Specific stereochemistry of OP-1074 disrupts estrogen receptor alpha helix 12 and confers pure antiestrogenic activity.

Complex tissue-specific and cell-specific signaling by the estrogen receptor (ER) frequently leads to the development of resistance to endocrine therapy for breast cancer. Pure ER antagonists, which completely lack tissue-specific agonist activity, hold promise for preventing and treating endocrine resistance, however an absence of structural information hinders the development of novel candidates. Here we synthesize a small panel of benzopyrans with variable side chains to identify pure antiestrogens in a uterotrophic assay. We identify OP-1074 as a pure antiestrogen and a selective ER degrader (PA-SERD) that is efficacious in shrinking tumors in a tamoxifen-resistant xenograft model. Biochemical and crystal structure analyses reveal a structure activity relationship implicating the importance of a stereospecific methyl on the pyrrolidine side chain of OP-1074, particularly on helix 12.

Recent advances in the discovery of small molecule therapies for HCV.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. This virus, first identified in the late-1980s, has been the target of aggressive efforts by the pharmaceutical industry to identify novel therapeutic agents. Current therapy requires the use of interferon-alpha in conjunction with ribavirin. Regrettably, the utility of this therapy is limited by both an unsuitable side effect profile and low efficacy. These shortcomings have maintained the pressure to identify more effective therapies. Several compounds that take advantage of different strategies for achieving an antiviral effect are currently in clinical trials. Several more classes of novel antiviral agents have been identified using a range of in vitro assays. The recently developed stable cell-based assay for HCV will be a highly useful tool in on-going discovery efforts.

Yeast and rat Coq3 and Escherichia coli UbiG polypeptides catalyze both O-methyltransferase steps in coenzyme Q biosynthesis.

Ubiquinone (coenzyme Q or Q) is a lipid that functions in the electron transport chain in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. Q-deficient mutants of Saccharomyces cerevisiae harbor defects in one of eight COQ genes (coq1-coq8) and are unable to grow on nonfermentable carbon sources. The biosynthesis of Q involves two separate O-methylation steps. In yeast, the first O-methylation utilizes 3, 4-dihydroxy-5-hexaprenylbenzoic acid as a substrate and is thought to be catalyzed by Coq3p, a 32.7-kDa protein that is 40% identical to the Escherichia coli O-methyltransferase, UbiG. In this study, farnesylated analogs corresponding to the second O-methylation step, demethyl-Q(3) and Q(3), have been chemically synthesized and used to study Q biosynthesis in yeast mitochondria in vitro. Both yeast and rat Coq3p recognize the demethyl-Q(3) precursor as a substrate. In addition, E. coli UbiGp was purified and found to catalyze both O-methylation steps. Futhermore, antibodies to yeast Coq3p were used to determine that the Coq3 polypeptide is peripherally associated with the matrix-side of the inner membrane of yeast mitochondria. The results indicate that one O-methyltransferase catalyzes both steps in Q biosynthesis in eukaryotes and prokaryotes and that Q biosynthesis is carried out within the matrix compartment of yeast mitochondria.

Characterization of the COQ5 gene from Saccharomyces cerevisiae. Evidence for a C-methyltransferase in ubiquinone biosynthesis.

Ubiquinone (coenzyme Q or Q) is a lipophilic metabolite that functions in the electron transport chain in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes. Q-deficient mutants of Saccharomyces cerevisiae fall into eight complementation groups (coq1-coq8). Yeast mutants from the coq5 complementation group lack Q and as a result are respiration-defective and fail to grow on nonfermentable carbon sources. A nuclear gene, designated COQ5 was isolated from a yeast genomic library based on its ability to restore growth of a representative coq5 mutant on media containing glycerol as the sole carbon source. The DNA segment responsible for the complementation contained an open reading frame (GenBankTM accession number Z49210Z49210) with 44% sequence identity over 262 amino acids to UbiE, which is required for a C-methyltransferase step in the Q and menaquinone biosynthetic pathways in Escherichia coli. Both the ubiE and COQ5 coding sequences contain sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases. A gene fusion expressing a biotinylated form of Coq5p retains function, as assayed by the complementation of the coq5 mutant. This Coq5-biotinylated fusion protein is located in mitochondria. The synthesis of two farnesylated analogs of intermediates in the ubiquinone biosynthetic pathway is reported. These reagents have been used to develop in vitro C-methylation assays with isolated yeast mitochondria. These studies show that Coq5p is required for the C-methyltransferase step that converts 2-methoxy-6-polyprenyl-1, 4-benzoquinone to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone.

Complementation of coq3 mutant yeast by mitochondrial targeting of the Escherichia coli UbiG polypeptide: evidence that UbiG catalyzes both O-methylation steps in ubiquinone biosynthesis.

Ubiquinone functions in the mitochondrial electron transport chain. Recent evidence suggests that the reduced form of ubiquinone (ubiquinol) may also function as a lipid soluble antioxidant. The biosynthesis of ubiquinone requires two O-methylation steps. In eukaryotes, the first O-methylation step is carried out by the Coq3 polypeptide, which catalyzes the transfer of a methyl group from S-adenosylmethionine to 3,4-dihydroxy-5-polyprenylbenzoate. In Escherichia coli, 2-polyprenyl-6-hydroxyphenol is the predicted substrate; however, the corresponding O-methyltransferase has not been identified. The second O-methylation step in E. coli, the conversion of demethylubiquinone to ubiquinone, is carried out by the UbiG methyltransferase, which is 40% identical in amino acid sequence with the yeast Coq3 methyltransferase. On the basis of the chemical similarity of the first and last methyl-acceptor substrates and the high degree of amino acid sequence identity between Coq3p and UbiG, the ability of UbiG to catalyze both O-methylation steps was investigated. The current study shows that the ubiG gene is able to restore respiration in the yeast coq3 mutant, provided ubiG is modified to contain a mitochondrial leader sequence. The mitochondrial targeting of O-methyltransferase activity is an essential feature of the ability to restore respiration and hence ubiquinone biosynthesis in vivo. In vitro import assays show the mitochondrial leader sequence present on Coq3p functions to direct mitochondrial import of Coq3p in vitro and that processing to the mature form requires a membrane potential. In vitro methyltransferase assays with E. coli cell lysates and synthetically prepared farnesylated-substrate analogs indicate that UbiG methylates both the derivative of the eukaryotic intermediate, 3,4-dihydroxy-5-farnesylbenzoate, as well as that of the E. coli intermediate, 2-farnesyl-6-hydroxyphenol. The data presented indicate that the yeast Coq3 polypeptide is located in the mitochondria and that E. coli UbiG catalyzes both O-methylation steps in E. coli.