Polo-like kinase 1 (Plk1) is one of the most attractive targets for anti-cancer therapy. Efforts to generate Plk1-specific inhibitors by targeting the catalytic activity of Plk1 have proven to be difficult due to similarities with the catalytic domains of other structurally related kinases. Here, we propose to develop a new class of mono-specific Plk1 inhibitors by employing a novel approach of targeting the non-catalytic, but functionally essential, PBD of Plk1. To this end, we have been taking advantage of the crystal structures of the Plk1 PBD in complex with a highly specific ligand, PLHSpT.
The first aim of the project is to develop PLHSpT-derived templates for drug design and subsequent modifications. Since the function of the N-terminal Pro-4 (number indicates the relative position of the residue from pT) residue of PLHSpT can be substituted by hydrophobic moieties and the side chain of the Leu-3 residue is not involved in interactions with surrounding PBD residues, we first generated N-substituted glycine (Nsg)-containing LHSpT or HSpT peptoidpeptide hybrids and their respective cyclic forms. Then, the Nsg residue of the hybrids was modified by covalently conjugating them with site-specifically synthesized hydrophobic moieties to fill in the intramolecular cavity present between the compound and the PBD. Since the His-2, Ser-1 and p-Thr residues are critical for the specificity and high affinity binding, these residues will not be modified. Peptide-derived inhibitors are commonly associated with problems in stability, lipophilicity, and transcellular permeability. Hence, the second aim of the project is to enhance the stability, membrane permeability, and tumor-specific targeting of the above compounds by generating innovative prodrugs. Selected compounds with a high PBD-binding affinity and specificity were converted to phosphatase-insensitive, non-hydrolyzable, p-Thr mimetic (Pmab) forms and then further modified to generate Ala- or Val-ester-conjugated phosphonic diamide prodrugs. The latter modification is not only to eliminate the electronegativity of the dianionic phosphonic acid moiety for better transcellular permeation but also to target the compound to the highly active PepT1 transporter for efficient cellular uptake. For the compounds that exhibit anti-Plk1 PBD activities at the cultured cell level, we plan to investigate whether the addition of a tumor-targeting RGD motif facilitates tumor-specific delivery of the compounds. As the third aim of the project, we will determine the potency and selectivity of the resulting compounds in the inhibition of Plk1-dependent cell proliferation activity in mouse tumor models. Because of the harsh chemical and enzymatic conditions of the gastrointestinal tract, intravenous injection into mouse-tail vein will be the choice of compound administration. Since peptide-derived inhibitors often exhibit physicochemical drawbacks, we also propose to take complementary approaches to isolate PBD-inhibitory compounds or moieties by screening the NCI natural products extract repository or by carrying out in silico screening. Structural analyses and computer modeling of the isolated small molecule compounds together with the above PLHSpT-derived inhibitors will allow us to perform site-specific replacements and modifications of the latter inhibitors to achieve enhanced in vivo stability and bioavailability. To this end, we will bring the expertise of our collaborators ranging from in vitro high throughput screening and cell-based assays, in silico screening and computer modeling, and X-ray crystallography. The ultimate goal of this multifaceted approach is to generate a new class of mono-specific anti-Plk1 therapeutic agents that have potential to treat various cancers in humans.
Showing the most recent 10 out of 18 publications
