target identefication

Drug receptor interaction
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Biochemistry. 2014 Jun 17;53(23):3790-5
Thermodynamics and mechanism of the interaction of
willardiine partial agonists with a glutamate receptor:
implications for drug development
AUTHORS: Martinez M1, Ahmed AH, Loh
AP, Oswald RE.

Abstract
1. Understanding the thermodynamics of binding of a lead
compound to a receptor can provide valuable information for
drug design. The binding of compounds, particularly partial
agonists, to subtypes of the α-amino-3-hydroxy-5-methyl-4-
isoxazole-propionic acid (AMPA) receptor is, in some cases,
driven by increases in entropy.,
2. Using a series of partial agonists based on the structure of the
natural product willardiine, we show that the charged state of
the ligand determines the enthalpy contribution to binding.
3. Willardiines have uracil rings with pKa values ranging from 5.5
to 10. The binding of the charged form is largely driven by
enthalpy, while that of the uncharged form is largely driven by
entropy. This is due at least in part to changes in the hydrogen
bonding network within the binding site involving one water
molecule.
4. This work illustrates the importance of charge to the
thermodynamics of binding of agonists and antagonists to
AMPA receptors and provides clues for further drug discovery

DNA as drug targets
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 Authors: Bipasha Mukherjee3, Nozomi Tomimatsu3, Kaushik
Amancherla3, Cristel V. Camacho, Nandini Pichamoorthy and
Sandeep Burma
 Department of Radiation Oncology, University of Texas
Southwestern Medical Center, Dallas, TX, USA
 NEOPLASIA Volume 14 Number 1 January 2012
pp. 34–43
The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Is a Potent
Inhibitor of ATM- and DNA-PKCs-Mediated DNA Damage
Responses1,2

Abstract
 Inhibitors of PI3K/Akt signaling are being actively developed for tumor
therapy owing to the frequent mutational activation of the PI3K-Akt-
mTORC1path way in many cancers , including glioblastomas(GBMs).NVP-
BEZ235 is anovel and potent dual PI3K/mTOR inhibitor that is currently in
phase 1/2 clinical trials for advanced solid tumors.
 Here, we show that NVP-BEZ235 also potently inhibits ATM and DNA-PKcs, the
two major kinases responding to ionizing radiation (IR)–induced DNA double-
strand breaks (DSBs).
 Consequently, NVP-BEZ235 blocks both non homologous end joining and
homologous recombination DNA repair pathways resulting in significant
attenuation of DSB repair.
 In addition , phosphorylation of ATM targets and implementation of
theG2/M cell cycle check points are also attenuated by this drug.
 As a result, NVP-BEZ235 confers an extreme degree of radio sensitization
and impairs DSB repair in a panel of GBM cell lines irrespective of their Akt
activation status.NVP-BEZ235 also significantly impairs DSB repair in a
mouse tumor model thereby validating the efficacy of this drug as a DNA
repair inhibitor invivo.Our results , showing that NVP-BEZ235 is a potent
and novel inhibitor of ATM and DNA-PKcs , have important implications for
the informed and rational design of clinical trials involving this drug and
also reveal the potential utility of NVP-BEZ235 as an effective radio
sensitizer for GBMs in the clinic.

introduction
The phosphatidylinositol 3-kinase (PI3K)–Akt-mTORC1 pathway is frequently
activated in a variety of human cancers, including glioblastomas (GBMs). Therefore,
inhibitors of PI3K/Akt signaling are being actively developed for tumor therapy
(reviewed in Liu et al. [1] and Garcia-Echeverria and Sellers [2]). Dual PI3K-mTOR
inhibitors are particularly effective in blocking Akt activation because they prevent
the feedback activation of PI3K signaling normally observed with mTORC1
inhibitors, such as Rapamycin. NVP-BEZ235 is a potent dual PI3K-mTOR inhibitor
[3] that has shown great efficacy in inhibiting tumor growth in preclinical mouse
models Because radiotherapy plays a key role in the treatment of GBM, we
investigated the potential utility of NVP-BEZ235 as a radio sensitizing agent against
human GBM lines. Surprisingly, we found that very low concentrations of this drug
conferred a high degree of radio sensitization that was significantly greater than that
previously reported with PI3K/Akt inhibition , and this correlated with attenuation of
DSB repair. Detailed experimentation revealed that NVP-BEZ235 potently inhibits
ATM and DNA- PKcs, thereby blocking both non homologous end joining (NHEJ)
and homologous recombination (HR), the two major pathway s of DSB repair . In
addition , phosphorylation of ATM targets and implementation of the G2/M cell cycle
checkpoint are also attenuated by this drug. The consequence is profound radio
sensitization at very low concentrations of NVP-BEZ235 (100nM). This radio
sensitizing effect is significantly more potent than that seen with much higher
concentrations (10 μm) of current inhibitors of DNA-PKcs [20] or ATM [21] that are
being optimized for clinical testing (reviewed in Ding et al. [22]). These results have
significant translational importance, both for the design of current clinical trials

Materials and methods
 Cell Culture and Drug Treatment:
The cell lines used in this study are U251, U118,
LN18, T98G, LN229, SF188, 1BR3, AT5, M059K,
and M059J
 Irradiation of Cells:
 Cells were irradiated with gamma rays from a
137Cs source
• Western Analyses and Immunofluorescence
Staining
• Colony Formation Assays:
Cells were plated in triplicate onto 60-mm
dishes(1000 cells per dish), treated with the

Cont.. method
 DSB Repair Assays:
 DSB repair rates were assessed by quantifying the
rates of dissolution of 53BP1 foci after irradiation of
cells with 1 Gy of gamma rays
• HR and NHEJ Assays:
 In the HR assay , GFP expression was quantified (by
flow-- cytometer) in MCF7-DRGFP cells transfected
with an I-SceI plasmid
• G2/M Checkpoint Assay
• DNA-PKcs Kinase Assays
• Mouse Tumor Studies
• Statistical Analyses

 . We found that NVP-BEZ235 could potently block NHEJ (no RFP signal after
transfection with an I-SceI–expressing plasmid) (Figure 4C).These results clearly
indicate that NVP-BEZ235 potently
 blocks both ATM and DNA-PKcs, resulting in a DSB repair defect that is more
striking than that seen on the inhibition of ATM or DNA-PKcs alone. Indeed,
blocking both ATM and DNA-PKcs by combining KU55933 and NU7026 resulted
in greater numbers of unrepaired DSBs, similar to that seen with NVP-BEZ235
alone, both in 1BR3 cells (Figure 4D) as well as in the panel of GBM cell lines
(Figure W2). Given the potential cross-talk between ATM and DNA-PKcs [36], we
investigated whether NVP-BEZ235 could attenuate IR-induced ATM activation in
DNA-PKcs-null M059J cells and DNA-PKcs activation inATM-null AT5
cells.Wefound inhibition of kinase activation in both cell lines, demonstrating that
this drug can
 independently block either kinase (Figure W3). We also examined ATM and
DNA-PKcs activation in the panel of glioma lines that were radiosensitized by
NVP-BEZ235 (Figure 1B) and observed inhibition of both ATM (Figure W4) and
DNA-PKcs (Figure W5) to varying extents. Taken together, these data implicate
the impairment of both HR and NHEJ repair pathways, due to inhibition of both
ATM and DNA-PKcs, as the underlying mechanism behind the profound
radiosensitization conferred by NVP-BEZ235. Finally, to examine the effect of
NVP-BEZ235 on DSB repair in tumors, we generated subcutaneous tumors in
Nu/Nu mice using U87 cells overexpressing EGFRvIII

Results
 NVP-BEZ235 could inhibit Akt activation and block DSB repair in
U87-EGFRvIII cells in culture (Figure W6). Next, tumor-bearing
mice were treated with a single dose of 45 mg/kg NVP-BEZ235
or with vehicle as control. Tumors were mock irradiated or
irradiated (2 Gy of x-rays) 2 hours later, collected at 0.5 and 24
hours after IR, and sectioned for IF. Tumors from NVP-BEZ235–
treated mice exhibited a marked reduction in the phosphorylation
of Akt (Ser473) and abrogation of phosphorylation of an mTOR
substrate, the ribosomal protein S6 (Ser235/236), thereby
confirming intra tumoral
 delivery of the drug and consequent inhibition of the PI3K-
AktmTOR pathway [7] (Figure 5A). Irradiated NVP-BEZ235– or
vehicle treated tumor sections were IF stained for 53BP1 foci as
described [19]. Vehicle-treated tumors were able to completely
repair radiation-induced DSBs by 24 hours after IR. Interestingly,
NVP-BEZ235–treated tumors exhibited higher levels of
unresolved 53BP1 foci at 24 hours after IR, indicating
attenuation of DSB repair(Figure5,BandC). These results unequi
vocally demonstrate that the striking inhibition of DSB repair by

Conclusions
 Using multiple approaches ,we find that NVP-
BEZ235, a drug already in clinical trials, can
inhibit IR-induced activation of ATM and DNA
PKcs, the two major kinases responding to DSBs.
This results in inhibition of DSB repair,
attenuation of cell cycle arrest, and profound
radio sensitization. In the original report
describing the compound, the authors examined
the effects of NVP-BEZ235 only on doxorubicin
induced phosphorylation of ATM(Ser1981)and
DNA-PKcs (Thr2609) and found attenuation only
at high concentrations of the drug

Ion channels as drug targets
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- - - -

 NATURE REVIEWS | DRUG DISCOVERY
VOLUME 10 | AUGUST 2011 | 601 -620
 Transient receptor potential channels as
therapeutic targets
 Magdalene M. Moran, Michael Allen Alexander,
Tamás Biro and Arpad Szallasi

Abstract
 Transient receptor potential (TRP) cation channels have been among
the most aggressively pursued drug targets over the past few years.
 Although the initial focus of research was on TRP channels that are
expressed by nociceptors, there has been an upsurge in the amount of
research that implicates TRP channels in other areas of physiology and
pathophysiology, including:
1. the skin,
2. Bladder
3. and pulmonary systems.
In addition, mutations in genes encoding TRP channels are the cause of
several inherited diseases that affect a variety of systems including:
1. the renal,
2. skeletal
3. and nervous system.
This Review focuses on recent developments in the TRP channel-related
field, and highlights potential opportunities for therapeutic intervention

Introduction
 Transient receptor potential (TRP) channels are being
ardently pursued as targets for drug discovery. There
are several factors that make TRP cation channels
appealing as drug targets:
I. particularly with voltage-gated sodium and calcium
channels. As members of the TRP family of
channels do not share much homology with one
another, the identification of subtype-selective
compounds is likely to be more attainable
II. TRP channels act as integrators of several well-
described signaling systems, including those that
are mediated by cell surface receptors (for
example, G protein-coupled receptors (GPCRs)
and growth factor receptors
III. mutations in many of the genes that encode TRP
channels are sufficient to cause disease in humans.

Cont.… introduction
 TRP channels are associated with several pathophysiological
processes, which include (but are not limited to)
I. pain,
II. Respiratory
III. reflex hypersensitivity,
IV. cardiac hypertrophy
V. and ischemic cell death
several gene association studies in humans have indicated that single-
nucleotide polymorphisms (SNPs) in the coding regions and/or promoters
of genes that encode TRP channels are either associated with an
increased risk of multifactorial diseases or they appear to be causative
factors in rare heritable conditions. Interestingly, when these mutated TRP
channels are expressed in recombinant systems, they generally display
enhanced activity, which suggests that blockade of these channels may
provide therapeutic benefit. ,
target validation of TRP channels has largely been generated via genetic
studies; by comparison, the identification of chemical modulators of TRP
channels is in its infancy. Several natural ligands (for example, capsaicin
and menthol) have provided valuable insights into the pharmacology of
TRP channels

TRP channels as analgesic
targets
 The role of TRP channels is best understood in the
pain area (FIG. 2). TRPV1 and TRPV3 antagonists
have already advanced to clinical trials whereas
TRPA1 antagonists are still in preclinical
development.
 TRPV1. As the desensitization of nociceptive neurons
to capsaicin has analgesic potential5, the cloning of
the capsaicin receptor, TRPV1 (REF. 9), has spurred
considerable efforts in the pharmaceutical community
to find TRPV1 antagonists. However, side effects
associated with the use of TRPV1 antagonists have
so far prevented any compounds from progressing
beyond testing in Phase II trials. Particular concerns
have surfaced around the effects of TRPV1
antagonism on the regulation of body temperature10
and in the detection of noxious heat (S. Eid, personal
communication).

TRPV1 antagonists and noxious heat perception in
humans.
 Clinical studies have confirmed the role of TRPV1 as a
noxious heat sensor in humans. Indeed, the threshold for
detecting painful heat was considerably elevated in non-
sensitized skin of healthy volunteers following oral
administration of 400 mg of SB-705498 per day
(Supplementary information with subsequent studies
reporting blunted heat perception in healthy human
subjects, which was not desensitized after repeated
dosing. This effect could potentially cause scalding injuries
during common activities such as taking a hot shower or
consuming hot food or beverages. Indeed, some subjects
taking MK-2295 perceived potentially harmful
temperatures as innocuous. In randomized clinical trials,
similar findings were reported using ABT-102 (which was
administered at a dose of up to 4 mg twice a day) and
AZD1386 (which was administered at a single daily dose
of 95 mg). Notably, there were no other relevant safety
findings in these two trials and the investigators felt that
AZD1386 may have clinical potential in relieving pain
associated with gastro_ esophageal reflux disease

 in the clinic. Topical TRPV1 agonists (for
example, capsaicin creams) have been used
clinically for many years to alleviate chronic
painful conditions such as diabetic neuropathy. An
occlusive high-concentration capsaicin patch
 (Qutenza ; Neuroges X) was recently approved
for the treatment of various pain conditions.
Injections of resiniferatoxin , an ultrapotent
capsaicin analogue , are being evaluated as a so-
called ‘molecular scalpel’ to achieve long-term
analgesia in patients with cancer who have
chronic, intractable pain
TRPV1 agonists (capsaicin and
resiniferatoxin)

TRP channels in bladder
disorders
 Several TRP channels are expressed in the
bladder — in the urothelium, nerve endings and
detrusor muscle where they are thought to
function as sensors of stretch and chemical
irritation Intravesical administration of TRPV1
agonists has been used in the management of
the overactive bladder for many years, largely on
an empirical basis The recent recognition of
disease state-related changes in the expression
of TRP channels has provided a new impetus to
investigate the roles of these channels in normal
bladder function and dysfunction

TRP channels in the skin
 Populations of non-neuronal cells within the skin
express many different types of TRP channels
which are thought to be involved in various key
cutaneous functions including skin-derived
pruritus, proliferation, differentiation, cancer and
inflammatory processes TRPV1 as a key
molecule in itch. TRPV1 is involved in the
development of skin-derived pruritus, which is
thought to occur through itch-specific
subpopulations of TRPV1-expressing sensory
afferent neurons TRPV1 is also expressed in non-
neuronal cell types of human skin74, and its
expression is elevated in epidermal keratinocytes

TRP channels in the skin
 Certain endogenous signaling molecules that potentiate TRPV1 activity
(including acids, ATP, lipoxygenase products, prostaglandins and
histamine) are also potent pruritogens. It is probable that on sensory
neurons, histamine indirectly activates TRPV1 through histamine H1
receptor-dependent
 Alopecia A type of pathological hair loss that mostly affects the scalp.
The most common forms of alopecia are alopecia universalis, alopecia
areata and alopecia androgenetica. Telogen Effluvium, which is
characterized by diffuse hair shedding, is a form of alopecia.
 Hirsutism Excessive and increased hair growth (especially in women)
on regions of the body where the occurrence of hair normally is minimal
or absent.
 Dermatitis A universal term describing inflammation of the skin. It can be
induced by various factors such as allergens (allergic dermatitis),
infections, eczema (atopic dermatitis) or external compounds (contact
dermatitis).
 synthesis of 12-hydroperoxyeicosatetraenoic acid, which is an
endogenous activator of TRPV1. Consistent with this finding, genetic
deletion of Trpv1 in mice substantially suppressed histamine induced
scratching behavior. In humans, TRPV1 mediates histamine-induced

Role of TRPV1 in the control of skin growth,
skin cell survival and cutaneous inflammation
 It has been suggested that TRPV1 participates in the
regulation of cutaneous growth and differentiation.
TRPV1-mediated calcium influx in cultured human
keratinocytes suppresses proliferation and promotes
apoptosis. In addition, activation of TRPV1 by either
capsaicin or heat alters the formation of the epidermal
permeability barrier in human skin in vivo. TRPV1 has
also been suggested to regulate cutaneous
inflammation. Capsaicin-induced activation of TRPV1
on human epidermal and hair follicle-derived
keratinocytes in vitro results in the release of several
pro-inflammatory cytokines. In addition, as ultraviolet
irradiation up regulates TRPV1 expression in human
skin, TRPV1 that is expressed on keratinocytes is a
specific mediator of heat shock-induced and
ultraviolet irradiation induced expression of matrix
metalloproteinase 1 an enzyme that is implicated in
skin inflammation and remodeling.

TRP channels in the pulmonary
system
 The mammalian respiratory tract is lined with a dense plexus of sensory
fibers, including those that express TRPA1 and TRPV1
 TRPA1. Many of the irritants that activate TRPA1 are:
I. air pollutants that are produced by the combustion of materials
(including tobacco products) that cause pronounced cutaneous,
ocular and respiratory irritation in humans.
II. Several classes of anesthetic molecules — including lidocaine,
propofol, etomidate and volatile gaseous anesthetics — also act as
TRPA1 agonists96. Although these data raise the possibility that
anesthesia may paradoxically increase postoperative pain, the more
immediate impact of these data is the identification of TRPA1 as a
possible mediator of the respiratory complications of gaseous
anesthetics, which can include coughing and laryngospasms. In
support of this hypothesis, the TRPA1 blocker HC-030031 has been
shown to prevent desflurane-induced increases in airway resistance
in guinea pigs.
III. Additional hazardous irritants — which include isocyanates, ozone,
chlorine and cigarette smoke extracts — activate overexpressed
TRPA1 and cause pulmonary nociceptor activation, respiratory
irritation and/ or neurogenic inflammation in a TRPA1-dependent

 Several TRP channels have been linked to cancer,
some as markers of biological behavior (such as
aggressive versus indolent phenotypes), whereas
others could be putative therapeutic targets (reviewed
in REF. 146). TRPM8 is a prime example of a marker
that is also a target. TRPM8 is overexpressed in
prostate cancer, and its level of expression correlates
with tumor severity147. At the same time, the TRPM8
agonist menthol reduces the proliferation and viability
of prostate cancer cell lines148. Notably, the synthetic
TRPM8 agonist D-3263, which reduces benign
prostatic hyperplasia in rats, is in clinical trials
(ClinicalTrials.gov identifier: NCT00839631). The
structure of this compound has not yet been
published. As patients with benign prostatic
hyperplasia often have prostate cancer, they could
conceivably benefit from TRPM8 agonist treatment,

Metabolic disorders
 Metabolic disorders. Genetic deletion of TRPM5, a
known taste sensor, results in impaired glucose
tolerance in mice. This phenotype may be due to the
loss of high-frequency calcium oscillations in
pancreatic β-cells, although the effects of TRPM5
deletion cannot be accounted for by simple changes
in membrane potential. In animal models, inactivation
of TRPV1 by genetic or pharmacological manipulation
has been shown to protect against the development
of type 1 diabetes and improve glucose tolerance in
type 2 diabetes In Trpv1–/– mice, both increased and
decreased body fat was reported, therefore the role of
TRPV1 in the regulation of body weight remains
controversial.

Conclusions
 The recent expansion of research into TRP channels
has resulted in the identification of numerous
potential drug targets beyond TRPV1, and has
elucidated roles for TRP channels in diverse
therapeutic areas including pain, pulmonary
indications, oncology, neurology and genetic
disorders. Interest is mounting as a result of emerging
data from animal models, human genetic disorders
and, in some cases, compounds entering clinical
trials. Indeed, at this early stage, with very limited
clinical data available regarding the effects of small-
molecule blockade of a single TRP channel (TRPV1),
it is deceptively easy to speculate on the therapeutic
potential — or the potential to cause mechanism-

Inducers of cellular senescence
Cell proliferation
(short telomeres)
DNA damage
Oncogenes
Strong mitogens/
stress
Potential Cancer Causing Events

 Am J Cancer Res
2014;4(3):304-311
 Anticancer drug FL118 is more than a survivin
inhibitor: where is the Achilles’ heel of cancer?
Author : Fengzhi Li

Abstract
 Can a solution be found that overcomes all chemotherapy and/or
radiation resistance resulting from different genetic and epigenetic
alternations in various cancer types? The answer is likely NO. However,
there are two ways that may be followed to approach this goal. One way
is through the use of poly-therapies that target multiple mechanisms to
kill cancer cells, which is the current state of the art. This approach
raises issues of high costs and/ or toxic limitations, since the toxicities of
each agent are often additive. This poly-pharmacy approach has not
proven to be a major success, although it has proven to be superior to
most current mono-pharmacy approaches. The other way to approach
the goal is to find a single anticancer drug that targets multiple different
treatment resistant mechanisms. In this regard, a small chemical
molecule (FL118) was recently discovered by serendipity during
targeted discovery of anticancer drugs using the survivin gene as a
target and biomarker. FL118 was found to not only inhibit multiple
antiapoptotic proteins (survivin, XIAP, cIAP2) in the inhibitor of apoptosis
(IAP) family, but to also inhibit the antiapoptotic protein Mcl-1 in the Bcl-
2 family, while inducing the pro-apoptotic proteins Bax and Bim
expression. Importantly, inhibition of these target genes and of tumor
growth by FL118 is independent of p53 status (wild type, mutant or null),
although mechanisms of action may be distinct among cells with

Introduction
 Eradication of cancer is an ultimate mission in the cancer
research field and clinical practice. One unsolved challenge for
realizing the mission is cancer treatment (chemotherapy,
radiation) resistance, which is a major cause of a high rate of
cancer recurrence after treatment. Treatment resistance and
cancer recurrence are responsible for the majority (if not all) of
cancer patient deaths. Such resistance therefore continues to
challenge the entire field. The question is where is the Achilles’
heel of cancer and can we overcome these challenges.
 Accumulated knowledge from cancer research and clinical trials
reveals that cancer treatment resistance results from multiple
different mechanisms, and the resistance to traditional cytotoxic
drugs and molecularly targeted agents shares similar
characteristics including genetic and/or epigenetic alternations,
induced and/or constitutive activation of pro-survival pathways to
evade cell death, and increased drug efflux via ATP-binding
cassette (ABC) transporters, to name some of the more
commonly encountered mechanisms of resistance [1]. Cancer is
a highly heterogeneous disease [2]; new studies indicate that
gene-expression signatures of favorable versus unfavorable
prognosis can be detected in different regions of the same tumor,

Cont.. introduction
 It is clear that such extensive intratumor
heterogeneity presents a new challenge for the
current concept of personalized cancer treatment
(personalized medicine) and biomarker development.
Since the new findings provide a rich seeding soil for
positive selection of resistant cancer cells during
treatment with current medicines, the current
medicine and approaches would not well resolve the
issue of cancer treatment resistance. New
approaches are needed. To face up to the continuing
challenge in treatment resistance, we must consider
the fact that treatment resistance results from diverse
molecular mechanisms. Based on the nature of
various anticancer agents that are currently available
for cancer treatment, we can use a defined treatment
regimen that contains multiple molecularly targeted

Cont.… introduction
 this approach will be too costly for cancer patients
or insurance coverage. So clinically, it is rare to
employ this approach for cancer treatment.
Alternatively, we can use a defined treatment regimen
that applies multiple traditional cytotoxic agents. This
approach would maintain affordable costs for patients,
while enjoying maximal control of cancer with
traditional cytotoxic drugs. The challenge of this
approach is the high toxicity to patients and thus
limited its application. To balance the above two
approaches, the trend in the current clinical practice is
to use one molecularly targeted agent plus one or two
traditional cytotoxic drugs asa combination regimen to
balance the issue of toxicity, efficacy and cost.
However, this approach is also unable to avoid
eventual escapes by the treated cancer in many
situations, as resistance usually develops during

Is FL118 a topoisomerase 1 (Top1) inhibitor?
 This question is raised at the beginning, because FL118 structurally has
similarity with irinotecan, SN-38 (active metabolite of irinotecan), and
topotecan, which are classified as camptothecin (CPT) derivatives
(Figure 1). It is known that the CPT analogs, irinotecan, SN-38 and
topotecan are Top1 inhibitors. We demonstrated that the antitumor
efficacy of FL118 is much superior to the antitumor efficacy of irinotecan
in animal model of both human colon and head-&-neck tumors [5].
Therefore, it is possible that FL118 may be a better Top1 inhibitor than
irinotecan. Irinotecan is a pro-drug and shows very low activity in the in
vitro experiment, we therefore used its active metabolite SN-38 to
compare their relative ability to inhibit Top1 activity for an answer. Our
studies indicated that even at a 1 µM level, which is the highest SN-38
dose that can be reached by irinotecan in vivo, FL118 shows poor ability
to inhibit Top1 activity (at most, half of those that SN-38 shows) [5].
However, FL118 can effectively inhibit cancer cell growth at far below a
nM level [5]. These observations suggest that inhibition of Top1 activity
by FL118 unlikely plays a major role in FL118-mediated inhibition of
cancer cell growth and induction of tumor regression.
 these observations indicate that although FL118 structurally has
similarity to topotecan, SN-38 and CTP (Figure 1), FL118’s
anticancer activity is unlikely through the inhibition of Top1
activity as its major mechanism of action. FL118 should have its
unique mechanisms of action that are different from the Top1
inhibitors, irinotecan, SN-38 and topotecan

What is the selectivity of FL118 to inhibit IAP and Bcl-2
family antiapoptotic proteins?
 Specifically, FL118 at a concentration of 1-10 nM can
effectively inhibit survivin promoter activity, while
FL118 at 10 nM shows no inhibitory effects on
promoter activity of the cell cycle regulator p21 gene,
the dihydrofolate reductase (DHFR) gene, the human
thrombin receptor (HTR) gene and the thymidine
kinase (TK) gene [5], indicating high selectivity
compared to those non-cancer related genes.
However, in addition to survivin, FL118 selectively
inhibits the expression of XIAP and cIAP2 (IAP
family), and Mcl-1 (Bcl-2 family), while inducing the
expression of prapoptotic proteins Bax and Bim in
various cancer cell types [5]. The inhibition of survivin,
Mcl1, XIAP, and cIAP2 by FL118 can be partially
explained by the similarity of the promoter region of
the survivin, Mcl-1, XIAP, and cIAP2 genes for the
transcription factor (TF) binding, which are distinct
from the promoter region of p21 and DHFR genes

Does FL118-mediated inhibition of survivin, Mcl-
1, XIAP, and cIAP2 play a role in FL118
efficacy?
 This is an important question. Without demonstration
of a role of these genes in FL118 function, we cannot
consider these genes as the downstream targets of
FL118. Our studies showed that genetic knockdown
of survivin increases FL118-mediated inhibition of
cancer cell growth and induction of apoptosis
(Annexin V positive cells) [5]; in contrast, Tet-on
induced survivin expression decreases FL118’s ability
to inhibit cancer cell growth and induce DNA
fragmentation (a hallmark of apoptosis) [7]. Similarly,
genetic knockdown of Mcl-1 increases the cleavage of
PARP, another hallmark of apoptosis .these studies
implicate the four FL118 downstream targets
(survivin, Mcl-1, XIAP, cIAP2) as downstream

What is the effect of p53 status on FL118 mediated
inhibition of its downstream targets and tumor growth?
 p53 is a pivotal tumor suppressor that can be
activated by various stress signals, such as DNA
damage. Activated p53 participates many important
cellular processes, including arrest of cell cycle and
induction of apoptosis or senescence. This is mainly
through control of p53 downstream target genes in
the p53 transcriptional networks [8]. Therefore, cancer
cells with wild type p53 is essential for efficacy of
many anticancer drugs that work through eventual
induction of apoptosis and/or senescence, especially
for those that interfere DNA synthesis, repair and cell
cycle. In other words, loss of functional p53 (p53
mutated or null) would make cancer cells acquire
treatment resistance to many chemotherapeutic drugs
Interestingly, our recent studies indicate that
cancer cells with null p53 are even more sensitive
to FL118 treatment than cancer cells with wild
type p53

What is the toxicology profile of FL118 in animal
models?
 This is another critical issue that needs to be
addressed before FL118 is moved into clinical trials.
While a complete profile of FL118 toxicology data is
under investigation, there is a basis for FL118 to have
a favorable toxicology profile. Several aspects support
this notion. Firstly, FL118 selectively inhibits cancer-
associated antiapoptotic proteins (survivin, Mcl-1,
XIAP, cIAP2). These proteins are well known to be
good therapeutic targets to avoid toxicity to normal
tissues, since these proteins, especially survivin, are
expressed at a very low or undetectable level in
normal tissue.
 Secondly, cancer cells usually require the
overexpression of these proteins for survival;
interference of two or more of these proteins would
effectively disrupt the survival balance and inhibit
tumor cell growth and induce apoptosis.

Is FL118’s core structure a good platform for generation
of safe and efficacious FL118 derivatives?
 The exceptional antitumor efficacy of FL118 triggers our
enthusiasm to explore the possibility that the core structure
of FL118 may represent a promising platform for the
generation of novel FL118 analogs. The FL118-derived
analogs may exhibit differential selectivity preferences for
cancers with different genetic and/or epigenetic
alternations. In this regard, we have demonstrated that the
exceptional anti-cancer activity of FL118 is highly
dependent on its primary structure and steric configuration.
In contrast to previous studies on prototype camptothecin
compounds, we found that maintenance of a free hydroxyl
group in the lactone ring of FL118 is critical for FL118
maintenance of its exceptional antitumor efficacy. Thus, we
confirmed FL118’s high potential for further development
toward clinical trials; meanwhile, the studies provided
the first evidence pointing to a possibility that FL118 is
a promising platform for the generation of novel FL118
analogs.

Concluding remarks
 the author employs FL118 as an example to
demonstrate the feasibility of developing a single
molecule that can target and/or bypass multiple
treatment resistant mechanisms. The author
propose that a versatile anticancer molecule can
better resolve the newly discovered challenging
issue of cancer treatment resistance for
personalized medicine and biomarker
development [2, 3], while keeping the treatment
at a relative low toxicity, low cost, and high
efficacy. FL118 and/or its core structure-derived
analogs are expected to make great contributions

target identefication

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