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Identification of #-lapachone analogs as novel MALT1 inhibitors to treat an aggressive subtype of diffuse large B-cell lymphoma Sang Min Lim, Yujeong Jeong, Suhyun Lee, Honggu Im, Hyun Seop Tae, Byung Gyu Kim, Hee Dong Park, Jonghoon Park, and Sungwoo Hong J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01415 • Publication Date (Web): 23 Oct 2015 Downloaded from http://pubs.acs.org on October 24, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Identification of β-lapachone analogs as novel MALT1 inhibitors to treat an aggressive subtype of diffuse large B-cell lymphoma Sang Min Lim, a Yujeong Jeong, a,b Suhyun Lee, a,b Honggu Im, a,b Hyun Seop Tae, a Byung Gyu Kim, c Hee Dong Park, c Jonghoon Park,*,c and Sungwoo Hong*,a,b a
Center for Catalytic Hydrocarbon Functionalizations, Institute of Basic Science (IBS), Daejeon 305-
701, Korea, bDepartment of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea, cDrug Discovery Institute, LG Life Sciences, Daejeon 305-738, Korea. RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) CORRESPONDING AUTHOR FOOTNOTE *To whom correspondence should be addressed. Fax: (+82) 42-350-2810; Tel: (+82) 42-350-2811; Email:
[email protected] (S. Hong); Fax: (+82) 42-866-2220; Tel: (+82) 42-861-2566; E-mail:
[email protected] (J. Park). ABSTRACT
The treatment of activated B cell–like DLBCL (ABC-DLBCL) is one of the urgent unmet medical needs because it is the most resistant DLBCL subtype to current therapies eagerly awaiting effective therapeutic strategies. Recently, the paracaspase MALT1 has emerged as a promising therapeutic target for the treatment of ABC-DLBCL. Herein, we report a new class of MALT1 inhibitors developed by ACS Paragon Plus Environment
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high-throughput screening and structure-based drug design. The original hit, 4-amino-1,2naphthoquinone series inhibited MALT1 activity but suffered from poor cellular activity. The extensive pharmacophore search led to the discovery of structurally similar β-lapachone that is a direct inhibitor of MALT1 and possesses favorable physicochemical properties. Molecular simulation studies suggested the possibility of the formation of a covalent bond between MALT1 and β-lapachone, which was corroborated by experimental wash-out studies. Inspired by this, we explored the structure-activity relationships by incorporating electron-withdrawing substituents at C8 position of β-lapachone. These MALT1 inhibitors exhibited potent anti-proliferative activity to OCI-LY3 cell line and inhibited the cleavage of CYLD mediated MALT1.
INTRODUCTION Diffuse large B-cell lymphoma (DLBCL) is the most common type among B-cell lymphomas, comprising approximately 35-40% of all cases in adulthood.1,2 DLBCL is a considerably heterogeneous disease in terms of patterns of gene expression, dependence on different oncogenic pathways and clinical outcomes of current therapies.3-5 The clinical response of each DLBCL subtype to current therapeutic regimens shows consistent differences, and activated B cell–like DLBCL (ABC-DLBCL) is the most resistant DLBCL subtype to current therapies, showing the worst overall survival rate, which urgently necessitates the development of new effective therapeutics for ABC-DLBCL.6,7 The hallmark of ABC-DLBCL is the constitutive activation of the proapoptotic NF-kB pathway resulting from several distinct molecular mechanisms such as mutations of CARD11,8 CD79,9 TNFAIP3/A20,10,11 and MYD88.12 A genetic screening study employing an RNA interference library demonstrated that the proliferation or survival of ABC-DLBCL cell lines was significantly compromised by knock-down of several genes in the NF-kB signaling pathway including IKBKB, CARD11, BCL10, and MALT1.13 Mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) is a crucial signaling mediator in B- and T-cell receptor signaling particularly due to its modulation of the NF-κB signaling pathway. Along with CARD11 and BCL10, MALT1 forms the “CBM” complex, which is a signaling ACS Paragon Plus Environment
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scaffold critical to maintaining the constitutive activation of the NF-kB pathway. Upon activation, the CBM complex recruits TRAF6, which subsequently ubiquitinates TAB2 and IKKγ. Then, TAB2 activates TAK1, which can phosphorylate IKKβ, eventually resulting in stimulation of the NF-kB signaling pathway. In addition to this scaffolding function, MALT1 also activates the NF-kB signaling pathway by its paracaspase activity. MALT1 cleaves and inactivates TNFAIP3/A20 and CYLD, both of which are negative regulators of NF-kB, thus potentiating the NF-kB signaling pathway.14,15 Indeed, it was demonstrated that inhibition of the paracaspase activity of MALT1 with an irreversible peptidebased MALT1 inhibitor, z-VRPR-fmk, significantly reduced the expression of the target genes of NFkB indicating suppression of the NF-kB pathway. In addition, treatment with the MALT1 inhibitor resulted in the on-target cytotoxicity to several ABC-DLBCL cell lines.16,17 Two recent studies reported the discovery of small molecule inhibitors of MALT1 protease activity: phenothiazine derivatives, reversible MALT1 inhibitors and MI-2, an irreversible MALT1 inhibitor (Figure 1).18,19 These compounds inhibit the catalytic activity and functions of MALT1 and are selectively cytotoxic to MALT1-dependent ABC-DLBCL cell lines. Furthermore, these compounds can suppress the growth of cancer in xenograft models in vivo. Therefore, the inhibition of MALT1 can be considered a promising and powerful approach for the treatment of ABC-DLBCL. Herein, we report our studies on the identification of a new class of potent β-lapachone-based MALT1 inhibitors through a combination of high-throughput screening and structure-based design strategies, and demonstrate the biological evaluation of these compounds in terms of anti-cancer effects on ABC-DLBCL cells.
Figure 1. Chemical structures of reported MALT1 inhibitors. ACS Paragon Plus Environment
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RESULTS AND DISCUSSION Discovery of New MALT1 Inhibitors. With the goal of developing a new structural class of potent MALT1 inhibitors, we initiated high-throughput screening to identify small molecules that can inhibit the paracaspase activity of MALT1. We set up a fluorescence-based assay employing Jurkat cell lysate and a fluorophore-conjugated substrate of MALT1, Ac-LRSR-AMC. After incubation of each screening compound with the peptide substrate, the degree of inhibition of the proteolytic activity of MALT1 was determined by measuring and comparing the fluorescence resulting from the release of AMC (7-amino4-methylcoumarin) after the cleavage of the tetrapeptide substrate by MALT1. We observed that the assay employing 15 µg of Jurkat lysate ensured the sufficient MALT1 activity window suitable for high-throughput screening (Figure S1). With this assay, we screened approximately 3400 small molecules composed of natural products, their derivatives, and in-house synthetic compounds. We designated hit compounds as those that inhibited MALT1’s activity by more than 40% at 100 µM compared to the control. Of particular significance is the observation that a profound effect on potency was displayed by 4-dimethylamino-1,2-naphthoquinone (1a, Figure 2a). Compound 1a was subjected to a subsequent full IC50 value determination resulting in an IC50 of 2.5 µM. To the best of our knowledge, the 1,2-naphthoquinone scaffold is not present in any of the MALT1 inhibitors reported so far. Considering its promising activity and low molecular weight of 201, compound 1a was selected as an initial scaffold from which much more potent MALT1 inhibitors can be derivatized. To obtain structural insight into the inhibitory mechanism of the identified inhibitor 1a, its binding mode was investigated by employing a MALT1 crystal structure (PDB ID: 3UO8)20 as a template. Figure 2b shows the lowest energy conformation of compound 1a as calculated with the Discovery Studio software. The two carbonyl groups of 1a appear to point toward the binding pocket region and maintain a key hydrogenbonding interaction with Cys 464 and His 465. Interestingly, both of the carbonyl groups of 1a appear to have close contact with residue Cys 464, implying the possibility of electrophilic carbonyl group being
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attacked by Cys 464. Compound 1a could be further stabilized in the paracaspase domain of MALT1 via hydrophobic interactions with residues His 415, Gly 416, Asp 462, Met 463, Phe 499 and Glu 500.
(a)
(b)
Figure 2. (a) Structure of the screening hit 4-dimethylamino-1,2-naphthoquinone (1a). (b) A calculated binding mode of compound 1a in the paracaspase domain of MALT1 (PDB ID: 3UO8).
To find more potent inhibitors, we focused our design attempts on the amino-1,2-naphthoquinone scaffold. In an initial effort to enhance binding affinity, a survey of the C4 group was conducted to evaluate the substituent space of 1a at the C4 position and our first round of analogues was focused on replacing the dimethylamino group with different types of amine appendages. The compound library was estabilished by two different facile routes allowing for rapid exploration of the SAR profile. Scheme 1 illustrates the general synthetic route of a two-step sequence for the preparation of compounds 1b-1k. Cerium-mediated conjugate addition between 1,2-naphthoquinone (2) and ethanol, followed by oxidation with NaIO321 provided 4-ethoxy-1,2-naphthoquinone (3) as a common synthetic intermediate. The intermediate can be easily equipped with a variety of substituted amino groups via
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addition and elimination processes.22 Interestingly, when installing glycine amide derivatives in order to syntheize compounds 1l-1n, we observed the direct reaction with 1,2-naphthoquinone was more effective (route B). Scheme 1. Synthesis of 4-amino-1,2-naphthoquinones.
The resulting compounds were tested in the Jurkat lysate assay to measure the potency of their inhibition of MALT1 activity (Table 1). Of particular significance was the observation that the ethylenediamine (1c, IC50 = 1.6 µM) could form favorable molecular interactions without causing a steric clash. We observed that the activity was reduced when the dimethylamino group was replaced with a methoxy group (1d, IC50 = 7.8 µM), indicating that the amine group at the ethylene appendage is critical in sustaining potency. Separately, although an ethylene amine group was relatively well tolerated, substitution with an ethylene phenyl group was deleterious to the inhibitory activity (1i, IC50 > 100 µM). Substitution with morpholine at this site also led to reduced activity (1e, IC50 = 21.9 µM). Interestingly, pyrrolidine (1j) and piperidine (1k) moieties were well tolerated, and a modest increase in activity was observed with pyrrolidine-containing compound 1j (IC50 = 1.5 µM). Similarly, glycine derivatives 1l, 1m, and 1n resulted in compounds that were generally of equivalent enzymatic potency, showing IC50 values between 3.0 and 4.1 µM. Molecular docking studies of compounds 1l-1n ACS Paragon Plus Environment
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consistently suggested that the amide moiety of the appendage plays an important role in determining activity by a hydrogen bonding interaction. Installation of 1,3-diaminopropane groups at the C4 position gave derivatives 1f and 1g, which featured a 5-10 fold reduction in potency compared with 1a. Table 1. Results of the Jurkat lysate assay for 4-amino-1,2-naphthoquinone compounds.
Compd
IC50 (µM)
Compd
1a
2.5
1h
14.9
1b
20.4
1i
>100
1c
1.6
1j
1.5
1d
7.8
1k
5.4
21.9
1l
3.0
1f
11.9
1m
4.1
1g
14.4
1n
3.1
1e
R
HN
N
R
IC50 (µM)
O
Data are mean values for two independent experiments performed in duplicate with standard error of 10
>10
>10
Data are mean values for two independent experiments performed in duplicate with standard error of 10
9.4
>10
4.0
>10
2.6
O O
HN
O
1d
Data are mean values for two independent experiments performed in duplicate with standard error of
