Overexpression of the serine/threonine polo-like kinase 1 (Plk1) is tightly associated with oncogenesis in several human cancers. Interference with Plk1 function induces apoptosis in tumor cells but not in normal cells. Accordingly, Plk1 is a potentially attractive anticancer chemotherapeutic target. Plk1 possesses a unique phosphopeptide-binding polo box domain (PBD), which functions by recognizing and binding to to phosphothreonine (pT)/phosphoserine (pS)-containing protein sequences. This recognition and binding is essential for intracellular localization and mitotic functions of Plk1. Unlike kinase domains, PBDs are only found among the Plks. Therefore, PBDs represent attractive targets for selectively down-regulating Plk function. We have been engaged in efforts to develop Plk1 PBD-binding inhibitors starting from the 5-mer phosphopeptide PLHSpT. We have previously identified peptidic inhibitors that showed from 1000- to more than 10,000-fold improved PBD-binding affinity. In collaboration with Dr. Michael Yaffe (MIT), X-ray co-crystal structures of these peptides bound to Plk1 PBD indicated unanticipated modes of binding that take advantage of a cryptic binding channel that is not present in the non-liganded PBD or engaged by the parent pentamer phosphopeptide. However, critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pT/pS-residues within the binding sequences. Yet, the use of pT residues in therapeutics is potentially limited by hydrolytic lability of their phosphate groups to cellular phosphatases. Therefore, we developed a series of phosphatase-stable pT mimetics and found that when we incorporated these into peptides, that certain of the resulting peptides retained PBD-binding affinities, which In the best case, equaled the parent pT-containing peptides. Most recently, we have developed new, modified phosphatase pT-mimetics and incorporated them into peptides and found that we can achieve significantly higher PBD-binding affinities than the parent pT-containing peptides. In separate work, we have been able to optimize ligand interactions within the cryptic binding pocket and in so doing, achieve several-fold enhancement in PBD-binding affinities. We have also explored the application of conformational constraint (in which binding entropy penalties are reduced by reducing ligand flexibility). We synthesized several several macrocyclic peptides and some of these exhibit reduced overall anionic charge relative to the parent pT-containing peptides, while showing improved binding affinities. Work continues, with an objective to achieve PBD-binding inhibitors that exhibit potent effects in whole cell systems. In further work we are developing proteins that merge properties of antibodies with biologically active small molecules. This work is being done in collaboration with Dr. Christoph Rader (Scripps Florida). One aspect of our approach employs monoclonal antibodies and antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec) as well as catalytic antibodies, which can be selectively covalently modified by azetidinone and beta-diketone-containing drug payloads. The resulting antibody drug conjugates (ADCs) are directed against a variety of targets by changing the peptide or small molecule to which they are conjugated.
Showing the most recent 10 out of 48 publications
