Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases.

PURPOSE: To investigate whether it is possible to specifically suppress the expression and function of endogenous canine P-glycoprotein (cPgp) in Madin-Darby canine kidney type II cells (MDCKII) transfected with hPGP and breast cancer resistance protein (hBCRP) by zinc finger nuclease (ZFN) producing sequence specific DNA double strand breaks. METHODS: Wild-type, hPGP-transfected, and hBCRP-transfected MDCKII cells were transfected with ZFN targeting for cPgp. Net efflux ratios (NER) of Pgp and Bcrp substrates were determined by dividing efflux ratios (basal-to-apical / apical-to-basal) in over-expressing cell monolayers by those in wild-type ones. RESULTS: From ZFN-transfected cells, cell populations (ko-cells) showing knockout of cPgp were selected based on genotyping by PCR. qRT-PCR analysis showed the significant knock-downs of cPgp and interestingly also cMrp2 expressions. Specific knock-downs of protein expression for cPgp were shown by western blotting and quantitative targeted absolute proteomics. Endogenous canine Bcrp proteins were not detected. For PGP-transfected cells, NERs of 5 Pgp substrates in ko-cells were significantly greater than those in parental cells not transfected with ZFN. Similar result was obtained for BCRP-transfected cells with a dual Pgp and Bcrp substrate. CONCLUSION: Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.

Design and synthesis of novel potent and selective integrin alphanubeta3 antagonists--novel synthetic routes to isoquinolinone, benzoxazinone, and quinazolinone acetates.

An unexpected ring contraction of benzazepinone based alpha(nu)beta(3) antagonists led to the design of quinolinone-type derivatives. Novel and efficient synthetic routes to isoquinolinone, benzoxazinone, and quinazolinone acetates were established. Nanomolar alpha(nu)beta(3) antagonists based on these new scaffolds were prepared. Moreover, benzoxazinones 15a and 15b exhibited high microsomal stability and good permeability.

A fluorescence-based in vitro assay for drug interactions with breast cancer resistance protein (BCRP, ABCG2).

PURPOSE: To establish a fluorescence-based assay for drug interactions with the ABC-export-protein BCRP (ABCG2). METHODS: BCRP expression was verified by immunostaining and Western blots in intact porcine brain capillaries, isolated endothelial cells (PBCECs) and in MDCKII-cells over-expressing human wild-type BCRP (MDCKII-hBCRP). Transport of fluorescent mitoxantrone across cells was determined to assess a preferred transport direction. Sensitivity of cultured cells versus mitoxantrone in the absence and in the presence of transport modulators was examined at increasing concentrations of the cytostatic using the AlamarBlue assay. In addition, cells were incubated with mitoxantrone in the absence and presence of increasing concentrations of different compounds with the potential to interact with BCRP. Intracellular fluorescence accumulation was measured using a flow cytometer. RESULTS: Isolated capillaries as well as 7-day old PBCECs showed expression of BCRP. Cell sensitivity to mitoxantrone significantly increased in the presence of the BCRP inhibitors KO143 and GF120918. Transport of mitoxantrone across PBCEC monolayers was directed with P(app) (apical to basolateral) 5.6 x 10(-6) cm s(-1) and with P(app) (basolateral to apical) 2.8 x 10(-5) cm s(-1). FACS analysis revealed a different extent of fluorescence accumulation dependent on the kind and concentration of BCRP modulating compounds. CONCLUSIONS: The mitoxantrone-based assay can be used as a rapid FACS screening system to assess drug interactions with BCRP at the blood-brain barrier and therefore represents a useful tool in drug profiling.

Synthesis and SAR of highly potent and selective dopamine D(3)-receptor antagonists: Quinolin(di)one and benzazepin(di)one derivatives. herve.geneste@abbott.com.

The synthesis and SAR of novel and selective dopamine D(3)-receptor antagonists based on a 3,4-dihydro-1H-quinolin-2-one, a 1,3,4,5-tetrahydro-benzo[b]azepin-2-one, 1H-quinoline-2,4-dione or a 3,4-dihydro-1H-benzo[b]azepine-2,5-dione scaffold are discussed. A706149 (2.15mg/kg, po) antagonizes PD 128907-induced huddling deficits in rat, a social interaction paradigm.

Synthesis and SAR of highly potent and selective dopamine D(3)-receptor antagonists: 1H-pyrimidin-2-one derivatives.

The synthesis and SAR of novel highly potent and selective dopamine D(3)-receptor antagonists based on a 1H-pyrimidin-2-one scaffold are described. A-690344 antagonized PD 128907-induced huddling deficits in rat (ED(50) 6.1mg/kg po), a social interaction paradigm.

Synthesis and SAR of highly potent and selective dopamine D3-receptor antagonists: variations on the 1H-pyrimidin-2-one theme.

In our efforts to further pursue one of the most selective dopamine D(3)-receptor antagonists reported to date, we now describe the synthesis and SAR of novel and highly selective dopamine D(3) antagonists based on a 1H-pyridin-2-one or on a urea scaffold. The most potent compounds exhibited K(i) values toward the D(3) receptor in the nano- to subnanomolar range and high selectivity versus the related D(2) dopamine receptor. Thus, 1H-pyridin-2-one 7b displays oral bioavailability (F=37%) as well as brain penetration (brain plasma ratio 3.7) in rat. Within the urea series, an excellent D(3) versus D(2) selectivity (>100-fold) could be achieved by removal of one NH group (compound 6), although bioavailability (rat) was suboptimal (F<10%). These data significantly enhance our understanding of the D(3) pharmacophore and are expected to lead to novel approaches for the treatment of schizophrenia.

Synthesis of highly potent and selective hetaryl ureas as integrin alpha(V)beta3-receptor antagonists.

Solid-phase synthesis and SAR of integrin alpha(V)beta3-receptor antagonists containing a urea moiety as non-basic guanidine mimetic are described. The most potent compounds exhibited IC(50) values towards alpha(V)beta3 in the nanomolar range and high selectivity versus related integrins like alpha(IIb)beta3. For selected examples efficacy in functional cellular assays is demonstrated.

Design and synthesis of 1,5- and 2,5-substituted tetrahydrobenzazepinones as novel potent and selective integrin alphaVbeta3 antagonists.

The design and synthesis of novel integrin alpha(V)beta(3) antagonists based on a 1,5- or 2,5-substituted tetrahydrobenzaezpinone core is described. In vitro activity of respective compounds was determined via alpha(V)beta(3) binding assay, and selected derivatives were submitted to further characterization in functional cellular assays. SAR was obtained by modification of the benzazepinone core, variation of the spacer linking guanidine moiety and core, and modification of the guanidine mimetic. These efforts led to the identification of novel alpha(V)beta(3) inhibitors displaying potency in the subnanomolar range, selectivity versus alpha(IIb)beta(3) and functional efficacy in relevant cellular assays. A method for the preparation of enantiomerically pure derivatives was developed, and respective enantiomers evaluated in vitro. Compounds 31 and 37 were assessed for metabolic stability, resorption in the Caco-2 assay and pharmacokinetics.

Highly potent and selective alphaVbeta3-receptor antagonists: solid-phase synthesis and SAR of 1-substituted 4-amino-1H-pyrimidin-2-ones.

Solid-phase synthesis and SAR of alpha(V)beta(3)-receptor antagonists based on a N(1)-substituted 4-amino-1H-pyrimidin-2-one scaffold are described. The most potent compounds exhibited IC(50) values towards alpha(V)beta(3) in the nano- to subnanomolar range and high selectivity versus related integrins like alpha(IIb)beta(3). For selected examples efficacy in functional cellular assays was demonstrated.

Synthesis and SAR of N-substituted dibenzazepinone derivatives as novel potent and selective alpha(V)beta(3) antagonists.

Synthesis and SARs of new integrin alpha(V)beta(3) antagonists based on an N-substituted dibenzazepinone scaffold are described. Variation of spacer and guanidine mimetic led to potent compounds exhibiting an IC(50) towards alpha(V)beta(3) in the nanomolar range, high selectivity versus integrin alpha(IIb)beta(3) and efficacy in functional cellular assays.