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Biomedical subjects

Laura Sepp-Lorenzino

Publications and source records attributed to Laura Sepp-Lorenzino.

12 recordsLinked to original sources
PubMed2006

An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis.

The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.

Antibiotics, Antineoplastic↗
PubMed2006

Development of 6-substituted indolylquinolinones as potent Chek1 kinase inhibitors.

Through a comparison of X-ray co-crystallographic data for 1 and 2 in the Chek1 active site, it was hypothesized that the affinity of the indolylquinolinone series (2) for Chek1 kinase would be improved via C6 substitution into the hydrophobic region I (HI) pocket. An efficient route to 6-bromo-3-indolyl-quinolinone (9) was developed, and this series was rapidly optimized for potency by modification at C6. A general trend was observed among these low nanomolar Chek1 inhibitors that compounds with multiple basic amines, or elevated polar surface area (PSA) exhibited poor cell potency. Minimization of these parameters (basic amines, PSA) resulted in Chek1 inhibitors with improved cell potency, and preliminary pharmacokinetic data are presented for several of these compounds.

Animals↗
PubMed2006

3-(Indol-2-yl)indazoles as Chek1 kinase inhibitors: Optimization of potency and selectivity via substitution at C6.

The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC(50)=0.30nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.

Cell Cycle↗
PubMed2006

Development and implementation of multiplexed cell-based imaging assays.

Fluorescence microscopy, image analysis, and automated screening technologies are some of the most powerful tools enabling cell biologists to investigate complex signaling pathways and compound or siRNA effects on cellular function in individual cells. Researchers can now use multiple fluorescent probes to quantify effects on intracellular molecular events, measure phenotypic changes, and provide contextual information about cellular pathways not discernible by traditional single-parameter, end point experiments. This chapter focuses on fluorescent labeling techniques and methods for designing image-based assays, multiplexed readouts, and image analysis routines. Case studies are presented describing the use of cell-based imaging assays for monitoring cell proliferation, cell cycle stage, and apoptosis.

Animals↗
PubMed2005

Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage.

The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.

Apoptosis↗
PubMed2005

Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling.

Extensive efforts are under way to identify antiangiogenic therapies for the treatment of human cancers. Many proposed therapeutics target vascular endothelial growth factor (VEGF) or the kinase insert domain receptor (KDR/VEGF receptor-2/FLK-1), the mitogenic VEGF receptor tyrosine kinase expressed by endothelial cells. Inhibition of KDR catalytic activity blocks tumor neoangiogenesis, reduces vascular permeability, and, in animal models, inhibits tumor growth and metastasis. Using a gene expression profiling strategy in rat tumor models, we identified a set of six genes that are selectively overexpressed in tumor endothelial cells relative to tumor cells and whose pattern of expression correlates with the rate of tumor endothelial cell proliferation. In addition to being potential targets for antiangiogenesis tumor therapy, the expression patterns of these genes or their protein products may aid the development of pharmacodynamic assays for small molecule inhibitors of the KDR kinase in human tumors.

Angiogenesis Inhibitors↗
PubMed2004

Potent N-(1,3-thiazol-2-yl)pyridin-2-amine vascular endothelial growth factor receptor tyrosine kinase inhibitors with excellent pharmacokinetics and low affinity for the hERG ion channel.

A series of N-(1,3-thiazol-2-yl)pyridin-2-amine KDR kinase inhibitors have been developed that possess optimal properties. Compounds have been discovered that exhibit excellent in vivo potency. The particular challenges of overcoming hERG binding activity and QTc increases in vivo in addition to achieving good pharmacokinetics have been acomplished by discovering a unique class of amine substituents. These compounds have a favorable kinase selectivity profile that can be accentuated with appropriate substitution.

Administration, Oral↗
PubMed2004

A novel orally bioavailable inhibitor of kinase insert domain-containing receptor induces antiangiogenic effects and prevents tumor growth in vivo.

A strategy for antagonizing vascular endothelial growth factor (VEGF) -induced angiogenesis is to inhibit the kinase activity of its receptor, kinase insert domain-containing receptor (KDR), the first committed and perhaps the last unique step in the VEGF signaling cascade. We synthesized a novel ATP-competitive KDR tyrosine kinase inhibitor that potently suppresses human and mouse KDR activity in enzyme (IC(50) = 7.8-19.5 nM) and cell-based assays (IC(50) = 8 nM). The compound was bioavailable in vivo, leading to a dose-dependent decrease in basal- and VEGF-stimulated KDR tyrosine phosphorylation in lungs from naïve and tumor-bearing mice (IC(50) = 23 nM). Pharmacokinetics and pharmacodynamics guided drug dose selection for antitumor efficacy studies. HT1080 nude mice xenografts were treated orally twice daily with vehicle, or 33 or 133 mg/kg of compound. These doses afforded trough plasma concentrations approximately equal to the IC(50) for inhibition of KDR autophosphorylation in vivo for the 33 mg/kg group, and higher than the IC(99) for the 133 mg/kg group. Chronic treatment at these doses was well-tolerated and resulted in dose-dependent inhibition of tumor growth, decreased tumor vascularization, decreased proliferation, and enhanced cell death. Antitumor efficacy correlated with inhibition of KDR tyrosine phosphorylation in the tumor, as well as in a surrogate tissue (lung). Pharmacokinetics and pharmacodynamics assessment indicated that the degree of tumor growth inhibition correlated directly with the extent of inhibition of KDR tyrosine phosphorylation in tumor or lung at trough. These observations highlight the need to design antiangiogenic drug regimens to ensure constant target suppression and to take advantage of PD end points to guide dose selection.

Administration, Oral↗
PubMed2004

Analysis of the activating mutations within the activation loop of leukemia targets Flt-3 and c-Kit based on protein homology modeling.

Molecular modeling provides a mechanistic hypothesis at the molecular level for the constitutive activation recently observed and reported for tyrosine protein kinases Flt-3 and c-Kit. Three-dimensional homology models for the active and inactive forms of these two kinases were made. Comparison of these models at the molecular level reveals that mutations of specific residues located in the activation loop (D835X and 836-deletion in Flt-3; D816V in c-Kit) as well as a 6-base pair (6-bp) insertion at residue 840 in Flt-3 operate in a similar way. Each mutation tends to weaken the forces that maintain the activation-loop folded inwards. None of the mutations are found to particularly stabilize the active state directly. The reason why the equilibrium is shifted towards the gate-open conformation of the protein is because, at least in these models, the mutations are found to critically destabilize the inactive conformational state of the kinase.

Catalytic Domain↗
PubMed2002

Antiangiogenic agents targeting vascular endothelial growth factor and its receptors in clinical development.

There is ample therapeutic opportunity for the use of antiangiogenic inhibitors in the clinic, as there are several human diseases that are dependent upon angiogenesis [1]. However, no disease has attracted as much attention as a target for antiangiogenic therapy as malignant disorders. There is a vast amount of literature acting as proof-of-principle for the use of angiogenic inhibitors as effective agents for blocking tumour-induced angiogenesis and subverting tumour growth and disease dissemination. One of the unique attractions of targeting tumour angiogenesis is that vascular endothelial cells are a genetically stable population in which acquisition of therapeutic resistance might be less efficient than in genetically unstable tumour cells [2,3]. This review covers inhibitors that target the tumour angiogenic agent vascular endothelial growth factor and its receptors as one such antiangiogenic approach. Many agents in this class are in clinical trials with limited reports of toxicity and some early evidence of clinical benefit.

Angiogenesis Inhibitors↗