Biomedical subjects

B Bienfait

Publications and source records attributed to B Bienfait.

16 recordsLinked to original sources

PubMed2000

Conformationally constrained analogues of diacylglycerol (DAG). 17. Contrast between sn-1 and sn-2 DAG lactones in binding to protein kinase C.

In previous work, we have obtained potent protein kinase C (PK-C) ligands with low-namomolar binding affinities by constructing diacylglycerol (DAG) mimetics in which the sn-2 carbonyl of DAG was constrained into a lactone ring. An additional structural element that helped achieve high binding affinity was the presence of branched acyl or alpha-alkylidene chains. In the present study, the effects of similarly branched chains on a different lactone system, where the lactone carbonyl is now equivalent to the sn-1 carbonyl of DAG, are investigated. In this new lactone template, the two chiral centers must have the S-configuration for enzyme recognition. As with the sn-2 DAG lactones, the branched chains were designed to optimize van der Waals contacts with a group of conserved hydrophobic amino acids located on the rim of the C1 domain of PK-C. The acyl and alpha-alkylidene chains were also designed to be lipophilically equivalent (8 carbons each). Eight new compounds (7-14) representing all possible combinations of linear and branched acyl and alpha-alkylidene were synthesized and evaluated. The sn-1 DAG lactones were less effective as PK-C ligands than the sn-2 DAG lactones despite having a similar array of linear or branched acyl and alpha-alkylidene chains

Diglycerides↗

PubMed2000

The lipophilicity of phorbol esters as a critical factor in determining the pattern of translocation of protein kinase C delta fused to green fluorescent protein.

Our previous study showed differential subcellular localization of protein kinase C (PKC) delta by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC delta. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13-dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13-dibutyrate and phorbol 12,13-dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nM to 10 microM). The highly lipophilic derivatives, phorbol 12,13-didecanoate and phorbol 12, 13-diundecanoate, at 1 microM caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC delta localization and thereby potentially to their different biological activities.

Animals↗

PubMed2000

Diacylglycerols with lipophilically equivalent branched acyl chains display high affinity for protein kinase C (PK-C). A direct measure of the effect of constraining the glycerol backbone in DAG lactones.

New synthetic diacylglycerols (DAGs) with equivalent branched acyl chains were compared with commercially available DAGs as PK-C ligands. The results support the view that there is a minimal lipophilic requirement provided by the equivalent acyl groups that results in high binding affinity. Locking the glycerol backbone of the most potent DAG into a five-member lactone resulted in a 10-fold increase in potency.

Diglycerides↗

PubMed2000

Conformationally constrained analogues of diacylglycerol (DAG). 16. How much structural complexity is necessary for recognition and high binding affinity to protein kinase C?

The design of potent protein kinase C (PK-C) ligands with low nanomolar binding affinities was accomplished by the combined use of pharmacophore- and receptor-guided approaches based on the structure of the physiological enzyme activator, diacylglycerol (DAG). Earlier use of the former approach, which was based on the structural equivalence of DAG and phorbol ester pharmacophores, identified a fixed template for the construction of a semirigid "recognition domain" that contained the three principal pharmacophores of DAG constrained into a lactone ring (DAG-lactones). In the present work, the pharmacophore-guided approach was refined to a higher level based on the X-ray structure of the C1b domain of PK-Cdelta complexed with phorbol-13-O-acetate. A systematic search that involved modifying the DAG-lactone template with a combination of linear or branched acyl and alpha-alkylidene chains, which functioned as variable hydrophobic "affinity domains", helped identify compounds that optimized hydrophobic contacts with a group of conserved hydrophobic amino acids located on the top half of the C1 domain where the phorbol binds. The hydrophilic/hydrophobic balance of the molecules was estimated by the octanol/water partition coefficients (log P) calculated according to a fragment-based approach. The presence of branched alpha-alkylidene or acyl chains was of critical importance to reach low nanomolar binding affinities for PK-C. These branched chains appear to facilitate important van der Waals contacts with hydrophobic segments of the protein and help promote the activation of PK-C through critical membrane interactions. Molecular modeling of these DAG-lactones into an empty C1b domain using the program AutoDock 2.4 suggests the existence of competing binding modes (sn-1 and sn-2) depending on which carbonyl is directly involved in binding to the protein. Inhibition of epidermal growth factor (EGF) binding, an indirect PK-C mediated response, was realized with some DAG-lactones at a dose 10-fold higher than with the standard phorbol-12, 13-dibutyrate (PDBU). Through the National Cancer Institute (NCI) 60-cell line in vitro screen, DAG-lactone 31 was identified as a very selective and potent antitumor agent. The NCI's computerized, pattern-recognition program COMPARE, which analyzes the degree of similarity of mean-graph profiles produced by the screen, corroborated our principles of drug design by matching the profile of compound 31 with that of the non-tumor-promoting antitumor phorbol ester, prostratin. The structural simplicity and the degree of potency achieved with some of the DAG-lactones described here should dispel the myth that chemical complexity and pharmacological activity go hand in hand. Even as a racemate, DAG-lactone 31 showed low namomolar binding affinity for PK-C and displayed selective antitumor activity at equivalent nanomolar levels. Our present approach should facilitate the generation of multiple libraries of structurally similar DAG-lactones to help exploit molecular diversity for PK-C and other high-affinity receptors for DAG and the phorbol esters. The success of this work suggests that substantially simpler, high-affinity structures could be identified to function as surrogates of other complex natural products.

4-Butyrolactone↗

PubMed1999

beta2-chimaerin is a novel target for diacylglycerol: binding properties and changes in subcellular localization mediated by ligand binding to its C1 domain.

The members of the chimaerin family of Rac-GTPase-activating proteins possess a single C1 domain with high homology to those present in protein kinase C (PKC) isozymes. This domain in PKCs is involved in phorbol ester and diacylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, beta2-chimaerin, binds phorbol esters with high affinity. In this study we analyzed the properties of beta2-chimaerin as a DAG receptor by using a series of conformationally constrained cyclic DAG analogues (DAG lactones) as probes. We identified analogs that bind to beta2-chimaerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycerol. The potencies of these analogs approach those of the potent phorbol ester tumor promoters. The different DAG lactones show some selectivity for this novel receptor compared with PKCalpha. Cellular studies revealed that these DAG analogs induce translocation of beta2-chimaerin from cytosolic (soluble) to particulate fractions. Using green fluorescent protein-fusion proteins for beta2-chimaerin we determined that this novel receptor translocates to the perinuclear region after treatment with DAG lactones. Binding and translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of beta2-chimaerin and the C1b domain of PKCdelta also was confirmed by modeling analysis. Our results demonstrate that beta2-chimaerin is a high affinity receptor for DAG through binding to its C1 domain and supports the emerging concept that multiple pathways transduce signaling through DAG and the phorbol esters.

Amino Acid Sequence↗

PubMed1998

Conformationally constrained analogues of diacylglycerol (DAG). 15. The indispensable role of the sn-1 and sn-2 carbonyls in the binding of DAG-lactones to protein kinase C (PK-C).

The binding mode of DAG-lactones to PK-C was investigated using the C1b domain from the X-ray structure of the phorbol ester/C1b complex of PK-C delta as a template. Modeling experiments revealed two binding alternatives in which one of the carbonyls of the DAG lactones remained uninvolved with the protein. Experimentally, however, the removal of either sn-1 or sn-2 carbonyls caused a dramatic drop in binding affinity towards PK-C. Although it was not possible to discriminate between the two binding alternatives of the DAG-lactones, the study demonstrates an important role for the additional carbonyl group. The function of this group could be equivalent to that of the C-9(OH)/C-13 (C = O) motif in phorbol esters, which also appears free of interactions in the phorbol ester/C1b complex. This role presumably reflects interaction with the phosholipid head groups required for high affinity binding under the conditions of the biological assays.

Diglycerides↗

PubMed1997

Checking the projection display of multivariate data with colored graphs.

Projection methods such as principal component analysis (PCA), nonlinear mapping (NLM), and the self-organizing map (SOM) are valuable algorithms for visualizing multidimensional data in a two-dimensional plane. Unfortunately, the reduction of the dimensionality involves distortions. In an attempt to graphically localize the distortions of the projected data, we suggest superposing colored graphs onto the 2D plots. The color of the edges of these graphs encodes the original high-dimensional distances between the connected points. The method is applied to a cluster analysis of 37 biologically active compounds and 471 molecules represented by a structural 3D descriptor.

Computer Graphics↗

PubMed1988