Challenges of high throughput screening against cell surface receptors.

Excessive or inappropriate activation of cell surface receptors can mediate the development of disease. Receptors, therefore, are a focus for drug discovery activities. Empirical screening is important in the search for novel compounds acting as receptors. Technical developments and the application of molecular biology have facilitated access to receptors of interest and have provided efficient screening methods capable of very high throughput. Reliability in high throughput screening requires the use of appropriate methodology, good screen design and effective validation and quality control processes. Validation should aim to establish that the basic experimental design is sound. In developing software to handle high throughput screening data, a fundamental requirement is to provide performance monitoring and error trapping facilities. Additional requirements are automatic data capture from instruments, on-line data reduction and analysis and transfer of results to central databases. As data volumes increase through effective high throughput screening, conventional interrogation methods become less appropriate and are being augmented by newer computing techniques referred to as knowledge mapping or database mining. Targeting cell surface receptors has been very successful as an approach to drug discovery. If the challenges of high throughput empirical screening are addressed effectively, cell surface receptors will provide new opportunities for improved therapy in the coming years.

New nonpeptide angiotensin II receptor antagonists. 3. Synthesis, biological properties, and structure-activity relationships of 2-alkyl-4-(biphenylylmethoxy)pyridine derivatives.

A novel series of nonpeptide angiotensin II (AII) receptor antagonists is reported, derived from linkage of the biphenylyltetrazole moiety found in previously described antagonists via a methyleneoxy chain to the 4-position of a 3-substituted 2,6-dialkylpyridine. When evaluated in an in vitro binding assay using a guinea pig adrenal membrane preparation, compounds in this series generally gave IC50 values in the range 0.005-0.5 microM. A variety of substituents was found to be effective at the 3-position of the pyridine ring. On intravenous administration in a normotensive rat model, the more potent compounds inhibited the AII-induced pressor response with ED50 values in the range 0.1-1.0 mg/kg. One of the compounds, 2-ethyl-5,6,7,8-tetrahydro-4-([2'-(1H-tetrazol-5-yl)biphenyl-4y l] methoxy)quinoline (26), demonstrated good oral activity in two rat models. At doses in the range 1-10 mg/kg po in AII-infused, conscious, normotensive rats, the compound exhibited a dose-related inhibition of the pressor response with a good duration of action at the higher doses. In a renal hypertensive rat model compound 26 showed a rapid and sustained lowering of blood pressure at a dose of 5 mg/kg po. Based on its profile, this compound, designated ICI D6888, has been selected for evaluation in volunteers.

New nonpeptide angiotensin II receptor antagonists. 2. Synthesis, biological properties, and structure-activity relationships of 2-alkyl-4-(biphenylylmethoxy)quinoline derivatives.

A novel series of nonpeptidic angiotensin II (AII) receptor antagonists is reported, derived from linkage of the biphenylcarboxylic acid or biphenylyltetrazole moiety found in previously described antagonists via a methyleneoxy chain to the 4-position of a 2-alkyl quinoline. When evaluated in an in vitro binding assay using a guinea pig adrenal membrane preparation, compounds in this series generally gave IC50 values in the range 0.01-1 microM. Structure-activity studies showed the quinoline nitrogen atom and a short alkyl chain at the quinoline 2-position to be essential for receptor binding. On intravenous administration in a normotensive rat model, the more potent compounds inhibited the AII-induced pressor response with ED50 values in the range 0.1-2.0 mg/kg. One of the compounds, 2-ethyl-4-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methoxy]quinoline (5g), demonstrated good oral activity in two rat models. At doses in the range 1-10 mg/kg in AII-infused, normotensive rats, the compound exhibited a dose-related inhibition of the pressor response with a good duration of action at the higher doses. In a renal hypertensive rat model, compound 5g showed a rapid and sustained lowering of blood pressure at a dose of 5 mg/kg. On the basis of its profile, this compound, designated ICI D8731, has been selected for clinical evaluation.

New nonpeptide angiotensin II receptor antagonists. 1. Synthesis, biological properties, and structure-activity relationships of 2-alkyl benzimidazole derivatives.

On the basis of an extension of the literature lead 1, a series of benzimidazoles have been synthesized and shown to be angiotensin II (AII) receptor antagonists. The structure-activity relationships of these new antagonists have been explored and the key binding interactions defined. Molecular mechanics calculations were carried out on analogues of imidazole AII antagonists and conformationally restricted analogues were synthesized. The benzimidazole antagonists displaced AII in binding studies in vitro with IC50 values in the range 10(-5)-10(-7) M and antagonized the hypertensive effects of AII in vivo (rats) following intravenous administration with ED50 values in the range of 5-20 mg/kg.

Inhibitors of human renin. Cyclic peptide analogues containing a D-Phe-Lys-D-Trp sequence.

Cyclic peptides containing a D-phenylalanine and a D-tryptophan residue have been synthesized and tested as inhibitors of human renin. Most of these are tripeptide derivatives of the type CO(CH2)3CO-D-Phe-Lys-D-Trp- or COCH2NHCH2CO-D-Phe-Lys-D-Trp- in which the individual side-chain methylene groups have been replaced with -CHMe-, -CMe2-, -CH(Ph)-, -CH(CH2Ph)-, or -CH[CH2)2CHMe2)-groups. The three amino acid residues and the size of the ring were very important features of these compounds. Reducing the ring size gave much less potent compounds. The most potent analogue of the series, CO(CH2)2CHPhCO-D-Phe-Lys-D-Trp-NH(CH2)2CHMe (14, IC50 = 26 nM), was obtained by substituting the methylene group nearer to the D-Phe residue by a -CHPh- group. Compound 14 was 15-fold more potent in inhibiting human renin than porcine renin.

Novel inhibitors of human renin. Cyclic peptides based on the tetrapeptide sequence Glu-D-Phe-Lys-D-Trp.

Cyclic peptide inhibitors of human renin based on a linear peptide, Boc-D-Phe-Cys(Acm)-D-Trp-Leu-OMe (1), were prepared by solution-phase methods. Potent inhibitors were obtained in one series of compounds, Z-Glu-D-Phe-Lys-D-Trp-Leu-OMe (3), in which the D-phenylalanine residue was incorporated in a 15-membered ring structure. Any reductions or enlargements of the ring size led to inactive or less potent peptides. The most potent inhibitor of human renin, Me3CCH2-Glu-D-Phe-Lys-D-Trp-NH(CH2)2CHMe2 (31) (IC50 6.3 x 10(-8) M), was obtained by changing N- and C-terminal parts of pentapeptide 3. It was about 650-fold more potent than linear tetrapeptide I and about 50-fold more potent than cyclic peptide 3. Compound 31 was also 112-fold more potent against human renin than against porcine renin.

1,2,4-Triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity. 2. Synthesis, biological properties and molecular modeling of hydroxyethylene isostere derivatives.

A series of inhibitors of human renin have been synthesized, derived from combination of a 2-(8-propyl-6-pyridin-3-yl-1,2,4-triazolo[4,3-a]pyrazin-3-yl)- 3-pyridin- 3-ylpropionic acid moiety 6c with the hydroxyethylene isostere of the scissile amide bond (2S,4S,5S)-5-amino-6-cyclohexyl-4-hydroxy-2-isopropylhexanoic acid (ChaOH--Val). The more potent members of this series showed good inhibitory activity against partially purified human renin, 7d, for example, having an IC50 of 0.2 nM. Structure-activity relationships for these compounds were consistent with their binding to the S4-S2' sites of human renin. Analogues 7e and 7h-k with a variety of substituents at the C-terminus all had in vitro IC50S less than 1 nM. In contrast with the majority of previously reported inhibitors of similar potency, these compounds contain no natural amino acid fragments. When administered intravenously to anesthetized, sodium-depleted marmosets at doses of 0.3-3.0 mg/kg, compound 7d caused a marked reduction in mean arterial pressure. Following oral administration at 30 mg/kg in the same animal model, 7d again elicited a significant fall in mean arterial pressure, accompanied by suppression of plasma renin activity lasting up to 3 h after dosing.

1,2,4-Triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity. 1. Synthesis and biological properties of alkyl alcohol and statine derivatives.

A series of 1,2,4-triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity, which incorporate (1S,2S)-2-amino-1,3-dicyclohexyl-1-hydroxypropane, statine (Sta), and (3S,4S)-4-amino-5-cyclohexyl-3-hydroxy-pentanoic acid (ACHPA) transition-state mimetics, have been prepared. Structure-activity relationships for renin inhibitory activity in the series are consistent with the 2-[8-isobutyl-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3-yl]-3-(3 pyridyl)propionic acid moiety 10b acting as a non-peptidic replacement for the P4-P2 (Pro-Phe-His) residues of the natural substrate angiotensinogen. Compounds 12m, 12o and 12q were potent inhibitors of partially purified human renin (IC50 values 1.7, 6.8, and 3.7 nM, respectively), and also effectively lowered blood pressure in anesthetized, sodium depleted marmosets following intravenous administration. On oral administration however, no blood pressure lowering activity could be detected, and absorption studies in bile duct cannulated rats indicate that this may be due primarily to poor oral absorption, rather than rapid biliary excretion. The reason for the observed poor oral activity is not clear, but it seems unlikely that poor aqueous solubility or metabolic instability to gut enzymes are rate-determining, and other factors such as high molecular weight may also be very important.

Kidney renin mRNA levels in the early and chronic phases of two-kidney, one clip hypertension in the rat.

The effect of clipping the left renal artery on left and right kidney renin mRNA levels during the early and chronic phases of two-kidney, one clip Goldblatt hypertension in the rat was studied. Renin mRNA levels were determined using northern and dot blotting. Four weeks after clipping, renin mRNA levels were sixfold higher in the left kidney and eightfold lower in the right kidney of the Goldblatt rats compared with the left kidney of the sham-operated rats. Similar analysis at 20 weeks after clipping showed a fourfold increase in the left kidney and a 16-fold suppression in the right kidney compared with age-matched sham-operated control rats. The study demonstrates the profound changes that occur in renin gene expression in the clipped and contralateral kidneys in this model of hypertension and shows that these changes persist into the chronic phase of the hypertension.

In vivo comparison of the renin inhibitor H77 with the angiotensin-converting enzyme inhibitor captopril.

Changes in blood pressure and heart rate in response to the renin inhibitor H77 and the angiotensin-converting enzyme inhibitor captopril were compared in conscious dogs during progressive sodium depletion. There were significant positive correlations between plasma renin activities immediately prior to administration of the inhibitors and the subsequent reductions in blood pressure produced by the inhibitors. The slopes and intercepts were similar for the two inhibitors, suggesting that both H77 and captopril were operating predominantly via inhibition of the renin-angiotensin system. Although the effects of H77 and captopril on heart rate and blood pressure were quantitatively similar, a small additional action of captopril was observed in dogs that had been sodium restricted for 12 days. Captopril injected during H77 infusion also had a small additional hypotensive action and caused a significant further increase in heart rate.

Non-specific inhibition of pressor agents in vivo by the renin inhibitor pepstatin A.

The specificity of pepstatin A as an inhibitor of the cardiovascular actions of renin injected into anaesthetized rats has been investigated. Pepstatin A 70 micrograms/kg/min partially inhibited the pressor response to injected renin without affecting the pressor responses to injected angiotensin II, phenylephrine or vasopressin. Pepstatin A 150 micrograms/kg/min also produced partial inhibition of injected renin, but in addition caused significant inhibition of the other pressor agents. This was in contrast to the effects of the angiotensin converting enzyme inhibitor captopril, 100 micrograms/kg i.v., which caused greater inhibition of the renin pressor response than pepstatin A without affecting the pressor response to injected angiotensin II, phenylephrine or vasopressin. Finally the direct acting vasodilator hydralazine was found to have a similar non-specific inhibitory effect to pepstatin A on the pressor responses to injected pressor agents. It is concluded that pepstatin A reduces the pressor responsiveness to injected pressor agents and that this non-specific cardiovascular activity limits the usefulness of pepstatin A as a pharmacological tool to inhibit renal renin in vivo.

Effects of the renin inhibitor H77 on the pressor responses to injected pig, rat and dog renins in vivo.

The potency of the substrate analogue inhibitor H77 has been investigated in vivo in anaesthetized rats and conscious dogs. ID50 values have been determined against rat, dog and pig renins injected into rats and against dog renin injected into dogs. In the rat H77 was most potent against injected dog renin (ID50 0.53 mg/kg/h), of intermediate potency against injected pig renin (ID50 1.1 mg/kg/h) and least potent against injected rat renin (ID50 40 mg/kg/h). H77 was more potent against dog renin injected into dogs than into rats (ID50 against dog renin in dogs 0.056 mg/kg/h). At infusion rates up to 0.5 mg/kg/h in dogs and 50.0 mg/kg/h in rats H77 was without effect on the pressor response to angiotensin I. Hence H77 appears to be a specific inhibitor of dog, rat and pig renins in vivo, the dose of H77 depending upon the type of renin injected and the species of the recipient animal.

The localization of a histamine H2-receptor adenylate cyclase system in canine parietal cells and its inhibition by prostaglandins.

A method is described for the preparation of parietal cell-enriched suspensions from dog gastric mucosa. Histamine and E type prostaglandins produce an elevation of cyclic AMP concentration in mixed cell preparations. Parietal cell-rich fractions respond to histamine but only weakly to prostaglandins whilst in fractions virtually free from parietal cells the converse is observed. Prostaglandins which are good antisecretory agents, PGE1, PGE2 and 16,16 dimethyl PGE2 are potent inhibitors of the histamine elevations of cyclic AMP in parietal cell-rich fractions, whilst PFG2alpha shows 1% of their potency. The experiments described support the view that histamine stimulates gastric acid secretion by excitation of an H2-receptor adenylate cyclase system in the plasma membrane of the parietal cell and that acid secretory inhibition by prostaglandins is a result of inhibition of that system.

Interactions of oestradiol-17beta and tamoxifen in the uterus of the pregnant rat.

Measurement of the uptake and retention of a radioactive post-coital antifertility agent tamioxifen, by reproductive tissues of the rat have shown that the ovary retained more radioactivity than did any other reproductive organ. Studies have also been made of the uptake and distribution of [3H]tamoxifen and [3H]oestradiol-17beta in the uterus of the pregnant rat on days 2-6 post coitum. Twenty-four hours after administration of tamoxifen, either i.v. or orally, 40-50% of the radioactivity was in the high speed pellet, 10-20% in the nuclear fraction, and 15-30% in the cytosol. An equivalent dose of [3H]oestradiol-17beta yielded distributions of 5%, 5% and 82% respectively. Fractionation of uteri from animals given 0-2 mg tamoxifen/kg on Day 2 of pregnancy followed by [3H]oestradiol 60 min before death showed little difference in total uptake of oestradiol or distribution in the subcellular fraction on Days 4,5 and 6. Although uptake of oestradiol by uterine nuclei was reduced on Day 3 by previous administration of tamoxifen on Day 2, appreciable quantities were still bound to the nuclear receptors. Treatment of ovariectomized animals with tamoxifen at doses up to 40 mug/rat (i.e. 0-2 mg/kg) led to the accumulation of oestrogen-receptor complex in the nucleus. It is concluded that the antifertility properties of tamoxifen (under the conditions of these experiments) cannot be ascribed to the suppression of uptake and binding of oestradiol by the uterus.