Fragment-Based Discovery of a Qualified Hit Targeting the Latency-Associated Nuclear Antigen of the Oncogenic Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8

Article abstract of DOI:10.1021/acs.jmedchem.8b01827

The latency-associated nuclear antigen (LANA) is required for latent replication and persistence of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8. It acts via replicating and tethering the virus episome to the host chromatin and exerts other functions. We conceived a new approach for the discovery of antiviral drugs to inhibit the interaction between LANA and the viral genome. We applied a biophysical screening cascade and identified the first LANA binders from small, structurally diverse compound libraries. Starting from a fragment-sized scaffold, we generated optimized hits via fragment growing using a dedicated fluorescence-polarization-based assay as the structure-activity-relationship driver. We improved compound potency to the double-digit micromolar range. Importantly, we qualified the resulting hit through orthogonal methods employing EMSA, STD-NMR, and MST methodologies. This optimized hit provides an ideal starting point for subsequent hit-to-lead campaigns providing evident target-binding, suitable ligand efficiencies, and favorable physicochemical properties.

Full text of DOI:10.1021/acs.jmedchem.8b01827

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Article
Fragment-based discovery of a qualified hit targeting the
Latency-associated Nuclear Antigen of the oncogenic Kaposi’s
Sarcoma-associated Herpesvirus/Human Herpesvirus 8
Philine Elea Kirsch, Valentin Jakob, Kevin Oberhausen, Saskia Stein,
Ivano Salvatore Cucarro, Thomas F. Schulz, and Martin Empting
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01827 • Publication Date (Web): 19 Mar 2019
Downloaded from http://pubs.acs.org on March 20, 2019
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Fragment-based discovery of a qualified hit targeting
the Latency-associated Nuclear Antigen of the
oncogenic Kaposi’s Sarcoma-associated
Herpesvirus/Human Herpesvirus 8
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Philine Kirsch†1,2,3, Valentin Jakob , Kevin Oberhausen , Saskia C. Stein , Ivano Cucarro ,
1,2
1,2
3,4
1,2
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,4
1,2,3*
Thomas F. Schulz , Martin Empting
1Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)-Helmholtz Centre for
Infection Research (HZI), Department of Drug Design and Optimization (DDOP), Campus E8.1,
6
6123 Saarbrücken, Germany
2Department of Pharmacy, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany
3German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig,
Saarbrücken, Germany
4Institute of Virology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover,
Germany
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ABSTRACT: The Latency-Associated Nuclear Antigen (LANA) is required for latent replication
and persistence of Kaposi´s Sarcoma-associated Herpesvirus (KSHV)/human herpesvirus-8
(HHV-8). It acts via replicating and tethering the virus episome to the host chromatin and exert
other functions. We conceived a new approach for the discovery of antiviral drugs to inhibit the
interaction between LANA and the viral genome. We applied a biophysical screening cascade and
identified the first LANA binders from small, structurally diverse compound libraries. Starting
from a fragment-sized scaffold, we generated optimized hits via fragment growing using a
dedicated fluorescence polarization-based assay as the structure-activity-relationship driver. We
improved compound potency to the double-digit micromolar range. Importantly, we qualified the
resulting hit through orthogonal methods employing EMSA, STD-NMR and MST methodologies.
This optimized hit provides an ideal starting point for subsequent hit-to-lead campaigns providing
evident target-binding, suitable ligand efficiencies and favorable physicochemical properties.
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Introduction
Kaposi’s sarcoma-associated herpesvirus (KSHV; taxonomic name human herpesvirus 8) is a
human gamma herpes virus and is classified as a carcinogenic agent Group I by the World
Health Organisation.1; 2 It was identified as the etiological agent of Kaposi Sarcoma (KS) and
lymphoproliferative disorders. After a first infection, it establishes a lifelong latent infection in
the host organism. KSHV is usually not pathogenic in healthy individuals, but AIDS-related
Kaposi's sarcoma (AIDS-KS), KS in transplant recipients and endemic KS in East/Central Africa
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cause significant morbidity and mortality in affected patients. In latently infected cells, KSHV
expresses only a limited set of proteins, which are important for the persistence. One of these is
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,5
the Latency-Associated Nuclear Antigen (ORF73/LANA). LANA plays an important role for
the latency and regulation of the viral genome in the host organism. Previous studies have shown
that LANA exerts several functions in the host cell like cell survival, transcriptional control,
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latent viral DNA replication and stable episome segregation during mitosis. The C-terminal
domain of LANA binds to the terminal repeat (TR) region of the viral genome in a
sequence-specific manner. 7; 2 The TR consists of three adjacent LANA binding sites (LBS),
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which are referred to as LBS1, LBS2 and LBS3 (Figures 1 and 2). The N-terminal domain of
LANA is very poorly structured and is tethered to the host nucleosome.8; 9 It is separated by a
large internal repeat sequence from the C-terminal DNA-binding domain (Figure 1).10; 11
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Figure 1. Illustration of molecular interactions between KSHV LANA, viral episome, and host
nucleosome, rationalizing the C-terminal DNA binding domain of KSHV LANA as an antiviral
drug target as it links viral DNA (yellow and orange) to host histones (dark green) and attached
host DNA (yellow and green). Three KSHV LANA dimers (blue) are shown and unordered
repeats are displayed as tubes. Illustration was modeled using coordinates of PDB entries 1zla,
4
uzb and 4uzc.
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To date, the options for treating KSHV-associated diseases are limited. While several inhibitors
of herpesviral DNA polymerases are active against KSHV productive replication, they are not
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effective against KS or other KSHV associated malignancies. LANA represents a very
promising target for the discovery and development of new anti-KSHV drugs that, in contrast to
currently available compounds, would interfere with the latent phase of the viral life cycle. Based
on the knowledge that LANA is involved in binding to viral latent episomal DNA and tethering
it to host nucleosomes7; 6 we conceived a new approach for the discovery of specific KSHV
inhibitors. Our concept aims at the inhibition of the interaction between LANA and the viral
genome. This should ultimately prevent latent persistence of KSHV, which could result in the
gradual loss of infected cells and in a decrease in viral load in infected individuals. In the present
work, we present our efforts to exploit this strategy through identification of the first functional
LANA-DNA-interaction inhibitors by using fragment-based drug design. As a first step, we
made use of different biological and biophysical methods to screen fragment libraries for
identification of LANA binders. Subsequently, we established a fluorescence polarization-based
assay to determine the inhibitory activity of our hits and we used it for further optimization steps.
Furthermore, we confirmed target binding of our best compound via orthogonal assays using
saturation-transfer difference (STD) NMR and microscale thermophoresis (MST)
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methodologies. Finally, we qualified the optimized hit scaffold for future lead-generation
campaigns in an orthogonal interaction inhibition assay, namely electrophoretic mobility shift
assay (EMSA). This provides an ideal starting point for subsequent medicinal-chemistry efforts
toward specific anti-KSHV agents. To the best of our knowledge, this study is the first report of
inhibitors targeting the DNA-binding domain of LANA. A similar approach, however, has been
previously applied to the EBNA1 protein, the functional homologue of LANA in Epstein-Barr
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virus (EBV). However, these conceptually related studies as well as experiments with a DNA-
binding site mutant of LANA provide a sufficient basis for the validity of this antiviral drug
target.14
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Figure 2. Model of the C-terminal DNA binding domain of three KSHV dimers bound to
adjacent LANA-binding sites LBS1, 2, and 3. Protein chains are shown as ribbon representations
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chain A: blue, chain B: light blue). Viral DNA (yellow orange) is shown in space-filling
representation. The model was generated using pdb entries 4uzb and 4uzc.
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Results and Discussion
Screening and Hit Identification. To discover the first small molecules, binding to LANA, we
used orthogonal biophysical methods to screen two different small-molecule libraries from
synthetic and natural sources.15; 16 For the primary selection, we used two protein binding assays,
surface plasmon resonance (SPR) and differential scanning fluorimetry (DSF) due to their high
sensitivity and low protein consumption. First, we conducted SPR screening at a constant
concentration to preselect putative LANA binders. Subsequently, we applied DSF (“Thermal
Shift Assay“ TSA) as a secondary filter.
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This methodology enabled us to select 20 compounds for further testing. In order to test for
functionality of our LANA binders in vitro, we established a fluorescence polarization-based
assay, which allows for the quantitative evaluation of the LANA-DNA-interaction and its
inhibition by small molecules. In this manner, we identified three promising small molecule hit
scaffolds for further consideration. In this report, we will focus on our hit optimization efforts,
starting from the fragment-sized Hit 1 (Figure 3).
SPR- and DSF-based Primary Screening. Two different libraries, containing a total of 720
highly structurally diverse set of hit-like small molecules with a molecular weight below
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98 g/mol were screened.15; 16 We started with SPR spectroscopy using the wild-type LANA
C-terminal domain (CTD) as ligand and the library compounds at a constant concentration of
00 µM. Compounds that showed a response higher than 9 µ-refractive index units were selected
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from this screening, which yielded 52 primary binders. (for detailed results see SI, Figure S1). In
a second step, we employed DSF experiments as a secondary filter. This assay quantifies a
change in thermal denaturation temperature of wild-type LANA C-terminal domain by binding
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to a compound. Generally, an increase of melting temperature indicates a stabilization of the
protein due to binding of a small molecule. Our experiments showed for almost all compounds a
decrease in melting temperature for LANA. A decrease of melting temperature may indicate a
destabilization of the protein by compound binding. Although usually an increase in protein
stability and, thus, increase in melting temperature is observed for target binders, also negative
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shifts are commonly considered as binding events. As a consequence, we selected 20
compounds, which showed a significant thermal shift T ≥ + 0.5 °C and T ≤ -1.0 °C for further
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investigations (for detailed results see SI, Figure S2).
Figure 3. Screening procedure using two different fragment libraries targeting LANA. In total
20 compounds were screened using SPR experiments, followed by DSF. A FP-based assay was
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used to identify promising interaction inhibitors, which resulted in three promising hits.
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Establishment of FP-Assay. For further characterization and optimization, our compounds were
tested for inhibition of the DNA-LANA interaction using a fluorescence polarization (FP) -based
competition assay. The FP assay is a rapid and quantitative method for identification of small
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molecular macromolecule-macromolecule interaction inhibitors. For this process we used a
mutant of the KSHV LANA C-terminal DNA binding domain (DBD) (aa1008-1146) that lacked
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the ability to form oligomers of LANA DBD dimers. In previous studies it was observed that
the wild-type LANA C-terminal domain (LANA CTD (aa934-1162)) precipitated readily
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following the addition of either specific or unspecific DNA. It has the ability to form higher-
order oligomers, which contribute to the low solubility in the presence of DNA. For avoiding
these solubility problems, multiple point mutations were inserted into the basic patch and the
oligomerization interface of LANA DBD; none of these mutations are located at the specific
DNA-binding site of LANA, while the basic patch mutations suppress unspecific DNA-binding.
This C-terminal LANA mutant with the following amino acid mutations: K1055E, K1138S,
K1140D, K1141D, R1039Q, R1040Q, A1121E, K1109A, and D1110A shows a high solubility
also in presence of oligonucleotides representing the viral LANA-binding sites (LBS) in the viral
terminal repeat subunit.19; 7; 14
A fluorescence-labeled DNA sequence was employed as competitive binding partner, which
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corresponds to LANA binding site 2 (LBS2) in a KSHV terminal repeat subunit. We chose
LBS2 as the fluorescence probe because of its lower affinity for the LANA DBD and used
varying concentrations of unlabeled LBS1, LBS2 and LBS3 to validate and optimize our assay
conditions (see SI, Figure S3 and S4). In accordance with previous reports, we obtained a
difference in affinity between the LBS sequences.7
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FP-based Functional Screening. In order to assess the effect of identified screening hits on
LANA-DNA-interaction inhibition we tested them in our FP-based assay using LBS2 as
fluorescent probe. Due to their high solubility, the compounds could be tested at high
concentrations (1 mM or 500 µM), allowing for the identification of even weak inhibitory effects
usually observed with fragment-like scaffolds. Each compound was measured in duplicates and
in two independent experiments. Three of the 20 tested compounds showed promising results
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(Table S9, SI). In this paper we will present the hit optimization and validation for hit 1. Despite
its fragment-like size with a molecular weight of only 159.08 g/mol, this compound showed an
inhibitory effect of 25 ± 9 % at 1 mM (Figure 3; SPR at 500 µM: 15.69 ± 9.3 RU; DSF at 500
µM: T -1.80 ± 1.41 °C). Considering the large interaction site between LANA and its target
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DNA sequence, this result was promising and encouraged medicinal chemistry optimization of
this fragment hit.
The fragment-like structure of hit 1 provided reasonable opportunities for fragment-growing
strategies toward generating drug like LANA-DNA inhibitors. Unfortunately, no X-ray or NMR
structure was available when starting this hit optimization endeavor. Hence, structure-guided
fragment-linking or -merging approaches were not feasible.
Chemistry. The synthesis of Hit 1 was carried out starting from commercially available 2-Iodo
nitrobenzene in two steps. Hit 1 and further imidazole derivatives 9-13 were synthesized via an
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Ullmann-type coupling reaction with a halogen nitrobenzene and the appropriate imidazole. In
a second step, the nitro group was reduced with tin(II)chloride to the amine to yield the target
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compounds 1 and 9-13. The N-acetyl derivative 8 was obtained by acetylation with acetyl
anhydride. Furthermore, the 2-methoxyphenyl imidazole (14) was synthesized via copper salt
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catalyzed coupling of imidazole with (2-methoxyphenyl)boronic acid and by cleaving the
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methyl group with aqueous HBr, affording the hydroxyl derivative 15. The synthesis of the
benzoic acid derivatives was done by copper catalyzed coupling with the appropriate halo-
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benzoic acid and 1H-imidaziole in a one step reaction (Scheme 1). Generation of 1-azido
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aniline (19) was achieved by copper catalyzed C-H activation of aniline. The azido
intermediates (20-24) were synthesized from the corresponding anilines using standard azidation
2
6
methods (conc. H SO or conc. HCl, NaNO , NaN in H O). Methyl azidobenzoate (25) was
2
4
2
3
2
2
7
synthesized by activation with thionyl chloride followed by treatment with methanol. The
,2,3-triazoles (26-50) were synthesized using standard copper(I)-catalyzed click reaction
1
conditions. The appropriate alkyne was dissolved in 1:1 tert-butanol:water and treated with
DIPEA, CuSO ·5H O and sodium ascorbate under argon atmosphere, followed by the addition
4
2
of the corresponding azide. Amino-phenyl-substituted compounds (51-55) were synthesized
from the appropriate amino-phenyl scaffold by treatment with different halogen alkyl analogues
under basic conditions in DMF (Scheme 2).
Scheme 1. Synthesis of Hit I and 1H-imidazole-1-yl derivatives.a
N
X
a
N
R2
R1
R1
2
: R1 = 2-NO2, R2 = H
HN
or HN
R2
3: R1 = 3-NO2, R2 = H
: R1 = 4-NO2, R2 = H
N
N
4
R1 = 2-NO2, 3-NO2,
-NO2; X = I, Br
R2 = H, Me, Br
5: R1 = 2-NO2, R2 = Me
6: R1 = 2-NO2, R2 = Br
4
7
: R1 = 2-NO2, Benzimidazole
N
1: R1 = 2-NH2, R2 = H
8: R1 = 2-NHAc, R2 = H
9: R1 = 3-NH2, R2 = H*
0: R1 = 4-NH2, R2 = H*
c
R2
N
b
R1
1
11: R1 = 2-NH2, R2 = Me
12: R1 = 2-NH2, R2 = Br
13: R1 = 2-NH2, Benzimidazole
OH
N
N
N
B
N
OH
d
e
O
O
OH
14
15
N
X
f
N
16: R3 = 2-COOH
17: R3 = 3-COOH
R3
R3
18: R3 = 4-COOH
R3 = 2-COOH, 3-COOH,
-COOH, X: I, Br
4
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2
2
2
2
2
2
3
3
3
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3
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3
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a
Reagents and conditions: (a) K PO , CuI abs. DMF, 110 °C, 24 h; (b) SnCl , EtOH, 80 °C,
3
4
2
3
0 min; (c) H SO , AcOH, Ac O, rt, 16 h; (d) Imidazole, CuCl, MeOH, 80 °C, 16 h; (e) 48% aq.
2 4 2
HBr, 100°C, 16 h; (f) K CO , CuCl, N,N'-dimethylmethanediamine, DMF, 120 °C, 24 h.
2
3
0
1
2
3
4
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8
9
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8
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Scheme 2. Synthesis of azide intermediates and reaction of aryl azides with different alkynes
using click chemistry and derivatives.a
N3
R1
NH2
R1
N3
b)
a)
c)
NH2
N3
NH2
X
X
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
19
2
2
2
0: R1 = H, X = CH
1: R1 = 2-COOH, X = CH
2: R1 = 3-COOH, X = CH
23: R1 = OH, X = CH
24: R1 = H, X = N
R1 = H, 2-COOH,
-COOH, OH
X = CH, N
3
N3
HOOC
MeOOC
25
N
26: R1 = H, R2 = 2-COOH, X = CH
39: R1 = CH2OH, R2 = 4-COOMe, X = CH
40: R1 = CH2OMe, R2 = 4-COOMe, X = CH
41
: R1 = 2-aniline, R2 = 4-COOH, X = CH
N
N
R1 27: R1 = H, R2 = 3-COOH, X = CH
28
R1
R2
N3
d)
R2
+
:
R1 = H, R2 = 4-COOH, X = CH
2
3
3
9: R1 = Ph, R2 = 2-COOH, X = CH
0: R1 = Ph, R2 = 3-COOH, X = CH
1: R1 = Ph, R2 = 4-COOH, X = CH
42: R1 = 3-aniline, R2 = 4-COOH, X = CH
43: R1 = 4-aniline, R2 = 4-COOH, X = CH
44: R1 = phenyl-4-methanol, R2 = 4-COOH,
X = CH
45: R1 = 4-anilole, R2 = 4-COOH, X = CH
46: R1 = 3-aniline, R2 = H, X = CH
47: R1 = 4-benzioc acid, R2 = 2-NH2, X = CH
48: R1 = 4-benzoic acid, R2 = 4-NH2, X = CH
X
X
R1 = TMS, Ph, CH2OH, CH2NH2,
CH2OMe, CH2OAc, COOMe,
cyclopentanol, C3H5, 2-aniline,
R
=
4
-
C
O
O
H
,
4
-
N
H
,
2
2
32: R1 = CH2OH, R2 = 4-COOH, X = CH
3: R1 = CH2NH2, R2 = 4-COOH, X = CH
34: R1 = CH2OMe, R2 = 4-COOH, X = CH
19 - 25
3
3
-aniline, 4-aniline, 4-anilole,
phenyl-4-methanol, 4-phenol,
-pyridine
3
3
3
3
5: R1 = CH2OAc, R2 = 4-COOH, X = CH
6: R1 = COOMe, R2 = 4-COOH, X = CH
7: R1 = cyclopentanol, R2 = 4-COOH, X = CH 49: R1 = 4-benzoic acid, R2 = 4-OH, X = CH
8: R1 = C3H5, R2 = 4-COOH, X = CH
2
50: R1 = 4-benzoic acid, R2 = H, X = N
H
N
N
N
N
N
R3
51: 2-NH, R3 = cyclohexyl
52: 3-NH, R3 = cyclohexyl
53: 4-NH, R3
= cyclohexyl
4: 3-NH, R3 = methylmorphline
55: 3-NH, R3 = methylbenzene
N
N
NH2
X
e)
+
R3
5
HOOC
HOOC
R3 = cyclohexyl,
4
1 - 43
methylmorpholine,
methylbenzene
X = Cl, Br
a
Reagents and conditions: (a) TMSN , CuBr, TBHP, MeCN, 0 °C → rt, 16 h; (b) conc. H SO or
3
2
4
conc. HCl, NaNO , NaN , H O, 0 °C, 1,5 h; (c) SOCl , MeOH, 0 °C→ rt, 16 h; (d)
2
3
2
2
CuSO ·5H O, Na-ascorbate, H O:tert-BuOH (1:1), DIPEA, rt, 16 h; (e) Cs CO , DMF, 5h, 90
4
2
2
2
3
°
C.
Stepwise Hit Optimization and Biological Evaluation. For measuring the inhibitory effect of
our compounds, we performed dose-response experiments using constant concentrations of the
mutated LANA DBD (aa1008-1146) and fluorescence-labeled LBS2 with varying concentrations
of test compound. To exclude false positives through interaction with DNA or via fluorescence
quenching, we conducted the dose-dependent experiments with and without addition of LANA.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We did not observe any noticeable assay-interfering effect for any of the compounds. The results
obtained with our FP-based interaction inhibition assay are listed in Table 1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Inhibitory activity of 1H-imidazole-1-yl derivatives in FP-assay at 1 mM.
N
R2
4
N
R1
2
3
inhibition [%]@
Cpd R1
R2
1 mM or IC [µM]
50
(LBS2)
1
1
1
1
8
9
1
2
H
2-NH2
2-NH2
2-NH2
25 ± 9 %
91 ± 8 %
13 ± 4 %
32 ± 9 %
32 ± 16 %
n. i.
1
2
3
Me
Br
Benzimidazole 2-NH2
H
H
H
H
2-NHAc
3-NH2
4-NH2
2-NO2
0
n. i.
74 ± 16 %
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1
1
H
H
H
H
H
H
H
3-NO2
4-NO2
2-OMe
2-OH
21 ± 16 %
22 ± 16 %
15 ± 12 %
51 ± 13 %
4
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16
17
2-COOH 333 ± 59 µM*
3-COOH 19 ± 9 %
4-COOH 13 ± 1 %
1
8
*
maximum effect was 50 % displacement..
The aim of this first series of derivatives was to identify possible growth vectors to increase size
and potency of the compound. Hence, substituents at the imidazole (R1) as well as the phenyl
ring (R2) were introduced. Notably, moieties of different size (11-13) were tolerated at R1. In
particular, the methyl derivative 11 showed an improved inhibitory effect of 91 ± 8 % at 1 mM.
We concluded that position R1 should be further explored (vide infra) as a possible growth
vector. Regarding R2, we first varied the position of the amino group at the phenyl ring and
investigated the effect of acetylation (compounds 8-10). However, these modifications did not
improve the inhibitory effect on the DNA-LANA-interaction significantly. Hence, we introduced
different hydrophilic moieties like nitro (2-4), methoxy (14), hydroxyl (15) and carboxy (16-18)
groups instead. To our surprise, the presence of a carboxylic acid on the aromatic moiety was
tolerated. For compound 16 we were able to plot a full sigmoidal inhibition curve providing an
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
IC value of 333 ± 59 µM, with a restriction that the maximum effect leveled out at 50%
5
0
displacement. Considering the rather basic interaction surface at the DNA-binding domain of
LANA, the effectiveness of the acidic moiety in compound 16 was indeed a plausible finding in
hindsight. Furthermore, it rendered an unfavorable compound-DNA-interaction as the cause for
the observed activity in the FP-assay very unlikely.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
8
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0
1
2
3
4
5
6
7
8
9
0
N
N
NH2
Hit 1
25 ± 9 % inhibition @ 1 mM
first
derivatives
N
N
N
N
Me
COOH
NH2
16
11
IC50 333 ± 59 µM
91 ± 8 % i. @ 1mM
combichem approach
using click chemisty
N
N
R1
N
R OOC
2
26 - 55
Figure 4. From our initial screening Hit 1 first derivatizations lead to compounds 11 and 16,
which served as a starting point for a combichem approach using click chemistry.
These initial findings inspired us to conduct a combinatorial chemistry approach exploiting the
copper(I)-catalyzed alkyne-azide cycloaddition as a straight-forward synthetic method for the
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rapid generation of a reasonable number of new derivatives. This prototypic click chemistry
provides very efficient and robust reactions under mild conditions and has become a powerful
2
8
tool in drug discovery. Assuming that the replacement of the imidazole moiety by a triazole
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
core is tolerated, this strategy would dramatically accelerate the establishment of
structure-activity-relationships. Further considerations for the design of the click library were the
envisioned fragment-growth in the direction of residue R1 (Figure 4) as well as the switch from
the amino to the carboxylic group in the western part of the molecule. Hence, we first checked
whether this strategy was valid by synthesizing compounds 26-31 (Table 2).
Table 2. Inhibition of first series of 1,2,3-triazole compounds in FP-assay.
N
N
R1
R2
4
N
2
3
Cpd R1
R2
IC (LBS2)
50
[µM] or
Inhibition [%]*
12 ± 10 %
98 ± 1 %
2
2
2
6
7
8
H
H
H
2-COOH
3-COOH
4-COOH
35 ± 3 %
2
9
2-COOH
13 ± 1 %
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
0
3-COOH
4-COOH
43 ± 17 %
31
232 ± 10 µM
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*Inhibition [%] at 1 mM.
Indeed, by comparing the carboxylic acid imidazole compounds (16-18) and the carboxylic acid
triazole analogs (26-28), the introduction of a 1,2,3-triazole was accepted, although the
preference for the orientation of the carboxylic acid was shifted from position 2 to 3. This trend
even continued when introducing a bulky phenyl moiety in the Eastern part of the molecule
(
29-31). In this case, position 4 was favored for the carboxylic group implying a shift in
interaction geometries and/or binding modes between LANA and the inhibitors when moving
from imidazolyl to triazolyl to enlarged triazolyl compounds. Derivative 31 showed reasonable
potency (IC value of 232 ± 10 µM) with a significant improvement over inhibitor 16 and
5
0
additionally provided the opportunity for further modifications replacing the newly introduced
bulky phenyl ring. Consequently, we chose this scaffold as the basis for the click library design
(Table 3) keeping the carboxylic acid in para position at the aromatic ring in the Western part
and varying the substituents on the Eastern side of the molecule.
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Table 3. Inhibitory activity of further 1,2,3-triazole derivatives in FP-assay.
N
N
R1
N
R2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC50
IC50
Cpd R1
R2
(LBS2)
[µM]
Cpd R1
R2
(LBS2)
[µM]
NH2
OH
3
3
3
3
2
3
4
5
COOH
COOH
COOH
COOH
COOH
79 ± 2
30 ± 2
210 ± 31
n. i.
42
43
44
45
46
COOH
COOH
COOH
COOH
H
109 ± 3
14 ± 1
NH2
NH2
O
O
27 ± 4
OH
20 ± 3 % *
O
O
O
NH2
36
163 ± 30
n. i.
n. i.
O
HN
OH
3
3
3
7
8
9
COOH
COOH
COOMe
159 ± 2
694 ± 41
84 ± 53
51
52
53
COOH
COOH
COOH
H
N
n. i.
n. i.
OH
N
H
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
6
H
N
O
N
40
COOMe
COOH
237 ± 33
28 ± 1
54
55
COOH
COOH
n. i.
n. i.
O
NH2
H
N
4
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*Inhibition [%] at 500 µM.
A rather general observation when varying the residue R1 was that introducing
hydrogenbond-donating groups gave a boost in potency. In detail, direct attachment of a primary
hydroxyl or amino group to the triazole core could improve the inhibitory effect by three- to
four-fold (32: IC 79 ± 2 µM, 33: IC 30 ± 2 µM). A methyl ether (34) or methyl ester (36), on
50
50
the other hand, showed just a small potency enhancement compared to compound 29, while the
acetylated analog 35 resulted in a complete loss of activity. Moving from the primary (32) to a
tertiary alcohol (37) by addition of a cyclopentyl motif led to a decrease in activity. However,
this derivative shows that obviously bulkier substituents could be tolerated at this position. Also,
a rather hydrophobic cyclopropyl residue (38) did not yield a potent compound. In parallel, we
synthesized two additional compounds with a methyl ester instead of the carboxylic acid at the
aromatic ring, 39 and 40. By comparing compound 32 with 39 and 34 with 40 it seems that also
a methyl ester is well accepted, which certainly provides opportunities for future optimization
efforts. Additionally, it becomes clear that the beneficial effect of the carboxylic group is not
fully relying on a possible ionic interaction with the protein surface. As laid out above, our aim
was to explore R1 as a growth vector. Hence, we synthesized aniline derivatives (41-43).
Introducing the amino group in ortho (41) or para (43) position resulted in the most active
compounds to date with IC values in the low double-digit micromolar range. A loss of activity
50
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was observed with meta aniline (42) and para anisole (45). Also, the phenyl methanol analog
(
44) showed a rather potent IC value of 27 ± 4 µM. Keeping the aniline residue and deleting de
50
carboxylic acid at the aromatic region we observed a total loss in activity (46). In order to further
grow the LANA inhibitor, compounds substituted at the aniline (51-55) were synthesized.
Unfortunately, none of these derivatives were active regardless of the position of the attached
substituent. Probably these residues incorporated in 37-41 are too bulky and are, thus, not
tolerated. Nevertheless, more research should be done in this area of the molecule when moving
the project into the lead-generation phase. Finally, we synthesized a series of compounds with an
inverted orientation of the 1,2,3-triazole core using 4-ethinyl carboxylic acid and an array of
azido benzenes. Compounds 47 and 48 were similar to the previous compounds 41 and 43
showing good IC values in the same range. Modifying the para aniline analogue to a para
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2
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2
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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phenol (49) resulted in slight loss of activity. Besides that, for the 2-pyridine derivative 50 we
observed an IC of 17 ± 1 µM (Table 4).
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
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Table 4. Inhibitory activity of compounds with inverted orientation of the 1,2,3-triazole core in
FP-assay.
N
N
N
R1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HOOC
IC (LBS2)
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Cpd
R1
[µM]
NH2
4
7
18 ± 3
48
49
50
19 ± 1
52 ± 3
17 ± 1
NH2
OH
N
Qualification of Obtained Optimized Hits.
Comparison of the Inhibition of the Interaction between LANA and LBS1, 2, and 3. In
order to investigate whether inhibitors of the LANA-LBS2 interaction would also interfere with
protein binding to the other LANA binding sites, we modified our FP assay protocol to also
include labeled LBS1 and LBS3 sequences as fluorescent probes. We selected six compounds
(37, 41-43, 47, 50) with differing activity against LBS2 for this assessment and the determined
IC values are summarized in Table 5. Notably, we observed IC values in a similar rage
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compared to the inhibition against LBS2 probe. Additionally, we calculated the logP value and
the ligand efficiency (LE) for these compounds to provide a metric for comparing the most
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potent hits taking potency and molecular weight into account. The logP value is defined as the
partition coefficient of a given compound between octanol and water. It provides information
about its hydrophobicity and logP values below 3 are found generally in aqueous medium (e.g.
1
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1
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2
2
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2
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3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
0
blood serum). The LE value is defined as the binding energy of a compound for its target
divided by its number of heavy atoms and, hence, it enables to identify those hits, which interact
efficiently with most of their atoms. In praxis, an LE of 0.3 or greater is considered to
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characterize a suitable hit for the optimization to a drug-like compound. Notably, compounds
41, 43, 47, and 50 displayed a LE value of 0.3 or higher and logP values below 3, hence, are
suitable scaffolds for further optimization efforts. Considering that these hits have to compete
with a macromolecule (DNA) upon binding to a rather flat interaction surface, these results are
encouraging.
Table 5. Comparison of IC values obtained by using LBS1, 2 or 3 as fluorescent probe, clogP
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and LE.
Cpd Structure
IC LBS1
IC LBS2
IC LBS3
clogP
1.80
LE
5
0
50
50
N
N
N
OH
37
104 ± 33 µM 159 ± 2 µM
136 ± 27 µM 109 ± 3 µM
39 ± 4 µM
0.26
HOOC
N
N
N
HOOC
NH2
42
106 ± 32 µM 2.78
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2
2
2
2
2
2
2
2
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3
4
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4
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6
N
N
N
NH2
HOOC
HOOC
4
4
4
5
1
3
7
0
25 ± 1 µM
28 ± 1 µM
14 ± 1 µM
18 ± 3 µM
17 ± 1 µM
26 ± 1 µM
12 ± 1 µM
26 ± 1 µM
19 ± 3 µM
2.78
2.78
2.78
2.00
0.30
0.32
0.32
0.33
N
N
N
19 ± 55 µM
25 ± 1 µM
20 ± 3 µM
0
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0
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0
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NH2
N
N
N
NH2
HOOC
HOOC
N
N
N
N
EMSA (Electrophoretic Mobility Shift Assay) as Orthogonal Interaction Inhibition Assay.
As an orthogonal interaction inhibition assay, EMSA was used to probe the ability of these six
selected compounds to inhibit the DNA-LANA interaction (Figure 5). In this assay, solutions of
protein, nucleic acid and inhibitor were combined and the resulting mixtures were subjected to
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electrophoresis under native conditions. We evaluated the effects of the compounds using fixed
concentrations of DNA probes of 20 nM, LANA DBD mutant of 200 nM and compounds of 500
µM. The probes were Dy-682-modified and a purified GST protein was used as a control. We
performed the EMSA with two different DNA probes: An oligonucleotide representing only
LBS1 for comparing the results with the results of our previous FP assay (Figure 5 A) and a
longer oligonucleotide containing both of the LBS1 and LBS2 sequences (Figure 5 B). The latter
also forms trimeric complexes with LANA (SI, Fig. S6). In both experiments, we observed that
the compounds with IC values in the triple-digit micromolar range have no specific effect on
5
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the DNA-LANA interaction at the concentration used. Importantly, the aniline analog having an
IC value in the lower double-digit micromolar range caused a significant decrease in the
50
intensity of the DNA-LANA complexes and an increase in the intensity of the band representing
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the free DNA probe. This clearly indicated that these compounds inhibit the interaction of the
LANA DBD mutant with LBS1 and/or LBS1+2.
The most effective inhibitor was the pyridine analogue (50, IC 17 ± 1 µM). The single LBS1
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probe (Fig. 5 A) could be displaced almost completely and the combined LBS1-LBS2 probe
Fig. 5 B) to a significant extent. Additionally, we performed further dose-dependent EMSA
(
experiments with the most efficient compound 50 using LBS1 as probe and the LANA DBD
mutant as well as wild-type LANA C-terminal domain (CTD) (aa934-1162) as protein (for more
information see SI, EMSA gels: Figure S9 and S10; calculated IC values see Figure S11, IC
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50
(
LANA DBD mutant) 426 ± 2 µM and IC (LANA CTD wild-type) 435 ± 6 µM).
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9
0
Figure 5. EMSA analysis of compounds 37, 41, 42, 43, 47, and 50 using (A) LBS1 and (B)
LBS1+2 as probes. Representative EMSA gels of one independent experiment are shown,
respectively, containing: unbound control: GST+LBS1 or LBS1+2, bound control:
DMSO+LANA+LBS1 or LBS1+2 and compounds: Cpd+LANA+LBS1 or LBS1+2. Bar graphs
are shown with normalized data points (inhibition from 0-100%) representing mean intensities of
top band values (LANA-LBS-complex) and bottom band values (single LBS). The experiment
was performed in duplicates and the standard deviation given, each compound was used at 500
µM, proteins at 200 nM and DNA probe concentration was 20 nM.
MST and STD NMR Studies for Further Characterization of Ligand Binding. We used
MST to quantify the binding affinity of compound 50 to LANA DBD mutant and determined the
3
3
dissociation constant K . The binding assay was performed using the labeled LANA DBD
D
mutant protein at a concentration of 50 nM and starting the dose-response curve with a ligand
concentration of 1 mM. The calculated K for the binding of compound 50 to LANA was
D
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determined to be 23 ± 1 µM. The detected binding curve is shown in Figure 6, A. We also
attempted crystallography of the LANA DBD-inhibitor-complex, but have so far not been
successful. With the aim to gather information on the mode of binding, we performed ligand-
observed STD NMR studies with compounds 41, 47 and 50. The STD NMR can provide
information on the putative orientation of a given binder to the target of interest in absence of
any structural data of the protein. In this assay, protons that are closest to the protein upon
binding, show the strongest STD effect. In our measurement, samples contained a 40-fold excess
of compound (1 mM) relative to LANA DBD mutant (25 µM) and were recorded at 298 K
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(
Figure 6, B). The STD effects (I/I ) were measured and calculated for each proton of the ligand.
0
The STD effect of compound 50 is shown in Figure 6, B. The overlaid spectra were normalized
to the signals of H-1 and H-2, which gave the strongest enhancement and, hence, can be assumed
to interact the most with the protein surface. These observable variations of the STD effects
suggest that the compound binds in a defined orientation to the protein where the pyridine
moiety faces the protein surface with its aromatic nitrogen. It can be assumed that this motif acts
as a hydrogen bond acceptor. STD effects of H-3 and H-4 (53% and 61% of H-3/H-4,
respectively) suggest, that these protons are not in direct contact with the protein and should, be
further investigated as potential growth vectors. Indeed, inactive compounds 51-55, were already
grown in this direction and lead to abolished activity.
However, the introduced residues were rather large leaving the option of smaller less bulky
substituents to be tried. The four protons referred to by H-8 and H-9 presumably divide into two
populations: Two hydrogens which are closer to the protein surface and two which are more
remote (see Figure 6, B). As these are indistinguishable in the STD NMR experiment, the
observed effect should be a mean of the signals from both populations. As a consequence,
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growth of the compound breaking this ambiguity is a potential path forward in future
optimization efforts.
For compound 41 and 47 we observed similar STD effects (see Figure S12, SI). The protons
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from the aniline next to the amino group gave the strongest enhancement. This also implies that
the NH plays an important role as a hydrogen bond acceptor or donor for binding.
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Figure 6. (A) Dose-dependent MST interaction curve of compound 50 with LANA DBD
mutant. (B) STD experiments of compound 50 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 signals for H-1 and H-2, which showed the strongest
enhancement.
Molecular Docking. In order to generate a possible binding mode of the optimized hit 50 to
LANA, we performed docking experiments taking the STD-NMR data into account. Importantly,
we specifically searched for target-ligand complex geometries, which are in line with the
gathered experimental data. As we demonstrated that this compound binds to LANA (FP assay,
MST, STD NMR) and is able to displace the DNA (FP-assay, EMSA), we directed our docking
experiment to the DNA-binding site of the target (Figure 7 A). This approach intentionally
neglects a possible allosteric mechanism of our compound, which we consider to be rather
unlikely due to the rigidity of the DNA-binding domain.
In order to identify the initial docking site, we selected those LANA residues, which were in
7
close proximity (4.5 Å) to the DNA atoms found in PDB entry 4uzb. Docking to this large
interaction surface yielded three distinct clusters (Figure 7 A). We searched for a binding pose of
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