Patent Application
20180055816
The presently-disclosed subject matter generally relates to pyranonaphthoquinone compounds, methods of forming pyranonaphthoquinone compounds, and methods of use thereof. In particular, certain embodiments of the presently-disclosed subject matter relate to pyranonaphthoquinone compounds, methods of forming pyranonaphthoquinone compounds, and methods for modulating cap-dependent translation and 4E-BP1 phosphorylation using pyranonaphthoquinone compounds.
Disease progression and drug resistance to anticancer therapies is often associated with mutational activation of multiple signaling pathways that promote aberrant cell growth and metastasis. For example, the aggressiveness of diseases such as metastatic colorectal cancer (CRC) is in part driven by the aberrant expression of oncoproteins. As such, these diseases are difficult to treat and patients have few long term effective therapeutic options. At the molecular level, cap-dependent translation of the precursor oncogenic mRNAs is frequently activated. Specifically, this occurs via 4E-BP1 phosphorylation which, when not phosphorylated, functions as a mRNA translation repressor downstream from mTOR.
Novel therapeutic approaches include pharmacologic inhibition of proteins within signaling pathways as well as converging nodes like mTOR, which is activated in many cancers. The inhibition of mTOR has been of interest because it integrates multiple signals. However, the clinical efficacy of mTOR inhibitor drugs (e.g., rapamycin analogs) is limited. It is widely believed that this is largely attributed to their weak capacity to prevent phosphorylation of 4E-BP1 (a key translational repressor) which, when phosphorylated by mTOR, relieves its inhibitory control on elF4E-initiated cap-dependent translation of oncogenic mRNAs that drive oncoprotein production. Existing mTOR kinase (ATP-competitive) inhibitors non-selectively inhibit 4E-BP1 phosphorylation but also modulate the function of other mTOR-associated targets that may contribute to unwanted toxicities.
Accordingly, there exists a need for effective, selective inhibition of 4E-BP1 in cancer treatment.
The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.
This summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.
In some embodiments, the presently-disclosed subject matter includes a method of selectively inhibiting 4E-BP1 phosphorylation comprising administering at least one pyranonaphthoquinone or pyranonaphthoquinone analog to a subject in need thereof. In one embodiment the at least one pyranonaphthoquinone or pyranonaphthoquinone analog includes a structure according to Formula I:
In another embodiment the at least one pyranonaphthoquinone or pyranonaphthoquinone analog is a frenolicin or frenolicin analog. In a further embodiment, the frenolicin analog includes a structure according to Formula II:
In still a further embodiment, the frenolicin analog includes a structure according to Formula III:
In on embodiment, the at least one pyranonaphthoquinone or pyranonaphthoquinone analog is a griseusin or griseusin analog. In another embodiment, the griseusin or griseusin analog includes a structure according to formula IV:
In some embodiments, administering the at least one pyranonaphthoquinone or pyranonaphthoquinone analog to the subject selectively inhibits 4E-BP1 phosphorylation. In some embodiments, administering the at least one pyranonaphthoquinone or pyranonaphthoquinone analog to the subject modulates 4E-BP1-regulated cap-dependent translation.
Also provided herein, in some embodiments, is a 4E-BP1 phosphorylation inhibitor comprising a pyranonaphthoquinone analog. In one embodiments, the pyranonaphthoquinone analog comprises a griseusin analog. In another embodiment, the griseusin analog includes a structure according to formula IV:
In one embodiment, the pyranonaphthoquinone analog comprises a frenolicin analog. In another embodiment, the frenolicin analog includes a structure according to Formula II:
In a further embodiment, the frenolicin analog includes a structure according to Formula III:
In some embodiments, the frenolicin analog is selected from the group consisting of an epi-frenolicin C1 analog, an epi-frenolicin ring A analog, an epi-frenolicin open D analog, and combinations thereof.
Further provided herein, in some embodiments, is a method of treating cancer comprising administering at least one pyranonaphthoquinone or pyranonaphthoquinone analog to a subject in need thereof. In one embodiment, the at least one pyranonaphthoquinone or pyranonaphthoquinone analog includes a structure according to Formula I:
In another embodiment, the pyranonaphthoquinone analog comprises a griseusin analog. In a further embodiment, the pyranonaphthoquinone analog comprises a frenolicin analog.
Further features and advantages of the presently-disclosed subject matter will become evident to those of ordinary skill in the art after a study of the description, figures, and non-limiting examples in this document.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:
The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.
Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9): 1726-1732).
Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
The presently-disclosed subject matter includes methods and compounds for inhibiting 4E-BP1 phosphorylation. In some embodiments, the compound includes a 4E-BP1 inhibitor. For example, in one embodiment, the inhibitor is a pyranonaphthoquinone or pyranonaphthoquinone analog. In another embodiment, the inhibitor is of the structure represented by Formula I:
where R1 includes, but is not limited to, H; C1-C6 alkyl (e.g., CH2CH3, CH2CH2CH3, CH(CH3)2); (CH2)nX, where n is between 0 and 6 and X is N3, CN, CH3, aryl (e.g., C6H5), triazole (e.g., C2H2N3), alkyl-substituted triazole, piperidyl (e.g., (CH2)5N), morpholinyl (e.g., O(CH2CH2)2N), tetrahydropyranyl (e.g., C5H9O), cyclohexyl (e.g., C6H11), halogen-substituted aryl (e.g., C6H4F, C6H3F2, C6H4Cl, C6H4Br,); alkyl-substituted aryl (e.g., C6H4(CH3)), alkoxyl-substituted aryl (e.g., C6H4(OCH3)), hydroxyl-substituted aryl (e.g., C6H4(OH)), amino-substituted aryl (e.g., C6H4(NH2)), pyridinyl (e.g., C5H4N), diazinyl (e.g., C4H3N2), triazinyl (e.g., C3H2N3), C(O)C6H5, or OCH2C6H5; alkenyl (e.g., CH2CHCH2); alkynyl (e.g., CH2CH2CCH); where R2 includes, but is not limited to, H, OH, alkoxy (e.g., OCH3), halogen, or amine; where R3 includes, but is not limited to, H, OH, or halogen; where R4 includes, but is not limited to, H or OH; and where R5 includes, but is not limited to, CH2Y, CH2CH2Z, or CH2C(O)Z, where Y is CN, CH2OR, CH2NHR, COOR, or CONHR, and where Z is a bond between R4 and R5, forming a five membered ring with the carbon atoms to which R4 and R5 are attached.
In certain embodiments, suitable structures corresponding to the chemical formulas disclosed for R1 above include, but are not limited to:
In some embodiments, suitable structures corresponding to the chemical formulas disclosed for R2 and R3 above include, but are not limited to:
Turning to R4 and R5, in some embodiments, as discussed above, the groups are separate and do not form a ring. One example of these embodiments includes UCF76-A (
where R1, R2, and R3, are as defined above with regard to Formula I, and R6 includes, but is not limited to, CH2 or CO. In one embodiment, the inhibitor according to Formula II includes frenolicin B (
As used herein, the term analog refers to any suitable combination of variable groups according to the Formulas disclosed herein. Additionally, in some embodiments, the term analog refers to stereoisomers of the inhibitor. For example, the down stereo bonds of R4 and/or R5 in the open configuration, or the down stereo bonds of the five member ring including R6, may be replaced with up stereo bonds in any one or more of the embodiments disclosed herein. Suitable structures according to Formula II, where R1 is attached through an up stereo bond, and Formula III, where R1 is attached through a down stereo bond, include, but are not limited to:
In some embodiments, the inhibitor of the instant disclosure includes a griseusin and/or griseusin analog. For example, in one embodiment, the griseusin and/or griseusin analog includes the structure according to Formula IV below:
Where each R1 independently includes, but is not limited to, H, O, OH, OCH3, OC(O)CH3, N3, NH2, halide, or C1-C6 alkyl (e.g., CH3); where R2 includes, but is not limited to, H, OH, alkoxy (e.g., OCH3), halogen, or amine; where R3 includes, but is not limited to, H, OH, or halogen; where R4 includes, but is not limited to, H or OH; and where R5 includes, but is not limited to, CH2Y, CH2CH2Z, or CH2C(O)Z, where Y is CN, CH2OR, CH2NHR, COOR, or CONHR, and where Z is a bond between R4 and R5, forming a five membered ring with the carbon atoms to which R4 and R5 are attached.
In one embodiment, the griseusen of Formula IV is griseusen C, which has the following structure:
In another embodiment, the griseusen of Formula IV is a griseusen C analog, which is a stereoisomer of griseusen C and/or has a structure according to Formula V below:
and stereoisomers thereof, where R2 and R3 are as discussed above with regard to Formula IV. In a further embodiment, griseusen C analogs include, but are not limited to:
Additionally or alternatively, in one embodiment, the griseusen of Formula IV is griseusen A, which has the following structure:
In another embodiment, the griseusen of Formula IV is a griseusen A analog, which is a stereoisomer of griseusen A and/or has a structure according to Formula VI below:
and stereoisomers thereof, where R2 and R3 are as discussed above with regard to Formula IV; X is OAc, OH, or O; and Y is H or SG. For example, one griseusen A analog includes 4′-deacetyl-GA, which has the following structure:
As will be appreciated by those skilled in the art, the structures discussed above are for illustration only and are not intended to limit the scope of the instant disclosure. Accordingly, other pyranonaphthoquinone and pyranonaphthoquinone analogs are expressly contemplated herein, including, but not limited to, frenolicin analogs, griseusin B, griseusin D, griseusin E, griseusin F, griseusin G, and analogs thereof.
In some embodiments, one or more of the pyranonaphthoquinones or pyranonaphthoquinone analogs described herein is arranged and disposed to bind and/or inhibit any suitable target. Suitable targets include, but are not limited to, targets which are overproduced in one or more cancers and/or which play a key role in one or more parasitic-type diseases. For example, in one embodiment, one or more of the pyranonaphthoquinones or pyranonaphthoquinone analogs described herein binds glutaredoxin, peroxiredoxin, or a combination thereof. As used herein, the terms “glutaredoxin” and “peroxiredoxin” include any suitable isoform thereof, such as, but not limited to, glutaredoxin 3, peroxiredoxin 1, peroxiredoxin 2, any other isoform thereof, or a combination thereof. In another embodiment, the binding of the one or more of the pyranonaphthoquinones or pyranonaphthoquinone analogs described herein inhibits glutaredoxin and/or peroxiredoxin. Without wishing to be bound by theory, it is believed that the compounds disclosed herein represent the first reported inhibitor of glutaredoxing to date. Again, without wishing to be bound by theory, it is believed that in certain embodiments the pyranonaphthoquinones or pyranonaphthoquinone analogs described herein also provide the most potent inhibitor of peroxiredoxin to date.
Additionally or alternatively, in some embodiments, as discussed above, the pyranonaphthoquinones or pyranonaphthoquinone analogs described herein form 4E-BP1 phosphorylation inhibitors. More specifically, in contrast to the previously held belief that pyranonaphthoquinones or pyranonaphthoquinone analogs were Akt inhibitors, the instant inventors surprisingly discovered that one or more of the pyranonaphthoquinones or pyranonaphthoquinone analogs disclosed herein inhibit 4E-BP1 phosphorylation through inhibition of Prx1 and/or Grx3, without or substantially without inhibiting Akt. That is, pyranonaphthoquinones or pyranonaphthoquinone analogs disclosed herein inhibit 4E-BP1 phosphorylation in a manner that is mechanistically distinct to existing mTOR inhibitors.
Without wishing to be bound by theory, it is believed that deregulation of cap-dependent translation downstream of mTOR at the level of 4E-BP1/elF4E is a key to tumor formation and metastatic progression. More specifically, translation of key oncogenic mRNAs is strongly dependent on the mRNA cap-binding protein elF4E (
In this regard, the instant inventors recently discovered that activated signaling via the PI3K/AKT and RAS/RAF/MEK/ERK pathways cooperate to promote CRC progression by convergent phosphorylation (inactivation) of 4E-BP1. Additionally, the instant inventors have demonstrated that 4E-BP1 phosphorylation-mediated oncogene translation functions as a critical node that integrates oncogenic signals of the AKT and ERK pathways for CRC tumorigenesis and metastasis. Moreover, the instant inventors have found that CRC resistance to upstream kinase targeted therapy is associated with incomplete inhibition of 4E-BP1 phosphorylation. That is, due to cooperation between the pathways, inhibition of Akt or Erk alone is insufficient to provide complete inhibition of 4E-BP1 phosphorylation, and therefore, is insufficient to suppress tumor growth and metastasis.
However, the pyranonaphthoquinones or pyranonaphthoquinone analogs disclosed herein form what is believed to be the first selective inhibitor of 4E-BP1 phosphorylation. Since the novel mechanism for inhibiting 4E-BP1 phosphorylation disclosed herein does not rely on Akt or Erk inhibition, the pyranonaphthoquinones or pyranonaphthoquinone analogs of the instant disclosure eliminate the redundant phosphorylation issues associated therewith. Additionally, pharmacoloqic activation of 4E-BP1, via pharmacological inhibition of 4E-BP1 phosphorylation, disrupts cap-dependent translation by blocking converging oncogenic signals at a key node. Therefore, in one embodiment, administering at least one pyranonaphthoquinone or pyranonaphthoquinone analog modulates 4E-BP1-regulated cap-dependent translation and provides a treatment strategy for advanced CRC and/or other major cancers. Thus, in certain embodiments, the pyranonaphthoquinones or pyranonaphthoquinone analogs disclosed herein form novel anticancer drugs, where direct targeting of 4E-BP1 phosphorylation-mediated oncogene translation represents a novel strategy for cancer drug development and therapy.
Accordingly, also provided herein, in some embodiments, is a method of treating cancer including administering at least one of the pyranonaphthoquinones and/or pyranonaphthoquinone analogs to a subject in need thereof. Suitable pyranonaphthoquinones or pyranonaphthoquinone analogs include any of the compounds disclosed herein, such as, but are not limited to, frenolicin B (FB), epi-FB, griseusin, analogs thereof, or a combination thereof. In one embodiment, the method provides selective inhibition of 4E-BP1 phosphorylation.
As used herein, the term “cancer” refers to all types of cancer or neoplasm or malignant tumors found in animals, including leukemias, carcinomas, melanoma, and sarcomas. For example, the cancer may include, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, colorectal cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer. In some particular embodiments, the cancer is colorectal cancer (CRC).
Further provided herein, in some embodiments, is a method of treating a parasitic-type disease. In some embodiments, the method of treating a parasitic-type disease includes administering at least one pyranonaphthoquinone or pyranonaphthoquinone analog to a subject in need thereof. Suitable pyranonaphthoquinones or pyranonaphthoquinone analogs include, but are not limited to, frenolicin B (FB), epi-FB, griseusin, an analog of FB, epi-FB, or griseusin, or a combination thereof.
As used herein, the term “parasitic-type disease” refers to any disease caused by or resulting from a parasite. For example, in one embodiment, parasitic-type disease includes malaria. In another embodiment, one or more of the pyranonaphthoquinones or pyranonaphthoquinone analogs described herein provide anti-parasitic-type disease function by inhibiting one or more of the targets disclosed herein. More specifically, in certain embodiments, one or more of the pyranonaphthoquinones or pyranonaphthoquinone analogs described herein inhibit peroxiredoxin, which provides potent anti-malarial function. This peroxiredoxin inhibition may, in some embodiments, provide increased antimalarial potency as compared to existing malaria drugs/treatments.
The presently-disclosed subject matter also includes, in some embodiments, method of forming a pyranonaphthoquinone or pyranonaphthoquinone analog. In one embodiment, the method includes an enantioselective syntheses of frenolicin analogs, as described in greater detail in the Examples below. In another embodiment, the method includes a divergent process for the synthesis of both FB and epi-FB. In a further embodiment, the method provides Lewis acid catalyst optimization to facilitate increased control over diastereoselectivity in the key oxa-Pictet-Spengler reaction, thereby providing access to all core stereoisomers. Additionally or alternatively, the method provides optimization of the culminating demethylation (a key deprotection step) to avoid epimerization that notably plagued prior syntheses. Methods for synthesizing griseusin and giseusin analogs are also provided herein.
Still further provided herein, in some embodiments, is a molecular probe that facilitates understanding of 4E-BP1 in tumorigenesis and metastasis. In one embodiment, the molecular probe binds to SEQ ID NO: 1 and/or SEQ ID NO: 2.
Enantioselective Syntheses of Frenolicin Analogs for SAR and Probes.
Referring to
A schematic example of the mechanism of pyranonaphthaquinone inhibition of 4E-CP1 phosphorylation and tumor progression in vivo is shown in
Synthesis and Preliminary Evaluation of Novel FB-Based Analogs.
Prompted by the potentially novel mechanism of action outlined above, the instant inventors developed an efficient divergent strategy for the synthesis of both FB and epi-FB to allow for further exploration of the pyranonaphthoquinone SAR (
Based on the preliminary mechanistic studies with naturally-occurring FB metabolites described above, an initial streamlined compound prioritization strategy that requires 90% inhibition of 4E-BP1 phosphorylation at 1 1.LM of test compound was selected as the initial filter. Compounds that pass this single dose filter are then tested in a CRC (HCT116) in vitro cytotoxicity assay and, in parallel, an in vitro assay to assess propensity for intracellular reactive oxygen species (ROS) production using the redox sensitive probe CeIIROX® Deep Red reagent (Invitrogen) by flow cytometry analysis (47). The rationale for inclusion of ROS induction as a key criterion stems from the well-established liabilities of quinones (namely, as oxidants and electrophiles) (48-50) and the demonstrated ability of ROS to activate key signaling pathways of relevance to cancer (51, 52). These data are then correlated as illustrated in
Using this general strategy, initial SAR analyses (
A representative member (b47) from this priority set was further studied in secondary assays as an additional assessment of the prioritization strategy. Specifically, b47 (4-fold more potent than FB against HCT116 but 2-fold less proficient at ROS production, (
Epi-FB-Based Analog Synthesis.
Chemistries directed toward further epi-FB-based analoging are first directed toward further assessment of the impact of C1 variation (
Synthesis and Preliminary Evaluation of Novel Griseusins.
In view of the data above, SAR was further expand with a particular emphasis on C1 modification of the C ring. Within this context, the griseucins (
Importantly, in contrast to prior reported synthetic approaches to the griseusins (62-66), the instant method (which draws conceptually on the inventors previously described work with the frenolicin series) stands as the first truly divergent enantioselective method and the most efficient strategy to date. Six griseusin analogs have been synthesized via this method, two of which have been further evaluated in in vitro assays (
Griseusin Analog Synthesis.
Using the established divergent strategy highlighted in
Altered redox status is a common feature of many cancers in which deregulation of cell signaling and metabolism by multiple genetic alterations often lead to increased generation of intracellular reactive oxygen species (ROS). Although intracellular ROS elevation contributes to tumor initiation and progression, it is believed that agents capable of increasing intracellular ROS beyond the cellular tolerability threshold may represent a potential selective anticancer therapeutic strategy. Within this context, this Example reports the identification of the molecular target and anticancer mechanism of frenolicin B (FB), a classical, but mechanistically undefined, pyranonaphthoquinone (PNQ) natural product commonly used as an anticoccidial feedstock additive.
Specifically, in this Example, FB is identified as a selective inhibitor of peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3), two antioxidant proteins overexpressed in many cancers that play key roles in maintaining cellular redox status. Inhibition of Prx1 and Grx3 by FB induces a concomitant elevation of intracellular ROS which activates the peroxisome-bound tuberous sclerosis complex and thereby inhibits mTORC1-mediated phosphorylation of the translation repressor 4E-BP1, a key effector of the oncogenic activation of the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways in tumorigenesis and metastasis. FB structure-activity relationship (SAR) studies reveal a positive correlation between inhibition of 4E-BP1 phosphorylation, intracellular ROS concentrations, cancer cell cytotoxicity and suppression of tumor growth in vivo. These findings establish FB as the most potent and novel Prx1/Grx3 inhibitor reported to date and also notably highlights 4E-BP1 phosphorylation status as a potential new predictive marker in response to oxidative stress-based therapies in cancer.
Dysregulation of cap-dependent translation through redundant phosphorylation of the translational repressor 4E-BP1 by multiple oncogenic pathways, such as PI3K/AKT/mTOR and RAS/RAF/MEK/ERK, is associated with malignant progression and therapeutic resistance. To determine the importance of 4E-BP1 dephosphorylation in the repression of cap-dependent translation and tumor progression, a non-phosphorylated 4E-BP1 mutant, 4E-BP1-4A, was generated. The four known phosphorylation sites, T37, T46, S65, and T70 (
Thus, targeting cap-dependent translation may overcome intra-tumor heterogeneity-mediated resistance and provide a promising strategy for improving cancer therapy. To identify microbial nature products (NPs) capable of targeting cap-dependent translation, crude extracts of prioritized microbes from the Ruth Mullins underground coal mine fire site14 were screened using a cap-dependent translation-based luciferase reporter assay5. This initial screen revealed that extracts of Streptomyces sp. RM-4-15, a strain previously identified to produce a series of known and novel pyranonaphthoquinones (PNQs)15, contain compounds capable of inhibiting cap-dependent translation (
To further probe the function of PNQs within the context of cap-dependent translation, the ability of FB and UCF76-A to modulate 4E-BP1 and p70S6 kinase phosphorylation was compared to that of representative mTOR inhibitors. The mTOR kinase complex 1 (mTORC1), a downstream target of both AKT and ERK signaling, is a well-characterized activator of cap-dependent translation through phosphorylation of 4E-BP1 and p70S6 kinase17. Rapamycin is an allosteric inhibitor of mTORC1 and can effectively inhibit p70S6K phosphorylation, but only weakly inhibits 4E-BP1 phosphorylation18, while second generation ATP-competitive mTOR kinase inhibitors such as AZD8055 that inhibit both mTORC1 and mTOR complex 2 (mTORC2) are more effective than rapamycin in inhibiting 4E-BP1 phosphorylation. Like AZD8055 but distinct from rapamycin, FB and UCF76-A effectively inhibited 4E-BP1 phosphorylation in HCT116 CRC cells (
Both rapamycin and AZD8055 potently inhibited phosphorylation of p70S6K and its substrate, the ribosomal protein S6, and AZD8055 also inhibited phosphorylation of the mTORC2 substrate AKT17. In contrast, FB or UCF76-A did not inhibit S6 or AKT phosphorylation, only weakly inhibited p70S6K phosphorylation, but were potent activators of caspase 3 with induction of the apoptotic marker cleaved PARP and dramatic suppression of HCT116 cell growth (
Prompted by the potential mechanistic novelty of PNQs, we evaluated additional FB-based PNQ synthetic analogs (
To identify the PNQ molecular target(s) responsible for the observed inhibition of 4E-BP1 phosphorylation and cancer cell cytotoxicity, a comparative affinity pulldown-based target identification strategy was employed. Specifically, guided by the SAR studies described, two FB-based biotinylated probes were synthesized. ‘Active’ probe 1 (d7) retained FB-like activity (inhibition of 4EBP1 phosphorylation and CRC cell line cytotoxicity), while the corresponding activities of structurally-related ‘inactive’ comparator probe 2 (d5) were notably suppressed (
Prx1 and Grx3 are antioxidant enzymes known to regulate oxidative stress. Prx1 catalyzes peroxide reduction of H2O2, whereas Grx3 functions as glutathione (GSH)-dependent oxidoreductase by participating in the conversion of reduced GSH to oxidized glutathione disulfide (GSSG). Biochemical inhibition studies using recombination Prx1 revealed FB or semi-synthetic 12 as potent inhibitors of Prx1 with observed KiS >60-fold lower than the most potent Prx1 inhibitor, conoidin A, reported to date (
Consistent with Prx1 and Grx3 as the molecular targets, it was found that FB and active surrogates (
To assess whether this unique anticancer mechanism translates to in vivo efficacy, we utilized an aqueous soluble phosphate prodrug 14 synthesized from 12 in two steps (65% overall yield,
FB is the prototypical PNQ-based NP first reported in the late 60's23 and has since been demonstrated to function as an effective anticoccidial and antimalarial24, the fundamental mechanism(s) for which remain undetermined. The current study highlights, for the first time, that FB and PNQ-based analogs function as inhibitors of Prx1 and Grx3 for cancer therapy by targeting ROS stress-response pathway in which 4E-BP1 phosphorylation functions as a major sensor to guide for the development of this new class of agents and predict their antitumor efficacy. Overexpression of Prx1 and Grx3 often occurs in a variety of cancers, and is associated with redox adaptation that promotes tumor progression and resistance to many anticancer agents and radiation. The use of agents such as PNQs as identified here to abrogate the adaptation mechanism due to the increased intracellular antioxidant capacity in combination with conventional chemotherapy, radiotherapy or target therapy could be an attractive new approach to improve therapeutic outcomes.
Methods
Methods and any associated references are available in the online version of the paper.
Chemistry.
Frenolicin B and UCF76-A were isolated from Streptomyces sp. RM-4-15 as previously described. The syntheses of 1-14 followed previously reported strategies22 and are detailed below. Compound purity for all studies was ≧95% based on HPLC and all compound stock solutions were standardized to reference standards based on HPLC and UV-vis.
General Chemistry Method.
1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Varian Unity Inova 400 MHz instrument (Palo Alto, Calif.). The chemical shifts were reported in δ (ppm) using the δ 7.26 signal of CDCl3, δ 1.94 signal of CD3CN and δ 2.50 signal of DMSO-d6 (1H NMR), the δ 77.16 signal of CDCl3, δ 1.32 signal of CD3CN and δ 39.52 signal of DMSO-d6 (13C NMR) as internal standards. The following abbreviations were used to explain the multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet. HR-ESI-MS experiments were carried out using AB SCIEX TripleTOF® 5600 System. HPLC analyses were performed using an Agilent 1260 system equipped with a DAD detector and a Phenomenex C18 column (4.6×150 mm, 0.5 μm). Semi-preparative/preparative HPLC separation was performed using a Varian Prostar 210 HPLC system equipped with a PDA detector 330 using a Supelco C18 column (25×21.2 mm, 10 μm; flow rate, 10 mL/min). Enantiomeric excess was determined by HPLC with a Chiralpak IC column, compared with racemic isomer. All commercially available reagents were used without further purification, purchased from Sigma-Aldrich, TCI America and Alfa-Aesar. The progress of the reactions was monitored by analytical thin-layer chromatography (TLC) from EMD Chemicals Inc. (Darmstadt, Germany) with fluorescence F254 indicator. And Silica gel (230-400 mesh) for column chromatography was purchased from Silicycle (Quebec City, Canada).
To a solution of (3aR,5S,11bR)-5-(3-azidopropyl)-6, 7,11-trimethoxy-3,3a,5,11b-tetrahydro-2H-benzo[g]furo[3,2-c]isochromen-2-one (reported intermediate 6 h1) (41 mg, 0.1 mmol) in a mixture of water (0.5 mL) and acetonitrile (1 mL) at 0° C., a solution of cerium ammonium nitrate (126 mg, 0.2 mmol) in H2O (0.5 mL) was added in dropwise fashion with stirring. The reaction mixture was stirred for 10 min before the addition of water (5 mL). The mixture was extracted with EtOAc (10 mL×2) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified on silica gel using hexane/EtOAc (1/1) to afford 6 as a yellow solid (31 mg, 80% yield). 1H NMR (400 MHz, CDCl3): δ=7.77-7.69 (m, 2H), 7.32 (d, J=8.0 Hz, 1H), 5.30 (s, 1H), 4.77 (d, J=9.6 Hz, 1H), 4.34 (d, J=7.2 Hz, 1H), 4.01 (s, 3H), 3.27 (t, J=6.4 Hz, 2H), 2.91 (dd, J=4.4, 17.6 Hz, 1H), 2.73 (d, J=17.6 Hz, 1H), 2.17-2.13 (m, 1H), 1.99-1.92 (m, 1H), 1.76-1.65 (m, 2H); 13C NMR (100 MHz, CDCl3) δ=183.4, 182.3, 174.5, 159.6, 151.3, 135.6, 133.7, 133.3, 120.3, 119.5, 118.3, 72.1, 71.3, 69.7, 56.7, 51.3, 37.4, 30.6, 24.7 ppm; HRMS (ESI) m/z [M+H]+ calcd for C19H18N3O6 384.1196, found 384.1199.
Compound 6 (25 mg, 0.065 mmol) was dissolved in CH2Cl2 (1 mL) and then cooled to −78° C. under argon. A solution of BCl3 (0.1 mL, 0.1 mmol, 1 N in CH2Cl2) was added to the mixture with stirring for 2 hours at −78° C. After quenching with saturated aqueous NH4Cl solution (1 mL), the reaction was diluted with H2O (5 mL) and EtOAc (5 mL). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified on silica gel using hexane/EtOAc (2/1) to afford 1 as an orange solid (19 mg, 80% yield). 1H NMR (400 MHz, CDCl3): δ=11.71 (s, 1H), 7.71-7.66 (m, 2H), 7.31 (dd, J=2.4, 7.2 Hz, 1H), 5.28 (t, J=2.0 Hz, 1H), 4.80-4.79 (m, 1H), 4.35 (dd, J=2.4, 4.4 Hz, 1H), 3.30 (t, J=6.4 Hz, 2H), 2.88 (dd, J=4.4, 17.6 Hz, 1H), 2.75 (d, J=17.6 Hz, 1H), 2.27-2.25 (m, 1H), 2.05-2.03 (m, 1H), 1.75-1.72 (m, 1H), 1.64-1.62 (m, 1H); 13C NMR (100 MHz, CDCl3) δ=188.6, 181.4, 174.2, 161.9, 148.9, 137.3, 136.7, 131.4, 125.1, 119.9, 115.1, 71.6, 71.1, 69.7, 51.2, 37.3, 31.1, 24.5 ppm; HRMS (ESI) m/z [M+H]+ calcd for C18H16N3O6 370.1039, found 370.1041.
The synthesis and characterization of 2 were previously reported.
This compound was synthesized according to the reported procedures1 except that AD-mix-α was employed to prepare the enantiomer of reported intermediate 5.1 1H NMR (400 MHz, CDCl3): δ=11.85 (s, 1H), 7.71-7.65 (m, 2H), 7.30 (dd, J=2.0, 8.0 Hz, 1H), 5.25 (t, J=3.2 Hz, 1H), 4.91 (dd, J=3.2, 10.4 Hz, 1H), 4.62 (dd, J=2.8, 5.2 Hz, 1H), 2.96 (dd, J=5.2, 17.6 Hz, 1H), 2.70 (d, J=17.6 Hz, 1H), 1.71-1.64 (m, 6H), 1.03 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=188.2, 181.6, 174.0, 162.0, 149.4, 137.3, 135.3, 131.6, 124.9, 119.8, 114.9, 69.7, 68.8, 66.3, 36.9, 33.8, 19.6, 13.6 ppm; HRMS (ESI) m/z [M+H]+ calcd for C18H17O6 329.1025, found 329.1025.
NBS (53 mg, 0.3 mmol) was added by portion to (3aR,5S,11bR)-6,7,11-trimethoxy-5-propyl-3,3a,5,11b-tetrahydro-2H-benzo[g]furo[3,2-c]isochromen-2-one (reported intermediate 6aα1) (111 mg, 0.3 mmol) in CH2Cl2 (3 mL) at room temperature and the resulting mixture was stirred overnight. After evaporating the volatiles, the residue was purified on silica gel using hexane/EtOAc (6/1) to afford the 4a (40 mg, 29%) and 5a (90 mg, 65%).
1H NMR (400 MHz, CDCl3): δ=7.75 (d, J=8.0 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 5.57 (s, 1H), 5.05 (d, J=6.0 Hz, 1H), 4.35 (s, 1H), 4.09 (s, 3H), 3.88 (s, 3H), 3.74 (s, 3H), 2.91 (dd, J=4.0, 18.4 Hz, 1H), 2.77 (d, J=17.6 Hz, 1H), 2.15-2.13 (m, 1H), 2.03-1.99 (m, 1H), 1.76-1.65 (m, 2H), 0.98 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=175.9, 153.7, 152.5, 147.7, 130.7, 129.8, 124.8, 120.2, 117.5, 107.3, 107.0, 73.2, 73.0, 71.1, 65.0, 62.2, 56.4, 38.5, 37.4, 18.4, 14.1 ppm.
1H NMR (400 MHz, CDCl3): δ=7.72 (d, J=8.0 Hz, 1H), 6.74 (d, J=8.0 Hz, 1H), 5.60 (s, 1H), 5.05 (d, J=6.0 Hz, 1H), 4.34 (s, 1H), 4.11 (s, 3H), 3.99 (s, 3H), 3.96 (s, 3H), 2.90 (d, J=18.4 Hz, 1H), 2.77 (d, J=17.6 Hz, 1H), 2.15-2.13 (m, 1H), 1.99-1.96 (m, 1H), 1.76-1.66k (m, 2H), 0.91 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=175.9, 156.0, 152.9, 149.5, 133.9, 129.6, 127.1, 126.7, 122.1, 107.8, 107.0, 73.1, 72.8, 71.1, 65.8, 61.8, 56.7, 38.7, 37.8, 18.5, 14.0 ppm.
Following the above deprotection protocol for 1, intermediate 4a (40 mg, 0.09 mmol) was used to obtain compound 4 (21 mg, 60% yield). 1H NMR (400 MHz, CDCl3): δ=12.38 (s, 1H), 7.95 (d, J=8.0 Hz, 1H), 7.56 (d, J=8.0 Hz, 1H), 5.26 (t, J=2.0 Hz, 1H), 4.77-4.75 (m, 1H), 4.33 (dd, J=2.4, 4.4 Hz, 1H), 2.90 (dd, J=4.4, 17.6 Hz, 1H), 2.74 (d, J=17.6 Hz, 1H), 2.00-1.90 (m, 2H), 1.44-1.42 (m, 1H), 1.28-1.25 (m, 1H), 0.90 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=188.8, 180.9, 174.4, 158.3, 149.6, 140.4, 136.7, 130.4, 120.1, 119.9, 115.5, 72.0, 71.0, 69.7, 37.4, 36.0, 18.4, 14.1 ppm; HRMS (ESI) m/z [M+H]+ calcd for C18H16BrO6 407.0130, found 407.0117.
Following the above deprotection protocol for 1, intermediate 5a (90 mg, 0.2 mmol) was used to obtain compound 5 (43 mg, 53% yield). 1H NMR (400 MHz, CDCl3): δ=12.28 (s, 1H), 7.83 (d, J=9.2 Hz, 1H), 7.11 (d, J=9.2 Hz, 1H), 5.33 (t, J=2.0 Hz, 1H), 4.75-4.73 (m, 1H), 4.37 (dd, J=2.4, 4.4 Hz, 1H), 2.94 (dd, J=4.4, 17.6 Hz, 1H), 2.73 (d, J=17.6 Hz, 1H), 2.03-2.00 (m, 1H), 1.89-1.85 (m, 1H), 1.44-1.42 (m, 1H), 1.29-1.27 (m, 1H), 0.88 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=187.9, 180.0, 174.4, 162.0, 148.6, 143.9, 136.9, 128.2, 125.6, 116.5, 113.7, 71.6, 71.1, 69.5, 37.2, 35.7, 18.4, 14.0 ppm; HRMS (ESI) m/z [M+H]+ calcd for C18H16BrO6 407.0130, found 407.0118.
The synthesis of 7 was previously reported.1 1H NMR (400 MHz, CDCl3): δ=7.81 (dd, J=1.2, 8.0 Hz, 1H), 7.72 (t, J=8.0 Hz, 1H), 7.32 (dd, J=1.2, 8.4 Hz, 1H), 5.25 (d, J=2.8 Hz, 1H), 4.87 (m, dd, J=3.2, 10.8 Hz, 1H), 4.60 (dd, J=3.2, 5.2 Hz, 1H), 4.03 (s, 3H), 2.94 (dd, J=5.2, 17.6 Hz, 1H), 2.69 (d, J=17.6 Hz, 1H), 1.83-1.81 (m, 1H), 1.66-1.57 (m, 3H), 1.00 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=182.7, 182.3, 174.3, 160.2, 150.6, 135.8, 133.8, 132.6, 119.7, 119.5, 118.3, 70.4, 69.1, 66.3, 56.7, 37.0, 33.7, 19.7, 13.6 ppm; HRMS (ESI) m/z [M+H]+ calcd for C19H19O6 343.1182, found 343.1180.
A solution of frenolicin B (10 mg, 0.03 mmol) in DMSO (1 mL) was added to HEPES buffer (8 mL, 50 mM, pH=9.5) in a dropwise fashion at room temperature. The resulting mixture was incubated at 37° C. overnight with brief shaking. After neutralization with 1N HCl to pH=7, the mixture was extracted with Et2O (10 mL×2). The collected organic layers were washed with brine, dried over Na2SO4, concentrated and purified on preparative HPLC (40%-100% CH3CN/H2O, 20 min, then 100% CH3CN, 5 min) to give 8 as a yellow solid (8 mg, 80%). 1H NMR (400 MHz, CDCl3): δ=11.91 (s, 1H), 7.65-7.63 (m, 2H), 7.28-7.62 (m, 1H), 4.86 (dd, J=2.0, 10.8 Hz, 1H), 4.69 (d, J=2.4 Hz, 1H), 4.31 (s, 1H), 3.49-3.46 (m, 1H), 2.91-2.89 (m, 2H), 2.65 (s, 1H), 1.74-1.65 (m, 6H), 1.02 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=189.0, 183.5, 175.4, 161.8, 146.6, 141.0, 136.8, 131.6, 125.0, 119.5, 114.9, 71.0, 67.1, 60.3, 35.4, 33.0, 19.8, 13.7 ppm; HRMS (ESI) m/z [M−H]− calcd for C18H17O7 345.0974, found 345.0971.
To a stirred solution of (3aR,5S,11bR)-6,7,11-trimethoxy-5-propyl-3,3a,5,11b-tetrahydro-2H-benzo[g]furo[3,2-c]isochromen-2-one (reported intermediate 6aα1) (93 mg, 0.25 mmol) in CH2Cl2 (2.5 mL) at −78° C. was added DIBAL-H (0.5 mL, 1 M in toluene). The reaction was quenched with saturated aqueous potassium sodium tartrate (1 mL) after 1 hour. The mixture was allowed to warm to room temperature with stirring, extracted with CH2Cl2 (10 mL×2), and the combined organics washed with brine, dried over Na2SO4, and concentrated. The residue was dissolved in CH2Cl2 containing trifluoroacetic acid (58 μL, 0.75 mmol) and cooled to −78° C. to which triethylsilane (119 μL, 0.75 mmol) was added in dropwise fashion. The resulting mixture was allowed to warm to room temperature with stirring overnight. After evaporating the volatiles, the residue was purified on silica gel using hexane/EtOAc (15/1) to obtain the tetrahydrofuran intermediate 9a (65 mg, 73% for two steps) as a colorless oil.
Following the above deprotection protocol for 1, 9a (65 mg, 0.18 mmol) was used to obtain 9 (28 mg, 50% yield). 1H NMR (400 MHz, CDCl3): δ=11.84 (s, 1H), 7.68-7.59 (m, 2H), 7.26-7.24 (m, 1H), 4.714.68 (m, 1H), 4.59 (t, J=2.0 Hz, 1H), 4.16-4.10 (m, 3H), 2.24-2.20 (m, 1H), 2.04-1.99 (m, 1H), 1.51-1.46 (m, 1H), 1.32-1.28 (m, 1H), 0.91 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=189.7, 182.7, 161.5, 148.0, 139.6, 136.7, 131.9, 124.4, 119.4, 115.1, 74.9, 72.2, 70.2, 67.7, 36.1, 33.6, 18.3, 14.2 ppm; HRMS (ESI) m/z [M+H]+ calcd for C18H19O5 315.1232, found 315.1224.
Following the above deprotection protocol for compound 1, (3aR,11bR)-6,7,11-trimethoxy-3,3a,5,11b-tetrahydro-2H-benzo[g]furo[3,2-c]isochromen-2-one (reported intermediate 141) (33 mg, 0.1 mmol) was used to obtain 10 (16 mg, 54% yield). 1H NMR (400 MHz, CDCl3): δ=11.71 (s, 1H), 7.70-7.68 (m, 2H), 7.30 (dd, J=2.0, 7.6 Hz, 1H), 5.26 (t, J=2.0 Hz, 1H), 4.95 (d, J=18.8 Hz, 1H), 4.47-4.39 (m, 2H), 2.95 (dd, J=2.0, 17.6 Hz, 1H), 2.76 (d, J=17.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ=188.0, 173.8, 161.9, 146.1, 137.5, 137.4, 135.8, 131.6, 124.9, 120.0, 114.7, 72.5, 69.1, 61.4, 37.0 ppm; HRMS (ESI) m/z [M+H]+ calcd for C15H11O6 287.0556, found 287.0546.
Following the above deprotection protocol for compound 1, (3aR,5S,11bR)-5-ethyl-6,7,11-trimethoxy-3,3a,5,11b-tetrahydro-2H-benzo[g]furo[3,2-c]isochromen-2-one (reported intermediate 6b1) (36 mg, 0.1 mmol) was used to obtain 11 (19 mg, 61% yield). 1H NMR (400 MHz, CDCl3): δ=11.75 (s, 1H), 7.68-7.66 (m, 2H), 7.30-7.28 (m, 1H), 5.27 (s, 1H), 4.74 (s, 1H), 4.34 (s, 1H), 2.90 (dd, J=4.0, 17.6 Hz, 1H), 2.75 (d, J=17.6 Hz, 1H), 2.14-2.03 (m, 2H), 0.89 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ=188.7, 181.5, 174.5, 161.8, 149.5, 137.2, 136.7, 131.5, 124.9, 119.7, 115.1, 72.8, 70.9, 69.8, 37.4, 27.1, 9.2 ppm; HRMS (ESI) m/z [M+H]+ calcd for C17H15O6 315.0869, found 315.0857.
To a solution of (4R,5R)-4-hydroxy-5-(1,4,5-trimethoxynaphthalen-2-yl)dihydrofuran-2(3H)-one (reported intermediate 51) (192 mg, 0.6 mmol) and 4,4,4-trifluorobutanal (152 mg, 1.2 mmol) in anhydrous CH2Cl2 at 0° C., Cu(OTf)2 (108 mg, 0.3 mmol) was added with stirring. The temperature was allowed to rise to room temperature and the mixture was stirred for 16 hours. After evaporating the volatiles, diastereoselectivity of the crude mixture was evaluated via NMR and then purified on silica gel using hexane/EtOAc (3/1-2/1) to give 12a as a colorless solid (200 mg, 81% yield, >20:1 dr ratio). 1H NMR (400 MHz, CDCl3): δ=7.73 (dd, J=1.2, 8.4 Hz, 2H), 7.46 (t, J=8.4 Hz, 1H), 6.95 (d, J=7.6 Hz, 1H), 5.58 (d, J=2.4 Hz, 1H), 5.12-5.10 (m, 1H), 4.37 (dd, J=2.4, 4.0 Hz, 1H), 4.09 (s, 3H), 4.02 (s, 3H), 3.75 (s, 3H), 2.92 (dd, J=4.4, 17.2 Hz, 1H), 2.77 (d, J=17.6 Hz, 1H), 2.632.60 (m, 1H), 2.31-2.20 (m, 2H), 2.06-2.02 (m, 1H); 13C NMR (100 MHz, CDCl3) δ=175.5, 156.3, 153.4, 149.6, 130.6, 127.0, 126.2, 126.0, 121.7, 119.4, 115.2, 107.7, 72.9, 71.9, 71.3, 64.6, 61.7, 56.4, 38.3, 29.6 (q), 27.7 ppm.
N-chlorosuccinamide (68 mg, 0.51 mmol) was added to a CH2Cl2 solution of 12a (200 mg, (0.47 mmol) at room temperature. The resulting mixture was heated to 80° C. with stirring for 24 hours. After evaporating the volatiles, the residue was purified on silica gel using hexane/EtOAc (5/1) to afford 12b (200 mg, 93% yield). 1H NMR (400 MHz, CDCl3): δ=7.48 (d, J=8.4 Hz, 2H), 6.83 (d, J=8.4 Hz, 1H), 5.58 (d, J=2.0 Hz, 1H), 5.12-5.09 (m, 1H), 4.35 (dd, J=2.4, 4.0 Hz, 1H), 3.98 (s, 3H), 3.97 (s, 3H), 3.7 (s, 3H), 2.92 (dd, J=4.4, 17.2 Hz, 1H), 2.76 (d, J=17.6 Hz, 1H), 2.62-2.56 (m, 1H), 2.28-2.24 (m, 2H), 2.06-2.00 (m, 1H); 13C NMR (100 MHz, CDCl3) δ=175.4, 171.2, 155.4, 153.2, 149.9, 130.2, 127.4, 126.8, 123.5, 122.0, 120.7, 107.7, 72.7, 71.6, 71.5, 65.7, 61.8, 56.7, 38.3, 29.5 (q), 28.0 ppm.
Following the above deprotection protocol for compound 1, 12b (200 mg, 0.43 mmol) was used to obtain 12 (120 mg, 67% yield). 1H NMR (400 MHz, CDCl3): δ=12.28 (s, 1H), 7.65 (d, J=9.2 Hz, 1H), 7.25 (d, J=9.2 Hz, 1H), 5.35 (t, J=1.6 Hz, 1H), 4.79-4.77 (m, 1H), 4.39 (dd, J=2.8, 4.4 Hz, 1H), 2.96 (dd, J=4.4, 17.6 Hz, 1H), 2.76 (d, J=17.6 Hz, 1H), 2.53-2.48 (m, 1H), 2.20-2.01 (m, 3H); 13C NMR (100 MHz, CDCl3) δ=187.7, 179.6, 173.8, 161.7, 146.6, 141.1, 137.9, 127.3, 126.7, 126.0, 115.9, 71.4, 70.3, 69.2, 37.1, 29.5 (q), 26.3 ppm; HRMS (ESI) m/z [M+NH4]+ calcd for C18H16ClF3NO6 434.0618, found 434.0615.
To a solution of 12 (84 mg, 0.2 mmol) and Na2CO3 (106 mg, 1 mmol) in acetone (1 mL) was added diethyl chlorophosphate (44 μL, 0.3 mmol). The resulting mixture was stirred at 35° C. for 6 hours. Upon completion, the reaction mixture was directly loaded to silica gel using hexane/EtOAc (1/1) to obtain 13 as a pale yellow liquid (100 mg, 91% yield). 1H NMR (400 MHz, CDCl3): δ=7.72 (d, J=9.2 Hz, 1H), 7.66 (d, J=9.2 Hz, 1H), 5.38 (t, J=2.0 Hz, 1H), 4.74-4.72 (m, 1H), 4.37-4.32 (m, 1H), 4.31-4.23 (m, 4H), 2.95 (dd, J=4.8, 17.6 Hz, 1H), 2.74 (d, J=17.6 Hz, 1H), 2.38-2.34 (m, 1H), 2.25-2.23 (m, 2H), 1.99-1.96 (m, 1H), 1.40-1.34 (m, 6H); 13C NMR (100 MHz, CDCl3) δ=182.2, 179.8, 173.8, 148.8, 148.2, 148.1, 138.5, 135.4, 131.3, 129.2, 128.3 (t), 125.7 (t), 71.8, 70.6, 68.7, 65.5 (d), 65.4 (d), 37.0, 29.7 (q), 25.6, 16.2, 16.1 ppm.
Iodotrimethylsilane (29 μL, 0.2 mmol) was added to a solution of 13 (100 mg, 0.18 mmol) in anhydrous CH2Cl2 (360 μL) with stirring under argon. The reaction was stirred at room temperature and monitored by HPLC analysis. Upon completion (typically 8-12 hours) the volatiles were evaporated and the residue was taken up in the mixture of Et2O (5 mL) and H2O (5 mL). The aqueous phase was collected and lyophilized to afford 14 as a yellow amorphous powder (64 mg, 72%). 1H NMR (400 MHz, DMSO-d6): δ=7.91 (d, J=8.8 Hz, 1H), 7.68 (dd, J=1.2, 9.2 Hz, 1H), 5.37 (t, J=2.0 Hz, 1H), 4.79-4.78 (m, 1H), 4.43 (dd, J=2.8, 4.8 Hz, 1H), 3.21 (dd, J=4.8, 17.6 Hz, 1H), 2.54 (d, J=17.6 Hz, 1H), 2.40-2.24 (m, 3H), 1.93-1.86 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ=181.7, 180.0, 175.2, 149.1, 148.8, 137.5, 134.8, 128.7, 127.8, 125.8, 125.7, 71.5, 69.7, 69.1, 36.5, 28.4 (q), 24.6 ppm; HRMS (ESI) m/z [M+H]+ calcd for C18H14ClF3O9P 497.0016, found 497.0008.
Cell Culture and Transfection.
Human colon (HCT116, DLD-1, T84, HCT15, RKO, SW620) and breast (MDA-MB-231) cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Va.) and cultured in the appropriate medium with supplements as recommended by ATCC. All the cell lines were tested for mycoplasma contamination via PCR (e-Myco Plus kit; iNtRON Biotechnology) and were found to be negative. In addition, all the cell lines are routinely checked for morphologic and growth changes, to probe for cross-contaminated, or genetically drifted cells. If any of these features occur, we use the short tandem repeat (STR) profiling service by ATCC to re-authenticate the cell lines. HCT116 cells with stable knockdown of 4E-BP1 and its control stable transfectants were generated as described previously {Ye, 2014 #1007}. For transient transfection, cells were transiently transfected with DNA using Lipofectamine 3000 according to the manufacturer's protocol (Life Technologies, Carlsbad, Calif.).
Generation of Stable Cells Using Lentiviral Infection.
The lentiviral-based shRNA (pLKO.1 plasmids) used to knock down expression of human Prx1 and Grx3, and the Non-Target Control shRNA (SHC002) were purchased from Sigma (St Louis, Mo.). On the basis of knockdown efficiency of Prx1 and Grx3 protein expression in HCT116 cells, we selected two shPrx1 and two shGrx3 clones for this study. The mature antisense sequences are as follows: 5′-GCTTT CAGTGATAGGGCAGAA-3′ (shPrx1_1) (SEQ ID NO: 3), 5′-GATGAGACTTTGAGACTAGTT-3′ (shPrx1_2) (SEQ ID NO: 4), 5′-CCTACCTATCCTCAGCTCTAT-3′ (shGrx3_1) (SEQ ID NO: 5), 5′-GAACGAAGTTATGGCAGAGTT-3′ (shGrx3_2) (SEQ ID NO: 6). To generate lentivirus-expressing shRNA for Prx1 and Grx3, we transfected 293T cells with pLKO.1-non-silence (for vector control virus), pLKO.1-shPrx1 or pLKO.1-shGrx3 with Lipofectamine 3000 transfection reagent. Twenty-four hours after transfection, the medium was changed, and then it was collected at 24-h intervals. The collected medium containing lentivirus was filtered through 0.45-mm filters. Cells were seeded at 50% confluence 24 h before infection, and the media were replaced with a medium containing lentivirus. After infection for 24 h, the medium was replaced with fresh medium and the infected cells were selected with 2 μg ml−1 puromycin for 7-10 days as described previously {Ye, 2014 #1007}.
Plasmids.
The human Prx1 and Grx3 were cloned into the pCMV6-Entry expression vector with C-terminal Myc-Flag Tag (PS100001, OriGene, Rockville, Md.) for transient transfection. Using the pCMV6-Prx1-Myc-Flag or pCMV6-Grx3-Myc-Flag as a template, the Prx1 mutant (C51A, C71A, C83A, C173A) and Grx3 mutant (C46A, C146A, C159A, C229A, C261A, C159A/C261A) constructs were generated using the QuikChange XLII site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). The primers used are listed in Supplementary Table 1. All constructs were confirmed using enzyme digestion and automated DNA sequencing.
Antibodies and Chemicals.
Antibodies for phospho-Akt (Ser473) (4060), phospho-p70S6 Kinase (Thr389) (9234), phospho-S6 (Ser235/236) (4858), phospho-4E-BP1 (Thr37/46) (2855), phospho-4E-BP1 (Ser65) (13443), phospho-4E-BP1 (Thr70) (13396), 4E-BP1 (9644), eIF4E (2067), Myc-tag (2276, 2278), cleaved caspase-3 (Asp175) (9661) and cleaved PARP (Asp214) (5625) were from Cell Signaling Technology (Danvers, Mass.). Peroxiredoxin 1 antibody (ab15571) was from Abcam (Cambridge, Mass.). PICOT (Grx3, sc-100601) antibody was from Santa Cruz Biotechnology (Dallas, Tex.). Biotin antibody (A150-109A) was from Bethyl Laboratories (Montgomery, Tex.) and β-actin antibody (A5411) was from Sigma. Rapamycin, AZD8055 and MK2206 were obtained from Selleck (Houston, Tex.).
Western Blot Analysis and Immunoprecipitation.
Cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol, protease and phosphatase inhibitor cocktail). Western blot analysis were performed using equivalent total protein loadings as described previously. For immunoprecipitation, the cell lysates were incubated with the indicated antibody overnight followed by incubation with a 50% slurry of protein G sepharose beads for 3 h at 4° C. The beads were washed three times with the lysis buffer, and the immunoprecipitated protein complexes were resuspended in 2× Laemmli sample buffer followed by western blot analysis.
Cap-Dependent Translation Assay.
Cells (8×104) were transfected with a bicistronic luciferase reporter plasmid (0.2 μg), pcDNA3-rLuc-PolioIRES-fLuc, which directs cap-dependent translation of the Renilla luciferase gene and cap-independent Polio IRES-mediated translation of the firefly luciferase gene. After 24 h transfection, cells were treated with indicated compounds for 12 h, and cell lysates were assayed for Renilla and firefly luciferase activities using a dual-luciferase assay kit (Promega, Madison, Wis.). Cap-dependent Renilla luciferase activity was normalized against cap-independent firefly luciferase activity as the internal control. The ratio of Renilla/firefly luciferase activity was calculated for cap-dependent translational activity as described previously. Each experiment was performed in triplicate and repeated at least three times.
Cap-Binding Assay.
Cap-binding assay was performed as described previously. Briefly, cell lysates (500 μg protein) as prepared in the NP-40 lysis buffer were incubated at 4° C. overnight with m7GTP Sepharose beads (GE Healthcare Life Sciences, Pittsburgh, Pa.) to capture eIF4E and its binding partners. Precipitates were washed three times with the lysis buffer and resuspended in 2× Laemmli sample buffer followed by western blot analysis.
Cell Growth and Apoptosis Assays.
Cell growth was assessed as described previously. Briefly, 5×104 cells were seeded in 6-well plates in triplicates. After 24 h, cells were treated with the indicated compounds and incubated at 37° C. The cells were cultured for 3 days and the number of viable cells was counted using the Vi-CELL XR 2.03 (Beckman Coulter, Brea, Calif.). For apoptosis, cells were analyzed by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's protocol (BD Biosciences, San Jose, Calif.).
Pull-Down and MS Analysis of FB-Bound Proteins.
To identify the target protein for FB, FB-based biotinylated active d7 and inactive d5 were synthesized as described in XXXX. HeLa cell pellets were purchased from National Cell Culture Center (Minneapolis, Minn.) and lysed in 20 ml of the NP-40 lysis buffer. The cell lysates (250 mg protein) were pre-cleared with 200 μl streptavidin beads (20353, Thermo Fisher Scientific, Grand Island, N.Y.) at 4° C. for 1 h. Binding reactions were performed by incubating the pre-cleared cell lysates (125 mg proteins/40 ml) with 2 μM d5 or d7 at 4° C. for 3 h, followed by adding 100 μl streptavidin beads and incubating the mixtures overnight at 4° C. After incubation, the beads were washed four times with the lysis buffer, and the bead-bound proteins were eluted, resolved by SDS-PAGE, and visualized by Coomassie blue staining. The protein-containing band in the gel was excised, followed by in-gel digestion and analysis by LC-MS/MS.
Immunofluorescence.
Cells grown on glass bottom culture dishes were incubated with 25 μM d5, 25 μM d7 or DMSO as control for 5 h. After treatment, cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized in 0.2% Triton X-100 and 0.5% BSA in PBS for 5 min and then blocked with 4% BSA in PBS for 10 min. The cells were incubated overnight at 4° C. with the rabbit polyclonal antibody against Prx1 (1:200, ab15571, Abcam) and mouse monoclonal anti-Biotin-FITC (1:200, 200-092-211, Jackson ImmunoResearch, West Grove, Pa.), or with the mouse monoclonal antibody against Grx3 (1:200, MAB7560, R&D Systems, Minneapolis, Minn.) and rabbit polyclonal anti-Biotin-FITC (1:200, ab53469, Abcam). After three washes with 0.05% Triton X-100 in PBS, cells were incubated with anti-rabbit secondary antibody conjugated with Texas-Red for Prx1 (1:500, 111-585-144, Jackson ImmunoResearch) or anti-mouse secondary antibody conjugated with Texas-Red for Grx3 (1:500, 111-585-144, Jackson ImmunoResearch) for 1 h. Cells were washed, mounted with UltraCruz DAPI containing mounting medium (Santa Cruz Biotechnology), viewed, and photographed under a FluoView 1200 confocal microscope (Olympus, Center Valley, Pa.).
Measurement of Cellular ROS and H2O2 Production.
The ROS production was determined using the CellROX Deep Red Flow Cytometry Assay Kit according to the manufacturer's protocol (Life Technologies). Briefly, cells were treated with 2 μM of the indicated compounds or DMSO as control for 1 h. After treatment, cells were incubated with 0.5 μM of CellROX Deep Red reagent for 1 h at 37° C., washed twice with PBS and immediately analyzed by a FACScan flow cytometer. For the measurement of cellular H2O2 level, cells were treated with 2 μM of the indicated compounds for 5 h.
Cellular Glutathione Assay.
Cells were treated with 2 μM of the indicated compounds or DMSO as control for 5 h. After treatment, a total number of 1×106 cells were lysed in 100 μl of ice-cold NP-40 lysis buffer for 10 min. The lysate was centrifuged for 10 min and the supernatant was used for glutathione assay using the ApoGSH glutathione detection kit according to the manufacturer's protocol (BioVision Research Products, Mountain view, CA). The total amount of GSH was measured using a fluorescence plate reader at Ex./Em.=380/460 nm.
Quantification of Glutathione Disulfide (GSSG).
Cells were treated with 2 μM of the indicated compounds or DMSO as control for 5 h. Quantification of GSSG was essentially performed using the manufactures instructions for a microplate assay for GSH/GSSG (Oxford Biomedical Research, Inc, Oxford, Mich.). A total number of 0.5×106 cells were collected in 1.5 ml centrifuge tubes containing ice-cold buffer with the thiol scavenger to keep GSSG in its oxidized form. The cells were homogenized with a Teflon pestle and the cell suspension sonicated in icy water for 2-3 minutes. Ice-cold metaphosphoric acid was added to deproteinate the samples. The samples were centrifuged at 1000×g at 4° C. and the supernatants were used for determining the GSSG concentration according to the manufactures protocol using a microplate reader with 405 nm filter. The change in GSSG levels in the indicated compound-treated samples was expressed as fold change compared to control (DMSO) treated samples.
Animal Experiments.
Male athymic nude mice (5-6 weeks old) were purchased from Taconic (Hudson, N.Y., USA). Experiments were carried out under a protocol approved by the University of Kentucky Institutional Animal Care and Use Committee. HCT116 and DLD-1 xenograft tumors were established by subcutaneously injecting 3×106 cells in a 1:1 mixture of media and Matrigel (BD Biosciences) into the right flank. For efficacy studies, mice were randomized among control and treated groups (n=8 per group) when tumors were well-established (˜120 mm3). Compound 14 was prepared freshly in saline and administered by intraperitoneal injection at 14 mg/kg once per day, Mon-Fri per week. Control mice received saline solution. Tumor dimensions were measured using a caliper and tumor volumes were calculated as mm3=π/6× larger diameter×(smaller diameter)2. Tumors were excised and snap frozen in liquid nitrogen, homogenized in 2% SDS lysis buffer and then processed for Western blot analysis as described previously5.
Statistical Analysis.
Results are expressed as the mean±s.e.m. where applicable. A two-tailed Student's t-test was used to compare between groups as outlined in each legend. Differences between groups were considered statistically significant at P<0.05.
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It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/371,123, filed Aug. 4, 2016, the entire disclosure of which is incorporated herein by this reference.
This invention was made with government support under grant numbers CA203257, CA175105, T32 DA016176, and CCTS UL1TRO00117 awarded by the National Institutes of Health (NIH), and grant number P30 CA177558 awarded by the National Cancer Institute (NCI). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62371123 | Aug 2016 | US |
