Discovery of Highly Selective Inhibitors of the Immunoproteasome Low Molecular Mass Polypeptide 2 (LMP2) Subunit

Article abstract of DOI:10.1021/acsmedchemlett.6b00496

Building upon the success of bortezomib (VELCADE) and carfilzomib (KYPROLIS), the design of a next generation of inhibitors targeting specific subunits within the immunoproteasome is of interest for the treatment of autoimmune disease. There are three catalytic subunits within the immunoproteasome (low molecular mass polypeptide-7, -2, and multicatalytic endopeptidase complex subunit-1; LMP7, LMP2, and MECL-1), and a campaign was undertaken to design a potent and selective LMP2 inhibitor with sufficient properties to allow for sustained inhibition in vivo. Screening a focused library of epoxyketones revealed a series of potent dipeptides that were optimized to provide the highly selective inhibitor KZR-504 (12).

Full text of DOI:10.1021/acsmedchemlett.6b00496

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Letter
The Discovery of Highly Selective Inhibitors of the Immunoproteasome
Low Molecular Mass Polypeptide 2 (LMP2) Subunit.
Henry W. B. Johnson, Janet L. Anderl, Erin K. Bradley, John Bui, Jeffrey Jones, Shirin Arastu-Kapur, Lisa M.
Kelly, Eric Lowe, David C. Moebius, Tony Muchamuel, Christopher Kirk, Zhengping Wang, and Dustin McMinn
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00496 • Publication Date (Web): 09 Mar 2017
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The Discovery of Highly Selective Inhibitors of the Immunopro-
teasome Low Molecular Mass Polypeptide 2 (LMP2) Subunit.
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Henry W. B. Johnson,*† Janet L. Anderl,† Erin K. Bradley,§ John Bui,§ Jeffrey Jones,§
Shirin Arastu-Kapur,§ Lisa M. Kelly,§ Eric Lowe,† David C. Moebius,§ Tony Muchamuel,† Christopher
Kirk,† Zhengping Wang,§ and Dustin McMinn†
†
Kezar Life Sciences, 300 Utah Ave. Suite 105, South San Francisco, CA 94080.
§Onyx Pharmaceuticals, An Amgen Subsidiary, 249 East Grand Ave., South San Francisco, CA 94080.
KEYWORDS: LMP2, immunoproteasome, constitutive proteasome, β1i, β1c
ABSTRACT: Building upon the success of bortezomib (VELCADE™) and carfilzomib (KYPROLIS™), the design of a next
generation of inhibitors targeting specific subunits within the immunoproteasome is of interest for the treatment of auto-
immune disease. There are three catalytic subunits within the immunoproteasome (low-molecular mass polypeptide-7, -
2, and multicatalytic endopeptidase complex subunit-1; LMP7, LMP2, and MECL-1) and a campaign was undertaken to
design a potent and selective LMP2 inhibitor with sufficient properties to allow for sustained inhibition in vivo. Screening
a focused library of epoxyketones revealed a series of potent dipeptides that were optimized to provide the highly selec-
tive inhibitor KZR-504 (12).
With the FDA approval of the peptide inhibitors borte-
zomib (1) and carfilzomib (2), inhibition of the pro-
teasome has proven an effective therapeutic strategy to
target the plasma cell neoplasm, multiple myeloma. In
most cells, the constitutive proteasome is responsible for
non-lysosomal intracellular protein degradation, mediat-
ed by the β1, β2, and β5 catalytic subunits.1 Cells of hem-
atopoietic origin (e.g. lymphocytes) contain proteasomes
expressing 3 distinct catalytic subunits, LMP7 (low-
molecular mass polypeptide-7), LMP2, and MECL-1 (mul-
ticatalytic endopeptidase complex-like 1); referred to
elsewhere as β5i, β1i, and β2i, respectively.2 This so-called
immunoproteasome is also expressed in non-
hematopoietic cells exposed to inflammatory cytokines
and plays an important role in antigen presentation, in-
fluencing cytokine production, and T-cell differentiation
and survival.3
units of the proteasome.5 Derivatization of the peptide
backbone of epoxyketone analogues has allowed for the
design of inhibitors that target specific subunits of the
proteasome. For example, ONX 0914 (3, formerly PR-957)6
and PR-924 (4)7 selectively target immunoproteasome
subunits while PR-893 (5)7 and PR-825 (6)6 selectively
target the β5 subunit of the constitutive proteasome (Fig-
ure 1). These compounds contain a epoxyketone function-
ality that covalently binds specifically to an N-terminal
threonine residue in the active site of proteasome catalyt-
ic subunits.8 Administration of ONX 0914 reduces cyto-
kine production in vitro and inflammation and tissue
damage in mouse models of lupus nephritis,9 arthritis,6
experimental autoimmune encephalomyelitis (EAE), and
colitis.10 Selective immunoproteasome inhibition also
promotes cardiac allograft acceptance in mice.11 Thus, the
immunoproteasome represents an intriguing target for
the treatment of autoimmune disorders.
Carfilzomib and bortezomib primarily inhibit with
nearly equal potency the chymotrypsin-like β5 and LMP7
proteasome subunits.4 Building upon the positive clinical
experience of these dual-inhibitors, it is quite intriguing
to consider the effect of selectively targeting various sub-
A first-generation, moderately-selective covalent inhibi-
tor of LMP2, UK-101 (7), induces cell death in prostate
cancer cell lines at a high concentration and suppresses in
vivo tumor growth in a xenograft mouse model of prostate
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cancer.12-13 Additional cell-permeable selective LMP2 in-
a high degree of sequence homology is observed between
hibitors in the epoxyketone (az-NC-001 (8)14 and LU-001i
(9)15 ) and boronate compound class have since been de-
signed.16
mouse and human proteasome subunits around the re-
spective catalytic sites. This allowed for an accurate hu-
man model to be built based upon the murine x-ray struc-
ture.21 In agreement with previous studies, modeling of
the non-covalent complex accurately aligned with ob-
served SAR within the epoxyketone series.22 Additionally,
and as expected, IC50 values for all subunits are similar
between species with the epoxyketone inhibitors tested
herein (Table 1, see supplemental Table 4 for mouse data).
Previously, the selectivity of LMP7 inhibitors has been
rationalized based on specific interactions within the S1-
S3 pockets (Figure 2a).15,21,23 However, when examining the
binding of 10, interactions with three residues (S48, S118,
and H114) within the S3 pocket appears most critical for
dictating LMP2 selectivity.
LMP2-/- mice exhibit defects in B and T helper (Th)
cell function and dendritic cell secretion of pro-
inflammatory cytokines.17 These results however have not
been confirmed with a selective LMP2 inhibitor. To gain a
better understanding of the immunoregulatory effects of
LMP2 inhibition both in vitro and in animal models, we
sought to characterize a selective tool compound with
suitable pharmaceutic properties to allow for sustained
target inhibition in vivo.
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An overlay of ONX 0914 and 10 within LMP2 reveals a
stark difference in how the 2-pyridone and morpholino
tail are configured (Figure 2a). This was the first indica-
tion that the selectivity of 10 could be driven by interac-
tions within the S3 pocket. The mouse co-crystal struc-
ture of ONX 0914 bound to β1 (PDB code: 3UNB) reveals
that two water molecules sit within the S3 pocket forming
an H-bonding network with the peptide backbone of
ONX 0914 (Figure 2b).24 Although these waters are not
explicitly observed in the murine LMP2 (PDB code:
3UNF) structure, the two sequences are identical in this
region, allowing us to deduce that a similar water network
is also formed in LMP2. Modeling of 10 reveals that one
water molecule in LMP2 is displaced with the 2-pyridone
oxygen while a hydrogen-bonding network including the
remaining water, S48, and S118 is maintained (Figure 2c).
A key structural difference between subunits in the S3
pocket is the D114H substitution from LMP7, MECL-1, β5,
and β2 to LMP2. An H-bond is formed between D114 and
the amide backbone of ONX 0914 that is absent with 10
(Figure 2d, 2e, 2f, and 2g). The low affinity of 10 for
LMP7, MECL-1, β5, and β2 is at least partially due to the
2-pyridone being unable to rotate within the S3 pocket to
interact with D114. Poor binding of 10 to β1 is thought to
come about from two amino acid substitutions from
LMP2 to β1: H114Y and A22T. For 10 to adopt a similar
binding mode in β1 as LMP2, these residues in the S3
pocket would clash with the 2-pyridone (Figure 2h). Col-
lectively, this data suggests that the 2-pyridone motif of
10 is critical for favorable binding to LMP2 over other
proteasomal subunits.
Figure 1. Covalent proteasome inhibitors.
Primary screening of an in-house focused library of di-
and tri-peptide epoxyketones was performed utilizing the
LMP2-selective fluorogenic substrate acetyl-Pro-Ala-Leu-
7-amino-4-methylcoumarin (Ac-PAL-AMC, see Supple-
mental Table 1).18 Further characterization utilizing a sub-
unit-specific ELISA (enzyme-linked immunosorbent as-
say), termed ProCISE, revealed that 10 (KZR-109) has ex-
cellent selectivity for LMP2 over other proteasomal subu-
nits (Table 1).
Although other reported dipeptide
epoxyketone derivatives have demonstrated low affinity
for β5 and LMP7, this motif is suitable for targeting
LMP2.19
It is known that the high clearance of peptide
epoxyketones is mediated predominantly by peptidase
cleavage and epoxide hydrolysis.25-26 Therefore in vitro
The high degree of homology between proteasomal
subunits and scarcity of structural data makes the design
of subunit-selective inhibitors a challenge.20 Fortunately,
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stability data was routinely collected with liver mouse,
10, at least in part, contributes to greater target inhibition
rat, and human S9 fraction in the absence of NADPH
(Table 1). Utilizing the ProCISE assay, inhibition of pro-
teasome active sites in blood was measured 1 h after intra-
venous administration of compound. The low in vivo tar-
get inhibition (83% activity remaining) with 10 is likely
related to its poor stability (Table 2). Therefore, another
derivative that could address the shortcomings of 10 was
sought.
in blood (Table 2). This could also be due to differences in
cell permeability/efflux, however, 12 exhibits significantly
lower permeability than 10 and, as further confirmation,
activity in whole cells vs. lysate is also compromised for 12
(see Supplemental Tables 2 and 3). Evaluating the inhibi-
tion of LMP2, and anti-targets LMP7 and β5, in mouse
tissues reveals that 12 is both selective and potent in vivo
with >50% target inhibition achieved at >1 mg/kg in all
tissues tested except brain (see Supplemental Figure 2).
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A hydrophobic interaction with the p-methoxyphenyl
group of 10 and the C48 residue leads to an increase in
affinity to the anti-target LMP7 (Figure 3a). However, in
LMP2 a more polar serine residue is present at this posi-
Although broad immunoproteasome inhibition with
ONX 0914 decreases pro-inflammatory cytokine produc-
tion, the effect of selective LMP2 inhibition was unknown
at the outset. A pro-inflammatory response in human
peripheral blood mononuclear cells (PBMCs) stimulated
with the endotoxin lipopolysaccharide (LPS) was ob-
served with ONX 0914. However, selective inhibition of
LMP2 with 10 or 12 appears to have little to no effect on
cytokine production (Table 3).
tion
(Figure
3b).
Replacement
of
the
p-
methoxyphenylalanine residue in 10 with either alanine
(11) or serine (12, KZR-504) diminishes this hydrophobic
interaction and results in a substantial increase in solubil-
ity and stability while maintaining selectivity (Table 1).
Based on the attractive potency and selectivity profile
of 12, it was evaluated in vivo. The greater stability of 12 vs.
Table 1. Optimization of 10.
Subunit-Specific Cell Lysate IC50 (µM)a
Solubil-
Liver S9
ity
(mg/m
L)c
(% remaining)b
Analogue
R
-
LMP2
β1
LMP7
β5
MECL1
β2
Mouse
Rat
Human
0.290 ±
0.062
0.039 ±
0.009
0.422 ±
0.090
0.911 ±
0.239
0.930 ±
0.276
106.0 ±
9.0
23.3 ±
3.9
0.461 ±
0.003
ONX 0914
10
>12.7
8.6 ± 2.4
1.1 ± 0.1
0.140 ±
0.038
91.072 ±
3.137
4.810 ±
0.488
18.226 ±
3.332
8.0 ±
0.8
0.166 ±
0.000
>250
>250
0.4 ± 0.1
0.112 ±
0.033
44.639 ±
5.914
42.468 ±
5.804
85.1 ±
2.5
58.8 ±
4.3
7.524 ±
0.402
11
H
>222
>250
>250
>250
>250
25.3 ± 2.4
81.5 ± 3.3
0.051 ±
0.005
46.274 ±
6.903
4.274 ±
0.638
6.894 ±
1.622
90.9 ±
1.5
92.3 ±
4.1
6.639 ±
0.647
12
aUtilizing MOLT-4 (human T cell leukemia) cellular lysate, a proteasome constitutive/immunoproteasome subunit ELISA
(ProCISE) was employed for quantitative assessment of subunit-specific activity. Data are reported as the mean ± SD (n ≥ 2).
bLiver S9 data collected at 90 min without NADPH present. Data are reported as the mean ± SD (n ≥ 3).
cSolubility data collected at pH 7.2. Data are reported as the mean ± SD (n = 2).
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Figure 2. (a) ONX 0914 (orange) and 10 (green) modeled within the human LMP2 structure (van der Waals surfaces). The phe-
nylalanine substituent of the peptide backbone of these inhibitors is buried below the surface within the S1 pocket. The shallow
S3 pocket created at the interface of LMP2 and MECl-1 is occupied by the 2-pyridone of 10. (b) ONX 0914 (orange) modeled
within the human LMP2 structure (grey) with mouse β1 (yellow, PDB code: 3UNF) overlaid. Based upon the high degree of se-
quence homology between mouse β1 and human LMP2 a similar water network is assumed to be present in both structures. (c)
The 2-pyridone of 10 (green) displaces a water molecule within the S3 pocket of LMP2 (grey) while maintaining a hydrogen
bonding network with S48 and S118. (d) Comparison of ONX 0914 (orange) and 10 (green) modeled in the human LMP7. (e)
ONX 0914 (orange) and 10 (green) modeled in human MECL-1 (f) ONX0914 (orange) and 10 (green) modeled in human β5 (g)
ONX 0914 (orange) and 10 (green) modeled in human β2. (h) 10 (green) modeled in LMP2, overlaid on the human β1 model. The
H114Y and A22T substitutions from LMP2 to β1 occupies the space in which the 2-pyridone would reside.
Table 2. Mouse pharmacodynamic data for ONX 0914, 10
and 12.a
Table 3. Cytokine data for ONX 0914, 10, and 12a
Analogue
Analogue
ONX 0914
0.12 ± 0.07
0.56 ± 0.25
0.24 ± 0.06
0.22 ± 0.14
1.1 ± 0.81
10
>5.6
>25
>15.8
>25
>25
>25
>25
12
ONX 0914
10
12
IL-12/23 p40
TNF-α
IL-6
>9.7
>25
>19.2
>25
>25
>25
>25
LMP2 95.0 ± 10.6 83.0 ± 4.2
31.1 ± 6.3
% Activity
Cytokine
IC50
102.5 ±
11.3
Remaining
in Blood
LMP7
β5
65.3 ± 0.4
99.8 ± 2.3
98.3 ± 2.5
90.7 ± 3.9
(μM)a
GMCSF
IL-8
Relative to
Controla
101.7 ±
13.2
IL-1b
1.5 ± 0.73
3.4 ± 1.7
aNominal doses: ONX 0914 = 1 mg/kg, 10 = 2 mg/kg, 12 = 1
Viability IC50 (ꢀM)a,b
mg/kg. Data are reported as the mean ± SD (n ≥ 3 animals).
aData are reported as the mean ± SD (n=4).
bViability IC50 was measured using CellTiter-Glo.
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Figure 3. Comparison of ONX 0914 (orange) and 10 (green) within the human LMP7 and LMP2 structures (van der Waals sur-
faces). (a) In LMP7, a hydrophobic interaction between the p-methoxyphenyl group and C48 is a critical selectivity-driver for
ONX 0914. (b) The LMP2 S2 pocket is more polar due to the C48S substitution. Replacement of the p-methoxyphenyl group on
10
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the
ligand
with
less
lipophilic
functionality
is
well-tolerated.
tidase complex subunit 1; ELISA, enzyme-linked immuno-
sorbent assay; PBMC, peripheral blood mononuclear cells
Although the warhead is identical between ONX 0914
and the dipeptide structures we’ve described, exclusively
changing the motif that binds within the S3 pocket is suf-
ficient to alter the selectivity of the ligand from favoring
LMP7, in the case of ONX 0914, to LMP2. Modeling was
further leveraged to refine the pharmacophore of this
dipeptide series and enable the design of derivatives with
significantly improved in vivo stability. A quantitative
measurement of proteasome active site occupancy was
generated using ProCISE. Selectivity and potency data for
12 is comparable to literature data for LU-001i, the most
selective analogue to date.15 Furthermore, 12 (KZR-504)
can be administered at a selective dose in vivo. Collective-
ly, the desirable properties of 12 make it an excellent addi-
tion to the armory of molecules available in the hunt for a
more complete understanding of proteasome biology.
REFERENCES
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ASSOCIATED CONTENT
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lim, K. W.; Lauer, C.; Sylvain, C.; Ring, E. R.; Shields, J.;
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Experimental procedures for the synthesis of all compounds,
assay conditions, screening data for select hits, Michaelis-
Menten saturation curves for characterization of the Ac-PAL-
AMC substrate, full-tissue-dose-response curves for 12,
whole-cell ProCISE and permeability for select compounds,
and modeling of 12 within LMP2 is included. This material is
available free of charge via the Internet at
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Lorens, J. B.; Micklem, D. R.; Puurs, P.; Sylvain, C.; Lu, Y.;
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AUTHOR INFORMATION
Corresponding Author
8. Schrader, J.; Henneberg, F.; Mata, R. A.; Tittmann, K.;
Schneider, T. R.; Stark, H.; Gleb, B.; Chari, A. The inhibi-
tion mechanism of 20S proteasomes enables next-
generation inhibitor design. Science, 2016, 353, 594-598.
9. Ichikawa, T. H.; Conley, T.; Muchamuel, T.; Jiang, J.; Lee,
S.; Owen, T.; Barnard, J.; Nevarez, S.; Goldman, B. I.; Kirk,
C. J.; Looney, J. R.; Anolik, J. H. Beneficial Effect of Novel
Proteasome Inhibitors in Murine Lupus via Dual Inhibi-
tion of Type I Interferon and Anutoantibody-Secreting
Cells. Arthritis Rheum. 2012, 64, 493.
* Phone: 1-650-822-5603. E-mail: hjohnson@kezarbio.com
ACKNOWLEDGMENT
The authors would like to thank Joe Kansopon for collecting
mouse ProCISE data.
ABBREVIATIONS
LMP2, low-molecular mass polypeptide-2; LMP-7, low mo-
lecular mass polypeptide 7; MECL-1, multicatalytic endopep-
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