Synthetic small molecule furin inhibitors derived from 2,5-dideoxystreptamine.

Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small molecule furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addition of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochemical cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Molecular modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-Lys-Arg-CH(2)Cl. The compounds presented here are bona fide synthetic small molecule furin inhibitors that exhibit potency in the nanomolar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furin-dependent diseases.

Selectively guanidinylated derivatives of neamine. Syntheses and inhibition of anthrax lethal factor protease.

A series of mono-, di-, and tri-guanidinylated derivatives of neamine were prepared via selective guanidinylation of neamine. These molecules represent a novel scaffold as inhibitors of anthrax lethal factor zinc metalloprotease. Methods for the synthesis of these compounds are described, and structure-activity relationships among the series are analyzed. In addition, initial findings regarding the mechanism of LF inhibition for these molecules are presented.

Guanidinylated 2,5-dideoxystreptamine derivatives as anthrax lethal factor inhibitors.

Anthrax lethal factor is a Zn(2+)-dependent metalloprotease and the key virulence factor of tripartite anthrax toxin secreted by Bacillus anthracis, the causative agent of anthrax. A series of guanidinylated 2,5-dideoxystreptamine derivatives were designed and synthesized as inhibitors of lethal factor, some of which show strong inhibitory activity against lethal factor in an in vitro FRET assay. Preparation and structure-activity relationships of these compounds are presented.

SU11752 inhibits the DNA-dependent protein kinase and DNA double-strand break repair resulting in ionizing radiation sensitization.

Loss of the DNA-dependent protein kinase (DNA-PK) results in increased sensitivity to ionizing radiation due to inefficient repair of DNA double-strand breaks. Overexpression of DNA-PK in tumor cells conversely results in resistance to ionizing radiation. It is therefore possible that inhibition of DNA-PK will enhance the preferential killing of tumor cells by radiotherapy. Available inhibitors of DNA-PK, like wortmannin, are cytotoxic and stop the cell cycle because they inhibit phoshatidylinositol-3-kinases at 100-fold lower concentrations required to inhibit DNA-PK. In an effort to develop a specific DNA-PK inhibitor, we have characterized SU11752, from a three-substituted indolin-2-ones library. SU11752 and wortmannin were equally potent inhibitors of DNA-PK. In contrast, inhibition of the phoshatidylinositol-3-kinase p110gamma required 500-fold higher concentration of SU11752. Thus, SU11752 was a more selective inhibitor of DNA-PK than wortmannin. Inhibition kinetics and a direct assay for ATP binding showed that SU11752 inhibited DNA-PK by competing with ATP. SU11752 inhibited DNA double-strand break repair in cells and gave rise to a five-fold sensitization to ionizing radiation. At concentrations of SU11752 that inhibited DNA repair, cell cycle progression was still normal and ATM kinase activity was not inhibited. We conclude that SU11752 defines a new class of drugs that may serve as a starting point for the development of specific DNA-PK inhibitors.

Design and synthesis of aminopropyl tetrahydroindole-based indolin-2-ones as selective and potent inhibitors of Src and Yes tyrosine kinase.

A novel series of substituted 3-[3-(aminopropyl)-4,5,6,7-tetrahydro-1H-indol-2-ylmethylene]-1,3-dihydro-indole-2-ones was discovered as potent inhibitors of the non-receptor tyrosine kinase Src and Yes. A structure-activity relationship was developed in order to optimize their potency and selectivity. Syntheses of these compounds are also described herein.

Novel pyrrolyllactone and pyrrolyllactam indolinones as potent cyclin-dependent kinase 2 inhibitors.

Cyclin-dependent kinases (CDKs) are essential in the control of cell cycle progression. Inhibition of CDKs represents a new approach for pharmacological intervention in the treatment of a variety of proliferative diseases, especially cancer. Based on the crystal structure of CDK2 in complex with an imidazole indolinone compound 1 (SU9516), lead optimization through modeling, synthesis, and SAR studies has led to the discovery of a novel series of pyrrolyllactone and pyrrolyllactam indolinones as potent CDK2 inhibitors.

A selective and oral small molecule inhibitor of vascular epithelial growth factor receptor (VEGFR)-2 and VEGFR-1 inhibits neovascularization and vascular permeability.

Vascular endothelial growth factor (VEGF) is a key driver of the neovascularization and vascular permeability that leads to the loss of visual acuity in diabetic retinopathy and neovascular age-related macular degeneration. Our aim was to identify an orally active, selective small molecule kinase inhibitor of vascular endothelial growth factor receptor (VEGFR)-2 with activity against both VEGF-induced angiogenesis and vascular permeability. We used a biochemical assay to identify 3-[5-methyl-2- (2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-proprionic acid (SU10944), a pyrrole indolinone, which is a potent ATP-competitive inhibitor of VEGFR-2 (Ki of 21 +/- 5 nM). In cellular assays, SU10944 inhibited VEGF-induced receptor autophosphorylation (IC50 of 227 +/- 80 nM) as well as downstream signaling (IC50 of 102 +/- 27 nM). In biochemical assays, SU10944 exhibits potent inhibitory activity against VEGFR-1; weak activity against other related subgroup members, including stem cell factor receptor (SCFR), platelet-derived growth factor receptor beta (PDGFRbeta), and fibroblast growth factor receptor-1 (FGFR-1); and no detectable activity against other protein tyrosine kinases such as epidermal growth factor receptor (EGFR), Src, and hepatocyte growth factor receptor. In cellular assays, the selectivity for SU10944 to inhibit VEGFR is maintained compared with other tyrosine kinases (IC50 for SCFR of 1.6 +/- 0.3 microM, for PDGFRbeta of 30.6 +/- 13.3 microM, for FGFR-1 of >50 microM, and for EGFR of >50 microM). Upon oral administration, SU10944 gave a clear dose response in the corneal micropocket model with an ED50 value for inhibition of neovascularization of approximately 30 mg/kg and a maximum inhibition of 95% at 300 mg/kg. Similarly, upon oral administration in the Miles assay, SU10944 potently inhibited VEGF-induced vascular permeability. Our data indicate that small molecule inhibitors of VEGFR signaling have the potential to ameliorate VEGF-induced neovascularization as well as vascular permeability.

Structure-based design and discovery of novel inhibitors of protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) are important in the regulation of signal transduction processes. Certain enzymes of this class are considered as potential therapeutic targets in the treatment of a variety of diseases such as diabetes, inflammation, and cancer. However, many PTP inhibitors identified to date are peptide-based and contain a highly charged phosphate-mimicking component. These compounds usually lack membrane permeability and this limits their utility in the inhibition of intracellular phosphatases. In the present study, we have used structure-based design and modeling techniques to explore catalytic-site directed, reversible inhibitors of PTPs. Employing a non-charged phosphate mimic and non-peptidyl structural components, we have successfully designed and synthesized a novel series of trifluoromethyl sulfonyl and trifluoromethyl sulfonamido compounds as PTP inhibitors. This is the first time that an uncharged phosphate mimic is reported in the literature for general, reversible, and substrate-competitive inhibition of PTPs. It is an important discovery because the finding may provide a paradigm for the development of phosphatase inhibitors that enter cells and modify signal transduction.

Discovery of 5-[5-fluoro-2-oxo-1,2- dihydroindol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase.

To improve the antitumor properties and optimize the pharmaceutical properties including solubility and protein binding of indolin-2-ones, a number of different basic and weakly basic analogues were designed and synthesized. 5-[5-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide (12b or SU11248) has been found to show the best overall profile in terms of potency for the VEGF-R2 and PDGF-Rbeta tyrosine kinase at biochemical and cellular levels, solubility, protein binding, and bioavailability. 12b is currently in phase I clinical trials for the treatment of cancers.

In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship.

One challenging aspect in the clinical development of molecularly targeted therapies, which represent a new and promising approach to treating cancers, has been the identification of a biologically active dose rather than a maximum tolerated dose. The goal of the present study was to identify a pharmacokinetic/pharmacodynamic relationship in preclinical models that could be used to help guide selection of a clinical dose. SU11248, a novel small molecule receptor tyrosine kinase inhibitor with direct antitumor as well as antiangiogenic activity via targeting the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), KIT, and FLT3 receptor tyrosine kinases, was used as the pharmacological agent in these studies. In mouse xenograft models, SU11248 exhibited broad and potent antitumor activity causing regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. To predict the target SU11248 exposure required to achieve antitumor activity in mouse xenograft models, we directly measured target phosphorylation in tumor xenografts before and after SU11248 treatment and correlated this with plasma inhibitor levels. In target modulation studies in vivo, SU11248 selectively inhibited Flk-1/KDR (VEGF receptor 2) and PDGF receptor beta phosphorylation (in a time- and dose-dependent manner) when plasma concentrations of inhibitor reached or exceeded 50-100 ng/ml. Similar results were obtained in a functional assay of VEGF-induced vascular permeability in vivo. Constant inhibition of VEGFR2 and PDGF receptor beta phosphorylation was not required for efficacy; at highly efficacious doses, inhibition was sustained for 12 h of a 24-h dosing interval. The pharmacokinetic/pharmacodynamic relationship established for SU11248 in these preclinical studies has aided in the design, selection, and evaluation of dosing regimens being tested in human trials.

Rational design of 4,5-disubstituted-5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones as a novel class of inhibitors of epidermal growth factor receptor (EGF-R) and Her2(p185(erbB)) tyrosine kinases.

A novel class of 4,5-disubstituted-5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones has been discovered as potent and selective inhibitors of the EGF-R tyrosine kinase family. These compounds selectively inhibit EGF-R kinase activity at low nanomolar concentration and tyrosine autophosphorylation in cells expressing EGF-R or Her2 (p185(erbB)). Structure-activity relationships (SARs) for this class of compounds are presented.