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STUIDENT NAME : LUBNA .M KHARABSHEH

LUBNA M. KHARABSHEH

Drug receptor interactionsiteitemitemitemitemitemitem

filterKey word:Free full textDate (5y)Article type:reviewSpecieshumanLanguage:englishPubMed39058138583968263240230Pubchem zerozerozerozerozerozerofilter Drug receptor interactionLess than1.5ANO MORENO MORESpecieshumanNO MOREBDB172---------------------13--------- FILTERKey word:

YEAR2015PUP-title:Bioorganic-and medicContent:journalAccess typeopenNo moreSCIENCE DIRECT395,074395,090395,094395,0845720--------Google scholar 400050400000300072--------------------

Biochemistry.2014 Jun 17;53(23):3790-5Thermodynamics 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

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 -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor is, in some cases, driven by increases in entropy., 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. 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. 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 targetssiteitemitemitemitemitemitemPubMed 4905840005350300290290filterKey wordFree full textDate (5y)Article type:reviewSpecieshumanLanguage:englishPubchem zerozerozerozerozerozerofilter Key wordYEAR2015PUP-title:Bioorganic-and medicContent:journalAccess typeopenlanguageScience direct62,26762,26562,26762,2001,0601000BDBZEROZEROZEROZEROZEROZERO

Authors: Bipasha Mukherjee3, Nozomi Tomimatsu3, Kaushik Amancherla3, Cristel V. Camacho, Nandini Pichamoorthy and Sandeep BurmaDepartment of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA NEOPLASIA Volume 14 Number 1 January 2012 pp. 3443The 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 m) 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 involving this drug and for the potential use of this drug as a powerful radio sensitizer in the clinic.

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 M059JIrradiation 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 indicated drugs

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 AssaysMouse Tumor Studies Statistical Analyses

Results

Results

. We found that NVP-BEZ235 could potently block NHEJ (no RFP signal after transfection with an I-SceIexpressing plasmid) (Figure 4C).These results clearly indicate that NVP-BEZ235 potentlyblocks 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 canindependently 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

ResultsNVP-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-BEZ235treated 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 tumoraldelivery 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-BEZ235treated 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 NVP-BEZ235 is also valid in a tumor setting.

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 targetssiteitemitemitemitemitemitem filterKey word :Free full textDate (5y)Article type:reviewSpecieshumanLanguage:EnglishPubMed 4005133129119117116Pubchem zerozerozerozerozerozeroBDBzerozerozerozerozerozerofilterKey wordYEAR2015PUP-title:Bioorganic-and medicContent: journalAccess typeopenlanguageenglishScience direct50684998300028891515filterKey wordEnglish----Google scholar 1095610940

NATURE REVIEWS | DRUG DISCOVERY VOLUME 10 | AUGUST 2011 | 601 -620

Transient receptor potential channels as therapeutic targets

Magdalene M.Moran, Michael Allen Alexander, Tams 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: the skin, Bladder 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: the renal, skeletal and nervous system. This Review focuses on recent developments in the TRP channel-related field, and highlights potential opportunities for therapeutic intervention

IntroductionTransient 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: 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 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 mutations in many of the genes that encode TRP channels are sufficient to cause disease inhumans.

Cont. introductionTRP channels are associated with several pathophysiological processes, which include (but are not limited to) pain, Respiratory reflex hypersensitivity, cardiac hypertrophy and ischemic cell deathseveral 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 therapeuticbenefit. , 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 PhaseII 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)

34

TRP channels in bladder disordersSeveral 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 of patients with prurigo nodularis

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 experimental itch, as well as pruritus in patients with seasonal allergic rhinitis. By contrast, histamine-independent itch caused by chloroquine or the endogenous pruritogen peptide BAM8-22 (originally isolated from bovine adrenal medulla) is predominantly mediated by TRPA1

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 invivo. TRPV1 has also been suggested to regulate cutaneous inflammation. Capsaicin-induced activation of TRPV1 on human epidermal and hair follicle-derived keratinocytes invitro 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: air pollutants that are produced by the combustion of materials (including tobacco products) that cause pronounced cutaneous, ocular and respiratory irritation in humans. 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 guineapigs. 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, which would both improve bladder function and reduce the risk ofcancer.

Metabolic disordersMetabolic 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 -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.

ConclusionsThe 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-based toxicological liabilities of TRP channel modifiers

Inducers of cellular senescenceCell proliferation(short telomeres)DNA damageOncogenesStrong 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 different p53 status. Therefore, FL118 may effectively control cancer that loses functional p53

IntroductionEradication 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, and a significant percentage of somatic mutations may not be detected across every tumor region

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 agents to target multiple different resistant mechanisms

Cont. introduction this app- roach 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 treatment.

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 M 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), FL118s 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 FL118s 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 targets of FL118.

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 FL118s 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 FL118s 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 to the current cancer treatment resistance.

Good people wont ask :p