Conformationally Constrained Analogues of Diacylglycerol (DAG). 16. How Much Structural Complexity Is Necessary for Recognition and High Binding Affinity to Protein Kinase C?

Abstract

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...

Characterization of the Interaction of Ingenol 3-Angelate with Protein Kinase C

“…Kinase Assay. Activation of purified PKC-␣ and PKC-␦ was assayed by measuring the incorporation of 33 P from [␥-33 P 3000 Ci/mmol]ATP (ICN) into their specific peptide substrate as described previously (42). PKC-␣ activation was measured using an assay mixture (50 l) consisting of 20 mM Tris-HCl (pH 7.5), 0.25 mg/ml BSA, 7.5 mM magnesium acetate, 0.1 mM CaCl 2 , 100 g/ml phospholipid (phosphatidylserine:phosphatidylcholine, 20:80 w/w, prepared by sonication), 0.03% Triton X-100, 600 nM PKC Selectide peptide (Calbiochem), ATP (50 M final concentration, 1 Ci) and ingenol 3-angelate or PMA (20 nM-40 M) from appropriate DMSO stocks, the final concentration of the diluent not exceeding 0.2%.…”

Rational Design of Drugs That Induce Human Immunodeficiency Virus Replication

“…DAG lactones were synthesized and purified as described previously (30,36). 12-Deoxyphorbol 13-phenylacetate (DPP) was purchased from LC Laboratories (Woburn, Mass.).…”

“…DAG lactones, in which the entropic penalty for ligand binding is reduced by constraining the glycerol backbone into a five-member lactone ring, have proven to be especially potent and selective as antitumor agents (27,30,36). Recent studies have correlated various cellular effects of these DAG lactones, such as induction of apoptosis, to the specific activation of PKC isotypes and their translocation to different cellular compartments (13).…”

E230Q mutation of the catalytic subunit of cAMP‐dependent protein kinase affects local structure and the binding of peptide inhibitor

“…[22][23][24][25] However, PKI was substantially larger than the ligands used in the mentioned experiments. Furthermore, the dynamic snapshots of the C-subunit had high flexibility.…”

“…8 Kinetic data showed that the E230Q mutation affected the binding of ATP with only 2-fold perturbation but induced a 200-fold reduction in the binding of the peptide substrate, Kemptide. 8 Another important feature of the E230Q mutant is the loss of the synergistic binding of MgATP and protein kinase inhibitor PKI (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) to the mutant. The mutant was unable to form ternary complex crystals in the presence of MgATP and PKI.…”