Timothy A. Subashi
Pfizer
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Timothy A. Subashi.
Nature Structural & Molecular Biology | 2007
David Cunningham; Dennis E. Danley; Kieran F. Geoghegan; Matthew C. Griffor; Julie Hawkins; Timothy A. Subashi; Alison H. Varghese; Mark Ammirati; Jeffrey S. Culp; Lise R. Hoth; Mahmoud N. Mansour; Katherine M McGrath; Andrew P. Seddon; Shirish Shenolikar; Kim Jonelle Stutzman-Engwall; Laurie C. Warren; Donghui Xia; Xiayang Qiu
Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with Kd = 170 nM at the neutral pH of plasma, but with a Kd as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.
Nature Structural & Molecular Biology | 2007
Xiayang Qiu; Anil Mistry; Mark Ammirati; Boris A. Chrunyk; Ronald W. Clark; Yang Cong; Jeffrey S. Culp; Dennis E. Danley; Thomas B. Freeman; Kieran F. Geoghegan; Matthew C. Griffor; Steven J. Hawrylik; Cheryl Myers Hayward; Preston Hensley; Lise R. Hoth; George A. Karam; Maruja E. Lira; David B. Lloyd; Katherine M McGrath; Kim Jonelle Stutzman-Engwall; Ann Subashi; Timothy A. Subashi; John F. Thompson; Ing-Kae Wang; Honglei Zhao; Andrew P. Seddon
Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-Å resolution, revealing a 60-Å-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.
Journal of Pharmacology and Experimental Therapeutics | 2008
Julie Hawkins; Michael D. Robbins; Laurie C. Warren; Donghui Xia; Stephen F. Petras; James J. Valentine; Alison H. Varghese; Ing-Kae Wang; Timothy A. Subashi; Lorraine D. Shelly; Bruce A. Hay; Katherine T. Landschulz; Kieran F. Geoghegan; H. James Harwood
Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 μM PF-429242 (Bioorg Med Chem Lett 17:4411–4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SRE-luciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC50 of 0.5 μM. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome.
Structure | 2003
Tom Pauly; J.L Ekstrom; D.A Beebe; B Chrunyk; D Cunningham; M Griffor; A Kamath; S.E Lee; R Madura; D Mcguire; Timothy A. Subashi; D Wasilko; P Watts; B.L Mylari; P.J Oates; Paul D. Adams; Virginia L. Rath
Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.
Journal of the American Chemical Society | 2012
Sreeman K. Mamidyala; Sanjay Dutta; Boris A. Chrunyk; Cathy Préville; Hong Wang; Jane M. Withka; Alexander McColl; Timothy A. Subashi; Steven J. Hawrylik; Matthew C. Griffor; Sung Kim; Jeffrey A. Pfefferkorn; David A. Price; Elnaz Menhaji-Klotz; Vincent Mascitti; M. G. Finn
The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.
Journal of Lipid Research | 2010
Ronald W. Clark; David Cunningham; Yang Cong; Timothy A. Subashi; George T. Tkalcevic; David B. Lloyd; James G. Boyd; Boris A. Chrunyk; George A. Karam; Xiayang Qiu; Ing-Kae Wang; Omar L. Francone
The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd ≥ 25 uM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-α normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis.
Acta Crystallographica Section D-biological Crystallography | 2009
Thomas J. McLellan; Eric S. Marr; Lillian M. Wondrack; Timothy A. Subashi; Paul A. Aeed; Seungil Han; Zuoyu Xu; Ing-Kae Wang; Bruce Maguire
A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 A resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested.
Bioorganic & Medicinal Chemistry Letters | 2013
Thomas V. Magee; Seungil Han; Sandra P. McCurdy; Thuy-Trinh Nguyen; Karl Granskog; Eric S. Marr; Bruce Maguire; Michael D. Huband; Jinshan Michael Chen; Timothy A. Subashi; Veerabahu Shanmugasundaram
A novel series of 3-O-carbamoyl erythromycin A derived analogs, labeled carbamolides, with activity versus resistant bacterial isolates of staphylococci (including macrolide and oxazolidinone resistant strains) and streptococci are reported. An (R)-2-aryl substituent on a pyrrolidine carbamate appeared to be critical for achieving potency against resistant strains. Crystal structures showed a distinct aromatic interaction between the (R)-2-aryl (3-pyridyl for 4d) substituent on the pyrrolidine and G2484 (G2505, Escherichia coli) of the Deinococcus radiodurans 50S ribosome (3.2Å resolution).
PLOS ONE | 2017
Justin Hall; Amy Brault; Fabien Vincent; Shawn Weng; Hong Wang; Darren S. Dumlao; Ann Aulabaugh; Dikran Aivazian; Dana Castro; Ming Chen; Jeffrey S. Culp; Ken Dower; Joseph Gardner; Steven J. Hawrylik; Douglas T. Golenbock; David Hepworth; Mark Horn; Lyn H. Jones; Peter Jones; Eicke Latz; Jing Li; Lih-Ling Lin; Wen Lin; David C. Lin; Frank Lovering; Nootaree Niljanskul; Ryan Nistler; Betsy S. Pierce; Olga Plotnikova; Daniel Schmitt
Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.
Analytical Biochemistry | 2009
Samuel P. Simons; Thomas J. McLellan; Paul A. Aeed; Richard P. Zaniewski; Charlene R. Desbonnet; Lillian M. Wondrack; Eric S. Marr; Timothy A. Subashi; Thomas J. Dougherty; Zuoyu Xu; Ing-Kae Wang; Peter K. LeMotte; Bruce Maguire
We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.