Identification of NAE inhibitors exhibiting potent activity in leukemia cells: Exploring the structural determinants of NAE specificity

Article abstract of DOI:10.1021/ml2000615

MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE) and has advanced into clinical trials for the treatment of both solid and hematological malignancies. In contrast, the structurally similar compound 1 (developed by Millennium: The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to understand the determinants of NAE selectivity. A series of compound 1 analogues were synthesized through iterative functionalization of the purine C6 position and evaluated for NAE specificity. Optimal NAE specificity was achieved through substitution with primary N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated. When assessed in vitro, inhibitors reduced the growth and viability of malignant K562 leukemia cells. Through this study, we have successfully identified a series of sub-10 nM NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE selectivity.

Full text of DOI:10.1021/ml2000615

LETTER
pubs.acs.org/acsmedchemlett
Identification of NAE Inhibitors Exhibiting Potent Activity in Leukemia
Cells: Exploring the Structural Determinants of NAE Specificity
Julie L. Lukkarila,†,^ Sara R. da Silva,†,^ Mohsin Ali,‡,^ Vijay M. Shahani,† G. Wei Xu,‡ Judd Berman,§
Andrew Roughton,§ Sirano Dhe-Paganon,|| Aaron D. Schimmer,*,‡ and Patrick T. Gunning*,†
†Department of Chemistry, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON, L5L1C6, Canada
‡Division of Hematology and Oncology, The Princess Margaret Hospital and the Ontario Cancer Institute, 610 University Avenue,
Toronto, ON, M5G2M9, Canada
§Dalton Medicinal Chemistry Inc., 349 Wildcat Road, Toronto, ON, M3J 2S3, Canada
Structural Genomics Consortium, University of Toronto, 100 College Street, Toronto, ON, M5G1L5, Canada
S Supporting Information
b
ABSTRACT: MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE)
and has advanced into clinical trials for the treatment of both solid and hematological
malignancies. In contrast, the structurally similar compound 1 (developed by Millennium:
The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin
activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed
as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to
understand the determinants of NAE selectivity. A series of compound 1 analogues were
synthesized through iterative functionalization of the purine C6 position and evaluated for
NAE specificity. Optimal NAE specificity was achieved through substitution with primary
N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated.
When assessed in vitro, inhibitors reduced the growth and viability of malignant K562
leukemia cells. Through this study, we have successfully identified a series of sub-10 nM
NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE
selectivity.
KEYWORDS: NAE inhibitors, leukemia, anticancer drugs, proteasomal inhibitors
biquitin (Ub) and ubiquitin-like (Ubl) proteins, such as
neural precursor cell-expressed developmentally downregu-
labeled.9,10 These CRLs are therefore responsible for the ubiqui-
tination and targeted degradation of a plethora of proteins within
the cell. Other important NEDD8 targets include the mouse
double minute 2 (MDM2) oncogene product,11 the p53 tumor
suppressor protein,12 the von Hippel-Lindau protein (pVHL),13
the epidermal growth factor (EGF) receptor,14 the breast cancer-
associated protein 3 (BCA3),15 and the estrogen receptor-R
(ERR).16 Elevated levels of NEDDylation have been observed in
aggressively proliferating malignant cells and have thus been
identified as a promising target for cancer therapeutics.17
Recent studies by Millennium: The Takeda Oncology Com-
pany reported an AMP mimetic, compound 1 (Figure 1), com-
posed of a purine base substituted at C6 and N9 with a
hydrophobic 2,3-dihydroindene moiety and a ribose sulfamate
substituent, respectively.18,19 Compound 1 strongly bound and
equipotently inhibited NEDD8 activating enzyme (NAE), ubi-
quitin activating enzyme (UAE), and SUMO activating enzyme
(SAE) with a low nanomolar potency (IC50 = 5 nM). Inhibition
of NAE by compound 1 is achieved through a NEDD8ꢀinhibitor
adduct formed in situ that inhibits the normal progression of the
U
lated 8 (NEDD8) and small ubiquitin-related modifier (SUMO),
are essential mediators of cellular function.1ꢀ3 Through a cascade
of three enzymatically catalyzed events, Ub/Ubls are conju-
gated to target proteins, designating them for various fates such
as degradation, translocation, signaling, and regulation of tran-
scriptional activity.4,5 In an ATP-dependent step, Ub/Ubl binds
to the Ub or Ubl activating enzyme (E1), resulting in Ub/Ubl
adenylation. Subsequent conformational changes in E1 structure
mediate intramolecular nucleophilic attack of the adenylated
Ub/Ubl by a cysteine residue on E1, covalently attaching Ub/Ubl
to E1 through a thioester linkage (Ub/Ubl-E1). A second Ub/
Ubl is adenylated by E1, resulting in a doubly Ub/Ubl loaded E1.
Next, Ub/Ubl is transferred to the Ub or Ubl conjugating
enzyme (E2) via transthiolation, which, in concert with a Ub
or Ubl ligase (E3), covalently attaches the Ub/Ubl to target
proteins.6,7
NEDD8-labeled proteins play a crucial role in the assembly
and function of the largest family of E3 Ub ligases, the cullin ring
ligases (CRL).8 NEDDylated cullin proteins serve as a scaffold
for the assembly of the CRL and enhance CRL efficiency by
increasing the recruitment of Ub-E2 complexes and assisting in
their most efficient positioning relative to the substrate to be
Received: February 26, 2011
Accepted: May 16, 2011
Published: May 16, 2011
r
2011 American Chemical Society
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Figure 1. (A) Chemical structures of compound 1 and MLN4924 (2) and (B) GOLD docking of compound 1 in the ATP binding pocket of NAE
(PDB: 3GZN).
Scheme 1a
a Reagents and conditions: (a) p-TsOH, acetone, room temperature, 3 h, 92ꢀ95%. (b) HNR1R2, DMSO, 120 ꢀC, 1ꢀ5 h, microwave assisted, 70ꢀ97%.
(c) (i) NaH, THF, N2, 0 ꢀC, 30 min; (ii) H2NSO2Cl, room temperature, 2ꢀ5 h, 63ꢀ88%. (d) TFA:H2O (3:1), room temperature, 1ꢀ1.5 h, 50ꢀ89%.
cycle by preventing transthiolation to E2. The nucleophilic
sulfamate attacksthethioester NEDD8-E1intermediate, resulting
in a covalently bound NEDD8ꢀinhibitor adduct.19 This adduct
effectively inhibited the activity of NAE, leading to increased
levels of CRL proteins in cells treated with compound 1.
MLN4924 (2, Figure 1),20 an analogous inhibitor also developed
by the Millennium group, is a similarly potent NAE inhibitor but
is significantly more specific for NAE than compound 1. The
backbone of MLN4924 is composed of a pyrrolopyrimidine base,
substituted at C6 and N9 with a hydrophobic 2,3-dihydroindene
moiety and a (hydroxycyclopentyl)methylsulfamate appendage,
respectively. In vitro, this compound reduces the growth and
viability of malignant cells including HCT-116 (colorectal),
Calu-6 (lung), SKOV-3 (ovarian), CWR22 (prostate), OCI-
LY19 (lymphoma), and AML (acute myeloid leukemia).20ꢀ23
In vivo studies of MLN4924 with solid tumor xenografts (HCT-
116, H522, and Calu-6) showed significant inhibition of tumor
growth, and AML xenografts showed disease regression.20,23
Currently, this drug is being evaluated for the treatment of
leukemia and lymphoma.
usinggenetic optimization for ligand docking (GOLD) software,24
showed that the 2,3-dihydroindene moiety (present in both
compound 1 and MLN4924) bound in the region above the
ATP pocket formed by residues Ile148, Gln 149, Ile 170, and
Trp174 in NAE (highlighted in green in Figure 1). This “gate-
keeper” region of the ATP pocket appeared to be an attractive
target for developing isoform-specific E1 inhibitors. Thus, with
particular emphasis on understanding the determinants of NAE
specificity, we prepared a series of 45 C6 N-alkylated adenosine-
sulfamate analogues, incorporating substituents of varying size,
flexibility, and functionality. From this focused library, we identi-
fied a series of NAE-specific inhibitors that mirror MLN4924's
potency toward NAE but have reduced activity against other E1s,
as compared with compound 1.
Briefly, C6 N-alkylated adenosine-sulfamate analogues, 8ꢀ51,
were prepared via a facile synthetic route outlined in Scheme 1.
First, the 20,30-ribose diol of 6-chloropurine riboside (3) was
ketal protected using acetone to furnish 4 (95%). Next, micro-
wave-assisted nucleophilic aromatic substitution of the 6-chloro-
purine with a series of primary and secondary amines produced
5(aꢀz, aaꢀas) in excellent yields (70ꢀ97%). Subsequent
deprotonation of the 50-hydroxyl group using NaH in
THF and reaction with a preprepared sulfamoyl chloride
reagent25 furnished the sulfamate 6(aꢀz, aaꢀas) in good yields
The structures of compound 1 and MLN4924 are similar, but
the molecules differ significantly in their specificity for NAE. Here,
we explored the determinants of NAE selectivity. Virtual docking
of compound 1 within the ATP pocket of NAE (PDB: 3GZN),
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Figure 2. In vitro evaluation of compound 1 analogues against E1 enzymes. Inhibitors were evaluated in cell-free enzymatic assays to determine their
effects on the activity of NAE (A), UAE (B), SAE (C), and UBA5 (D). Increasing concentrations of analogues were added to the appropriate E1, E2, and
Ub/Ubl substrate along with ATP. After 1.5 h of incubation at room temperature, products were fractioned on SDS-PAGE gels followed by
immunoblotting. Concentration of inhibitor: lane 0, 0 μM; lane 1, 0.001 μM; lane 2, 0.01 μM; lane 3, 0.1 μM; lane 4, 1 μM; and lane 5, 10 μM.
Millennium's compound 1 and MLN4924 (2).
(63ꢀ88%). Finally, removal of the ketal protecting group was
achieved under acidic conditions using TFA:H2O to yield
products 8ꢀ51 (50ꢀ89%). Compound 7 was synthesized in
an analogous manner to that described in Scheme 1, starting from
adenosine instead of 6-chloropurine riboside.
Inhibitors were principally assessed for effects on Ub/Ubl-E2
conjugate formation via a modified enzymatic assay, described
previously26 and in the Supporting Information. Briefly, purified
human recombinant E1 proteins were incubated with their
respective Ub/Ubl and E2 proteins in a buffer containing
50 mM HEPES, pH 7.4, 5 mM MgCl2, 1ꢀ20 μM ATP, and
increasing concentrations of compounds 7ꢀ51. The protein
complexes generated were separated on a 10ꢀ20% gradient
nondenaturing sodium dodecyl sulfateꢀpolyacrylamide gel elec-
trophoresis (SDS-PAGE), followed by immunoblotting. The
His6-tagged UbꢀUbcH6 complex was detected using a mouse
monoclonal primary antibody (1:2500) specific for His6. Rabbit
polyclonal primary antibodies were used for the detection of
other E2ꢀUbl complexes [anti-SUMO-1 (1:500) for SAE1/
SAE2, anti-NEDD8 (1:500) for APPBP1-UBA3, and anti-UFM1
(1:1000) for UBA5]. Ub/UblꢀE2 complexes were revealed
using enhanced chemiluminescent detection. As a control, we
demonstrated that the NEDD8 linkage with the E1 and E2
loading was sensitive to reducing agents (Supporting In-
formation). Densitometry was used to semiquantify the efficacy
of the inhibitors in the gel-based assays.
The effect of substituting the C6 position of compound 1 with
a variety of N-substituted amines on E1 enzymatic activity was
examined (representative examples shown in Figure 2 and in the
Supporting Information). In general, we found that the C6
analogues were selective for NAE over UAE, SAE, and UFM1
activating enzyme (UBA5) (Figure 2), regardless of the C6
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when 13 was substituted with a methyl group, as in 22, the
compounds equipotently inhibited NAE (Figure 2). We believe
the incorporation of the optimal alkyl chain overcomes the
requirement for minimal steric bulk around the C6 nitrogen.
When C6 was substituted with a series of tertiary amines, no
inhibition of NAE activity was observed at concentrations <10 nM
(i.e., 26ꢀ30, 34, 46, 47, and 50; see 27, 30, 34, and 50 in Figure 2;
see 26 and 47 in the Supporting Information; 28, 29, and 46 data
not shown). At higher concentrations, inhibitory effects were
observed for tertiaryamineanalogues;however, thecorresponding
secondary amines all elicit higher potency than their more sub-
stituted counterparts (11 > 26, 15 > 28, and 33 > 34, data not
shown). These data suggested that the peripheral region of the
ATP-bindingsite isstericallyconstrained. Insupportof this theory,
the unsubstituted 6-aminopurine (7) and N-methylated 6-amino-
purine (8) both showed promising activity (7, <16% inhibition at
1 nM, and 8, <76% inhibition at 10 nM; Supporting Information).
We also noted that cyclic aliphatic compounds, employed to
reduce entropic binding costs and enhance binding efficiency,
were less potent inhibitors of NAE than analogous straight chain
alkyl groups (12 > 31, 13 > 38, 14 > 48, Supporting Information).
Interestingly, we observed higher disruption of both NAE and
UAE activity by increasing the ring size of the cyclic alkyl
appendage (i.e., 5- < 6- < 7- < 8-membered rings) with compound
49 (an 8-membered ring) showing similar activity to 13 against
NAE. However, increasing the ring size beyond eight members,
using a 2-methyl-15-crown-5 appendage in 51, resulted in lower
activity against both NAE and UAE, relative to 49.
We next evaluated a series of heterocyclic derivatives. The
most potent inhibitors in this class of compounds were identified
as the furan-3-ylmethanamine (33) and thiophen-3-ylmethana-
mine (35), which equipotently inhibited NAE activity (∼100%
inhibition at 10 nM). However, both heterocyclic analogues were
less potent than our N-hexyl derivative (13). Comparative
inhibition of E1 activity by 13 and 35 is shown in Figure 2.
Additionally, we confirmed that active compounds 1 and 13
inhibited loading of NEDD8 onto UBA3 and APPB1 (Supporting
Information).
Our SAR identified determinants of selectivity for NAE as
compared to UAE and SAE. However, AMP mimetics may
exhibit promiscuity for other enzymes with ATP binding sites,
such as kinases. Therefore, we evaluated the specificity of our lead
NAE inhibitor, 13, against a panel of ATP-dependent kinases
including JAK1, JAK3, ABL, and EGFR (Supporting In-
formation). Encouragingly, 13 showed no activity against the
selected kinases at a concentration of 100 nM.
Figure 3. Low energy GOLD-docked 13 (green) in the ATP binding
pocket of NAE (PDB: 3GZN).
amino substituent. The addition of sterically hindered substitu-
ents at C6 had detrimental effects on the potency of molecules
against all E1 isoforms; however, these modifications were better
tolerated by NAE (greater inhibition of activity) than UAE or
SAE (Figure 2). We also noted that all molecules showed no
effect against UBA5, even when tested at 10 μM. This suggests
that the NAE specificity can be achieved with larger, more
sterically hindered purine substrates.
We were able to identify a number of interesting structureꢀ
activity relationship (SAR) trends, which help further explain the
basis for NAE selectivity. Moreover, these SAR trends may be
useful for the development of new E1-targeting scaffolds. First,
the data revealed that compounds incorporating hydrophobic,
straight chain, N-alkyl substituents at C6 were among the most
potent NAE inhibitors, that is, 13, where R = n-hexyl, NAE IC50 <
10 nM (Figure 2). Interestingly, the length of the N-alkyl chain
was shown to play an important role in NAE binding potency.
For example, at 1 nM, when R = methyl (8) or n-propyl (9), there
was no reduction in the formation of the UBC12ꢀNEDD8
complex. However, when extended to either an n-butyl (11) or
n-pentyl (12) chain, we observed <58 and <62% NAE inhibition,
respectively, at 1 nM (Supporting Information). Moreover,
further extension using an n-hexyl substituent (13) showed
>70% inhibition of NAE activity at the same concentration. At
this chain length, we believe that the alkyl group optimally
occupies a hydrophobic region composed of residues Ile148,
Gln149, Ile 170, and Trp174 (Figure 3). Further extension using
an n-heptyl appendage (14) reduced NAE inhibition (no inhibi-
tion at 1 nM, Figure 2), suggesting that chain lengths >6 carbons
are not well tolerated by NAE.
As mentioned previously, incorporating steric bulk proximal
to the C6 nitrogen negatively impacted inhibition of NAE
activity. For example, in the straight chain alkyl series of
compounds, decreased potency was observed when the carbon
R to the C6 nitrogen was substituted with either a methyl
(21, where R = 2-butyl, <25% inhibition of human
UBC12ꢀNEDD8 complex at 10 nM cf. 9, where R = n-propyl,
<63% inhibition human UBC12ꢀNEDD8 complex at 10 nM,
Figure 2) or a gem-dimethyl group (20, R = 1,1,3,3-tetramethyl-
butylamine, no inhibition of UBC12ꢀNEDD8 complex at
10 nM cf. 19, where R = 3,3-dimethylbutylamine, <29% inhibition
of UBC12ꢀNEDD8 complex at 10 nM, Figure 2). Interestingly,
Chemical and genetic inhibition of NAE reduces the prolif-
eration of malignant cells in culture. Therefore, we evaluated the
effects of our analogues on the growth and viability of leukemia
cells. K562 leukemia cells were treated with increasing concen-
trations of selected analogues for 72 h. After incubation, cell
growth and viability were measured by the CellTiter-Glo Lumi-
nescent Cell Viability assay (Table 1). Encouragingly, the ability
of compounds to inhibit the proliferation of K562 matched their
ability to inhibit NAE.
We also determined the effects of 13 on the activity of NAE in
vitro. K562 cells were treated with increasing concentrations of
13 for 24 h. After incubation, cells were harvested, and total
cellular protein was isolated. The presence of NEDD8ꢀcullin
conjugates was evaluated by immunoblotting. Compound 13
inhibited the enzymatic activity of NAE as evidenced by reduc-
tions in NEDD8ꢀcullin conjugates (Figure 4). Moreover, the
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Table 1. Growth Inhibition (EC50) of K562 Leukemia Cells by Compound 1 Analogues (n = 2)
* The backbone for compound 2 (MLN4924) differs from the other compounds listed. See Figure 1 for the complete structure.
’ ASSOCIATED CONTENT
S
Supporting Information. Full synthetic details, experi-
b
mental procedures, and copies of H and 13C NMR spectra.
This material is available free of charge via the Internet at http://
pubs.acs.org.
1
’ AUTHOR INFORMATION
Figure 4. Inhibition of NEDD8 conjugation in K562 cells by 13.
Corresponding Author
concentration of 13 required to inhibit NAE matched the
concentration required to inhibit cellular proliferation. Although
13 inhibited NEDD8 activity at concentrations associated with
and times that preceded loss of cell growth and viability, we
cannot exclude additional off-target effects that may contribute to
cytotoxicity. Likewise, we cannot exclude additional targets for
the other analogues tested in the cell growth and viability assays.
*Tel: 416-946-2838. Fax: 416-946-6545. E-mail: aaron.schimmer@
utoronto.ca (A.S). Tel: 905-828-5354. Fax: 905-569-5425.
E-mail: patrick.gunning@utoronto.ca (P.G.).
Author Contributions
^These three authors contributed equally to this work.
Funding Sources
This work was supported by the Ontario Ministry for
Innovation (P.T.G.) and by an Ontario Postdoctoral Fellowship
(J.L.L.).
’ CONCLUSIONS
Our preliminary SAR study of the purine C6 position identi-
fied selective and highly potent NAE inhibitors via substitution
with N-aliphatic R groups. In particular, 13, which contains an N-
hexyl R group, exhibited impressive inhibition with a potency
that rivals MLN4924 (Figure 2). Finally, compounds containing
furan and thiophene moieties (33 and 35, respectively), which
could provide increased metabolic stability, showed minor
reductions in potency, as compared to 13. Thus, by under-
standing the determinants of NAE selectivity, additional NAE
inhibitors may be developed with different stability and meta-
bolic profiles and offer insight into the design of selective
inhibitors that target UAE and SAE.
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