Complementary cell-based high-throughput screens identify novel modulators of the unfolded protein response.

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes.

Elucidation of natural product biosynthetic pathways provides important insights into the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here, we show full reconstitution, in vitro, of an unusual multi-step oxidative cascade for post-assembly-line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and a flavin adenine dinucleotide-dependent oxidase (TamL), which act co-dependently through the repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a previously unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C10 oxidation of TirE.

Identification of the tirandamycin biosynthetic gene cluster from Streptomyces sp. 307-9.

The structurally intriguing bicyclic ketal moiety of tirandamycin is common to several acyl-tetramic acid antibiotics, and is a key determinant of biological activity. We have identified the tirandamycin biosynthetic gene cluster from the environmental marine isolate Streptomyces sp. 307-9, thus providing the first genetic insight into the biosynthesis of this natural product scaffold. Sequence analysis revealed a hybrid polyketide synthase-nonribosomal peptide synthetase gene cluster with a colinear domain organization, which is entirely consistent with the core structure of the tirandamycins. We also identified genes within the cluster that encode candidate tailoring enzymes for elaboration and modification of the bicyclic ketal system. Disruption of tamI, which encodes a presumed cytochrome P450, led to a mutant strain deficient in production of late stage tirandamycins that instead accumulated tirandamycin C, an intermediate devoid of any post assembly-line oxidative modifications.

Isolation and characterization of tirandamycins from a marine-derived Streptomyces sp.

The novel dienoyl tetramic acids tirandamycin C (1) and tirandamycin D (2) with activity against vancomycin-resistant Enterococcus faecalis were isolated from the marine environmental isolate Streptomyces sp. 307-9, which also produces the previously identified compounds tirandamycins A (3) and B (4). Spectroscopic analysis of 1 and 2 indicated structural similarity to 3 and 4, with differences only in the pattern of pendant oxygenation on the bicyclic ketal system. The isolation of these putative biosynthetic intermediates was enabled by their sequestration on an adsorbent resin during early stationary-phase fermentation.

Syntheses of conjugated pyrones for the enzymatic assay of lovastatin nonaketide synthase, an iterative polyketide synthase.

[reaction: see text] Lovastatin nonaketide synthase (LovB, LNKS) is an iterative type I polyketide synthase (PKS) from Aspergillus terreus that produces dihydromonacolin L (2), the biosynthetic precursor of lovastatin (1), from acetyl CoA, malonyl CoA, and S-adenosylmethionine (SAM) if the accessory protein LovC is present. In the absence of LovC, LNKS forms the conjugated pyrones 3 and 4 as truncated PKS products. Short syntheses of these pyrones provide material for the assay of LNKS activity by HPLC and radioisotope dilution analysis.