From PARP1 to TNKS2 inhibition: A structure-based approach

Article abstract of DOI:10.1021/acsmedchemlett.9b00654

Tankyrases (TNKSs) have recently gained great consideration as potential targets in Wnt/β-catenin pathway-dependent solid tumors. Previously, we reported the 2-mercaptoquinazolin-4-one MC2050 as a micromolar PARP1 inhibitor. Here we show how the resolution of the X-ray structure of PARP1 in complex with MC2050, combined with the computational investigation of the structural differences between TNKSs and PARP1/2 active sites, provided the rationale for a structure-based drug design campaign that with a limited synthetic effort led to the discovery of the bis-quinazolinone 5 as a picomolar and selective TNKS2 inhibitor, endowed with antiproliferative effects in a colorectal cancer cell line (DLD-1) where the Wnt pathway is constitutively activated.

Full text of DOI:10.1021/acsmedchemlett.9b00654

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Letter
From PARP1 to TNKS2 Inhibition: A Structure-Based Approach
Stefano Tomassi, Julian Pfahler, Nicola Mautone, Annarita Rovere, Chiara Esposito, Daniela Passeri,
Roberto Pellicciari, Ettore Novellino, Martin Pannek, Clemens Steegborn,* Alessandro Paiardini,*
Antonello Mai,* and Dante Rotili*
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ABSTRACT: Tankyrases (TNKSs) have recently gained great
consideration as potential targets in Wnt/β-catenin pathway-
dependent solid tumors. Previously, we reported the 2-
mercaptoquinazolin-4-one MC2050 as a micromolar PARP1
inhibitor. Here we show how the resolution of the X-ray structure
of PARP1 in complex with MC2050, combined with the
computational investigation of the structural differences between
TNKSs and PARP1/2 active sites, provided the rationale for a
structure-based drug design campaign that with a limited synthetic
effort led to the discovery of the bis-quinazolinone 5 as a picomolar and selective TNKS2 inhibitor, endowed with antiproliferative
effects in a colorectal cancer cell line (DLD-1) where the Wnt pathway is constitutively activated.
KEYWORDS: Tankyrases, adenosine subpocket ligands, selective inhibition, quinazolinones, antitumor
DP-ribosylation consists in the catalytic transfer of the
ADP-ribose moiety from the nicotinamide adenine
development of inhibitors for PARP1/2, two enzymes engaged
in DNA break repair.6 Recently, some of these compounds
moved forward to clinical applications as chemosensitizers in
cytotoxic therapeutic blueprints or as single agents in BRCA-
defected tumors.7−9 The sustained interest in PARPs as
anticancer targets together with the therapeutic potential of
the above-mentioned TNKS functions increased the appeal of
TNKS1/2 in medicinal chemistry.3,10 Indeed, TNKS inhibitors
especially as Wnt/β-catenin pathway modulators have solid
perspectives in oncology, particularly in colorectal cancer
(CRC) and nonsmall cell lung cancer (NSCLC).3−5,10,11 In
addition, tackling TNKS-mediated ADP-ribosylation may
provide a valuable alternative in viral infections and fibrotic
diseases treatments.3,5,10,11 Until now, most TNKS inhibition
approaches have focused on occupying the NAD+ binding site,
formally divided into nicotinamide (NAM) and adenosine (AD)
subsites, which is outlined by the donor D-loop, the glycine-rich
G-loop and the Phe-containing F-loop.7,12 Leveraging inter-
actions in the NAM subpocket represented a validated strategy
to inhibit ADP-ribosylation, and different chemotypes were
found active in this sense.6,7,11 Unfortunately, the high grade of
sequence homology and the spatial organization in this region
renders NAM-mimicking compounds promiscuous PARP
A
dinucleotide (NAD+) cofactor to acceptor proteins and is
mediated by the 17-membered family of ADP-ribosyltrans-
ferases diphtheria toxin-like (ARTDs), formerly known as
poly(ADP-ribose) polymerases (PARPs).1,2 Although all family
members catalyze the initial attachment of ADP-ribose
monomers on different amino acid side chains, only a subset
of them can further elongate and/or branch the ADP-ribose tail
by polymerization (PARP1/2 and PARP5a/b).2 Among these,
tankyrase 1 (TNKS1, PARP-5a) and tankyrase 2 (TNKS2,
PARP5b) are peculiar since, in addition to the conserved C-
terminal catalytic polyADP-ribosylating domain, they encom-
pass two distinct protein−protein interaction domains: a sterile
α-motif and a five ankyrin repeat cluster, mediating self-
multimerization and target protein recognition, respectively.3
Tankyrases (TNKSs) are ubiquitously expressed and have a
tissue-specific cytoplasmic sublocalization according to their
cellular functions. Although TNKS1 was initially identified as a
negative regulator of telomeric repeat binding factor-1, a
component of telomere-end architecture, the most investigated
function of TNKSs is the regulatory effect on the Wnt/β-catenin
signaling pathway.4 Indeed, ADP-ribosylation triggers protea-
somal degradation of axin proteins, the process-limiting and core
component of β-catenin destruction complex, allowing β-
catenin to evade degradation and to prime the downstream
transcription of Wnt pathway genes.3,4 In addition to this cancer
related mechanism, TNKSs play roles in membrane trans-
location of the GLUT4 transporter, in the assembly of
microtubules at spindle poles, and viral replication.3,5 The
chemical modulation of ADP-ribosylation started with the
Special Issue: In Memory of Maurizio Botta: His
Vision of Medicinal Chemistry
Received: December 26, 2019
Accepted: February 3, 2020
Published: February 3, 2020
https://dx.doi.org/10.1021/acsmedchemlett.9b00654
© 2020 American Chemical Society
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Letter
inhibitors rather than selective chemical tools.12 Recently, a
fruitful approach to elicit selectivity, especially in TNKS
inhibition, consisted in targeting the AD subsite or both NAM
and AD subsites simultaneously.13,14 This strategy benefited
from the isoform-specific chemical environment within the AD
pocket. Moreover, spanning the length of the whole NAD+
binding cleft may provide further interaction sites between
active site and inhibitors. The D-loop in TNKSs is in fact shorter
than the homologue in PARP1/2, contains bulkier hydrophobic
amino acids, and offers a reduced space to host ligands in this
region.15
Previously, we reported the 2-mercaptoquinazolin-4-one
derivative MC2050 as a (sub)micromolar PARP1 inhibitor
selective over PARP2, and correlated this activity with the
cytoprotective effect in neuroblastoma SH-SY5Y cells and with
the modulation of viral gene expression in EBV-infected
Burkitt’s lymphoma cells.16,17 Here, our aim has been to
develop a series of MC2050 analogues able to potently inhibit
TNKSs while sparing PARP1/2 (Figure 1).
Figure 2. (A) Octahedral binding of Ni2+ (green sphere) in PARP1,
with 2.2 0.1 Å coordination distances. Red spheres represent water
molecules. The anomalous density is contoured at 5σ. (B) Active site of
the hPARP1c/MC2050 complex. The inhibitor (magenta) is overlaid
with Fo − Fc omit density contoured at 3σ. (C and D) Binding mode of
MC2050 in the active site of hPARP1c. (E) Structural overlay of PARP
complexes with the inhibitors olaparib (green, 5DS3), rucaparib
(orange, 4RV6), niraparib (yellow, 4R6E), and MC2050 (magenta).
The NAM-binding site is indicated by the red dashed circle.
inhibitor MC2050 (Figure 2B). The ligand is mainly bound
through the quinazolinone group, which mimics the nicotina-
mide moiety of the NAD+ substrate. N3 and the keto group of
the 4-quinazolinone nucleus form H-bonds to Gly863 and
Ser904, respectively (Figure 2C, D), while His862 and Tyr907
render the pocket suitable to harbor the phenyl ring of this
NAM-mimetic moiety. Besides the H-bond connecting N1 of
the pyridine group to Asp770, the piperazine and pyridine
moieties lack H-bond interactions, but appear also tightly
bound, as indicated by the absence of fragmented or smeared
density that would indicate flexibility or second conformations.
Rigid binding is achieved by hydrophobic interactions to the
side chains of Glu763, Asp766, Asn767, Leu769, Asp770,
Asn868, Arg878, and Ile879, which create a binding cleft
stabilized through a H-bond network. Thus, MC2050 is
dominantly bound via π-stacking and other hydrophobic
interactions, as observed for almost all structurally characterized
PARP inhibitors.12,14,15
However, a comparison of the binding site interactions of
MC2050 and of olaparib, rucaparib, and niraparib visualizes
differences for our parent compound (Figure 2E, PDB: 5DS3,18
4RV6, and 4R6E19). All these chemotypes share the same
interaction pattern within the PARP1 NAM-binding site,
establishing H-bonds to Gly863 and Ser904. Although the
cyclopropane of olaparib lies in the AD subsite, like for the
pyridine tail of MC2050, the connecting piperazinyl and
fluorophenyl portions fill a different active site area (next to
Tyr896) than the MC2050 linker (next to Asn767). Deviating
even more from the binding pose of MC2050 and extending
toward Arg878, the distal [(methylamino)methyl]phenyl of
rucaparib and 3-phenylpiperidine of niraparib occupy a different
pocket comprising Gln759, Val762, Glu763, and Tyr889.
Based on the experimental data on MC2050 interactions with
hPARP1c, we evaluated potential interactors of the binding site
for designing selective TNKS inhibitors.
Figure 1. Analogues of MC2050 investigated in the study.
We started by shedding light on the MC2050 binding in the
PARP1 catalytic domain by X-ray crystallography. These
insights, combined with structural differences between TNKSs
and PARP1/2, set the stage for a structure-based drug design
campaign. We kept intact the NAM-mimicking 2-mercaptoqui-
nazolin-4-one scaffold and synthesized a set of derivatives
protruding out of the NAM-binding site to elicit further and
specific interactions with the TNKS AD subpocket. Biological
evaluation on a panel of different PARP enzymes led to the
identification of a couple of selective (sub)nanomolar TNKS
inhibitors endowed with antiproliferative effect in DLD-1
human colon carcinoma cells.
The structure of human PARP1 (hPARP1c) in complex with
MC2050 was solved at 2.0 Å resolution and refined to R/Rfree
values of 22.6/26.5% (Table S1). A significant anomalous signal
was detected during processing, and the anomalous electron
density revealed six Ni2+ ions from the crystallization solution,
two inside each protein molecule in the asymmetric unit and two
between their interfaces (Figure 2A). A nickel dependency of
PARP1 has not been described and a potential physiological
relevance of the ion sites remains to be clarified. Inspection of
the active site revealed well-defined electron density for the
We superposed the crystal structures of the human TNKS
catalytic domains (residues 1105−1315 of TNKS1, PDB:
863
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4MSG and residues 956−1161 of TNKS2, PDB: 3U9Y14) with
aromatic ring with a flexible spacer (i.e., 2−4 methylene units
or similar) in order to attain a dual effect for TNKSs selectivity:
removing an important structural element for PARP1
interaction and averting a steric clash with Phe from TNKS1/
2. Moreover, this linker by projecting from the NAM-binding
site, where the 2-mercaptoquinazolin-4-one is harbored, could
orientate proper aromatic moieties into the AD pocket. Indeed,
using a computational scaffold hopping approach with a library
of aromatic rings, we selected a small series of best hits
(compounds 1−5 in Figure 1) to be connected through the
spacer to the thioethylene-piperazinyl moiety of MC2050 and
predicted to exploit the structural differences in the AD pockets
of PARP1 and TNKS1/2. With the only exception of compound
6, designed by an alternative strategy relying on the fusion of
both pyridine and piperazine moieties into a rigid spiro
tetracyclic system, the piperazine ring as connecting moiety
between the NAM- and AD-mimetic portions was kept in all
compounds because it offers the proper geometry to connect
these two portions and at the same time may act as inner
solubility group.
The general synthetic route for the preparation of final
compounds 1−6 is illustrated in Scheme 1. The commercially
available 4-phenylphenol and N′-hydroxybenzimidamide were
first converted into the corresponding alkyl bromides (7 and 9)
by alkylation with 1,3-dibromopropane and cyclization with 4-
bromobutanoyl chloride, respectively, and then treated with
commercial 2-(piperazin-1-yl)ethan-1-ol affording the related
intermediates 8 and 10. These alcohols then underwent
Mitsunobu reaction with commercial 2-mercaptoquinazolin-4-
one in the presence of diisopropyl azodicarboxylate and
trimethyl phosphine under microwave irradiation to provide
final derivatives 1 and 2 (Scheme 1A and 1B). The condensation
between the commercially available 2-hydroxyacetophenone
and tert-butyloxycarbonylpiperidin-4-one followed by N-Boc
deprotection provided the spiro-4-oxocromane intermediate 11,
which was then acylated with bromoacetyl chloride to give the
intermediate 12 that was finally converted into 3 by alkylation
with 2-(piperazin-1-yl)ethan-1-ol followed by reaction of the
intermediate 13 with 2-mercaptoquinazolin-4-one under the
same Mitsunobu conditions as for 1 and 2 (Scheme 1C). Final
compounds 4 and 5 were both prepared starting from the
commercially available 2-(piperazin-1-yl)ethanol (Scheme 1D).
In the former case the alcohol was first acylated with
benzyloxycarbonyl chloride and then converted into the
corresponding bromide (15) that was finally alkylated with 2-
mercaptoquinazolin-4-one to provide 4. The symmetric
compound 5 was instead obtained by Mitsunobu reaction of
two equivalents of 2-mercaptoquinazolin-4-one with the diol 16
previously prepared by alkylation of 2-(piperazin-1-yl)ethanol
with 2-bromoethanol. The final compound 6 was synthesized by
coupling between the spiropiperidine intermediate 18 and the
quinazolinone-containing propionic acid derivative 20 in the
presence of the 1-ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide/hydroxybenzotriazole system (Scheme 1E). The
intermediate 18 was prepared by conversion of the commercial
3-amino-2-naphthoic acid into the corresponding amide 17
followed by condensation of this intermediate with the piperid-
4-one under acidic conditions. The 3-((4-oxo-3,4-dihydroqui-
nazolin-2-yl)thio)propanoic acid 20 was obtained by alkylation
of 2-mercaptoquinazolin-4-one with commercially available
ethyl 3-bromopropionate followed by hydrolysis of the ethyl
ester 19 under basic conditions.
the hPARP1c/MC2050 crystal structure (Figure 3). It revealed
Figure 3. Structural comparison between the crystal structures of the
human TNKS2 ARTD catalytic domain (light gray) and MC2050
bound to the active site of PARP1 (cyan). The structural superposition
underscores differences in key residues in the AD-binding site, in
particular Asp770 and Arg878 in PARP1 (cyan) are replaced by
Phe1188/Phe1035 and His1201/His1048 in TNKS1/2 enzymes
(residue numbering refers to PDB codes 4MSG20 and 3U9Y,14
respectively).
a high degree of similarity at the 2-mercaptoquinazolin-4-one
binding site but also substantial differences in key residues in the
AD subsite, which accommodates the MC2050 pyridine moiety.
In particular, Asp770 and Arg878 in the PARP1 AD cavity are
replaced by Phe and His (Phe1188 and His1201 in TNKS1 and
Phe1035 and His1048 in TNKS2) suggesting that (1) removing
the pyridin-2-yl ring could induce the loss of interaction with
Asp770 and reduce inhibition of PARPs; (2) in order to interact
with TNKSs, MC2050 should avoid the steric clash with
Phe1188/Phe1035 and adopt a different conformation of the
ethylene linker and piperazine ring, pointing toward His1201/
His1048; (3) increasing the length of the linker between the
piperazine and a proper not-basic (hetero)aromatic ring could
allow the aromatic tail of potential MC2050 analogues to
establish a favorable stacking interaction with His1201/His1048
in TNKSs, and at the same time could lead to a clash with the
bulkier side-chain of Arg878 in PARP1.
Given the interaction details of hPARP1c/MC2050, we
decided to leverage the PARP1/TNKSs structural differences
from our in-silico comparison to design a small library of
analogues of our lead (Figure 1). To explore the molecular space
in the AD cavity of the ARTD catalytic domain, we maintained
the 2-mercaptoquinazolin-4-one as a NAM-mimetic scaffold
since it evokes favorable H-bonds with Gly863 and Ser904 in
PARP1, corresponding to Gly1185-Ser1221 and Gly1032-
Ser1068 in TNKS1/2, respectively. Additionally, the π-staking
between the phenyl ring of the 4-quinazolinone nucleus and
Tyr907 and His862 of PARP1 might be preserved in TNKS1/2
(with Tyr1224-His1184 and Tyr1071-His1031). Since the
thioethylene-piperazinyl moiety adapts into a molecular gorge
covering the space between Glu763/Asn868 and Leu769/
Arg878 in the hPARP1c/MC2050 structure, we reasoned that
this portion would span this length also in TNKS1/2. Our
crystallographic investigation also showed that the MC2050
pyridin-2-yl group plays a relevant role in PARP1 binding by H-
bonding the Asp770 side chain. This evidence and our
computational modeling studies suggested to replace this
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a
Scheme 1. Synthetic Routes to Compounds 1−6
a
Reagents and conditions: (a) 1,3-dibromopropane, K2CO3, dry CH3CN, reflux; (b) 2-(piperazin-1-yl)ethan-1-ol, K2CO3, NaI, dry DMF, 60 °C;
(c) 2-mercapto-4(3H)-quinazolinone, DIAD, 1 M trimethylphosphine in toluene, dry DMF, microwave, 40 °C; (d) (i) 4-bromobutanoyl chloride,
TEA, dry DCM, rt, (ii) toluene, reflux; (e) tert-butyl 4-oxopiperidine-1-carboxylate, pyrrolidine, dry MeOH, reflux; (f) 4 M HCl in dioxane, dry
THF; (g) bromoacetyl chloride, TEA, dry DCM, rt; (h) benzyloxycarbonyl chloride, TEA, dry THF, rt; (i) CBr4, triphenylphosphine, dry THF, rt;
(j) 2-mercapto-4(3H)-quinazolinone, K2CO3, NaI, dry DMF, rt; (k) 2-bromoethan-1-ol, K2CO3, dry CH3CN, reflux; (l) NH3 aq., EDCI, HOBt, 4-
methylmorpholine, dry THF, rt; (m) piperidin-4-one hydrochloride, concentrated H2SO4, CH3COOH, rt; (n) EDCI, HOBt, TEA, dry DMF, rt;
(o) 2 M potassium hydroxide, EtOH, rt.
Table 1. Inhibitory Activity of Derivatives 1−6 on PARP1/2 and TNKS1/2
a
IC50 (nM)
Compd
PARP-1
9.3 0.09
2040 61
1480 58
1280 49
356 14
89.6 2.2
26% inhib. (@200 μM)
9.1 0.09
NI (@200 μM)
PARP-2
549 12
340 11
370
347 13
963 39
37.8 1.1
132000 987
14.7 0.4
TNKS1
8
673 28
37.4 0.4
552 18
398 14
6.1 0.07
TNKS2
193 7
588 19
MC2050
1
2
3
4
5
6
218
8
11.7 0.3
103
4
318 13
0.3 0.005
NI (@200 μM)
52.7 1.2
b
NI (@200 μM)
39.2 0.9
PJ34
IWR-1
NI (@200 μM)
303
9
29.2 0.8
a
b
Values are means SD determined from at least two independent experiments, each one done in triplicate. NI, no inhibition.
The capability of 1−6 to inhibit poly(ADP)ribosylating
and of the selective TNKS inhibitor IWR-114 (Table 1). All
synthesized compounds substantially lost PARP1 inhibitory
potency and selectivity over PARP2 in comparison with
MC2050 and, with the sole exception of 6 (substantially
activity was assessed in vitro against PARP1/2 and TNKS1/2,
and it was compared with the activity of the parent PARP1
inhibitor MC2050,16 of the unselective PARP inhibitor PJ34,21
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inactive against all enzymes), displayed a submicromolar activity
against TNKS1 (IC50 ranging from 673 to 6.1 nM) and TNKS2
(IC50 ranging from 588 to 0.3 nM). Significant differences were
observed among compounds 1 and 2, that are characterized by a
not-condensed bicyclic system linked to the piperazine ring
through a polymethylene spacer. Indeed, the presence of a 4-(4′-
biphenyl) moiety (1) induced a loss of activity in comparison
with MC2050 not only against PARP1 but also versus TNKS1/
2, whereas the introduction of a 3-phenyl-1,2,4-oxadiazol-5-yl
moiety (2) produced a significant increase of inhibitory potency
against TNKSs (IC50 = 37.4 nM on TNKS1 and IC50 = 11.7 nM
on TNKS2) joined to selectivity over PARP1 (IC50 = 1480 nM)
and, to a lesser extent, PARP2 (IC50 = 370 nM). The
introduction on the spacer between the piperazine and the
aromatic ring pointing toward the AD subpocket of a carbonyl
function (carboxamide or carbamate) joined to either a bulky
spiro tricyclic system (3) or a simple benzene ring (4) caused an
almost complete loss of selectivity for TNKSs over PARP1/2
leading to the unselective (sub)micromolar PARP inhibitors 3
and 4. Interestingly, when the pyridine ring of MC2050 was
substituted with a 2-mercaptoquinazolin-4-one linked to the
central piperazine through an ethylene spacer providing the
symmetrical compound 5, a massive increase in activity on
TNKSs and selectivity over PARP1/2 was re-established. In
particular, endowed with subnanomolar inhibitory potency
against TNKS2 (IC50 = 0.3 nM) and with an important
selectivity not only over PARP1/2 (more than 100-fold) but also
over the closely related TNKS1 (more than 20-fold), 5 displayed
a significant increase of TNKS2 inhibitory potency and
selectivity not only in comparison with the pan-PARP inhibitor
PJ34 (TNKS2 IC50 = 52.7 nM) but also with the TNKS selective
inhibitor IWR-1 (TNKS2 IC50 = 29.2 nM). Overall these results
confirmed that leveraging favorable interactions within the AD
subpocket, as previously reported,13 may be advantageous also
to gain isoform selectivity within the TNKS subfamily of PARPs.
A retrospective structural investigation of the interaction
mode of 5 into the AD binding cleft of PARP1 and TNKS2 using
computational docking suggested a possible explanation for the
observed selectivity (Figure S1). In the energetically minimized
geometry of 5, the second quinazolinone ring is placed in a
perfect stacking interaction with His1048, and at the same time
is predicted to be stabilized by favorable interactions with the
residues of the TNKS2 D-loop (Phe1035, Ala1038, Lys1042,
Asp1045). Conversely, the same geometry would sterically clash
with Arg878 of PARP1.
Figure 4. (A) Bar diagram of antiproliferative effects on DLD-1 CRC
cells of 2 and 5. (B) Colony forming assay on DLD-1 cancer cells. (A)
untreated cells; (B) compound 2 at 1 μM; (C) compound 2 at 10 μM;
(D) compound 5 at 0.1 μM; (E) compound 5 at 1 μM; (F) IWR-1 at 1
μM.
molecular tools. The establishment of accessory interplays
with the regulatory α-helical subdomain next to the cofactor-
binding site in PARP1/2 (and lacking in TNKSs) led to
development of selective inhibitors, and some of them are now
on the market.8 Similarly, exploring the AD subpocket as well as
exploiting the unique structural features of the D-loop, such as
the hydrophobic nature of the sequence and the narrower space,
resulted in a more selective inhibition of TNKSs. Moreover, the
simultaneous targeting of different portions of the cofactor
donor site, i.e. by dual binders, might not only lead to selectivity
among PARPs, but might also prevent inhibition of other NAD+
dependent enzymes, possibly reducing unpredictable off-target
effects.7,11−15
Here we took advantage of the combination of crystallo-
graphic and computational techniques to generate a small
collection of quinazolinone-based compounds able to span the
length of the NAD+ binding groove. The inspection of the X-ray
structure of the PARP1 inhibitor MC2050 in complex with
hPARP1c revealed that its quinazolinone ring is embedded in
the NAM subsite of the cofactor-binding pocket engaging π−π
stacking interactions and H-bonds. Superimposition with
TNKSs catalytic domains uncovered that the quinazolinone
H-bonding could be preserved and paved the way for the
rational design of a focused library of compounds in which the 2-
mercaptoquinazolin-4-one scaffold was functionalized with
different aromatic systems installed through a proper spacer
on the piperazine moiety of the parent compound MC2050.
Biological tests highlighted 2 and 5 as (sub)nanomolar TNKS2
inhibitors with a significant selectivity over PARP1/2, inverting
the selectivity profile of MC2050. 5 showed a dramatic increase
of activity on TNKSs (up to 600-fold). Moreover, it exhibited a 2
orders of magnitude higher potency on TNKS2 with respect to
PARP1 and PARP2 and a selectivity of about 20-fold on the
closely related TNKS1. Compounds 2 and 5 also showed dose-
dependent antiproliferative effects in colony forming assays,
comparable to the TNKSs inhibitor IWR-1, confirming the
efficaciousness of TNKS inhibition in affecting cancer cell
growth.
Since endowed with (sub)nanomolar inhibitory potency
against TNKSs with a significant selectivity over PARP1/2,
compounds 2 and 5 were selected to be further evaluated in a
colony forming assay on DLD-1 human CRC cells, a cell line
characterized by a truncated form of the APC protein, one of the
components of the β-catenin destruction complex, that shows a
constitutive activation of the canonical Wnt signaling pathway
and is frequently used to validate TNKS inhibitors in cells.4,13,22
The selectedcompounds were testedat 1 and 10 μM (2) or at 0.1
and 1 μM (5) depending on the relative potency in enzyme
assays (see Table 1). As shown in Figure 4, compounds 2 and 5
were able to inhibit formation of DLD-1 cell colonies in a dose-
dependent manner, with compound 5 showing a strong
antiproliferative effect at 1 μM, better than that of the reference
TNKS inhibitor IWR-1 at the same concentration.
In conclusion, herein we reported a structure-based computa-
tionally driven approach, that with a limited, but extremely
focused, synthetic effort led to the discovery of the bis-
quinazolinone-based NAM-AD dual-binder 5 as a picomolar
and selective TNKS2 inhibitor, that has been selected as lead
compound for further optimization studies.
The development of PARP inhibitors historically focused on
the NAM subpocket of the NAD+ binding cavity but mostly
afforded polypharmacological agents rather than selective
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Notes
ASSOCIATED CONTENT
■
The authors declare no competing financial interest.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00654.
ACKNOWLEDGMENTS
■
This work was supported by funds from PRIN 2015
(prot.20152TE5PK) (A.M.), AIRC IG 2016 (n. 19162)
(A.M.), and AIRC MFAG2017 (n. 20447) (A.P. and C.E.).
Experimental procedures for the synthesis and phys-
icochemical characterization of all new compounds (1−
20); materials and methods for enzyme inhibition assays,
crystallographic (PDB: 6XVW) and in silico studies, and
cell-based assays (PDF)
́
Thanks are due to Dr. Sebastien Moniot for helpful discussion.
We thank Swiss Light Source (SLS; Villigen, Switzerland) for
beamtime and support. The authors dedicate the work to the
memory of Prof. Maurizio Botta, a great friend and continuous
source of inspiration for med-chem studies.
AUTHOR INFORMATION
■
Corresponding Authors
ABBREVIATIONS
■
Dante Rotili − Department of Chemistry and Technology of
Drugs, ″Sapienza” University of Rome, 00185 Rome, Italy;
Phone: +39-0649913237; Email: dante.rotili@uniroma1.it
Antonello Mai − Department of Chemistry and Technology of
Drugs, ″Sapienza” University of Rome, 00185 Rome, Italy;
orcid.org/0000-0001-9176-2382; Phone: +39-
0649913391; Email: antonello.mai@uniroma1.it
Alessandro Paiardini − Department of Biochemical Sciences “A.
Rossi Fanelli″, ″Sapienza” University of Rome, 00185 Rome,
Italy; Phone: +39-0649917700; Email: alessandro.paiardini@
uniroma1.it
Clemens Steegborn − Department of Biochemistry and Research
Center for Bio-Macromolecules, University of Bayreuth, 95440
Bayreuth, Germany; orcid.org/0000-0002-0913-1467;
Phone: +49-0921557831; Email: clemens.steegborn@uni-
bayreuth.de
AD, adenosine; ARTDs, ADP-ribosyltransferases diphtheria
toxin-like; CRC, colorectal cancer; NAD+, nicotinamide adenine
dinucleotide; NAM, nicotinamide; NSCLC, nonsmall cell lung
cancer; PARPs, poly(ADP-ribose) polymerases; TNKSs, tank-
yrases
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Authors
Stefano Tomassi − Department of Pharmacy, University of
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4467
Julian Pfahler − Department of Biochemistry and Research Center
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Nicola Mautone − Department of Chemistry and Technology of
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Annarita Rovere − Department of Chemistry and Technology of
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Chiara Esposito − Department of Biochemical Sciences “A. Rossi
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