Crystal structure of NRAS Q61K with a ligand-induced pocket near switch II.

The RAS isoforms (KRAS, HRAS and NRAS) have distinct cancer type-specific profiles. NRAS mutations are the second most prevalent RAS mutations in skin and hematological malignancies. Although RAS proteins were considered undruggable for decades, isoform and mutation-specific investigations have produced successful RAS inhibitors that are either specific to certain mutants, isoforms (pan-KRAS) or target all RAS proteins (pan-RAS). While extensive structural and biochemical investigations have focused mainly on K- and H-RAS mutations, NRAS mutations have received less attention, and the most prevalent NRAS mutations in human cancers, Q61K and Q61R, are rare in K- and H-RAS. This manuscript presents a crystal structure of the NRAS Q61K mutant in the GTP-bound form. Our structure reveals a previously unseen pocket near switch II induced by the binding of a ligand to the active form of the protein. This observation reveals a binding site that can potentially be exploited for development of inhibitors against mutant NRAS. Furthermore, the well-resolved catalytic site of this GTPase bound to native GTP provides insight into the stalled GTP hydrolysis observed for NRAS-Q61K.

Structure and dynamics of a pentameric KCTD5/CUL3/Gßγ E3 ubiquitin ligase complex.

Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gßγ and reduces Gßγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gß1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gßγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gßγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gß and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gßγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.

Immobilized enzyme cascade for targeted glycosylation.

Glycosylation is a critical post-translational protein modification that affects folding, half-life and functionality. Glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. We describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro enabled by immobilized enzymes produced with a one-step immobilization/purification method. We reconstruct a reaction cascade mimicking a glycosylation pathway where promiscuity naturally exists to humanize a range of proteins derived from different cellular systems, yielding near-homogeneous glycoforms. Immobilized ß-1,4-galactosyltransferase is used to enhance the galactosylation profile of three IgGs, yielding 80.2-96.3% terminal galactosylation. Enzyme recycling is demonstrated for a reaction time greater than 80 h. The platform is easy to implement, modular and reusable and can therefore produce homogeneous glycan structures derived from various hosts for functional and clinical evaluation.

Discovery of OICR12694: A Novel, Potent, Selective, and Orally Bioavailable BCL6 BTB Inhibitor.

B cell lymphoma 6 (BCL6), a highly regulated transcriptional repressor, is deregulated in several forms of non-Hodgkin lymphoma (NHL), most notably in diffuse large B-cell lymphoma (DLBCL). The activities of BCL6 are dependent on protein-protein interactions with transcriptional co-repressors. To find new therapeutic interventions addressing the needs of patients with DLBCL, we initiated a program to identify BCL6 inhibitors that interfere with co-repressor binding. A virtual screen hit with binding activity in the high micromolar range was optimized by structure-guided methods, resulting in a novel and highly potent inhibitor series. Further optimization resulted in the lead candidate 58 (OICR12694/JNJ-65234637), a BCL6 inhibitor with low nanomolar DLBCL cell growth inhibition and an excellent oral pharmacokinetic profile. Based on its overall favorable preclinical profile, OICR12694 is a highly potent, orally bioavailable candidate for testing BCL6 inhibition in DLBCL and other neoplasms, particularly in combination with other therapies.

High-Throughput Evaluation of Emission and Structure in Reduced-Dimensional Perovskites.

High-throughput experimentation (HTE) seeks to accelerate the exploration of materials space by uniting robotics, combinatorial methods, and parallel processing. HTE is particularly relevant to metal halide perovskites (MHPs), a diverse class of optoelectronic materials with a large chemical space. Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals, allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs. We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP, methoxy-phenethylammonium lead iodide ((4-MeO-PEAI)2-PbI2). We then synthesize 16 000 MHP single crystals and measure their photoluminescence to study the effects of synthesis parameters and compositional engineering on the emission intensity of 54 distinct MHPs: we achieve an acceleration factor of more than 100 times over previously reported HTE MHP synthesis and characterization methods. Using insights derived from this analysis, we screen an existing database for new, potentially emissive MHPs. On the basis of the Tanimoto similarity of the bright available emitters, we present our top candidates for future exploration. As a proof of concept, we use one of these (3,4-difluorophenylmethanamine) to synthesize an MHP which we find has a photoluminescence quantum yield of 10%.

Structures of RGL1 RAS-Association Domain in Complex with KRAS and the Oncogenic G12V Mutant.

Ral Guanine Nucleotide Dissociation Stimulator Like 1 (RGL1) is a RAS effector protein that activates Ral GTPase by stimulating nucleotide exchange. Most structures of RAS-effector complexes are for the HRAS isoform; relatively few KRAS-effector structures have been solved, even though KRAS mutations are more frequent in human cancers. We determined crystal structures of KRAS/RGL1-RAS-association (RA) domain complexes and characterized the interaction in solution using nuclear magnetic resonance spectroscopy, size-exclusion chromatography combined with multi-angle light scattering and biolayer interferometry. We report structures of wild-type KRAS and the oncogenic G12V mutant in complex with the RA domain of RGL1 at < 2 Å resolution. KRASWT/RGL1-RA crystallized as a 1:1 heterodimer, whilst KRASG12V/RGL1-RA crystallized as a heterotetrameric structure in which RGL1-RA dimerized via domain-swapping the C-terminal beta-strand. Solution data indicated that KRASWT and KRASG12V in complex with RGL1-RA both exist predominantly as 1:1 dimers, while tetramerization occurs through very slow association. Through detailed structural analyses, the distance and angle between RAS α1 helix and RBD/RA α1 helix were found to differ significantly among RAS and RBD/RA complexes. The KRAS/RGL1-RA structures possess some of the largest α1RAS/α1Effector distances (21.7-22.2 Å), whereas the corresponding distances in previously reported RAS/RAF complexes are significantly shorter (15.2-17.7 Å). Contact map analysis identified unique structural signatures involving contacts between the ß1-ß2 loop of RA and the α1 helix of RAS, clearly distinguishing the KRAS/RGL1-RA (and other RAS/RA complexes) from RAS/RBD complexes. These results demonstrate that RAS effectors employ an assortment of finely-tuned docking surfaces to achieve optimal interactions with RAS.

Fungal-Selective Resorcylate Aminopyrazole Hsp90 Inhibitors: Optimization of Whole-Cell Anticryptococcal Activity and Insights into the Structural Origins of Cryptococcal Selectivity.

The essential eukaryotic chaperone Hsp90 regulates the form and function of diverse client proteins, many of which govern thermotolerance, virulence, and drug resistance in fungal species. However, use of Hsp90 inhibitors as antifungal therapeutics has been precluded by human host toxicities and suppression of immune responses. We recently described resorcylate aminopyrazoles (RAPs) as the first class of Hsp90 inhibitors capable of discriminating between fungal (Cryptococcus neoformans, Candida albicans) and human isoforms of Hsp90 in biochemical assays. Here, we report an iterative structure-property optimization toward RAPs capable of inhibiting C. neoformans growth in culture. In addition, we report the first X-ray crystal structures of C. neoformans Hsp90 nucleotide binding domain (NBD), as the apoprotein and in complexes with the non-species-selective Hsp90 inhibitor NVP-AUY922 and three RAPs revealing unique ligand-induced conformational rearrangements, which reaffirm the hypothesis that intrinsic differences in protein flexibility can confer selective inhibition of fungal versus human Hsp90 isoforms.

Random mutagenesis reveals residues of JAK2 critical in evading inhibition by a tyrosine kinase inhibitor.

BACKGROUND: The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2. METHODOLOGY: In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. CONCLUSIONS: Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens.

Cutting-hedge research into bacterial invasion.

Glycoside hydrolases are the tools that pathogenic bacteria use to cut through the defensive glycan structure on host cell surfaces. In this issue of Structure, Pluvinage et al. (2011) report how a bacterial polypeptide with more than one hydrolase module broadens the effective substrate specificity.

Synthesis of a biologically active isomer of kotalanol, a naturally occurring glucosidase inhibitor.

The syntheses of an isomer of kotalanol, a naturally occurring glucosidase inhibitor, and of kotalanol itself are described. The target compounds were synthesized by nucleophilic attack of PMB-protected 1,4-anhydro-4-thio-d-arabinitol at the least hindered carbon atom of two 1,3-cyclic sulfates, which were synthesized from d-mannose. Methoxymethyl ether and isopropylidene were chosen as protecting groups. The latter group was critical to ensure the facile deprotection of the coupled products in a one-step sequence to yield kotalanol and its isomer. The stereoisomer of kotalanol, with the opposite stereochemistry at the C-6' stereogenic centre, inhibited the N-terminal catalytic domain of intestinal human maltase glucoamylase (ntMGAM) with a K(i) value of 0.20+/-0.02microM; this compares to a K(i) value for kotalanol of 0.19+/-0.03microM. The results indicate that the configuration at C-6' is inconsequential for inhibitory activity against this enzyme.

Structural investigation of the binding of 5-substituted swainsonine analogues to Golgi alpha-mannosidase II.

Golgi alpha-mannosidase II (GMII) is a key enzyme in the N-glycosylation pathway and is a potential target for cancer chemotherapy. The natural product swainsonine is a potent inhibitor of GMII. In this paper we characterize the binding of 5alpha-substituted swainsonine analogues to the soluble catalytic domain of Drosophila GMII by X-ray crystallography. These inhibitors enjoy an advantage over previously reported GMII inhibitors in that they did not significantly decrease the inhibitory potential of the swainsonine head-group. The phenyl groups of these analogues occupy a portion of the binding site not previously seen to be populated with either substrate analogues or other inhibitors and they form novel hydrophobic interactions. They displace a well-organized water cluster, but the presence of a C(10) carbonyl allows the reestablishment of important hydrogen bonds. Already approximately tenfold more active against the Golgi enzyme than the lysosomal enzyme, these inhibitors offer the potential of being extended into the N-acetylglucosamine binding site of GMII for the creation of even more potent and selective GMII inhibitors.

Potent glucosidase inhibitors: de-O-sulfonated ponkoranol and its stereoisomer.

Ponkoranol, a glucosidase inhibitor isolated from the plant Salacia reticulata, comprises a sulfonium ion with an internal sulfate counterion. An efficient synthetic route to de-O-sulfonated ponkoranol and its 5'-stereoisomer is reported, and it is shown that these compounds are potent glucosidase inhibitors that inhibit a key intestinal human glucosidase, the N-terminal catalytic domain of maltase glucoamylase, with K(i) values of 43 +/- 3 and 15 +/- 1 nM, respectively.

Synthesis and biological evaluation of heteroanalogues of kotalanol and de-O-sulfonated kotalanol.

The synthesis of nitrogen and selenium analogues of kotalanol and de-O-sulfonated kotalanol, naturally occurring sulfonium-ion glucosidase inhibitors isolated from Salacia reticulata, and their evaluation as glucosidase inhibitors against the N-terminal catalytic domain of human maltase glucoamylase (ntMGAM) are described.

A long-wavelength fluorescent substrate for continuous fluorometric determination of alpha-mannosidase activity: resorufin alpha-D-mannopyranoside.

A simple and reliable continuous assay for measurement of alpha-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin alpha-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK(a) of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi alpha-mannosidases. The assay performed using recombinant Drosophila Golgi alpha-mannosidase (dGMII) has been shown to give the kinetic parameters K(m) of 200 microM and V(max) of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known alpha-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment.

Human lysosomal alpha-mannosidases exhibit different inhibition and metal binding properties.

Two structurally-related members of the lysosomal mannosidase family, the broad substrate specificity enzyme human lysosomal alpha-mannosidase (hLM, MAN2B1) and the human core alpha-1, 6-specific mannosidase (hEpman, MAN2B2) act in a complementary fashion on different glycosidic linkages, to effect glycan degradation in the lysosome. We have successfully expressed these enzymes in Drosophila S2 cells and functionally characterized them. hLM and hEpman were significantly inhibited by the class II alpha-mannosidase inhibitors, swainsonine and mannostatin A. We show that three pyrrolidine-based compounds designed for selective inhibition of Golgi alpha-mannosidase II (GMII) exhibited varying degrees of inhibition for hLM and hEpman. While these compounds inhibited hLM and GMII similarly, they inhibited hEpman to a lesser extent. Further, the two lysosomal alpha-mannosidases also show differential metal dependency properties. This has led us to propose a secondary metal binding site in hEpman. These results set the stage for the development of selective inhibitors to members of the GH38 family, and, henceforth, the further investigation of their physiological roles.

The molecular basis of inhibition of Golgi alpha-mannosidase II by mannostatin A.

Mannostatin A is a potent inhibitor of the mannose-trimming enzyme, Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway. Inhibition of this enzyme provides a route to blocking the transformation-associated changes in cancer cell surface oligosaccharide structures. Here, we report on the synthesis of new Mannostatin derivatives and analyze their binding in the active site of Drosophila GMII by X-ray crystallography. The results indicate that the interaction with the backbone carbonyl of Arg876 is crucial to the high potency of the inhibitor-an effect enhanced by the hydrophobic interaction between the thiomethyl group and an aromatic pocket vicinal to the cleavage site. The various structures indicate that differences in the hydration of protein-ligand complexes are also important determinants of plasticity as well as selectivity of inhibitor binding.

Structural analysis of Golgi alpha-mannosidase II inhibitors identified from a focused glycosidase inhibitor screen.

The N-glycosylation pathway is a target for pharmaceutical intervention in a number of pathological conditions including cancer. Golgi alpha-mannosidase II (GMII) is the final glycoside hydrolase in the pathway and has been the target for a number of synthetic efforts aimed at providing more selective and effective inhibitors. Drosophila GMII (dGMII) has been extensively studied due to the ease of obtaining high resolution structural data, allowing the observation of substrate distortion upon binding and after formation of a trapped covalent reaction intermediate. However, attempts to find new inhibitor leads by high-throughput screening of large commercial libraries or through in silico docking were unsuccessful. In this paper we provide a kinetic and structural analysis of five inhibitors derived from a small glycosidase-focused library. Surprisingly, four of these were known inhibitors of beta-glucosidases. X-ray crystallographic analysis of the dGMII:inhibitor complexes highlights the ability of the zinc-containing GMII active site to deform compounds, even ones designed as conformationally restricted transition-state mimics of beta-glucosidases, into binding entities that have inhibitory activity. Although these deformed conformations do not appear to be on the expected conformational itinerary of the enzyme, and are thus not transition-state mimics of GMII, they allow positioning of the three vicinal hydroxyls of the bound gluco-inhibitors into similar locations to those found with mannose-containing substrates, underlining the importance of these hydrogen bonds for binding. Further, these studies show the utility of targeting the acid-base catalyst using appropriately positioned positively charged nitrogen atoms, as well as the challenges associated with aglycon substitutions.

Functionalized pyrrolidine inhibitors of human type II alpha-mannosidases as anti-cancer agents: optimizing the fit to the active site.

Refining the chemical structure of functionalized pyrrolidine-based inhibitors of Golgi alpha-mannosidase II (GMII) to optimize binding affinity provided a lead molecule that demonstrated nanomolar competitive inhibition of alpha-mannosidases II and an optimal fit in the active site of Drosophila GMII by X-ray crystallography. Esters of this lead compound also inhibited the growth of human glioblastoma and brain-derived endothelial cells more than the growth of non-tumoral human fibroblasts, suggesting their potential for anti-cancer therapy.

Golgi alpha-mannosidase II cleaves two sugars sequentially in the same catalytic site.

Golgi alpha-mannosidase II (GMII) is a key glycosyl hydrolase in the N-linked glycosylation pathway. It catalyzes the removal of two different mannosyl linkages of GlcNAcMan(5)GlcNAc(2), which is the committed step in complex N-glycan synthesis. Inhibition of this enzyme has shown promise in certain cancers in both laboratory and clinical settings. Here we present the high-resolution crystal structure of a nucleophile mutant of Drosophila melanogaster GMII (dGMII) bound to its natural oligosaccharide substrate and an oligosaccharide precursor as well as the structure of the unliganded mutant. These structures allow us to identify three sugar-binding subsites within the larger active site cleft. Our results allow for the formulation of the complete catalytic process of dGMII, which involves a specific order of bond cleavage, and a major substrate rearrangement in the active site. This process is likely conserved for all GMII enzymes-but not in the structurally related lysosomal mannosidase-and will form the basis for the design of specific inhibitors against GMII.

Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant.

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.