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E Jacoby

Publications and source records attributed to E Jacoby.

At least 19 recordsLinked to original sources
PubMed2005

The Novartis compound archive -- from concept to reality.

As HTS technologies come of age, pharmaceutical companies are focusing increasingly on the quality of their screening collections. Storage conditions and their influence on compound stability and solubility are debated intensely. At Novartis, a strategy was developed that is different to most other companies: (1) compounds unsuitable for storage in solution are excluded by computational methods; (2) compounds are stored at 4 degrees C/20% relative humidity in a DMSO/water mixture to avoid freeze-thaw cycles and water uptake and to allow rapid plate replication; (3) resolubilisation of compounds at regular intervals.

Automation↗
PubMed2004

Molecular diversity management strategies for building and enhancement of diverse and focused lead discovery compound screening collections.

This publication describes processes for the selection of chemical compounds for the building of a high-throughput screening (HTS) collection for drug discovery, using the currently implemented process in the Discovery Technologies Unit of the Novartis Institute for Biomedical Research, Basel Switzerland as reference. More generally, the currently existing compound acquisition models and practices are discussed. Our informatics, chemistry and biology-driven compound selection consists of two steps: 1) The individual compounds are filtered and grouped into three priority classes on the basis of their individual structural properties. Substructure filters are used to eliminate or penalize compounds based on unwanted structural properties. The similarity of the structures to reference ligands of the main proven druggable target families is computed, and drug-similar compounds are prioritized for the following diversity analysis. 2) The compounds are compared to the archive compounds and a diversity analysis is performed. This is done separately for the prioritized, regular and penalized compounds with increasingly stringent dissimilarity criterion. The process includes collecting vendor catalogues and monitoring the availability of samples together with the selection and purchase decision points. The development of a corporate vendor catalogue database is described. In addition to the selection methods on a per single molecule basis, selection criteria for scaffold and combinatorial chemistry projects in collaboration with compound vendors are discussed.

Computational Biology↗
PubMed2001

S 14506: novel receptor coupling at 5-HT(1A) receptors.

S 14506 is chemically related to the inverse agonist at 5-HT(1A) receptors, spiperone, but S 14506 behaves as one of the most potent agonists known at these receptors, both in vitro and in vivo. In hippocampal membranes, the specific binding of [(3)H]-S 14506 (K(d)=0.79+/-0.2 nM; B(max)=400+/-32 fmol/mg protein) to 5-HT(1A) receptors resembled that of an antagonist in that it was increased by GppNHp, whereas GppNHp reduced the binding of the classic agonist [(3)H]-8-OH-DPAT (K(d)=1.5+/-0.5 nM; B(max)=303+/-20 fmol/mg protein). Manganese, magnesium and calcium reduced the binding of [(3)H]-S 14506 to 5-HT(1A) receptors whereas the binding of [(3)H]-8-OH-DPAT was increased. Further, sodium markedly reduced the binding of [(3)H]-8-OH-DPAT, without affecting the binding of [(3)H]-S 14506. [(3)H]-S 14506 also bound with high affinity to h 5-HT(1A) receptors stably expressed in membranes of CHO cells (K(d)=0.13+/-0.05 nM; B(max)=2.99+/-0.60 pmol/mg protein): the B(max) was double that of [(3)H]-8-OH-DPAT. GppNHp strongly decreased [(3)H]-8-OH-DPAT binding but scarcely changed [(3)H]-S 14506 binding; calcium, magnesium and manganese had little effect on [(3)H]-S 14506 binding in CHO cells. Antagonists (WAY 100635, WAY 100135) and inverse agonists (spiperone and metitepine) displaced [(3)H]-S 14506 binding with high affinity and Hill slopes close to unity, whereas agonists (5-HT and 5-CT) displayed low affinity with low Hill slopes: partial agonists (buspirone, ipsapirone) showed intermediate properties. In fusion proteins of h 5-HT(1A) receptors with G(ialpha1) the compound potently increased high-affinity GTPase, with a steeper Hill slope than for 5-HT, which may indicate positive cooperativity. The maximum response for S 14506 in these assays was equivalent to 5-HT, indicating it to be a full agonist.In molecular modelling studies, using a three-site model of the 5-HT(1A) receptor, S 14506 spanned between the 5-HT recognition site and the "arginine switch" (DRY microdomain) postulated to activate the interaction of the receptor with the G protein. Thus it is possible to synthesise ligands at G-protein-coupled receptors which are highly potent agonists, but which are structurally related to inverse agonists and show some features of antagonist/inverse agonist binding.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2000

Substrate specificity and inhibition studies of human serotonin N-acetyltransferase.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetyl-CoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[(3)H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.

Acyl Coenzyme A↗
PubMed1998

Interaction of the anxiogenic agent, RS-30199, with 5-HT1A receptors: modulation of sexual activity in the male rat.

RS-30199 has been shown previously to have atypical interactions at 5-HT1A receptors. RS-30199 and RS-64459, an analogue of buspirone with a buspirone side chain, were compared with the classic, partial agonist at 5-HT1A receptors, 8-hydroxy-2 (di-n-propylamino) tetralin (8-OH-DPAT) and buspirone. At human (h) 5-HT1A receptors in CHO cells, RS-30199-193 (racemate) and its enantiomers (-197, -198) inhibited [3H]-8-OH-DPAT binding (RS-30199-198, ki, 29.7 +/- 11.7 nM; RS-30199-197, ki, 74.1 +/- 11.7 nM) as did RS-64459 (ki, 24.9 +/- 6.0 nM), but RS-30199-197 and -198 were almost full agonists in a [35S]-GTPgammaS binding assay, whereas RS-64459 was a partial agonist, resembling buspirone and 8-OH-DPAT. RS-64459 and the enantiomers of RS-30199 had weaker affinity for 5-HT2C and 5-HT7 receptors. These compounds did not induce the 5-HT behavioural syndrome in male rats. However, in a model where naive male rats were introduced to estrogen-progesterone primed, sexually receptive female rats, RS-30199-197 (0.1, 1, 10 mg/kg, s.c.) had a profound inhibitory effect on sexual behaviour score. Neither buspirone nor 8-OH-DPAT reduced the sexual behaviour score. Unlike 8-OH-DPAT, which shortens intromission latency, RS-30199 prolonged intromission latency. RS-30199 (10 mg/kg s.c.) fully inhibited the facilitation of sexual behaviour caused by the alpha2-adrenoceptor antagonist, delequamine (0.1 mg/kg, p.o.). In contrast, RS-64459 (100, 250, 1000 and 4000 microg/kg, s.c.) failed to modify the sexual behaviour score and did not modify intromission latency. The differences between the effects of RS-30199 and RS-64459 in binding and functional experiments are supported by molecular models of the receptor-ligand interaction, where the compounds interact in different ways with the receptor; a model is proposed for the allosteric interaction of different agents with the receptor, resulting in different functional profiles. RS-30199 can be considered an atypical agonist at 5-HT1A receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1997

A structural rationale for the design of water soluble peptide-derived neurokinin-1 antagonists.

Molecular models of a pharmacophore for NK1 neurokinin antagonists and of ligand-receptor complexes for the human NK1 G protein-coupled receptor are presented. The models develop a structural rationale for the discovery of the recently described highly potent peptidomimetic NK1 antagonists S18523 and S19752 which were designed to be water soluble. Water solubility was conferred on these compounds by introduction of an anionic butyl-tetrazole substituent on the scaffold of dipeptide-derived NK1 antagonist analogues. The models provide convincing evidence that the anionic butyl-tetrazole moieties of S18523 and S19752 protrude outside the membrane-spanning domain of the receptor and do not interfere significantly with the core of the antagonist binding site. It is emphasized that this result could only be obtained through the combination of the two modelling approaches. The result suggest a general way to modify the transport properties of the peptidomimetic antagonists without altering the receptor-binding interaction, and it outlines the potential of including the combination of pharmacophore models and crude models of receptor-ligand complexes early in the drug design process.

Animals↗
PubMed1996

Structure and dynamics of a protein assembly. 1H-NMR studies of the 36 kDa R6 insulin hexamer.

The structure and dynamics of the R6 human insulin hexamer are investigated by two- and three-dimensional homonuclear 1H-NMR spectroscopy. The R6 hexamer, stabilized by Zn2+ and phenol, provides a model of an allosteric protein assembly and is proposed to mimic aspects of receptor recognition. Despite the large size of the assembly (36 kDa), its extreme thermal stability permits high-resolution spectra to be observed at 55 degrees C. Each spin system is represented uniquely, implying either 6-fold symmetry or fast exchange among allowed protomeric conformations. Dramatic changes in chemical shifts and long-range nuclear Overhauser enhancements (NOEs) are observed relative to the spectra of insulin monomers. Complete sequential assignment is obtained and demonstrates native secondary structure with distinctive R-state N-terminal extension of the B-chain alpha-helix (residues B1 to B19). The distance-geometry structure of an R-state promoter is similar to those of R6 crystal structures. Specific long-range intra- and intersubunit NOEs, assigned by stepwise analysis of engineered insulin monomer and dimers, demonstrate that tertiary and quaternary contacts are also similar. Although the hexamer is well-ordered in solution, binding of phenol to an internal cavity occurs within milliseconds, implying the existence of "gatekeeper" residues whose flexibility provides a portal of entry and release. Changes in 1H-NMR chemical shifts on hexamer assembly are readily rationalized by analysis of aromatic ring-currents and provide sensitive probes for sites of protein-protein interaction and phenol binding. Our results provide a foundation for the interaction and phenol binding. Our results provide a foundation for the studies of insulin analogues (such as "designed" insulins of therapeutic interest) under conditions of clinical formulation and for the investigation of the effects of protein assembly on the dynamics of individual protomers.

Circular Dichroism↗
PubMed1996

Simulation of a complex protein structural change: the T <--> R transition in the insulin hexamer.

The T <--> R transition in the insulin hexamer is an outstanding model for protein structural changes in terms of its extent and complexity: the limiting structures T(6), T(3)R(3) and R(6) have been defined by X-ray crystallography. The transition occurs cooperatively within trimers. It involves displacements of >30 A and a secondary structural rearrangement of 15% of the peptide chain between extended and helical conformations. Experimental data for the transition are plentiful. Theoretical methods to simulate pathways without constraints would never succeed with such substantial transitions. We have developed two approaches, targeted energy minimization (TEM) and targeted molecular dynamics (TMD). Previously successful in simulating the T <--> R transition of the insulin monomer, these procedures are also shown here to be effective in the hexamer. With TMD, more conformational space is explored and pathways are found at 500 kJ/mol lower energy than with TEM. Because the atoms have to meet distance constraints in sum rather than individually, a high degree of conformational freedom and independence is implied. T(6) --> T(3)R(3) and T(3)R(3) --> T(6) pathways do not coincide because the transformation is directed. One subunit enters a dead end pathway in one direction of the TMD simulation, which shows that constraint and freedom are critically balanced. The ensemble of productive pathways represents a plausible corridor for the transition. A video display of the transformations is available.

Crystallography, X-Ray↗
PubMed1995

Acute childhood diarrhoea and maternal time allocation in the northern central Sierra of Peru.

Interventions to improve child health depend, at least implicitly, on changing maternal knowledge and behaviour and a reallocation of maternal time. There have been few studies, however, of the time cost involved in the adoption of new health technologies and even fewer that examine changes in maternal activities in response to child illness. The present study examines maternal daytime activities and investigates changes that occur when children are ill. We examine the impact of acute childhood diarrhoea episodes on the activity patterns of the mother/caretaker in this setting. The results show that mothers alter their usual activity patterns only slightly in response to acute diarrhoea episodes in their children. They continue to perform the same variety of activities as when the children are healthy, although they are more likely to perform them with the child 'carried' on their back. There is some indication that diarrhoea perceived to be more severe did result in the mother acting as caretaker more frequently. These findings have important implications for health interventions that depend on changing the amount of maternal or caretaker time spent for child health technologies, but the implications may vary depending on the reasons for the observed lack of changes in caretaker activities.

Child Care↗
PubMed1994

Modeling of G-protein coupled receptors with bacteriorhodopsin as a template. A novel approach based on interaction energy differences.

The structure of bacteriorhodopsin was used as a template to generate a model for G-protein coupled receptors. However, these receptors and the template are not related by sequence homology. Therefore a pragmatic and reproducible approach was developed to achieve an energetically favourable accommodation of receptor sequences to the backbone structure of bacteriorhodopsin. Improved interaction energy differences are used in a two step procedure analogous to a hypothetical folding mechanism for integral membrane proteins. The resulting model is in good agreement with existing data from structure-function studies.

Amino Acid Sequence↗
PubMed1993

Distinction of structural reorganisation and ligand binding in the T<==>R transition of insulin on the basis of allosteric models.

Two allosteric models are presented for the T<==>R transition of insulin hexamers in the presence of phenolic ligands which are based on existing experimental information. The transition mainly involves residues 1-8 of the B-chain, i.e. 15% of the molecule, which are extended in the T- and helical in the R-state. The main facts to be accounted for are: 1) the transition is undergone trimer-wise; 2) the transition of the second trimer is disadvantaged compared to the first one; 3) the subunits of a trimer undergo transition in a cooperative process; 4) binding sites for phenolic ligands only exist in R3 trimers; 5) ligands shift the equilibrium by arresting the R-state; 6) the ligand is accommodated in a pocket made up between two adjacent subunits; 7) binding one ligand molecule extends the lifetime of the two other binding sites of a trimer; 8) only ligand-free trimers can undergo transitions. The two models allowed for CD spectroscopic titrations of zinc and cobalt insulin with phenol and m-cresol to be assessed in terms of structural reorganisation and ligand binding, and for the respective standard free energy differences to be calculated. delta G degrees for the reorganisation of the first timer in zinc-insulin is about 8 kJ/mol, and for that of the second trimer, 21kJ/mol. The corresponding values for cobalt-insulin are 12 and 24 kJ/mol, respectively. For both zinc- and cobalt-insulin, the delta G degrees for phenol and m-cresol binding is about -18 kJ/mol. Both models are equally compatible with the titration data.

Allosteric Regulation↗
PubMed1992

The T<-->R structural transition of insulin; pathways suggested by targeted energy minimization.

The transition of insulin between its crystallographically defined states T and R is connected with considerable change even of backbone structure: the N-terminal B chain (residues B1-B8) refolds from extended conformation in T into helical in R, and vice versa. Although hitherto observed only in hexamers the transition of the monomer was adequate for developing and testing the method of 'targeted energy minimization' (TEM), capable of coping with conformational changes of such extent at moderate computational expenditure. The simulation is performed in a predetermined number of steps consisting of two atomic displacements each, one by force in the direction of the target structure, the second by energy minimization releasing the constraint caused in the first. The transition pathway is represented by the string of energy minimized transient structures. Due to the directedness of the algorithm the simulated pathway for R-->T is not the reversal of that for T-->R. It is, therefore, not pretended that the minimum energy pathway was identified. In the T-->R direction the N-terminal B chain first swivels while remaining largely stretched and then winds up extending the pre-existing helix B9-B19. The A chain advances into the space abandoned and withdraws from it in the R-->T simulation. In the latter the extended helix first kinks at B8/B9, and then the B1-B8 segment is unwound and stretched. The helical H-bonds of that segment are formed late in T-->R and are maintained during almost half of R-->T. The AN helix is less stable and more involved in the transitions than helix AC.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed1991

A model for the C5a receptor and for its interaction with the ligand [corrected].

A model of the C5a receptor was built based on the assumption that the seven membrane-spanning helices of known inward/outward direction are in an arrangement roughly similar to that in bacteriorhodopsin. Guidelines for the positioning of the helices were cysteine pairing, 'ridges into grooves' interdigitation of side chains and aromatic cluster formation. The chain segments protruding from the membrane are too short for folding into an independent ectodomain. The only longer segment (179-202) is tied down in its centre onto the membrane by a disulphide bridge and, thereby, made into two short loops as well. Ideas of the interaction of the C5a receptor with its ligand were derived mainly from the search for accommodation of the functionally essential arginine residues 40 and 74 of C5a. Asp82 is the only charged residue in a pocket approximately 20 A below the receptor surface and is conserved in the rhodopsin superfamily. It commends itself for binding Arg74 which is the tip of the flexible C-terminal chain of C5a, and rules out Arg40 in the structurally well-defined part of the molecule. The latter may bind to Glu180 at the bottom of a more shallow pocket which happens to resemble the substrate-binding site of trypsin.

Amino Acid Sequence↗
PubMed1984

Decreased placental monooxygenase activities associated with birth defects.

We have measured monooxygenase activities in placentas from 82 women who smoked throughout their pregnancies and correlated these with the presence or absence of major somatic anomalies. Monooxygenase activities toward benzo(a)pyrene and ethoxyresorufin in placentas from 18 abnormal infants were compared with activities in placentas from 64 concurrently studied normal infants. Placentas from normal infants were found to have high levels of monooxygenase activities and low apparent Kms toward ethoxyresorufin (10(-7) M), reflecting induction of cytochrome P-450 enzymes usually associated with maternal cigarette smoking. Placentas from the abnormal infants, however, had significantly lower monooxygenase activities and higher apparent Kms toward ethoxyresorufin (10(-5) M), indicating that induction of specific cytochrome P-450 systems occurred less frequently among placentas from abnormal infants. The reasons for this association are unclear. Apparent lack of induction of monooxygenase activity occurred most frequently in placentas from anencephalic infants but was neither exclusively nor consistently found with this defect. No specific maternal condition or environmental exposure associated with lack of monooxygenase induction was identified.

Anencephaly↗