Biomedical subjects

David R Light

Publications and source records attributed to David R Light.

14 recordsLinked to original sources

PubMed2007

A novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) - part I: pharmacological characterization.

We have discovered a novel small-molecule (3-phosphinoylpropionic acid) inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa), BX 528, which had an IC (50) of 2 nM in an enzymatic assay and 50 nM in an in-vitro clot lysis assay, with 3,500- to 35,000-fold selectivity against other carboxypeptidases, such as CPN, CPZ and CPD, and 5- and 12-fold selectivity against CPE (CPH) and CPB, respectively. At 10 micro M, BX 528 had no significant activity (<50% inhibition or antagonism) in a panel of 137 enzymes and receptors. It had no effects on blood coagulation and platelet aggregation up to 300 and 10 micro M, respectively. The plasma half-life following intravenous administration was 0.85 hours in rats and 4.5 hours in dogs. No significant metabolism was detected in human, dog or rabbit hepatic microsomes, and no significant inhibition of cytochrome P450 3A4 and 2D6 up to 30 micro M. No cytotoxic or cell proliferative effects were found in three hepatic and renal cell lines up to 300 micro M and no mutagenic activity was seen in the Ames II screen. There were no significant hemodynamic effects in rats and dogs up to 100 and 30 mg/kg with peak plasma drug concentrations of approximately 1,000 and 300 micro M, respectively. In an in-vivo complement activation model in guinea pigs, BX 528 showed minimal inhibition of plasma CPN activity up to 60 mg/kg with peak plasma concentrations up to 250 micro M. Thus, these data demonstrate that BX 528 is a novel, potent, selective and safe TAFIa inhibitor.

Animals↗

PubMed2006

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

Animals↗

PubMed2006

Amplified anticoagulant activity of tissue factor-targeted thrombomodulin: in-vivo validation of a tissue factor-neutralizing antibody fused to soluble thrombomodulin.

Tissue factor (TF) exposure is a potent pro-thrombotic trigger that initiates activation of the coagulation cascade, while thrombomodulin (TM) is a potent anticoagulant protein that limits the extent of activation. Both TF neutralizing antibodies and soluble TM (sTM) are effective anticoagulants. We have developed a novel anticoagulant fusion protein, Ab(TF)-TM, by fusing a TFneutralizing single-chain antibody, Ab(TF), to an active fragment of TM. Ab(TF)-TM is a novel anticoagulant targeting to sites of TF exposure with a dual mechanism of action. The Ab(TF) portion of the molecule inhibitsTF/factorVIIa mediated activation of FIX and FX, and the TM portion of the molecule acts as a cofactor for activation of protein C. In-vitro coagulation assays show that Ab(TF)-TM more potently inhibits TF-initiated coagulation (prothrombin time) than can its individual components, Ab(TF) (20-fold) and sTM (80-fold) alone, or in combination (10-fold). In contrast, the potency of Ab(TF)-TM in the activated partial thromboplastin and thrombin clotting time assays was similar to sTM alone. In a rat model of disseminated intravascular coagulation (DIC), intravenous injection of a human TF-containing thromboplastin reagent (0.5 ml/kg) resulted in an immediate death in approximately 60% of the animals and a clinical score of approximately 2.5. Pre-injection of Ab(TF)-TM or Ab(TF) and sTM, given alone or in combination, showed dose-dependent efficacy. At a dose of 0.7 nmol/kg, Ab(TF)-TM completely prevented death and reduced clinical scores by 79%, while neitherAb(TF) nor sTM, given alone or in combination, showed significant therapeutic effects. Calculated effective doses that reduced mortality by 50% relative to that in the control group (ED(50), nmol/kg) were 0.21 for Ab(TF)-TM, 3.2 for an equimolar mixture of Ab(TF) and sTM, 4.3 for sTM and 20 for Ab(TF). Thus, Ab(TF)-TM presented 10- to 100-fold enhancement of the anticoagulant potency, relative to the ED(50) in Ab(TF) and sTM given either alone or in combination, in a rat DIC model.

Animals↗

PubMed2006

Anti-EphA2 antibodies decrease EphA2 protein levels in murine CT26 colorectal and human MDA-231 breast tumors but do not inhibit tumor growth.

The EphA2 receptor tyrosine kinase has been shown to be over-expressed in cancer and a monoclonal antibody (mAb) that activates and down-modulates EphA2 was reported to inhibit the growth of human breast and lung tumor xenografts in nude mice. Reduction of EphA2 levels by treatment with anti-EphA2 siRNA also inhibited tumor growth, suggesting that the anti-tumor effects of these agents are mediated by decreasing the levels of EphA2. As these studies employed human tumor xenograft models in nude mice with reagents whose cross reactivity with murine EphA2 is unknown, we generated a mAb (Ab20) that preferentially binds, activates, and induces the degradation of murine EphA2. Treatment of established murine CT26 colorectal tumors with Ab20 reduced EphA2 protein levels to approximately 12% of control tumor levels, yet had no effect on tumor growth. CT26 tumor cell colonization of the lung was also not affected by Ab20 administration despite having barely detectable levels of EphA2. We also generated and tested a potent agonistic mAb against human EphA2 (1G9-H7). No inhibition of humanMDA-231 breast tumor xenograft growth was observed despite evidence for >85% reduction of EphA2 protein levels in the tumors. These results suggest that molecular characteristics of the tumors in addition to EphA2 over-expression may be important for predicting responsiveness to EphA2-directed therapies.

Animals↗

PubMed2005

The discovery of fluoropyridine-based inhibitors of the Factor VIIa/TF complex.

The activated Factor VII/tissue factor complex (FVIIa/TF) plays a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. An X-ray crystal structure of a fluoropyridine-based FVIIa/TF inhibitor bound in the active site of the enzyme complex suggested that incorporation of substitution at the 5-position of the hydroxybenzoic acid side chain could lead to the formation of more potent inhibitors through interactions with the S1'/S2' pocket.

Binding Sites↗

PubMed2005

Solid-phase synthesis of naphthylamidines as factor VIIa/tissue factor inhibitors.

Reductive amination followed by acylation of polymer-linked formyl aryl amidines generate combinatorial libraries of aryl amidines 8-13. Potent small molecule naphthylamidine inhibitors 12 (Ki<100 nM) of FVIIa/TF have been discovered and their activity against other serine proteases in the coagulation cascade is reported.

Amidines↗

PubMed2005

Fasudil, a Rho-kinase inhibitor, attenuates angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice by inhibiting apoptosis and proteolysis.

BACKGROUND: Angiotensin II (Ang II) accelerates atherosclerosis and induces abdominal aortic aneurysm (AAA) in an experimental mouse model. Agonism of a G protein-coupled receptor by Ang II activates Rho-kinase and other signaling pathways and results in activation of proteolysis and apoptosis. Enhanced proteolysis and smooth muscle cell apoptosis are important mechanisms associated with AAA. In this study, we tested the hypothesis that fasudil, a Rho-kinase inhibitor, could attenuate Ang II-induced AAA formation by inhibiting vascular wall apoptosis and extracellular matrix proteolysis. METHODS AND RESULTS: Six-month-old apolipoprotein E-deficient mice were infused with Ang II (1.44 mg x kg(-1) x d(-1)) for 1 month. Animals were randomly assigned to treatment with fasudil (136 or 213 mg x kg(-1) x d(-1) in drinking water) or tap water. Ang II infusion induced AAA formation in 75% of the mice, which was accompanied by an increase in proteolysis detected by zymographic analysis and quantified by active matrix metalloproteinase-2 activity, as well as apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and quantified by both caspase-3 activity and histone-associated DNA fragmentation. The level of DNA fragmentation in the suprarenal aorta correlated with AAA diameter. Ang II also increased atherosclerotic lesion area and blood pressure. Fasudil treatment resulted in a dose-dependent reduction in both the incidence and severity of AAA. At the higher dose, fasudil decreased AAA by 45% while significantly inhibiting both apoptosis and proteolysis, without affecting atherosclerosis or blood pressure. CONCLUSIONS: These data demonstrate that inhibition of Rho-kinase by fasudil attenuated Ang II-induced AAA through inhibition of both apoptosis and proteolysis pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2005

Inhibition of Rho-kinase by fasudil attenuated angiotensin II-induced cardiac hypertrophy in apolipoprotein E deficient mice.

Recent evidence indicates that the GTPase activated Rho/Rho-kinase pathway contributes angiotensin II-induced cardiac hypertrophy and vascular remodeling. We tested this hypothesis in vivo by determining the effects of fasudil, a Rho-kinase inhibitor, on angiotensin II-induced cardiac hypertrophy, coronary vascular remodeling, and ventricular dysfunction. Six-month-old apolipoprotein E deficient (apoE-KO) mice were subcutaneously infused with angiotensin II (1.44 mg/kg/day) using an osmotic mini-pump. Mice were randomly assigned to either vehicle or fasudil (136 or 213 mg/kg/day in drinking water) group. Infusion of angiotensin II for 4 weeks resulted in cardiac enlargement, myocyte hypertrophy, and myocardial interstitial and coronary artery perivascular fibrosis. These changes were accompanied by reduced aortic flow velocity and acceleration rate. Cardiac gene expression levels of atrial natriuretic peptide (ANP) and collagen type III detected by real-time reverse transcriptase polymerase chain reaction were significantly increased in angiotensin II-infused mice. Treatment with fasudil dose-dependently attenuated angiotensin II-induced cardiac hypertrophy, prevented perivascular fibrosis, blunted the increase in ANP and collagen type III expression, and improved cardiac function, without changing blood pressure. These data are consistent with a role for Rho-kinase activation in angiotensin II-induced cardiac remodeling and vascular wall fibrosis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2005

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Amino Acid Sequence↗

PubMed2003

Conformation of a bound inhibitor of blood coagulant factor Xa.

13C[(15)N] and (13)C[(19)F] rotational-echo double-resonance NMR have been used to characterize the enzyme-bound structure of ZK-816042, an amidine-imidazoline inhibitor of human factor Xa (FXa). The NMR experiments were performed on a lyophilized FXa-inhibitor complex. The complex was formed in solution in the presence of stabilizing excipients and frozen after gradual supercooling prior to lyophilization. The results indicate that the inhibitor binds with a distribution of orientations of the imidazoline ring.

Amidines↗

PubMed2003

Discovery and characterization of a potent and selective non-amidine inhibitor of human factor Xa.

Benzothiophene-anthranilamide 1 (3-chloro-N-[2-[[(4-fluorophenyl)amino]carbonyl]-4-methylphenyl]benzo[b]thiophene-2-carboxamide) was discovered by high throughput screening to be a highly potent and selective non-amidine inhibitor of human factor Xa with a K(i) of 15+/-4nM. Compound 1 is a selective inhibitor of human factor Xa as suggested by the K(i)((app)) determined for nine other human serine proteases and bovine trypsin. The activity of reconstituted human prothrombinase complex was inhibited by compound 1 when assayed in physiological concentrations of the substrate prothrombin. However, 27-fold higher inhibitor concentrations were needed to achieve the same level of inhibition than were required for the inhibition of free factor Xa, due in part to non-specific binding of the inhibitor to phospholipid under the assay conditions. Failure to demonstrate enzymatic cleavage of compound 1 suggests that compound 1 is solely an inhibitor rather than a substrate for factor Xa. The inhibition of factor Xa by compound 1 was reversible upon dilution of the enzyme/inhibitor mixture. Analyses of the inhibition mechanism with Dixon, Cornish-Bowden, and Lineweaver-Burk plots showed that compound 1 is a linear mixed-type inhibitor with 5-fold higher affinity for free factor Xa than the factor Xa/substrate complex. The linear mixed-type inhibition suggests that compound 1 binds to the active site region of factor Xa, but its binding cannot be fully displaced by the substrate S2222 (1:1 mixture of N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide and N-benzoyl-Ile-Glu(gamma-OMe)-Gly-Arg-p-nitroanilide hydrochloride). Thus, the inhibition mechanism for compound 1 is novel compared to most serine protease inhibitors including amidine-containing factor Xa inhibitors, which rely on binding to the S1 pocket of the enzyme active site. Compound 1 represents an attractive, novel structural template for further development of efficacious, safe, and potentially orally active human factor Xa inhibitors.

Anticoagulants↗

PubMed2003

Human factor Xa bound amidine inhibitor conformation by double rotational-echo double resonance nuclear magnetic resonance and molecular dynamics simulations.

Double rotational-echo double resonance (double REDOR) NMR was used to investigate the conformation of a (13)C-, (15)N-, and (19)F-labeled inhibitor (Berlex Biosciences compound no. ZK-806299) bound to human factor Xa. Conformationally dependent carbon-fluorine dipolar couplings were measured by (13)C[(19)F] REDOR. Natural abundance carbon signals in the full-echo spectra were removed by (13)C[(15)N] REDOR. Major and minor binding modes were suggested by the NMR data, but only the former had adequate signal to noise for distance determinations. Molecular dynamics simulations restrained by double-REDOR-determined intramolecular (13)C-(19)F distances revealed two models for the dominant binding mode that are consistent with the NMR data. We conclude that ZK-806299 binds similarly to both FXa. Moreover, it appears to bind to FXa in a fashion previously demonstrated for ZK-807834, a more selective FXa inhibitor.

Amidines↗

PubMed2002

Crystal structures of two potent nonamidine inhibitors bound to factor Xa.

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.

Amidines↗

PubMed2002

Human in vitro pharmacodynamic profile of the selective Factor Xa inhibitor ZK-807834 (CI-1031).

ZK-807834 (also known as CI-1031) is a small molecule that potently and selectively inhibits Factor Xa. Studies in animals have shown that ZK-807834 attenuates thrombosis and thrombus progression after fibrinolysis and exhibits an increased antithrombotic-to-bleeding risk ratio compared with conventional agents. The present study describes the human in vitro anticoagulant and pharmacodynamic profile ZK-807834. Consistent with its selective inhibition of Factor Xa, ZK-807834 in the range of 0.3-0.5 microM prolonged prothrombin time (PT) and activated partial thromboplastin time (aPPT) twofold without affecting thrombin time (TT). Intersubject variability of in vitro anticoagulant activity was nominal and gender-independent. ZK-807834 inhibited Factor Xa in clot-bound prothrombinase with an average IC50 of 10+/-7 (S.D.) nM. ZK-807834 exhibited no direct effect on ADP- or collagen-induced platelet aggregation. Based on the potency and specificity, ZK-807834 may represent an important advance in the development of selective and safe antithrombotics.

Amidines↗