68
W. L. Cody et al. / Bioorg. Med. Chem. 13 (2005) 59–68
CNX, and model/ligand building was performed with
the program QUANTA. The final model includes 668 res-
idues, 175 water molecules, and two molecules (2) with
an R factor of 23.4% and an Rf of 26.5%.
in 2h of a 30mg/kg oral dose of either CI-992 or com-
pound 2. At the nadir, MABP was normalized, but
returned to baseline hypertensive levels approximately
6–8h later.
3.5. In vitro renin IC50 determinations
References and notes
The renin assay utilized a tandem Green Flourescent
Protein (tGFP) substrate (175nM) that was hydrolyzed
by renin human (50.4IU/well). The tGFP substrate con-
tained a nine amino acid (Ile-His-Pro-Phe-His-Leu-Val-
Ile-His) recognition sequence for human renin flanked
by two GPF proteins (W1B and Topaz). Human renin
cleaves the leucine–valine site of the substrate linker.
Tandem GFP Fret assays were carried out in a reaction
buffer containing 50mM Hepes (pH7.4), 1.0mM
EDTA, 1% PEG (MW800), 1.0mM DTT, and 0.10%
BSA. Once cleaved, the emission ratio changes. The
change was monitored by the ratio of 530nM (topaz)
over 475nM (W1B) with the excitation set at 432nM
and the cutoff at 515nM. The assay used a 384 well plate
format that was read using a Gemini XS fluorometric
plate reader (Molecular Devices). Compounds were
screened at a starting concentration of 10lM and used
a fourfold eleven-point dilution regiment.
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3.6. In vivo efficacy studies
Blood pressure data was obtained by telemetry in con-
scious, free moving three to four month old double
transgenic mice that expressed both human angiotensi-
nogen and renin. The double transgenic mice were de-
rived from a founder colony of five male mice
expressing human angiotensinogen (h-Ang 204/1) and
six female mice expressing human renin (h-Ren 9) ob-
tained from the University of Iowa. Mice expressing
both transgenes were obtained through a breeding pro-
gram conducted at Charles River Laboratories (Wilm-
ington, MA). The double transgenic mice were
hypertensive with mean arterial blood pressures
(MABP) of 140mmHg. Both males and females were
used. MABP was measured via a radiotransmitter (mod-
el TA11PA-C20, Data Sciences International, Saint
Paul, Minnesota) implanted subcutaneously, between
the left fore and hind limbs. The radiotransmitter cath-
eter was placed in the left carotid artery. To obtain base-
line blood pressure data the mice were dosed via oral
gavage with vehicle (3% volume dimethyl acetamide,
97% sulfobutylether-beta-cyclodextrin (40% w/v)) in
50nM lactic acid) for two consecutive days. On the third
day, either CI-992 (N-(4-morpholinylsulfonyl)-L-pheny-
lalanyl-3-(2-amino-4-thiazolyl)-N-[(1S,2R,3S)-1-(cyclo-
hexylmethyl)-2,3-dihydroxy-5-methylhexyl]-(HCl))11,12
or compound 2 was administered at 30mg/kg. Delta
MABP was obtained by subtracting CI-992 or com-
pound 2 dosed blood pressure from baseline blood pres-
sure. Animals were allowed food and water ad libitum.
The maximum antihypertensive response occurred with-
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