Laurel R. Barbieri

Discovery of lactoquinomycin and related pyranonaphthoquinones as potent and allosteric inhibitors of AKT/PKB: mechanistic involvement of AKT catalytic activation loop cysteines

The serine/threonine kinase AKT/PKB plays a critical role in cancer and represents a rational target for therapy. Although efforts in targeting AKT pathway have accelerated in recent years, relatively few small molecule inhibitors of AKT have been reported. The development of selective AKT inhibitors is further challenged by the extensive conservation of the ATP-binding sites of the AGC kinase family. In this report, we have conducted a high-throughput screen for inhibitors of activated AKT1. We have identified lactoquinomycin as a potent inhibitor of AKT kinases (AKT1 IC50, 0.149 ± 0.045 μmol/L). Biochemical studies implicated a novel irreversible interaction of the inhibitor and AKT involving a critical cysteine residue(s). To examine the role of conserved cysteines in the activation loop (T-loop), we studied mutant AKT1 harboring C296A, C310A, and C296A/C310A. Whereas the ATP-pocket inhibitor, staurosporine, indiscriminately targeted the wild-type and all three mutant-enzymes, the inhibition by lactoquinomycin was drastically diminished in the single mutants C296A and C310A, and completely abolished in the double mutant C296A/C310A. These data strongly implicate the binding of lactoquinomycin to the T-loop cysteines as critical for abrogation of catalysis, and define an unprecedented mechanism of AKT inhibition by a small molecule. Lactoquinomycin inhibited cellular AKT substrate phosphorylation induced by growth factor, loss of PTEN, and myristoylated AKT. The inhibition was substantially attenuated by coexpression of C296A/C310A. Moreover, lactoquinomycin reduced cellular mammalian target of rapamycin signaling and cap-dependent mRNA translation initiation. Our results highlight T-loop targeting as a new strategy for the generation of selective AKT inhibitors. [Mol Cancer Ther 2007;6(11):OF1–11]

Spiroxins, DNA cleaving antitumor antibiotics from a marine-derived fungus

The spiroxins (1–5) were purified from the culture extract of a marine-derived fungus. Their unique bisnaphthospiroketal structures were established by NMR spectroscopy. In addition to cytotoxicity, these compounds showed antibiotic activity and were active in a mouse xenograft model against human ovarian carcinoma. The mechanism of action of these compounds was shown to be due, in part, to their effect on DNA.

Discovery and Assembly Line Biosynthesis of the Lymphostin Pyrroloquinoline Alkaloid Family of mTOR Inhibitors in Salinispora Bacteria

The pyrroloquinoline alkaloid family of natural products, which includes the immunosuppressant lymphostin, has long been postulated to arise from tryptophan. We now report the molecular basis of lymphostin biosynthesis in three marine Salinispora species that maintain conserved biosynthetic gene clusters harboring a hybrid nonribosomal peptide synthetase-polyketide synthase that is central to lymphostin assembly. Through a series of experiments involving gene mutations, stable isotope profiling, and natural product discovery, we report the assembly-line biosynthesis of lymphostin and nine new analogues that exhibit potent mTOR inhibitory activity.

The lecanindoles, nonsteroidal progestins from the terrestrial fungus Verticillium lecanii 6144.

Four new indolosesquiterpenes, lecanindoles A-D (1-4), were isolated from fermentations of the terrestrial fungus Verticillium lecanii 6144. The structures of compounds 1-4 were elucidated from analysis of spectroscopic data. Compound 2 was reduced to give 4 and its isomer 5. Compound 4 was found to be a potent and selective progesterone receptor agonist with an EC50 of 1.1 +/- 0.4 nM in a cell-based luciferase reporter assay.

Absolute stereochemistry of the spiroxins

The exciton coupled CD method has been applied to determine the absolute configuration of the diepoxins, spiroketaI-linked naphthodiepoxydecaIinones of fungal origin. The CD spectra of the bis-dimethylaminobenzoate derivatives of the diepoxins 71, land ~c, reveal a positive chiral twist between the two substituted hydroxyl groups and thus infer the

Structures of the Muraymycins, Novel Peptidoglycan Biosynthesis Inhibitors

configuration at both of these stereogenic centers. The absolute configuration of the remaining chiral centers is deduced from their relative configurations as established by X-ray diffraction of diepoxin ~c. The twist boat conformation of the epoxycyclohexanone ring and the continued axial orientation of the substituents at C-4 and C-5 after dimethylaminobenzoate derivatization