Targeting Protein- Kinase (PK)-Dependent Signaling: Aberrant PK-depenent signaling is associated with the etiology of several cancers. For this reason, pharmacological agents are being developed to modulate kinase-dependent signaling as potential new anticancer therapeutics. We are developing PK-dependent signaling inhibitors that function by: (1) Blocking protein-protein associations mediated by recognition and binding of the polobox binding domain (PBD) of polo-like kinase 1 (Plk1)to phosphothreonine (pThr)/phosphoserine (pSer)-containing protein sequences and (2) Blocking the removal of phosphotyrosyl (pTyr) phosphoryl groups by cellular protein-tyrosine phosphatases (PTPs).(1) Polo-like Kinase 1 (Plk1) Polo Box Domain Binding Inhibitors: 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 phosphopeptidebinding polo box domain (PBD) that is essential for its intracellular localization and mitotic functions. Unlike kinase domains, PBDs are found only in the four members of Plks. Therefore, they represent ideal targets for selectively inhibiting the function of Plks. By examining various PBD-binding phosphopeptides, our NCI collaborator, Dr. Kyung Lee, previously found that the 5-mer phosphopeptide PLHSpT specifically interacts with the Plk1 PBD with high affinity, whereas it fails to significantly interact with the PBDs of two closely-related kinases, Plk2 and Plk3. Starting from this peptide, we employed an iterative sequential process of structural refinement to arrive at new agents, which bind with high affinity to the Plk1 PBD. In collaboration with Dr. Thorsten Berg (The University of Leipzing, Germany), we have been able to show that the Plk1 PBD binding affinity is increased by over 3 - orders of magnitude, while retaining high selectivity for the Plk1 PBD relative to the related Plk2 or Plk3 PBDs. Three distinct classes of high affinity-binding inhibitors were discovered, which contain new amino acid analogues. In collaboration with Dr. Michael Yaffe (MIT) X-ray co-crystal structures of these peptides bound to Plk1 PBD protein were solved. These reveal an unanticipated mode of binding. The work resulted in the development of new amino acid analogues and their application to three classes of selective, high affinity Plk1 PBD inhibitors. The binding modes exhibited by these inhibitors define a new genre of PBD-binding interactions that could greatly impact the field of PBD-directed inhibitors. Further work has been directed at modification of the pThr/pSer residues, which form key components of ligand recognition. Although critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues, the use of these residues in therapeutics is potentially limited by their lability in the presence of cellular phosphatases and by their poor cellular uptake due to their high anionic charge. To date, there has been little examination of pThr/pSer replacements within a PBD context. Accordingly, we have examined the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements. This work has shed new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics.(2) PTP Inhibitors: Synthetic small molecule inhibitors are being developed against the YopH PTP, which is a pathogenic component of the potential bioterrosim agent Yersinia pestis. This work is being done in collaboration with Drs. Robert Ulrich (USAMRIID) and David Waugh (CCR, NCI). A focused library approach has been used wherein two aromatic fragments are joined together by a series of linker segments. This has led to the development of a low-nanomolar non-promiscuous inhibitors that showed significant inhibition of intracellular Y. pestis replication at a non-cytotoxic concentration. To compliment this 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 (CCR, NCI). Our approach employs antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec), which are directed against a variety of targets by changing the peptide or small molecule to which they are conjugated. In one aspect of our work, we are employing click chemistry to attach biologically-cleavable linkers that allow release of cargo once delivery to the target has been achieved. We have developed versatile hetero-bifunctional linkers that are compatible with multiple types of Cu-free click reagents and that incorporate biologically cleavable bonds. These linkers contain both targeting functionality and drug payloads. In one aspect of our work involving the potently cytotoxic peptide, monomethyl auristatin F (MMAF), we are examining bio-cleavable linkers, which can be conjugated to the Fc-Sec protein by nucleophilic alkylation reactions. This work has involved developing new synthetic routes to key components of the MMAF peptide.
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