Hit-to-lead optimization of a latency-associated nuclear antigen inhibitor against Kaposi's sarcoma-associated herpesvirus infections

Article abstract of DOI:10.1016/j.ejmech.2020.112525

The Latency-associated nuclear antigen (LANA) plays a central role for the latent persistence of the Kaposi's Sarcoma Herpesvirus (KSHV) in the human host and helps to establish lifelong infections. Herein, we report our efforts towards hit-to-lead generation starting from a previously discovered LANA-DNA inhibitor. By tethering the viral genome to the host nucleosomes, LANA ensures the segregation and persistence of the viral DNA during mitosis. LANA is also required for the replication of the latent viral episome during the S phase of the cell cycle. We aim to inhibit the interaction between LANA and the viral genome to prevent the latent persistence of KSHV in the host organism. Medicinal chemistry-driven optimization studies and structure-activity-relationship investigation led to the discovery of an improved LANA inhibitor. The functional activity of our compounds was evaluated using a fluorescence polarization (FP)-based interaction inhibition assay and electrophoretic mobility shift assay (EMSA). Even though a crystal structure of the ligand protein complex was not available, we successfully conducted hit optimization toward a low micromolar protein-nucleic acid-interaction inhibitor. Additionally, we applied STD-NMR studies to corroborate target binding and to gain insights into the binding orientation of our most potent inhibitor, providing opportunities for further rational design of more efficient LANA-targeting anti KSHV agents in future studies.

Full text of DOI:10.1016/j.ejmech.2020.112525

Journal Pre-proof
Hit-to-lead optimization of a latency-associated nuclear antigen inhibitor against
Kaposi's sarcoma-associated herpesvirus infections
Philine Kirsch, Saskia C. Stein, Aylin Berwanger, Julia Rinkes, Valentin Jakob,
Thomas F. Schulz, Martin Empting
PII:
S0223-5234(20)30497-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112525
EJMECH 112525
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 February 2020
Revised Date: 25 May 2020
Accepted Date: 30 May 2020
Please cite this article as: P. Kirsch, S.C. Stein, A. Berwanger, J. Rinkes, V. Jakob, T.F. Schulz, M.
Empting, Hit-to-Lead Optimization of a Latency-associated Nuclear Antigen Inhibitor against Kaposi’s
Sarcoma-associated Herpesvirus Infections, European Journal of Medicinal Chemistry, https://
doi.org/10.1016/j.ejmech.2020.112525.
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© 2020 The Author(s). Published by Elsevier Masson SAS.

Highlights:
•
•
Role of the latency-associated nuclear antigen (LANA) during a latent infection of the Kaposi’s
Sarcoma-associated Herpesvirus (KSHV).
Inhibition of the interaction between LANA and the viral DNA will prevent the latent persistence
of KSHV.
•
•
•
Chemistry-driven hit-to-lead optimization studies on a previous discovered LANA-DNA Inhibitor.
Structure-activity-relationship (SAR) of LANA-DNA inhibitors are highlighted.
Development of a potent LANA-DNA inhibitor showing IC50 values in the lower micromolar range
against wild-type LANA in EMSA experiments.

Hit-to-Lead Optimization of a Latency-associated Nuclear Antigen Inhibitor
against Kaposi’s Sarcoma-associated Herpesvirus Infections
Philine Kirsch,1,2,3 Saskia C. Stein,3,4 Aylin Berwanger,1,2,3 Julia Rinkes,1,2,3 Valentin Jakob,1,2,3 Thomas F.
Schulz3,4 and Martin Empting*,1,2,3
1Department of Drug Design and Optimization (DDOP), Helmholtz-Institute for Pharmaceutical
Research Saarland (HIPS) - Helmholtz Centre for Infection Research (HZI), Campus E8.1, 66123
Saarbrücken, Germany.
2Department of Pharmacy, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany.
3German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 66123
Saarbrücken, Germany.
4Institute of Virology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
Abstract
The Latency-associated nuclear antigen (LANA) plays a central role for the latent persistence of the
Kaposi’s Sarcoma Herpesvirus (KSHV) in the human host and helps to establish lifelong infections.
Herein, we report our efforts towards hit-to-lead generation starting from a previously discovered
LANA-DNA inhibitor. By tethering the viral genome to the host nucleosomes, LANA ensures the
segregation and persistence of the viral DNA during mitosis. LANA is also required for the replication
of the latent viral episome during the S phase of the cell cycle. We aim to inhibit the interaction
between LANA and the viral genome to prevent the latent persistence of KSHV in the host organism.
Medicinal chemistry-driven optimization studies and structure-activity-relationship investigation led
to the discovery of an improved LANA inhibitor. The functional activity of our compounds was
evaluated using
a fluorescence polarization (FP)-based interaction inhibition assay and
electrophoretic mobility shift assay (EMSA). Even though a crystal structure of the ligand protein
complex was not available, we successfully conducted hit optimization toward a low micromolar
protein-nucleic acid-interaction inhibitor. Additionally, we applied STD-NMR studies to corroborate
target binding and to gain insights into the binding orientation of our most potent inhibitor,

providing opportunities for further rational design of more efficient LANA-targeting anti KSHV agents
in future studies.
Keywords: hit-to-lead optimization, latency-associated nuclear antigen (LANA), Kaposi’s Sarcoma
Herpesvirus (KSHV), fluorescence polarization (FP)-based interaction inhibition assay, electrophoretic
mobility shift assay (EMSA), CuAAC, STD-NMR
Highlights:
•
•
•
Role of the latency-associated nuclear antigen (LANA) during a latent infection of the
Kaposi’s Sarcoma-associated Herpesvirus (KSHV).
Inhibition of the interaction between LANA and the viral DNA will prevent the latent
persistence of KSHV.
Chemistry-driven hit-to-lead optimization studies on a previous discovered LANA-DNA
Inhibitor.
•
•
Structure-activity-relationship (SAR) of LANA-DNA inhibitors are highlighted.
Development of a potent LANA-DNA inhibitor showing IC50 values in the lower micromolar
range against wild-type LANA in EMSA experiments.
Introduction
Kaposi’s Sarcoma Herpesvirus (KSHV) is a human gamma herpesvirus and establishes a lifelong latent
infection in B-cells and endothelial cells.1,2 The virus was identified as the etiological agent of Kaposi’s
Sarcoma (KS) and is involved in two other neoplastic diseases, multicentric Castleman’s disease and
pleural effusion lymphoma.1,3 In healthy individuals, KSHV-associated diseases are rare. However, in
immunosuppressed patients, e.g., transplant recipients or patients with the acquired
immunodeficiency syndrome (AIDS), KSHV is highly oncogenic.4,5 However, classic KS mainly can also
occur in elderly men especially from KSHV-endemic areas and endemic KS in East and Central Africa.6
The main key player for the establishment and maintenance of the latent infection is the latency-
associated nuclear antigen (LANA).7–9 It is an origin-binding protein, whose C-terminal domain binds
to the viral genome and whose N-terminal region interacts simultaneously with host nucleosomes.10–
12
This allows the segregation of latent viral episomes during mitosis and their partitioning to
daughter cells.13 LANA has also additional functions like latent viral replication, transcriptional control
and survival in the host cell.14–16 The C-terminal DNA-binding domain (DBD) of LANA binds the viral
genome in a sequence-specific manner.17 Located on the terminal repeats (TRs) are three specific
LANA binding sites (LBS), LBS1, LBS2 and LBS3. LBS1 has a hundred fold higher affinity to LANA
compared to LBS2 and LBS3.17 In the majority of KSHV-associated cancer cells the viral genome is
present and LANA is expressed.18 It has been shown, that the persistence of viral DNA is affected by

disturbing or influencing LANA.15 The inhibition of the interaction between LANA and viral DNA could
lead to a reduction or loss of viral genomes in the infected cells. Today’s treatment of KSHV and
KSHV-associated diseases is difficult and still limited.19,20 It is clear, that there is an urgent need for
specific drugs, which interfere with novel steps in the KSHV lifecycle. In view of its central role during
latent viral persistence, LANA is considered to be a very promising target for the development of
specific antiviral therapeutics against KSHV.
In a study previously published by us in 2019, we described the discovery of first inhibitors, which
interfere with the LANA-DNA interaction.11 Further inhibitor scaffolds have been identified using a
functional screen and an in-house compound library.21 Starting with a fragment-based drug discovery
approach, we successfully developed a fragment-sized inhibitor I capable to compete with the viral
DNA (Figure 1). For the evaluation of functional activity of our compounds, we used a
fluorescence-polarization (FP)-based assay and electrophoretic mobility shift assay (EMSA)
experiments. For our most promising fragment-sized inhibitor I, we observed an IC50 value of 17 ±
1 µM in our FP-assay using a LANA DNA binding domain (DBD) mutant and 435 ± 6 μM in the EMSA
studies using the wild-type LANA C-terminal domain (CTD).
Figure 1: Previously described LANA-DNA interaction inhibitor I and its predicted binding mode which provides
the basis for structural optimization by rational design and growth vector exploration.
We confirmed target binding using microscale thermophoresis (MST) and saturation transfer
difference (STD)-NMR experiments. Additionally, the STD-NMR experiments and molecular docking
studies provided important information on the putative orientation of Inhibitor I when bound to
LANA. Based on the STD-NMR studies and docking results we suggested that the nitrogen at the
pyridine core acts as a hydrogen bond acceptor and protons 2, 3 and 4 are not in direct interaction
with the protein surface, hence these positions should be further investigated as potential growth
vectors. Furthermore, two glutamines are presumably involved in hydrogen-bond interactions with

the carboxyl group. However, it was not clear whether the carboxylic acid function is necessary for
binding.11
Based on these findings, we embarked on structure-activity relationship (SAR) studies and further
medicinal chemistry optimization to improve the potency of our hit compounds. Herein, we report
our recent advances in improving our LANA-DNA-interaction inhibitors using compound I as a
starting point. Unfortunately, our efforts in solving a co-crystal structure of inhibitor I in complex
with LANA have not been successful to date. This renders unambiguous experiment-supported
structure-based optimization unfeasible. Therefore, we systematically investigated the LANA-DNA-
interaction inhibition of new synthesized compounds using FP-based competition assay and EMSA
experiments as the SAR drivers.
Design Concept
Based on the previously applied STD-NMR and docking studies we modified Inhibitor I in a step-by-
step manner. Inhibitor I was divided in two regions, the benzoic acid part A and the pyridine core B
(Figure 1). The triazole core was not yet modified in order to exploit the robust and facile Copper(I)-
catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry. First, region A was modified and
variations of the carboxylic acid were introduced. As a second step, we have modified the pyridine
moiety, region B. From our previous results we assumed that the nitrogen at the pyridine motif is
essential for binding and functions as hydrogen bond acceptor. STD-NMR data revealed that Proton 1
interacts tightly with LANA while proton 2 is also in close proximity to the protein surface. However,
our docking studies suggested that the latter might be at least partially solvent exposed. In contrast,
protons at position 3 and 4 did not show direct contact with the LANA surface according to their
weak STD-NMR effects. These observations inspired us to investigate positions 2, 3 and 4 as potential
growth vectors in the presented study.
Results and Discussion
Chemistry
Modifications of region A. 3-azidopyridine 2 was generated by a standard azidation method using 3-
aminopyridine 1, NaNO2 and NaN3 in a mixture of EtOAc and 6M HCl.11,22 In a second step, as
depicted in scheme 1, various commercially available ethynylbenzene derivatives were used in a
standard copper-catalyzed CuAAC reaction with 3-azidopyridine to provide the triazoles 3 - 5, 8, 10,
11, and 13.11

Scheme 1. Modification of region A.a
aReagents and conditions: a) NaNO2, NaN3, EtOAc, 6M HCl, 0°C → rt, yield 50%, 2 h; b) corresponding ethynylbenzene,
CuSO4•5H20, Na-Ascorbate, DIPEA, MeOH, H2O, rt, 16 h, yield 60–82%; c) 1. SOCl2, DMF, 60°C, 1 h, 2. EtOH, DIPEA, rt, 16 h,
yield 53%; d) 1. SOCl2, DMF, 60°C, 1 h, 2. NH4OH, rt, 16 h, yield 27%; e) 2 M NaOH, MeOH, rt, 16 h, yield 66%; f) Actyl
chloride, Et3N, DCM, DMF, rt, 16 h, yield 14%.
The ethyl ester 6 and amide 7 analogue were generated from the carboxylic acid 3 by thionyl
chloride-mediated activation and subsequent treatment with ethanol or aq. ammonia solution. The
hydrolysis of 3-chloro-4-methylester intermediate 8 with NaOH in ethanol produced the
corresponding acid 9. The N-acetyl analogue 12 was synthesized from amine 11 with acetyl chloride
under basic conditions.
Modifications of region B. Compound 16 bearing an additional CH2-linker between triazole and
pyridine core was synthesized starting from (bromomethyl)benzene 14, which was converted to the
azide 15 using NaN3 in DMSO23, followed by a click reaction with 4-ethynylbenzoic acid. Different
arylazides 17a-n and 20 decorated with various substitutions were generated by reaction of the
corresponding commercially available amines 18a-n and 21 with NaNO2 and NaN3 in 6 M HCl and
EtOAc (Scheme 2). The subsequent CuAAC click reaction with 4-ethynylbenzoic acid provided the
target molecules 19a-n and 22. The hydroxypyridine 19o analogue was generated from the
methoxypyridine 19k by treating with 48% aqueous HBr solution at 80 °C.
Scheme 2: Azide synthesis and CuAAC click reaction.a

aReagents and conditions: a) NaN3, Et3N, DMSO, rt, 16 h, yield 78%; b) 4-ethynylbenzoic acid, CuSO4•5H20, Na-Ascorbate,
DIPEA, MeOH, H2O, rt, 16 h, yield 20-90%; c) NaNO2, NaN3, EtOAc, 6M HCl, 0°C → rt, 2 h, yield 4-98%; d) 48% aq. HBr, 80°C,
12 h, yield 86%.
As depicted in Scheme 3, the syntheses of target compounds via Suzuki coupling was achieved using
two different synthetic routes. In route 1, Suzuki coupling with different commercially available
boronic acids and halogenated pyridine-3-amines 23, 27 and 28 in presence of Pd(P(Ph3)4 achieved
phenyl-substituted pyridine amines 24a-b and 29a-b in the first step. Subsequently, the amines were
converted to the corresponding azides 25a-b and 30a-b followed by a CuAAc click reaction with
4-ethynylbenzoic acid to obtain the target compounds 26a-b and 31a-b. In parallel, the alternative
route 2 was established for late stage modifications via Suzuki coupling. First, halogenated pyridine-
3-amines 32a-b were converted to the corresponding azide 33a-b, followed by click reaction with
4-ethynylbenzoate to obtain the corresponding triazole intermediates 34a-b. Subsequently, phenyl-
substituted compounds 35a-k were achieved via Suzuki coupling using corresponding boronic acids
and Pd(P(Ph3)4.
Scheme 3: Synthesis of target compounds via Suzuki coupling using two different routes.a

aReagents and conditions: a) corresponding boronic acid, Na2CO3, Pd(P(Ph3)4, 1,4-dioxan, H2O, 90 °C, 16h, yield 21-75%; b)
NaNO2, NaN3, EtOAc, 6M HCl, 0°C → rt, 2 h, yield 86-99%; c) 4-ethynyl benzoic acid, CuSO4•5H20, Na-Ascorbate, DIPEA,
MeOH, H2O, rt, 16 h, yield 36-76%; d) NaNO2, NaN3, EtOAc, 6M HCl, 0°C → rt, 2 h, yield 34-80%; e) 4-ethynyl benzoate,
CuSO4•5H20, Na-Ascorbate, DIPEA, MeOH, H2O, rt, 16 h, yield 63-80%; f) corresponding boronic acid, Na2CO3, Pd(P(Ph3)4,
1,4-dioxan, H2O, 90 °C, 16h, yield 20-90%; g) 2 M NaOH, MeOH, rt, 16 h, yield 16-83%.
Finally, the hydrolysis of the esters with NaOH in methanol produced the target carboxylic acid
compounds 36a-k. As depicted in Scheme 4, for the synthesis of the series of pyridine-phenoxy target
compounds 40a-e, cupper-catalyzed Ullmann reaction was used in the first step using 6-bromo-4-
methylpyridin-3-amine 37, the corresponding phenol derivative or thiophenol, Cs2CO3 and CuI to
obtain the aminopyridine-phenoxy intermediates 38a-e.
Scheme 4: Synthesis of target compounds via Ullmann Reaction.a

aReagents and conditions: a) corresponding phenol or thiophenol, Cs2CO3, CuI, DMF, 130 °C, 16 h, yield 28-94%; b) NaNO2,
NaN3, EtOAc, 6M HCl, 0°C → rt, 2 h, yield 70-99%; c) 4-ethynyl benzoic acid, CuSO4•5H20, Na-Ascorbate, DIPEA, MeOH, H2O,
rt, 16 h, yield 45-93%.
The obtained amines were transformed into the corresponding azides 39a-e as described above. Last
step was a CuAAC reaction of azides with 4-ethynylbenzoic acid to obtain the target compounds
40a-e. The isoquinoline 43 and quinoline 46 analogue were synthesized starting from isoquinoline-4-
amin 41 and quinoline-3-amin 44 by standard azidation to 42 and 45, followed by CuAAC click
reaction with 4-ethynylbenzoic acid. Further isoquinoline derivatives 50a-c were synthesized in a 3
step procedure (Scheme 5). A direct transformation of bromo isoquinolines 47a-c into the
corresponding azides using NaN3, Cu(I) and Na2CO3 at 85°C over night as described in literature was
not efficient.24 LCMS-guided reaction monitoring showed the formation the primary amine and other
side products. For this reason, we extended the reaction time until we detected full conversion into
the corresponding primary amine 48a-c with the aim to subsequently transform these intermediates
into the corresponding azides. Indeed, we achieved successful azidation (intermediates 49a-c) and
CuAAC coupling, respectively, using amines 48a-c and the conditions described above yielding the
desired isoquinoline products 50a-c.

Scheme 5: Synthesis of isoquinoline derivatives.a
aReagents and conditions: a) NaNO2, NaN3, EtOAc, 6M HCl, 0°C → rt, 2 h, yield 68-91%; b) 4-ethynyl benzoic acid,
CuSO4•5H20, Na-Ascorbate, DIPEA, MeOH, H2O, rt, 16 h, yield 25-90%; c) NaN3, Na2CO3,CuSO4•5H20, Na-Ascorbate, L-
proline, DMF, H2O, 85 °C, 24 h, yield 88-97%
Functional Evaluation using LANA-DNA Interaction Inhibition Assays and SAR Studies.
The target compounds were tested for functional activity in the FP-based LANA-DNA interaction
inhibition assay using LBS2 as the probe and an oligomerization-deficient LANA DBD mutant.11 This
oligomerization-deficient C-terminal LANA mutant (aa1008-1146) has nine amino acid point
mutations: K1055E, K1138S, K1140D, K1141D, R1039Q, R1040Q, A1121E, K1109A, and D1110A. For
this mutant, also in presence of oligonucleotides, which represent the viral LANA-binding sites LBS1,
LBS2 or LBS3, a high water solubility was shown.11,10,17,25 All compounds showing an IC50 values less
than 250 µM were further tested in an orthogonal LANA-DNA interaction inhibition assay employing
EMSA methodology, the same LANA DBD mutant and LBS1 as probe. As described above, the latter
oligo has a higher affinity to the target rendering the EMSA experiment a more stringent read out for
compound efficacy.

For the first series of compounds, we investigated the significance of the carboxylic acid in the
Western part of the molecule (region A) by varying its position, attaching additional groups or
substituting it by other polar functional groups capable of participating in hydrogen bonding. The
results are shown in Table 1.
Moving the carboxylic acid from para (inhibitor I) to meta position (4) decreases the activity
significantly. Also an additional chlorine atom attached in meta position (9) lead to a complete loss of
activity. The replacement of the carboxylic acid by a methyl ester (5), ethyl ester (6) or amide (7) was
also detrimental. Furthermore, moving from the carboxylic acid to the methyl alcohol (10), amine
(11), acetamide (12) or nitrile (13) also resulted in inactive compounds. These results indicate that
the carboxylic acid in para position in region A is essential for inhibitory activity. Therefore, we kept
the p-carboxylic acid in region A fixed for further optimization studies and focused on the
modifications at the pyridine core in region B. First, we examined the effect on inserting a short
linker between the triazole and the pyridine core (16).
Table 1: Inhibition activities of compounds with modification in region A.
FP
Assa
y
FP
Ass
ay
(LBS
2)a
(LBS
2)
Cp
d
Cpd
R
IC50
R
IC50
17 ±
1
Inhibi
tor I
9
n.i.
µM
>250
µM
4
5
10
n.i.
>25
0
n.i.b 11
µM
6
7
n.i.
n.i.
12
13
n.i.
>25
0

µM
b
aFluorescence-polarization assay using LBS2 as probe, data representing average of duplicates ± standard deviation; No
inhibition at 500 µM.
This, however, resulted in loss of activity. As described before, from previous STD-NMR and
molecular docking experiments we expected, that growing the fragment-sized Inhibitor I in different
positions at the pyridine core (region B) would potentially increase potency.
To explore the influence of larger structural motifs at the pyridine core in position 4 we introduced a
variety of residues. As listed in Table 2, growing in this position is accepted and resulted in moderate
to potent inhibitory effects in FP assay ranging from IC50 values of 86 ± 6 µM (19a) to 18 ± 4 µM
(19c). The size of the introduced residue seems to play an important role. While a small methyl group
is not favorable, but accepted (19a, IC50 86 ± 6 µM), further increasing the size from chlorine (19b) to
phenyl (19c) improves IC50 values to 29 ± 1 µM µM and 18 ± 4 µM, respectively. This observation
might hint at a steric ortho effect. The additional bulky phenyl ring strongly hinders the rotation of
the bond between triazole and pyridine and, therefore, might fix the nitrogen in the pyridine core in
a more favorable orientation. In EMSA experiments, 4-substituted compounds 19b (EMSA: 94%
inhibition @ 500 µM) and 19c (EMSA: 100% inhibition @ 500 µM) showed a higher efficiency
compared to Inhibitor I (EMSA: 83% inhibition @ 500 µM).11 Additionally, we shifted the nitrogen of
the pyridine core from meta (19a) to para position (22) which resulted in an inactive compound. The
improvements in the EMSA assay for compounds 19b-c over our initial hit compound I were not
perfectly mirrored by the FP IC50 values, which presumably is rooted in the usage of different DNA
probes (LBS1 vs LBS2, respectively).
Table 2. Inhibitory activities of analogues modified in position 4 – observing higher efficiency for 4-
substituted compounds
FP Assay (LBS2)a
IC50
EMSA (LBS1)b
cpd
R
inhibition @ 500 µM
Inhibitor I
17 ± 1 µM
>250 µM
86 ± 6 µM
82 %
n.d.d
39 %
16
19a

19b
29 ± 1 µM
94 %
19c
22
18 ± 4 µM
100 %
n.d.
n.i.c
aFluorescence-polarization assay using LBS2 as probe, data representing average of duplicates ± standard deviation;
bElectrophoretic mobility shift assay using LBS1 as probe; cNo inhibition at 500 µM; dNot determined.
Nevertheless, the results for compounds 19b and 19c were a major step towards achieving LANA
inhibitors suitable for cellular assays and encouraged us to explore the potential of growing the hit
scaffold in this direction even further.
In the next series of compounds, Inhibitor I was grown in position 3 at the pyridine core by
introducing a variety of aromatic rings. As listed in table 3, a small methyl residue in position 3 (19d,
IC50 of 45 ± 5 µM; EMSA: 78% inhibition @ 500 µM) is tolerated, but the fluorinated analogue 19j
and most of the phenyl substituted compounds 36a-d, 36f-i, and 31a showed a complete loss or only
moderate activity.
Table 3. Inhibitory activities of analogues modified in position 3. Most derivatives substituted in this position
(R2) showed a significant decrease in activity.
FP Assay (LBS2)
IC50
EMSA (LBS1)
cpd
R1
R2
Inhibition @ 500 µM
Inhibitor I
H
H
17 ± 1 µM
45 ± 5 µM
n. i.
82 %
78 %
n. d.
19d
19j
H
H
Me
F
36a
36f
H
H
>250 µM
n.i.
n. d.
n. d.

36g
H
>250 µM
n. d.
36b
36c
H
H
n. i.
n. d.
n. i.
110 ± 32 µM
36d
36i
H
H
n. i.
n. i.
n. d.
n. .d
36e
31a
H
H
153 ± 7 µM
>250µM
20 %
n. d.
36h
H
>250 µM
n. d.
36j
H
19 ± 2 µM
38 ± 3 µM
34 %
31b
Cl
100 %
aFluorescence-polarization assay using LBS2 as probe, data representing average of duplicates ± standard deviation;
bElectrophoretic mobility shift assay using LBS1 as probe; cNo inhibition at 500 µM; dNot determined.
However, compounds with an additional hydroxyl function attached to the phenyl ring (36e, IC50 of
153 ± 7 µM, EMSA: 20% inhibition @ 500 µM) showed moderate activity.
Moving from a phenyl 36a to a smaller and more polar furanyl residue 36j the potency was restored
(IC50 of 19 ± 2 µM). Unfortunately, in EMSA experiments we observed only a weak effect (34%
inhibition @ 500 µM) for this compound. Interestingly, by attaching an additional chlorine atom in
position 4 and having a phenyl in position 3 (31b) resulted in a highly potent compound with IC50 of
38 ± 3 µM and full inhibition in FP and EMSA assays, respectively. These results further corroborate
the notion of a beneficial ortho effect.
To explore the influence of growing inhibitor I at the pyridine core in position 2, a set of different
target compounds was synthesized (Table 3 and 4). The direct attachment of a nitrile group to the
pyridine was tolerated (19e: IC50 52 ± 37 µM, EMSA: n. i.). Moving to chlorine, hydroxy or methoxy

group we observed a significant loss in activity (19f: IC50 >250 µM; 19o: IC50 214 ± 24 µM and 19k: IC50
218 ± 192 µM). Also in EMSA experiments 19j (75% inhibition @ 500µM) and 19k (11% inhibition @
500µM) did not show a significant effect. An increase in activity was observed by introducing bulkier
substituents and an additional methyl group for R1. In detail, an unpolar bulky phenyl or p-
chlorophenyl was accepted in position 2 and we observed IC50 values of 36 ± 5 µM for 36k and 58 ± 7
µM for 36l and moderate inhibition in EMSA. Analogues 26a and 26b with polar hydroxyl groups
attached at the phenyl showed good potency with IC50 values of 21 ± 3 µM and 25 ± 1 µM,
respectively. Furthermore, the efficiency of these two analogues in our EMSA studies was high with a
full inhibition @ 500 µM. By attaching methylamine (19g), isopropylamine (19h) and anilino (19i) at
position 2 we observed an increase in activity from small to bigger size, whereby the methylamine
compound 19g was completely inactive and the anilino analogue 19i showed a moderate activity of
IC50 of 110 µM and 29% inhibition in EMSA.
Additionally, a series of compounds was synthesized with a more flexible and bulky phenoxy group in
position 2 (Table 5). The phenoxy analogue 19l, similar to the aminophenyl compound 19i, was
inactive, indicating that an amino linker between pyridine and phenyl is more suitable for activity
compared to the oxygen linker. By attaching an additional methylgroup in position 4 (R1) at the
pyridine core 40a an increase in activity compared to 19l was observed leading to a moderate IC50 of
198 ± 8 µM. The fluorinated analogues 40b-d also showed moderate activities like compound 40a
while the o- fluoro analogue 40b possessed the best IC50 of 64 ± 2 µM. For the m- (40c) and p-fluoro
(40d) derivatives IC50s of 122 ± 3 µM and 134 ± 2 µM were observed, respectively. Unfortunately, all
these compounds showed no effect in EMSA experiments.
Table 4. Inhibitory activities of analogues modified in position 2. Attaching polar hydroxyl benzene groups
increases inhibitory activity.
FP Assay (LBS2)
IC50
EMSA (LBS1)
cpd
R1
R2
Inhibition @ 500 µM
Inhibitor I
H
H
17 ± 1 µM
52 ± 37 µM
>250 µM
82 %
n.i.
19e
19f
H
CN
Cl
Me
n.d.

19o
19k
36k
36l
H
H
OH
214 ± 24 µM
218 ± 192 µM
36 ± 5 µM
75 %
11 %
36 %
30 %
OMe
Me
Me
58 ± 7 µM
26a
Me
21 ± 3 µM
100 %
26b
19g
19h
Me
H
25 ± 1 µM
n.i.
100 %
n.d.
H
>250 µM
n.d.
19i
H
110 ± 20 µM
29 %
aFluorescence-polarization assay using LBS2 as probe, data representing average of duplicates ± standard deviation;
bElectrophoretic mobility shift assay using LBS1 as probe; cNo inhibition at 500 µM; dNot determined.
Exchanging the oxygen linker by a sulfur (40e, IC50 175 ± 10 µM) was tolerated (compare with 40a,
IC50 198 ± 8 µM). As expected, by removing the nitrogen in the pyridine core resulted in an inactive
compound (19m). Astonishingly, moving the nitrogen to the phenoxy residue (19n) yielded a highly
potent compound with an IC50 of 19 ± 1 µM showing also full inhibition in the EMSA experiments.
Table 5. Inhibitory activities of Phenoxy analogues. Shifting nitrogen to the phenoxy residue improves
inhibitory efficiency.
FP Assay (LBS2)
IC50
EMSA (LBS1)
cpd
R2
inhibition @ 500 µM
Inhibitor I
17 ± 1 µM
n. i.
82 %
n. d.
19l
40a
198 ± 8 µM
64 ± 2 µM
n. i.
n. i.
40b

40c
40d
40e
122 ± 3 µM
134 ± 2 µM
175 ± 10 µM
n. i.
n. i.
n. i.
19m
19n
n.i.
n. d.
19 ± 1 µM
100 %
aFluorescence-polarization assay using LBS2 as probe, data representing average of duplicates ± standard deviation;
bElectrophoretic mobility shift assay using LBS1 as probe; cNo inhibition at 500 µM; dNot determined.
Intrigued by the notion that fragment growing in position 3 was possible in combination with ortho-
substituents, we focused our efforts on further exploring these two positions by installing a
connected structural motif. To this end, we designed and synthesized isochinoline analogues (Table
6). In general, isoquinoline analogues were pleasingly effective. The unsubstituted isochinoline 43
showed an FP IC50 value of 33 ± 1 µM and 96% inhibition in EMSA experiments. Moving from
isoquinoline to quinoline 46 resulted in a slight loss in activity compared to 43 (46, IC50 70 ± 34 µM,
61% inhibition in EMSA).
Table 6. Inhibitory activities of Isoquinoline derivatives. Adding an annulated ring structure in direction of
identified growth vector results in the most efficient inhibitors to date.
FP Assay (LBS2)
IC50
EMSA (LBS1)
Cpd
R
inhibition @ 500 µM
Inhibitor I
17 ± 1 µM
82 %
96%
43
46
33 ± 1 µM
70 ± 34 µM
61%

50a
50b
8 ± 1 µM
100%
100%
17 ± 1 µM
50c
>250 µM
n. d.
aFluorescence-polarization assay using LBS2 as probe, data representing average of duplicates ± standard deviation;
bElectrophoretic mobility shift assay using LBS1 as probe; cNo inhibition at 500 µM; dNot determined.
Attaching an additional methyl (50a) or chlorine (50b) at the isoquinoline motif was beneficial for the
inhibitory effect. Noteworthy, 50a showed the lowest FP IC50 of 8 ± 1 µM reported to date, while 50b
also possessed a decent IC50 of 17 ± 1 µM. Furthermore, in EMSA experiments 100% inhibition was
detected for both compounds at 500 µM. Finally, an isoquinoline methylester analogue 50c was
inactive, however.
Further Characterization and EMSA Studies using wild-type LANA.
For further evaluation and characterization the most promising compounds were selected. On the
bases of our results, we chose compounds 19c, 31b, 26a-b, 19n, 50a, and 50b, which possessed the
best IC50 values in the FP-based assay and showed strong inhibitory effects at 500 µM in EMSA using
the oligomerization-defizient LANA DBD mutant (aa1008-1146).
First, these compounds were initially tested for inhibition at 250 µM using LANA DBD mutant (Figure
2, A) to see if they are also able to disturb the LANA-DNA interaction at a lower concentration in
EMSA and compared these results also with inhibitor I. Inhibitor I and the compounds 19c, 31b, and
26b showed no inhibitory effect on the LANAmut-DNA interaction at 250 µM. However, strong
inhibitory effects were observed at this concentration for compounds 26a, 19n, 50a, and 50b as
observed by the disappearance of the bands for the LANAmut-DNA complex (upper band, Figure 2A).

Figure 2: EMSA gels with inhibitor I, 19c, 31b, 26a-b, 19n, 50a-b. Compounds were tested at a final
concentration of 250 µM and LBS1 was used as probe. (A) Using an oligomerization-deficient LANA DBD
mutant, a strong inhibitory effect (disappearance of LANA-DNA complex band) was observed for compounds
26a, 19n and 50a-b (B) Using wild-type LANA CTD, a significant inhibitory effect for compound 50a was
observed.
We also determined the inhibitory activity of our best compounds against the interaction between
wild-type LANA CTD (aa934-1162) and viral LBS1 (Figure 2, B) in EMSA. Our wild-type LANA CTD
construct is longer compared to the LANA DBD mutant and has no mutations and still shows a
sufficient solubility in aqueous medium also in presence of viral LBS1. The compounds were also
tested at 250 µM. Unfortunately, no inhibitory effect was observed for compounds I, 19c, 31b, 26a-b,
19n, and 50b. However, Compound 50a showed a significant effect and was able to inhibit the
interaction between wild-type LANA CTD and LBS1.
Furthermore, we titrated the compounds showing an effective inhibition in EMSA using the LANA
DBD mutant (Figure 2, A), in EMSA experiments using the LANA DBD mutant and LBS1 as a probe to
determine the IC50 values. The results are listed in Table 7 and detailed information can be found in
the supporting information.
Table 7: Comparison of most efficient LANA-DNA inhibitors.
FP-Assaya
IC50 (LBS2)
FP-Assay
FP-Assay
EMSAb
Cpd
Structure
IC50 (LBS1)
IC50 (LBS3)
IC50 (LBS1)
(LANAmut
)
(LANAmut
)
(LANAmut
)
(LANAmut)

Inhibitor I
19c
17 ± 1 µM
18 ± 4 µM
20 ± 3 µM
52 ± 2 µM
19 ± 3 µM
42 ± 3 µM
426 ± 2 μM
n.i. at 250 µMc
31b
38 ± 3 µM
55 ± 7 µM
45 ± 4 µM
n.i. at 250 µM
26a
26b
19n
50a
21 ± 3 µM
25 ± 1 µM
19 ± 1 µM
8 ± 1 µM
30 ± 2 µM
64 ± 1 µM
15 ± 1 µM
9 ± 2 µM
34 ± 3 µM
63 ± 8 µM
25 ± 1 µM
8 ± 1 µM
156 ± 27 µM
n.i. at 250 µM
64 ± 12 µM
53 ± 43 µM
50b
17 ± 1 µM
14 ± 1 µM
15 ± 1 µM
93 ± 8 µM
aFluorescence-polarization assay using LBS1, LBS2 and LBS3 as probe, data representing average of duplicates ± standard
deviation; bElectrophoretic mobility shift assay using LBS1 as probe; cNo inhibition at 250 µM.
As reported earlier by us, inhibitor I showed an IC50 in FP assay (LBS2) of 17 ± 1 µM and an IC50 in
EMSA of 426 ± 2 μM using LANA DBD mutant.11 The observed IC50 values using LBS2 for the most
promising inhibitors were basically in the same range. Additionally, we also tested the most
promising inhibitors in FP assay using LBS1 and LBS3, respectively. Compound 50a showed a 2-fold
better IC50 of 8−9 µM against all LBS compared to I. Furthermore, we could increase the inhibitory
activity in EMSA experiments using LBS1 by 7-fold. As a consequence compounds 19n and 50a are
the most potent LANA-LBS1-inhibitors reported so far (IC50 values of 64 ± 12 µM and 53 ± 3 µM).
Interestingly, compounds showing increased IC50 values of 50-60 µM against LBS1 and LBS3 in FP
assay were also not effective in EMSA at 250 µM. Excepted is however inhibitor I, which showed also
IC50 values around 20 µM against LBS1 and LBS3, but no effect at 250 µM in EMSA.
As described above, only compound 50a showed an inhibitory effect at a concentration of 250 µM in
EMSA using wild-type LANA CTD (Figure 3 A). A dose-response EMSA experiment with wild-type
LANA CTD yielded an IC50 value of 60 ± 4 µM (Figure 3 B).

Figure 3: (A) Dose-dependent EMSA experiment using wild-type LANA CTD, LBS1 as probe and Compound 50a.
(B) Curve shows normalized data points (inhibition from 0 - 100%) representing intensities of LANA-DNA-
complex bands (Figure 3 (A), upper bands, marked in red) from dose-dependent EMSA experiment. IC50 value
was calculated using a four-parameter dose-response model. (C) Dose-dependent EMSA experiment using
LANA DBD mutant, a combination of LBS1+2 as probe and Inhibitor I and Compound 50a. (D) Dose-dependent
EMSA experiment using LANA DBD mutant, a combination of LBS2+1+3 as probe and Inhibitor I and Compound
50a.
These results indicate that compound 50a is equally potent against wild-type LANA CTD and the
oligomerization-deficient LANA DBD mutant. In comparison to inhibitor I (IC50 of 435 ± 6 μM against
wild-type LANA CTD in EMSA) these results represent a huge potency improvement.11 Furthermore,
we tested if compound 50a is also able to disturb the LANA-DNA interaction when using longer
oligonucleotides comprising LBS1+2 as well as LBS2+1+3, in an arrangement that is present on the
viral KSHV genome.17 Each LBS is able to associate with one LANA dimer. Hence, LANA and the
LBS1+2 oligomer can form a trimeric complex while the LBS2+1+3 oligomer gives rise to a quaternary
complex. In order to test the efficacy of our inhibitors against the formation of these higher-order
aggregates, dose–dependent EMSA experiments using LBS1+2 and LBS2+1+3 with the LANA DBD
mutant were performed (Figure 3, C and D). We compared the effects of inhibitor I and Compound

50a in this setup. As expected, inhibitor I showed no inhibitory effects in both experiments. However,
Compound 50a was still able to significantly inhibit the LANA LBS1+2 interaction at a concentration of
62.5 µM (Figure 3, C) and additionally inhibited the LANA LBS2+1+3 interaction at 125 µM (Figure 3,
D). These results provide a basis for testing these inhibitors in cell based assays in the future.
The similar IC50 values of compound 50a observed against wild-type LANA and LANA DBD mutant
corroborates our hypothesis that our inhibitor binds at the DNA binding interface and is able to
compete with the DNA. The DBD mutant involves nine point mutations, which are all located outside
of the DNA binding site.25 The goal of generating and using this mutant was to disturb the higher
oligomerization in solution, which resulted in improved handling characteristics of the protein and
better solubility of LANA-DNA complexes. Figure 4 illustrates the LANA DBD surface (blue) and its
single point mutations (yellow) bound to double-stranded DNA (red). The distribution of these
mutations distant from the DNA-interaction interface combined with the observed similar IC50 values
of our compound against wild-type and DBD mutant provide strong evidence, that we are targeting
the LANA DNA interaction interface and that we do not unintentionally target one of the mutated
regions on the LANA surface.
Figure 4: Illustration of the nine single point mutations (yellow) of oligomerisation interface and basic patch
LANA mutant (blue) bound to DNA (red) assuming that our inhibitors bind at the LANA-DNA interaction
interface.
Taken together, compound 50a is the most effective inhibitor against LANA DBD mutant and wild-
type LANA CTD reported to date. To complement our studies, we applied STD-NMR experiments in
order to identify further growth vectors.
STD-NMR Studies for Insight on Ligand Binding Modes

Finally, we complemented our lead generation campaign by STD-NMR experiments with the aim to
gather information on the binding orientation and to identify further growth vectors. In parallel, we
attempted to solve co-crystal structures of our inhibitors in complex with LANA, unfortunately
without success. However, we could successfully confirm previous STD-NMR and molecular docking
results for inhibitor I by the obtained SAR in the present study and we were able to enlarge inhibitor I
in the suggested direction. In order to inform our next optimization steps, we again performed STD-
NMR experiments of our best inhibitor 50a.
The protons of the tested compound, which are in closest proximity to the protein surface upon
binding, are showing the strongest STD effects. In Figure 5 an overlay of the on- (red) and off- (black)
resonance STD-NMR spectra of the aromatic region are shown. The spectra were normalized to the
strongest signal, which was observed for proton 1. The STD effects were calculated for each proton
of inhibitor 50a (EffectSTD = I/I0). The results suggest that compound 50a has a binding orientation to
LANA similar to that of inhibitor I.11
Figure 5: STD experiments of compound 50a in complex with LANA DBD mutant. The reference spectrum is
displayed in black (STD-off) and STD difference spectra (STD-on) in red. Overlaid spectra were normalized to
the signal for 1-H, which showed the strongest enhancement.
The nitrogen of the pyridine ring presumably acts as a proton acceptor and thereby anchors proton 1
close to the LANA surface. The proton in position 2 showed also a strong STD effect of 89%. The
corresponding proton in inhibitor I showed a slightly stronger effect of 100%.11 The bulky

isoquinoline moiety and the additional methyl group cause an ortho effect, which hinders the
rotation of the bond between the triazole core and the isoquinoline. We hypothesize, that this effect
brings the nitrogen in an even more favorable orientation to the LANA surface and in turn leaves
proton 2 now slightly more exposed. Furthermore, proton 6 located in the triazole core shows a
stronger STD effect of 85% (47% in inhibitor I),11 which leads us to suspect that the triazole is also
now interacting more favourably with LANA. The four protons at the benzoic acid moiety, 7-H and 8-
H, were found under one signal displaying a prominent STD effect of 89%. In contrast, the protons at
the second ring of the isoquinioline motif showed a significantly lower STD effect (3-H: 34%, 4-H: 28%
and 5-H: 49%). Hence, we conclude that these protons are not in direct contact with the protein and
should be further investigated as potential secondary growth vectors. Unfortunately, the STD effect
of the methyl group could not be determined, because signals of buffer ingredients were in the same
chemical shift range. For future medicinal chemistry optimization studies, further growing of the
isoquinoline analogues in positions 3, 4 and 5 should be investigated. Additionally, a combination of
the isoquinoline 50a and compounds elongated in position 2 (see 19n and 26a) should be attempted.
Conclusion
In this study we synthesized a series of new derivatives of our LANA inhibitor I11 and evaluated them
for LANA-DNA interaction inhibition in an FP-assay and in EMSA experiments in order to generate
more potent LANA-DNA inhibitors. Based on the previously discovered fragment-sized inhibitor I,
medicinal chemistry optimization lead to new inhibitors with improved potency. In particular, the
replacement of the pyridine core (inhibitor I) by a methyl-isoquinoline (50a) led to an increase of
inhibitory potency of 7-fold against the wild-type LANA CTD interaction with viral LBS1. Moreover,
compound 50a was also able to inhibit the interaction between LANA DBD mutant and LBS1+2 and
LBS2+1+3, respectively, in the lower micromolar range. Additionally, step-by-step modification
studies gave new and important SAR insights for future medicinal chemistry optimizations towards
lead structures. Furthermore, STD-NMR measurements of the most potent inhibitor 50a in complex
with LANA revealed important details about the binding orientation and allowed for the
identification of a new potential growth vector. The ability to inhibit the wild-type LANA DNA
interaction in a low micro molar range (IC50 60 ± 4 µM) with such a small molecule scaffold is striking
as such a macromolecule-macromolecule-interaction is usually considered to be highly challenging, if
not “undruggable”. Unexpectedly, we were able to significantly improve the inhibitory effects of our
inhibitors using a chemistry-driven approach without having any structural information from a co-
crystal about the binding mode and location on the LANA surface. Our results pave the way for the
generation of a LANA-targeting anti-KSHV agent.

Experimental Section
All reagent-grade chemicals were purchased from commercial suppliers and were used as received.
The purifications were performed using automated column flash chromatography (CombiFlash Rf+,
Teledyne ISCO, Lincoln, NE, USA) on silica gel 0.04 – 0.063 mm (RediSep Rf Kartuschen, Axel Semrau,
Spocklhövel, Germany) or using preparative high performance liquid chromatography (HPLC,
Ultimate 3000 UHPLC+ focused, Thermo Scientific) on a reversed-phase column (C18 column, 5 μm,
Macherey-Nagel, Germany). The solvents used for column flash chromatography were EtOAc and
cyclohexane or DCM and MeOH. The solvents used for HPLC were water (containing 0.05% [v/v] FA)
and MeCN (containing 0.05% [v/v] FA) (gradient elution, MeCN:H2O 1:9 ꢀ 9:1). Reaction progress
was monitored by TLC on TLC Silica Gel 60 F254 plates (Merk, Darmstadt, Germany) or by a reversed-
phase liquid chromatography mass spectrometer (LCMS). 1H and 13C NMR were recorded on a Bruker
Fourier spectrometers (500 or 126 MHz). Chemical shifts (δ) were reported in parts per million (ppm)
relative to the corresponding reference solvent. The chemical shifts recorded as δ values in ppm
units by reference to the hydrogenated residues of the deuterated solvent as the internal standard.
Coupling constants (J) are given in hertz (Hz) and splitting patterns are designated as follows: s,
singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet; br., broad signal. Purity of all final
compounds was measured on the UV trace recorded at a wavelength of 254 nm and was determined
to be >95% by a reversed-phase liquid chromatography mass spectrometer (LCMS). Representative
1H and 13C spectra of all final compounds can be found in the supporting information. High resolution
mass spectra of all final compounds were measured on a Thermo Scientific Q Exactive Focus
(Germany) equipped with a DIONEX ultimate 3000 UHPLC+ focused and can be found in the
supporting information. The reactions and purification steps were not optimized regarding yields.
General procedure for azide formation (GP1) for Compounds 2, 18a-n, 21, 25a-b, 30a-b, 33a-b,
39a-e, 42, 45, 49a-c: The appropriate aryl amine (1 eq.) was dissolved in EtOAc, cooled to 0 °C and 6
M HCl was added. Sodium nitrite (1.7 eq.) was dissolved in water and added slowly. The reaction
mixture was stirred for 30 min at 0 °C. Subsequently, sodium azide (1.7 eq.) in water was added
slowly at 0 °C. The mixture was stirred at room temperature for 2 h. TLC control indicated full
conversion and the mixture was basified with saturated NaHCO3 solution and was extracted with
EtOAc (2×). The combined organic layers were dried over sodium sulfate and concentrated under
reduced pressure to give the crude. The crude product was used as obtained in the next step without
further purifications. Compound 2 is presented as an example. 3-azidopyridine (2): The azide was
synthesized according to GP1 using pyridin-3-amine 1 (376 mg, 4 mmol), sodium nitrite (1.7 eq., 469

mg, 6.8 mmol), sodium azide (1.7 eq., 442 mg, 6.8 mmol), EtOAc (8 mL), 6 M HCl (5 mL). The crude
product (245 mg, 2 mmol, 50%) was used as obtained in the next step without further purifications.
Rf = 0.37 (PE/EtOAc 7:3)
General procedure for synthesis of amino isoquinolin derivatives (GP2) for compounds 48a-c:
Under argon atmosphere the appropriate bromo isoquinolin (1 eq.), L-proline (0.1 eq.), sodium azide
(1.3 eq.) and sodium carbonate (1.3 eq.) was dissolved in a 2:1 mixture of DMF and water.
Subsequently, sodium ascorbate (1.3 eq.) and copper sulfate hepta hydrate (1 eq.) were added and
the reaction mixture was stirred over night at 85 °C. After full conversion (LCMS control) the mixture
was cooled to room temperature and EtOAc and sat. aqueous NaHCO3 solution were added. The
mixture was extracted with EtOAc (3x), the combined organic layers were dried over sodium sulfate
and concentrated under reduced pressure to obtain the crude. The obtained products were used as
obtained without further purification. Compound 48a is presented as an example. 6-
methylisoquinolin-4-amine (48a): The amino isoquinolin was synthesized according to GP2 using 4-
bromo-6-methylisoquinoline 47a (100 mg, 0.46 mmol), L-proline (0.1 eq., 0.05 mmol, 5 mg), sodium
azide (1.3 eq., 0.60 mmol, 34 mg), sodium carbonate (1.3 eq., 0.60 mmol, 64 mg), sodium ascorbate
(1.3 eq., 0.60 mmol, 119 mg), copper sulfate heptahydrate (1 eq., 0.46 mmol, 115 mg), DMF (4 mL)
and water (2 mL). The crude product (70 mg, 0.44 mmol, 97%) was used as obtained in the next step
without further purifications. MS (ESI+) m/z 159 (M + H).
General procedure for copper catalyzed click reaction (GP3) for compounds 3-11, 13, 16, 19a-n, 22,
26a-b, 31a-b, 34a-b, 40a-e, 50a-c: Under argon atmosphere the appropriate alkyne (1 eq.) was
suspended in a 1:1 mixture of water and MeOH. Subsequently, DIPEA (2.0 eq.), copper sulfate hepta
hydrate (0.5 eq.) and sodium ascorbate (0.5 eq.) were added. After addition of the corresponding
azide (1.2 eq.) the mixture was stirred for 16 h at room temperature. After full conversion (LCMS
control) the mixture was acidified with 1 M HCl and the product was precipitated. The solids were
collected, washed with water, and dried under vacuum to obtain the crude product. The products
were purified using preparative HPLC. The solvents used were water (containing 0.05% [v/v] FA) and
MeCN (containing 0.05% [v/v] FA) (gradient elution, MeCN:H2O 1:9 ꢀ 9:1). Compound 4 is presented
as an example. 3-(1-(pyridin-3-yl)-1H-1,2,3-triazol-4-yl)benzoic acid (4): The triazole was synthesized
according to GP3 using 3-ethynylbenzoic acid (92 mg, 0.64 mmol) and 3-azidopyridine 2 (1.3 eq., 100
mg, 0.83 mmol) as starting materials. The crude was obtained as a white solid (110 mg, 0.41 mmol,
64%). Purification was done using preparative HPLC. 1H NMR (500 MHz, DMSO-d6) δ ppm 7.66 (br. s.,
1 H) 7.68 - 7.79 (m, 1 H) 7.88 - 8.06 (m, 1 H) 8.19 (d, J=7.63 Hz, 1 H) 8.41 (d, J=8.24 Hz, 1 H) 8.53 (s., 1
H) 8.76 (s., 1 H) 9.25 (s., 1 H) 9.55 (s, 1 H) 13.23 (br. s., 1 H); 13C NMR (126 MHz, DMSO-d6) δ ppm
120.56, 124.96, 127.82, 129.40, 141.24, 146.80, 149.81, 167.30

General procedure for Suzuki coupling (GP4) for compounds 24a-b, 29a-b, 35a-l: Under argon
atmosphere the appropriate aryl halide 1 (eq.) was dissolved in water and 1,4-dioxane (1:1). Sodium
carbonate (3 eq.), the corresponding boronic acid (1.2 eq.) and tetrakis (triphenylphosphine)
palladium (0.1 eq.) were added. The reaction mixture was heated to 90 °C for 16 h. After full
conversion (LCMS control) the mixture was cooled to room temperature and EtOAc and sat. aqueous
NaHCO3 solution were added. The mixture was extracted with EtOAc (3x), the combined organic
layers were dried over sodium sulfate and concentrated under reduced pressure to obtain the crude.
The purification was done using automated flash chromatography (cyclohexane/EtOAc 1:0 ꢀ 0:1).
Compound 24a is presented as an example. 2-(5-amino-4-methylpyridin-2-yl)phenol (24a): The
coupling was done according to GP4 using 6-bromo-4-methylpyridin-3-amine 23 (130 mg, 0.69
mmol), (2-hydroxyphenyl)boronic acid (1.2 eq., 113 mg, 0.83 mmol), sodium carbonate (3 eq., 218
mg, 2.08 mmol, ) and tetrakis (triphenylphosphine) palladium (0.1 eq., 78 mg, 0.07 mmol) in 1,4-
dioxan:water (1:1, 6 mL). 24b was obtained as yellow solid (103 mg, 0.52 mmol, 75%). MS (ESI+) m/z
201 (M + H).
General procedure for hydrolysis of methyl ester (GP5) for compounds 36a-l: The appropriate
methyl ester was dissolved in MeOH and aqueous 0.5 M NaOH solution (1:1). The mixture was stirred
at room temperature for 16 h. After full conversion (LCMS control) the mixture was acidified with 1
M HCl and the product was precipitated. The solids were collected, washed with water, and dried
under vacuum to obtain the crude product. The products were purified using preparative HPLC.
Compound 36a is presented as an example. 4-(1-(5-phenylpyridin-3-yl)-1H-1,2,3-triazol-4-yl)benzoic
acid (36a): The synthesis was done according to GP5 using methyl 4-(1-(5-phenylpyridin-3-yl)-1H-
1,2,3-triazol-4-yl)benzoate 35a (20 mg, 0.06 mmol). The crude was obtained as a white solid (13 mg,
0.04 mmol, 66%). Purification was done using preparative HPLC.1H NMR (500 MHz, DMSO-d6) δ ppm
7.49 - 7.54 (m, 1 H) 7.56 - 7.61 (m, 2 H) 7.84 - 7.95 (m, 2 H) 8.02 - 8.16 (m, 4 H) 8.65 (t, J=2.21 Hz, 1 H)
9.07 (d, J=1.98 Hz, 1 H) 9.22 (d, J=2.29 Hz, 1 H) 9.64 (s, 1 H); 13C NMR (126 MHz, DMSO-d6) δ ppm
121.41, 125.29, 125.57, 127.30, 128.97, 129.30, 130.22, 133.38, 133.97, 135.68, 136.49, 139.93,
146.65, 147.70, 167.01
General procedure for Ullmann reaction (GP6) for compounds 38a-e: Under argon atmosphere 6-
bromo-4-methylpyridin-3-amine (1 eq.) was dissolved in DMF and the appropriate phenol derivative
(1.2 eq.), cesium carbonate (3 eq.) and CuI (0.05 eq.) were added. The mixture was stirred for 16 h at
130 °C. LCMS control indicated full conversion and the mixture was cooled to room temperature.
EtOAc and sat. aqueous NaHCO3 solution were added. The mixture was extracted with EtOAc (3x),
the combined organic layers were dried over sodium sulfate and concentrated under reduced
pressure to obtain the crude. Compound 38a is presented as an example. 4-methyl-6-

phenoxypyridin-3-amine (38a): The aryl ether was synthesized according to GP6 using 6-bromo-4-
methylpyridin-3-amine 37 (100 mg, 0.53 mmol), phenol (1.2 eq., 0.60 mmol, 70 mg), cesium
carbonate (3 eq., 1.61 mmol, 523 mg) and CuI (0.1 eq., 0.05 mmol, 9 mg) in DMF (3 mL). The product
was purified using automated flash chromatography (DCM/MeOH 1:0 ꢀ 9:1). Yield: (30 mg, 0.15
mmol, 28%) MS (ESI+) m/z 201 (M + H).
Protein expression and purification: The expression and purification of His-tagged oligomerization-
deficient LANA DNA binding domain (DBD; aa1008−1146) mutant and GST-tagged KSHV LANA C-
terminal domain (CTD; aa934-1162) were described previously and the protocol was adopted.10,11
Fluorescence Polarization (FP) assay: The FP assay was performed, analyzed and evaluated as
described previously.11
Electrophoretic mobility shift assay (EMSA): The EMSA was performed, analyzed and evaluated as
described previously.11
Saturation-Transfer Difference (STD) NMR: The STD experiments were recorded at 298 K on a Bruker
Fourier spectrometer (500 MHz). The samples contained 10 µM (final concentration) His-tagged
oligomerization-deficient LANA DBD (aa1008−1146) mutant and a final compound concentraꢀon of
500 µM. The control spectra were recorded under the same conditions containing the free
compound to test for artifacts. The STD buffer for experiments consists of 10 mM HEPES, 150 mM
NaCl, pH 7.4 in D2O containing 10% [v/v] DMSO-d6. The experiments were recorded with a carrier set
at -1 ppm for the on-resonance and −40 ppm for the off-resonance irradiation. Selective protein
saturation was carried out at 2 s by using a train of 50 ms Gauss-shaped pulses, each separated by a
1 ms delay.
Declaration of competing interest
The authors declare no competing financial interest.
Corresponding Author
*E-mail: Martin.Empting@helmholtz-hzi.de. Phone: +(49) 681 988062031
Acknowledgments