The products of the Escherichia coli umuDC operon are required for translesion synthesis, the mechanistic basis of most mutagenesis caused by UV radiation and many chemicals. The UmuD protein shares homology with LexA, the repressor of SOS-regulated loci, and similarly undergoes a facilitated autodigestion on interaction with the RecA͞single-stranded DNA nucleoprotein filaments formed after a cell experiences DNA damage. This cleavage, in which Ser-60 of UmuD acts as the nucleophile, produces UmuD, the form active in translesion synthesis. Expression of the noncleavable UmuD(S60A) protein and UmuC was found to increase survival after UV irradiation, despite the inability of the UmuD(S60A) protein to participate in translesion synthesis; this survival increase is uvr ؉ dependent. Additional observations that expression of the UmuD(S60A) protein and UmuC delayed the resumption of DNA replication and cell growth after UV irradiation lead us to propose that the uncleaved UmuD protein and UmuC delay the resumption of DNA replication, thereby allowing nucleotide excision repair additional time to repair the damage accurately before replication is attempted. After a UV dose of 20 J͞m 2 , uncleaved UmuD is the predominant form for approximately 20 min, after which UmuD becomes the predominant form, suggesting that the umuDC gene products play two distinct and temporally separated roles in DNA damage tolerance, the first in cell-cycle control and the second in translesion synthesis over unrepaired or irreparable lesions. The relationship of these observations to the eukaryotic DNA damage checkpoint is discussed.

The export protein CRM1 is required for the nuclear export of a wide variety of cancer-related ''cargo'' proteins including p53, c-Abl, and FOXO-3A. Leptomycin B (LMB) is a highly specific inhibitor of CRM1 with significant in vitro potency but limited in vivo efficacy due to toxicity. We now report a series of semisynthetic LMB derivatives showing substantially improved therapeutic windows. Exposure of cancer cells to these compounds leads to a rapid and prolonged block of nuclear export and apoptosis. In contrast to what is observed in cancer cells, these agents induce cell cycle arrest, but not apoptosis, in normal lung fibroblasts. These new nuclear export inhibitors (NEI) maintain the high potency of LMB, are up to 16-fold better tolerated than LMB in vivo, and show significant efficacy in multiple mouse xenograft models. These NEIs show the potential of CRM1 inhibitors as novel and potent anticancer agents. [Cancer Res 2009;69(2):510-7]

B-RAF V600E ͉ hypothemycin ͉ irreversible kinase inhibitor ͉ Michael adduct R esorcylic acid lactones (RALs) are polyketide natural products with a large macrocyclic ring fused to resorcylic acid. Some RALs contain an ␣,␤-unsaturated ketone in the macrocycle, as exemplified by the cis-enone RALs hypothemycin, 5Z-7-oxozeaenol, and L-783,277 (Fig. 1). The cis-enone RALs have been shown to inhibit mammalian cell proliferation and tumor growth in animals (1-3). Furthermore, several reports have indicated that cis-enone RALs inhibit certain protein kinases, such as mitogenactivated protein (MAP) kinase (MAPK) kinase (MEK)1 (4), TGF-␤-activated kinase 1 (TAK1) (5) and platelet-derived growth factor receptor (PDGFR) (6), but not others, such as RAF, PKA, PKC, endothelial growth factor receptor (EGFR), FGF receptor, ZAP70, MEK kinase 4, and lymphoid-specific Tyr kinase p56kk (LCK) (3, 4, 6). Where tested, targets inhibited by the cis-enone RALs were not affected by trans-enone RALs or RAL analogs lacking the ␣,␤-unsaturated ketone (4, 5).L-783,277 was shown to be a potent in vitro inhibitor of MEK1, competitive with ATP, that became even more potent upon preincubation (4). The apparent time-dependent inhibition caught our attention because it is a hallmark of affinity-directed covalent bond formation between enzyme and inhibitor, and the ␣,␤-unsaturated ketone moiety is an effective Michael acceptor of protein nucleophiles, particularly Cys thiolate.Off-target inhibition by a kinase inhibitor is usually unpredictable, and assessment of complete specificity usually requires screening of the entire kinome (7). In the present work, we identified a Cys residue that is conserved in the ATP site of kinase targets reported to be inhibited by cis-enone RALs but absent from those that are not. Furthermore, a structure-bioinformatics approach revealed that this Cys is present in a subset of some 46 protein kinases in the kinome that include such important targets as mitogen receptor Tyr kinases, MEK, and ERK. We show that hypothemycin forms stable covalent adducts with this Cys residue and is highly efficacious in inhibiting growth of cells dependent on the target kinases, in particular, cells dependent on mitogen receptor RAL targets or harboring B-RAF V600E mutations that drive the ERK pathway. ResultsBioinformatics. Sequence alignment of the kinases reported to be inhibited by cis-enone RALs revealed a conserved Cys residue (Cys-166 in human ERK2) adjacent to the completely conserved Asp that is involved in binding the Mg 2ϩ complexed to ATP; kinases that were reportedly not inhibited by a RAL had no Cys residue at that position. Interrogation of the human kinome sequence database revealed that some 46 of 510 identified kinases contained the target Cys and were therefore considered candidates for RAL inhibition (Table 1).Of the 46 Cys-containing RAL targets, 38 exist in 8 evolutionarily related clusters, 7 of which lie within 5 major branches of the human kinase tree (Fig. 2) (8). The largest branch of Ϸ90 Tyr kinases contains...

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.

Escherichia coli is an attractive candidate as a host for polyketide production and has been engineered to produce the erythromycin precursor polyketide 6-deoxyerythronolide B (6dEB). In order to identify and optimize parameters that affect polyketide production in engineered E. coli, we first investigated the supply of the extender unit ( 2S)-methylmalonyl-CoA via three independent pathways. Expression of the Streptomyces coelicolor malonyl/methylmalonyl-CoA ligase ( matB) pathway in E. coli together with methylmalonate feeding resulted in the accumulation of intracellular methylmalonyl-CoA to as much as 90% of the acyl-CoA pool. Surprisingly, the methylmalonyl-CoA generated from the matB pathway was not converted into 6dEB. In strains expressing either the S. coelicolor propionyl-CoA carboxylase (PCC) pathway or the Propionibacteria shermanii methylmalonyl-CoA mutase/epimerase pathway, methylmalonyl-CoA accumulated up to 30% of the total acyl-CoA pools, and 6dEB was produced; titers were fivefold higher when strains contained the PCC pathway rather than the mutase pathway. When the PCC and mutase pathways were expressed simultaneously, the PCC pathway predominated, as indicated by greater flux of (13)C-propionate into 6dEB through the PCC pathway. To further optimize the E. coli production strain, we improved 6dEB titers by integrating the PCC and mutase pathways into the E. coli chromosome and by expressing the 6-deoxyerythronolide B synthase (DEBS) genes from a stable plasmid system.