Biomedical subjects

K F Koehler

Publications and source records attributed to K F Koehler.

10 recordsLinked to original sources

PubMed2000

Functional probing of the human glucocorticoid receptor steroid-interacting surface by site-directed mutagenesis. Gln-642 plays an important role in steroid recognition and binding.

To elucidate which amino acids in the glucocorticoid receptor ligand-binding domain might be involved in determining steroid binding specificity by interaction with the D-ring of glucocorticoids, we have performed site-directed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-739 based on their proximity to the steroid in a model structure. Mutations of these residues affected steroid binding affinity, specificity, and/or steroid-dependent transactivation. The results indicate that these residues are located in close proximity to the ligand and appear to play a role in steroid recognition and/or transactivating sensitivity, possibly by changes in the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. Mutation of Gln-642 resulted in a marked decrease in affinity for steroids containing a 17alpha-OH group. This effect was alleviated by the presence of a 16alpha-CH(3) group to a varying degree. Thr-739 appears to form a hydrogen bond with the 21-OH group of the steroid, as well as possibly forming hydrophobic interactions with the steroid. Met-560 and Met-639 appear to form hydrophobic interactions with the D-ring of the steroid, although the nature of these interactions cannot be characterized in more detail at this point.

Animals↗

PubMed1995

Development of a comprehensive pharmacophore model for the benzodiazepine receptor.

A unified pharmacophore model of the benzodiazepine receptor (BzR) has been developed using the techniques of chemical synthesis, radioligand binding, and receptor mapping. This model is based on 136 different ligands spanning ten structurally diverse classes of compounds and qualitatively accounts for the relative affinities, efficacies, and functional effects (agonism vs. antagonism vs. inverse agonism) displayed by various ligands at the BzR. In addition, the model is expanded to account for the pharmacology of a recently discovered BzR receptor subtype termed the 'Diazepam-Insensitive' (DI) BzR. Moreover, the unified model described here is compared and contrasted with other published pharmacophore models. As previously reported, the synthesis of both partial agonists and partial inverse agonists has been achieved by using parts of this model. Partial agonists are of interest as potentially improved agents for treatment of anxiety disorders, while the partial inverse agonists may furnish important clues for the treatment of age-associated memory impairment.

Benzodiazepines↗

PubMed1994

Synthesis of benzo-fused benzodiazepines employed as probes of the agonist pharmacophore of benzodiazepine receptors.

The synthesis and in vitro evaluation of benzo-fused benzodiazepines 1-6 are described. These "molecular yardsticks" were employed to probe the spatial dimensions of the lipophilic pocket L2 in the benzodiazepine receptor (BzR) cleft and to determine the effect of occupation of L2 with respect to agonist activity. Of the new analogs synthesized, the 7,8-benzo-fused benzodiazepine 6 displayed moderately high affinity for the BzR (IC50 = 55 nM) and exhibited both anticonvulsant (ED50 approximately 15 mg/kg) and muscle relaxant (ED50 approximately 15 mg/kg) activity. As expected, 2 and 4 interacted with the repulsive regions of interaction, S1 and S2, and exhibited low affinities for BzR. The rigid nature of these molecular yardsticks (especially 6, Figure 7) has been employed to probe the depth of L2. Moreover, in the case of 6 full occupation of L2 has resulted in an increase in the muscle relaxant effect at the expense of the anticonvulsant/anxiolytic effect.

Animals↗

PubMed1993

Synthetic and computer-assisted analysis of the structural requirements for selective, high-affinity ligand binding to diazepam-insensitive benzodiazepine receptors.

Several 1,4-diazepines were recently reported to bind with high affinities to the "diazepam-insensitive" (DI) isoform of the benzodiazepine receptor (BzR) (Korpi, E.R.; Uusi-Oukari, M.; Wegelius, K. Eur. J. Pharm. 1992, 213, 323-329. Wong, G.; Skolnick, P. Eur. J. Pharmacol. Mol. Pharm. Sec. 1992, 225, 63-68). However, only the putative ethanol antagonist 1 (Ro 15-4513) displayed modest selectivity for the DI site compared to other "diazepam-sensitive" (DS) BzR isoforms. In order to probe the requirements for selective, high-affinity binding to the DI site, the affinities of 47 benzodiazepines have been determined at both DI and DS BzR sites. In addition, single X-ray crystallographic analyses for three of these derivatives, 5 (Ro 17-1812), 6 (Ro 16-6028), and 42 (Ro 14-5974), are reported. The radioligand binding studies reveal that modifications to the 3-, 7-, and 8-positions of 6-oxoimidazo[1,5-alpha] [1,4]benzodiazepines have a marked influence on the Ki(DI)/Ki(DS) ratios. In order to more precisely determine the structural requirements for both high affinity and selectivity at DI BzR relative to DS, 3D-QSAR analyses were carried out on ligand affinities at both of these BzR isoforms. This analysis was based, in part, on the new X-ray crystallographic data. Satisfactory cross-validated regression equations were obtained individually for the logarithms of ligand affinities at DI and DS as well as for the differences of the logarithms of their affinities at these two isoforms (cross-validated R2 > 0.70 for all three regression equations). The steric and electrostatic 3D-QSAR DI and DS maps are in qualitative accord with the structure-activity relationship (SAR) data. Furthermore, the DI and DI/DS maps may be useful in the design of ligands with enhanced DI affinity and DI/DS selectivity, respectively.

Animals↗

PubMed1992

Predictive binding of beta-carboline inverse agonists and antagonists via the CoMFA/GOLPE approach.

The synthesis and affinities of six new 3-substituted beta-carbolines (6-10, 12) for the benzodiazepine receptor (BzR) are described. These analogs were used both to probe the dimensions of the hydrophobic pocket in the benzodiazepine receptor and to test the predictive ability of a previously reported 3D-QSAR regression model. Of the new analogs synthesized, the gamma-branched derivatives (isobutoxy, 7, IC50 = 93 nM; isopentoxy, 9, IC50 = 104 nM) display significantly higher affinity for the BzR than either the beta-branched (sec-butoxy, 6, IC50 = 471 nM; tert-butyl ketone, 12, IC50 = 358 nM) or delta-branched (isopentoxy, 8, IC50 = 535 nM) analogs. An exception to this rule is the gamma-branched 3-benzyloxy derivative 10 (IC50 > 1000 nM) which appears to have a chain length that is too long to be accommodated by the BzR. The standard error of prediction for these six new beta-carbolines using the original regression model is significantly lower than the standard error estimate of the cross validation runs on the training set, hence the predictions made using this model are much better than expected. In order to obtain more credible predictions, a new procedure called GOLPE (generating optimal linear PLS estimates) was used to eliminate irrelevant electrostatic and steric descriptors from the regression equation. A substantial reduction in the standard error estimate resulted. The predictions from this new regression equation were somewhat less accurate than the ones obtained with the original regression equation; however the standard error of prediction and the standard error estimate are in much closer agreement. Finally, to probe the effect that the quality of the steric and electrostatic potentials has on 3D-QSAR analyses, the semiempirical MNDO parallel PRDDOE geometries and Mulliken charges used in the original analyses were replaced with ab initio 3-21G parallel 6-31G* geometries and electrostatic potential fit charges. A modest decrease in the standard error estimate and increase in cross validated R2 resulted.

Animals↗

PubMed1991

Synthetic and computer assisted analysis of the pharmacophore for agonists at benzodiazepine receptors.

In order to employ rational drug design in the discovery of selective benzodiazepine receptor agonists and inverse agonists, pharmacophore/receptor models for both these activities must first be established. Recently, a pharmacophore for the inverse agonist site has been formulated employing the most recent receptor mapping techniques (22). The continuation of this approach to the pharmacophore for agonist ligands has permitted a definition of this site independently of the inverse agonist model. The agonist pharmacophore/receptor contains two hydrogen bond donating sites of interaction (H1 and H2) located about 6.5 A from each other, as well as three areas of lipophilic interaction (L1-L3). The areas L1 and L2 are critical for agonist activity; moreover, some ligands also require an interaction in a third lipophilic area termed L3. This is in agreement with previous work (12-23). In addition, an area of negative steric interaction (S1) between the ligand and receptor-binding protein is defined. In regard to the pharmacophore, it was established that the alignment rule for agonist beta-carbolines is different from that which elicits inverse agonist activity. Consideration of the pharmacophore has resulted in the synthesis of a new beta-carboline 16 which elicits agonist activity. This ligand 16 not only satisfied the requirements of the pharmacophore, but more importantly it elicited both anticonvulsant and anxiolytic activity, but was devoid of the myorelaxant/ataxic properties associated with the benzodiazepines.

Animals↗

PubMed1990

Synthetic and computer-assisted analyses of the pharmacophore for the benzodiazepine receptor inverse agonist site.

The structural requirements for ligand binding to the benzodiazepine receptor (BzR) inverse agonist site were probed through the synthesis and in vitro evaluation of 3-substituted beta-carbolines 6, 7, 11, 12, gamma-carboline 13, and diindoles 18-21, 23-25, 27, 28, and 34. On the basis of the apparent binding affinities of these and other analogues, a hydrogen bond acceptor site (A2) on the receptor is proposed to interact with the N(9) hydrogen atom of the beta-carbolines or the N(7) hydrogen nuclei of the diindoles. Likewise, a proposed hydrogen bond donating site (H1) interacts with the N(2) nitrogen atom of the beta-carbolines or the N(5) nitrogen atom of the diindoles. It appears that interaction with both sites is a prerequisite for high affinity since analogues which have either one or both of these positions blocked exhibit substantial reduction in affinity. Moreover, H1 appears to be capable of engaging in a three-centered hydrogen bond with appropriately functionalized ligands, which explains the increase in potency observed in the following series of 3-substituted beta-carbolines: the n-butyl (12, IC50 = 245 nM), n-propoxy (9, IC50 = 11 nM), and propyl ketone (11, IC50 = 2.8 nM) congeners. In addition to H1 and A2, there appears to be a relatively narrow hydrophobic pocket in the binding cleft that can accommodate substituents at the 3-position of the beta-carbolines which have chain lengths less than or equal to C5. There is a 1 order of magnitude decrease in affinity between n-propoxy analogue 9 (IC50 = 11 nM, chain length = 4) and n-butoxy derivative 7 (IC50 = 98 nM, chain length = 5). Furthermore, alpha- and gamma-branching [e.g. ethoxycarbonyl (2), IC50 = 5 nM and tert-butoxycarbonyl (31) IC50 = 10 nM] but not beta- and delta-branching [e.g. isopropoxy (6), IC50 = 500 nM and (neopentyloxy) carbonyl (48), IC50 = 750 nM] at position 3 are tolerated. Occupation of this hydrophobic pocket is clearly important for high affinity as evidenced by the relatively low affinity of 30, a beta-carboline which possesses a hydrogen atom at the 3-position. This same hydrophobic pocket is partially filled by the D and E rings of the diindoles, which accounts for the high affinity of several members of this series. An excluded volume analysis using selected 3-substituted beta-carbolines and ring-E substituted pyridodiindoles is consistent with the presence of this hydrophobic pocket (see Figure 1).(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1988

Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C.

The bryostatins are macrocyclic lactones that represent an additional structural class of potent activators of protein kinase C. These marine animal biosynthetic products are of unusual interest because they induce only a subset of the biological responses induced by the phorbol esters. We have now determined the binding affinities of naturally occurring and semisynthetic bryostatins for protein kinase C by competition analysis with [26-3H]bryostatin 4 as the radioactive ligand. Esterification of the hydroxyl group at C26 caused dramatic loss of activity as did inversion of the asymmetric center at this position. In contrast, neither of the ester groups at C7 and C20 had a major influence on activity. Computer modeling of the phorbol esters, related diterpenes, and indole alkaloids suggested that the C20, C9, and C4 oxygens of phorbol represented critical elements of the phorbol ester pharmacophore. The C26 oxygen of the bryostatins, together with the C1 and C19 oxygens, gave an excellent spatial correlation with this model, with a root-mean-square deviation of 0.16 A (compared to 0.10-0.35 A among phorbol-related diterpenes). The extension of the phorbol ester pharmacophore model to the bryostatins and its agreement with the structure-activity relations for the bryostatin class of compounds provide additional support for the validity of the model.

Animals↗

PubMed1986

Analysis of the phorbol ester pharmacophore on protein kinase C as a guide to the rational design of new classes of analogs.

The diterpene diester phorbol 12-myristate 13-acetate and the alkaloid teleocidin B are structurally unrelated natural products that display similar potent irritant and tumor-promoting activities. Computer modeling of these and other structural classes of tumor promoters show a marked similarity in the relative positions of certain heteroatoms and hydrophobic groups. For phorbol this mapping consists of the C-4, C-9, and C-20 hydroxyl groups as well as a hydrophobic region filled by a long-chain acyl functionality attached to either the C-12 or the C-13 positions. Diacylglycerols, thought to be the endogenous activators of the major phorbol ester receptor protein kinase C likewise fit this model in a stereospecific fashion. As an initial test of the utility of the model, members of a new and simplified class of activators were synthesized that possess the predicted essential structural features. These compounds all inhibited specific phorbol ester binding to protein kinase C, albeit with low affinity (10-60 microM); further analysis of one derivative, decylhydroxylindole, confirmed that the inhibition of phorbol ester binding was competitive. This same derivative inhibited epidermal growth factor binding in intact Swiss 3T3 cells and studies with another derivative showed phosphorylation of a 40-kDa protein in platelets. Both of these in vivo responses are characteristic of phorbol esters.

Blood Platelets↗

PubMedDate not supplied

Molecular design using the minireceptor concept.

Explicit molecular binding pockets were constructed and optimized around sets of superimposed ligands using the minireceptor concept. The resulting binding sites incorporate the properties of the different ligands and were shown to be suitable for the design of molecules presenting novel interaction patterns. Two applications of minireceptor construction and/or optimization, followed by molecular design are described. In the pursuit of new ligands mimicking the action of paclitaxel, a minireceptor was constructed using the primary amino acid sequence of the target protein as a guide. The active site extracted from a homology-based model of the serotonin 5-HT1A receptor was optimized around a set of three ligands using the same approach.

Antineoplastic Agents, Phytogenic↗