April 11, 2012
Mosley RT, Edwards TE, Murakami E, Lam AM, Grice RL, Du J, Sofia MJ, Furman PA, Otto MJ.
J Virol. 2012 Apr 11.
Replication of the hepatitis C viral genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization incompetent state. Removal of an autoinhibitory β-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory β-hairpin loops including bovine viral diarrhea virus, dengue virus, and West Nile virus.
March 13, 2012
Green LS, Chun LE, Patton AK, Sun X, Rosenthal GJ, Richards JP.
Biochemistry. 2012 Mar 13;51(10):2157-68.
N6022 is a novel, first-in-class drug with potent inhibitory activity against S-nitrosoglutathione reductase (GSNOR), an enzyme important in the metabolism of S-nitrosoglutathione (GSNO) and in the maintenance of nitric oxide (NO) homeostasis. Inhibition of GSNOR by N6022 and related compounds has shown safety and efficacy in animal models of asthma, chronic obstructive pulmonary disease, and inflammatory bowel disease [Sun, X., et al. (2011) ACS Med. Chem. Lett. 2, 402-406]. N6022 is currently in early phase clinical studies in humans. We show here that N6022 is a tight-binding, specific, and fully reversible inhibitor of GSNOR with an IC(50) of 8 nM and a K(i) of 2.5 nM. We accounted for the fact that the NAD(+)- and NADH-dependent oxidation and reduction reactions, catalyzed by GSNOR are bisubstrate in nature in our calculations. N6022 binds in the GSNO substrate binding pocket like a competitive inhibitor, although in kinetic assays it behaves with a mixed uncompetitive mode of inhibition (MOI) toward the GSNO substrate and a mixed competitive MOI toward the formaldehyde adduct, S-hydroxymethylglutathione (HMGSH). N6022 is uncompetitive with cofactors NAD(+) and NADH. The potency, specificity, and MOI of related GSNOR inhibitor compounds are also reported.
February 3, 2012
Park SJ, Ahmad F, Philp A, Baar K, Williams T, Luo H, Ke H, Rehmann H, Taussig R, Brown AL, Kim MK, Beaven MA, Burgin AB, Manganiello V, Chung JH
Cell. 2012 Feb 3;148(3):421-33.
Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKβ-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.
December 31, 2011
Davies DR, Begley DW, Hartley RC, Staker BL, Stewart LJ.
Methods Enzymol. 2011;493:91-114.
Fragment screening using X-ray crystallography is a method that can provide direct three-dimensional readouts of the structures of protein-small molecule complexes for lead development and fragment-based drug discovery. With current technology, an amenable crystal form can be screened crystallographically against a library of 1000-2000 fragments in 1-2 weeks. We have performed over a dozen crystallographic screening campaigns using our own compound collection called Fragments of Life™ (FOL). While the majority of our fragment screening campaigns have generated multiple hits, some unexpectedly turned out to be nonproductive, either yielding no bound ligands, or only those thought to be inadequate for lead development. In this chapter, we have attempted to identify one or more parameters which could be used to predict whether a crystallized protein target would be a good candidate for fragment hit discovery. Here, we describe the parameters of crystals from 18 fragment screening campaigns, including six unsuccessful targets. From this analysis, we have concluded that there are no parameters that are absolutely predictive of fragment screening success. However, we do describe a parameter we have termed pocket factor which provides a statistically significant variance between nonproductive targets and productive targets shown to bind fragments. The pocket factor is calculated using a novel method of consensus scoring from three distinct pocket-finding algorithms, and the results may be used to prioritize targets for fragment screening campaigns based on an initial crystal structure.
December 31, 2011
Gurney, ME, Burgin, AB, Magnusson, OT, and Stewart, LE
Handbook of Experimental Pharmacology. 2011;(204):167-92.
Phosphodiesterase 4 (PDE4) inhibitors have shown benefit in human clinical trials but dosing is limited by tolerability, particularly because of emesis. Novel cocrystal structures of PDE4 catalytic units with their regulatory domains together with bound inhibitors have revealed three different PDE4 conformers that can be exploited in the design of novel therapeutic agents. The first is an open conformer, which has been employed in the traditional approach to the design of competitive PDE4 inhibitors. The second is an asymmetric dimer in which a UCR2 regulatory helix from one monomer is placed in a closed conformation over the opposite active site in the PDE4 dimer (trans-capping). Only one active site can be closed by an inhibitor at a time with the consequence that compounds exploiting this conformer only partially inhibit PDE4 enzymatic activity while retaining potency in cellular and in vivo models. By placing an intrinsic ceiling on the magnitude of PDE4 inhibition, such compounds may better maintain spatial and temporal patterning of signaling in cAMP microdomains with consequent improved tolerability. The third is a symmetric PDE4 conformer in which helices from the C-terminal portion of the catalytic unit cap both active sites (cis-capping). We propose that dual-gating of PDE4 activity may be further fine tuned by accessory proteins that recognize open or closed conformers of PDE4 regulatory helices.
December 31, 2011
Gurney ME, Burgin AB, Magnusson OT, Stewart LJ.
Handb Exp Pharmacol. 2011;(204):167-92. Review.
Phosphodiesterase 4 (PDE4) inhibitors have shown benefit in human clinical trials but dosing is limited by tolerability, particularly because of emesis. Novel cocrystal structures of PDE4 catalytic units with their regulatory domains together with bound inhibitors have revealed three different PDE4 conformers that can be exploited in the design of novel therapeutic agents. The first is an open conformer, which has been employed in the traditional approach to the design of competitive PDE4 inhibitors. The second is an asymmetric dimer in which a UCR2 regulatory helix from one monomer is placed in a closed conformation over the opposite active site in the PDE4 dimer (trans-capping). Only one active site can be closed by an inhibitor at a time with the consequence that compounds exploiting this conformer only partially inhibit PDE4 enzymatic activity while retaining potency in cellular and in vivo models. By placing an intrinsic ceiling on the magnitude of PDE4 inhibition, such compounds may better maintain spatial and temporal patterning of signaling in cAMP microdomains with consequent improved tolerability. The third is a symmetric PDE4 conformer in which helices from the C-terminal portion of the catalytic unit cap both active sites (cis-capping). We propose that dual-gating of PDE4 activity may be further fine tuned by accessory proteins that recognize open or closed conformers of PDE4 regulatory helices.
October 13, 2011
Edwards TE, Abramov AB, Smith ER, Baydo RO, Leonard JT, Leibly DJ, Thompkins KB, Clifton MC, Gardberg AS, Staker BL, Van Voorhis WC, Myler PJ, Stewart LJ.
BMC Struct Biol. 2011 Oct 13;11:39.
BACKGROUND:
Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis.
RESULTS:
Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD) phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB.
CONCLUSION:
The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether.
September 27, 2011
Yates SP, Edwards TE, Bryan CM, Stein AJ, Van Voorhis WC, Myler PJ, Stewart LJ, Zheng J, Jia Z.
Biochemistry. 2011 Sep 27;50(38):8103-6.
Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). On the basis of the recently determined structure of the AceK-ICDH complex from Escherichia coli, we have classified the structures of homodimeric NADP(+)-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH that exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria.
September 13, 2011
Complete Title: Monoolein Lipid Phases as Incorporation and Enrichment Materials for Membrane Protein Crystallization
PLoS One. 2011;6(8):e24488. Epub 2011 Aug 31.
Wallace E, Dranow D, Laible PD, Christensen J, Nollert P.
ABSTRACT
The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial insitu enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase.
September 1, 2011
Smith ER, Begley DW, Anderson V, Raymond AC, Haffner TE, Robinson JI, Edwards TE, Duncan N, Gerdts CJ, Mixon MB, Nollert P, Staker BL, Stewart LJ.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Sep 1;67(Pt 9):1015-21.
The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications.