Conformational Analysis of Chirally Deuterated Tunicamycin as an Active Site Probe of UDP-N-Acetylhexosamine:Polyprenol-PN-Acetylhexosamine-1-P Translocases†

Biochemistry ◽  
2004 ◽  
Vol 43 (42) ◽  
pp. 13248-13255 ◽  
Author(s):  
Lin Xu ◽  
Michael Appell ◽  
Scott Kennedy ◽  
Frank A. Momany ◽  
Neil P. J. Price
Keyword(s):  
Conformational Analysis ◽  
Active Site ◽  
Active Site Probe

Conformation–activity relationships and the mechanism of action of penicillin

1988 ◽  
Vol 66 (11) ◽  
pp. 2733-2750 ◽  
Author(s):  
Saul Wolfe ◽  
Kiyull Yang ◽  
Maged Khalil
Keyword(s):  
Conformational Analysis ◽  
Carbonyl Group ◽  
Active Site ◽  
Global Minimum ◽  
Peptide Bond ◽  
Biologically Active ◽  
Penicillin G ◽  
Side Chain ◽  
Geometrical Parameters ◽  
Carboxyl Group

Using the MMPEN parameters of Allinger's MMP2(85) force field, a conformational analysis has been performed on four biologically active penicillins; D-ampicillin, L-α-phenoxyethylpenicillin, penicillin G, and penicillin V, and on five biologically inactive or much less active penicillins: L-ampicillin, D-α-phenoxyethylpenicillin, N-methylpenicillin G, 6α-methylpenicillin G, and bisnorpenicillin G. Antibacterial activity is found to be associated with the existence of a global minimum having a compact structure, whose convex face is accessible to a penicillin binding protein (PBP), with the C3-carboxyl group and the side-chain N-H exposed on this face. Using the MMPEP parameters of MMP2(85), a conformational analysis has been performed on phenylacetyl-D-Ala-D-Ala-O−, a peptide model of the normal substrate of a PBP. Labischinski's global minimum has been reproduced, along with structures that correspond to Tipper and Strominger's proposal that the N4—C7 bond of a penicillin corresponds to the Ala–Ala peptide bond, and to Hasan's proposal that the N4—C5 bond of penicillin corresponds to the peptide bond. For both models, conformations of the peptide related to the pseudoaxial and pseudoequatorial conformations of the thiazolidine ring of penicillin G have been examined. It is concluded that penicillin is not a structural analog of the global minimum of the peptide; however, comparisons based on unbound conformations of PBP substrates are unable to determine which model is more appropriate, or which conformation of penicillin G is the biologically significant one. Using the ECEPP/MMPEP strategy, a model of the active site of a PBP has been obtained, following a search of 200,000 structures of the peptide Ac-NH-Val-Gly-Ser-Val-Thr-Lys-NH-Me. This peptide contains the sequence at the active site of a PBP of Streptomyces R61, for which it is also known that the C3-carboxyl group of penicillin binds to the ε-amino group of lysine, and the β-lactam reacts chemically with the serine OH. The lysine and serine side chains and the C-terminal carbonyl group are found to occupy the concave face of the active site model.A strategy for the docking of penicillins or peptides to this model, with full minimization of the conformational energies of the complexes, has been devised. All active penicillins bind through strong hydrogen bonds to the C3-carboxyl group and the side-chain N-H, and with a four-centered relationship between the O-H of serine and the (O)C-N of the β-lactam ring. The geometrical parameters of this relationship are reminiscent of those found in the gas phase transition state of neutral hydration of a carbonyl group. When the energies of formation and geometries of the pseudoaxial and pseudoequatorial penicillin G complexes are examined, there is now a clear preference for the binding of the pseudoaxial conformation, which is the global minimum of the uncomplexed penicillin in this case. A similar examination of the peptide complexes reveals that only the conformation of the peptide that corresponds to Tipper and Strominger's model, and is based on the pseudoaxial conformation of penicillin G, can form a complex with a geometry and energy comparable to those of a biologically active penicillin.

scholarly journals Functional consequences of an arginine180 to glutamine mutation in factor IX Hilo

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1540-1544 ◽  
Author(s):  
DM Monroe ◽  
DM McCord ◽  
MN Huang ◽  
KA High ◽  
RL Lundblad ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Ix ◽  
Affinity Column ◽  
Extrinsic Pathway ◽  
Control Value ◽  
Amino Terminal ◽  
Active Site Probe ◽  
Covalently Linked

Abstract Factor IX Hilo is a variant factor IX molecule that has no detectable coagulant activity. The defect in factor IX Hilo arises from a point mutation in the gene such that in the protein Arg180 is converted to a Gln. Activation of factor IX Hilo by factor Xla was monitored using the fluorescent active site probe p-aminobenzamidine. Normal factor IX showed complete activation in one hour as determined by measuring the increase in fluorescence when p-aminobenzamidine bound to activated factor IX. Factor IX Hilo showed no increase in fluorescence even after 24 hours, indicating that the active site was not exposed. Polyacrylamide gel electrophoresis showed that factor IX Hilo was cleaved to a light chain plus a larger peptide with a molecular weight equivalent to a heavy chain covalently linked to an activation peptide. Amino terminal amino acid sequencing of factor IX Hilo cleaved by factor Xla showed cleavage only at Arg145-Ala146, indicating that the Gln180-Val181 bond was not cleaved and that the active site was thus not exposed. The presence of factor IX Hilo in patient plasma was responsible for the patient having a very long ox brain prothrombin time characteristic of severe hemophilia Bm. Patient plasma had an ox brain prothrombin time of 100 seconds using a Thrombotest kit, significantly prolonged over the normal control value of 45 seconds. When factor IX Hilo was depleted from patient plasma using an immunoaffinity column, the ox brain prothrombin time decreased to 41 seconds. When factor IX Hilo was added back to depleted patient plasma, to normal plasma depleted of factor IX by the same affinity column, or to plasma from a CRM- hemophilia B patient, the ox brain prothrombin time was significantly prolonged. We conclude that the Arg180 to Gln mutation in factor IX Hilo results in a molecule that cannot be activated by factor Xla. Further, our data suggest that the mutation results in a molecule that interacts with components of the extrinsic pathway to give a prolonged ox brain prothrombin time.

Targeting Btk in Lymphoma: PCI-32765 Inhibits Tumor Growth in Mouse Lymphoma Models and a Fluorescent Analog of PCI-32765 Is an Active-Site Probe That Enables Assessment of Btk Inhibition In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1592-1592 ◽  
Author(s):  
Lee A. Honigberg ◽  
Ashley M. Smith ◽  
Jun Chen ◽  
Patti Thiemann ◽  
Erik Verner
Keyword(s):  
Oral Dose ◽  
B Cell ◽  
Single Oral Dose ◽  
Active Site ◽  
In Vivo Studies ◽  
Cell Lysates ◽  
Bcr Signaling ◽  
Btk Inhibitor ◽  
Active Site Probe

Abstract There is increasing evidence indicating that B-cell-receptor (BCR) signaling is required for survival of non-Hodgkin’s lymphoma (NHL) cells. Bruton’s tyrosine kinase (Btk) is required for BCR signaling and mutations that inactivate human Btk cause X-linked-agammaglobulinemia, a B-cell immunodeficiency. Although Btk functions selectively in B cells, the Btk active site is structurally similar to the active site in several Src and Abl kinases and as a result, there have been few highly selective small molecule inhibitors of Btk. We have developed a series of covalent Btk inhibitors that target Cys-481 in Btk and this approach results in increased potency and selectivity over related kinases that lack a Cys residue at this position (ChemMedChem 15:58). PCI-32765 is a Cys-481 targeting Btk inhibitor that has been optimized for potency, selectivity and pharmacokinetics. In cellular assays, PCI-32765 inhibits BCR-stimulus induced phosphorylation of Phospholipase-C-gamma, a Btk substrate, as well as downstream phosphorylation of Erk (IC50 < 100 nM). In addition, PCI-32765 induces apoptosis and inhibits proliferation in a subset of NHL cell lines including DHL-4, DHL-6, WSU-DLCL2, OCI-Ly10 and DOHH2 (IC50s = 0.6–1.6uM). We have used RNAi knockdown in DOHH2 cells as an independent method to confirm that Btk is required for lymphoma cell proliferation. In vivo, orally dosed PCI-32765 (50mg/kg) inhibits growth of DOHH2 and WSU-DLCL2 xenografts. PCI-32765 also prevents disease progression in a mouse collagen-induced arthritis model (12.5mg/kg PO), indicating that other B cell lineage diseases are sensitive to Btk inhibition. In order to further characterize the selectivity and in vivo potency of PCI-32765, we have developed PCI-33380, an active-site probe consisting of a covalent Btk inhibitor linked to the fluorophore Bodipy-FL. PCI-33380 binds to Btk and can be detected by flow cytometry or by denaturing gel electrophoresis of cell lysates. In cell lysates, the probe labels a single predominant band of the same molecular weight as Btk and this band is absent in cells from xid mice. Labeling of this band is inhibited (IC50=10nM) by a brief pre-treatment of cells with PCI-32765, indicating that the probe can be used to assess occupancy of Btk by a covalent inhibitor. We have used the probe to quantitate the inhibition of Btk by PCI-32765 in vivo. A single oral dose of PCI-32765 (10mg/kg) delivered to mice leads to rapid and complete inhibition of Btk in spleen. In addition, a single oral dose of PCI-32765 fully inhibits Btk in xenograft tumors and peripheral blood cells and this inhibition is maintained for up to 24hr. The Btk probe provides pharmacodynamic measurements that may allow optimization of dosing and schedule for in vivo studies and we are currently adapting the probe assays for use in monitoring the inhibition of Btk in human clinical trials.

Meizothrombin: Zymogen or Proteinase? Slow, Ligand-Dependent Equilibration Between Equally Populated Zymogen-Like and Proteinase-Like Forms Explains Its Selectively Anticoagulant Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 533-533
Author(s):  
Harlan Bradford ◽  
Sriram Krishnaswamy
Keyword(s):  
Active Site ◽  
Structural Integrity ◽  
Equilibrium Distribution ◽  
Conflicts Of Interest ◽  
Second Phase ◽  
Covalent Linkage ◽  
Soluble Thrombomodulin ◽  
Carboxyglutamic Acid ◽  
Active Site Probe ◽  
Anticoagulant Function

Abstract Abstract 533 Prothrombinase activates prothrombin by catalyzing its ordered proteolysis at Arg320 followed by cleavage at Arg271. Initial cleavage at Arg320 yields the proteinase meizothrombin (mIIa), which accumulates abundantly as an intermediate before its conversion to thrombin (IIa). Although mIIa is a proteinase, it only acts on a limited subset of substrates cleaved by IIa. mIIa is considered an anticoagulant proteinase because it functions efficiently in protein C activation while exhibiting poor clotting activity or reactivity towards antithrombin III. This limited substrate repertoire of mIIa has remained enigmatic and is generally considered to lie in the retention of covalent linkage to the fragment 1.2 (F12) domain allowing for membrane binding. Instead, our recent findings with IIa, illustrating its ligand dependent interconversions between zymogen-like and proteinase-like states, predict that covalent linkage of F12 to the proteinase domain in mIIa would impart it with zymogen-like properties. We produced stable and active mIIa (mIIaQQQ) using a recombinant prothrombin variant in which the bonds susceptible to autolysis were rendered uncleavable by substitution of Arg at 155, 271 and 284 with Gln. Fluorescence stopped flow studies were pursued with the probes dansyl arginine 3-ethyl piperidine amide (DAPA) or Nα-dansyl-(p-guanidino)-L-phenylalanine-piperidide (I-2581) to characterize the binding of ligands to the active site of mIIaQQQ and IIa. Binding to IIa was rapid and consistent with a rate limiting, bimolecular interaction between probe and the active site of the proteinase. In contrast, traces with mIIaQQQ were distinctly biphasic with ∼50% of the fluorescence change occurring on the millisecond timescale followed by a slow second phase (∼50%) that occurred over several seconds. Global fitting indicated that the findings were consistent with a pre-equilibrium between two forms of mIIa, one which binds the active site probe with μM affinity and a second that binds with nM affinity. The two forms interconvert with forward and reverse rate constants of ∼2 s−1. We surmise that these reflect zymogen-like and proteinase-like forms that are equally populated and interconvert slowly with each other in a ligand-dependent fashion. Accordingly, the distributions of the two forms could be altered by ligands established to affect the transition of IIa between zymogen-like and proteinase-like states. The equilibrium distribution was altered to favor the zymogen-like form by decreasing Na+ to 0 at constant ionic strength. In contrast, soluble thrombomodulin (sTM) drove the equilibrium towards the proteinase-like state in a manner consistent with a 1:1 interaction between mIIaQQQ and sTM. Surprisingly, the pre-equilibrium was heavily dependent on covalent linkage with fragment 1 (F1) or its structural integrity. Proteolytic removal of F1, chelation of Ca2+ with EDTA or elimination of 4-carboxyglutamic acid modifications had a profound effect on forcing the enzyme into the proteinase-like state. Thus, the equilibrium distribution of mIIa between zymogen-like and proteinase-like forms is affected by F1 and its Ca2+-stabilized conformation despite the fact that this domain is expected to be distant from the catalytic site. Our findings shed unexpected light into the mechanisms underlying the peculiar activity profile of mIIa relative to IIa. Its ability to interconvert slowly and reversibly between equally populated zymogen-like and proteinase-like states lies at the heart of its properties. By driving it to proteinase, thrombomodulin imparts full activity to mIIa allowing for efficient function in the anticoagulant pathway. In contrast, more weakly binding substrates, inhibitors or ligands will be less effective at perturbing the equilibrium thereby allowing mIIa to persist in blood with reduced activity towards procoagulant substrates. The F1 domain participates in an unexpected way in enforcing these unique features of mIIa. By virtue of its essential role in modulating the equilibrium distribution between zymogen-like and proteinase-like states, we document a new function for F1 in its role as a zymogenizer of mIIa. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe

2010 ◽  
Vol 8 (1) ◽  
pp. 5 ◽  
Author(s):  
Ragunath Singaravelu ◽  
David R Blais ◽  
Craig S McKay ◽  
John Pezacki
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Active Site ◽  
Hepatoma Cells ◽  
Protein Profiling ◽  
Active Site Probe

scholarly journals A Caspase Active Site Probe Reveals High Fractional Inhibition Needed to Block DNA Fragmentation

2004 ◽  
Vol 279 (27) ◽  
pp. 27905-27914 ◽  
Author(s):  
Nathalie Méthot ◽  
John P. Vaillancourt ◽  
JingQi Huang ◽  
John Colucci ◽  
Yongxin Han ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Active Site ◽  
Active Site Probe ◽  
Fractional Inhibition

Epoxidation of arachidonic acid as an active-site probe of cytochrome P-450 2B isoforms

1994 ◽  
Vol 1206 (1) ◽  
pp. 42-48 ◽  
Author(s):  
Ronald M. Laethem ◽  
James R. Halpert ◽  
Dennis R. Koop
Keyword(s):  
Arachidonic Acid ◽  
Active Site ◽  
Active Site Probe ◽  
Cytochrome P 450

Synthesis of a new substrate analog of firefly luciferin. Active-site probe

Biochemistry ◽  
1973 ◽  
Vol 12 (10) ◽  
pp. 1845-1852 ◽  
Author(s):  
Lemuel J. Bowie ◽  
Vaclav Horak ◽  
Marlene DeLuca
Keyword(s):  
Active Site ◽  
Substrate Analog ◽  
Firefly Luciferin ◽  
Active Site Probe
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