Use of neomycin as a structured amino-containing side chain motif for phenanthroline-based G-quadruplex ligands and telomerase inhibitors

DR LIANG XUE (Orcid ID : 0000-0003-4206-2800)

Article type

: Research Article

Use of neomycin as a structured amino-containing side chain motif for

phenanthroline based G-quadruplex ligands and telomerase inhibitors

1

2

Mandeep Singh, Siwen Wang, Hyun Joo, Zhihan Ye, Krege M. Christison, Ryan Hekman, Craig

2

1

Vierra, and Liang Xue *

¹Department of Chemistry, University of the Pacific, 3601 Pacific Avenue, Stockton, California

9

5211 (USA)

²Department of Biological Sciences, University of the Pacific, Stockton, California 95211 (USA)

*

Corresponding author:

Tel: 1-209-946-2360; Fax: 1-209-946-2607; Email: lxue@pacific.edu

Keywords: G-quadruplex DNA, DNA binding ligands, phenanthroline, neomycin, telomerase

inhibition

Abbreviations

G4 ligands, G-quadruplex ligands; BIBR, 2-[[(2E)-3-(2-naphthalenyl)-1-oxo-2-butenyl-1-

yl]amino]benzoic acid; TMPyP4, meso-tetra(N-methyl-4-pyridyl)porphyrin tetratosylate; BRACO-

1

9, N,N′-(9-(4-(Dimethylamino)phenylamino)acridine-3,6-diyl)bis(3-(pyrrolidin-1-yl)propanamide);

DMF, dimethylformamide; TCDP, 1,1’-thiocarbonyldi-1(1H)-pyridone; TO, thiazole orange; TLC,

thin-layer chromatography; CD, circular dichroism; ITC, isothermal titration calorimetry; TRAP,

telomeric repeat amplification protocol; FID, fluorescent intercalator displacement

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process, which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

1

0.1111/CBDD.13741

Abstract

In this paper, we report the synthesis of a phenanthroline and neomycin conjugate (7).

Compound 7 binds to a human telomeric G-quadruplex (G1) with a higher affinity compared with its

parent compounds (phenanthroline and neomycin), which is determined by several biophysical

studies. Compound 7 shows good selectivity for G-quadruplex (G4) DNA over duplex DNA. The

binding of 7 with G1 is predominantly enthalpy-driven, and the binding stoichiometry of 7 with G1 is

one for the tight-binding event as determined by ESI mass spectrometry. A plausible binding mode is

a synergistic effect of end-stacking and groove interactions, as indicated by docking studies.

Compound 7 can inhibit human telomerase activity at low micromolar concentrations, which is more

potent than previously reported 5-substituted phenanthroline derivatives.

1

. Introduction

G-quadruplex (G4) DNA is a unique DNA secondary structure formed via self-assembly of

guanines in a G-rich sequence under physiological conditions (1, 2). The formation of G4s occurs in

vivo (3). Initial surveys suggested that over 300,000 putative G-quadruplex forming sequences are

present in the human genome (4, 5), and recent studies predicted an even higher G4 prevalence when

considering G4s with long loops (6) and G4s in some large genes (7), implying that a structure-

function relationship may exist. In recent years, G4s have emerged as a target of considerable interest

because they are not native substrates for binding of nucleic acid processing enzymes (e.g.,

telomerase and polymerase); therefore, inducing G4 formation has been recognized as a promising

approach to regulate enzymatic functions (8-10), for instance, inhibition of human telomerase activity

(

11-13) and regulation of oncogene expression (14, 15). In addition, G4s could activate DNA damage

responses (16).

Small molecules known as G4 ligands facilitate the formation and stabilization of G4s (17,

8). Most of the G4 ligands developed so far contain a large aromatic surface that effectively stacks

1

with G-quartet(s) at the ends of G4s (end-stacking mode) (19-21). Phenanthroline derivatives are

examples of G4 ligands that have been extensively studied (11, 22-27). 1,10-Phenanthroline (Phen)

2

+

2+

alone does not bind to G4s. When Phen coordinates with metal ions such as Ni and Pt , the resulting

complex becomes a potent G4 ligand due to the enlarged aromatic stacking surface. In a recent study

of 5-substituted phenanthrolines, we reported that aromatic side chains (arylsulfanyl group) at the 5-

position of Phen provide the best selectivity for G4 over duplex DNA (11). On the other hand, amino-

containing side chains (piperidine, pyrrolidine) showed enhanced binding affinity but decreased

binding selectivity for G4 over duplex DNA. Similar results have also been reported in our study of

thiazole orange derivatives as G4 ligands (12) and by others (28, 29). The decreased selectivity could

result from non-specific interactions between positively charged amino groups and DNA backbones.

We believe that it is possible to create more selective and potent phenanthroline-based G4 ligands

using structured side chains containing positive charges such as neomycin. Neomycin contains six

amino groups that are protonated or partially protonated under physiological conditions (30). More

importantly, these amino groups are spatially arranged via saccharide linkage to create a structured

binding motif. Masking all six amino groups of neomycin with a tert-butoxycarbonyl (Boc) group

drastically reduces its binding ability to nucleic acids (unpublished data in the group). Neomycin is

known to bind to the A site of 16S rRNA (31) and various A-form-like nucleic acid structures (32). It

has also been reported that neomycin binds to an Oxytricha nova G-quadruplex via groove binding,

but the binding affinity is rather moderate (33). A series of neomycin-capped macrocyclic molecules

have been studied for G-quadruplex DNA recognition (34). Arya’s group has done pioneering work

by developing a series of neomycin conjugates that show a significant synergistic effect on binding to

various DNA structures, including triplex DNA (35-37) and G-quadruplex DNA (38, 39). As a

continuation of our study on Phen-based G4 ligands, we herein report the synthesis of a

phenanthroline-neomycin conjugate and evaluation of its binding to a telomeric G-quadruplex and

inhibition of human telomerase activity.

2

. Experimental section

2

.1. Materials and General Methods

All the chemicals for synthesis were purchased from Sigma-Aldrich or Fisher Scientific and

used without further purification. DNA oligonucleotides were synthesized on an ABI 392 DNA/RNA

synthesizer using standard phosphoramidite protocols and purified using polyacrylamide gel

electrophoresis. All reagents and solid-support columns for DNA synthesis were purchased from Glen

Research. The concentrations of DNA solutions were determined by UV spectroscopy, using the

-1

molar extinction coefficients (in units of mol of strand/L cm ) obtained from OligoAnalyzer

(

www.idtdna.com). ε₂₆₀= 237,000 for G1 [5’-AGGG(TTAGGG) T-3’]; ε = 259,100 for S1 (5’-

3 260

ATGAAGGACGTAACCGGCTCTGAACG-3’);

ε₂₆₀

=

167,400

238,300 for TS primer (5’-

261,000 for RP (5’-

151,800 for NT (5’-

for TSNT (5’-

for

S2

(5’-

CGTTCAGAGCCGGTTACGTCCTTCAT-3’); ε₂₆₀

AATCCGTCGAGCAGAGTT-3’); ε₂₆₀

GCGCGGCTTACCCTTACCCTTACCCTAACC-3’);

ATCGCTTCTCGGCCTTTT-3’); ε₂₆₀

=

ε₂₆₀

=

370,100

AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3’). H NMR and ¹³C NMR spectra

were collected on a JEOL ECA 600 MHz FT-NMR spectrometer. Mass spectra of the synthesized

compounds were analyzed on an Agilent 1290 UHPLC coupled to an Agilent 6230 Time-of-Flight

mass spectrometer. Mass spectra of ligand-DNA complexes were collected on a Thermo Fisher

Orbitrap Fusion Tribrid Mass Spectrometer. UV spectra were recorded on a Varian Cary 100 Bio UV-

1

Vis spectrophotometer equipped with a thermoelectrically controlled 6 × 6 cell holder. Circular

dichroism spectra were recorded on a JASCO J-810 spectropolarimeter using a quartz cuvette with a 1

mm or 1 cm optical path length. Isothermal microcalorimetric measurements were performed on a TA

Instruments Affinity ITC LV. T4 polynucleotide kinase was obtained from New England Biolabs. [-

3

2

32

P]-ATP was purchased from MP Biochemicals. Quantification of 5’ P-labeled oligonucleotides

was carried out using a Storm 860 phosphorimager and ImageQuant 5.1 software. Cell medium and

supplements (fetal bovine serum, L-glutamine, antimycotic) were acquired from Invitrogen. HeLa

cells were maintained in advanced DMEM/F12 medium supplemented with 5% fetal bovine serum,

L-glutamine, and antimycotic at 37 °C in a humid atmosphere containing 5% CO . DNA labeling was

2

3

2

performed by incubating [- P]-ATP (30 Ci) and T4 polynucleotide kinase (20 units) in the

3

2

presence of an oligonucleotide (10 pmol) at 37C for 30 min. Unreacted [γ- P]-ATP was removed

using a MicroSpin G-25 column.

2

.2. Synthesis of 6

In 25 mL round-bottomed flask, 5 (46.8 mg, 0.0385 mmol) was dissolved in anhydrous

pyridine (5 mL). An anhydrous pyridine solution (1 mL) of compound 9 (10.4 mg, 0.0350 mmol) was

added dropwise into the flask with a syringe. The reaction mixture was stirred overnight at room

temperature under N and concentrated under vacuum. Flash chromatography of the residue [silica

2

gel, CH OH:CH Cl :NH OH = 8:92:0.8 (v/v/v)] yielded compound 6 (R = 0.30; 23.7 mg, 40.7 %). IR

3

2

4

f

-1

1

(

KBr, cm ) 3340, 2976, 2920, 2851, 1685, 1521, 1384, 1252, 1174, 1031. H NMR (600 MHz,

1

CD OD): H NMR (600 MHz, CD OD): δ 9.11 (dd, J = 4.2, 1.8 Hz, 1H), 9.00 (dd, J = 4.2, 1.8 Hz,

3

1

-

H), 8.83 (dd, J = 8.4, 1.8 Hz, 1H), 8.41 (dd, J = 7.8, 1.8 Hz, 1H), 8.35 (s, 1H), 7.82 (q, J = 4.2 Hz,

H), 7.76 (q, J = 4.2 Hz, 1H), 6.59 - 6.94 (m, 2H), 5.51 (s, 1H), 5.03 (s, 1H), 4.24 - 4.55 (m, 2H), 3.68

4.21 (m, 8H), 3.69 - 3.79 (m, 2H), 3.45 - 3.64 (m, 6H), 3.06 - 3.22 (m, 3H), 1.88 - 1.99 (m, 1H), 1.27

+

-

1.57 (m, 54H). HRMS (ESI) calcd for C H N O S [M+H] : 1511.6901, found 1511.6894.

6

8

107 10 24 2

2

.3. Synthesis of 7

Compound 6 (6.00 mg, 0.00397 mmol) was dissolved in trifluoroacetic acid (2 mL) and stirred

for 15 min. The reaction mixture was concentrated on a rotavapor under vacuum. The residue was

dissolved in water (1 mL), and the aqueous solution was washed with dichloromethane (3 × 3 mL).

-1

Lyophilization yielded compound 7 (5.00 mg, 78.9%). IR (KBr, cm ) 3431, 2920, 2850, 1624, 1500,

1

8

384, 1040. H NMR (600 MHz, D O): δ 9.09 (d, J = 4.3 Hz, 1H), 8.89 - 9.03 (m, 2H), 8.76 (d, J =

2

.4 Hz, 1H), 8.18 (s, 1H), 8.02 (dd, J = 8.4, 5.4 Hz, 1H), 7.97 (dd, J = 8.4, 4.8 Hz, 1H), 5.90 (d, J = 3

Hz, 1H), 5.27 (d, J = 3 Hz, 1H), 5.12 (s, 1H), 4.65 (s, 2H), 4.31 (t, J = 4.8 Hz, 1H), 4.02 - 4.26 (m,

3

1

9

2

H), 3.97 (t, J = 10.2 Hz, 1H), 3.50 - 3.92 (m, 8H), 3.05 – 3.50 (m, 11H), 2.35 (dt, J = 8.4, 2.4 Hz,

+

H), 1.75 (q, J = 13.2 Hz, 1H). HRMS (ESI) calcd for C H N O S [M+H] : 911.3755, found

3

8

60 10 12 2

11.3754.

.4. Synthesis of 9

In a 25 mL round-bottomed flask, compound 8 (80.0 mg, 0.314 mmol) and DMAP (catalytic

amount) were dissolved in DMF (10 mL) under N . A DMF solution (1 mL) of TCDP (109 mg, 0.471

2

mmol) was added dropwise into the flask using a syringe and the resulting mixture was stirred

overnight at room temperature. The completion of the reaction was monitored by TLC [R = 0.52,

f

CH OH:CH Cl :NH OH=8:92:0.5 (v/v), silica gel 60 F , EMD Millipore] under UV. The reaction

3

2

4

254

mixture was concentrated under vacuum. Flash chromatography of the residue [silica gel,

CH OH:CH Cl = 5:95 (v/v)] yielded compound 9 as a pale yellow oil (52.0 mg, 55.5%). IR (KBr,

3

2

-1

cm ) 3173, 3018, 2924, 2187, 2122, 2086, 1674, 1652, 1590, 1560, 1502, 1418, 1344, 1292, 1284,

1

240, 1142, 1103, 1079, 942, 874, 824. H NMR (600 MHz, CD OD): δ 9.1 (dd, J = 4.2, 1.8 Hz, 1H),

3

9

.03 (dd, J = 4.8, 1.8 Hz, 1H), 8.89 (dd, J = 7.8, 4.2 Hz, 1H), 8.38 (dd, J = 7.8, 4.2 Hz, 1H), 8.11 (s

H), 7.82 (q, J = 4.2 Hz, 1H), 7.73 (q, J = 4.2 Hz, 1H), 3.79 (t, J = 6 Hz, 2H), 3.40 (t, J = 6.6 Hz,

1

3

2

H). C NMR (150 MHz, CD OD) δ151.16, 151.10, 146.91, 146.14, 137.35, 135.20, 133.55, 132.34,

3

+

1

30.36, 129.92, 125.13, 124.79, 45.48, 35.21. HRMS (ESI) calcd. for C H N S [M+H] : 298.0473,

1

5

13

3 2

found 298.0459.

2

.5. MS analysis procedures of synthesized compounds

The samples were diluted in methanol to approximately 1 M concentration. A sample solution

1 µL) was introduced by flow injection analysis (FIA) without a chromatographic column except for

(

compound 7 where 2 µL was injected. The solvent system was 1:1 methanol:water with 0.1% formic

acid. Ions were delivered to the mass spectrometer with an electrospray ionization (ESI) source. The

nebulizer pressure was 50 PSIG. at 325 °C and nitrogen drying gas flow was 12 mL/min. The

capillary voltage was 3000 V for all samples. For compound 6 (Boc protected), the fragmentor

voltage was 150 V. Data were collected in the m/z range of 100-3200. Spectra were collected at a rate

of 2 spectra/s allowing for 4903 transients/spectrum. For compound 7, the fragmentor voltage was 70

V. Data were collected in the m/z range of 500-2000. Spectra were acquired at a rate of 1 spectra/s

allowing for 9978 transients/spectrum.

2

.6. Preparation of G-quadruplex DNA and duplex DNA for CD and UV experiments

The G-quadruplex DNA strand (G1, 100 M) was dissolved in a mixture (100 L) of lithium

cacodylate buffer (10 mM, pH 7.0) and KCl (50 mM) and incubated at 90 °C for 10 min, slowly

cooled to 25°C, and incubated at 4 C overnight. Similarly, duplex D1 (complementary strands of S1

and S2, 100 M each) was dissolved in a mixture of lithium cacodylate buffer (10 mM, pH 7.0) and

KCl (150 mM) and incubated at 90 °C for 10 min, slowly cooled to 25°C, and incubated at 4 C

overnight. All solutions were stored at 4 C for further use.

2

.7. Preparation of G-quadruplex DNA for ESI mass spectrometry experiments

G1 (100 µM) in 150 mM NH OAc solution was heated to 95 °C for 5 min followed by

4

cooling slowly to room temperature and incubated at 4°C overnight.

2

.8. Thermal denaturation analysis monitored by CD

The mixture solutions (200 μL) of G1 (5 µM) in lithium cacodylate buffer (10 mM, pH 7.0)

and KCl (50 mM) in the absence or presence of a ligand (Phen, neomycin, and 7, 10 µM) were

prepared. The CD spectra were recorded at 290 nm as a function of increasing temperature (25-85 ˚C)

at a heating rate of 0.5 ˚C/min. Melting profiles were converted into a folded faction based on a

published procedure (40), and the melting temperatures were determined at the 0.5 folded fraction.

2

.9. CD titration

Aliquots of a stock solution containing compound 7 were gradually added into a solution (2.0

mL) of G1 (5 M) in lithium cacodylate (10 mM, pH 7.0) and 50 mM KCl at 20 C. The final molar

ratios of 7 to G1 were varied from 0 to 5. After each addition, the solution was gently mixed and

incubated for 5 min for equilibrium before collecting the spectrum. The overall volume change during

the titration was kept less than 1% of the initial volume. CD spectra were recorded as a function of

wavelength (200-400 mm, 100 nm/min scan speed, 0.5 nm data pitch) using a 1 cm pathlength quartz

cuvette. Each spectrum was averaged over three scans.

2

.10. Thermal denaturation analysis monitored by UV

The mixture solutions (1 mL) of D1 (1 µM) in lithium cacodylate buffer (10 mM, pH 7.0) and

KCl (150 mM) in the absence or presence of a ligand (Phen, neomycin, and 7, 10 µM) were prepared.

The UV melting curves were recorded at 260 nm as a function of temperature (20-85 C, heating rate:

0

.5 C/min). Melting profiles were converted into a folded faction, and the melting temperatures were

determined at the 0.5 folded fraction.

2.11. Fluorescent intercalator displacement assay

A reaction solution containing 0.25 μM DNA (G-quadruplex (G1) or duplex (D1) in lithium

cacodylate (10 mM, pH 7.0) and KCl (50 mM) was incubated with 0.5 μM thiazole orange (TO) for

0 min in the dark. Aliquots of a ligand were added to this mixture solution and equilibrated for 5 min

in the dark before measurement. The fluorescence spectra (Ex: 501 nm, slit width: 5.0 nm, scan speed

20 nm/min) were recorded from 510-650 nm after each addition. The percent fluorescence

displacement (%F) was calculated at λ_m_a_xby using the formula %FI. displacement=100-

(FA/FA )×100], where FA is the initial fluorescence intensity in the absence of a ligand, and FA is

3

1

[

0

the fluorescence intensity upon addition of a ligand.

2

.12. Isothermal Titration Calorimetry (ITC)

In a typical experiment, 2 µL aliquots of compound 7 (200 μM) were injected from a 264 µL

rotating syringe (125 rpm) into an isothermal sample cell containing 185 μL of a telomeric G-

quadruplex (G1) (20 µM/strand) or a duplex DNA (D1) solution (10 µM/strand) at 25 °C. The

corresponding control experiment was carried out by injecting 2 µL aliquots of compound 7 (200 μM)

into a solution of buffer alone. The duration of each injection was 4 s, and the delay between

injections was 120 s. The initial delay prior to the first injection was 100 s. A heat burst curve

(microcalories per second vs. seconds) was generated from each injection. The area under each curve

was calculated using the NanoAnalyzer software (Version 3.8.0) to yield a measure of the heat

associated with that injection. The heat of ligand binding to DNA associated with that injection was

obtained by subtracting the heat associated with each ligand-buffer injection from the corresponding

heat associated with each ligand-DNA injection.

2

.12. Preparation of cell lysate for TRAP assay

7

Approximately 1 x 10 HeLa cells were suspended in CHAPS buffer (1 mL) and incubated on

ice for 30 min. The supernatant was collected after spinning the sample at 12,000 x g for 10 min and

stored at -80℃.

2

.13. TRAP assay

3

2

The 5′-[ P] end-labeled TS primer (0.1 μg) was incubated in a reaction mixture (50 μL)

buffer: Tris-HCl (20 mM, pH 8.3), MgCl (1.5 mM), KCl (63 mM), 0.05% Tween 20, EGTA (1

[

2

mM)] with cell lysate (1 μL), RP (0.1 μg, reverse primer for telomerase extended products), NT (0.1

μg, primer for polymerase as internal standard), TSNT (0.01 amol, template for NT primer), 2 units of

Taq polymerase, dNTP mix (50 μM), and ligand (3.125-100 μM) at 30℃ for 30 min and 94℃ for 2

min to deactivate telomerase. The reaction mixture was then incubated using a 3-step PCR method

(94 ℃ for 20 s, 59 ℃ for 30 s, and 72 ℃ for 30 s) with a total of 25 cycles. The residues were

concentrated and resuspended in formamide loading buffer (7 μL). Analytical separation was carried

out using 10% denaturing polyacrylamide gel [5% crosslink, 45% urea w/w].

2

.14. Docking procedure

The geometry optimization of compound 7 was carried out at the hybrid density functional

B3LYP level (41) with 6-31G(d) (42, 43) basis set using Gaussian’09 (44) program package. The

Natural bond order (NBO) analysis (45, 46) was carried out to calculate the atomic charges on the

optimized geometries. G1 structure for docking simulation was modeled after the solution structure of

complex of platinum(II) based ligand with human telomeric G-quadruplex (PDB id: 5Z80) (47)

whose structure was obtained from RCSB protein data bank (48). In the complex structure, the bound

ligand molecule was removed. The resulting template has two more A residues at the 5’-end and one

additional A at the 3’-end in comparison to G1. In the NMR structure, Adenine-25 was mutated to

thymine, and residues Adedine-1, 2, and 26 were truncated to yield the structure of G1 for docking

studies. The flexible ligand docking experiments were carried out using the DOCK 6.9 program (49,

5

0). Analyses of docked structures and docked image creations were carried out using UCSF Chimera

program (51).

2

.14. ESI-MS analysis of complex formation between G1 and 7

G1 (20 µM) and compound 7 at various concentrations (0, 20, 40, 60, and 80 µM) were mixed

as 50% methanol-water solutions. The mixed solutions were injected into a Thermo Fisher Orbitrap

Fusion Tribrid Mass Spectrometer at a rate of 5 µL/min. The ion spray voltage was set to -2,250 V

and ion transfer tube temperature to 300 °C. Nitrogen sheath and auxiliary gas flows were 45 and 0

arbitrary units, respectively. MS spectra were recorded in the m/z range from 1400 to 1800 with an

average of 150 scans. Data were collected and analyzed using the Xcalibur 4.0 software.

3

. Results and Discussion

3

.1. Synthesis of phenanthroline-neomycin conjugate 7

As shown in Scheme 1, neomycin precursor 5 was synthesized based on published procedures

(52). The C5” position of the ring III of neomycin (Scheme 1) was used for conjugation. The

modification of this site has a minimal effect on the structure of neomycin and its binding affinity to

several nucleic acid structures as previously reported (52, 53). All six amino groups of neomycin (1)

were protected using di-tert-butyl dicarbonate to yield compound 2. Conversion of the 5” primary

hydroxyl group of 2 into 2,4,6-triisopropylbenzenesulfonylate followed by reaction with sodium azide

yielded compound 4. The synthesis of compound 9 was accomplished by reacting phenanthroline

amine 8 (54) with TCDP in DMF. Coupling of 9 with 5 yielded Boc-protected phenanthroline-

neomycin conjugate 6. Removal of Boc groups of 6 in the presence of trifluoroacetic acid yielded the

desired compound 7.

Insert Scheme 1

3

.2. Fluorescence Intercalator Displacement (FID) assay

The binding of a telomeric G-quadruplex (G1) or a duplex (D1) with 7, phenanthroline (Phen),

and neomycin was first assessed using the Fluorescence Intercalator Displacement (FID) Assay (55).

In this assay, thiazole orange (TO) was first bound to G-quadruplex DNA (G1), and the pre-bound

TO was successively displaced out of DNA by the addition of a ligand of interest. The amount of

G4

ds

ligand needed to displace 50% of pre-bound TO is defined as DC for G-quadruplex and DC for

5

0

50

duplex DNA. Low DC values represent the high binding affinity of the ligand towards DNA.

Neomycin is known to coordinate with metal ions (56). An initial study of the binding of 7 to G1 in

2

+

the presence of Ni did not show enhanced binding compared with that of 7 alone (Figure S1 of the

2

+

supporting information). Therefore, it is possible that Ni could not effectively coordinate with the

Phen moiety of 7 due to the interference of neomycin at the concentration range used. Hence, in this

2

+

report, we are presenting data with Ni -free conditions in the following studies. As shown in Figure

1

, the corresponding rank order of the ^G⁴DC values was 7 < Phen-Ni < neomycin << Phen. The

50

G4

DC value for 7 (0.9 µM) was impressively low, 50-fold less than that of neomycin (49.8 µM) and

5

0

more than 500-fold less than that of phenanthroline alone (> 500 μM). The low ^G⁴DC values for 7

5

0

suggested a strong displacement of TO from G1 by 7. It is noteworthy that these values are much

lower than those obtained for other 5-substituted phenanthroline derivatives (10 and 11, Scheme 1)

that contain amino-side chains. The ^G⁴DC₅₀values for 10 and 11 are 83.2 µM and 72.3 µM,

respectively, as previously reported (11). The significant difference in ^G⁴DC values amongst 7, 10,

5

0

and 11 suggested that the structured amino-containing binding motif (neomycin) can significantly

ds

50

enhance the binding with G-quadruplex DNA. Using the same conditions, the DC values for these

ds

50

compounds were also measured with a DNA duplex (D1). The DC values for 7 and neomycin are

.4 µM and 5.7 µM, respectively. The relative selectivity of G-quadruplex over duplex DNA was

3

ds

G4

determined by the ratio of DC / DC . The rank order for such selectivity was 7 (3.8) > neomycin

5

0

50

(0.11). It is clear that compound 7 binds more preferentially to G4 than duplex DNA. Interestingly,

ds

50

G4

neomycin has a smaller DC value (5.7 µM) than its DC value (49.8 µM). Phen binds to both

5

0

duplex and G-quadruplex DNA very weakly based on the FID data. Both of the ^G⁴DC and DC

ds

50

5

0

values for Phen are over 500 μM; therefore, its selectivity of G4 over duplex DNA is negligible. An

G4

FID experiment of a mixture of neomycin and Phen (1:1 molar ratio) was also carried out. The DC

5

0

value for the mixture is 9.0 µM, much smaller than those of neomycin (49.8 µM) and Phen (> 500

µM), indicating that the simultaneous binding of neomycin and Phen to G-quadruplex DNA has a

minor synergistic effect. As a comparison, this value is 10-fold greater than that of 7 (0.9 µM). Hence,

conjugation of neomycin with Phen via covalent bonds provides a more significant enhancement of

binding with G-quadruplex DNA than merely mixing two ligands. It is noteworthy that 7 shows more

ds

50

remarkably enhanced binding with G-quadruplex DNA when compared to duplex DNA. The DC

value (4.5 µM) of the mixture of neomycin and Phen is similar to the values of neomycin (5.7 µM)

and 7 (3.4 µM). The binding of duplex DNA by 7 could mainly result from the non-specific binding

by electrostatic interactions of cationic charges of the neomycin moiety.

Insert Figure 1

3

.3. Interactions of 7 with DNA monitored by CD and UV

The ability of 7 to stabilize G1 was confirmed using thermal denaturation of G1 monitored by

circular dichroism. As shown in Figure 2A, the representative folded fractions of G1 in the absence

and presence of a ligand (10 μM) are plotted as a function of temperature. The melting temperature

(T_m) of G1 was measured to be 61.0  0.6 °C. In the presence of neomycin or Phen at 10 μM, the T_m

values were 60.6  0.3 and 60.8  0.5 °C, respectively. Both neomycin and Phen could not stabilize

G1 under this condition. Phen did not increase the T value of G1, which is consistent with previous

m

studies (11). In the presence of 7 at 10 μM, the T value was 66.6  0.3 °C, a 5.6 °C increment as

m

compared with that of G1. When incubating G1 with neomycin (10 μM) and Phen (10 μM), no

increment of T (61.0  0.8 °C) was observed. Therefore, the stabilization effect of 7 toward G1 must

m

result from a synergistic enhancement of binding affinity by conjugating neomycin and

phenanthroline together. Similar results were obtained from thermal denaturation monitored by UV at

2

95 nm (Figure S2 of the supporting information).

Evidence of the interaction between 7 and G1 also came from monitoring the CD spectral

changes when titrating a solution of G1 with 7 at various concentrations. As shown in Figure 2B, the

+

native folding of G1 in K adopts a hybrid-type topology with a maximum (+) around 292 nm, a

shoulder (+) centered around 270 nm, a weak band (+) with a maximum around 250 nm, and a band (-

)

with a maximum around 235 nm, which is consistent with previous reports (57, 58). Titration of G1

with 7 did not lead to an overall topological change of G1. Up to a ligand to DNA molar ratio of 3, a

gradual increase in the intensity of the band (-) at 235 nm, the band (+) at 250 nm, and the band (+)

around 292 nm was observed, respectively, suggesting that a further stabilization of the folded

conformation (59) induced by interactions of 7 with G1. When the ligand to DNA molar ratio was

above 3, a slight decrease in the intensities of these three bands occurred. In addition, the band (+) at

2

92 showed a redshift up to 300 nm during the course of titration. It is known that the lateral or

diagonal loops of G-quadruplexes greatly affect the shape of their CD spectra (57). As indicated by

the docking studies in the following section, the phenanthroline moiety of compound 7 could form π-

π stacking interaction with an adenine in the loop (Figure 5B). At high ligand to DNA molar ratios, it

is plausible that the neomycin moiety of several molecules of 7 could interact with the loops of G1 via

non-specific interactions, causing further redshift in band maxima.

The stabilization effect of neomycin, 7, and Phen on a duplex DNA (D1) was investigated

using thermal denaturation monitored by UV (Figure S3 of the supporting information). None of them

showed a noticeable stabilization effect on the melting of D1, suggesting a weak binding of

compound 7 with duplex DNA.

Insert Figure 2

3

.4. Binding stoichiometry determined by ESI mass spectrometry

Mixtures of 7 and G1 at a molar ratio ranging from 1:1 to 1:4 were analyzed using

electrospray ionization (ESI) mass spectrometry, and a solution of G1 was used as control. A mass

spectrum representing a mixture of G1 and 7 ([G1]:[7] = 1:2) is shown in Figure 3. The formation of

the G-quadruplex structure was confirmed by the peak at m/z 1459.85, representing the ion

+

+ 5-

+

[

G1+2NH -7H ] . The inclusion of two NH ions is a distinct indication of the presence of three G-

4

quartets in the G-quadruplex (60). Compound 7 was found to form a 1:1 complex with G1, indicated

+

+ 5-

by the ion [G1+2NH +7-7H ] at m/z 1642.13. The relative abundance of the complex is higher than

4

those of free intact G-quadruplex when the molar ratio of 7 to G1 is larger than 1, suggesting the

strong tenancy of 7 to bind with G-quadruplex. It is noteworthy that varying the molar ratios of 7 and

G1 did not lead to different binding stoichiometries (Figure S4 of the supporting information).

Insert Figure 3

3

.5. Thermodynamic parameters determined by Isothermal Titration Calorimetry (ITC)

The thermodynamics of a ligand (7 or neomycin) binding to a telomeric G-quadruplex (G1)

and a 26-mer duplex (D1) were determined using isothermal titration calorimetry (ITC) (61, 62).

Figure 4 shows the representative ITC profiles (upper panels) resulting from a series of injections of 7

+

(

200 µM) into a solution (20 μM) of G1 (A) and D1 (B) in K at 25 °C. Calorimetric datum from

+

injections of 7 into K phosphate buffer under the same experimental conditions is used as blank for

the heat of dilution (Figure S5 of the supporting information). The dotted lines (Figure 4, lower

panels) show the resulting corrected injection heats (by subtracting the blank data for corrections of

dilution effects) plotted as a function of the [7]/[DNA] molar ratio. Both binding isotherms can be

fitted with different binding models provided in the TA Instrument’s NanoAnalyze software (version

3

.8.0). At 25 °C, the binding isotherm of 7 with G1 (Figure 4A, lower panel) reveals a complex

binding process, indicative of two binding events. The data best fit a multiple-sites model with a 90%

8

-1

5

confidence level and result in two association constants (K s), (8.92.4)×10 M and (3.5 0.5)×10

a

-1

M . Based on the K values, the first binding event is a tight binding with a sharp transition, and the

a

second binding event shows a gradual slope towards the binding saturation of G1 by 7, suggesting a

relatively weak binding. Binding enthalpies for the two events were determined to be ΔH [-

1

(

37.31.6) kJ/mol] and ΔH [-(32.30.5) kJ/mol], indicative of highly enthalpically favored binding

2

for both events. The first binding event is entropy favorable with a TΔS value of +13.8 kJ/mol.

According to a survey of thermodynamic signatures for ligand-DNA binding mode (63), groove

binding is predominantly entropically driven, while intercalation is enthalpically driven. Our

observation suggests that the tight-binding event could involve both groove-binding and π-π stacking,

implying a synergetic binding effect between Phen and neomycin. The second binding event is

slightly entropy unfavorable with a TΔS value of -0.7 kJ/mol. The presence of the first binding event

was highlighted by a separate ITC titration experiment in which a series of injections of 7 (200 µM)

was titrated into a solution (40 μM) of G1 to reach a final mole ratio of approximately 3 (Figure S6 of

the supporting information).

+

The overall stoichiometry for binding of 7 to G1 in the K solution was determined to be

approximately five ligands per G-quadruplex DNA. The sizeable binding stoichiometry suggests the

second (weak) binding event may be associated with non-specific binding. It is noted that only a 1:1

binding stoichiometry was obtained from the ESI/MS experiments, as mentioned above. This

discrepancy could result from the stability variations of ligand-DNA complexes in the solution phase

and the gas phase of experimental conditions; therefore, the binding of 7 to weaker binding sites (non-

specific binding) might not be observed in the ESI/MS experiments. At 25 °C, the binding isotherm of

7

with D1 (Figure 4B, lower panel) best fits an independent (single-site) model with a 90%

4

-1

confidence level and result in an association constant of (5.90.3)×10 M . It is evident that the

binding of 7 with D1 is much weaker than both binding events of 7 with G1 when comparing K_a

values. The results obtained here are consistent with the aforementioned biophysical studies, in which

compound 7 binds preferentially to telomeric G4 over duplex DNA. The binding of neomycin with

3

-1

G1 at 25 °C shows a much lower association constant of (6.10.2)×10 M (Figure S7 of the

supporting information). Given that Phen does not bind to G4s and weak binding of neomycin with

G1, the strong binding of 7 with G1 must come from covalently tethering neomycin and Phen.

Insert Figure 4

3

.6. Molecular docking

Plausible binding modes of 7 with human telomeric G-quadruplex DNA was investigated

using the DOCK 6.9 program. Because the G4 structure of the exact sequence used this work is not

available in the data bank, we modeled G1 structure using the NMR solution structure (PDB id:

5

Z80) of the complex formed by a platinum ligand and a human telomeric G-quadruplex as the initial

point. It is noted that the G-quadruplex-forming regions [AGGGTT(AGGG) ] of G1 and 5Z80 are

3

identical. Upon manual removal of the platinum ligand and modification of a few residues at the 5’

and 3’-positions, the resulting G4 structure has the same sequence as G1 and is a hybrid-type

topology. The simple docking results show that compound 7 can bind in the original binding site of

the hybrid G-quadruplex, where the platinum ligand was found in the solution NMR structure. The

most energetically favorable binding mode (Figure 5A) of 7 has the phenanthroline ring stack on top

of the base pair in the G-quadruplex through π-π stacking and the neomycin moiety in the groove of

the G-quadruplex. Further analysis shows π-π stacking between the phenanthroline ring of 7 and

residues of adenine-7 and guanine-8 (Figure 5B) and hydrogen bonding interactions of the neomycin

moiety with guanine-21 and the phosphate backbone of residues 19-21 (Figure 5C). The proposed

binding mode is cohesive with the synergistic effect observed from biophysical studies. Using

docking simulation, we also found several binding modes that do not interfere with each other. A

representative binding mode of G1 with three molecules of 7 is presented in Figure S9 of the

supporting information. In addition, non-specific interactions between G1 and 7 are plausible due to

the electrostatic interactions of the neomycin moiety with DNA backbones.

Insert Figure 5

3

.7. Inhibition of human telomerase activity by compound 7

Upon confirming the interactions between 7 and the telomeric G4, inhibition of human

telomerase activity in vitro was investigated using a modified TRAP assay. As shown in Figure 6, a

known telomerase inhibitor BIBR (64) (50 μM) was used as a control. The extension product bands in

the lane for BIBR were negligible compared to those in the positive control lane (no ligand) while the

PCR product band was clearly observed, suggesting that BIBR inhibited telomerase but not

polymerase in the reaction. In contrast to BIBR, TMPyP4, a potent G4 ligand (65, 66), fully inhibited

the activities of telomerase and polymerase. No telomerase extension and PCR product bands (IS

band disappeared) could be observed. The large stacking surface of TMPyP4 could intercalate into

duplex DNA at this high concentration (50 μM) and thus block polymerase activity. These results

were consistent with previously reported data (11, 12). As shown in Figure 6, compound 7 selectively

inhibits the telomerase activity while it has no effect on the polymerase activity in the concentration

range of 1.6 to 12.5 µM. The inhibition of telomerase activity by 7 could be unambiguously observed

at 3.1 µM. Such inhibition is clearly concentration-dependent. At 12.5 µM, the activity of telomerase

is greatly inhibited while the polymerase activity remains unaffected (IS band observed). It is

noteworthy that the observed inhibition concentration (3.1 µM) is much lower than those of other 5-

substituted phenanthrolines such as 10 and 11, both of which did not show any noticeable inhibition

effect even at concentrations as high as 50 μM (11). Furthermore, the potent inhibition of telomerase

activity by compound 7 should not result from the effect of the neomycin moiety because neomycin

does not show any inhibition effect even at a concentration of 100 µM (data not shown).

Insert Figure 6

4

. Conclusion

In summary, the data here demonstrate that structured amino-containing side chains such as

neomycin can be used as a suitable binding motif for 5-substituted phenanthroline based G4 ligands

and telomerase inhibitors. The observed enhancement is significant, considering that Phen alone

cannot effectively bind to G4s due to its relatively small stacking surface compared with the surface

area of a G-quartet. Following the same concept, such side chains could also be used to conjugate

with other G4 stacking ligands. Further investigations of the binding of 7 with other biologically

relevant G4s such as c-Myc G-quadruplex DNA will be carried out in due course. This approach may

open a new direction for designing novel telomerase inhibitors.

Acknowledgments

We are grateful for financial support from the University of the Pacific and support for

purchasing an isothermal titration calorimeter from the National Science Foundation (MRI-1828179).

We also thank Professor William K. Chan’s lab (University of the Pacific) for the HeLa cell lysate.

Note

The authors declare no conflict of interest.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the

article and its supplementary materials.

Supplementary data

NMR spectra of compounds 6, 7 and 9, FID titration profiles, mass spectra of G1 and 7

mixtures, ITC profiles, and UV thermal denaturation profiles are presented.

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Figure Legends

Scheme 1. Synthesis of phenanthroline derivatives (7 and 9). a) (Boc) O, DMF-H O, 60 °C, 78.0%;

2

b) TPS-Cl, pyridine, r.t., 60.0%; c) NaN , DMF, 2h, 95.0%; d) H , Pd/C, Ethanol, overnight, quant.;

3

2

e) 9, pyridine, overnight, r.t., 45.0%; f) TFA, 30 min, 69.0%; g) TCDP, DMF, r.t., overnight, 56.0%.

ds

G4

Figure 1. The DC and DC values for compound 7, neomycin (neo), Phen, a mixture of Phen

5

0

50

and neomycin (1:1 molar ratio) from the FID assay. Buffer: 10 mM lithium cacodylate, 50 mM KCl,

pH 7.0.

Figure 2. The CD melting profiles of G1 in the absence and presence of neomycin and 7 (10 µM) in

5

0 mM KCl. [G1] = 5 µM. Buffer: 10 mM lithium cacodylate, pH 7.0.

Figure 3. A representative mass spectrum of a mixture solution of G1 and 7 ([G1]:[7] = 1:2). The

peak with the maximum intensity was set as the base peak (100%).

Figure 4. upper panel: ITC profiles of G1 (20 µM) (A) and D1 (10 µM) (B) titrated with 7 (200 µM)

in a 10 mM potassium phosphate buffer with 133 mM KCl, pH 7.0 at 25 °C. lower panel: The dotted

line represents the corrected injection heats plotted as a function of the [7]/[G1] (A) and [7]/[D1] (B)

molar ratio. The smooth solid line reflects the calculated fit of the data using the binding models

(multiple-sites for G4 and independent for duplex) from the software NanoAnalyze. Upward peaks

represent an exothermic event.

Figure 5. A) Docked structure of compound 7 with a human telomeric G-quadruplex (G1). G-

quadruplex is represented as surface colored by electrostatic potential, and the ligand is represented as

ball and stick. B) π-π stacking interactions of the phenanthroline moiety with the top G-quartet of G1.

C) Binding interactions of the neomycin moiety with the groove of G1. The distances are in Å.

Figure 6. TRAP reactions in the presence of 7 at concentrations of 12.5, 6.25, 3.13, and 1.56 μM

(from left to right). Reactions in the presence of BIBR (50 μM) and TMPyP4 (50 μM) were carried

out as comparisons. The reaction in the first left lane has no telomerase. IS: PCR product as internal

standard (TSNT as template).

Scheme 1

Figure 1

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Figure 2

Figure 3

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Figure 4

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Figure 5

Figure 6

Article Doi

Use of neomycin as a structured amino-containing side chain motif for phenanthroline-based G-quadruplex ligands and telomerase inhibitors

DOI: 10.1111/cbdd.13741

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Article abstract of DOI:10.1111/cbdd.13741

Full text of DOI:10.1111/cbdd.13741

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