Rational Design of Antitumor and Antiviral Agents Ultrapotent Ligands for PKC and Other Phorbol Ester Receptors

One of the principal goals of this research was understanding the geometry of bioequivalent pharmacophores-present in diacylglycerol (DAG) and in the more potent phorbol esters that appeared to be responsible for binding to the regulatory C1 domains of protein kinase C (PKC) and other target proteins. These domains, which act as a molecular hydrophobic switch, represent regulatory modules in several families of proteins involved in signal transduction, including the PKC, chimaerin, and RasGRP families. Many of the features that characterize the interaction between ligands and PKC can be understood at the level of the isolated C1 domain. A receptor-guided approach, which used as its basis the x-ray structure of the C1b domain of PKC-delta in complex with phorbol-13-O-acetate, helped us identify the 'recognition domain' for DAG, which consisted of a similar network of hydrogen bonds as those utilized by phorbol. The application of this model to DAGs and to the more potent DAG-lactones that were designed later suggested that both classes of compounds could bind to the C1 domain in two distinct orientations, so-called sn-1 and sn-2 orientations. Since in each of these orientations the hydrophobic groups or 'affinity domains' of the ligand are directed into rather different directions, the interactions of these groups with a cluster of conserved hydrophobic amino acids located on the top half of the C1 domain were incorporated into the model. The optimization of binding to this hydrophobic cluster produced ligands that displayed low nanomolar binding affinities for PKC and related C1 domain-containing protein targets. The first subnanomolar binding ligand for beta-2 chimaerin, which surpasses even the phorbol esters in binding potency, was developed. Furthermore, some of these novel ligands showed potent antitumor activities in the NCI 60-cell line in vitro screen. A combination of small library approaches and molecular dynamics simulations contributed to our understanding of the nature of these important hydrophobic interactions.

Conformationally Locked Nucleosides

Nucleosides and nucleotides are inherently flexible molecules. Hence, one of the main obstacles in interpreting structure-activity correlations has been this high level of flexibility. In 1993, our laboratory described the first synthesis of a conformationally locked bicyclo[3.1.0]hexane nucleoside. Although other types of locked nucleosides had been devised by others, only the bicyclo[3.1.0]hexane pseudosugar could successfully mimic both antipodal North and South conformations typical of conventional nucleosides. Significant differences in the biological activity of locked nucleoside antipodes, which depend on the precise interaction of these rigid molecules with specific target enzymes, had been clearly demonstrated. The principal theme in this area was the study of 'shape' in the recognition of individual nucleosides and nucleotides by specific enzymes (e.g., adenosine deaminase, reverse transcriptase, HSV-1 thymidine kinase, etc.). This concept was then extended to the study of DNA segments incorporating locked nucleotide units. It is well known that the binding of proteins to DNA elicits a unique conformational response of the double helix, which is tied to the function of the associated protein. Studies with cytosine (C5)-methyltransferase, an important enzyme in the control of gene expression, revealed important differences between target abasic sites locked in both antipodal conformations. Furthermore, the incorporation of conformationally locked nucleotides into DNA is a powerful tool to reinforce or disrupt typical B- or A-DNA forms associated with South and North conformations, respectively.