Discovery of Potent and Selective Inhibitors for ADAMTS-4 through DNA-Encoded Library Technology (ELT)

Article abstract of DOI:10.1021/acsmedchemlett.5b00138

The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-encoded triazine library by affinity selection. Structure-activity relationship studies based on the selection information led to the identification of potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC₅₀ of 10 nM against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE, and ADAMTS-13. These inhibitors have no obvious zinc ligand functionality.

Full text of DOI:10.1021/acsmedchemlett.5b00138

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Discovery of Potent and Selective Inhibitors for ADAMTS-4 Through
DNA-encoded Library Technology (ELT)
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Yun Ding,*,† Heather O’Keefe,† Jennifer L. DeLorey,§ David I. Israel,† Jeffrey A. Messer,† Cynthia
H. Chiu,† Steven R. Skinner,† Rosalie E. Matico,# Monique F. Murray-Thompson,# Fan Li,⊥ Mat-
thew A. Clark,‡ John W. Cuozzo,‡ Christopher Arico-Muendel,† Barry A. Morganφ
†GlaxoSmithKline, Platform Technology & Science, ELT-Boston, 830 Winter street, Waltham, MA 02451
§Tedor Pharma, Inc., 400 Highland Corporate Drive, Cumberland, RI 02864
‡X-Chem Inc., 100 Beaver Street, Waltham, MA 02453
#GlaxoSmithKline, Biological Reagent and Assay Development, 709 Swedeland Road, King of Prussia, PA 19406
⊥Tufts Healthcare Institute, 136 Harrison Ave, Boston, MA 02111
φCenter for Drug Discovery, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030
KEYWORDS. DNA-Encoded library, ADAMTS-4, inhibitors
ABSTRACT: The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for
osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-
encoded triazine library by affinity selection. SAR studies based on the selection information led to the identification of
potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-
methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC50 of 10 nM
against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE and ADAMTS-13. These inhibitors have no
obvious zinc ligand functionality.
Type II collagen and aggrecan are two major compo-
nents of cartilage matrix.1 Disregulation of proteolytic
enzymes can result in cartilage destruction, leading to
joint injury and diseases such as rheumatoid and osteoar-
selective and potent inhibitors, preferably without obvi-
ous metal chelating groups was our goal. Unfortunately
there are limited examples of ADAMTS-4 inhibitors with
potent activity and high selectivity reported in the litera-
ture.9-10
thritis.2
Currently
NSAIDS
(non-steroidal
anti-
inflamatories) are used to alleviate the symptoms of oste-
oarthritis. However, this treatment offers little, if any,
effect on the progress of the disease. One expectation is
that inhibiting aggrecanase should directly slow cartilage
breakdown as compared to current treatment for joint
disease.3-4 One choice for such therapeutic intervention is
the aggrecanase family of proteins (ADAMTS). This fami-
ly of zinc metalloproteases is characterized by degrada-
tion of aggrecan.5 Two closely related members,
ADAMTS-4 (Aggrecanase-1) and ADAMTS-5 (Aggre-
canase-2), cleave aggrecan at the same site (Glu373-
Ala374), which makes them attractive targets for the
treatment of osteoarthritis.6
DNA-encoded library technology has been developed
over a decade both in academia and industry. It has been
successfully applied in the discovery of small molecule
hits for various targets.11-16 This makes it possible to create
and screen a library containing large numbers of com-
pounds through affinity binding and provides an alterna-
tive solution in drug discovery along with current tech-
niques such as high throughput screening. Many potent
inhibitors for various targets have been successfully dis-
covered through this technology, including inhibitors for
ADAMTS-5.17-21 This paper will discuss the discovery of
low nanomolar, highly selective inhibitors of ADAMTS-4
using ELT.
Most metalloprotease (MMP) inhibitors were reported
to contain moieties that bind to the zinc, such as carbox-
ylate, hydroxamate, hydantoin and sulfhydryl groups.7
Even though such MMP inhibitors like Marimastat, Bati-
mastat, CGS-27023A and Prinomastat demonstrated sig-
nificant pre-clinical efficacy in vivo, they exhibited mus-
culoskeletal side-effects in humans.8 The identification of
As we reported earlier, a DNA-encoded library based on
a triazine scaffold and containing 800 million compounds
has been synthesized and has already delivered potent
inhibitors for both Aurora A kinase and p38 MAP kinase.12
In this library, 192 Fmoc amino acids were employed in
chemistry cycle 1, followed by installation of triazine with
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BB4
FIGURE 1. Selection of library against ADAMTS-4. (a) The triazine library. (b) The cube corresponding to 4-
(aminomethyl)benzoic acid as cycle 2 is shown in detail. The copy number of the selected species is indicated by the continuous
color and size of the points (darker and bigger points indicate higher copy number). The structure shown at bottom represents
the family of compounds defined by a line along the BB1 axis (arrow).
TABLE 1. (a) Full-length off-DNA molecules and their activities; (b) SAR at cycle 1 position.
cycle 1
R1
N
N
R
R
cycle 4
N
N
N
H
N
cycle 2
cycle 3
O
1: R = OMe
2: R = H
a
Compd
R1
R
IC50
Compd
R1
R
IC50
(nM)
(nM)
1a
OMe
60
1b
OMe
163
1c
1e
1f
OMe
OMe
OMe
181
60
30
1d
2a
1g
OMe
H
284
61
b
OMe
102
1h
1j
OMe
OMe
22
10
1i
OMe
H
23
6
2b
1k
1l
OMe
OMe
14
2c
H
H
8
88
2d
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and as such, provide a handle that could be used to im-
cyanuric chloride. Then 32 amino acids were incorporated
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2
3
4
5
6
7
8
in cycle 2, followed by substitution of chlorine with 340
amines in cycle 3. In cycle 4, 384 amines were acylated
with cycle 2 acid, yielding a 4-cycle library size of
802,160,640 (Figure 1a). Short double-stranded DNA tags
encoding each building block were ligated to the opposite
side of the headpiece at each stage of the small molecule
chemistry.
prove the physicochemical properties of the inhibitors.
We next turned our attention to the cycle 3 position on
triazine (Table 2). Changing tetrahydroisoquinoline to
tetrahydroquinoline (3) and isoindoline (4c), the activity
dramatically dropped from 10 nM to 5~6 µM. Alternative
substituents, such as F, at 6-position (4a) and 7-position
(4b) lost about 30-fold activity. A simple piperidine deriv-
ative (4e), which was included in the library, but was not
selected, resulted in a complete loss of activity. However,
with (3,4-dimethoxyphenyl)methanamine at R3 position,
the compound still maintained excellent activity (4d).
The affinity selection was performed on His-6 labeled
ADAMTS-4 protein immobilized on nickel resin. The li-
brary was passed over the bound protein for 1 hour and
the non-binders were washed away. The protein was de-
natured and the binding population was recovered. Two
additional rounds of selection were then performed, using
fresh protein at each round. After final PCR amplification
and DNA sequencing, we obtained 88242 sequences,
which encompassed 36061 unique structures. After re-
moving all sequences that occurred only once, the re-
maining families of chemotypes all contained 4-
(aminomethyl)benzoic acid as selected synthon at cycle 2.
Preferences of the other chemistry cycles can be visual-
ized in a 3D scatter plot (Figure 1b). There was a highly
populated plane, which corresponds to 6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline along the cycle 3 axis.
Within this plane, there was a line corresponding to a
family defined by the presence of N-ethyl-3-methylaniline
as the cycle 4 synthon, with no observable preference for
cycle 1. Notably, there was another less populated plane,
which corresponds to 1,2,3,4-tetrahydroisoquinoline along
the cycle 3 axis. The similarity between these two selected
cycle 3 provided additional support to the authenticity of
the chemotype.
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Table 2. SAR at cycle 3 position.
Compd
R3
IC50 (µM)
6.5
3
4a
4b
0.37
0.333
To confirm the selected features, a representative set of
the fully elaborated library warheads molecules were syn-
thesized (Table 1-a) using the protocol in Scheme 1. The
building blocks at cycle 1 were the decarboxy analogs of
the cycle 1 amino acids. The biochemical assay showed
these compounds (1a-1e, 2a) had activity ranging from 60
nM to 284 nM.
4c
5.6
4d
0.158
Additional truncations and modifications were per-
formed at the cycle 1 position on triazine (Table 1-b). In
the case of 6,7-dimethoxy-1,2,3,4- tetrahydroisoquinoline
derivative, truncation of the R1 to methyl amine improved
activity (1f). The chlorine intermediate (1g) also showed
good activity. Both the hydrolyzed byproduct (1h) and the
complete deletion of cycle 1 (1i) yielded better activity
than the amine derivatives (1a-1e). To increase the solu-
bility of the compounds, we installed diamines and gly-
cine at the cycle 1 position and they all showed potent
4e
>10
After exploring cycle 1 and cycle 3, we next turned our
attention to cycle 4 (Table 3). From the selection data,
besides N-ethyl-3-methylaniline which was strongly se-
lected for that cycle, we also observed other secondary
amines, including both aniline derivatives and aliphatic
secondary amines. To understand why primary amines
were not selected at this position, we synthesized the
compound with m-toluidine as cycle 4 synthon (5a). The
activity dropped about 50-fold. Further truncation to me-
thylamide (5b), free carboxylic acid (5c) and hydrogen
(5d) led to even more loss of the activity. When we trun-
cated the cycle 4 position to methylethylamide (5e), the
activity was improved about 45-fold compared with me-
thylamide (5b). Though its activity was about 6-fold less
compared with N-ethyl N-(m-tolyl) amide (1j), N-
activity (1j, 1k, 1l).
In the case of 1,2,3,4-
tetrahydroisoquinoline as cycle 3 synthon, both the dia-
mine derivatives (2b, 2c) and the complete deletion of
cycle 1 (2d) yielded similar or better activity compared to
the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline deriva-
tives. All of the compounds confirmed the selection result
that the cycle 1 was not necessary for the activity. Since
cycle 1 was also the position linked to the DNA, we pro-
posed that it might be the position exposed to the solvent
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Scheme 1. Synthesis of ADAMTS-4 off-DNA hits.a
3
4
5
6
7
8
NHBoc
TFA
NH2
R4
NHBoc
R4
R4
(A)
= R2NH2
HOOC
EDCI, DMAP
i
O
II
R3
O
I
ii
R4: cycle4 amine
Cl
9
Cl
R2NH2
iv
R1
iii
Cl
N
N
Cl
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N
N
N
N
N
N
R2
N
R1
R2
Cl
N
R1
N
N
v
N
N
R1
H
Cl
H
V
R1: amines
III
IV
R3 = H, OH or amines
R2NH2
Cl
N
Cl
R1
vii
N
N
(B)
N
N
R2
N
N
N
N
R1
R2
vi
H
N
N
Cl
R1: amines
V
H
IV
R3
N
R3
Cl
R3
N
N
N
R1
iii
R3
Cl
N
Cl
N
N
R4
N
N
N
R1
(C)
N
N
N
R1
N
N
R1
H
v, viii
R4
O
iv
Cl
N
i
Cl
N
R1
H
R1: amines
Cl
HOOC
VI
V
III
IV
a
reagents and conditions: (i) amine (1 equiv), EDCI (1.25 equiv), DMAP (0.2 equiv), CH2Cl2, 0 °C – rt; (ii) TFA (50% in
CH2Cl2), room temperature, 20 mins; (iii) amine (1 equiv), CH3CN/H2O (1/1), pH 9~10, 0 °C; (iv) amine (R2NH2 or R3) (1 equiv),
CH3CN/H2O (1/1), pH 9~10, room temperature overnight; (v) R3 = H: Pd/C, H2, room temperature 1 hour; R3 = OH: HCl (6 N, 10%
v/v), CH3CN/H2O (1/1), 80 °C overnight; R3 = amines: amine (5~10 equiv), CH3CN/H2O (1/1), 80 °C; (vi) amine (1 equiv), DIEA (2.5
equiv), NMP, 0 °C ; (vii) amine (5 equiv), NMP, 80 °C for 1 hour; (viii) 1N NaOH, room temperature overnight.
ethyl N-methyl amide (5e) had better molecular proper-
ties with respect to both molecular weight and clogP.
Since cycle 1 was the most tolerant position, next, we ex-
plored varying cycle 1 while fixing cycle 4 as N-ethyl N-
methyl amide. With N1,N1-dimethylethane-1,2-diamine at
cycle 1, the activity of the molecule (5f) was similar to that
of compound 5e. However, without an R1 substituent, the
activity dropped to 1.8 µM (5g). Substituting R1 with a
tertiary amine, the activity dropped dramatically (5h).
Replacing 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
with 1,2,3,4-tetrahydroisoquinoline at cycle 3, all the N-
ethyl N-methyl amide derivatives with diversity at the
cycle 1 position yielded similar activity (6a-6c). The above
data showed that, with a fine tuning at the R1 position, it
is possible to truncate the m-tolyl group while retaining
potency. It should be noted that the optimized com-
pounds with N-ethyl N-methyl amide at cycle 4 did not
originate from the library itself, but were identified under
the guidance of the selection SAR.
Compd
R1
R4
IC50
(µM)
5a
0.518
5b
Same as 5a
3
5c
5d
5e
Same as 5a
Same as 5a
Same as 5a
>10
3
Table 3. SAR at cycle 4 position.
0.064
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at 10 µM. Overall, the compound had >1000 fold selectivi-
5f
5g
5h
6a
6b
6c
0.081
1.817
8.5
1
2
3
4
5
6
7
8
ty for inhibition of ADAMTS-4 over ADAMTS-5, MMP-13,
ADAMTS-13 and TACE. A few other compounds 1b, 1d, 1e
and 1f also had excellent selectivity for inhibition of
ADAMTS-4 over TACE (see Supporting Information).
Compound 1j does not contain any obvious zinc ligand
functionality, like hydroxamate, which may explain its
specific activity towards ADAMTS-4.
Table 4. Selectivity Data for Compound 1j
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21
22
IC50 (µM)
0.01
Fold of selectivity
0.043
1.7
ADAMTS-4
ADAMTS-5
ADAMTS-13
MMP-13
-
>10
>1000
>1000
>1000
>1000
>10
>10
TACE
>10
5.5
In summary, we have described the discovery of a novel
and potent class of inhibitors for ADAMTS-4 using DNA-
encoded library technology. The selection data identified
numerous obligatory structural components such as the
N-alkyl amide and the tetrahydroisoquinoline that were
confirmed by off-DNA synthesis. In general, we did not
observe a clear correlation between the number of copies
of individual warheads sequenced and their correspond-
ing off-DNA potency. This could result from a number of
factors, including differing potency “on” and “off” DNA,
building block dependent variation in the chemistry dur-
ing library synthesis, as well as the low (~1%) sequencing
coverage of the library selection output. Nonetheless, tak-
en in aggregate, the selection-based enrichment profile is
predictive of off-DNA SAR, enabling additional chemistry
and optimization. The ELT selection output generated
ADAMTS-4 inhibitors that were highly selective with re-
spect to ADAMTS-5 and other MMPs and without any
obvious zinc ligand functionality. Further optimization of
these hits will be published elsewhere in the future.
The illustrated off-DNA compounds were synthesized
either via route A, B or C in Scheme 1. In route A, the cy-
cle 2,4 disynthon was synthesized through the acylation
between Boc-protected cycle 2 aminoacids and cycle 4
amine, followed by deprotection of the Boc group. Start-
ing from cyanuric chloride, two chlorines were substitut-
ed in turn with amine and cycle 2,4 disynthon under in-
creasing temperature yielding the monochloro intermedi-
ate. Final displacement of the last chlorine was conducted
under various conditions to yield the desired substituted
compounds or de-chloro compound. Similar to route A,
route B started with 2,4-dichloro-1,3,5-triazine, followed
by subsequent substitution of two chlorines with amines.
In route C, cyanuric chloride was subsequently substitut-
ed with two amines and an aminoester. After hydrolysis of
the ester, the corresponding acid was acyclated with
amine. Displacement of the last chlorine yielded the de-
sired final compounds.
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The selection data suggested that the cycle 1 synthon
was not selected and the cycle 2, 3, 4 synthons were high-
ly selected against ADAMTS-4. The synthesis of off-DNA
small molecules confirmed the selection data. Further-
more, under the guidance of the selection data, we found
that the cycle 4 can tolerate some level of truncation, as
long as it is a tertiary amide. Simple methylethyl amide is
enough for the potent activity. We propose that the ter-
tiary amide may have participated in the interaction with
the target. Though the R1 position was the most tolerant
position, while truncating R4 to smaller amides, there is a
preference for some substituents, such as secondary
amine instead of tertiary amine.
ASSOCIATED CONTENT
Supporting Information. Synthesis and characterization
data for the new compounds. This information is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Phone: 781-795-4290. Email: yun.x.ding@gsk.com.
Notes
The authors declare no competing financial interest.
Finally, we assessed the selectivity of these triazine hits
against other related zinc metalloproteinases. The com-
pound 1j was measured against ADAMTS-5, MMP13,
ADAMTS-13 and TACE (Table 4). For ADAMTS-5, we only
observed about 30% inhibition at a concentration of 10
µM. For all the other targets, the compound was inactive
ABBREVIATIONS
ELT, encoded library technology; DEL, DNA-encoded library;
TACE, tumor necrosis factor α-converting enzyme; MMP,
matrix metalloproteinase; SAR, structure activity relation-
ship.
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