ABSTRACT
Inhibition of Poly(ADP-ribose) Polymerase1 (PARP1) impairs DNA damage repair, and early generation PARP1/2 inhibitors (olaparib, niraparib, etc.) have demonstrated clinical proof of concept for cancer treatment. Here, we describe the development of the novel PARP inhibitor E7449, a potent PARP1/2 inhibitor that also inhibits PARP5a/5b, otherwise known as tankyrase1 and 2 (TNKS1 and 2), important regulators of canonical Wnt/ß-catenin signaling. E7449 inhibits PARP enzymatic activity and additionally traps PARP1 onto damaged DNA; a mechanism previously shown to augment cytotoxicity. Cells deficient in DNA repair pathways beyond homologous recombination were sensitive to E7449 treatment. Chemotherapy was potentiated by E7449 and single agent had significant antitumor activity in BRCA-deficient xenografts. Additionally, E7449 inhibited Wnt/ß-catenin signaling in colon cancer cell lines, likely through TNKS inhibition. Consistent with this possibility, E7449 stabilized axin and TNKS proteins resulting in ß-catenin de-stabilization and significantly altered expression of Wnt target genes. Notably, hair growth mediated by Wnt signaling was inhibited by E7449. A pharmacodynamic effect of E7449 on Wnt target genes was observed in tumors, although E7449 lacked single agent antitumor activity in vivo, a finding typical for selective TNKS inhibitors. E7449 antitumor activity was increased through combination with MEK inhibition. Particularly noteworthy was the lack of toxicity, most significantly the lack of intestinal toxicity reported for other TNKS inhibitors. E7449 represents a novel dual PARP1/2 and TNKS1/2 inhibitor which has the advantage of targeting Wnt/ß-catenin signaling addicted tumors. E7449 is currently in early clinical development.
Subject(s)
Cell Proliferation/drug effects , DNA Repair/drug effects , Isoquinolines/pharmacology , Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Quinazolinones/pharmacology , Wnt Signaling Pathway/drug effects , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Azo Compounds , Blotting, Western , Carboplatin/administration & dosage , Cell Line, Tumor , Cell Proliferation/genetics , DNA Repair/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dacarbazine/administration & dosage , Dacarbazine/analogs & derivatives , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/drug effects , Isoquinolines/chemistry , Mice, Inbred C57BL , Mice, Nude , Mice, SCID , Neoplasms/genetics , Neoplasms/pathology , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Poly(ADP-ribose) Polymerase Inhibitors/administration & dosage , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Quinazolinones/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Tankyrases/antagonists & inhibitors , Tankyrases/genetics , Tankyrases/metabolism , Temozolomide , Tumor Burden/drug effects , Wnt Signaling Pathway/genetics , Xenograft Model Antitumor AssaysABSTRACT
10-((4-Hydroxypiperidin-1-yl)methyl)chromeno[4,3,2-de]phthalazin-3(2H)-one (E7016), an inhibitor of poly(ADP-ribose) polymerase, is being developed for anticancer therapy. One of the major metabolites identified in preclinical animal studies was the product of an apparent oxidation and ring opening of the 4-hydroxypiperidine. In vitro, this oxidized metabolite could not be generated by incubating E7016 with animal or human liver microsomes. Further studies revealed the formation of this unique metabolite in hepatocytes. In a NAD(P)(+)-dependent manner, this metabolite was also generated by liver S9 fractions and recombinant human flavin-containing monooxygenase (FMO) 5 that was fortified with liver cytosol fractions. In animal and human liver S9, this metabolic pathway could be inhibited by 4-methylpyrazole, bis-p-nitrophenylphosphate (BNPP), or a brief heat treatment at 50°C. Based on these results, the overall metabolic pathway was believed to involve a two-step oxidation process: dehydrogenation of the secondary alcohol in liver cytosol followed by an FMO5-mediated Baeyer-Villiger oxidation in liver microsomes. The two oxidation steps were coupled via regeneration of NAD(P)(+) and NAD(P)H. To further confirm this mechanism, the proposed ketone intermediate was independently synthesized. In an NAD(P)H-dependent manner, the synthetic ketone intermediate was metabolized to the same ring-opened metabolite in animal and human liver microsomes. This metabolic reaction was also inhibited by BNPP or a brief heat treatment at 50°C. Methimazole, the substrate/inhibitor of FMO1 and FMO3, did not inhibit this reaction. The specificity of FMO5 toward catalyzing this Baeyer-Villiger oxidation was further demonstrated by incubating the synthetic ketone intermediate in recombinant enzymes.
Subject(s)
Benzopyrans/metabolism , Hepatocytes/metabolism , Liver/metabolism , Microsomes, Liver/metabolism , Oxygenases/metabolism , Phthalazines/metabolism , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Benzopyrans/chemistry , Benzopyrans/pharmacology , Biocatalysis , Dogs , Female , Hepatocytes/enzymology , Humans , Liver/enzymology , Male , Microsomes, Liver/enzymology , Oxidation-Reduction , Phthalazines/chemistry , Phthalazines/pharmacology , RatsABSTRACT
The separation of R,R-, S,S-, and meso-Koga bases on derivatized amylose chiral stationary phases (CSP) has been studied using different alcohol and alcohol-hexane mixtures as eluant. Straight-chain and branched alcohols with carbon numbers from one to four were investigated. The carbon number and geometry of the alcohol impacts the separation of Koga bases. The optimal separations were obtained using a mixture of methanol with linear or branched alcohol. Also, the elution order of meso- and R,R-Koga base was switched as content of branched alcohol increases in cosolvent. The study of acidic and basic additive effects demonstrated that maintaining analytes in the free base state is crucial in order to achieve retention and separation. TEA alone or TEA and TFA mixture were used in the studies.
Subject(s)
Alkalies/chemistry , Amylose/chemistry , Chromatography, High Pressure Liquid/instrumentation , Solvents/chemistry , Acids/chemistry , StereoisomerismABSTRACT
A convergent synthesis of the marine natural product (+)-peloruside has been reported. This target has been assembled through the successive application of two methyl ketone boron aldol addition reactions to the latent C(7)-C(11) dialdehyde synthon. This approach afforded a 22-step synthesis of this natural product. The influence of resident stereocenters on aldol reaction diastereoselection has been examined in detail.
