Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Ronald W. Sarver is active.

Publication


Featured researches published by Ronald W. Sarver.


Structure | 2002

X-ray crystal structure of Staphylococcus aureus FemA.

Timothy E. Benson; D. Bryan Prince; Veronica T. Mutchler; Kimberly A. Curry; Andrea M. Ho; Ronald W. Sarver; Jeanne C. Hagadorn; Gil H. Choi; Robert L. Garlick

The latter stages of peptidoglycan biosynthesis in Staphylococci involve the synthesis of a pentaglycine bridge on the epsilon amino group of the pentapeptide lysine side chain. Genetic and biochemical evidence suggest that sequential addition of these glycines is catalyzed by three homologous enzymes, FemX (FmhB), FemA, and FemB. The first protein structure from this family, Staphylococcus aureus FemA, has been solved at 2.1 A resolution by X-ray crystallography. The FemA structure reveals a unique organization of several known protein folds involved in peptide and tRNA binding. The surface of the protein also reveals an L-shaped channel suitable for a peptidoglycan substrate. Analysis of the structural features of this enzyme provides clues to the mechanism of action of S. aureus FemA.


Journal of Protein Chemistry | 2003

Spontaneous Aggregation and Cytotoxicity of the β-Amyloid Aβ1–40: A Kinetic Model

Bruce Taylor; Ronald W. Sarver; Gregory J. Fici; Roger A. Poorman; Barry S. Lutzke; Antonio Molinari; Thomas T. Kawabe; Karl Kappenman; Allen E. Buhl; Dennis E. Epps

The time dependency of the spontaneous aggregation of the fibrillogenic β-Amyloid peptide, Aβ1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Aβ antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 μM amyloid peptide. The anti–Aβ antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length Aβ was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Aβ1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Aβ1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Aβ1–40in vitro and possibly in vivo.


Journal of Medicinal Chemistry | 2008

Thermodynamic and Structure Guided Design of Statin Based Inhibitors of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

Ronald W. Sarver; Elizabeth Bills; Gary Louis Bolton; Larry D. Bratton; Nicole Caspers; James B. Dunbar; Melissa S. Harris; Richard Henry Hutchings; Robert Michael Kennedy; Scott D. Larsen; Alexander Pavlovsky; Jeffrey A. Pfefferkorn; Graeme Bainbridge

Clinical studies have demonstrated that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) inhibitors, are effective at lowering mortality levels associated with cardiovascular disease; however, 2-7% of patients may experience statin-induced myalgia that limits compliance with a treatment regimen. High resolution crystal structures, thermodynamic binding parameters, and biochemical data were used to design statin inhibitors with improved HMGR affinity and therapeutic index relative to statin-induced myalgia. These studies facilitated the identification of imidazole 1 as a potent (IC 50 = 7.9 nM) inhibitor with excellent hepatoselectivity (>1000-fold) and good in vivo efficacy. The binding of 1 to HMGR was found to be enthalpically driven with a Delta H of -17.7 kcal/M. Additionally, a second novel series of bicyclic pyrrole-based inhibitors was identified that induced order in a protein flap of HMGR. Similar ordering was detected in a substrate complex, but has not been reported in previous statin inhibitor complexes with HMGR.


Proteins | 2009

Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim

Holly Heaslet; Melissa S. Harris; Kelly Fahnoe; Ronald W. Sarver; Henry Putz; Jeanne Chang; Chakrapani Subramanyam; Gabriela Barreiro; J. Richard Miller

Dihydrofolate reductase (DHFR) is the enzyme responsible for the NADPH‐dependent reduction of 5,6‐dihydrofolate to 5,6,7,8‐tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. Because of its importance in multiple cellular functions, DHFR has been the subject of much research targeting the enzyme with anticancer, antibacterial, and antimicrobial agents. Clinically used compounds targeting DHFR include methotrexate for the treatment of cancer and diaminopyrimidines (DAPs) such as trimethoprim (TMP) for the treatment of bacterial infections. DAP inhibitors of DHFR have been used clinically for >30 years and resistance to these agents has become widespread. Methicillin‐resistant Staphylococcus aureus (MRSA), the causative agent of many serious nosocomial and community acquired infections, and other gram‐positive organisms can show resistance to DAPs through mutation of the chromosomal gene or acquisition of an alternative DHFR termed “S1 DHFR.” To develop new therapies for health threats such as MRSA, it is important to understand the molecular basis of DAP resistance. Here, we report the crystal structure of the wild‐type chromosomal DHFR from S. aureus in complex with NADPH and TMP. We have also solved the structure of the exogenous, TMP resistant S1 DHFR, apo and in complex with TMP. The structural and thermodynamic data point to important molecular differences between the two enzymes that lead to dramatically reduced affinity of DAPs to S1 DHFR. These differences in enzyme binding affinity translate into reduced antibacterial activity against strains of S. aureus that express S1 DHFR. Proteins 2009.


Journal of Biomolecular Screening | 2002

Determination of ligand-murB interactions by isothermal denaturation: Application as a secondary assay to complement high throughput screening

Ronald W. Sarver; Joseph M. Rogers; Dennis E. Epps

We used a temperature-jump isothermal denaturation procedure with various methods of detection to evaluate the quality of putative inhibitors of MurB discovered by high-throughput screening. Three optical methods of detection—ultraviolet hyperchromicity of absorbance, fluorescence of bound dyes, and circular dichroism—as well as differential scanning calorimetry were used to dissect the effects of two chemical compounds and a natural substrate on the enzyme. The kinetics of the denaturation process and binding of the compounds detected by quenching of flavin fluorescence were used to quantitate the dose dependencies of the ligand effects. We found that the first step in the denaturation of MurB is the rapid loss of flavin from the active site and that the two chemical inhibitors appeared to destabilize the interaction of the cofactor with the enzyme but stabilize the global unfolding. The kinetics of the denaturation process as well as the loss of flavin fluorescence on binding established that both compounds had nanomolar affinities for the enzyme. We showed that coupling of the various detection methods with isothermal denaturation yields a powerful regimen to provide analytical data for assessing inhibitor specificity for a protein target.


Chemical Biology & Drug Design | 2010

The use of biochemical and biophysical tools for triage of high-throughput screening hits - A case study with Escherichia coli phosphopantetheine adenylyltransferase.

J. Richard Miller; Venkataraman Thanabal; Michael Melnick; Manjinder S. Lall; Charles Francis Donovan; Ronald W. Sarver; Doh-Yeel Lee; Jeff Ohren; Don Emerson

High‐throughput screening is utilized by pharmaceutical researchers and, increasingly, academic investigators to identify agents that act upon enzymes, receptors, and cellular processes. Screening hits include molecules that specifically bind the target and a greater number of non‐specific compounds. It is necessary to ‘triage’ these hits to identify the subset worthy of further exploration. As part of our antibacterial drug discovery effort, we applied a suite of biochemical and biophysical tools to accelerate the triage process. We describe application of these tools to a series of 9‐oxo‐4,9‐dihydropyrazolo[5,1‐b]quinazoline‐2‐carboxylic acids (PQ) hits from a screen of Escherichia coli phosphopantetheine adenylyltransferase (PPAT). Initial confirmation of specific binding to phosphopantetheine adenylyltransferase was obtained using biochemical and biophysical tools, including a novel orthogonal assay, isothermal titration calorimetry, and saturation transfer difference NMR. To identify the phosphopantetheine adenylyltransferase sub‐site bound by these inhibitors, two techniques were utilized: steady‐state enzyme kinetics and a novel 19F NMR method in which fluorine‐containing fragments that bind the ATP and/or phosphopantetheine sites serve as competitive reporter probes. These data are consistent with PQs binding the ATP sub‐site. In addition to identification of a series of PPAT inhibitors, the described hit triage process is broadly applicable to other enzyme targets in which milligram quantities of purified target protein are available.


Combinatorial Chemistry & High Throughput Screening | 2003

Multi-Selective One Dimensional Proton NMR Experiments for Rapid Screening and Binding Affinity Measurements

Claudio Dalvit; Daneen T.A. Hadden; Ronald W. Sarver; Andrea M. Ho; Brian J. Stockman

High-throughput ligand-based proton NMR screening performed in the presence of a spy molecule and a control molecule is a valuable tool for identifying drug leads. A limitation of the technique is represented by the severe overlap encountered in the screening of large chemical mixtures. An approach for overcoming this overlap problem is the use of multi-selective R(1) filtered and COSY or TOCSY experiments. Application of this methodology to compounds binding to the Sudlow site I of human serum albumin is presented. The screening is performed by simply monitoring the intensity of two signals. The precise measurement of the relative intensity of the two resonances permits determination of the binding constant of the NMR-hit. For a simple competition binding mechanism, the rapidly-derived NMR binding constants are in good agreement with the values derived from full-titration ITC and fluorescence spectroscopy measurements.


Analytical Biochemistry | 2002

Physical methods to determine the binding mode of putative ligands for hepatitis C virus NS3 helicase.

Ronald W. Sarver; Joseph M. Rogers; Brian J. Stockman; Dennis E. Epps; Jack DeZwaan; Melissa S. Harris; Eric T. Baldwin

Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K(d)s) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.


Bioorganic & Medicinal Chemistry Letters | 2003

Inhibition of bacterial IF2 binding to fMet-tRNA(fMet) by aminoglycosides

Jonathan M. Evans; B.A Turner; S Bowen; Andrea M. Ho; Ronald W. Sarver; E Benson; C.N Parker

Screening for inhibitors of bacterial protein synthesis Initiation Factor 2 (IF2) binding to N-formyl-Methionyl-transfer RNA (fMet-tRNA((fMet))) identified a series of aminoglycosides, that included amikacin and kanamycin A1, as inhibitors of this interaction. Subsequent testing revealed that aminoglycosides displayed a wide range of inhibitory activity. However, the failure of these compounds to completely inhibit binding of IF2 to fMet-tRNA((fMet)), the known ability of aminoglycosides to bind RNA, and the ability of the aminoglycosides to displace PicoGreen bound to fMet-tRNA((fMet)) suggest these compounds act by binding fMet-tRNA((fMet)). This hypothesis is further supported by isothermal denaturation experiments that failed to show any interaction between the IF2 protein and the aminoglycosides.


Journal of Biological Chemistry | 2003

Identification of a Mutant Amyloid Peptide That Predominantly Forms Neurotoxic Protofibrillar Aggregates

Isam Qahwash; Katherine L. Weiland; Yifeng Lu; Ronald W. Sarver; Rolf F. Kletzien; Riqiang Yan

Collaboration


Dive into the Ronald W. Sarver's collaboration.

Researchain Logo
Decentralizing Knowledge