHD1441 (WT H37Rv producing Pup-GFP) is treated with macrocycles 6, 9, or 10 at the designated concentrations. Optical density at 580 nm and
relative fluorescence units (RFU; at 495 nm excitation, 510 nm emission) are measured on day 4 post treatment. RFU values are normalized by optical
density and fold changes are calculated by normalizing to the value of dimethyl sulfoxide (DMSO)-treated MHD1441. Data shown are representative
of at least three independent experiments. (B) Mtb strains (Pup-Glu and dop−/−) are treated with macrocycle 6 at the indicated concentrations.
Carfilzomib (CFZ) at 50 μM is used as a positive control. Pupylated proteins are probed with anti-Pup antibody and anti-PrcB is used as a loading
control. (C) Killing of Mtb Erdman in Sauton’s medium with 0.5 mM NaNO2 at pH 5.5, which generates NO.
groups in the S1−S3 tunnel (Figure 5F). Although the P2 and
P4 groups were linked to a cyclic form in this study, the binding
mode of P2−P4 in macrocycle 6 still resembles that of DPLG-2,
i.e., the P2 and P4 project into the solvent and only partially
interact with the S2 and S4 sites. As such, changing the linkers of
P2 and P4 did not significantly change the inhibitory potency, as
seen in macrocycles 6, 9, and 10.
Macrocycle 6 Induces Accumulation of Pupylated
Proteins and Kills Mtb under Nitrosative Stress. Host
macrophages produce nitric oxide and other reactive nitrogen
intermediates to control infection by M. tuberculosis. The Mtb
proteasome serves as a defense for the pathogen against the
nitrosative stress.2 We tested macrocycle 6 against non-
replicating Mtb under nitrosative stress, as reported.16,17 We
found that macrocycle 6 showed marked, dose-dependent
mycobactericidal activity (Figure 6C). To investigate whether
the bacterial killing was due to inhibition of intracellular Mtb20S
by macrocycle 6, we assayed the pupylome level in an Mtb strain
in which proteins cannot be depupylated (CDC1551
dop::MycoMarT7 expressing pupGGE).33 This strain does not
have the depupylase Dop but produces Pup-GGE, a C-terminal
Pup variant that does not require Dop-mediated deamidation
prior to substrate ligation by PafA.33 Without depupylation
activity, the pupylome will only increase when Mtb20S core
particles are inhibited. We treated the strain with macrocycle 6 at
1, 3, or 10 μM for 48 h. As shown in Figure 6B, macrocycle 6
dose dependently promoted the accumulation of pupylated
proteins, suggesting that macrocycle 6 penetrated Mtb and
inhibited Mtb20S. Carfilzomib (CFZ), a proteasome inhibitor
approved for the treatment of multiple myeloma, also led to the
accumulation of pupylated proteins, at a concentration of 50
μM.
Accumulation of Pup-GFP, a Novel Intracellular
Reporter, by Macrocyclic Mtb20S Inhibitors. One major
obstacle to studying Mtb20S inhibition in Mtb is the dual
function of Dop in the Pup−proteasome system.31,32 Dop
deamidates the C-terminal glutamine of Pup to convert it to
glutamate, which is required for pupylation to take place.
However, Dop also removes Pup from pupylated proteins.31,32
To test whether macrocycle 6 and other macrocyclic peptides
can penetrate Mtb and engage the intrabacterial target, we used
the Mtb strain MHD1441 (H37Rv WT + pSCK1). This strain
produces Pup-GFP, in which Pup is linearly fused to a green
fluorescent protein (GFP) at its C-terminus. Pup-GFP is
therefore constitutively pupylated independently of Dop
deamidation activity and cannot be depupylated by Dop
because it is linked to GFP by a peptide bond. Therefore, the
inhibition of Mtb20S core particles in this Mtb strain causes
Pup-GFP accumulation, which can be quantified by fluorescence
(Ex495 nm/Em510 nm). Using this novel intracellular reporter
strain, we screened 136 in-house synthesized macrocycles and
identified 10 macrocycles whose addition to Mtb cultures
resulted in the accumulation of Pup-GFP (Figure S3).
Macrocycles 6, 9, and 10 further showed a dose-dependent
increase of RFU (Figure 6A), strongly suggesting that these
compounds penetrated the Mtb cell wall to inhibit intracellular
proteasomal activity.
Macrocycles are Stable in Plasma and Well Tolerated
by Human Cells. The passive permeability of selected
macrocycles (macrocycles 6, 9, and 10) was assessed using the
parallel artificial membrane permeability assay (PAMPA). All
three compounds showed high passive permeability (Table 3).
Replacing the biphenyl ether of macrocycle 6 with the
monophenyl tether of macrocycle 10 decreased the hydro-
phobicity (cLogP) from 6.0 to 4.9, which translated into
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dramatically improved solubility, while maintaining potency
against Mtb20S. All three macrocycles were stable in human
blood plasma, with ≥86% of the parent remaining intact after 2 h
incubation at 37 °C. We found no appreciable cytotoxicity
toward human liver carcinoma cells (HepG2) for macrocycles 6,
9, and 10 at 30 μM, indicating a promising safety profile of this
series of Mtb-selective proteasome inhibitors. However, macro-
cycles 6, 9, and 10 were evaluated in microsome stability assays,
which showed that all three compounds were rapidly
metabolized by both human and mouse liver microsome
(CLint > 768 μL/min/mg).
EXPERIMENTAL SECTION
■
The fluorogenic substrates Suc-LLVY-AMC, Z-LLE-AMC, and Z-
VLR-AMC were from Boston Biochem (Cambridge, MA). Human
immunoproteasomes (i-20S, isolated from peripheral blood mono-
nuclear cells), human constitutive proteasomes (c-20S, isolated from
red blood cells), and recombinant human PA28 activator α subunit
(PA28α) were from Boston Biochem Inc. Mtb20SOG was expressed
and purified as reported.27 Mouse and human liver microsomes were
purchased from Sekisui XenoTech, LLC (Kansas City, KS). The purity
of the compounds was determined on a Waters Acquity UPLC/MS
system equipped with a C18 column (100 × 2.1 mm2, 1.7 μm), coupled
with a PDA detector. The purity of all final compounds was >95%
except macrocycle 13. The purity of macrocycle 13 was 93%.
IC50 Determination. The IC50 values of compounds against
Mtb20SOG, β5i, β2i, and β1i of i-20S and β5c, β2c, and β1c of c-20S
were determined from tests in a 96-well format as reported.16 One
microliter of a compound in a 3× series dilution in DMSO at
concentrations from 100 to 0.0017 μM was spotted to the bottom of
wells in a black 96-well plate. Reaction buffer (100 μL; 20 mM 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.5 mM
ethylenediaminetetraacetic acid (EDTA), pH 7.5) containing enzyme
and substrate were dispensed into each well and the plate was then spun
in a simple benchtop plate centrifuge at 1000 rpm for 1 min, followed by
shaking on an orbital shaker for 1 min. The time course of hydrolysis in
each well was plotted by recording the fluorescence of the product
AMC (λex 360 nm and λem 460 nm) on a SpectraMax M5 plate reader
over 1.5−2 h. The initial reaction velocity of each well was fit to a dose-
dependent inhibition equation using PRISM to determine the IC50.
Suc-LLVY-AMC was used for Mtb20SOG activity at 50 μM, for c-
20S β5c at 25 μM, and for i-20S β5i at 15 μM. Z-LLE-AMC was used for
c-20S β1c and i-20S β1i at 50 μM. Z-VLR-AMC was used for c-20S β2c
and i-20S β2i at 50 μM. Final concentrations of Mtb20S, β5i, and β5c
were 2, 0.4, and 0.2 nM, respectively. Sodium dodecyl sulfate was used
as an activator at 0.02% for human β1i, β1c, β5i, and β5c. PA28α was
used as an activator at 6 nM for human β2i and β2c.
Protein Expression and Purification. Cloning and expression of
Mtb20SOG have been described.27 Briefly, a PACYCDuet-1 vector
(Novagen, Madison, WI) consisting of the Mtb prcAOG (encoding
truncated-PrcA) and prcB genes was expressed in the BL21(DE3) strain
of Escherichia coli. Cells were grown at 37 °C to OD600 = 0.5−0.6 before
being induced with 0.2 mM IPTG. The Mtb proteasome was expressed
at 37 °C for 18 h to ensure full processing of β-subunits. Cells were
harvested by centrifugation, resuspended in 10 mM potassium
phosphate (pH 8.0), 10 mM imidazole, and 0.25 M NaCl, and lysed
by passing through a Microfluidizer cell disruptor. The homogenate was
clarified by spinning at 27 000g, and the supernatant was applied to a
HiTrap-Ni column (GE Healthcare) pre-equilibrated with the lysis
buffer. His-tagged proteins were eluted with a 10−300 mM imidazole
gradient in 10 mM potassium phosphate (pH 8.0) and 0.25 M NaCl.
Eluted fractions containing Mtb proteasomes were applied to a Hi-Trap
Q column pre-equilibrated with 10 mM Tris (pH 8.0) and 50 mM NaCl
and were eluted with a 50−500 mM NaCl gradient. Mtb20SOG was
further purified with a Superdex 200 column (16 × 600 mm2, GE
Healthcare) in buffer containing 10 mM HEPES (pH 7.5), 1 mM
dithiothreitol, and 0.15 M NaCl. For crystallization, the purified
Mtb20SOG complex was concentrated to 10 mg/mL.
CONCLUSIONS
■
There have been only five broad classes of antibiotic targets:
inhibition of synthesis of nucleic acids, proteins, cell walls and
folate, and disruption of membranes. Following the success of
proteasome inhibitor drugs for the treatment of hematopoietic
malignancy, our recent work has validated a fifth class: inhibition
of a protein degradation pathway.15 Our work has led to the
discovery by others of pathogen-selective proteasome inhibitors
that kill the protozoal agents of malaria, Chagas disease, and
leishmaniasis.34,35 Targeting another ancient, ATP-dependent
compartmentalized protease, ClpP, in bacteria has also made
tremendous progress.36−38
There is an intense effort to identify and target the Mtb genes
that are essential for its survival in the host. Recent studies have
shown that Mtb relies on several enzymatic pathways to survive
host stresses while entering a nonreplicative state. Among these
is the Pup−proteasome system discovered over several years,
beginning in 2003.2,7−9,11−13,39,40 In that system, Pup is
deaminated by Dop, conjugated to a client protein by PafA (a
Pup ligase)39−41 and fed by the Mtb20S activator Mpa39,42,43
into the Mtb proteasome for degradation.8,15,44 Although the
Mtb proteasome is dispensable under standard growth
conditions, it has been validated as a drug target both genetically
and pharmacologically: the knock-out strain dies in the chronic
phase of disease in mice. We have focused on targeting the Mtb
proteasome because it is the most downstream of the five known
enzymes in the system and we have succeeded in developing
both irreversible and reversible Mtb20S inhibitors that are
species-selective and kill nonreplicating Mtb.15,16
In this study, we developed a series of macrocyclic Mtb
proteasome inhibitors. A structure-guided study of their
structure−activity relationships led to improved potency and
species selectivity. We found that (1) targeting the S3 subpocket
with a bulky phenyl group at the α-position of P3 pyrrolidine
boosted both potency against Mtb20S and selectivity over
human proteasomes; (2) a 16-membered alkyl ether linker, a
3,4′-biphenyl ether linker, and a monophenyl ether linker were
accommodated in the P2 and P4 pockets, parts of which were
exposed to solvent; and (3) P5 amides of the rigid macrocyclic
peptide were not well tolerated at the P5 pocket of Mtb20S.
The macrocycles 6, 9, and 10 have low-nanomolar potency
against Mtb20S and >10 000-fold selectivity over human c-20S
and i-20S. Macrocycle 6 is stable in plasma, nontoxic to human
cells, and active against Mtb20S in a time-dependent manner. It
enters Mtb and prevents protein degradation by Mtb20S,
leading to the accumulation of pupylated proteins and bacterial
death under nitrosative stress. Given these characteristics,
further improvement on metabolic stability of the macrocycles
identified in this report may lead to viable anti-TB drug
candidates.
Kinetic Solubility. Two microliter 10 mM DMSO stock solution of
test compounds was added to 98 μL of phosphate-buffered saline (PBS)
buffer at pH 6.8. After incubation for 24 h, the samples were filtered
through filter plates to remove an undissolved compound. The filtrates
were then analyzed on an Acquity UPLC/MS system, equipped with a
PDA detector, for solubility quantification. The kinetic solubility (in
μg/mL) was calculated by dividing peak area of sample filtrate by peak
area of standard (10 μM test compound in acetonitrile/water (1:1, v/
v)) and multiplied by the target concentration (in μg/mL, 100 × MW/
1000 μg/mL).
Liver Microsomal Stability Assay. Test compounds (1 μM) and
microsomes (0.2 mg protein/mL) were mixed in phosphate buffer at
pH 7.4. The reactions were initiated by adding an NADPH generating
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system (a mixture of MgCl2, NADP+, glucose-6-phosphate, and
glucose-6-phosphate dehydrogenase) to the mixtures in a final volume
of 25 μL. The concentration of organic solvents was 1% (0.01% DMSO
and 0.99% acetonitrile) in the final incubation system. The mixtures
were incubated at 37 °C for 0, 15, and 30 min in duplicates. The
reactions were stopped at each time point by adding 75 μL of
acetonitrile containing internal standard. For the T0 min samples, 75 μL
of acetonitrile containing internal standard was added to mixtures
before adding an NADPH generating system. After the samples were
mixed and centrifuged, the supernatants were analyzed by LC/MS-MS.
The intrinsic clearance (CLint, in μL/min/mg) was calculated by
dividing rate constant (k, in min−1) by microsomal protein
concentration (0.2 mg/mL). The rate constant (k, in min−1) was
calculated based on the exponential function of the percentage of
compound remaining vs time curve on the assumption that it is the first-
order elimination. Flutamide was included as the positive control
compound in each experiment.
9, and 10). The mixtures were incubated at 37 °C for 0, 60, 120 min.
The reactions were stopped at each time point by adding 200 μL of ice-
cold methanol containing 50 μM internal standard. For the T0 min
samples, 200 μL of ice-cold methanol containing internal standard was
added to human plasma prior to the addition of the test compound.
Precipitated proteins were pelleted by centrifuging at 13 500 rpm for 20
min at 4 °C. The supernatants were collected and analyzed on an
Acquity UPLC/MS sysem, coupled with a PDA detector. Column
chromatography was carried out on a C18 Column, 130 Å, 1.7 μm, 2.1
mm × 100 mm. The percentage of compounds remaining at each time
point relative to starting concentration were calculated using integrated
UV peak areas normalized to the internal control. Procaine was
included as the positive control compound in each experiment.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00296.
Molecular Docking. Molecular docking was performed in the
Maestro (version 12.2) workspace using modules (Protein Preparation
̈
Synthesis and NMR spectra of final compounds and key
intermediates and experimental procedures for the
biological assays (PDF)
Docking pose of macrocycle 5 at Mtb20SOG (PDB ID
5TS0) and overlay of macrocycle 5 to parent PKS-2208 at
Mtb20SOG (PDB ID 5TS0) (PDB)
Wizard, LigPrep, and Glide) in the Schrodinger software package
(release 2019-4). The X-ray crystal structures of Mtb20SOG (PDB ID
5TS0) were retrieved from the Protein Data Bank. The protein
structures were prepared using the Protein Preparation Wizard by
assigning bond orders, adding hydrogens, creating disulfide bonds,
converting selenomethionines into methionines, and filling in missing
side chains. Water molecules in the entire system were removed.
PROPKA was used to assign H-bonds at pH 7.0. Restrained
minimization was then performed using OPLS3 force field. The
docking grid centered on the cocrystallized ligand PKS-2208 was
created using the Receptor Grid Generation using default settings
without any constraints. Ligands were built using the 2D Sketcher and
Molecular formula strings (CSV)
Accession Codes
Atomic coordinates have been deposited in RCSB with PDB ID
6WNK.
prepared using LigPrep by generating ionization states at pH 7.0
2
AUTHOR INFORMATION
Corresponding Author
■
and tautomers to generate suitable 3D conformations for docking. The
prepared ligands were docked using Glide in standard precision (SP)
mode with flexible ligand sampling. The docked results were visualized
and analyzed in Maestro. The best binding poses were chosen
according to the glide docking scores.
Gang Lin − Department of Microbiology & Immunology, Weill
Cornell Medicine, New York 10065, United States;
orcid.org/0000-0003-4797-7073; Email: gal2005@
med.cornell.edu
Crystallization and Structure Determination. Mtb20SOG was
crystallized at 4 °C by the hanging-drop vapor diffusion method using
60 mM sodium citrate (pH 6.2) and 15% PEG-3350 as precipitant. To
obtain the inhibitor−proteasome complex, Mtb20SOG crystals were
transferred stepwise to the cryo-protectant containing 60 mM sodium
citrate (pH 6.2), 15% PEG-3350, 35% dimethylformamide, and 1 mM
macrocycle 6 inhibitor. After incubation at 4 °C for 20 h, crystals were
flash-frozen in liquid nitrogen. Diffraction data were collected at the
Life Sciences Collaborative Access Team (LS-CAT) beamline of APS
and were processed with Mosflm. The space groups of inhibitor-soaked
Mtb20SOG crystals were all P21. Molecular replacement was carried
out to solve the macrocycle 6-bound Mtb20SOG structures using the
program PHASER.45 The DPLG-2-Mtb20SOG complex structure
(PDB ID 5TRG) was used as an initial search model. After building the
model of macrocycle 6 in COOT,46 refinements were performed using
Phenix-refine,47 and the refinement and modeling statistics are
provided in Table S2.
Impact of Mtb20S Inhibitors on Pup-GFP in MHD1441. Two
microliters of Mtb20S inhibitor solution in DMSO were added to the
inner wells of a 96-well black wall/clear bottom tissue culture plate.
MHD1441 (H37Rv WT + pSCK1) was grown to OD 1.6 and adjusted
to OD 0.05 with 7H9 medium. We dispensed 200 μL of MHD1441 in
7H9 into each well and incubated at 37 °C for 4 days. Optical density at
580 nm (OD580 nm) and relative fluorescence unit (RFU; excitation, 495
nm and emission, 510 nm) values were measured on day 4. RFU values
were then normalized by OD. We screened 255 compounds
(phenylimidazoles, benzimidazoles, and macrocyclic peptides) at final
concentrations of 100, 33, and 11 μM, respectively. The dose-
dependent impacts on Pup-GFP in Mtb of macrocycles 6, 9, and 10
were measured at final concentrations of 33, 11, 3.7, 1.2, 0.4, and 0.1
μM.
Authors
Hao Zhang − Department of Microbiology & Immunology,
Weill Cornell Medicine, New York 10065, United States;
orcid.org/0000-0002-1187-5844
Hao-Chi Hsu − Department of Structural Biology, Van Andel
Institute, Grand Rapids, Michigan 49503, United States
Shoshanna C. Kahne − Department of Microbiology, New York
University School of Medicine, New York 10016, United States
Ryoma Hara − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Wenhu Zhan − Department of Microbiology & Immunology,
Weill Cornell Medicine, New York 10065, United States;
orcid.org/0000-0002-7095-7081
Xiuju Jiang − Department of Microbiology & Immunology,
Weill Cornell Medicine, New York 10065, United States
Kristin Burns-Huang − Department of Microbiology &
Immunology, Weill Cornell Medicine, New York 10065,
United States
Tierra Ouellette − Department of Microbiology & Immunology,
Weill Cornell Medicine, New York 10065, United States
Toshihiro Imaeda − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Rei Okamoto − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Masanori Kawasaki − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Mayako Michino − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Human Plasma Stability Assay. To 100 μL of human plasma
(H3667, Sigma-Aldrich) was added 1 μL of 10 mM test compound (6,
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Tzu-Tshin Wong − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Akinori Toita − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Takafumi Yukawa − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Discovery Institute (TDI), a 501(c)(3) organization. TDI
receives financial support from Takeda Pharmaceutical
Company, TDI’s parent institutes (Memorial Sloan Kettering
Cancer Center, The Rockefeller University, and Weill Cornell
Medicine), from a generous contribution from Lewis Sanders,
and from other philanthropic sources.
Francesca Moraca − Schrödinger, Inc., New York 10036,
United States; orcid.org/0000-0003-0248-9834
Jeremie Vendome − Schrödinger, Inc., New York 10036,
United States
Priya Saha − Department of Microbiology & Immunology, Weill
Cornell Medicine, New York 10065, United States
Kenjiro Sato − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Kazuyoshi Aso − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
John Ginn − Tri-Institutional Therapeutics Discovery Institute,
New York 10065, United States
Peter T. Meinke − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Michael Foley − Tri-Institutional Therapeutics Discovery
Institute, New York 10065, United States
Carl F. Nathan − Department of Microbiology & Immunology,
Weill Cornell Medicine, New York 10065, United States
K. Heran Darwin − Department of Microbiology, New York
University School of Medicine, New York 10016, United States
Huilin Li − Department of Structural Biology, Van Andel
Institute, Grand Rapids, Michigan 49503, United States;
orcid.org/0000-0001-8085-8928
ABBREVIATIONS
■
Mtb20S, M. tuberculosis 20S proteasome; i-20S, immunopro-
teasome; c-20S, constitutive proteasome; three-letter codes,
amino acids
REFERENCES
■
(1) Russell, D. G.; Barry, C. E.; Flynn, J. L. Tuberculosis: what we
don’t know can, and does, hurt us. Science 2010, 328, 852−856.
(2) Darwin, K. H.; Ehrt, S.; Gutierrez-Ramos, J.-C.; Weich, N.;
Nathan, C. F. The proteasome of Mycobacterium tuberculosis is required
for resistance to nitric oxide. Science 2003, 302, 1963−1966.
(3) Gandotra, S.; Schnappinger, D.; Monteleone, M.; Hillen, W.; Ehrt,
S. In vivo gene silencing identifies the Mycobacterium tuberculosis
proteasome as essential for the bacteria to persist in mice. Nat. Med.
2007, 13, 1515−1520.
(4) Lin, G.; Tsu, C.; Dick, L.; Zhou, X. K.; Nathan, C. Distinct
specificities of Mycobacterium tuberculosis and mammalian proteasomes
for N-acetyl tripeptide substrates. J. Biol. Chem. 2008, 283, 34423−
34431.
(5) Gold, B.; Nathan, C. Targeting phenotypically tolerant
Mycobacterium tuberculosis. Microbiol. Spectr. 2017, 5, 317−360.
(6) Nathan, C.; Gold, B.; Lin, G.; Stegman, M.; de Carvalho, L. P. S.;
Vandal, O.; Venugopal, A.; Bryk, R. A philosophy of anti-infectives as a
guide in the search for new drugs for tuberculosis. Tuberculosis 2008, 88,
S25−S33.
(7) Darwin, K. H.; Lin, G.; Chen, Z.; Li, H.; Nathan, C. F.
Characterization of a Mycobacterium tuberculosis proteasomal ATPase
homologue. Mol. Microbiol. 2005, 55, 561−571.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00296
Author Contributions
H.Z. and H.-C.H. contributed equally. The manuscript was
written through the contributions of all authors. All authors have
given approval to the final version of the manuscript.
(8) Lin, G.; Hu, G.; Tsu, C.; Kunes, Y. Z.; Li, H.; Dick, L.; Parsons, T.;
Li, P.; Chen, Z.; Zwickl, P.; et al. Mycobacterium tuberculosis prcBA
genes encode a gated proteasome with broad oligopeptide specificity.
Mol. Microbiol. 2006, 59, 1405−1416.
Funding
This work is supported by NIH grants R01 AI143714 (to G.L.),
R21 AI144552 (to G.L.), and R01 AI070285 (to H.L.), the Tri-
Institutional Therapeutics Discovery Institute and Weill Cornell
Medicine Matching Fund (to G.L.), and by the Milstein
Program in Translational Medicine. S.C.K. is supported in part
by a Public Health Service Institutional Research Training
Award T32 AI007180.
(9) Pearce, M. J.; Mintseris, J.; Ferreyra, J.; Gygi, S. P.; Darwin, K. H.
Ubiquitin-like protein involved in the proteasome pathway of
Mycobacterium tuberculosis. Science 2008, 322, 1104−1107.
(10) Imkamp, F.; Rosenberger, T.; Striebel, F.; Keller, P. M.; Amstutz,
B.; Sander, P.; Weber-Ban, E. Deletion of dop in Mycobacterium
smegmatis abolishes pupylation of protein substrates in vivo. Mol.
Microbiol. 2010, 75, 744−754.
(11) Burns, K. E.; Pearce, M. J.; Darwin, K. H. Prokaryotic ubiquitin-
like protein provides a two-part degron to Mycobacterium proteasome
substrates. J. Bacteriol. 2010, 192, 2933−2935.
Notes
The authors declare no competing financial interest.
(12) Hu, K.; Jastrab, J. B.; Zhang, S.; Kovach, A.; Zhao, G.; Darwin, K.
H.; Li, H. Proteasome substrate capture and gate opening by the
accessory factor PafE from Mycobacterium tuberculosis. J. Biol. Chem.
2018, 293, 4713−4723.
(13) Bai, L.; Hu, K.; Wang, T.; Jastrab, J. B.; Darwin, K. H.; Li, H.
Structural analysis of the dodecameric proteasome activator PafE in
Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 2016, 113,
E1983−E1992.
(14) Gandotra, S.; Lebron, M. B.; Ehrt, S. The Mycobacterium
tuberculosis proteasome active site threonine is essential for persistence
yet dispensable for replication and resistance to nitric oxide. PLoS
Pathog. 2010, 6, No. e1001040.
(15) Lin, G.; Li, D.; De Carvalho, L. P. S.; Deng, H.; Tao, H.; Vogt, G.;
Wu, K.; Schneider, J.; Chidawanyika, T.; Warren, J. D.; et al. Inhibitors
selective for mycobacterial versus human proteasomes. Nature 2009,
461, 621−626.
(16) Lin, G.; Chidawanyika, T.; Tsu, C.; Warrier, T.; Vaubourgeix, J.;
Blackburn, C.; Gigstad, K.; Sintchak, M.; Dick, L.; Nathan, C. N, C-
ACKNOWLEDGMENTS
■
X-ray diffraction data were collected at the Life Sciences
Collaborative Access Team (LS-CAT) beamline of the
Advanced Photon Source (APS). LS-CAT was supported by
the Michigan Economic Development Corporation and the
Michigan Technology Tri-Corridor (Grant No. 085P1000817).
The APS is a U.S. Department of Energy (DOE) Office of
Science User Facility operated for the DOE Office of Science by
Argonne National Laboratory under Contract No. DE-AC02-
06CH11357. The Department of Microbiology and Immunol-
ogy is supported by the William Randolph Hearst Foundation.
The authors thank David Nadziejka (Van Andel Institute) for
the technical editing of this manuscript. The authors gratefully
acknowledge the support to the project (not to the non-TDI
labs) generously provided by the Tri-Institutional Therapeutics
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Article
Capped dipeptides with selectivity for mycobacterial proteasome over
human proteasomes: role of S3 and S1 binding pockets. J. Am. Chem.
Soc. 2013, 135, 9968−9971.
(33) Cerda-Maira, F. A.; Pearce, M. J.; Fuortes, M.; Bishai, W. R.;
Hubbard, S. R.; Darwin, K. H. Molecular analysis of the prokaryotic
ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium
tuberculosis. Mol. Microbiol. 2010, 77, 1123−1135.
(34) Khare, S.; Nagle, A. S.; Biggart, A.; Lai, Y. H.; Liang, F.; Davis, L.
C.; Barnes, S. W.; Mathison, C. J.; Myburgh, E.; Gao, M.-Y.; et al.
Proteasome inhibition for treatment of leishmaniasis, Chagas disease
and sleeping sickness. Nature 2016, 537, 229−233.
(35) Li, H.; O’Donoghue, A. J.; van der Linden, W. A.; Xie, S. C.; Yoo,
E.; Foe, I. T.; Tilley, L.; Craik, C. S.; da Fonseca, P. C.; Bogyo, M.
Structure-and function-based design of Plasmodium-selective protea-
some inhibitors. Nature 2016, 530, 233−236.
(36) Gavrish, E.; Sit, C. S.; Cao, S.; Kandror, O.; Spoering, A.; Peoples,
A.; Ling, L.; Fetterman, A.; Hughes, D.; Bissell, A.; et al. Lassomycin, a
ribosomally synthesized cyclic peptide, kills Mycobacterium tuberculosis
by targeting the ATP-dependent protease ClpC1P1P2. Chem. Biol.
2014, 21, 509−518.
(37) Schmitz, K. R.; Handy, E. L.; Compton, C. L.; Gupta, S.; Bishai,
W. R.; Sauer, R. T.; Sello, J. K. Acyldepsipeptide antibiotics and a
bioactive fragment thereof differentially perturb Mycobacterium tuber-
culosis ClpXP1P2 activity in vitro. ACS Chem. Biol. 2020,
DOI: 10.1021/acschembio.9b00454.
(38) Conlon, B. P.; Nakayasu, E. S.; Fleck, L. E.; LaFleur, M. D.;
Isabella, V. M.; Coleman, K.; Leonard, S. N.; Smith, R. D.; Adkins, J. N.;
Lewis, K. Activated ClpP kills persisters and eradicates a chronic biofilm
infection. Nature 2013, 503, 365−370.
(39) Striebel, F.; Hunkeler, M.; Summer, H.; Weber-Ban, E. The
mycobacterial Mpa-proteasome unfolds and degrades pupylated
substrates by engaging Pup’s N-terminus. EMBO J. 2010, 29, 1262−
1271.
(40) Striebel, F.; Imkamp, F.; Sutter, M.; Steiner, M.; Mamedov, A.;
Weber-Ban, E. Bacterial ubiquitin-like modifier Pup is deamidated and
conjugated to substrates by distinct but homologous enzymes. Nat.
Struct. Mol. Biol. 2009, 16, 647−651.
(17) Zhan, W.; Hsu, H.-C.; Morgan, T.; Ouellette, T.; Burns-Huang,
K.; Hara, R.; Wright, A. G.; Imaeda, T.; Okamoto, R.; Sato, K.; et al.
Selective phenylimidazole-based inhibitors of the Mycobacterium
tuberculosis proteasome. J. Med. Chem. 2019, 62, 9246−9253.
(18) Cummings, M. D.; Sekharan, S. Structure-based macrocycle
design in small-molecule drug discovery and simple metrics to identify
opportunities for macrocyclization of small-molecule ligands. J. Med.
Chem. 2019, 62, 6843−6853.
(19) Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. The exploration
of macrocycles for drug discoveryan underexploited structural class.
Nat. Rev. Drug Discovery 2008, 7, 608−624.
(20) Berthelot, A.; Piguel, S.; Le Dour, G.; Vidal, J. Synthesis of
macrocyclic peptide analogues of proteasome inhibitor TMC-95A. J.
Org. Chem. 2003, 68, 9835−9838.
(21) Kaiser, M.; Milbradt, A. G.; Siciliano, C.; Assfalg-Machleidt, I.;
Machleidt, W.; Groll, M.; Renner, C.; Moroder, L. TMC-95A analogues
with endocyclic biphenyl ether group as proteasome inhibitors. Chem.
Biodiversity 2004, 1, 161−173.
(22) Groll, M.; Götz, M.; Kaiser, M.; Weyher, E.; Moroder, L. TMC-
95-based inhibitor design provides evidence for the catalytic versatility
of the proteasome. Chem. Biol. 2006, 13, 607−614.
(23) Koguchi, Y.; Kohno, J.; Nishio, M.; Takahashi, K.; Okuda, T.;
Ohnuki, T.; Komatsubara, S. TMC-95A, B, C, and D, novel proteasome
inhibitors produced by Apiospora montagnei Sacc. TC 1093 taxonomy,
production, isolation, and biological activities. J. Antibiot. 2000, 53,
105−109.
(24) Li, H.; Tsu, C.; Blackburn, C.; Li, G.; Hales, P.; Dick, L.; Bogyo,
M. Identification of potent and selective non-covalent inhibitors of the
Plasmodium falciparum proteasome. J. Am. Chem. Soc. 2014, 136,
13562−13565.
(25) Zhan, W.; Zhang, H.; Ginn, J.; Leung, A.; Liu, Y. J.; Michino, M.;
Toita, A.; Wong, T. T.; Imaeda, T.; Hara, R.; Yukawa, T.; Chelebieva,
S.; Tumwebaze, P. K.; Lafuente-Monasterio, M. J.; Martinez-Martinez,
M. S.; Vendome, J.; Beuming, T.; Sato, K.; Aso, K.; Rosenthal, P. J.;
Cooper, R. A.; Meinke, P. T.; Nathan, C. F.; Kirkman, L. A.; Lin, G.
Development of a highly selective Plasmodium falciparum proteasome
inhibitor with anti-malaria activity in humanized mice. Angew. Chem.,
Int. Ed. 2021, 60, 9279−9283.
(26) Neilsen, P. M.; Pehere, A. D.; Pishas, K. I.; Callen, D. F.; Abell, A.
D. New 26S proteasome inhibitors with high selectivity for
chymotrypsin-like activity and p53-dependent cytotoxicity. ACS
Chem. Biol. 2013, 8, 353−359.
(27) Hsu, H.-C.; Singh, P. K.; Fan, H.; Wang, R.; Sukenick, G.;
Nathan, C.; Lin, G.; Li, H. Structural basis for the species-selective
binding of N, C-capped dipeptides to the Mycobacterium tuberculosis
proteasome. Biochemistry 2017, 56, 324−333.
(28) Raymer, B.; Bhattacharya, S. K. Lead-like Drugs: A Perspective:
Miniperspective. J. Med. Chem. 2018, 61, 10375−10384.
(29) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic,
J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.;
et al. Glide: a new approach for rapid, accurate docking and scoring. 1.
Method and assessment of docking accuracy. J. Med. Chem. 2004, 47,
1739−1749.
(41) Sutter, M.; Damberger, F. F.; Imkamp, F.; Allain, F. H. T.;
Weber-Ban, E. Prokaryotic ubiquitin-like protein (Pup) is coupled to
substrates via the side chain of its C-terminal glutamate. J. Am. Chem.
Soc. 2010, 132, 5610−5612.
(42) Wang, T.; Darwin, K. H.; Li, H. Binding-induced folding of
prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal
ATPase targets substrates for degradation. Nat. Struct. Mol. Biol. 2010,
17, 1352−1357.
(43) Wang, T.; Li, H.; Lin, G.; Tang, C.; Li, D.; Nathan, C.; Darwin, K.
H.; Li, H. Structural insights on the Mycobacterium tuberculosis
proteasomal ATPase Mpa. Structure 2009, 17, 1377−1385.
(44) Hu, G.; Lin, G.; Wang, M.; Dick, L.; Xu, R. M.; Nathan, C.; Li, H.
Structure of the Mycobacterium tuberculosis proteasome and mechanism
of inhibition by a peptidyl boronate. Mol. Microbiol. 2006, 59, 1417−
1428.
(45) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.
D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl.
Crystallogr. 2007, 40, 658−674.
(46) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular
graphics. Acta Crystallogr., Sect. D 2004, 60, 2126−2132.
(47) Afonine, P. V.; Grosse-Kunstleve, R. W.; Echols, N.; Headd, J. J.;
Moriarty, N. W.; Mustyakimov, M.; Terwilliger, T. C.; Urzhumtsev, A.;
Zwart, P. H.; Adams, P. D. Towards automated crystallographic
structure refinement with phenix.refine. Acta Crystallogr., Sect. D 2012,
68, 352−367.
(30) Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye,
L. L.; Pollard, W. T.; Banks, J. L. Glide: a new approach for rapid,
accurate docking and scoring. 2. Enrichment factors in database
screening. J. Med. Chem. 2004, 47, 1750−1759.
(31) Burns, K. E.; Cerda-Maira, F. A.; Wang, T.; Li, H.; Bishai, W. R.;
Darwin, K. H. “Depupylation” of prokaryotic ubiquitin-like protein
from mycobacterial proteasome substrates. Mol. Cell 2010, 39, 821−
827.
̈
(32) Imkamp, F.; Striebel, F.; Sutter, M.; Ozcelik, D.; Zimmermann,
N.; Sander, P.; Weber- Ban, E. Dop functions as a depupylase in the
prokaryotic ubiquitin-like modification pathway. EMBO Rep. 2010, 11,
791−797.
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