Disruption of angiogenesis and tumor growth with an orally active drug that stabilizes the inactive state of PDGFRbeta/B-RAF.

Kinases are known to regulate fundamental processes in cancer including tumor proliferation, metastasis, neovascularization, and chemoresistance. Accordingly, kinase inhibitors have been a major focus of drug development, and several kinase inhibitors are now approved for various cancer indications. Typically, kinase inhibitors are selected via high-throughput screening using catalytic kinase domains at low ATP concentration, and this process often yields ATP mimetics that lack specificity and/or function poorly in cells where ATP levels are high. Molecules targeting the allosteric site in the inactive kinase conformation (type II inhibitors) provide an alternative for developing selective inhibitors that are physiologically active. By applying a rational design approach using a constrained amino-triazole scaffold predicted to stabilize kinases in the inactive state, we generated a series of selective type II inhibitors of PDGFRbeta and B-RAF, important targets for pericyte recruitment and endothelial cell survival, respectively. These molecules were designed in silico and screened for antivascular activity in both cell-based models and a Tg(fli1-EGFP) zebrafish embryogenesis model. Dual inhibition of PDGFRbeta and B-RAF cellular signaling demonstrated synergistic antiangiogenic activity in both zebrafish and murine models of angiogenesis, and a combination of previously characterized PDGFRbeta and RAF inhibitors validated the synergy. Our lead compound was selected as an orally active molecule with favorable pharmacokinetic properties which demonstrated target inhibition in vivo leading to suppression of murine orthotopic tumors in both the kidney and pancreas.

In vitro gene transfer efficacies and serum compatibility profiles of novel mono-, di-, and tri-histidinylated cationic transfection lipids: a structure-activity investigation.

Recently, we demonstrated that covalent grafting of an endosome-disrupting single histidine functionality in the headgroup region imparts high gene transfer properties to cationic amphiphiles (Kumar, V. V., et al. Gene Ther. 2003, 10, 1206-1215). However, whether covalent attachment of multiple histidine functionalities in the headgroup region are capable of further enhancing the gene transfer efficacies of cationic amphiphiles remains to be explored. To this end, herein, we report on the design, syntheses, physicochemical characterizations, in vitro gene transfer properties, and serum compatibilities of three novel nontoxic cationic transfection amphiphiles containing mono-, di-, and tri-histidine functionalities in their headgroup regions (lipids 1-3) in multiple cultured cells. Significantly, findings in both the reporter gene expression assay and the whole cell histochemical X-gal staining assay support the notion that there is no linear correlation between the in vitro transfection efficacies and the number of histidine functionalities in the polar headgroup regions for histidinylated cationic amphiphiles. The relative gene transfer efficiencies, as well as the serum compatibilities, of the present histidinylated cationic amphiphiles were found to be strikingly dependent on the medium of lipoplex formation. Most importantly, high serum compatibilities (up to 50% added serum) of the lipoplexes of lipids 1 and 3 make them promising nonviral transfection vectors for future systemic applications.

In vitro gene transfer efficacies of N,N-dialkylpyrrolidinium chlorides: a structure-activity investigation.

Inspired by the previously reported superior gene transfer efficacies of amine headgroup-containing cationic lipids to their hydroxy counterparts, in the present structure-activity investigation we have compared the relative in vitro gene transfer efficacies of eight newly synthesized structural analogues of our previously reported lipids 1-4, namely the four 3,4-diaminopyrrolidinium chloride structural analogues (lipids 9-12, Chart 1) and the N-BOC-protected precursors of these amine analogues (lipids 5-8, Chart 1) with our previously reported lipids 1-4 (Chart 1) in five cultured cell lines. In contrast to the above-mentioned earlier reports, except for the superior or comparable transfection efficacies of the diaminopyrrolidinium lipids with distearyl and stearyloleyl chains (lipid 11 and 12 respectively, Chart 1) in MCF-7 and HEK293T cells, the relative transfection efficacies of the other diamino analogues were found to be much lower than their dihydroxy counterparts. The results of the DNase I sensitivity assays indicate that enhanced degradation of DNA associated with lipids 9-12 by cellular DNase I might play an important role behind their seriously compromised transfection efficacies. In addition, the present structure-activity investigation revealed a strikingly cell tropic transfection behavior of lipid 6 (Chart 1). While lipids 5, 7, and 8 were found to be either poor or essentially incompetent in transfecting all the five cells, lipid 6 was remarkably efficacious in transfecting kidney cells (COS-1 and HEK293T cells) at lipid:DNA charge ratios 3:1 and 1:1 when used in combination with equimolar amounts of DOPE and DOPC.

Example of fatty acid-loaded lipoplex in enhancing in vitro gene transfer efficacies of cationic amphiphile.

Herein, we report on the design and synthesis of a novel nontoxic cationic amphiphile N,N-di-n-tetradecyl-N-[2-[N',N'-bis(2-hydroxyethyl)amino]ethyl]-N-(2-hydroxyethyl)ammonium chloride (lipid 1) whose in vitro gene transfer efficacies in CHO, COS-1, MCF-7, and HepG2 cells are remarkably enhanced when used in combination with 30 mole percent added myristic acid. Reporter gene expression assay using p-CMV-SPORT-beta-gal reporter gene revealed poor gene transfer properties of the cationic liposomes of lipid 1 and cholesterol (colipid). However, the in vitro gene delivery efficacies of lipid 1 were found to be remarkably enhanced when the cationic liposomes of lipid 1 and cholesterol were prepared in the presence of 30 mole percent added myristic acid (with respect to lipid 1) as the third liposomal ingredient. The whole cell histochemical X-gal staining of representative CHO cells further confirmed the significantly enhanced gene transfer properties of the fatty acid-loaded cationic liposomes of lipid 1 and cholesterol. Electrophoretic gel patterns in the gel mobility shift assay supports the notion that better DNA release from fatty acid lipoplexes might play a role in their enhanced gene transfer properties. In addition, such myristic acid-loaded lipoplexes of lipid 1 were also found to be serum-compatible up to 30% added serum. Taken together, our present findings demonstrate that the transfection efficacies of fatty acid-loaded lipoplexes are worth evaluating particularly when traditional cationic liposomes prepared with either cholesterol or DOPE colipids fail to transfect cultured cells.

Enhanced intravenous transgene expression in mouse lung using cyclic-head cationic lipids.

Herein, we report enhanced intravenous mouse lung transfection using novel cyclic-head-group analogs of usually open-head cationic transfection lipids. Design and synthesis of the new cyclic-head lipid N,N-di-n-tetradecyl-3,4-dihydroxy-pyrrolidinium chloride (lipid 1) and its higher alkyl-chain analogs (lipids 2-4) and relative in vitro and in vivo gene transfer efficacies of cyclic-head lipids 1-4 to their corresponding open-head analogs [lipid 5, namely N,N-di-n-tetradecyl-N,N-(2-hydroxyethyl)ammonium chloride and its higher alkyl-chain analogs, lipids 6-8] have been described. In stark contrast to comparable in vitro transfection efficacies of both the cyclic- and open-head lipids, lipids 1-4 with cyclic heads were found to be significantly more efficient (by 5- to 11-fold) in transfecting mouse lung than their corresponding open-head analogs (5-8) upon intravenous administration. The cyclic-head lipid 3 with di-stearyl hydrophobic tail was found to be the most promising for future applications.