Michael Bell and Stephen M. Testa
Department of Chemistry, University of Kentucky
We have developed a novel catalytic RNA that can remove, in a sequence-specific fashion, designated disease causing mutations from RNA model systems in vitro.
Ashley K. Johnson, Jesse Tye, Dana Baum, and Stephen M. Testa
Department of Chemistry, University of Kentucky
We have developed a combinatorial assay for the analysis of the initial molecular recognition event between an RNA enzyme and its substrate.
F. H. Ebetino, M. Rogers, J. Dunford, X. Liu, R. Phipps, R. G. G. Russell, B. Barnett, P. Correa, and G. Mieling
Procter & Gamble Pharmaceuticals, Mason, OH 45040
Bisphosphonates (BPs) have now been successfully utilized clinically for a number of years. Recently, the nitrogen-containing bisphosphonates (N-BPs) were found to be potent inhibitors of farnesyl diphosphate (FPP) synthase, an enzyme in the mevalonate pathway. FPP synthase appears to be the major target enzyme of N-BPs, since minor chemical changes that alter the potency for inhibiting FPP synthase also alter anti-resorptive potency. This confirms that N-BPs interact with their target via a stereochemical recognition event, predicted from structure-activity relationships evaluated in our laboratories for a number of years. On close analysis of active and inactive structurally-related pairs of BPs, we propose a potentially more specific inhibition due to binding at a predicted IPP binding site in the enzyme.
Frank H. Ebetino, David L. Soper, Michelle J. Dirr, Mark W. Lundy, Glen Mieling, Paula Chmielewski, Cheryl Froman, Ralph Farmer, John A. Wos and Mitchell A. deLong
Procter & Gamble Pharmaceuticals, Mason, OH 45040
Prostaglandin F analogs have attracted considerable recent interest as superior agents for the lowering of Intraocular Pressure (IOP) pursuant to glaucoma management and, more recently, as potential bone anabolic agents. Early generation anabolic agents in this class are characterized by relatively short half-lives, likely due in part to the C-1 carboxylic acid, which is subject to beta oxidation and may serve as the recognition element for active metabolism. Guided by molecular modeling, we investigated the replacement of the C-1 acid by various suitable functional groups. Most of these replacements resulted in a significant loss of activity at the human FP receptor, the cellular mediator of the effects of the F analogs. The selectivity of these compounds observed for the FP receptor was unique compared to corresponding carbon analogs, and was predicted as a component of the original drug design, due to differences among the various prostaglandin receptors in the binding region of the C-1 carboxylic acid.
Jason D. Ehrick, Sapna K. Deo, Leonidas G. Bachas, and Sylvia Daunert
Department of Chemistry, University of Kentucky, Lexington, KY, 40506-0055
Reversibly controlled hydrogel microactuators incorporated in implantable drug delivery systems have several applications in the area of BioMEMS. A responsive protein-immobilized hydrogel has been developed to simultaneously sense and dispense drugs for controlled release applications. Calmodulin (CaM), a calcium binding protein, was employed in these studies since it undergoes a large conformational change upon binding calcium, certain peptides, and the phenothiazine class of drugs. Chemically modified CaM has been immobilized in conjunction with a phenothiazine derivative within an acrylamide polymer network. Phenothiazine binds CaM in the presence of calcium providing additional cross-linking within the network. The volume of the hydrogel changes when the non-covalent cross-linking between the CaM and phenothiazine breaks due to competitive binding of free chlorpromazine in the phenothiazine binding sites on CaM, and also by removal of calcium from the system. Reversible swelling and shrinking was observed and monitored by measuring the diameter change of the hydrogel with a CCD camera. Our studies have demonstrated that the protein-immobilized hydrogel can be utilized to detect the presence of anti-depressant drugs. Studies incorporating variable amounts of cross-linker and binding other phenothiazine class of drugs have also been investigated.
Jeffrey S. Lenihan1, Vasilis Gavalas1, Jianquan Wang1, Rodney Andrews2, and Leonidas Bachas1.
1 Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055,
2 Center for Applied Energy Research, Advanced Carbon Materials Center, University of Kentucky
With properties such as high tensile strength, excellent stiffness towards bending, high electrical conductivity, and a large surface area, carbon nanotubes are an attractive platform for the design of bioreactors and biosensors. In this work, investigations into factors affecting the activity of proteins immobilized onto nanotubes have been performed. Through the use of a biological molecular adapter, the effects of random versus controlled immobilization of sensing proteins onto the nanotube surface have been examined. Additionally, it is known that the processes currently used to synthesize carbon nanotubes also result in the production of amorphous carbon impurities on the nanotube surface. Investigations have been performed into the effects of removing these impurities, via standard purification techniques, on the immobilization of proteins onto the tube surface. As a result of these experiments, biosensors have been developed by which analyte detection can be performed by both optical and electrochemical methods.
Libby G. Puckett, Jennifer C. Lewis, Sylvia Daunert, and Leonidas G. Bachas
Department of Chemistry, University of Kentucky
Bacterial resistance to b-lactam antibiotics occurs through the production of b-lactamases. b-lactamase is an enzyme that catalyzes the cleavage of the b-lactam ring of antibiotics such as penicillin and ampicillin. To study this hydrolysis, a fusion protein was constructed between enhanced green fluorescent protein (EGFP) and b-lactamase. When the b-lactam ring is cleaved, there is a decrease in the local pH. Subsequently, the fluorescence of EGFP decreases which can be correlated to the amount of antibiotic present. Other hydrolytic enzymes can also be conjugated to EGFP. This EGFP/b-lactamase fusion protein has also been used to study a common b-lactamase inhibitor - sulbactam sodium. It is an irreversible competitive inhibitor of b-lactamase that is commonly combined with b-lactam antibiotics in order to prevent bacterial resistance. Upon addition of the inhibitor, sulbactam, b-lactamase is unable to hydrolyze b-lactam antibiotics at the same rate as previously observed.
Lorna Putnam and Robert C. Dickson
Department of Chemistry, University of Kentucky
Polymerase chain reaction (PCR) was carried out in the presence of Mn2+ and dITP, mutagens, to try and generate a temperature sensitive (ts) allele of SUR4. This ts allele will regulate the synthesis of C26 fatty acids of sphingolipids and provide more information about their function.
Alessandra Castegna, Christopher M. Lauderback, and D. Allan Butterfield
Department of Chemistry and Center of Membrane Sciences, University of Kentucky
The present study investigated the inactivation of the enzyme flippase by HNE and acrolein, reactives alkenals, the level of which is increased in Alzheimer's disease brain.
Jennifer Drake, Jaroslaw Kanski, Sridhar Varadarajan, Maria Tsoras, and D. Allan Butterfield
Department of Chemistry and Center of Membrane Sciences, University of Kentucky
The compound g-glutamylcysteinyl ethyl ester elevates brain GSH levels, thereby protecting synaptosomes from in-vitro peroxynitrite-induced oxidative stress.
Sean R. Klopfenstein*, Adam Golebiowski, Julita Jozwik, Xia Shao and Jack Chen
Procter & Gamble Pharmaceuticals, Mason, OH 45040
We have been working for several years on the design and solid-phase synthesis of reverse turn peptide mimetics for lead generation in early drug discovery. These efforts have resulted in the development of three separate synthetic routes to scaffolds which can produce rigid, bicyclic b-turn mimetics in a parallel fashion on a medium to ultra-high throughput (10's to 10,000's) scale. This presentation will highlight the chemistry developments as well as the design benefits and limitations to each of the proposed scaffolds and how this research has evolved to its current state.
Chava B. Pocernich and D. Allan Butterfield
Department of Chemistry, University of Kentucky
Alzheimer's (AD) brain has increased protein and lipid oxidation, along with mitochondrial dysfunction and loss of energy. Acrolein, a lipid peroxidation product found to be increased in AD brain, binds and inhibits the mitochondrial enzyme pyruvate dehydrogenase which could lead to the mitochondrial dysfunction and loss of energy.
Kazuyuki Hattori and Robert B. Grossman
Department of Chemistry, University of Kentucky
Fused bicyclic a-amino acids can be prepared by a double Michael reaction of p-anisyl ethynyl ketone and a tethered diacid, cyclization via hydrogenation or hydration of a CN or NO2 group, and oxidation of the p-anisyl group. The substitution level of the a-amino acids can be adjusted by decyanation or decarboxylation of the intermediates. Bicyclic a-amino acids prepared in this way include cis- and trans-perhydroisoquinoline-3-carboxylic acids, cis-perhydro-2-pyrindine-3-carboxylic acids, and trans-perhydroindole-2-carboxylic acids of various substitution and oxidation levels. The bicyclic a-amino acids may be regarded as analogs of proline, pipecolic acid, 2-minoadipic acid, or glutamic acid.
Robert S. Goforth and Robert B. Grossman
Department of Chemistry, University of Kentucky
Tethered diacids have been used as precursors for the double Michael reaction. The only acidifying groups used to date have been cyanos, esters, ketones, sulfones and nitros. The exploration of heterocyclic acidifying groups on tethered diacids is a new approach in this ongoing research. Many medicinally active compounds contain both a heterocycle and a rigid lipophilic group. Further methods will be discussed.
Jos P. Varghese, Vijaya N. Desai, and Robert B. Grossman
Department of Chemistry, University of Kentucky
A double Michael reaction, an acid-promoted ring-closing condensation of a CN group and a ketone, a hydrogenation of an enamide, and a desulfonation are the key steps in the synthesis of the DE ring system of the Yohimba alkaloids. Factors affecting the stereochemical course of the key reactions will be discussed.
ChangWoo Lee, Inhae Ji and Tae H. Ji
Department of Chemistry, University of Kentucky
Luteinizing hormone receptor is a G protein-coupled receptor and consists of two halves: the N-terminal extracellular half (exodomain) and C-terminal membrane associated half (endodomain). Hormone binds to the exodomain and the resulting hormone/exodomain complex modulates the endodomain to generate signals. There are mutations, which impair either hormone binding or signal generation. We report that the coexpression of a binding defective mutant and a signal defective mutant rescues signal generation to produce cAMP. This rescue requires both types of mutant receptors and is dependent on the hCG dose, surface concentration of mutant receptors, and the amino acid position of mutations. Furthermore, random collisions among mutant receptors are not involved in the rescue. Our observations provide new insights into the mechanisms of the functional and structural relationship of the exodomain and endodomain, signal transduction, and receptor genetics, in particular for defective heterozygotes.
ChangWoo Lee, Inhae Ji and Tae H. Ji
Department of Chemistry, University of Kentucky
G protein-coupled receptors (GPCRs) accommodate a wide spectrum of activators from ions to glycoprotein hormones. The mechanism of activation for this large and clinically important family of receptors is poorly understood. While initially thought to function as monomers, there is a growing body of evidence that GPCR dimers form, and in some cases, these dimers are essential for signal transduction. Here we describe a novel mechanism of intermolecular GPCR activation, which we refer to as trans-activation, in the luteinizing hormone receptor, a GPCR that does not form stable dimers. The luteinizing hormone receptor consists of a 350 amino acid amino terminal domain, which is responsible for high affinity binding to human chorionic gonadotropin, followed by seven transmembrane domains and connecting loops. This seven transmembrane domain bundle transmits the signal from the extracellular amino terminus to intracellular G proteins and adenylyl cyclase. Here, we show that binding of hormone to one receptor can activate adenylyl cyclase through its transmembrane bundle, intramolecular activation (cis-activation), as well as trans-activation through the transmembrane bundle of an adjacent receptor, without forming a stable receptor dimer. Coexpression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescues hormone-activated cAMP production. Our observations provide new insights into the mechanism of receptor activation mechanisms, and have implications for the treatment of inherited disorders of glycoprotein hormone receptors.
Richard J. Isaacs and H. Peter Spielmann
Department of Biochemistry, University of Kentucky
NMR spectroscopy has been used to solve the solution structures and high frequency internal dynamics of DNA oligomers containing G-T mismatches, a psoralen adduct, and their undamaged counterparts. The patterns of structural and dynamic differences for the DNA lesions compared to normal DNA are distinctly different, perhaps providing insight into why psoralen adducts are recognized by the nucleotide excision repair (NER) system, while G-T mismatches are not.
Chandra N. Patel,2 David Koh1, Yun Li1, Myron K. Jacobson1, and Marcos A. Oliveira2
1 Arizona Cancer Center and College of Pharmacy, University of Arizona
2 College of Pharmacy, Markey Cancer Center and Center for Structural Biology, University of Kentucky
One component of the cellular genomic surveillance system involves poly(ADP-ribose) polymerase (PARP) and its partner poly (ADP-ribose) glycohydrolase (PARG). PARP is activated in response to DNA damage covalently attaching ADP-ribose (ADPR) polymers to itself and other DNA repair proteins. The rapid increase in ADPR polymer is followed by its degradation within minutes by PARG, suggesting a close coordination between PARP and PARG. ADPR polymer is thought to induce DNA decondensation through the ADP-ribosylation of histones and act as a platform for the formation of DNA repair complexes (base excision repair (BER)), at the site of DNA lesion. The discovery of a growing number of PARP's with specialized functional roles suggests that presence of a similar family of PARG proteins. In order to enable the identification distantly related sequences with glycohydrolase activity we have: (i) investigated the secondary and tertiary structural prediction for PARG as tool in fold identification and (ii) used site directed mutagenesis to identify key functional residues of PARG catalytic fragment. The combination of structure prediction and mutagenesis allows us to define a signature sequence that we have used to identify homologues and/or related PARG's. Our results suggest the hypothesis that PARG may be a distant relative of PARP containing a core ADP-ribosyltransferease fold. In addition we have mapped acidic residues essential for catalytic activity as well as mapped residues involved in polymer recognition.
Jing Chen and Arthur Cammers-Goodwin
Department of Chemistry, University of Kentucky
A molecule that partitions into three conformations was used as a probe to study the stability of p-stacking in aqueous and mixed aqueous solvents. Conformer C hid the most hydrogen atom solvent accessible surface area (SASA) while F exposed the most hydrogen atom SASA. The probe preferred C when fluoroalkanol was used as cosolvent, but with alkanol cosolvent, conformer F was most stable. Collectively these studies comprise a solvent parameter that unexpectedly places water between two classes of hydrophobic solvents. The implications to protein and peptide conformation in mostly aqueous solvents are discussed.
Yan Zhu and Arthur Cammers-Goodwin
Department of Chemistry, University of Kentucky
This study reports synergism between tripodal polycationic host molecules and polymolybdate that gave rise to discrete spherical assemblies. The control exerted on the aggregate by the multipartite host was inferred from the spherical nanostructures observed by transmission electron microscopy. Comparative studies with a series of structurally analogous tripodal and bipodal hosts probed the relationship between the structure and formation of nanostructures. The structured polyoxoanion and the coulombic interactions between it and the multipartite cationic shell provided a motif with which angstrom-scale changes in structure were multiplied to nanometer-scale changes in structure. The analogy between these structures and spherical viruses was striking.
Sihui Long, Marlon Jones, and Arthur Cammers-Goodwin
Department of Chemistry, University of Kentucky
Cationic peptide antibiotics show great potential against pathogenic bacteria through a unique and interesting mode of action. Unfortunately, peptide drugs such as these are rarely successful due to their rapid systemic degradation and elimination. This effort seeks to develop chiral oligoureas as bioactive peptide mimics. The probable global conformation of this new foldamer and the challenges in the solution-phase synthetic endeavor utilizing various C2-symmetric, 1,2-diamines as building blocks are discussed.
Vinit K. Rastogi, Conrad F. Diven, Brian W. Howard and Kevin G. Peters
Procter & Gamble Pharmaceuticals, Mason, OH 45040
HCPTPA, one of the low molecular weight (ca. 19 kDa) Human Cytoplasmic Protein Tyrosine Phosphatase, is involved in signaling for regulation of cell function. A smart approach combining NMR and bench-top biological assay provides a rapid and reliable method for screening of small molecules against drug-targets. We have utilized a suite of 3D NMR experiments to obtain the sequence specific assignments of backbone resonances (1H, 15N, and 13C). The NMR assignments are used for screening compounds by recording and comparing 2D-15N1H-HSQC experiments in the absence and presence of ligands. NMR-based screening is useful for lead identification and elimination of false positives. The NMR hits provide important leads for structure-based drug design.
Emine Yikilmaz1, Anush Karapatyan1, Carrie K. Vance1, Tim Jackson2, Thomas Brunold2 and Anne-Frances Miller1
1 Department of Chemistry, University of Kentucky, Lexington KY 40506-0055
2 Department of Chemistry, University of Wisconsin, Madison, WI 53706
We propose that coordinated solvent molecules may serve a widespread role as adaptors enabling proteins to exploit their exquisite control over protons to indirectly control the Ems and thus electrons of bound metal ions. To test this model, we have generated two mutants of FeSOD in which the active site Gln that supplies the only hydrogen bond from protein to the coordinated solvent molecule has been mutated to Glu in one case and His in the other. In both, the Em is altered by hundreds of mV, despite the fact that these are only second sphere mutations. We describe thermodynamic and spectroscopic measurements elucidating the basis for these large shifts in Em and thus the mechanism of Em tuning in SOD.
Nielsen, K. E., Petersen, M., Singh, S. K., Hakansson, A. E., Wengel, J., Jacobsen, J. P.
University of Southern Denmark, Odense, Denmark
We have solved the solution structures of two decamer DNA duplexes, each containing four LNA (locked nucleic acid) or a-L-LNA nucleotides, hybridized with complementary DNA. The structure of the LNA:DNA duplex was shifted towards more A-type characteristics whereas the a-L-LNA:DNA duplex retained the overall B-type structure characteristic of native DNA:DNA sequences.
Bobby Barnett, Michelle Dunaway, Longyin Chen, Tim Rydel, Mike Natchus, Glen Mieling, Fei Gu, Matt Pokross, Artem Evdokimov
Procter & Gamble Pharmaceuticals, Mason, OH 45040
Matrix metalloproteinase (MMP) inhibitors are potential therapeutic agents for various diseases including cancer and osteoarthritis. Recent data from clinical trials with MMP inhibitors indicate that there is a great need for selective inhibitors. Here at P&G, we have used X-ray crystallography as a tool to help understand specific binding interactions of inhibitors to various MMPs. Large conformational changes have been noted when comparing the structures of the active MMP3 catalytic domain and the one inhibited by a hydroxamic acid inhibitor. Both soaking and co-crystallization methods were used to generate the MMP-3/inhibitor complex crystals for data collection. The same inhibitor has also been co-crystallized with MMP-1 and MMP-13. Comparisons of the structures of three inhibited enzymes, MMP-1, 3, and 13 show that MMP-3 and 13 are extremely similar. There are major differences in the binding pockets, especially in the S1' pocket between MMP-1 and MMP-3/13. These studies can help design more selective inhibitors that can be used as therapeutic agents with improved safety profile.
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