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A convergent asymmetric synthesis of the antinoeplastic macrolide antibiotic cytovaricin has been achieved through the synthesis and coupling of the illustrated spiroketal and polyol glycoside subunits. All absolute steroechemical relationships within the target structure were ultimately controlled by the use of asymmetric aldol, alkylation, or epoxidation methodology. Union of the two subnits was accomplished by Julia-Lythgoe trans olefination, providing direct access to a suitable macrolactonization substrate

ACK1 is a multidomain non-receptor tyrosine kinase that is an effector of the Cdc42 GTPase. Members of the ACK family have a unique domain ordering and are the only tyrosine kinases known to interact with Cdc42. In contrast with many protein kinases, ACK1 has only a modest increase in activity upon phosphorylation. We have solved the crystal structures of the human ACK1 kinase domain in both the unphosphorylated and phosphorylated states. Comparison of these structures reveals that ACK1 adopts an activated conformation independent of phosphorylation. Furthermore, the unphosphorylated activation loop is structured, and its conformation resembles that seen in activated tyrosine kinases. In addition to the apo structure, complexes are also presented with a non-hydrolyzable nucleotide analog (adenosine 5'-(beta,gamma-methylenetriphosphate)) and with the natural product debromohymenialdisine, a general inhibitor of many protein kinases. Analysis of these structures reveals a typical kinase fold, a pre-organization into the activated conformation, and an unusual substrate-binding cleft.

Structural biology is an invaluable tool in modern drug discovery, providing key insights into the interactions of small-molecule drugs with their protein targets. As in many aspects of the drug discovery process, significant synergies can be realized in structural biology by the contemporaneous pursuit of many target proteins from a single structural and functional class. We will review some of those synergies here using the example of the protein kinases – an important class of drug targets that has recently been the subject of intensive study. We conclude by discussing some of the technical advances in X-ray crystallography that have enabled implementation of high-throughput structural biology as applied to drug lead optimization.

Serpins exhibit a range of physiological roles and can contribute to certain disease states dependent on their various conformations. Understanding the mechanisms of the large-scale conformational reorganizations of serpins may lead to a better understanding of their roles in various cardiovascular diseases. We have studied the serpin, plasminogen activator inhibitor 1 (PAI-1), in both the active and the latent state and found that anionic halide ions may play a role in the active-to-latent structural transition. Crystallographic analysis of a stable mutant form of active PAI-1 identified an anion-binding site between the central beta-sheet and a small surface domain. A chloride ion was modeled in this site, and its identity was confirmed by soaking crystals in a bromide-containing solution and calculating a crystallographic difference map. The anion thus located forms a 4-fold ligated linchpin that tethers the surface domain to the central beta-sheet into which the reactive center loop must insert during the active-to-latent transition. Timecourse experiments measuring active PAI-1 stability in the presence of various halide ions showed a clear trend for stabilization of the active form with F(-)Cl(-)Br(-)I(-). We propose that the "stickiness" of this pin (i.e., the electronegativity of the anion) contributes to the energetics of the active-to-latent transition in the PAI-1 serpin.

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.

Two new imidazole alkaloids were isolated from a Micronesian sponge, Leucetta sp. Kealiiquinone (2) is a naphtoquinone which appears to be biogenetically derived from 1, which is a dibenzyl-substituted imidazole. Pyronaamidine (1) is cytotoxic against KB cells, MIC=5 μg/mL

In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutants of D221 except cysteine abolish activity. We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N. In a complex with dUMP and the antifolate 10- propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual. CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is less ordered than in wild-type complexes. The side chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of CB3717, which must be in the enol form. In contrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7,8-tetrahydrofolate shows the cofactor bound in two partially occupied, nonproductive binding sites. In both binding modes, the cofactor has a closed imidazolidine ring and adopts the solution conformation of the unbound cofactor. In one of the binding sites, the pterin ring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the cofactor. This orientation blocks the conformational change required for forming covalent ternary complexes. Taken together, the two crystal structures suggest that the hydrogen bond between the side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding, where it enforces the correct orientation of the pterin ring. Proper orientation of the cofactor appears to be a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-state intermediate in catalysis.

The crystal structure of PfPK5, a cyclin-dependent kinase from Plasmodium falciparum, is the first CDK structure determined from a nonhuman source and represents a potential new target for anti-malarial drug development.

Erratum to ‘‘Discovery of XL413, a potent and selective CDC7 inhibitor’’ [Bioorg. Med. Chem. Lett. 22 (2012) 3727–3731] Elena S. Koltun ⇑, Amy Lew Tsuhako, David S. Brown, Naing Aay, Arlyn Arcalas, Vicky Chan, Hongwang Du, Stefan Engst, Kim Ferguson, Maurizio Franzini, Adam Galan, Charles R. Holst, Ping Huang, Brian Kane, Moon H. Kim, Jia Li, David Markby, Manisha Mohan, Kevin Noson, Arthur Plonowski, Steven J. Richards, Scott Robertson, Kenneth Shaw, Gordon Stott, Thomas J. Stout, Jenny Young, Peiwen Yu, Cristiana A. Zaharia, Wentao Zhang, Peiwen Zhou, John M. Nuss, Wei Xu, Patrick C. Kearney

Mutations within exons encoding the kinase domain of EGFR confer increased sensitivity of non-small cell lung carcinomas to the tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib. Unfortunately, all patients whose disease responds initially to these therapies eventually develop acquired resistance. In ∼50% of cases, resistance is associated with acquisition of a second-site ‘gatekeeper’ mutation within EGFR, T790M. More potent ‘2nd generation’ EGFR inhibitors theoretically able to overcome T790M-mediated resistance have displayed limited clinical success. In preclinical models of acquired resistance, many of these TKIs (HKI-272, BIBW2992, PF00299804) actually select for the emergence of EGFR T790M-harboring clones. To determine if EGFR TKIs in general lead to the emergence of T790M, we modeled acquired resistance in vitro with the reversible TKI, XL647 (Symphony Evolution, Inc., Rockville, MD), the irreversible EGFR TKI, BIBW2992 (synthesized at MSKCC), and erlotinib. EGFR mutant TKI-sensitive PC-9 cells (lung adenocarcinoma, del E746-A750) were grown with increasing concentrations of XL647 for ∼160 days. Polyclonal resistant (XL-R) cells were derived that proliferated in drug concentrations ∼50-fold higher than the initial IC50. No other secondary mutations within EGFR were detected by direct sequencing, and the T790M mutation was not found using more sensitive mutation detection methods (e.g. SNaPShot; limit of detection ∼5%). By contrast, both BIBW2992- and erlotinib-resistant PC-9 cells (PC-9 BI-R and PC-9 E-R) harbored T790M, as shown by direct sequencing. No resistant cells displayed MET amplification, another known resistance mechanism. Crystal structure data revealed that XL647 binds reversibly within the ATP binding pocket of EGFR T790M with a similar orientation to erlotinib. Therefore, structural differences alone do not readily explain the differential selective pressure for EGFR T790M with gefitinib/erlotinib vs. XL647. Interestingly, PC-9 XL-R cells remained intermediately sensitive to erlotinib. Consistent with the in vitro observations, early clinical trial data revealed that only 1 of 6 patients with EGFR mutant lung cancer harbored T790M after developing acquired resistance to XL647. Furthermore, at least 2 of these patients subsequently derived clinical benefit from single-agent erlotinib. Collectively, these data suggest that the propensity to develop T790M-mediated resistance varies among different TKIs. We are currently exploring why the selective pressure for resistance conferred by XL647 is different from that of other EGFR TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1774.

The three-dimensional structure of the unusual fatty acid metabolite neohalicholactone, isolated from the marine sponge Halinchondria okadai Kadota, has been determined by X-ray crystallography.

The structures of four new drimane-type sesquiterpenes 1–4 produced in liquid cultures of the fairy ring fungus, Marasmiusoreades, have been determined by a combination of chemical and spectroscopic methods. The structure assigned to one of these sesquiterpenes (1) has been confirmed by an X-ray crystallographic study. An unprecedented feature of all four sesquiterpenes is the biosynthetic elaboration of C8–C12 and C15 of the drimane skeleton to a dioxabicyclooctane moiety. In addition, two of the sesquiterpenes possess an uncommon α orientation of the hydroxyl groups at C3. Keywords: sesquiterpenes, fungal metabolites, drimanes, fairy ring mushroom, crystal structure.

Duodenal ulcers were produced in rats by subcutaneous infusion for 48 hr of either of the following secretagogues: histamine, carbachol, pentagastrin. When any two of these secretagogues were administered together, synergism was observed in the ulcerogenic effect. Synergism, or potentiation, is defined as an effect produced by two agents given together that is greater than the sum of the effects produced by each agent when given separately. The ulcers were located opposite to the mesenteric attachment, were often multiple, and extended as far as 4 cm from the pylorus. Their severity ranged from shallow erosions to perforation with peritonitis. When two secretagogues were infused at the same time, the incidence of animals with ulcers, their severity, number, and distance from the pylorus were related to the doses given. Pentagastrin, when administered alone, was the least ulcerogenic agent, but when pentagastrin was given with either histamine or carbachol, the ulcerogenic effect was potentiated. Although the duodenum was consistently ulcerated, the stomach remained intact, except in some animals receiving high doses of secretagogues and dying or dead from peritonitis. It is concluded that: (1) the rat can develop duodenal ulcers; and (2) secretagogues, when infused for 48 hr and especially when combined, can produce such ulcers; (3) the mechanism by which these ulcers develop remains to be elucidated.

Introduction: EGFR- mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs). Unfortunately, they develop resistance, often due to acquisition of a second-site mutation (T790M). Current EGFR TKIs select for T790M in preclinical models of acquired resistance. We explored whether all EGFR TKIs similarly select for the T790M mutation using data from early clinical trials and established in vitro models of acquired resistance. Methods: We analyzed the clinical characteristics of eight patients with metastatic EGFR-mutant lung adenocarcinoma who were treated first-line with XL647 and then progressed. XL647 is an ATP-competitive inhibitor of EGFR, HER2, KDR, and EPHB4. Additional molecular preclinical studies were performed to characterize resistance. Results: Four patients displayed confirmed partial responses (PRs), three patients had unconfirmed PRs, and one patient displayed stable disease. Only one of five patients' tumor samples available for analysis after disease progression harbored the T790M mutation. Eight patients subsequently received erlotinib, with ( n = 3) or without ( n = 5) chemotherapy. Three of five patients treated with single-agent erlotinib derived additional benefit, staying on drug up to 9 months. EGFR-mutant PC-9 cells with acquired resistance to XL647 did not harbor the T790M mutation, displayed a distinct mRNA profile from PC-9 cells with T790M-mediated resistance, and were moderately sensitive to erlotinib in growth inhibition assays. Crystal structure analyses of XL647/EGFR T790M did not reveal a different binding mode from that of erlotinib. Conclusions: The findings of this exploratory study suggest that different EGFR TKIs may select for distinct mechanisms of resistance. These results raise the possibility that different EGFR TKIs could be sequentially used to improve outcomes in patients with EGFR -mutant lung cancer. Further work investigating this hypothesis is warranted.