Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated in response to a variety of endoplasmic reticulum stresses implicated in numerous disease states. Evidence that PERK is implicated in tumorigenesis and cancer cell survival stimulated our search for small molecule inhibitors. Through screening and lead optimization using the human PERK crystal structure, we discovered compound 38 (GSK2606414), an orally available, potent, and selective PERK inhibitor. Compound 38 inhibits PERK activation in cells and inhibits the growth of a human tumor xenograft in mice.

GW572016 (Lapatinib) is a tyrosine kinase inhibitor in clinical development for cancer that is a potent dual inhibitor of epidermal growth factor receptor (EGFR, ErbB-1) and ErbB-2. We determined the crystal structure of EGFR bound to GW572016. The compound is bound to an inactive-like conformation of EGFR that is very different from the activelike structure bound by the selective EGFR inhibitor OSI-774 (Tarceva) described previously. Surprisingly, we found that GW572016 has a very slow off-rate from the purified intracellular domains of EGFR and ErbB-2 compared with OSI-774 and another EGFR selective inhibitor, ZD-1839 (Iressa). Treatment of tumor cells with these inhibitors results in down-regulation of receptor tyrosine phosphorylation. We evaluated the duration of the drug effect after washing away free compound and found that the rate of recovery of receptor phosphorylation in the tumor cells reflected the inhibitor off-rate from the purified intracellular domain. The slow off-rate of GW572016 correlates with a prolonged down-regulation of receptor tyrosine phosphorylation in tumor cells. The differences in the off-rates of these drugs and the ability of GW572016 to inhibit ErbB-2 can be explained by the enzyme-inhibitor structures.

The three-dimensional structure of BirA, the repressor of the Escherichia col biotin biosynthetic operon, has been determined by x-ray crystallography and refined to a crystallographic residual of 19.0% at 2.3-A resolution. BirA is a sequence-specific DNA-binding protein that also catalyzes the formation of biotinyl-5'-adenylate from biotin and ATP and transfers the biotin moiety to other proteins. The level of biotin biosynthetic enzymes in the cell is controlled by the amount of biotinyl-5'-adenylate, which is the BirA corepressor. The structure provides an example of a transcription factor that is also an enzyme. The structure ofBirA is highly asymmetric and consists of three domains. The N-terminal domain is mostly a-helical, contains a helix-turn-helix DNA-binding motif, and is loosely connected to the remainder of the molecule. The central domain consists of a seven-stranded mixed 13-sheet with a-helices covering one face. The other side of the sheet is largely solventexposed and contains the active site. The C-terminal domain comprises a six-stranded, antiparallel 13-sheet sandwich. The location of biotin binding is consistent with mutations that affect enzymatic activity. A nearby loop has a sequence that has been associated with phosphate binding in other proteins. It is inferred that ATP binds in this region, adjacent to the biotin. It is proposed that the binding of corepressor to monomeric BirA may promote DNA binding by facilitating the formation of a multimeric BirA-corepressor-DNA complex. The structural details of this complex remain an open question, however.The biotin operon repressor, BirA, is a 33.5-kDa protein that regulates transcription of the Escherichia coli biotin operon (1-3). BirA is bifunctional, serving both as the biotin (vitamin H)-activating enzyme and as a transcriptional regulator. It catalyzes the formation of biotinyl-5'-adenylate from biotin and ATP and transfers biotin to a specific lysine residue on the biotin carboxyl carrier protein, a subunit of acetyl-CoA carboxylase (4,5). If all the biotin-accepting proteins in the cell have been biotinylated, the BirA-biotinyl-5'-AMP complex accumulates and binds to the 40-base-pair bio operator, repressing transcription of the biotin biosynthetic genes (4, 6-8). Thus BirA synthesizes its own corepressor, a unique property among known DNA-binding proteins. BirA represses transcription when biotinyl-5'-AMP is bound to the enzyme, suggesting that binding of corepressor helps form the BirA-DNA complex. Structure DeterminationCrystals of BirA were grown as described (9) and equilibrated with 2.05 M phosphate, pH 6.5/5% (vol/vol) glycerol. Native and derivative data sets were collected by using film or a Xuong-Hamlin (10) area detector ( Table 1). Inclusion of anomalous data in cross-phased difference Fourier maps showed the space group to be P43212 rather than its enantiomorph.Refined heavy-atom parameters were employed to compute multiple isomorphous replacement phases to 3.0-A resolution. The mean figure of merit, including an...

Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (USP). We provide evidence for a second ecdysteroid signaling pathway mediated by DHR38, the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. DHR38 also heterodimerizes with USP, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either DHR38 or USP. X-ray crystallographic analysis of DHR38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans.