Discovery of a Selective, Covalent IRAK1 Inhibitor with Antiproliferative Activity in MYD88 Mutated B-Cell Lymphoma

Article abstract of DOI:10.1021/acsmedchemlett.0c00378

Interleukin 1 (IL-1) receptor-Associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating the innate immune response against foreign pathogens. Additionally, dysregulation of IRAK1 signaling plays a role in neoplastic di

Full text of DOI:10.1021/acsmedchemlett.0c00378

pubs.acs.org/acsmedchemlett
Letter
Discovery of a Selective, Covalent IRAK1 Inhibitor with
Antiproliferative Activity in MYD88 Mutated B‑Cell Lymphoma
John M. Hatcher, Guang Yang, Li Wang, Scott B. Ficarro, Sara Buhrlage, Hao Wu, Jarrod A. Marto,
Steven P. Treon, and Nathanael S. Gray*
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ABSTRACT: Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in
initiating the innate immune response against foreign pathogens. Additionally, dysregulation of IRAK1 signaling plays a role in
neoplastic disorders. For example, IRAK1 was shown to be important for survival and proliferation in many B-cell lymphomas,
̈
including Waldenstrom’s macroglobulinemia (WM) and ABC subtype Diffused Large B-cell Lymphoma (DLBCL) cells. Here, we
report the discovery of a highly potent and selective covalent inhibitor of IRAK1, JH-X-119-01. Intact protein MS labeling studies
confirmed that JH-X-119-01 irreversibly labels IRAK1 at C302. This compound exhibited cytotoxic activity at single digit
micromolar concentrations in a panel of WM, DLBCL, and lymphoma cell lines expressing MYD88. Cotreatment of JH-X-119-01
with the BTK inhibitor ibrutinib resulted in synergistic killing effects in these systems. Taken together, JH-X-119-01 represents a
highly selective probe of IRAK1 for further development.
̈
KEYWORDS: IRAK1, MYD88, Waldenstrom’s macroglobulinemia, Diffused Large B-cell Lymphoma, Kinase inhibitor
Subsequent phosphorylation on IRAK1 by upstream signals or
nterleukin 1 (IL-1) receptor-associated kinases (IRAKs) are
Iserine/threonine kinases that play critical roles in initiating through autophosphorylation is the key post-translational
modification and hallmark of its activation, which allows
IRAK1 to bind to TRAF6 resulting in release of the IRAK1
homodimer from MYD88 and downstream NF-κB activation.4
The participation of IRAK1 in signaling networks of the innate
immune response makes it a critical regulator of inflamma-
tion,5 antiviral response,6 and subsequent activation of the
adaptive immune response.7 Consequently, an extensive
investigation into physiological and pathological functions of
IRAK1 in regulating these processes has been performed. In
particular, these studies have implicated IRAK1 inhibition as
the innate immune response against foreign pathogens. IRAKs
are involved in Toll-like receptors (TLRs) and IL-1R signaling
pathways and are recruited by activated MYD88 to the
receptor-signaling complex upon receptors activation. Alto-
gether there are four IRAK kinases: IRAK1 and IRAK4, which
are catalytically active kinases, and IRAK2 and IRAK3, which
are believed to be catalytically inactive and are hence classified
as “pseudokinases.”1 IRAK1 is ubiquitously expressed with its
highest expression observed in blood and immune tissues (for
example, bone marrow, lymph nodes, thymus, and peripheral
blood) and in hematological malignancies.2 IRAK signaling
contributes to multiple signaling pathways downstream of the
Toll-interleukin receptors (TIRs) that ultimately regulate NF-
κB and IFN regulatory factors (IRFs).3 In the case of NF-κB,
IRAK1 mediates the downstream signals of TIRs through an
interaction with MYD88 which is rapidly recruited to the
receptor upon ligand binding to either IL-1R or a TLR.
Received: July 8, 2020
Accepted: October 6, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00378
© XXXX American Chemical Society
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potential treatment for myocardial contractile dysfunction
following burn,8 autoimmune conditions associated with
hyperinflammation,9,10 myocardial dysfunction,11 microbial
septic response,12 human myelodysplastic syndrome
(MDS),13 and acute myeloid leukemia (AML).14 IRAK1 is
also overexpressed and hyperphosphorylated in a subset of
breast cancers: in particular, triple-negative breast cancer
(TNBC).15 Furthermore, in many B-cell lymphomas, including
of S(10) = 0.01 at a concentration of 1 μM (Figure 1). JH-X-
119-01 exhibited off-target inhibition of only two additional
kinases, YSK4 and MEK3. Dose response analysis revealed an
IC50 of 57 nM for YSK4. Biochemical assays for MEK3 were
not commercially available at the time of writing this
manuscript. Based on COBALT26 sequence alignment, there
are no cysteine residues in YSK4 or MEK3 in a similar location
to C302 or C307 of IRAK1 indicating that the binding of JH-
X-119-01 to YSK4 and MEK3 is likely reversible. LC-MS
analysis of labeled IRAK1 demonstrated JH-X-119-01 formed a
covalent bond with the protein. Subsequent digestion and
nanoflow LC-MS/MS27,28 confirmed this hybrid compound
irreversibly labeled IRAK1 preferentially at C302 (95%) vs
C307 (5%) (Figure 2).
̈
Waldenstrom’s macroglobulinemia (WM) and ABC subtype
Diffused Large B-cell Lymphoma (DLBCL) cells, the MYD88
L265P somatic mutation is highly prevalent and responsible for
malignant growth through activation of nuclear factor NF-κB.
Genetic knockdown of either BTK or IRAK1 leads to modest
cell killing,15 while co-inhibition of both BTK and IRAK1/4
results in synergistic tumor cell growth suppression.16
A
number of groups, including Amgen,17 Astellas,18 AstraZene-
ca,19 Genentech,20 Merck,21 Pfizer,22 and UCB,23 have
reported potent dual inhibitors of IRAK1/4. In addition,
several more dual IRAK1/4 inhibitors have recently been
reviewed.24 Notably, a highly selective IRAK4 inhibitor
developed by Nimbus with over 1000-fold selectivity for
IRAK4 over IRAK1 also showed synergistic killing of ABC
DLBCL cells with the BTK inhibitor ibrutinib.25 However,
medicinal chemistry efforts directed at the development of
selective inhibitors of IRAK1 have not been reported. Herein,
we describe the development of JH-X-119-01, a highly potent
and selective inhibitor of IRAK1.
The dual IRAK1/JNK inhibitor THZ-2-118 (Figure 1)
served as a lead compound for development of selective IRAK1
Figure 2. JH-X-119-01 (1) forms a covalent bond with IRAK1
preferentially at C302. (A) Mass spectra (left) and zero-charge mass
spectra (right) for IRAK1 protein treated with DMSO (top) or JH-X-
119-01 (1). Unlabeled protein peaks are labeled with orange glyphs,
while labeled protein peaks are indicated with green ones. After
treatment with JH-X-119-01, the protein shows an increase in mass
consistent with covalent labeling. (B) MS/MS spectrum acquired
during nanoflow LC-MS/MS analysis of trypsin digested, JH-X-119-
01 (1) treated IRAK1. Ions of type b and y are designated with blue
and red glyphs, respectively. Inhibitor specific ions are labeled with
green glyphs. Both y and b ion series localize the modification site to
C302. A small amount of of C307 (∼5%) was also detectable
Figure 1. Evolution of JH-X-119-01 starting from THZ-2-118 and
JH-I-25 and KinomeScan characterization of all three compounds at 1
μM showing the remarkable improvement in kinome selecvitivty for
JH-X-119-01. The portions of THZ-2-118 and JH-I-25 that were
fused are highlighted by pink and blue areas.
inhibitors. THZ-2-118 potently inhibits IRAK1 with an
apparent IC50 of 14.2 nM in a fixed time point assay by
covalently labeling cysteine residue C302. THZ-2-118 is
selective for IRAK1 over IRAK4; however, its potent inhibition
of JNK1/2/3 kinases limits its utility as an IRAK1 probe. We
sought to modify THZ-2-118 to remove JNK inhibition and
improve the overall kinome selectivity. In order to achieve
improved selectivity, we merged features of THZ series, which
selects IRAK1 over IRAK4, with the excellent kinome
selectivity of the dual IRAK1/4 inhibitor JH-1-2523 (Figure
1). Our resulting hybrid compound JH-X-119-01 (Figure 1)
inhibited IRAK1 biochemically with an apparent IC50 of 9 nM
while exhibiting no inhibition of IRAK4 at concentrations up
to 10 μM. The compound showed exceptional kinome
selectivity, as measured by a KINOMEScan selectivity score
The initial SAR of this series focused changing the trajectory
of the covalent warhead to target either C302 or C307 (Figure
4A). To accomplish this, we prepared a series of regioisomers
of the benzamide linkers shown in Table 1.
The representative preparation of JH-X-119-01 is presented
in Scheme 1. The 1,4−1,4 regioisomer (2) was less potent
against IRAK1 with an apparent enzymatic IC50 of 47 nM.
Interestingly, nanoflow LC-MS/MS analysis revealed this
compound was selective for C307. MS labeling studies
revealed (3) targeted both C302 and C307 (∼3:1 ratio).
The 1,3−1,3 regioisomer (4) was more potent than 2 and 3 in
the IRAK1 biochemical assay with an IC50 of 25 nM but was
still less potent than JH-X-119-01, and showed a strong
preference for C302 by nanoflow LC-MS/MS experiments. We
also prepared 5, the reversible version of JH-X-119-01 with a
B
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Table 1. SAR of the JH-X-119-01 Series
IRAK1
IRAK4
compd
IC50 (nM)
IC50 (nM)
labeling Site
Cys 302
Cys 307
Cys 302 and Cys 307
(3:1)
JH-X-119-01 (1)
9
47
55
>10 000
>10 000
>10 000
2
3
4
5
6
25
259
52
>10 000
>10 000
>10 000
Cys 302
N/A
N/A
Figure 3. (A) Cellular EC50’s for compounds 1−6. Cells were
incubated with inhibitors for 72 h and monitored via CellTiter-Glo
assay. (B) Design of desthiobiotin probe JH-XIII-39-1. (C) Western
blots for IRAK1 following pulldown assay by JH-XIII-39-01 at 5 μM
after the pretreatment of BCWM.1 WM cells with 1, 4, or 6 at the
indicated concentrations.
a
Scheme 1. Preparation of JH-X-119-01
moderate cell killing effects.15 We were initially concerned that
some of the cell killing effects could be due to potency against
YSK4. Therefore, we checked our gene expression profiling
data for primary tumor cells from patients with WM and for
healthy donor peripheral blood B-cells and confirmed that
YSK4 expression levels were very low in both cells. We also
searched the public protein atlas database for this kinase
expression and found the similar results that YSK4 expression
are barely detectable in general in B-cells or other hematologic
cells except dendritic cells. In addition, in a study using shRNA
library to screen multiple Defuse Large B-cell Lymphoma cell
lines, YSK4 was not on the list of genes that have impact on
cell survival.29 Based on this data, we do not believe YSK4
inhibition contributes to the efficacy of JH-X-119-01 in
MYD88 mutated B-cell lymphoma cells.
a
Reagents and conditions: (a) HATU, DIEA, DCM; (b) TFA, DCM;
(c) oxalyl chloride, 3-((tert-butoxycarbonyl)amino)benzoic acid,
DCM, DMF, pyridine; (d) acryloyl chloride, THF, NaHCO3, H2O;
(e) Pd(dppf)Cl2, t-BuXPhos, 1-(tetrahydro-2H-pyran-2-yl)-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Na2CO3,
1,4-dioxane, H2O, 100 °C.
propyl amide in place of the acrylamide warhead, which
showed a 25-fold loss in potency, supporting the importance of
the covalent bond formation for potency. In addition,
compound 6 with a dimethylamino group attached to the
warhead was prepared to improve the physicochemical
properties of JH-X-119-01, however, compound 6 showed a
5-fold loss in potency.
To understand how JH-X-119-01 binds to IRAK1, we
initiated a molecular modeling study (Figure 4) using the
We then tested compounds 1−6 in a panel of WM cells,
DLBCL cells, and lymphoma cells expressing mutant MYD88.
As seen in Figure 3A, JH-X-119-01 was the most potent,
showing moderate cell killing effects in these cells with EC50’s
ranging from 0.59 to 9.72 μM. Interestingly, despite having
apparent enzyme IC50’s that are 2−5-fold less than that of JH-
X-119-01, compounds 2 and 3 were much less potent in many
of these cells. In many cases, cellular EC50’s could not be
generated due to the lack of potency as indicated by N/A
(>1000 μM) in Figure 3A. EC50 curves for all compounds are
available in the Supporting Information (SI; Figure S1).
However, compound 6 was within 2−5-fold potency in these
cells as JH-X-119-01 despite having an apparent enzyme IC50
that is 5-fold less. This suggests that 6 might have better cell
permeabilility due to the dimethylamino solubilizing group.
Additionally, the reversible version of JH-X-119-01, compound
5 was much less potent highlighting the importance of the
covalent bond formation. Overall, these results are in
agreement with what has previously been observed with
IRAK1 genetic knockdowns, where IRAK1 knockdown had
Figure 4. (A) Molecular model of 1 in complex with IRAK1. (B)
Molecular model of 1 in complex with IRAK1 with only the
interacting residues shown. Interactions are shown with dashed lines.
Distances are in Ångstroms.
previously reported cocrystal structure of JH-I-25 with
IRAK1.30 See the SI for details regarding construction of the
molecular modeling study. Our molecular modeling study
predicted that JH-X-119-01 forms the same single hinge
contact with the carbonyl oxygen of L291. The pyridine ring
participates in a π−π stacking interation with Y288. In
addition, the central phenyl ring participates in a π−π stacking
C
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interation with F290. As previously reported,30 Y288
participates in a polar interaction network with the catalytic
lysine K239, E259, and D358 from the Asp-Phe-Gly (DFG)
motif to stabilize the DFG-inactive conformation. The pyrazole
moiety of 1 interacts with this network by forming a hydrogen
bond with the carboxylate group of D358. We also observed an
additional hydrogen bonding interaction with the amide linker
of 1 and D298. Consistent with our MS study, the acrylamide
warhead of 1 is in closer to C302 than C307 and is therefore
predicted to form a covalent bond with C302 instead of C307.
To understand the selectivity of JH-X-119-01 for IRAK1
over IRAK4, we superimposed the cocrystal structure of JH-I-
25 in complex with IRAK1 with the cocrystal structure of JH-I-
25 in complex with IRAK4 (Figure 5). JH-I-25 is able to bind
Figure 6. Combination index (CI) values for the determination of
synergistic (CI < 1.0) or additive (CI = 1.0) effects when combining
ibrutinib and JH-X-119-01 at indicated concentrations in (A) MYD88
mutated BCWM.1 WM and (B) TMD8 ABC DLBCL cells.
C302 as confirmed by mass spectrometry. Characterization of
1 in a panel of WM cells, DLBCL cells, and lymphoma cells
expressing MYD88 revealed that 1 exhibited moderate cell
killing effects in these cells. Compound 1 exhibits excellent
kinase selectivity as assessed by KinomeScan (468 kinases)
with only two off targets YSK4 and MEK3. Further
optimization of this chemotype especially in regard to in
vivo pharmacokinetics will be reported in due course.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00378.
■
sı
Figure 5. Structural analysis of JH-X-119-01 docked into the overlay
of the structure of IRAK1 and IRAK4.
Full Ambit profiling data for JH-X-119-01, experimental
details, EC50 curves, and MS labeling data for
compounds 2−4 (PDF)
both IRAK1 and IRAK4 due to the conserved ATP pocket of
IRAK1 and IRAK4. However, IRAK4 has a smaller ATP front
pocket (Figure 5 inset). Due to the smaller ATP front pocket,
JH-X-119-01 likely clashes with D278 of IRAK4. Therefore,
IRAK4 cannot accommodate JH-X-119-01.
AUTHOR INFORMATION
Corresponding Author
■
Nathanael S. Gray − Department of Cancer Biology, Dana-
Farber Cancer Institute, Boston, Massachusetts 02115, United
States; Department of Biological Chemistry & Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts
02115, United States; orcid.org/0000-0001-5354-7403;
Phone: 617-582-8590; Email: Nathanael_Gray@
dfci.harvard.edu
Nevertheless, to confirm cellular target engagement, we
designed the desthiobiotinylated version of JH-X-119-01, JH-
XIII-39-01 (7), by appending a linker from the solvent-
directed acrylamide moeity (Figure 3B). To ensure the
viability of 7 as a useful probe, we tested it in an enzyme
assay and found that it maintained inhibition of IRAK1
enzyme with an apparent enzyme IC50 of 70 nM. We then
conducted pull-down experiments (see the SI for experimental
details) using BCWM.1 WM cells (Figure 3C). All three
compounds were able to block IRAK1 pull-down by the
desthiobiotin compound 7, thereby confirming cellular target
engagement. However, our lead compound, JH-X-119-01 (1),
as well as compound 4 significantly blocked IRAK1 pull-down
at 10 μM and less so at 5 μM, as compared to 6, which
effectively blocked IRAK1 pull-down at 5 μM. These
differences are most likely due to better cell permeability of
6, resulting from the presence of the dimethylamino
solubilizing group or faster covalent bond formation of 6
with IRAK1. As BTK inhibition has been previously shown to
synergize with IRAK inhibition, we cotreated JH-X-119-01
with ibrutinib, a covalent BTK inhibitor in BCWM.1 and
TMD8 cells, which resulted in increased killing compared to
JH-X-119-01 alone (Figure 6).
Authors
John M. Hatcher − Department of Cancer Biology, Dana-
Farber Cancer Institute, Boston, Massachusetts 02115, United
States; Department of Biological Chemistry & Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts
02115, United States
Guang Yang − Bing Center for Waldenstrom’s
Macroglobulinemia, Dana Farber Cancer Institute, Boston,
Massachusetts 02115, United States; Department of Medical
Oncology, Dana Farber Cancer Institute and Harvard Medical
School, Boston, Massachusetts 02115, United States
Li Wang − Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts
02115, United States; Program in Cellular and Molecular
Medicine, Boston Children’s Hospital, Boston, Massachusetts
02115, United States
Scott B. Ficarro − Department of Cancer Biology, Department
of Oncologic Pathology, Blais Proteomics Center, Dana-Farber
Cancer Institute, Boston, Massachusetts 02115, United States;
Department of Pathology, Brigham and Women’s Hospital and
In summary, we have discovered that (1) is a potent and
remarkably selective inhibitor of IRAK1 kinase activity.
Compound 1 is a covalent inhibitor of IRAK1 and labels
D
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Letter
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Harvard Medical School, Boston, Massachusetts 02115, United
States
Sara Buhrlage − Department of Cancer Biology, Dana-Farber
Cancer Institute, Boston, Massachusetts 02115, United States;
Department of Biological Chemistry & Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts
02115, United States; orcid.org/0000-0003-4562-1823
Hao Wu − Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts
02115, United States; Program in Cellular and Molecular
Medicine, Boston Children’s Hospital, Boston, Massachusetts
02115, United States
Jarrod A. Marto − Department of Cancer Biology, Department
of Oncologic Pathology, Blais Proteomics Center, Dana-Farber
Cancer Institute, Boston, Massachusetts 02115, United States;
Department of Pathology, Brigham and Women’s Hospital and
Harvard Medical School, Boston, Massachusetts 02115, United
States; orcid.org/0000-0003-2086-1134
Steven P. Treon − Bing Center for Waldenstrom’s
Macroglobulinemia, Dana Farber Cancer Institute, Boston,
Massachusetts 02115, United States; Department of Medical
Oncology, Dana Farber Cancer Institute and Harvard Medical
School, Boston, Massachusetts 02115, United States
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00378
Author Contributions
Conceptualization: J.M.H., S.B., and N.S.G. Compound
synthesis: J.M.H. Cellular experiments: G.Y. MS studies:
S.B.F. and L.W. All authors have given approval to the final
version of the manuscript.
(11) Thomas, J. A.; Haudek, S. B.; Koroglu, T.; Tsen, M. F.; Bryant,
D. D.; White, D. J.; Kusewitt, D. F.; Horton, J. W.; Giroir, B. P.
IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects
against contractile dysfunction. American Journal of Physiology-Heart
and Circulatory Physiology 2003, 285 (2), H597−H606.
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E. A.; Thomas, J. A.; Spolarics, Z. IRAK1-Dependent Signaling
Mediates Mortality in Polymicrobial Sepsis. Inflammation 2013, 36
(6), 1503−1512.
(13) Rhyasen, G. W.; Bolanos, L.; Fang, J.; Jerez, A.; Wunderlich,
M.; Rigolino, C.; Mathews, L.; Ferrer, M.; Southall, N.; Guha, R.;
Keller, J.; Thomas, C.; Beverly, L. J.; Cortelezzi, A.; Oliva, E. N.;
Cuzzola, M.; Maciejewski, J. P.; Mulloy, J. C.; Starczynowski, D. T.
Targeting IRAK1 as a Therapeutic Approach for Myelodysplastic
Syndrome. Cancer Cell 2013, 24 (1), 90−104.
(14) Rhyasen, G.; Bolanos, L.; Fang, J.; Rigolino, C.; Cortelezzi, A.;
Oliva, E. N.; Cuzzola, M.; Starczynowski, D. T. Inhibition of IRAK1
As a Novel Therapeutic Strategy in Acute Myeloid Leukemia and
Myelodysplastic Syndrome. Blood 2011, 118 (21), 612.
(15) Wee, Z. N.; Yatim, S.; Kohlbauer, V. K.; Feng, M.; Goh, J. Y.;
Yi, B.; Lee, P. L.; Zhang, S. J.; Wang, P. P.; Lim, E.; Tam, W. L.; Cai,
Y.; Ditzel, H. J.; Hoon, D. S. B.; Tan, E. Y.; Yu, Q. IRAK1 is a
therapeutic target that drives breast cancer metastasis and resistance
to paclitaxel. Nat. Commun. 2015, 6, 8746.
(16) Yang, G.; Zhou, Y. S.; Liu, X.; Xu, L.; Cao, Y.; Manning, R. J.;
Patterson, C. J.; Buhrlage, S. J.; Gray, N.; Tai, Y. T.; Anderson, K. C.;
Hunter, Z. R.; Treon, S. P. A mutation in MYD88 (L265P) supports
the survival of lymphoplasmacytic cells by activation of Bruton
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Notes
The authors declare the following competing financial
interest(s): N.S.G. is a founder, science advisory board
member (SAB), and equity holder in Gatekeeper, Syros,
Petra, C4, B2S, Aduro, Inception, Allorion, Jengu and Soltego
(board member). The Gray lab receives or has received
research funding from Novartis, Takeda, Astellas, Taiho,
Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi.
S.P.T. lab receives or has received funding from Pharmacy-
clics/Abbvie, Janssen Pharmaceuticals, Beigene, and Bristol
Myers Squibb. J.A.M. serves on the SAB of 908 Devices. The
Marto lab receives sponsored research support from Vertex
and Astra-Zeneca. S.B. is a scientific advisory board member of
Adenoid Cystic Carcinoma Foundation. H.W. is a co-founder,
SAB member, and equity holder of Ventus Therapeutics.
ACKNOWLEDGMENTS
■
The authors wish to thank The David and Janet Bingham
Research Fund and Yang Family Fund of the International
Waldenstrom’s Macroglobulinemia Foundation, the Leukemia
and Lymphoma Society (Grant: R6507-18), the NIH SPORE
in Multiple Myeloma (Grant: 2P50CA100707-16A1), and
Peter S. Bing M.D. The authors also acknolwedge generous
financial support from the NIH: R01 CA233800, R01
CA222218 (to J.A.M.), and R03 TR002933; R37 AI050872
(to H.W.).
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