Structural bioinformatics studies of fluorinated balanol analogues as protein kinase C inhibitors

(-)-Balanol, a fungal natural product, is a non-selective inhibitor for protein kinase A (PKA) and protein kinase C (PKC) isozymes, binding to the adenosine triphosphate (ATP) site. Improving selectivity of balanol to a specific kinase is vital because most PKC isozymes can act as tumour promoters or suppressors depending on the cancer type. Designing selective inhibitors is a big challenge due to high conservation of ATP sites among PKC isozymes. Many structure-activity relationship studies on balanol have be en carried out but did not achieve substantial selectivity improvement. More recently, our group successfully performed stereospecific fluorinations on balanol and generated some analogues with an analogue showing improved selectivity for PKCε. This PKC isozyme is consistently implicated in tumorigenesis and a potential target for anticancer drugs. Thus, understanding the origin to this fluorine-based selectivity will be valuable for designing better balanol-based ATP-mimicking inhibitors. The overall objective of this thesis is to gain such understanding using structural b ioinformatics methods. Analysis data and conclusions drawn are documented to facilitate the design of potential novel balanol-based inhibitors. Molecular docking simulations of balanol and fluorinated balanol analogues (referred as balanoids) were carried out to the crystal structure of mouse PKA, which shares high sequence identity with human PKA , and to a homology model of PK Cε. These approaches provided information of static binding modes of balanoids in both kinases. This investigation provides preliminary results to decipher fluorine-based selectivity that stereospecific fluorination has local and remote effect, which is protein-dependent, to control balanol conformation in the homologous ATP sites. Molecular dynamics (MD) simulation approach can provide a deeper understanding of the binding of balanoids to PKA, PKCε, and other PKC isozymes since it allows the investigation of intermolecular interaction dynamics between the ligand and residues at the binding site. At the outset, a comprehensive exploration of the charge states of different balanoids was carried out. This study identified specific charge states of balanoids that correlate well with experiment and these charge states were adopted in subsequent MD simulations. Moreover, the study suggests that both fluorination and the local enzymatic environment of the ATP site can influence the charge states of balanol analogues. With these charge states, the sensitivity of PKCε over PKA toward stereospecific fluorination on balanol was investigated by MD simulations. The study showed that a single disparate residue situated in the ribosesubsite and near the a denine subsite, Thr 184 in PKA and Ala549 in PKCε, is crucial for global fluorine effect in the binding response of the two kinases to balanoids. Additionally, the study showed that an in variant Lys has a major contribution in the binding of balanoids to both kinases. The subsequent MD study addresses the improved binding affinity and selectivity of the C5( S )-fluorinated balanol analogue for PKCε over the other PKC isozymes. The study showed that although PKC isozymes share high residue conservation at their ATP sites, their unique dynamic features lead to di verse responses to this balanoid. The balanoid fits the dynamics of PKCε and yields optimal interactions leading to imp roved binding affinity and selectivity. The overall results provide a thorough understanding of how ste reospecific fluorination on balanol can improve binding affinity as well as selectivity to PKCε, which is useful for further development of balanol-based inhibitor targeting specif ic isozyme. Additionally, different dynamics on the homologous ATP sites of PKC isozymes is valuable information for designing selective inhi bitors for cancer therapy