Discovery and optimization of a series of small-molecule allosteric inhibitors of MALT1 protease

Article abstract of DOI:10.1016/j.bmcl.2019.126743

We describe a series of potent and highly selective small-molecule MALT1 inhibitors, optimized from a High-Throughput Screening hit. Advanced analogues such as compound 40 show high potency (IC₅₀: 0.01 μM) in a biochemical assay measuring MALT1 enzymatic activity, as well as in cellular assays: Jurkat T cell activation (0.05 μM) and IL6/10 secretion (IC₅₀: 0.10/0.06 μM) in the TMD8 B-cell lymphoma line. Compound 40 also inhibited cleavage of the MALT1 substrate RelB (IC₅₀: 0.10 μM). Mechanistic enzymology results suggest that these compounds bind to the known allosteric site of the protease.

Full text of DOI:10.1016/j.bmcl.2019.126743

Journal Pre-proofs
Discovery and optimization of a series of small-molecule allosteric inhibitors
of MALT1 protease
Tianbao Lu, Peter J. Connolly, Ulrike Philippar, Weimei Sun, Maxwell D.
Cummings, Kent Barbay, Luc Gys, Luc Van Nuffel, Nigel Austin, Mariette
Bekkers, Fang Shen, Ann Cai, Ricardo Attar, Lieven Meerpoel, James Edwards
PII:
S0960-894X(19)30706-1
DOI:
https://doi.org/10.1016/j.bmcl.2019.126743
BMCL 126743
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 March 2019
2 October 2019
6 October 2019
Please cite this article as: Lu, T., Connolly, P.J., Philippar, U., Sun, W., Cummings, M.D., Barbay, K., Gys, L., Van
Nuffel, L., Austin, N., Bekkers, M., Shen, F., Cai, A., Attar, R., Meerpoel, L., Edwards, J., Discovery and
optimization of a series of small-molecule allosteric inhibitors of MALT1 protease, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126743
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery and optimization of a series of small-molecule allosteric inhibitors of
MALT1 protease
Tianbao Lu  a, Peter J. Connollya, Ulrike Philipparb, Weimei Suna, Maxwell D. Cummingsa, Kent Barbaya,
Luc Gysb, Luc Van Nuffelb, Nigel Austinb, Mariette Bekkersb, Fang Shena, Ann Caia, Ricardo Attara,
Lieven Meerpoelb, James Edwardsa
aJanssen Research & Development, LLC, 1400 McKean Road, Spring House, PA 19477, United States of America
bJanssen Research & Development, Turnhoutseweg 30, B-2340, Beerse, Belgium
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
We describe a series of potent and highly selective small-molecule MALT1 inhibitors, optimized
from a High-Throughput Screening hit. Advanced analogues such as compound 40 show high
potency (IC50: 0.01 µM) in a biochemical assay measuring MALT1 enzymatic activity, as well as
in cellular assays: Jurkat T cell activation (0.05 µM) and IL6/10 secretion (IC50: 0.10/0.06 µM) in
the TMD8 B-cell lymphoma line. Compound 40 also inhibited cleavage of the MALT1 substrate
RelB (IC50: 0.10 µM). Mechanistic enzymology results suggest that these compounds bind to the
known allosteric site of the protease.
Keywords: MALT1; paracaspase; allosteric
inhibitors; autoimmune diseases; discovery and
optimization;
2018 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +1-215-628-7090; fax: +1-215-540-4612; e-mail: tlu3@its.jnj.com

The canonical NF-kB signaling pathway regulates the activation,
proliferation and survival of T- and B-lymphocytes downstream of
antigen recognition.1-4 Mouse associated lymphoid tissue
lymphoma translocation protein 1 (MALT1) serves a role in the
activation of NF-kB. Stimulation of T- or B- cell receptors leads
to recruitment of MALT1 into the CARD11(CARMA1)-BCL10-
MALT1 (CBM) signalosome complex,5 where it serves both
scaffolding and proteolytic roles. In its scaffolding role, MALT1
binds to TRAF and TAK1, leading to activation of the IκB kinase
complex and ultimately activation of the transcription factor NF-
κB and cytokine secretion. MALT1 also contains a paracaspase
(cysteine protease) domain. Upon activation, the protease cleaves
a number of protein substrates that are part of a feedback loop
controlling termination of the NF-κB response.6-7
Scheme 1 illustrates the synthesis of 1-heteroarylpiperidine-4-
carboxamides using compound 1 as a representative example.
First, 4-chlorothieno[3,2-c]pyridine (I) was treated with methyl
piperidine-4-carboxylate (II) and cesium carbonate in DMF to
give methyl 1-(thieno[3,2-c]pyridin-4-yl)piperidine-4-carboxylate
(III) in 70% yield. Next, compound III was hydrolyzed to 1-
(thieno[3,2-c]pyridin-4-yl)piperidine-4-carboxylic acid (IV) using
sodium hydroxide in methanol. Compound IV was subsequently
reacted with 3-chloro-4-methoxyaniline (V), HATU, and DIEA in
DMF to give final compound 1 in 42% yield. All compounds in
the SAR Tables 1-5 were prepared using a similar procedure.
We began structure-activity relationship (SAR) studies by
investigating bicyclic replacements for 4-thieno[3,2-c]pyridine
(Table 1, see supplementary data for assay conditions). In our
study, the most potent N-aryl group is 4-thieno[3,2-c]pyridine (1),
with an IC50 of 0.69 µM. Among the isomeric thieno-fused
analogues, 7-thieno[2,3-c]pyridine (3) is 2-fold less potent than
compound 1 and 2-methyl-4-thieno[3,2-c]pyridine (4) is 25-fold
less potent than 1. N-methylation of the secondary amide (2) led
to complete loss of activity. Although 1-pyrrolo[2,1-c]pyrazine
(7) was almost equipotent to compound 1, other heteroaryls
attached to the piperidine at the carbon adjacent to the ring juncture
such as 4-thiazolo[4,5-c]pyridine (5), 8-imidazo[2,1-c]pyrazine
(6), 1-isoquinoline (9), and 4-quinazoline (10) are all less potent.
Interestingly, when the piperidine attachment is shifted by one
atom, as in 2-quinoline (8), MALT1 protease inhibitory activity is
completely abrogated.
MALT1 protease inhibitors may find application in the treatment
of B-cell lymphomas driven by constitutive NF-κB activation.
Protease function of the paracaspase is also required for optimal
activation of T- and B- cells, suggesting that inhibitors may also
be useful in the treatment of autoimmune diseases. MALT1
protease activity can be inhibited by small-molecules acting at the
active site, with most reports describing peptide-based protease
inhibitors that contain a reactive warhead.8-10 Allosteric inhibitors
of MALT1 protease have also been disclosed.11-13 Small-molecule
inhibitors14-15of MALT1 protease have demonstrated efficacy in
preclinical models of ABC-DLBCL, further supporting
a
pharmaceutical role of inhibitors on this protease.16-19 In this work
we describe the discovery and optimization of a series of allosteric
small-molecule inhibitors of MALT1 protease.20
High-Throughput Screening was conducted on a library of ~560K
compounds from the Janssen collection using an enzymatic
biochemical assay to find MALT1 protease inhibitors.21 We
identified compound 1, a molecule that lacks an obvious cysteine-
reaction warhead but nevertheless shows sub-micro molar activity
against MALT1 (Figure 1). Because NF-κB signaling regulates the
secretion of multiple cytokines, including IL6 and IL10, secretion
of IL6 and IL10 by TMD8 ABC-DLBCL cells was measured using
a mesoscale assay.22 Compound 1 shows single digit micro molar
levels of inhibition of on IL6 and IL10 secretion. Compound 1
also blocks cleavage of the MALT1 substrate, RelB. Encouraged
by these promising findings we started lead optimization of these
class of compounds.
Table 1. SAR for fused bicyclic heteroaryls
O
Ar
N
N
O
R1
Cl
Cpd #
1
Ar
R1
MALT1 IC50 (µM)
0.69
N
N
N
H
CH3
H




S
S
2
3
4
>30
1.3
N
O
N
HN
O
S
Cl
S
1
N
H
17.8
MALT1 IC50 0.69 µM
IL6/IL10 secretion IC50 1.70/1.95 µM
RelB cleavage IC50 1.55 µM
S
S
Figure 1. Structure and properties of hit compound 1
N
5
6
7
H
H
H
12.9
20.4
1.1



N
Cs2CO3
DMF
NaOH
N
O
O
O
O
Cl
N
+
HN
N
70%
MeOH/ H2O
100%
S
S
N
II
I
III
Cl
N
H2N
O
N
O
Cl
N
O
V
N
N
N
HN
O
OH
N
HATU, DIEA
DMF, 42%
S
S
1
N
IV
Scheme 1. Synthesis of compound 1

N
N
8
9
H
H
>30
2.3
19
20
3.39
0.55
N



N

Cl
N
21
22
17.8
4.27
N
10
H
4.0


N

N
O
Because fused heteroaryls did not improve potency, we turned
our attention to substituted mono(hetero)aryls (Table 2).
Replacing 4-thieno[3,2-c]pyridine with mono-substituted
pyridines, we found that 3-methyl (11), 3-chloro (12), 3-bromo
(13), 3-methylthio (14), 3-methoxy (15), 3-trifluoromethyl (16),
and 3-ethyl (17) each have equal or improved potency compared
to 1, with 3-bromo (14) being more than 10-fold better. By
contrast, 3-methylsulfonyl (18) is 9-fold less potent than
compound 1. Unsubstituted pyridine (19) is 5-fold less potent
than 1 and disubstituted 5-chloro-2-methyl-6-pyridine (20) is
equally potent, but 3,4-dimethyl-2-pyridine (21) is 25-fold less
potent. Larger substituents, such as in compounds 22 and 23
give reduced potency. 2-Chlorophenyl (24) is 3-fold more potent
than compound 1 but 2-fold less potent than the corresponding
pyridine (13).
N
N
23
24
13.5
0.23

N
N

Cl
Based on its balanced properties including acceptable MALT1
potency, reasonable molecular weight (359.9), and good ligand
efficiency (0.37), compound 12 was selected for pharmacokinetic
(PK) study in mice. Although the compound is bioavailable (F =
35%), it shows high clearance (70 mL/min/mg) and a very short
half-life (0.25 h) after IV dosing. In vitro ADME studies -show
compound 12 to be highly unstable in mouse liver hepatocyte and
microsomal preparations (Clint: 132 µL/min/mg), Stability in
mouse plasma is also poor, due to amide bond cleavage (~20%
remaining after 4 h). Metabolite ID studies in mouse and human
liver microsomes indicated that the major metabolic liability is
amide hydrolysis. In an attempt to solve this issue, we explored
SAR around piperidine substitutions that might stabilize the
amide bond against enzymatic hydrolysis through steric effects
(Table 4). In term of MALT1 activity, we found that 2-methyl
substitution leads to either a 2-fold potency improvement
(compare 25 and 12) or equal potency (compare 26 and 14). On
the other hand, incorporation of 3-methyl (27) or 2-ethyl (28)
reduces potency by 2-fold. 4-Methyl substitution (29) and
spirocyclization of the amide (30) results in significant loss of
potency or complete inactivity. Unfortunately, plasma stability
for the equipotent compound 25 (15% remaining at 7 h) was
disappointing. Compound 28 (12% remaining at 7 h) is also
poorly stable in mouse plasma. Only the 2 fold weaker 2-methyl
compound 27 is stable in mouse plasma (100% remaining at 7
h).23
Table 2. SAR for mono(hetero)aryls
O
Ar
N
HN
O
Cl
Cpd #
11
Ar
N
MALT1 IC50 (µM)
0.10


N
12
13
14
0.10
0.05
0.98
Cl
N


Br
S
N
N
15
0.35


Table 3. SAR for substituted piperidines
O
N
16
17
0.41
0.83
N
R2
O
R3
Cl
CF3
N

R3
R2
Cpd #
25
MALT1
IC50 (µM)
0.05
O
CH3
N

N
18
6.03

HN

SO2

O
HN
O
N
26
27
28
Br
0.05
0.23
0.29
36
37
0.13
0.02
N



N
N
N


N



O
Cl
CH3
CH3
Cl
O
HN
O
N
HN
Cl


O
HN


Rac
Cl
N
O
N
29
30
CH3
CH3
>30
38
39
1.0


NH
N

N
N
H
Cl
Cl
O
11.2
N
O
O
0.54

N
HN

Cl

Next, we expanded our SAR investigation to the RHS amide
substituent, limiting substitution on the LHS to mono-
Compound 26 was first separated into cis and trans isomers by
column chromatography on silica gel and then separated into 4
enantiomerically pure diastereomers using chiral chromatography
(Table 5)24. In the MALT1 biochemical assay, the cis
diastereomer 40 is greater than10-fold more potent than the other
3 diastereomers (41-43). Unfortunately, in vitro ADME testing
showed compound 40 still highly unstable in mouse liver
hepatocytes (Clint: 31 µL/min/mg), mouse microsomal
preparation (Clint: 262 µL/min/mg) and mouse plasma (40%
remaining after 4 h).
(hetero)aryls, specifically 3-halogen-substituted pyridin-2-yl.
Combination of 3-chloropyridin-2-yl on the LHS and 3-chloro-2-
trifluoromethoxy-phenyl on RHS led to compound 31, which is
more than 10-fold less potent than its direct methoxy analog 12.
The cyclized benzofuran analog 32 maintains the same potency
as compound 12, while replacing 3-chloropyridin-2-yl with 3-
bromopyridin-2-yl 33 results in a 3-fold potency increase.
Continuing with the lower molecular weight 3-chloropyridin-2-yl
group as the LHS substituent, additional RHS exploration led to
several compounds more potent than 12: 3-chloro-2-
cyclopropoxy-5-pyridine (35), ~3-fold more potent; 8-chloro-2H-
6-benzo[b][1,4]oxazin-3(4H)-one (37), 5-fold more potent; 4-
chloro-5-methoxy-2-pyridine (34) and 3-chloro-2-(pyridin-3-
yloxy)-5-pyridine (36), both equipotent. However, substitution
of MeO by MeNH gave 38, 10-fold less potent than 13, while
removing a chloro from compound 37 results in more than 25
fold potency loss (compound 39)
Table 5. SAR of four diastereomers of compound 26
Cpd #
40
Structures
MALT1 IC50
(µM)
O
0.01
O
N
N
N
N
N
H
N
Table 4. SAR for RHS
Cl
O
Br
Ar
N
(cis diastereomer)
HN R4
41
42
43
O
0.18
0.15
17.4
O
R4
Cpd #
31
Ar
MALT1 IC50
(µM)
N
N
H
Cl
Br
CF3
O
N
1.74



2 (trans diastereomer)
O
Cl
Cl
N
O
32
0.10
N
N
H
Cl

O
O
O
Br
Cl
N
(trans diastereomer)
Cl
N
33
34
35
0.03
0.13
0.04
O
O


N
N
H
Br
Cl
Cl
N
N
Br



(cis diastereomer)
Cl
Cl
The cellular activities of selected MALT1 inhibitors were
evaluated. Inhibition of secretion of both IL6 and IL10 was tested
in TMD8 cells, and inhibition of cleavage of a MALT1 substrate,
RelB, was examined in OCI-Ly3 cells. Additionally, selected
compounds were also tested for inhibition of PMA/ionomycin-
stimulated IL2 production in Jurkat T cells to assess the effects of
MALT1 inhibitors on T cell activation. The tested compounds
show potencies for inhibiting IL6 and IL10 secretion and IL2
N
N

O
Cl
Cl

production with IC50 values from 0.05 – 1 µM, and also inhibited
RelB in the range of 0.07 – 1.55 µM (Table 6).
Figure 2. MALT1 mutant E397A vs MALT1 wt (top figure);
inhibition mechanism of compound 40 (bottom figure)
Table 6. In vitro activity for selected compounds
In summary, a series of potent and highly selective small-
molecule MALT1 inhibitors was discovered starting from an
HTS hit. Biochemical assay of an allosteric site mutant of
MALT1 (E397A), mechanism of inhibition studies, and the
Trp580 fluorescence quenching assay suggest that these
compounds are allosteric site binders. Advanced leads such as
compound 40 show high potency in the biochemical enzyme
assay as well as in cellular assays. Compound 40 also inhibited
cleavage of MALT1 substrates RelB and BCL10 in OCI-Ly3
cells and was shown to be highly selective for MALT1 versus
other proteases and kinases.
Cpd #
MALT1 IC50
(µM)
11
0.10
25
0.05
26
0.05
40
0.01
1
0.69
MALT1 E397A
IC50 (µM)
12.0
0.46
0.06
0.32
0.21
IL6/IL10, TMD8
cells IC50 (µM)
IL2, Jurkat PMA
IC50 (µM)
1.05/ 0.50/ 0.25/ 0.10/
1.70/
1.95
2.34
0.76
0.41
0.30
0.06
0.05
1.02
0.05
0.02
Rel B, OCI-Ly3
cells, IC50 (µM)
1.55
0.60
0.20
0.11
0.07
ABBREVIATIONS
MALT1: Mouse-associated lymphoid tissue lymphoma
translocation protein 1; ABC: activated B-cell; DLBCL: diffuse
large B-cell lymphoma; IL: interleukin; TMD8: Tokyo Medical
and Dental university 8; HTS: high throughput screening.
To address any potential off target protease activities, compound
40 was tested against a panel of 63 enzymes (Reaction Biology),
revealing no activity at 10 µM for any protease. Compound 40
also shown no activity against kinases (DiscoverX, <50%
inhibition of 109 kinases at 1 µM) and did not broadly inhibit
proliferation of cancer cells (Oncolead, IC50 >1 µM across a
panel of 91 tumor cell lines).
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/
It was reported MALT1 has two binding sites for inhibitors: the
paracaspase active site and allosteric binding site.11 In order to
understand why our compounds are so selective, we first
evaluated their ability to inhibit E397A mutant MALT1 protein25
(glutamic acid 397 forms a key part of the allosteric binding
pocket8), in order to confirm activity similar to that seen with
known allosteric site inhibitors such as phenothiazine
References and notes
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2. Martinez-Climent
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dervatives.11 In our MALT1-E397A assay, inhibitory potency of
compound 40 was reduced by about 4-fold (Figure 2). Second, a
detailed kinetic study26 indicated that the behavior of compound
40 best fit to a non-competitive inhibition mechanism (Figure 2).
Data analysis yielded the binding affinity (Ki) for the free
enzyme to be 9.6 ± 2.3 nM. Affinity for the enzyme-substrate
complex was 2.8-fold weaker than that for the free enzyme.
Third, an assay measuring fluorescence of an allosteric site
tryptophan residue (Trp580)11 showed fluorescence quenching
reaching saturation with an increasing amount of the compound
40 (data not shown). All these findings suggest that our inhibitors
bind to an allosteric pocket at the interface of the caspase
catalytic domain and the Ig3 domain.
3. Vincendeau M, Nagel D, Eitelhuber AC, Krappmann D, Int.
J. Hematol. Onco., 2013, 2(5), 409-417
4. Thome M, Nature Rev. Immunol., 2008, 8(7), 495-500
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Chennamadhavuni S, Gabas LF, Qiao Q, Du G, PCT Int.
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MALT1
120
WT
100
E397A
80
60
40
20
0
10. Wu G, Wang H, Zhou W, Zeng B, Mo W, Zhu K, Liu R, Zhou
J, Chen C, Chen H, Bioorg. Med. Chem., 2018, 26, 3321-3344
11. Schlauderer F, Lammens K, Nagel D, Vincendeau M,
Eitelhuber AC, Verhelst SHL, Kling D, Chrusciel A, Ruland
J, Krappmann D, Hopfiner KP, Angew. Chem. Int. Ed. 2013,
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-4
-3
-2
-1
0
1
log [Cpd 40] (M)
12. Pissot Soldermann C, Quancard J, Schlapbach A, Simic O,
[Cpd 40] (M)
Tintelnot-Blomley
M,
Zoller
T,
PCT
Int.
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0
Appl. (2015), WO 2015181747
0.00411
0.0123
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Vyavahare VP, Kulkarni KC, Sinha N, Palle VP, Kamboj RJ,
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0.037
0.111
0.333
1
500
1000
1500
2000
14. Fontan L, Yang C, Kabaleeswaran V, Volpon L, Osborne MJ,
Beltran E, Garcia M, Cerchietti L, Shaknovich R, Yang SN,
[Ac-LRSR-AMC] (M)

Fang F, Gascoyne RD, Martinez-Climent JA, Glickman JF,
Borden K, Wu H, Melnick A, Cancer Cell 2012, 22, 812-824;
50 minutes at room temperature (RT). Substrate was added
subsequently, and the reaction was then incubated for 4 hours
at RT, after which fluorescence was measured.
15. Nagel D, Spranger S, Vincendeau M, Grau M, Raffegerst S,
Kloo B, Hlahla D, Neuenschwander M, Peter von Kries J,
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22. TMD8 cells were treated with diluted compounds for
24 hours at 37°C and 5% CO2. After 24 hours of incubation,
50 µL of the supernatant was transferred to an MSD plate
(V-Plex Proinflammation Panel 1 [human] kit) and incubated
for 2 hours at RT followed by a 2-hour incubation with
IL-6/10 antibody solution. Plates were read on a SECTOR
imager.
16. Fontan L, Melnick A, Clin. Cancer Res., 2013, 19(24), 6662-
6668
17. Jaworski M, Thome M, Cell. Mol. Life Sci., 2016, 73(3), 459-
473
23. Note that compounds 25-28 were tested as mixtures of 4
diastereomers;
18. Hailfinger S, Lenz G, Thome M, Cur. Opin. Chem. Biol.,
24. The absolutely configurations are unknow, the steric centers
are assigned arbitrarily.
2014, 23, 47-55
19. Demeyer A, Staal J, Beyaert R, Trends Mol Med 2016, 22,
25. Dose dependence inhibition studies were performed in the
presence of 300 µM substrate Ac-LRSR-AMC and 5 nM
MALT1 enzyme. The steady-state reaction velocity was
measured under all conditions and the percent of activity
remaining was calculated by comparing to an uninhibited
reaction.
135-150
20. A related series of compounds was recently published by
Schlapbach A, Revesz L, Soldermann CP, Zoller T, Régnier
CH, Bornancin F, Radimerski T, Blank J, Schuffenhauer A,
Renatus M, Erbel M, Melkko S, Heng R, Simic O, Endres R,
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26. The steady-state activity of MALT1 was determined as a
function of the concentration of substrate, peptide Ac-LRSR-
AMC and 4.8 nM enzyme in the presence of 0, 0.00411,
0.0123, 0.037, 0.111, 0.333 and 1 µM of compound 40.
21. MALT1 protease activity was assessed in an in vitro assay
using a tetrapeptide substrate and full-length MALT1 protein
(Strep-MALT1(1-824)-His) purified from baculovirus-
infected insect cells. The tetrapeptide LRSR is coupled to
7-amino-4-methylcoumarin (AMC) and provides a quenched,
fluorescent substrate for the MALT1 protease (SM
Biochemicals). Cleavage of AMC from the arginine residue
results in an increase in coumarin fluorescence measured at
460 nm (excitation 355 nm). The final assay was performed
in the presence of 10 nM full-length MALT1 protein. Diluted
compounds were pre-incubated with MALT1 enzyme for
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Discovery and optimization of a series of sma
of MALT1 protease
Declaration of interests
Tianbao Lu  a, Peter J. Connollya, Ulrike Philipparb, Weime
Barbaya, Luc Gysb, Luc Van Nuffelb, Nigel Austinb, Mariet
Attara, Lieven Meerpoelb, James Edwardsa
☒ The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
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O
N
O
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N
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:
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MALT1 IC50: 0.10 M
Human IL6/IL10: 1.05/0.76 M
MALT1 IC50: 0.69 M
Human IL6/IL10: 1.70/1.95 M