Telomere maintenance as a target for drug discovery

Telomere Maintenance as a Target for Drug Discovery
Vijay Sekaran, Joana Soares, and Michael B. Jarstfer*
Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599, United States
ABSTRACT: The observation that the enzyme telomerase is up-regulated in 80−90% of
cancer cells isolated from primary human tumors but is absent in neighboring cells of
healthy tissue has resulted in significant efforts to validate telomerase as an anticancer drug
target and to develop effective approaches toward its inhibition. In addition to inhibitors
that target the enzymatic function of telomerase, efforts toward immunotherapy using
peptides derived from its catalytic subunit hTERT and hTERT-promoter driven gene
therapy have made significant advances. The increased level of telomerase in cancer cells
also provides a potential platform for cancer diagnostics. Telomerase inhibition leads to
disruption of a cell’s ability to maintain the very ends of the chromosomes, which are called
telomeres. Thus, the telomere itself has also attracted attention as an anticancer drug target.
In this Perspective, interdisciplinary efforts to realize the therapeutic potential of targeting
telomere maintenance with a focus on telomerase are discussed.
■ INTRODUCTION
Pathways exhibiting differential activity in cancer cells when
compared to healthy cells are attractive targets for therapeutic
intervention. Accordingly, the observation that the enzyme
telomerase is up-regulated in 80−90% of all cancer cells isolated
from primary human tumors but is absent in neighboring cells
of healthy tissue has resulted in significant efforts to validate
telomerase as an anticancer drug target and to develop effective
approaches toward its inhibition.1
In addition to inhibitors that
target the enzymatic function of telomerase, efforts toward
immunotherapy using peptides derived from the catalytic
telomerase subunit hTERT (human telomerase reverse tran-
scriptase)2
and hTERT-promoter driven gene therapy3
have
made significant advances. The increased level of telomerase
activity in cancer cells also provides a potential platform for
cancer diagnostics.4
Telomerase inhibition leads to disruption
of a cell’s ability to maintain the very ends of the chromosomes,
which are called telomeres. Thus, the telomere itself has also
attracted attention as an anticancer drug target. Here, we
discuss the interdisciplinary efforts to realize the therapeutic
potential of targeting telomere maintenance with a specific
focus on telomerase.
■ TELOMERE MAINTENANCE AND TELOMERASE IN
HUMAN CANCER CELLS
Telomeres are the physical ends of linear chromosomes, and
their importance to human health is underscored by the Nobel
Prize in Physiology or Medicine awarded to Elizabeth
Blackburn, Jack Szostak, and Carol Grieder in 2009 for their
work on telomere biology and biochemistry. Telomeres are so
important because proper telomere maintenance is an absolute
requirement for continued cellular proliferation. Functional
telomeres require a specific and repetitive DNA sequence and
several telomere-specific proteins.5,6
These telomere-associated
proteins function in two primary capacities: maintaining the
structural and functional integrity of the telomere and ensuring
complete replication of telomeric DNA. In the absence of
functional telomeres, chromosome ends are recognized as DNA
damage, and the ensuing DNA damage response generally
results in either senescence or apoptosis, depending on the cell
type.7
Mammalian telomeric DNA contains several thousand
base pairs of repetitive DNA with the sequence 5′-
TTAGGG/3′-AATCCC and a single stranded 3′ overhang of
the G-rich sequence.8,9
The canonical DNA polymerases
responsible for DNA replication cannot efficiently replicate
the very ends of linear DNA templates. As a result, proliferative
cells must employ additional pathways to overcome this end-
replication problem. In human cells, this additional activity
resides primarily in telomerase. Telomerase is a ribonucleo-
protein complex that contains several protein subunits and one
RNA subunit.10,11
Human telomerase RNA (hTER) is a highly
structured 451-nucleotide long RNA that contains several
functional domains in addition to a templating sequence used
to code for the G-rich telomeric DNA strand.12
The protein
subunit hTERT functions as a reverse transcriptase by using the
templating portion of hTER to synthesize telomeric DNA.13
In
the majority of cancer cells, hTERT is up-regulated, increasing
the telomerase activity required for continuous proliferation.8,14
In contrast, telomerase activity is down-regulated in most
normal human diploid cells, with the exception of germline and
stem cells, primarily owing to inhibition of hTERT expression
during cellular differentiation.
Because telomerase activity is absent in most differentiated
human cells, telomeres erode during the cellular aging process.
This erosion allows telomere length to function like a mitotic
hourglass that regulates cellular replication and arguably
functions as a tumor suppressor mechanism.5
Cells lacking
Received: April 11, 2013
Perspective
pubs.acs.org/jmc
© XXXX American Chemical Society A dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXX

telomerase activity experience erosion of their telomeric DNA
when they divide. At a critical length, the shortened telomeres
are recognized as DNA damage and form a telomere
dysfunction induced focus (TIF). Typically, cells respond to
TIFs through senescence or apoptosis. Importantly, the
telomere-length dependence of proliferation appears to be a
result of the structure and not the length of the telomeric DNA
per se. This is evidenced in part by experiments in the de Lange
lab showing that the negative consequences of short telomeres
can be overcome by the overexpression of the telomere-binding
protein TRF2.15
It is likely that as telomeres erode in cells
lacking an active telomere maintenance pathway, the telomere
structure, predicted to be a lasso-like fold back structure termed
a t-loop,16
becomes increasingly difficult to sustain eventually
leading to TIFs and senescence or apoptosis if telomere
function is not reestablished.6
Telomere maintenance directly impacts cancer in two
specific ways. The most obvious is the absolute requirement
for telomeric DNA maintenance to offset telomere loss from
the end-replication problem, processing, and degradation. Most
cancer cells up-regulate expression of hTERT, in order to
increase telomerase activity.17
In 10−15% of cancers, a
recombination based telomere lengthening pathway called
alternative lengthening of telomeres (ALT) supplants the need
for telomerase.18
The ALT pathway is particularly prevalent in
astrocytic brain tumors and osteosarcomas.19
A second
connection between telomere biology and neoplasias is the
effect of overall length of telomeric DNA and the actions of
telomere-associated proteins on genetic stability. Telomere
erosion has been argued to serve as a tumor suppression
mechanism because it can activate the senescence pathway,
which suppresses neoplastic transformation.5
However, even in
the presence of telomere erosion, senescence is not activated in
the absence of the Rb/p16 or p53 pathways, leading to genetic
instability and increased potential for transformation. These
pivotal roles of telomere maintenance in tumorigenicity are
evidenced by the ability to inhibit tumor growth by inhibiting
telomerase in cancer cells and by the ability to convert primary
human cells to cancer cells by combining constitutive
telomerase expression with oncogene expression and/or loss
of a tumor repressor gene. For example, the simian virus 40
large-T oncoprotein together with an oncogenic allele of H-ras
and ectopic expression of hTERT has been demonstrated to
transform a variety of cell types.20
For more comprehensive
discussions on the roles of telomerase and telomere
maintenance on tumor biology, several recent reviews are
recommended.21−24
■ TARGETING TELOMERE MAINTENANCE FOR
CANCER THERAPY
When considering anticancer drug targets, several advantages
for targeting telomerase and telomere maintenance exist when
compared to other targets and pathways. Most important is the
apparently universal requirement for telomere maintenance in
cancer cells including putative cancer stem cells.25,26
In
addition, normal human cells including stem cells express
lower telomerase activity and generally maintain telomeres at
longer lengths when compared to cancer cells.27,28
These
features provide a level of specificity with telomerase inhibition.
Telomerase activity itself was validated as an anticancer drug
target in 1999 when two independent studies reported that
overexpressing dominant-negative-hTERT mutants resulted in
telomerase inhibition, telomere shortening, morphological
changes, growth arrest, and apoptosis in a variety of tumor
cell lines.29,30
Since that time, several strategies for targeting
telomere maintenance and telomerase-positive cancer cells have
been explored (Figure 1). The initial approach explored was the
direct inhibition of telomerase’s enzymatic activity. As
validation of the approach, the telomerase inhibitor imetelstat
(1, GRN163L) has been explored in anticancer therapy in
several clinical trials.31
Direct targeting of the telomere itself has
also been accomplished with specific DNA binding agents that
bind to and stabilize the G-quadruplex structures formed by
telomeric DNA.32,33
The high activity of the hTERT promoter
in cancer cells has resulted in the exploration of hTERT-
promoter driven expression of cancer killing genes and
viruses,34
and one clinical trial for hTERT-driven replication
of an oncolytic virus is underway.35
Immunotherapy targeting
hTERT-derived peptides has also been explored leading to
several clinical trials.36
Each of these platforms will be discussed
further below.
■ CONSEQUENCE OF TELOMERASE INHIBITION
AND TELOMERE DISRUPTION
If telomere maintenance is to be considered a viable anticancer
drug discovery platform, it is paramount to understand the
consequences of disrupting telomere maintenance for both
healthy and cancerous cells. The simplest and best explored
telomere-targeted anticancer drug platform is the inhibition of
telomerase enzymatic activity. When telomerase activity is
inhibited, DNA replication attending cellular proliferation
results in telomeric DNA erosion.29,37
Erosion of telomeric
DNA eventually results in dysfunctional telomeres that initiate
TIF formation.5,6
Generally, proliferating human cells lose 50−
200 base pairs of telomeric DNA during each cell cycle in the
absence of telomerase activity.38
Therefore, a lag between the
phenotypic consequences of telomerase inhibition and the
initiation of telomerase inhibition therapy is likely to occur. The
duration of this phenotypic lag depends on both the initial
length of telomeric DNA, which ranges from 5000 to 15 000
base pairs in human cancer cells, and the rate of erosion.
Importantly, it is suspected that the length of a few telomeres,
Figure 1. Many ways to target the overexpression of telomerase in
cancer cells have been developed.
Journal of Medicinal Chemistry Perspective
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not the average length of all telomeres, dictates the cellular
response to telomerase inhibition.39
This suggests that some
tumor types may be especially sensitive to telomerase
inhibition. Once telomeres erode to a critically short length,
they cannot function properly,40
perhaps because they cannot
be folded into the proper protected state, presumably a t-
loop.16
In most cancer cells, the loss of telomere function
results in apoptosis, whereas in normal cells, telomere loss
results in cell cycle arrest. Notably, telomerase inhibition has
also been observed to promote the ALT phenotype in tumors
concomitant with up-regulation of the PGC-1β, a master
regulator of mitochondrial biogenesis.41
This report offers
critical insight into the mechanisms of adaptation that may be
in play when tumors are subjected to telomerase inhibition.
Inhibition of telomerase by some methods, for example,
antisense oligonucleotides targeting the hTERT mRNA,42
can
result in immediate effects that are not preceded by the
phenotypic lag characteristic of telomerase activity inhibitors
and are not coupled to erosion of telomeric DNA. While a
precise explanation remains missing, one intriguing possibility is
that hTERT has several noncanonical properties that are
separate from its enzymatic activity, and inhibition of these may
have immediate effects.43
Proposed additional activities of
telomerase include telomere capping, inhibition of apoptosis,
activation of the Wnt pathway, and activation of the NFκB
pathway.44,45
Thus, down-regulation of hTERT can potentially
lead to immediate antiproliferative effects. Understanding why
some cells are immediately impacted by the loss of hTERT and
others are not remains to be determined. Perhaps some cancer
cells become oncogenically addicted to hTERT and require
hTERT to maintain elevated growth signaling.
Currently, one of the most studied small-molecule
telomerase inhibitors are G-quadruplex stabilizers.46
This class
of molecules can impact telomere functions in at least two
primary mechanisms. One is by inhibiting the enzymatic
activity of telomerase by preventing telomerase from binding to
its primer or by interacting with the nascent DNA telomerase
product. The second is to dissociate or block telomere-binding
proteins from the telomere, disrupting the formation of the
protective cap. As expected, the former mechanism results in a
delayed but highly telomerase-expression-specific effect while
the second mechanism is more immediate but may suffer from
reduced specificity because formation of proper telomere
structure is required in all cells including normal somatic and
stem cells. Early evidence suggests that the therapeutic index
for telomere disrupters is surprisingly high at least in a mouse
model.47,48
One potential reason that telomere disruption may
be well tolerated in normal cells is that the DNA damage
response at the telomere may be transient and reversible.
Evidence for this hypothesis comes from experiments using a
ligand-stabilized version of the telomere binding protein
Protection of Telomeres 1 (POT1). POT1 is essential for
telomere protection, and the ligand-stabilized POT1 allowed
POT1 to be inhibited transiently and reversibly. In cancer cells,
POT1 inhibition resulted in cell death. But in normal cells,
POT1 inhibition resulted in arrest that was reversed by
reactivating POT1.49
■ SMALL-MOLECULE TELOMERASE INHIBITORS
Once the importance of telomerase activity for cancer cell
growth was recognized, several academic and pharmaceutical
groups initiated programs to discover small molecule
telomerase inhibitors. Approaches toward the identification of
effective telomerase inhibitors have included repurposing of
viral reverse transcriptase inhibitors, screening of natural
products, high throughput screens of diverse compound
libraries, and more recently, the beginnings of structure-based
discovery and optimization. Despite these efforts, small
molecule telomerase inhibitors have yet to reach the clinic. In
this section, the varieties of approaches toward direct
telomerase activity inhibition with small molecules will be
discussed.
■ NUCLEOSIDE ANALOGUES
Nucleoside analogues developed as viral reverse transcriptase
inhibitors have been investigated as telomerase inhibitors
(Figure 2). Nucleoside analogues can hypothetically affect the
telomere through several mechanisms, including telomerase
inhibition and chain termination that can lead to telomere
shortening and perturbation of telomere sequence that can lead
to disruption of telomere function. The chain terminator 3′-
azido-2′,3′-dideoxythymidine (2, AZT), the first nucleoside
reverse transcriptase inhibitor approved for the treatment of
HIV-1, inhibits telomerase in vitro and in vivo, giving rise to
telomere shortening.50,51
In cultured cells, treatment with 2
resulted in telomere shortening, cell-cycle perturbation, and
increased p14ARF expression,52
and 2 exhibited synergistic
effects when combined with other chemotherapeutic
drugs.53−55
Several nucleoside HIV reverse transcriptase
inhibitors including stavudine, tenofovir, didanosine, and
abacavir also inhibit telomerase activity and lead to telomere
erosion in cultured cells, but non-nucleoside HIV RT inhibitors
did not inhibit telomerase in these studies.56
HIV patients have
been taking these and other nucleoside reverse transcriptase
inhibitors for decades, leading to possible effects from long-
term telomerase inhibition. This potential side effect has been
investigated in a small patient population, and it was found that
telomere length was inversely correlated with total time
patients were exposed to nucleoside reverse transcriptase
inhibitors.57
Although other nucleoside triphosphate analogues,
including arabinofuranylguanosine and 6-thio-7-deaza-2′-deox-
yguanosine 5′-triphosphate, ddGTP, ddATP, and ddTTP,
produced substantial inhibition of telomerase and telomere
shortening, they have not been further tested in cell cultures or
in animal models and are likely to be nonspecific, thus limiting
Figure 2. Representative nucleoside analogues identified as telomerase inhibitors.
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their clinical utility.51,58
As telomerase structural biology
increases, it may be possible to rationally design nucleoside
analogues with the requisite telomerase specificity for this
platform to be viable.
■ NON-NUCLEOSIDE ANALOGUES
Non-nucleoside, small-molecule telomerase inhibitors have
been identified by a variety of approaches. High throughput
screening of chemical libraries has been used to identify
telomerase inhibitors (Figure 3). The isothiazolone derivative
2-[3-(trifluoromethyl)phenyl]isothiazolin-3-one (3, IC50 = 1
μM) was identified from a screen of a 16 000-member, chemical
library using rat telomerase (Figure 3).59
The reducing agent
dithiothreitol was found to protect telomerase from inhibition
by 3, suggesting that inhibition resulted from interaction
between the isothiazolone moiety of 3 with the sulfhydryl
group of key cysteine residue(s).59
The nitrostyrene derivative
3-(3,5-dichlorophenoxy)nitrostyrene (4, IC50 = 0.4 μM)60
and
the quinoxaline derivative 2,3,7-trichloro-5-nitroquinoxaline
(5),61
two noncompetitive telomerase inhibitors, were also
identified by screening chemical libraries. At low concentrations
(1 μM), 4 was not acutely cytotoxic to HeLa cells, but chronic
exposure led to progressive telomere erosion followed by the
induction of senescence consistent with telomerase inhibition.60
Long-term treatment of the breast cancer cell line MCF7 with 5
(1 μM) also resulted in telomere erosion, chromosome
abnormalities, and senescence.61
The crystal structure of TERT from the beetle Tribolium
castaneum62
has been used in limited structure based design of
telomerase inhibitors. Because 2H-pyrazole and dihydropyr-
azole derivatives have been shown to have strong anticancer,
antmalarial, antiviral, and anti-inflammatory activity, they were
selected as a starting scaffold for telomerase inhibition.63−65
Aryl-2H-pyrazoles with 1,3-benzodioxoles and electron with-
drawing aryls groups and sulfonyl-4,5-dihydropyrazoles dis-
played telomerase inhibition with IC50 values ranging from 0.8
to 10 μM in a variety of cancer cells (Figure 4).63−65
Since
previous studies have shown that tetracyclic coumarins have
notable anti-HIV activities and that the HIV and telomerase
reverse transcriptase proteins display structural homology,
dihydropyrazolepiperidine derivatives containing coumarin
moieties were also synthesized.63,66
These molecules showed
anticancer activity against gastric cancer and the most potent
inhibited telomerase in crude cell extracts with IC50 values in
the 1−2 μM range.63,66
Results from docking studies were used
to suggest that these molecules bind the hTERT active site
through key intermolecular hydrogen bonding and π−cation
interactions with invariant residues Lys189, Asp254, and
Gln308 and by projecting aryl and dihyrdopyrazole rings into
the hydrophobic dNTP-binding pocket.63,65,66
The π−cation
interaction is consistent with the structure−activity relationship
(SAR) trend in which electron withdrawing groups on the
aromatic ring attenuate inhibition, whereas electron donating
groups increased inhibition. 3D quantitative SAR (QSAR) was
used to rationalize the proposed binding mechanism, and a
correlation coefficient of r2
= 0.966 was found for predicted and
observed IC50 for a small, chemically similar test set.65
Other
telomerase inhibitors proposed to bind telomerase using
docking studies include 2-chloropyridines and 2-aminomethyl-
5-(quinolin-2-yl)-1,3,4-oxadiazole-2(3H)-thionequinoline de-
rivatives, although these studies did not offer strong validation
of the binding models.67,68
Several natural products have been identified as telomerase
inhibitors as well (Figure 5). Helenalin (6), a natural
sesquiterpene lactone, was found to inhibit telomerase
apparently by reaction with a cysteine residue similar to 3.69
o-Quinone containing compounds, for example, tanshinone 2A
(7), a plant diterpene known to exhibit diverse pharmacological
activities, also inhibit telomerase (IC50 = 3 μM). The quinone
was fully responsible for inhibition that was time- and DTT-
dependent. It was also found that 7 undergoes redox cycling to
produce hydrogen peroxide. The data suggest that quinone
redox cycling inhibits telomerase, perhaps by oxidation of
conserved cysteine residues.70
Another apparently reversible
inhibitor is 8 (U-73122), commonly used as an inhibitor of
Figure 3. Examples of small-molecule telomerase inhibitors identified
by screening small molecule libraries.
Figure 4. Telomerase inhibitors reported to bind the telomerase active site based on docking studies.
Figure 5. Natural product and natural product derivatives identified as
telomerase inhibitors.
dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXXD

phospholipase C, which was reported to be a potent (IC50 = 0.2
μM) telomerase inhibitor.71
Inhibition was shown to be
dependent on the pyrrole-2,5-dione functional group, suggest-
ing potential alkylation of telomerase. β-Rubromycin (9), a
quinone antibiotic, inhibited telomerase with an IC50 of 3 μM.72
Mechanistically, 9 appears to be a competitive inhibitor versus
the telomerase primer, it also inhibits retroviral reverse
transcriptases, mammalian DNA polymerases, and terminal
deoxynucleotidyl transferase,73
indicating that rubromycins are
not telomerase specific.
The search for more biologically effective telomerase
inhibitors has encouraged innovative yeast-based screens.
Natural products chrolactomycin (10) and UCS1025A (11)
were identified as direct telomerase inhibitors using a forward
chemical genetics screen in a yeast system (Figure 6).74
A yeast
strain with artificially shortened telomeres was used to screen a
microbial metabolite library. Since telomeres were shortened,
the strain was more sensitive to telomerase inhibition than wild
type. Therefore, compounds with selective activity in the
telomere shortened strain were more likely to be telomerase
inhibitors. With this assay, two new telomerase inhibitors 10
(IC50 = 0.5 μM) and 11 (IC50 = 1.3 μM) were identified. In
addition, the Hsp90 inhibitor radicicol was also identified as a
telomerase maintenance inhibitor in screen.74
The synthesis of
11 has been accomplished, but structure−activity relationships
of analogues have not been reported.75,76
More recently, an
alternative yeast based assay was developed using recombinant
human telomerase. Expression of human telomerase activity in
yeast results in a growth delay; therefore, potential inhibitors of
human telomerase could rescue the transfected cells. By use of
this screen, several potential telomerase inhibitors with
moderate potency (IC50 values of ∼1−10 μM) were identified
to confirm the screening platform.77
The most potent
compound identified in the screen was the dichlorothiophene
12 (CD11359).
One promising class of small molecule telomerase inhibitors
is the potent enoylaminobenzoic acids represented by 13
(BIBR1532, IC50 = 0.093 μM) and 14 (BIBR1591, IC50 = 0.47
μM) that function as selective mixed-type, noncompetitive
telomerase inhibitors and were developed from systematic
structure−activity correlations (Figure 7).37
Limited structure−
activity relationship data are publically available and show that
the α−β unsaturated amide is essential and modifications to the
naphthyl group are not well tolerated.78
The mechanism of
inhibition by 13 is distinct from those by other non-nucleoside
compounds or antisense oligonucleotides, as it does not cause
chain termination events but rather inhibits the formation of
long reaction products. Compound 13 seems to act allosteri-
cally to impair the unique ability of telomerase to catalyze
addition of multiple telomeric repeats onto a DNA primer.79
Acute treatment with high concentrations of 13 caused direct
cytotoxic effects on malignant cells of the hematopoietic
system, resulting in end to end fusions, phosphorylated p53,
and decreased expression of TRF2, consistent with a telomere-
associated effect.80
Compound 13 is also capable of sensitizing
drug-resistant cells to chemotherapeutic agents.81
Chronic
Figure 6. Small molecules identified as telomerase inhibitors utilizing
yeast-based assays.
Figure 7. Treatment with 14 reversibly inhibits telomere length maintenance. (A) Structures of 13 and 14. (B) Chronic treatment of NCI-H460
cells with 14 results in growth arrest that can be reversed after ending the treatment. Open circles are untreated cells. Filled triangles are cells treated
with 14. Treatment was halted at day 220; note the return to proliferative growth. (C) Southern blot analysis of average telomere length of
telomerase inhibitor treated cells and cells released from telomerase inhibition. Treatment with 14 resulted in loss of telomere length and exhibited
∼1.5 kb of telomeric DNA (lanes 1 and 2). Removal of 14 resulted in telomere elongation to ∼3 kb of telomeric DNA (lane 3). Untreated cells are
shown in lane 4 for comparison and have 3 kb of telomeric DNA. Parts B and C are reprinted by permission from Macmillan Publishers Ltd.: The
EMBO Journal (http://www.nature.com/emboj/index.html) (Damm, K.; Hemmann, U.; Garin-Chesa, P.; Hauel, N.; Kauffmann, I.; Priepke, H.;
Niestroj, C.; Daiber, C.; Enenkel, B.; Guilliard, B.; Lauritsch, I.; Muller, E.; Pascolo, E.; Sauter, G.; Pantic, M.; Martens, U. M.; Wenz, C.; Lingner, J.;
Kraut, N.; Rettig, W. J.; Schnapp, A. EMBO J. 2001, 20, 6958−6968),37
copyright 2001.
dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXXE

exposure to lower doses of 14 resulted in progressive erosion of
telomeric DNA leading to senescence.37
However, the lag
phase between telomerase inhibition and the impact on
proliferative capacity under the chronic treatment condition is
a major concern,37
as is the apparently high protein binding
evidenced by the significantly decreased potency in crude cell
extracts compared to purified telomerase.78
More than 120
days, approximately 135 population doublings, was necessary
for treatment with 13 to inhibit cell proliferation of NCI-H460
lung cancer.82
Interestingly, washout of 14 in telomerase
positive cells allowed recovery of telomere length (Figure 7).37
■ SMALL MOLECULES THAT TARGET hTER AND THE
hTER-TELOMERIC DNA HETERODUPLEX
In addition to targeting the enzymatic activity of telomerase,
exploratory efforts in identifying ligands that target hTER
suggest that RNA-binding ligands may be a productive platform
for targeting telomerase. In two separate reports, oligonucleo-
tides were used to identify regions of hTER that can be
specifically targeted to prevent proper telomerase assem-
blage.83,84
Specifically, the CR4-CR5 and pseudoknot domains
of hTER are susceptible. Detailed analysis demonstrated that
discrete domains of hTER are blocked without losing the ability
of hTERT to associate with hTER.84
This suggests that the
CR4-CR5 and pseudoknot domain targeting oligonucleotides
effectively cause formation of a dysfunctional telomerase
complex. Subsequently, small molecules that bind hTER have
been identified,85−87
and several of these including amino-
glycosides and the bisbenzimide Heochst 33258 have been
shown to inhibit telomerase,86
presumably by binding hTER
and perturbing proper assemblage of the holoenzyme complex
(Figure 8). Efforts to target the telomerase template RNA−
DNA primer/nascent product heteroduplex have also been
reported.88,89
In these studies, several intercalators were
identified that inhibit telomerase and bind preferentially to
the RNA−DNA heteroduplex as opposed to G-quadruplex
DNA. Advances in this area include second generation
intercalators and bis-intercalators that are fairly potent
telomerase inhibitors in cell free assays. A small library of bis-
ethidium derivatives with varying linkers was screened.
Surprisingly, the library exhibited a narrow range of moderately
potent telomerase inhibition without significant improvement
over the parent monointercalator.88
Data for cell based assays
using small molecule targeting of hTER or the hTER/DNA
heteroduplex have not yet been reported, so it is still too early
to tell if this approach will be clinical viable.
■ TELOMERASE INHIBITION BY INDIRECT MEANS
hTERT Transcription Regulators. Cellular telomerase
activity is regulated at various levels including transcription,
mRNA splicing, post-transcriptional and post-translational
modifications of hTER and hTERT, transport and subcellular
localization, assembly of active telomerase ribonucleoprotein,
and telomeric DNA accessibility. Transcriptional regulation of
hTERT is thought to be the major mechanism of telomerase
regulation.90
The hTERT promoter contains binding sites for
several transcription factors, suggesting that the regulation of
hTERT expression may be subject to multiple levels of control
by various factors and dependent on cellular contexts.91
A
primary activator of hTERT is the c-Myc oncogene, a known
regulator of cell proliferation, growth, and apoptosis.92
Targets
of the Myc family are involved in various aspects of cellular
functions, including cell cycle, growth, differentiation, and life
span. c-Myc expression correlates with hTERT, and both are
Figure 8. Ligands that inhibit telomerase by binding to the RNA subunit hTER (Hoechst 33258 and neomycin) and the telomerase DNA/RNA
heteroduplex (bis-ethidium compounds).
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up-regulated in highly proliferative and immortal cells but
down-regulated during terminal differentiation and in non-
proliferative cells. Therefore, it is not surprising that inhibitors
of c-Myc, butein,93
gamboic acid,94
and genistein,95
also inhibit
telomerase. Additionally, genistein treatment resulted in down-
regulation of the protein kinase Akt and thus hTERT
phosphorylation, indicating that genistein can inhibit telomer-
ase by at least two mechansisms: decreased hTERT tran-
scription and decreased posttranslational activation of
hTERT.95
Second generation tyrosine kinase inhibitors and the lipid-
derived signaling molecule ceramide have been shown to inhibit
hTERT promoter activity by altering the functions of Sp1 and
Sp3.96,97
Sp1 has been shown to collaborate with c-Myc in
order to initiate hTERT transcription.98
Imatinib, dasatinib, and
nilotininb decrease telomerase activity and hTERT expression
by decreasing Sp1 nuclear levels and activation and reducing
the nuclear translocation of hTERT via suppression of Akt
phosphorylation.96
Agents that act on hormonal pathways have
also been linked to telomerase activity regulation. For example,
estrogen activates telomerase in hormone-sensitive tissues such
as mammary and ovary epithelial cells,99,100
while the selective
estrogen receptor modulator tamoxifen reduces telomerase
activity in cells where it functions as an estrogen antago-
nist.101,102
Progesterone also has an antagonistic effect on
estrogen-induced hTERT expression.103
These observations
highlight the fact that the polypharmacological effects of many
anticancer drugs are anticipated to include inhibition of
telomere maintenance as a mechanism of their anticancer
effects.
Negative Regulators of hTERT Transcription. The lack
of telomerase activity in somatic cells appears to be a
consequence of transcriptional repression of the hTERT
expression. The loss of transcriptional repression results in
the up-regulation of hTERT expression and telomerase activity
as seen during carcinogenesis and cellular immortalization.
Several transcriptional repressors of hTERT have been
identified. One such negative regulator of hTERT transcription
is Mad1. Mad1, c-Myc, and Max are part of a family of
transcription factors that bind to E-box-containing promoters
to either down-regulate or up-regulate gene expression. Mad1 is
up-regulated in telomerase negative somatic cells, c-Myc is up-
regulated in cancer cell lines, and Max is expressed
ubiquitously.104
Consistent with its role in regulating
telomerase, overexpression of Mad1 resulted in decreased
hTERT promoter activity in gene reporter assays.105
Another negative regulator of hTERT transcription is the
tumor suppressor protein p53, which induces cell cycle arrest or
apoptosis in response to DNA damage and other cellular
stresses to inhibit tumor formation. p53 has been shown to
inhibit telomerase activity through the repression of hTERT
transcription.106
Repression of telomerase activity was also
observed by overexpressing pRB in human squamous cell
carcinomas107
and E2F1 in head and neck squamous cell
carcinomas.108
pRB and E2F1 have the potential to transcrip-
tionally repress hTERT activity either independently or in
tandem with one another. Another tumor suppressor involved
in the transcriptional repression of hTERT is Wilm’s tumor
protein 1 (WT1). WT1 has also been shown to significantly
decrease hTERT mRNA and telomerase activity in HEK293
cells.109
The role of WT1 in hTERT repression is cell type
dependent, as the WT1 gene is expressed only in specific cells
such as the kidney, gonad, and spleen.
Other negative regulators of hTERT transcription include
the myeloid cell-specific zinc finger protein 2, interferon-α, and
TGF-β. Interferon-α- and -γ-induced sensitization to apoptosis
is associated with repressed transcriptional activity of the
hTERT promoter in multiple myeloma.110
Transforming
growth factor β, a potent tumor suppressor, inhibits telomerase
through the transcription factors SMAD3 and E2F.111,112
TGF-
β activated kinase 1 (TAK1) has been shown to repress
transcription of the human telomerase reverse transcriptase
gene. TAK1-induced repression was caused by the recruitment
of histone deacetylase to Sp1 at the hTERT promoter.113
T-cell
acute lymphoblastic leukemia protein 1 (TAL1) is also a
negative regulator of the hTERT promoter. Overexpression of
the proto-oncogenic protein TAL1 leads to a decrease in
hTERT mRNA levels and consequently reduced telomerase
activity.114
Histone deacetylase (HDAC) inhibitors, such as
trichostatin A, have been shown to induce apoptosis and down-
regulate telomerase activity via suppression of hTERT
expression in leukemic U937 cells.115−117
Dynamic assembly
of the E2F−pocket protein−HDAC complex plays a central
role in the regulation of hTERT. For example, a small molecule,
CGK 1026, inhibits the recruitment of HDAC into E2F-pocket
protein complexes assembled on the hTERT promoter.118
These data underscore the fact that several anticancer treatment
strategies are likely to result in telomerase inhibition indirectly.
Several reports demonstrate the indirect effects on
telomerase activity that are possible with small molecules.
Tea catechins such as (−)-epigallocatechin 3-gallate (15)
prevent growth of a variety of cancer cell lines and inhibit
telomerase activity in a concentration dependent manner by
decreasing hTERT mRNA levels.110,119
The changes in hTERT
mRNA levels can be attributed to chromatin remodeling due to
the hypoacetylation of histones at the hTERT gene promoter
and decreased methylation of the hTERT promoter at the E2F-
1 binding site.119
Consistent with the observed decrease activity
of the hTERT promoter, 15-treated U937 monoblastoid
leukemia and HT29 colon adenocarcinoma cells experienced
decreased proliferation potential commensurate with telomere
shortening. In addition, 15 was also shown to induce a severe
reduction in cell growth with enhanced membrane blebbing,
cell swelling, apoptosis, and necrosis in laryngeal squamous cell,
small-cell lung, and breast cancers.119−121
Further studies
showed that the tea catechin derivative MST-312 (N,N′-
bis(2,3-dihydroxybenzoyl)-1,2-phenylenediamine) appears to
inhibit telomere maintenance by directly targeting telomerase
(IC50 = 0.67 μM), resulting in telomere erosion at a 20-fold
lower dose than 15 in the monoblastic leukemia cell line
U937.122
GSK3 inhibition by 6-bromoindirubin-3′-oxime caused
suppression of hTERT expression, telomerase activity, and
telomere length in various cancer cell lines and decreased
hTERT expression in ovarian cancer xenografts.123
Other
repressors of hTERT transcription are retinoids, derivatives of
vitamin A, which have been shown to down-regulate telomerase
leading to telomere erosion.124
Retinoic acid receptor α and
retinoid-X receptor-specific agonists synergistically down-
regulated hTERT and induced tumor cell death.125
Posttranslational Regulators of hTERT. Perturbing
posttranslational modifications represents another mechanism
for targeting telomerase. Because telomerase activity and
cellular location is controlled by its phosphorylation state,
phosphatase and kinase inhibitors can have direct effects on
telomerase. For example, the phosphatase 2A inhibitor okadaic
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acid prevents telomerase inhibition by phosphatase 2A in breast
cancer cells.126
Protein kinase C (PKC) has been shown to
enhance telomerase activity in certain cell types, and PKC
inhibitors have been shown to inactivate telomerase in
nasopharyngeal127,128
and cervical129
cancer cells among others.
Imatinib mesylate (Gleevec), a tyrosine kinase inhibitor, also
down-regulates telomerase activity and inhibits proliferation in
telomerase expressing cells.130
The protein kinase Akt has well-
established roles in telomerase activation. Akt phosphorylates
hTERT and activates telomerase in vitro, while the nonspecific
Akt inhibitor wortmannin causes loss of telomerase activity.127
The tyrosine kinase c-Abl is also capable of phosphorylating
hTERT. The SH3 domain of c-Abl interacts with and
phosphorylates hTERT resulting in a decrease in telomerase
activity.131
These observations demonstrate that telomerase
activity can be directly regulated by protein phosphorylation
independent of transcriptional regulation and highlight that
telomerase inhibition may contribute to the observed
anticancer effects of several drugs. In addition to regulation
by kinases, it has recently been determined that hTERT is a
substrate of caspases-6 and −7.132
Interestingly, the caspase
sites are unique and the cleavage products appear remarkably
stable.
Hsp90 Inhibitors. The telomerase ribonucleoprotein
complex requires the Hsp90 chaperone complex, including
p23, Hsp70, p60, and Hsp40/γdl for proper assemblage. Hsp90
associates directly with hTERT and is required to maintain
telomerase in an active conformation at least under some
conditions.133,134
Hsp90 inhibitors cause inactivation, destabi-
lization, and degradation of Hsp90 target proteins including
hTERT.46,133
Since several oncogenic proteins including c-Myc,
Akt, and mutated p53 require Hsp90 for their activity,
inhibition of this chaperone should block multiple oncogenic
pathways. Hsp90 inhibitors such as novobiocin, radicicol,
geldanamycin, and 17-AAG were also shown to inhibit
telomerase activity.134−137
Recently, curcumin has been
shown to inhibit nuclear localization of telomerase by
dissociating the Hsp90 co-chaperone p23 from hTERT.93
Curcumin treatment limited the association between p23 and
hTERT while not affecting Hsp90 interaction with telomerase.
■ G-QUADRUPLEX STABILIZERS
The most advanced area of small-molecule drug discovery
directed at the telomere is G-quadruplex-stabilizing ligands. G-
quadruplexes are unique structures formed by folding G-rich
nucleic acid sequences. These structures are heterogeneous
with a variety of structural topologies.138
Folding topologies are
influenced by sequence, buffer conditions including identity of
cations present, molecular crowding, and other parameters.
Significant effort toward determining the structures of human
telomeric DNA138,139
and the biological functions of G-
quadruplex DNA at the telomere and in other G-rich sequences
embedded in the chromosome140
has been reported. Because
telomerase must hybridize to its DNA primer to afford
telomeric DNA extension, it was proposed that G-quadruplex
formation would render a primer inert to telomerase.141
Seminal studies by Zahler et al. showing that telomerase
could not extend a folded G-quadruplex were consistent with
this hypothesis and initiated the field of G-quadruplex binding
ligands as a platform for telomerase inhibition.141
This report
was quickly followed by the cardinal work of the Hurley and
Neidle labs providing the first G-quadruplex ligands that
effectively inhibited telomerase.142,143
Since these reports, a
wide variety of pharmacophores have been explored as G-
quadruplex ligands, and most of these are polycationic aromatic
hydrocarbons (Figure 9).144
Since these initial studies
demonstrating a relationship between telomeres and G-
quadruplexes, these structures have become much more
complex as drug targets than originally assumed, as they are
predicted to be present in a wide variety of genomic locations
with increased density in promoter regions.145
Several of these
promoters, including those for hTERT,146
c-kit,147,148
and c-
myc,149,150
have verified abilities to fold into G-quadruplex
structures. Much of the work on G-quadruplex structure,
Figure 9. Representative G-quadruplex ligands that inhibit telomerase. (A) 16 (BRACO19), a 3,6,9 trisubstituted acridine derivative. (B) RHPS4, a
pentacyclic acridine derivative. (C) and (D) are models of 16 bound to a G-quadruplex formed by a dimer of TTAGGGTTAGGG. The structure
was rendered from PDB 3CE5. 16 is shown in red. dG residues are shown in cyan, and dA and dT residues are shown in gray.
dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXXH

biological relevance, and small-molecule targeting has been
recently and extensively reviewed, and readers are directed to
these.144,151,152
Here, we will simply highlight the major
concerns with targeting G-quadruplexes as a specifically
telomere-directed anticancer drug modality and highlight recent
approaches toward advancing telomere-binding ligands to the
clinic.
The first G-quadruplex ligands explored as telomerase
inhibitors were disubstituted anthraquinone derivatives.153
Since that time, several other polycyclic heteroaromatic
scaffolds and macrocycles have been explored allowing the
development of potent telomerase inhibitors with increasing
specificity for G-quadruplex DNA compared to duplex DNA,
and these ligands are available at the G-quadruplex ligands
database (http://www.g4ldb.org/ci2/index.php).154
Impor-
tantly, high resolution crystal and NMR structures of human
telomeric G-quadruplexes can inform rational design to
improve G-quadruplex affinity and specificity.152
From
structural studies, it is clear that critical features known to
enhance G-quadruplex binding include a delocalized π-electron
system capable of stacking onto G-tetrad a positive charge that
can rest in the center of the G-quartets to interact with the
carbonyl oxygens from guanine that form the core of the G-
quartets, and positively charged substituents that can interact
with the grooves of the G-quadruplex (Figure 9c and Figure
9d).138
From a purely biochemical view, the most significant
current issues in G-quadruplex ligand design are specificity for
G-quadruplex DNA compared to duplex DNA and specificity
for G-quadruplex type, i.e., telomeric versus other DNA and
RNA quadruplexes that may be present.155
From a
pharmaceutical standpoint, the major issue concerning trans-
lation of G-quadruplex ligands is the poor pharmacokinetic
profile associated with the typically hydrophilic and positively
charged molecules.156
One of the most significant recent advances toward selective
G-quadruplex targeting is the ability to rationally design
molecules using structure-based approaches. Structures of G-
quadruplexes formed by the human telomeric DNA sequence
by X-ray crystallography and NMR spectroscopy have provided
templates for rational design of G-quaduplex ligands.157,158
These advances have allowed for the development of rationally
designed molecules with increased.159,160
These promising
examples suggest that G-quadruplex specificity is achievable.
The more challenging hurdle that still remains is the poor
pharmacokinetic and pharmacodynamic properties of promis-
ing G-quadruplex compounds. These hurdles may not be
insurmountable, as evidenced by the G-quadruplex ligand and
fluoroquinolone derivative quarfloxin, which binds to nucleo-
lin/rDNA and inhibits Pol 1 transcription, thus inducing
apoptosis in adenocarcinoma cells, and which has had some
clinical success.161
■ DISRUPTION OF TELOMERE MAINTENANCE
USING ANTISENSE AND RELATED APPROACHES
Ribozymes. Hammerhead ribozymes are small catalytic
RNA molecules that have the capacity to specifically cleave
target RNA after NUH sequences, where N is any nucleotide,
U is uridine, and H is any nucleotide except G.162
Design of
hammerhead ribozymes is relatively simple, and specificity can
be achieved by incorporating sequences complementary to the
target. Because of this nature, ribozymes have been investigated
as a platform for telomerase inhibition via targeted RNA
cleavage of hTER or hTERT mRNA.
The template region of hTER is an ideal target for ribozyme
cleavage because it has the requisite target sequence for
Hammerhead or HDV ribozymes, is essential for telomerase
activity, and it may be solvent exposed even in the telomerase
holoenzyme. Studies using melanoma,163
hepatoma, colon
cancer,164
and endometrial cancer cells165
have correlated
ribozyme expression with a marked reduction in telomerase
activity resulting from diminished telomerase RNA levels.
While telomere lengths were greatly attenuated in some
carcinomas, other types did not display this phenotype.163,165
Treatment with template-targeting ribozymes also resulted in
significant growth retardation with proliferation rates reduced
compared to parent cells.166
Furthermore, treated cells
experienced telomere shortening in concert with altered or
dendritic morphologies, which are indicative of senescence,166
G0/G1 growth arrest, and increased apoptotic rate.164
Circularization and addition of a six nucleotide poly(A)
sequence to these ribozymes increased target RNA cleavage
while conferring protection from cellular exoribonuclease
degradation.167
TERT mRNA levels have been targeted by a Hammerhead
ribozyme targeting the initial nucleotides in hTERT mRNA,
which may be essential for the proper binding of the translation
initiation complex to the 5′ mRNA cap structure.168
Ribozyme
cleavage in the middle of hTERT mRNA has also demonstrated
decreased telomerase activity while also influencing rapid cell
death (4 days) even without diminished telomere length,
consistent with potentially nontelomeric roles of hTERT in
some cancer cells.169
Oligonucleotides and Template Antagonists. Antisense
(AS) oligonucleotides (ODNs) can inhibit telomerase by
several mechanisms including blocking hTERT translation,
reduction of hTERT mRNA and hTER levels, and sterically
blocking the association of telomerase with the telomere
(template antagonists).42
However, oligonucleotides present
many challenges toward achieving efficient intratumoral
telomerase inhibition. Nonetheless, studies have demonstrated
that unmodified antisense oligonucleotides can in fact potently
inhibit telomerase activity under in vitro conditions.170−172
One
of these was a genetic approach using a plasmid borne 125
nucleotide hTR-antisense RNA, which inhibited tumor growth
in mouse xenografts.170
To make up for the shortcomings of unmodified ODNs, a
spectrum of ODNs with chemically modified backbones have
been utilized to enhance the therapeutic potential of these
antisense strategies. Phosphorothioate (PS) ODNs composed
of telomere repeat sequences ranging from 6 to 24 bases and
targeting the template of hTER showed significant telomerase
inhibition in various cancer cell lines.173,174
While longer PS-
ODNs did not have any effect on tumor growth, a 6-mer PS-
ODN decreased cellular growth in Burkett’s lymphoma,
increased cell population doubling times, induced dramatic
levels of apoptosis, and reduced human xenograft tumors in
mice to undetectable sizes.174
2′-O-Methyl-PS chimeric ODNs
targeting the template sequence of hTER demonstrated potent
telomerase inhibition with IC50 values in the nanomolar
range.175,176
Similar ODNs blocked up to 95% of telomerase
activity after 1 day of treatment when delivered with a cationic
lipid delivery system and induced telomere erosion and
subsequent limited proliferation commensurate with marked
apoptosis in long-term studies with chronic treatment.177,178
Cellular delivery with chitosan-coated PLGA nanoparticles
afforded even higher cellular uptake and the similar phenotype
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of progressive telomere shortening with an EC50 value of 10
nM.179
Direct addition of 2′-O-methoxyethyl-PS chimeric
ODNs decreased telomere length and proliferation and growth
rates in MCF-7 (breast)180
and DU145181
(prostate) cancer
cells in culture and xenograft tumor models.180,181
Synergistic
effects were seen in DU145 prostate cancer cells upon addition
of chemotherapeutic agents such as cisplatin or carboplatin.181
Peptide nucleic acids (PNAs) completely covering the
template region of hTER were 10−50 times more potent
than similar PS-ODNs with IC50 values in the nanomolar
range.182
The potent telomerase inhibition is due to high
affinity of the PNA to the single stranded RNA template and
suggests that affinity is not limited to base pairing but may also
include interactions between hTERT and the peptide back-
bone.182
Both cellular permeabilization and lipid-mediated
delivery demonstrated similar IC50 values in DU145 (prostate
cancer)83
and HME50-5 (immortal breast epithelial)182
cells.
Conjugation of short peptide sequences to PNAs targeting the
hTER template displayed increased telomerase inhibition with
IC50 values 10-fold lower than PNAs alone.183
Targeting of
hTER by 2-5A ODNs has led to a remarkable inhibition of
telomerase activity concomitant with rapid hTER degradation
and a greater than 50% decrease in cell viability in a time
dependent manner.41,184−186
2-5A ODNs targeting hTER can
also arrest cells in G2/M phase187
and induce apoptosis via
activation of the caspase signaling cascade.186
Direct admin-
istration of hTER-targeting 2-5A ODNs and combination
therapy with interferon-β has demonstrated antiproliferative
effects including a reduction in colony formation and a
significant retardation in tumor growth and volume over
time.184,185,188
In 2003, Asai et al. described a novel class of chemically
modified N3′→P5′-thiophosphoramidates (NPS), which con-
ferred high stability and sequence specific potency.42,189
The
efforts led to the development of 17 (GRN163), a 13-
nucleotide long NPS-ODN with sequence 5′-TAGGGTT-
AGACAA-3′ that targets the template region of hTER by
overlapping and extending four nucleotides beyond the 5′
template boundary (IC50 = 0.14 μM).189
Treatment of various
cancer cell lines with 17 significantly reduced telomerase
activity, promoted critical telomere loss, and instigated
apoptosis while concomitantly up-regulating caspase activity.189
These results combined with impressive data from long-term
experiments and its high potency and specificity indicated that
17 may be an effective anticancer agent.189
In order to improve its bioavailability, lipophilicity, and
cellular uptake, a 5′ terminal palmitoyl group was attached
through an amide bond to α-aminoglycerol of the thiophos-
phoramidate 17 to produce the clinical candidate 1 (Figure
10).190
Comparison studies with 17 proved that 1 was just as
potent at inhibiting telomerase in various cancers, could inhibit
telomerase activity effectively without the use of a transfection
agent, and demonstrated greater tumor uptake.190−192
Since
then, many studies have been published revealing the extensive
anticancer potential of this drug in a variety of cancers. A single
treatment of 1 μM 1 yielded complete inhibition of telomerase
activity in various cancers within hours of dosing and near
complete inhibition could be maintained for up to 6 days, while
chronic treatment allowed sustain inhibition with undetectable
telomerase activity.193−198
Continued treatment for 2 weeks
elicited morphological changes such as rounding and increased
extension of filliopodia-like structures,193,198
reduced colony
formation,194,196
and attenuated metastasis invasion.194
Longer
periods of treatment with 1 triggered decreased proliferation,196
decline in cell viability,197,199
disruption of focal adhesions and
actin filament organization,193
substantial telomere short-
ening,194−196
reduced capacity to form neurospheres and
mammospheres,191,199
down-regulation of cell cycle regula-
tors,192
G0/G1 cell cycle arrest,191,198
up-regulation of APG5
and DAPK2 expression (genes implicated in the induction of
apoptosis),197
disrupted membrane intergrity,191
and promo-
tion of apoptotic cell death.191,197
1 also promoted a higher
percentage of anaphase bridges, hallmarks of telomere
dysfunction, and increased the frequency of γ-H2AX and
53bp1 foci, known molecular markers that localize to sites of
DNA strand breaks and dysfunctional telomeres.200
In tumor
models, 1 prevented tumor formation and metastasis from
xenografts,194,195
significantly reduced growth and tumor
volume,191,200,201
and enhanced antitumor effects in combina-
tion therapy with doxorubicin, temozolomide, and irradia-
tion.191,200
The successful preclinical experiments demonstrated
the antitumor effectiveness of 1, and it remains the only
telomerase inhibitor to have been tested in clinical trials.
Lessons learned from clinical trials of 1 are essential for the
field, as this represents the first telomerase inhibitor to reach
the clinic. Currently, six phase I and phase I/II trials of 1 as a
single agent and three combination trials are ongoing. Major
objectives of these studies are safety, tolerability, and maximum
tolerated dose. Early reports indicate that 1 is well tolerated,
but nothing further has been reported.
RNAi. RNA interference or silencing RNA (RNAi or siRNA)
is double stranded RNAs that take advantage of a sequence-
specific and natural mechanism that mediates gene silenc-
ing.42,202
As an RNA that is complicit for telomerase activity,
hTER is an excellent target for siRNA therapies. Transfection
and intravenous injection of partially double-stranded PS-
ODNs targeting the template region of hTER reduced cell
proliferation in human cervical carcinoma and, more
importantly, strongly reduced the number of metastases in
murine malignant melanoma cells.203
siRNAs targeting
nucleotides 11−19 of hTER expressed in various cancer cell
lines including HCT116 colon cancer, LOX melanoma, HeLa
cervical cancer, T24 bladder cancer, MCF-7 and BT424 breast
cancers, and LNCaP prostate cancer, via lentiviral-vector
expression, caused rapid growth inhibition and apoptosis
while displaying negligible cellular cytotoxicity independent of
Figure 10. Clinical tested telomerase inhibitor. (A) 1 is a modified oligonucleotide that contains a 5′-palmotyl group. The sequence is
complementary to the hTER template domain. (B) The N3′→P5′-thiophosphoramidate linkage present in 1.
dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXXJ

telomere length.204
Cancer cells exhibited knockdown of
endogenous hTER as well as down-regulation of cell cycle
progression genes (cyclin G2 and Cdc27) and specific genes
that play pivotal roles in tumor growth, angiogenesis, and
metastasis (integrin αV and Met proto-oncogene), suggesting a
role for these genes in the hTER-mediated cell growth
inhibition.204
Expression of siRNAs targeting hTERT mRNA in various
cancers has caused decreased hTERT mRNA levels, reduced
hTERT expression, and notable telomerase inhibition within 24
h of dosing and lasting up to 4 days.120,176,205−209
Stable
transfection of these siRNAs led to complete suppression of
telomerase activity in 3 days concomitant with the absence of
hTERT mRNA expression.205,207
Cancers treated with siRNAs
that deplete hTERT experienced decreased cell viability,120
prolonged population doubling times,209
suppression of
anchorage-dependent proliferation,206
significant growth inhib-
ition,208
and morphological changes indicative of senescence.205
Esophageal and colon adenocarcinomas displayed extensive
telomere shortening,205,207
leading to chromosomes with
telomere free ends suggestive of complete telomere erosion.205
Genetic silencing of hTERT led to an up-regulation of genes
involved in DNA damage recognition and repair, p53 family
proteins and their regulators, cell cycle inhibitors, proapoptotic
proteins, and caspases.199,205,210
At the same time, hTERT
depletion significantly attenuated the expression of integrin αV
and β3 subunits and c-Met, proteins that are known to play
important roles in cell differentiation, adhesion, motility, and
tumor metastasis.206
In tumor models, hTERT siRNAs
disrupted cell adhesion, migration, and invasion, promoted
slower tumor growth,206
decreased cell viability and colony
formation, induced cell cycle arrest,199
reduced tumor sizes and
volumes in xenografts,206,208
and demonstrated additive effects
upon combination therapy with doxorubicin and intereferon-
γ.120,199
Interestingly, while expression of siRNAs targeting
both hTER and hTERT concomitantly did decrease mRNA,
RNA, and protein levels of telomerase components, cell
proliferation, and xenograft tumor growth and promote
pyknotic nuclear chromatin, the effect of silencing hTER
alone was more pronounced than silencing either hTERT or
both together.211
■ IMMUNOTHERAPY
The realization that hTERT is overexpressed in the vast
majority of cancers prompted the investigation of hTERT as a
tumor antigen. Soon after the discovery of widespread hTERT
expression in tumors, hTERT peptide fragments were identified
as tumor-associated antigens functioning through the major
histocompatibility complex class I and class II pathways. In
several cancer types, CD8+ cytotoxic T-cells specific for
hTERT peptides, primarily hTERT-I540 (ILAKFLHWL), are
detectable suggesting active hTERT-directed immune surveil-
lance for these cancers.212
Subsequently, over 20 hTERT-
derived peptides have been employed to boost antitumor
immunity.2
The majority of these elicit a CD8+ cytotoxic T-cell
response, while the others activate a CD4+ response. Several
clinical trials of hTERT-based cancer vaccines have demon-
strated the safety of this approach.213,214
Unfortunately, the
impact on disease progression reported to date is modest. Some
patients respond with significant tumor regression, but more
generally only a low percentage of patients respond and
responses tend to be transient. This is despite the fact that
vaccination with hTERT-derived peptides generally does result
in hTERT-specific T cell production, indicative of an
immunological response. Current efforts involve understanding
the differences between responsive and nonresponsive patients,
optimization of antigenic hTERT epitopes, and optimization of
specific immunotherapy platforms for telomerase-targeted
treatment strategies.
■ GENE DELIVERY
One of the main concerns with cancer gene therapy is the
selective expression of target genes in tumor cells while
avoiding expression in normal tissues. Because activation of
telomerase activity in cancer cells is largely driven by increased
expression of hTERT, it is reasonable to speculate that hTERT
promoter activity is commensurately increased. Taking
advantage of this increased promoter activity, several groups
have investigated the potential of the hTERT promoter for
cancer-selective gene therapy. The hTERT promoter is
extremely attractive for the selective expression of transgenes
for anticancer gene therapy because of the near universal
expression of hTERT in cancers and the much greater activity
of the promoter in cancer cells compared to normal cells, even
those that express telomerase. Most reports on the use of the
hTERT promoter for cancer gene therapy attempt to control
expression of anticancer genes in tumor cells. For example,
proapoptotic genes (FADD and TRAIL) and a suicide gene
(HSVtk) have been tested.215−217
In cell-based assays and in
mouse models, tumor-specific transgene expression has been
achieved and promising results in mouse studies highlight early
successes. Because these approaches utilize replication-deficient
virus in order to decrease harmful side effects, these approaches
may suffer from limited tumor-mass distribution. A different
and very promising approach that overcomes this limitation is
to utilize the hTERT promoter not for transgene expression
but instead viral replication, in essence establishing an
anticancer virus. One hTERT-promoter driven adenovirus,
telomelysin, has entered clinical trials in a small, 16 patient,
phase I trial in which the patients had a wide variety of cancer
diagnoses.35
Early data suggest that telomelysin was well
tolerated, but clinical response was limited, owing perhaps to
the single viral infection, which may have limited the viral titer.
Most promising was the response of one patient with a
significant, 56%, reduction in metastatic tumor size.35
■ PERSPECTIVE AND FUTURE DIRECTIONS
Much progress has been made toward translating telomere
research to the clinic since the seminal studies, demonstrating
the importance of telomeres, the identification of telomerase
and telomere-binding proteins, and the validation of telomere
length maintenance as a requirement of cancer cells. Early
clinical trials with telomerase inhibitors have shown that the
approach is well tolerated and does reduce telomerase activity.
However, the absence of reported clinical efficacy suggests
limited progress. Future trials examining which patients might
be best suited for telomerase inhibitor therapy and current trials
testing telomerase inhibitors in combination with other
traditional chemotherapeutics can be expected to advance the
field. We can also expect continued advances in gene therapy by
taking advantage of the robust activity of the hTERT promoter
in cancer cells as viral vectors are optimized. Likewise,
immunotherapy targeting hTERT antigens is expected to
continue its progress through clinical trials and arguably holds
dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXXK

the most promise currently for telomerase-targeting therapeu-
tics.
Surprisingly, small-molecule inhibitors of telomerase have
not fared well. This may change as advances in telomerase
structural biology; for example, the crystal structure of an insect
TERT,62
structures of several hTER domains,218,219
models of
hTERT based on the beetle crystal structure,220
and the recent
three-dimensional model of human telomerase based on
electron microscopy images221
increase our understanding of
telomerase structure and provide templates for rational small-
molecule drug design. Rationally designed telomerase inhibitors
may allow increased specificity, bioavailability, and inhibition
mechanisms that have greater effects in a clinical setting than
those small molecules that are currently available. In addition,
the observation that G-quadruplex ligands can directly disrupt
telomere structure evidently by blocking binding of telomere-
binding proteins such as TRF2 and POT1 suggests that there
are more telomere-associated targets to be exploited, and we
can expect that near future studies will determine the
effectiveness of directly targeting the telomere as an anticancer
drug platform. Targeting telomere biology as a means to
improve human health remains an important objective that
drives discoveries in telomere biology and telomerase
biochemistry. The interdisciplinary approaches toward targeting
telomere biology have led to three discrete areas of clinical
trials: inhibition of telomerase, hTERT-directed immunother-
apy, and hTERT-promoter gene/viral therapy. It seems that the
future of pharmaceutical benefit from telomere-targeting agents
remains promising, and our increased understanding of
telomere biochemistry and biology is sure to inform more
effective approaches toward developing telomere biology
targeted agents.
■ AUTHOR INFORMATION
Corresponding Author
*Phone: 919-966-6422. E-mail: jarstfer@unc.edu.
Notes
The authors declare no competing financial interest.
Biographies
Vijay Sekaran is a postdoctoral fellow at the University of Colorado at
Boulder working with Drs. James Goodrich and Jennifer Kugel. He
received his B.S. in Biomedical Engineering from the Georgia Institute
of Technology in 2005 and his Ph.D. in Pharmaceutical Sciences from
the University of North Carolina at Chapel Hill under the guidance of
Michael Jarstfer in 2012. His dissertation focused on understanding
the structure and function of telomerase RNAs as well as
characterizing the effects of small molecules that bind to human
telomerase RNA and inhibit telomerase activity.
Joana Soares is a research scientist at Genezyme. She received her B.S.
in Chemistry from The College of William and Mary, VA, in 2004 and
her Ph.D. in Pharmaceutical Sciences from the University of North
Carolina at Chapel Hill in 2010 under the supervision of Michael
Jarstfer. Her dissertation focused on elucidating the mechanism of
telomerase activation or repression and its impact on cellular pathways
and elucidating the mechanism of action of new potential telomerase
inhibitors. Her research interrogated the relationship between
telomerase, aging, and cancer progression.
Michael B. Jarstfer is an Associate Professor of Chemical Biology and
Medicinal Chemistry in the Eshelman School of Pharmacy at the
University of North Carolina at Chapel Hill. He received his B.A. in
Biochemistry from Trinity University, San Antonio TX, and his Ph.D.
from the University of Utah under the guidance of Dale Poulter. He
conducted postdoctoral research in the laboratory of Thomas Cech at
the University of Colorado in Boulder. His research interests include
structure and function of telomerase, development of telomere
targeting agents, the roles of telomere biology in human health, and
the molecular basis for social motivation.
■ ABBREVIATIONS USED
ALT, alternative lengthening of telomeres; AS, antisense;
hTERT, human telomerase reverse transcriptase; HDAC,
histone deacetylase; hTER, human telomerase RNA; NPS,
N3′→P5′-thiophosphoramidates; ODN, oligonucleotide; PKC,
protein kinase C; PNA, peptide nucleic acid; POT1, Protection
of Telomeres 1; PS, phosphorothioate; QSAR, quantitative
structure−activity relationship; RNAi, RNA interference; SAR,
structure−activity relationship; siRNA, silencing RNA; TIF,
telomere dysfunction induced focus; TAK1, TGF-β activated
kinase 1; TAL1, T-cell acute lymphoblastic leukemia protein 1;
WT1, Wilm’s tumor protein 1
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