The structure of human GCN2 reveals a parallel, back-to-back kinase dimer with a plastic DFG activation loop motif.

When activated by amino acid starvation, the stress sensing protein kinase GCN2 phosphorylates the eukaryotic initiation factor 2 alpha, inhibiting translation to conserve energy and facilitate cell survival. Amino acid starvation, particularly of tryptophan and arginine, affects immune tolerance by suppressing differentiation and proliferation of T-cells via activation of GCN2 kinase. In addition, the GCN2 pathway mediates cancer survival directly within the context of metabolic stress. Here, we report the first crystal structures of the human GCN2 kinase domain (KD) in complex with two inhibitors of different size, shape, and chemical scaffold. Three novel activation loop conformations representative of different activation states of the kinase are described. In addition, a novel dimerization organization for GCN2 is observed. This arrangement is consistent with the hypothesis that the GCN2 KD forms an antiparallel inactive dimer until uncharged tRNA binds to it and triggers conformational changes that shift the equilibrium to the active parallel dimer.

Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells.

USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or α-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated ß-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect ß-catenin, demonstrating USP14 but not UCHL5 has a specific effect on ß-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides ß-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.

Structural dynamics as a contributor to error-prone replication by an RNA-dependent RNA polymerase.

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity.

RNA virus population diversity, an optimum for maximal fitness and virulence.

The ability of an RNA virus to exist as a population of genetically distinct variants permits the virus to overcome events during infections that would otherwise limit virus multiplication or drive the population to extinction. Viral genetic diversity is created by the ribonucleotide misincorporation frequency of the viral RNA-dependent RNA polymerase (RdRp). We have identified a poliovirus (PV) RdRp derivative (H273R) possessing a mutator phenotype. GMP misincorporation efficiency for H273R RdRp in vitro was increased by 2-3-fold that manifested in a 2-3-fold increase in the diversity of the H273R PV population in cells. Circular sequencing analysis indicated that some mutations were RdRp-independent. Consistent with the population genetics theory, H273R PV was driven to extinction more easily than WT in cell culture. Furthermore, we observed a substantial reduction in H273R PV virulence, measured as the ability to cause paralysis in the cPVR mouse model. Reduced virulence correlated with the inability of H273R PV to sustain replication in tissues/organs in which WT persists. Despite the attenuated phenotype, H273R PV was capable of replicating in mice to levels sufficient to induce a protective immune response, even when the infecting dose used was insufficient to elicit any visual signs of infection. We conclude that optimal RdRp fidelity is a virulence determinant that can be targeted for viral attenuation or antiviral therapies, and we suggest that the RdRp may not be the only source of mutations in a RNA virus genome.

Discovery of a selective, substrate-competitive inhibitor of the lysine methyltransferase SETD8.

The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure-activity relationships (SAR) of this series.

Novel Approaches for the Accumulation of Oxygenated Intermediates to Multi-Millimolar Concentrations.

Metalloenzymes that utilize molecular oxygen as a co-substrate catalyze a wide variety of chemically difficult oxidation reactions. Significant insight into the reaction mechanisms of these enzymes can be obtained by the application of a combination of rapid kinetic and spectroscopic methods to the direct structural characterization of intermediate states. A key limitation of this approach is the low aqueous solubility (< 2 mM) of the co-substrate, O2, which undergoes further dilution (typically by one-third or one-half) upon initiation of reactions by rapid-mixing. This situation imposes a practical upper limit on [O2] (and therefore on the concentration of reactive intermediate(s) that can be rapidly accumulated) of ∼1-1.3 mM in such experiments as they are routinely carried out. However, many spectroscopic methods benefit from or require significantly greater concentrations of the species to be studied. To overcome this problem, we have recently developed two new approaches for the preparation of samples of oxygenated intermediates: (1) direct oxygenation of reduced metalloenzymes using gaseous O2 and (2) the in situ generation of O2 from chlorite catalyzed by the enzyme chlorite dismutase (Cld). Whereas the former method is applicable only to intermediates with half lives of several minutes, owing to the sluggishness of transport of O2 across the gas-liquid interface, the latter approach has been successfully applied to trap several intermediates at high concentration and purity by the freeze-quench method. The in situ approach permits generation of a pulse of at least 5 mM O2 within ∼ 1 ms and accumulation of O2 to effective concentrations of up to ∼ 11 mM (i.e. ∼ 10-fold greater than by the conventional approach). The use of these new techniques for studies of oxygenases and oxidases is discussed.

Discovery of an in vivo chemical probe of the lysine methyltransferases G9a and GLP.

Among epigenetic "writers", "readers", and "erasers", the lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9me2) and nonhistone proteins, have been implicated in a variety of human diseases. A "toolkit" of well-characterized chemical probes will allow biological and disease hypotheses concerning these proteins to be tested in cell-based and animal models with high confidence. We previously discovered potent and selective G9a/GLP inhibitors including the cellular chemical probe UNC0638, which displays an excellent separation of functional potency and cell toxicity. However, this inhibitor is not suitable for animal studies due to its poor pharmacokinetic (PK) properties. Here, we report the discovery of the first G9a and GLP in vivo chemical probe UNC0642, which not only maintains high in vitro and cellular potency, low cell toxicity, and excellent selectivity, but also displays improved in vivo PK properties, making it suitable for animal studies.

Small-molecule ligands of methyl-lysine binding proteins: optimization of selectivity for L3MBTL3.

Lysine methylation is a key epigenetic mark, the dysregulation of which is linked to many diseases. Small-molecule antagonism of methyl-lysine (Kme) binding proteins that recognize such epigenetic marks can improve our understanding of these regulatory mechanisms and potentially validate Kme binding proteins as drug-discovery targets. We previously reported the discovery of 1 (UNC1215), the first potent and selective small-molecule chemical probe of a methyl-lysine reader protein, L3MBTL3, which antagonizes the mono- and dimethyl-lysine reading function of L3MBTL3. The design, synthesis, and structure-activity relationship studies that led to the discovery of 1 are described herein. These efforts established the requirements for potent L3MBTL3 binding and enabled the design of novel antagonists, such as compound 2 (UNC1679), that maintain in vitro and cellular potency with improved selectivity against other MBT-containing proteins. The antagonists described were also found to effectively interact with unlabeled endogenous L3MBTL3 in cells.

Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain.

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a K(d) of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.

Discovery of Novel Small Molecule Mer Kinase Inhibitors for the Treatment of Pediatric Acute Lymphoblastic Leukemia.

Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at sub-nanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.

Small-molecule ligands of methyl-lysine binding proteins.

Proteins which bind methylated lysines ("readers" of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small-molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first cocrystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design.

Four-electron oxidation of p-hydroxylaminobenzoate to p-nitrobenzoate by a peroxodiferric complex in AurF from Streptomyces thioluteus.

The nonheme di-iron oxygenase, AurF, converts p-aminobenzoate (Ar-NH(2), where Ar = 4-carboxyphenyl) to p-nitrobenzoate (Ar-NO(2)) in the biosynthesis of the antibiotic, aureothin, by Streptomyces thioluteus. It has been reported that this net six-electron oxidation proceeds in three consecutive, two-electron steps, through p-hydroxylaminobenzoate (Ar-NHOH) and p-nitrosobenzoate (Ar-NO) intermediates, with each step requiring one equivalent of O(2) and two exogenous reducing equivalents. We recently demonstrated that a peroxodiiron(III/III) complex (peroxo- -AurF) formed by addition of O(2) to the diiron(II/II) enzyme ( -AurF) effects the initial oxidation of Ar-NH(2), generating a mu-(oxo)diiron(III/III) form of the enzyme (mu-oxo- -AurF) and (presumably) Ar-NHOH. Here we show that peroxo- -AurF also oxidizes Ar-NHOH. Unexpectedly, this reaction proceeds through to the Ar-NO(2) final product, a four-electron oxidation, and produces -AurF, with which O(2) can combine to regenerate peroxo- -AurF. Thus, conversion of Ar-NHOH to Ar-NO(2) requires only a single equivalent of O(2) and (starting from -AurF or peroxo- -AurF) is fully catalytic in the absence of exogenous reducing equivalents, by contrast to the published stoichiometry. This novel type of four-electron N-oxidation is likely also to occur in the reaction sequences of nitro-installing di-iron amine oxygenases in the biosyntheses of other natural products.

A long-lived, substrate-hydroxylating peroxodiiron(III/III) intermediate in the amine oxygenase, AurF, from Streptomyces thioluteus.

The amine oxygenase AurF from Streptomyces thioluteus catalyzes the six-electron oxidation of p-aminobenzoate (pABA) to p-nitrobenzoate (pNBA). In this work, we have studied the reaction of its reduced Fe(2)(II/II) cofactor with O(2), which results in generation of a peroxo-Fe(2)(III/III) intermediate. In the absence of substrate, this intermediate is unusually stable (t(1/2) = 7 min at 20 degrees C), allowing for its accumulation to almost stoichiometric amounts. Its decay is accelerated approximately 10(5)-fold by the substrate, pABA, implying that it is the complex that effects the two-electron oxidation of the amine to the hydroxylamine. The nearly quantitative conversion of pABA to pNBA by solutions containing an excess of the intermediate suggests that it may also be competent for the two subsequent two-electron oxidations leading to the product.