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1.
Brain Commun ; 2(1): fcaa002, 2020.
Article in English | MEDLINE | ID: mdl-32954276

ABSTRACT

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood-brain barrier-permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.

2.
Cancer Res ; 78(8): 2065-2080, 2018 04 15.
Article in English | MEDLINE | ID: mdl-29358171

ABSTRACT

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065-80. ©2018 AACR.


Subject(s)
Enzyme Activators/therapeutic use , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/enzymology , Protein Phosphatase 2C/drug effects , Small Molecule Libraries/therapeutic use , Animals , Cell Line, Tumor , Enzyme Activators/pharmacology , Heterografts , Humans , Male , Mice , Mice, SCID , Phosphoproteins/metabolism , Protein Phosphatase 2C/metabolism , Proteomics , RNA, Messenger/genetics , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Small Molecule Libraries/pharmacology
3.
J Clin Invest ; 127(6): 2081-2090, 2017 Jun 01.
Article in English | MEDLINE | ID: mdl-28504649

ABSTRACT

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Activators/pharmacology , Protein Phosphatase 2/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm , Enzyme Activation , Enzyme Activators/chemistry , Humans , Male , Mice, Inbred BALB C , Mice, Nude , Mice, Transgenic , Protein Binding , Protein Phosphatase 2/chemistry , Signal Transduction , Tumor Burden , Xenograft Model Antitumor Assays
4.
FEBS J ; 283(6): 1004-24, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26507691

ABSTRACT

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in the regulation of many cellular processes. A confirmed tumor suppressor protein, PP2A is genetically altered or functionally inactivated in many cancers highlighting a need for its therapeutic reactivation. In this review we discuss recent literature on PP2A: the elucidation of its structure and the functions of its subunits, and the identification of molecular lesions and post-translational modifications leading to its dysregulation in cancer. A final section will discuss the proteins and small molecules that modulate PP2A and how these might be used to target dysregulated forms of PP2A to treat cancers and other diseases.


Subject(s)
Protein Phosphatase 2/chemistry , Protein Phosphatase 2/metabolism , Catalytic Domain , Enzyme Inhibitors/pharmacology , Gene Deletion , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Models, Molecular , Mutation , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/therapy , Phosphorylation , Protein Phosphatase 2/genetics , Protein Processing, Post-Translational , Protein Subunits , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
5.
J Biol Chem ; 291(8): 4079-90, 2016 Feb 19.
Article in English | MEDLINE | ID: mdl-26703475

ABSTRACT

We previously showed that an HLA-DR variant containing arginine at position 74 of the DRß1 chain (DRß1-Arg74) is the specific HLA class II variant conferring risk for autoimmune thyroid diseases (AITD). We also identified 5 thyroglobulin (Tg) peptides that bound to DRß1-Arg74. We hypothesized that blocking the binding of these peptides to DRß1-Arg74 could block the continuous T-cell activation in thyroiditis needed to maintain the autoimmune response to the thyroid. The aim of the current study was to identify small molecules that can block T-cell activation by Tg peptides presented within DRß1-Arg74 pockets. We screened a large and diverse library of compounds and identified one compound, cepharanthine that was able to block peptide binding to DRß1-Arg74. We then showed that Tg.2098 is the dominant peptide when inducing experimental autoimmune thyroiditis (EAT) in NOD mice expressing human DRß1-Arg74. Furthermore, cepharanthine blocked T-cell activation by thyroglobulin peptides, in particular Tg.2098 in mice that were induced with EAT. For the first time we identified a small molecule that can block Tg peptide binding and presentation to T-cells in autoimmune thyroiditis. If confirmed cepharanthine could potentially have a role in treating human AITD.


Subject(s)
Alkaloids/pharmacology , Antigen Presentation/drug effects , HLA-DRB1 Chains/immunology , Thyroiditis, Autoimmune/immunology , Alkaloids/chemistry , Animals , HLA-DRB1 Chains/genetics , Humans , Lymphocyte Activation/drug effects , Mice , Mice, Transgenic , Peptides/genetics , Peptides/immunology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Thyroglobulin/genetics , Thyroglobulin/immunology , Thyroiditis, Autoimmune/genetics , Thyroiditis, Autoimmune/pathology
6.
Bioorg Med Chem ; 23(19): 6528-34, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-26372073

ABSTRACT

The phenothiazine and dibenzazepine tricyclics are potent neurotropic drugs with a documented but underutilized anti-cancer side effect. Reengineering these agents (TFP, CPZ, CIP) by replacing the basic amine with a neutral polar functional group (e.g., RTC-1, RTC-2) abrogated their CNS effects as demonstrated by in vitro pharmacological assays and in vivo behavioral models. Further optimization generated several phenothiazines and dibenzazepines with improved anti-cancer potency, exemplified by RTC-5. This new lead demonstrated efficacy against a xenograft model of an EGFR driven cancer without the neurotropic effects exhibited by the parent molecules. Its effects were attributed to concomitant negative regulation of PI3K-AKT and RAS-ERK signaling.


Subject(s)
Antineoplastic Agents/chemistry , Heterocyclic Compounds, 3-Ring/chemistry , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Survival/drug effects , Dibenzazepines/chemistry , Heterocyclic Compounds, 3-Ring/pharmacology , Heterocyclic Compounds, 3-Ring/therapeutic use , Humans , Mice , Neoplasms/drug therapy , Neoplasms/metabolism , Phenothiazines/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Transplantation, Heterologous , Vesicular Monoamine Transport Proteins/antagonists & inhibitors , Vesicular Monoamine Transport Proteins/metabolism
8.
Antimicrob Agents Chemother ; 56(4): 1797-809, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22252828

ABSTRACT

SQ109, a 1,2-diamine related to ethambutol, is currently in clinical trials for the treatment of tuberculosis, but its mode of action remains unclear. Here, we demonstrate that SQ109 disrupts cell wall assembly, as evidenced by macromolecular incorporation assays and ultrastructural analyses. SQ109 interferes with the assembly of mycolic acids into the cell wall core of Mycobacterium tuberculosis, as bacilli exposed to SQ109 show immediate inhibition of trehalose dimycolate (TDM) production and fail to attach mycolates to the cell wall arabinogalactan. These effects were not due to inhibition of mycolate synthesis, since total mycolate levels were unaffected, but instead resulted in the accumulation of trehalose monomycolate (TMM), the precursor of TDM and cell wall mycolates. In vitro assays using purified enzymes showed that this was not due to inhibition of the secreted Ag85 mycolyltransferases. We were unable to achieve spontaneous generation of SQ109-resistant mutants; however, analogs of this compound that resulted in similar shutdown of TDM synthesis with concomitant TMM accumulation were used to spontaneously generate resistant mutants that were also cross-resistant to SQ109. Whole-genome sequencing of these mutants showed that these all had mutations in the essential mmpL3 gene, which encodes a transmembrane transporter. Our results suggest that MmpL3 is the target of SQ109 and that MmpL3 is a transporter of mycobacterial TMM.


Subject(s)
Adamantane/analogs & derivatives , Antitubercular Agents/pharmacology , Bacterial Proteins/drug effects , Cell Wall/metabolism , Cord Factors/metabolism , Ethylenediamines/pharmacology , Membrane Transport Proteins/drug effects , Mycobacterium tuberculosis/metabolism , Mycolic Acids/metabolism , Acyltransferases/metabolism , Adamantane/pharmacology , Aerobiosis , Antigens, Bacterial/metabolism , Cell Wall/drug effects , Chromatography, Thin Layer , Drug Resistance, Bacterial/genetics , Lipid Metabolism , Microbial Sensitivity Tests , Microscopy, Electron , Mutation/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/ultrastructure
9.
Bioorg Med Chem Lett ; 20(9): 2722-5, 2010 May 01.
Article in English | MEDLINE | ID: mdl-20381346

ABSTRACT

The synthesis and evaluation of a key series of analogs of duocarmycin SA, bearing a single substituent at the C5' position of the DNA binding subunit, are described.


Subject(s)
Alkylating Agents/chemical synthesis , Indoles/chemistry , Alkylating Agents/chemistry , Alkylating Agents/toxicity , Animals , Cell Line, Tumor , DNA/chemistry , Duocarmycins , Indoles/chemical synthesis , Indoles/toxicity , Mice , Pyrroles/chemical synthesis , Pyrroles/chemistry , Pyrroles/toxicity , Structure-Activity Relationship
10.
Tetrahedron Lett ; 51(1): 197-200, 2010.
Article in English | MEDLINE | ID: mdl-20161438

ABSTRACT

A concise synthesis of (14)C labeled meropenem prepared from (14)C dimethylamine hydrochloride is described. Using a similar reaction sequence, the meropenem nucleus was also attached to biotin providing a probe for protein interaction studies.

11.
J Am Chem Soc ; 128(49): 15683-96, 2006 Dec 13.
Article in English | MEDLINE | ID: mdl-17147378

ABSTRACT

Complementary to studies that provided the first yatakemycin total synthesis resulting in its structure revision and absolute stereochemistry assignment, a second-generation asymmetric total synthesis is disclosed herein. Since the individual yatakemycin subunits are identical to those of duocarmycin SA (alkylation subunit) or CC-1065 (central and right-hand subunits), the studies also provide an improvement in our earlier total synthesis of CC-1065 and, as detailed herein, have been extended to an asymmetric total synthesis of (+)-duocarmycin SA. Further extensions of the studies provided key yatakemycin partial structures and analogues for comparative assessments. This included the definition of the DNA selectivity (adenine central to a five-base-pair AT sequence, e.g., 5'-AAAAA), efficiency, relative rate, and reversibility of ent-(-)-yatakemycin and its comparison with the natural enantiomer (identical selectivity and efficiency), structural characterization of the adenine N3 adduct confirming the nature of the DNA reaction, and comparisons of the cytotoxic activity of the natural product (L1210, IC50 = 5 pM) with those of its unnatural enantiomer (IC50 = 5 pM) and a series of key partial structures including those that probe the role of the C-terminus thiomethyl ester. The only distinguishing features between the enantiomers is that ent-(-)-yatakemycin alkylates DNA at a slower rate (krel = 0.13) and is reversible, whereas (+)-yatakemycin is not. Nonetheless, even ent-(-)-yatakemycin alkylates DNA at a faster rate and with a greater thermodynamic stability than (+)-duocarmycin SA, illustrating the unique characteristics of such "sandwiched" agents.


Subject(s)
Alkylating Agents/chemical synthesis , Antibiotics, Antineoplastic/chemical synthesis , DNA/chemistry , Indoles/chemical synthesis , Pyrroles/chemical synthesis , Streptomyces/chemistry , Adenine/chemistry , Alkylation , Base Pairing , Base Sequence , Duocarmycins , Hydrogen-Ion Concentration , Kinetics , Models, Chemical , Stereoisomerism , Temperature
12.
J Am Chem Soc ; 126(27): 8396-8, 2004 Jul 14.
Article in English | MEDLINE | ID: mdl-15237994

ABSTRACT

The total synthesis of the reported structure 2 for yatakemycin, an exceptionally potent, naturally occurring antitumor agent disclosed in 2003, and its lack of correlation with the natural product are detailed. On the basis of spectroscopic distinctions between 2 and yatakemycin, the natural product structure was reformulated as 3, now bearing a thiomethyl ester versus thioacetate in the left-hand subunit. Total synthesis of 3 provided a compound nearly identical to but still subtly distinct from the natural product. A second reformulation of the yatakemycin structure as 1, incorporating the alternatively substituted right-hand subunit as well as the initial thiomethyl ester reformulation, was confirmed by total synthesis of both (+)- and ent-(-)-1 in studies that also unambiguously established the absolute configuration of the natural product.


Subject(s)
Indoles/chemistry , Indoles/chemical synthesis , Pyrroles/chemistry , Pyrroles/chemical synthesis , Duocarmycins , Stereoisomerism , Streptomyces/chemistry
13.
J Org Chem ; 69(7): 2284-9, 2004 Apr 02.
Article in English | MEDLINE | ID: mdl-15049620

ABSTRACT

A short, asymmetric synthesis of the 1,2,9,9a-tetrahydrocyclopropa[c]benzo[e]indol-4-one (CBI) analogue of the CC-1065 and duocarmycin alkylation subunits is detailed that employs an effective enzymatic desymmetrization reaction of prochiral diol 12 using a commercially available Pseudomonas sp. lipase. The optically active monoacetate (S)-13 is furnished in exceptional conversions (88%) and optical purity (99% ee) and serves as an intermediate for the preparation of either enantiomer of CBI. Similarly, the Pseudomonas sp. lipase resolved the racemic intermediate 19, affording advanced intermediates of CBI in good conversions and optical purity (99% ee), and provided an alternative approach to the preparation of optically active CBI derivatives.


Subject(s)
Antibiotics, Antineoplastic/chemical synthesis , Combinatorial Chemistry Techniques , Cyclopropanes/chemical synthesis , Indoles/chemistry , Indoles/chemical synthesis , Alkylation , Antibiotics, Antineoplastic/analysis , Catalysis , Cyclopropanes/analysis , Duocarmycins , Indoles/analysis , Lipase/metabolism , Molecular Structure , Pseudomonas/enzymology , Stereoisomerism
14.
J Org Chem ; 68(23): 8984-90, 2003 Nov 14.
Article in English | MEDLINE | ID: mdl-14604371

ABSTRACT

An efficient eight-step synthesis (53% overall) and the evaluation of 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]-3-azaindol-4-one (CBA) and its derivatives containing an aza variant of the CC-1065/duocarmycin alkylation subunit are detailed. This unique deep-seated aza modification provided an unprecedented 2-aza-4,4-spirocyclopropacyclohexadienone that was characterized chemically and structurally (X-ray). CBA proved structurally identical with CBI, the carbon analogue, including the stereoelectronic alignment of the key cyclopropane, its bond lengths, and the bond length of the diagnostic C3a-N2 bond, reflecting the extent of vinylogous amide (amidine) conjugation. Despite these structural similarities, CBA and its derivatives were found to be much more reactive toward solvolysis and hydrolysis, much less effective DNA alkylating agents (1000-fold), and biologically much less potent (100- to 1000-fold) than the corresponding CBI derivatives.


Subject(s)
Cyclopropanes/chemical synthesis , Indoles/chemical synthesis , Pyrrolidinones/chemical synthesis , Alkylation , Crystallography, X-Ray , Cyclopropanes/chemistry , Duocarmycins , Indoles/chemistry , Molecular Structure , Pyrrolidinones/chemistry
15.
J Am Chem Soc ; 125(36): 10971-6, 2003 Sep 10.
Article in English | MEDLINE | ID: mdl-12952479

ABSTRACT

Yatakemycin represents the newest and now most potent member of a class of naturally occurring antitumor compounds that includes CC-1065 and the duocarmycins, which derive their biological properties from a characteristic DNA alkylation reaction. Herein, the first description of the yatakemycin DNA alkylation properties is detailed, constituting the first such study of a naturally occurring "sandwiched" member of this class. Thus, the event, sequence selectivity, relative rate and efficiency, and reversibility of the DNA alkylation reaction of yatakemycin are described.


Subject(s)
DNA/metabolism , Indoles/pharmacology , Pyrroles/pharmacology , Alkylation/drug effects , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , DNA/chemistry , DNA/drug effects , Duocarmycins , Indoles/chemistry , Kinetics , Models, Molecular , Pyrroles/chemistry , Stereoisomerism
16.
Bioorg Med Chem ; 11(17): 3815-38, 2003 Aug 15.
Article in English | MEDLINE | ID: mdl-12901927

ABSTRACT

An extensive series of CBI analogues of the duocarmycins and CC-1065 exploring substituent effects within the first indole DNA binding subunit is detailed. In general, substitution at the indole C5 position led to cytotoxic potency enhancements that can be >/=1000-fold providing simplified analogues containing a single DNA binding subunit that are more potent (IC(50)=2-3 pM) than CBI-TMI, duocarmycin SA, or CC-1065.


Subject(s)
DNA/metabolism , Indoles/chemistry , Indoles/toxicity , Pyrroles/chemistry , Pyrrolidinones/chemistry , Alkylation , Amides/chemical synthesis , Amides/chemistry , DNA/drug effects , Duocarmycins , Indoles/chemical synthesis , Indoles/metabolism , Kinetics , Models, Molecular , Pyrroles/chemical synthesis , Pyrroles/metabolism , Pyrrolidinones/chemical synthesis , Pyrrolidinones/metabolism , Sulfones/chemical synthesis , Sulfones/chemistry
17.
Org Lett ; 5(14): 2577-9, 2003 Jul 10.
Article in English | MEDLINE | ID: mdl-12841785

ABSTRACT

[structure: see text] An efficient eight-step synthesis (54% overall) and the subsequent X-ray characterization of 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]-3-azaindol-4-one (CBA) containing an aza variant of the CC-1065/duocarmycin alkylation subunit are detailed. Despite the unique deep-seated aza modification providing an unprecedented and stable 2-aza-4,4-spirocyclopropacyclohexadienone, CBA proved to be structurally identical with CBI, the carbon analogue, in terms of the stereoelectronic alignment of the key cyclopropane, its bond lengths, and the length of the diagnostic C3a-N2 bond reflecting the extent of vinylogous amide conjugation.


Subject(s)
Cyclohexanes/chemistry , Spiro Compounds/chemistry , Spiro Compounds/chemical synthesis , Crystallography, X-Ray , Cyclohexanes/chemical synthesis
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