Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents against MYC-Driven Pediatric Cancer.

Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. SIGNIFICANCE: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

Targeting ALK in pediatric RMS does not induce antitumor activity in vivo.

PURPOSE: The anaplastic lymphoma kinase (ALK) has been demonstrated to be a valid clinical target in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. Recent studies have indicated that ALK is overexpressed in pediatric rhabdomyosarcoma (RMS) and hence we hypothesized that this kinase may be a suitable candidate for therapeutic intervention in this tumor. METHODS: We evaluated the expression of ALK in a panel of pediatric RMS cell lines and patient-derived xenografts (PDX), and sensitivity to ALK inhibitors was assessed both in vitro and in vivo. RESULTS: Essentially, all RMS lines were sensitive to crizotinib, NVP-TAE684 or LDK-378 in vitro, and molecular analyses demonstrated inhibition of RMS cell proliferation following siRNA-mediated reduction of ALK expression. However, in vivo PDX studies using ALK kinase inhibitors demonstrated no antitumor activity when used as single agents or when combined with standard of care therapy (vincristine, actinomycin D and cyclophosphamide). More alarmingly, however, crizotinib actually accelerated the growth of these tumors in vivo. CONCLUSIONS: While ALK appears to be a relevant target in RMS in vitro, targeting this kinase in vivo yields no therapeutic efficacy, warranting extreme caution when considering the use of these agents in pediatric RMS patients.

Optimization of a Neural Stem-Cell-Mediated Carboxylesterase/Irinotecan Gene Therapy for Metastatic Neuroblastoma.

Despite improved survival for children with newly diagnosed neuroblastoma (NB), recurrent disease is a significant problem, with treatment options limited by anti-tumor efficacy, patient drug tolerance, and cumulative toxicity. We previously demonstrated that neural stem cells (NSCs) expressing a modified rabbit carboxylesterase (rCE) can distribute to metastatic NB tumor foci in multiple organs in mice and convert the prodrug irinotecan (CPT-11) to the 1,000-fold more toxic topoisomerase-1 inhibitor SN-38, resulting in significant therapeutic efficacy. We sought to extend these studies by using a clinically relevant NSC line expressing a modified human CE (hCE1m6-NSCs) to establish proof of concept and identify an intravenous dose and treatment schedule that gave maximal efficacy. Human-derived NB cell lines were significantly more sensitive to treatment with hCE1m6-NSCs and irinotecan as compared with drug alone. This was supported by pharmacokinetic studies in subcutaneous NB mouse models demonstrating tumor-specific conversion of irinotecan to SN-38. Furthermore, NB-bearing mice that received repeat treatment with intravenous hCE1m6-NSCs and irinotecan showed significantly lower tumor burden (1.4-fold, p = 0.0093) and increased long-term survival compared with mice treated with drug alone. These studies support the continued development of NSC-mediated gene therapy for improved clinical outcome in NB patients.

Tumour-selective targeting of drug metabolizing enzymes to treat metastatic cancer.

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of ester-containing xenobiotics. This hydrolysis reaction results in the formation of the corresponding carboxylic acid and alcohol. Due to their highly plastic active site, CEs can hydrolyze structurally very distinct and complex molecules. Because ester groups significantly increase the water solubility of compounds, they are frequently used in the pharmaceutical industry to make relatively insoluble compounds more bioavailable. By default, this results in CEs playing a major role in the distribution and metabolism of these esterified drugs. However, this can be exploited to selectively improve compound hydrolysis, and using specific in vivo targeting techniques can be employed to generate enhanced drug activity. Here, we seek to detail the human CEs involved in esterified molecule hydrolysis, compare and contrast these with CEs present in small mammals and describe novel methods to improve drug therapy by specific delivery of CEs to cells in vivo. Finally, we will discuss the development of such approaches for their potential application towards malignant disease.

Carboxylesterases: General detoxifying enzymes.

Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents).

Targeting the DNA repair pathway in Ewing sarcoma.

Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

Targeting oxidative stress in embryonal rhabdomyosarcoma.

Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

Modulation of esterified drug metabolism by tanshinones from Salvia miltiorrhiza ("Danshen").

The roots of Salvia miltiorrhiza ("Danshen") are used in traditional Chinese medicine for the treatment of numerous ailments including cardiovascular disease, hypertension, and ischemic stroke. Extracts of S. miltiorrhiza roots in the formulation "Compound Danshen Dripping Pill" are undergoing clinical trials in the United States. To date, the active components of this material have not been conclusively identified. We have determined that S. miltiorrhiza roots contain potent human carboxylesterase (CE) inhibitors, due to the presence of tanshinones. K(i) values in the nM range were determined for inhibition of both the liver and intestinal CEs. As CEs hydrolyze clinically used drugs, the ability of tanshinones and S. miltiorrhiza root extracts to modulate the metabolism of the anticancer prodrug irinotecan (CPT-11) was assessed. Our results indicate that marked inhibition of human CEs occurs following incubation with both pure compounds and crude material and that drug hydrolysis is significantly reduced. Consequently, a reduction in the cytotoxicity of irinotecan is observed following dosing with either purified tanshinones or S. miltiorrhiza root extracts. It is concluded that remedies containing tanshinones should be avoided when individuals are taking esterified agents and that patients should be warned of the potential drug-drug interaction that may occur with this material.

Inhibition of human carboxylesterases hCE1 and hiCE by cholinesterase inhibitors.

Carboxylesterases (CEs) are ubiquitously expressed proteins that are responsible for the detoxification of xenobiotics. They tend to be expressed in tissues likely to be exposed to such agents (e.g., lung and gut epithelia, liver) and can hydrolyze numerous agents, including many clinically used drugs. Due to the considerable structural similarity between cholinesterases (ChE) and CEs, we have assessed the ability of a series of ChE inhibitors to modulate the activity of the human liver (hCE1) and the human intestinal CE (hiCE) isoforms. We observed inhibition of hCE1 and hiCE by carbamate-containing small molecules, including those used for the treatment of Alzheimer's disease. For example, rivastigmine resulted in greater than 95% inhibition of hiCE that was irreversible under the conditions used. Hence, the administration of esterified drugs, in combination with these carbamates, may inadvertently result in decreased hydrolysis of the former, thereby limiting their efficacy. Therefore drug:drug interactions should be carefully evaluated in individuals receiving ChE inhibitors.

ATP-dependent mitochondrial porphyrin importer ABCB6 protects against phenylhydrazine toxicity.

Abcb6 is a mammalian mitochondrial ATP-binding cassette (ABC) transporter that regulates de novo porphyrin synthesis. In previous studies, haploinsufficient (Abcb6(+/-)) embryonic stem cells showed impaired porphyrin synthesis. Unexpectedly, Abcb6(-/-) mice derived from these stem cells appeared phenotypically normal. We hypothesized that other ATP-dependent and/or -independent mechanisms conserve porphyrins. Here, we demonstrate that Abcb6(-/-) mice lack mitochondrial ATP-driven import of coproporphyrin III. Gene expression analysis revealed that loss of Abcb6 results in up-regulation of compensatory porphyrin and iron pathways, associated with elevated protoporphyrin IX (PPIX). Phenylhydrazine-induced stress caused higher mortality in Abcb6(-/-) mice, possibly because of sustained elevation of PPIX and an inability to convert PPIX to heme despite elevated ferrochelatase levels. Therefore, Abcb6 is the sole ATP-dependent porphyrin importer, and loss of Abcb6 produces up-regulation of heme and iron pathways necessary for normal development. However, under extreme demand for porphyrins (e.g. phenylhydrazine stress), these adaptations appear inadequate, which suggests that under these conditions Abcb6 is important for optimal survival.

Requirements for mammalian carboxylesterase inhibition by substituted ethane-1,2-diones.

Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many clinically used drugs. Hence, the activity of these agents is likely dependent upon the levels and location of CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study, we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different classes of molecules were assayed: one containing different atoms vicinal to the carbonyl carbon atom and the benzene ring [PhXC(O)C(O)XPh, where X=CH2, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibitory potency (when X=N), or conversion of the compounds into substrates for the enzymes (when X=S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Subsequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH3 to C8H17] and 1-phenyl-2-alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH3 to C6H13], demonstrated that the potency of enzyme inhibition directly correlated with the hydrophobicity (clogP) of the molecules. We conclude from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group.

Nerve agent hydrolysis activity designed into a human drug metabolism enzyme.

Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.

Organ-specific carboxylesterase profiling identifies the small intestine and kidney as major contributors of activation of the anticancer prodrug CPT-11.

The activation of the anticancer prodrug CPT-11, to its active metabolite SN-38, is primarily mediated by carboxylesterases (CE). In humans, three CEs have been identified, of which human liver CE (hCE1; CES1) and human intestinal CE (hiCE; CES2) demonstrate significant ability to hydrolyze the drug. However, while the kinetic parameters of CPT-11 hydrolysis have been measured, the actual contribution of each enzyme to activate the drug in biological samples has not been addressed. Hence, we have used a combination of specific CE inhibition and conventional chromatographic techniques to determine the amounts, and hydrolytic activity, of CEs present within human liver, kidney, intestinal and lung specimens. These studies confirm that hiCE demonstrates the most efficient kinetic parameters for CPT-11 activation, however, due to the high levels of hCE1 that are expressed in liver, the latter enzyme can contribute up to 50% of the total of drug hydrolysis in this tissue. Conversely, in human duodenum, jejunum, ileum and kidney, where hCE1 expression is very low, greater than 99% of the conversion of CPT-11 to SN-38 was mediated by hiCE. Furthermore, analysis of lung microsomal extracts indicated that CPT-11 activation was more proficient in samples obtained from smokers. Overall, our studies demonstrate that hCE1 plays a significant role in CPT-11 hydrolysis even though it is up to 100-fold less efficient at drug activation than hiCE, and that drug activation in the intestine and kidney are likely major contributors to SN-38 production in vivo.

Improved, selective, human intestinal carboxylesterase inhibitors designed to modulate 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (Irinotecan; CPT-11) toxicity.

CPT-11 is an antitumor prodrug that is hydrolyzed by carboxylesterases (CE) to yield SN-38, a potent topoisomerase I poison. However, the dose limiting toxicity delays diarrhea that is thought to arise, in part, from activation of the prodrug by a human intestinal CE (hiCE). Therefore, we have sought to identify selective inhibitors of hiCE that may have utility in modulating drug toxicity. We have evaluated one such class of molecules (benzene sulfonamides) and developed QSAR models for inhibition of this protein. Using these predictive models, we have synthesized a panel of fluorene analogues that are selective for hiCE, demonstrating no cross reactivity to the human liver CE, hCE1, or toward human cholinesterases, and have K(i) values as low as 14 nM. These compounds prevented hiCE-mediated hydrolysis of the drug and the potency of enzyme inhibition correlated with the clogP of the molecules. These studies will allow the development and application of hiCE-specific inhibitors designed to selectively modulate drug hydrolysis in vivo.

Planarity and constraint of the carbonyl groups in 1,2-diones are determinants for selective inhibition of human carboxylesterase 1.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the detoxification of xenobiotics, including numerous clinically used drugs. Therefore, the selective inhibition of these proteins may prove useful in modulating drug half-life and bioavailability. Recently, we identified 1,2-diones as potent inhibitors of CEs, although little selectivity was observed in the inhibition of either human liver CE (hCE1) or human intestinal CE (hiCE). In this paper, we have further examined the inhibitory properties of ethane-1,2-diones toward these proteins and determined that, when the carbonyl oxygen atoms are cis-coplanar, the compounds demonstrate specificity for hCE1. Conversely, when the dione oxygen atoms are not planar (or are trans-coplanar), the compounds are more potent at hiCE inhibition. These properties have been validated in over 40 1,2-diones that demonstrate inhibitory activity toward at least one of these enzymes. Statistical analysis of the results confirms the correlation (P < 0.001) between the dione dihedral angle and the preferential inhibition of either hiCE or hCE1. Overall, the results presented here define the parameters necessary for small molecule inhibition of human CEs.

Analysis of the inhibition of mammalian carboxylesterases by novel fluorobenzoins and fluorobenzils.

We have synthesized and assessed the ability of symmetrical fluorobenzoins and fluorobenzils to inhibit mammalian carboxylesterases (CE). The majority of the latter were excellent inhibitors of CEs however unexpectedly, the fluorobenzoins were very good enzyme inhibitors. Positive correlations were seen with the charge on the hydroxyl carbon atom, the carbonyl oxygen, and the Hammett constants for the derived K(i) values with the fluorobenzoins.

Selective inhibition of carboxylesterases by isatins, indole-2,3-diones.

Carboxylesterases (CE) are ubiquitous enzymes thought to be responsible for the metabolism and detoxification of xenobiotics. Numerous clinically used drugs including Demerol, lidocaine, capecitabine, and CPT-11 are hydrolyzed by these enzymes. Hence, the identification and application of selective CE inhibitors may prove useful in modulating the metabolism of esterified drugs in vivo. Having recently identified benzil (diphenylethane-1,2-dione) as a potent selective inhibitor of CEs, we sought to evaluate the inhibitory activity of related 1,2-diones toward these enzymes. Biochemical assays and kinetic studies demonstrated that isatins (indole-2,3-diones), containing hydrophobic groups attached at a variety of positions within these molecules, could act as potent, specific CE inhibitors. Interestingly, the inhibitory potency of the isatin compounds was related to their hydrophobicity, such that compounds with clogP values of <1.25 were ineffective at enzyme inhibition. Conversely, analogs demonstrating clogP values>5 routinely yielded Ki values in the nM range. Furthermore, excellent 3D QSAR correlates were obtained for two human CEs, hCE1 and hiCE. While the isatin analogues were generally less effective at CE inhibition than the benzils, the former may represent valid lead compounds for the development of inhibitors for use in modulating drug metabolism in vivo.

Tumor-targeted enzyme/prodrug therapy mediates long-term disease-free survival of mice bearing disseminated neuroblastoma.

Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.

Analysis of mammalian carboxylesterase inhibition by trifluoromethylketone-containing compounds.

Carboxylesterases (CE) are ubiquitous enzymes that hydrolyze numerous ester-containing xenobiotics, including complex molecules, such as the anticancer drugs irinotecan (CPT-11) and capecitabine and the pyrethroid insecticides. Because of the role of CEs in the metabolism of many exogenous and endogenous ester-containing compounds, a number of studies have examined the inhibition of this class of enzymes. Trifluoromethylketone-containing (TFK) compounds have been identified as potent CE inhibitors. In this article, we present inhibition constants for 21 compounds, including a series of sulfanyl, sulfinyl, and sulfonyl TFKs with three mammalian CEs, as well as human acetyl- and butyrylcholinesterase. To examine the nature of the slow tight-binding inhibitor/enzyme interaction, assays were performed using either a 5-min or a 24-h preincubation period. Results showed that the length of the preincubation interval significantly affects the inhibition constants on a structurally dependent basis. The TFK-containing compounds were generally potent inhibitors of mammalian CEs, with Ki values as low as 0.3 nM observed. In most cases, thioether-containing compounds were more potent inhibitors then their sulfinyl or sulfonyl analogs. QSAR analyses demonstrated excellent observed versus predicted values correlations (r2 ranging from 0.908-0.948), with cross-correlation coefficients (q2) of approximately 0.9. In addition, pseudoreceptor models for the TKF analogs were very similar to structures and models previously obtained using benzil- or sulfonamide-based CE inhibitors. These studies indicate that more potent, selective CE inhibitors, containing long alkyl or aromatic groups attached to the thioether chemotype in TFKs, can be developed for use in in vivo enzyme inhibition.