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 by targeting four critical components: (1) Protein-protein associations mediated by recognition and binding of src homology 2 (SH2) domains to phosphotyrosyl (pTyr) residues;(2) The removal of the pTyr phosphoryl group by cellular protein-tyrosine phosphatases (PTPs). (3) Src homology 3 (SH3) domain-mediated protein-protein associations;(4) Polobox binding domain of polo-like kinase 1. (1) SH2 Domain-Binding Inhibitors. High affinity growth factor receptor-bound protein 2 (Grb2)-binding antagonists are being prepared as potential new therapeutics for erbB-2 and c-Met dependent cancers. As part of a collaborative effort with NCI clinical investigators (Drs. Don Bottaro and Marston Linehan), our Grb2 signaling inhibitors are being examined in cellular studies, where certain of these agents have been shown to block hepatocyte growth factor (HGF)-induced cell migration in Met containing fibroblasts at nanomolar concentrations and to inhibit tubule formation potentially involved in angiogenesis. Using one of our agents, our collaborators have demonstrated inhibition of metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. This supports the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establishes a critical role for Grb2 SH2 domainmediated interactions in the metastatic process. (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 (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 identification of low micromolar affinity inhibitors that are undergoing further optimization. A parallel approach to inhibitor development is being conducted that relies on the optimization of YopH substrates to provide structural starting points for inhibitor development. The approach is unique in its use of nitrophenylphosphate substrates that allow the monitoring of substrate release by the simple measurement of yellow color derived reaction product nitrophenols. Final inhibitors are obtained by replacing the phosphate esters with hydrolytically-stable bioisosteres. This work has yielded low-micromolar non-promiscuous inhibitors. (3) SH3 Domain-Binding Inhibitors. More recently, we have undertaken the development of peptide-based inhibitors that block the critical association of Grb2 with its constitutive binding partner, Son-of-Sevenless (SOS). This work involves the synthesis of peptides and peptide mimetics that bind to the Grb2 Src homology 3 (SH3) domain. Ring-closing metathesis (RCM) has been used to prepare macrocyclic peptide that exhibit enhanced ability to block the formation of cognate Grb2-SOS complexes in cell lysates. (4) 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 a 5mer 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 a previously reported 5 - amino acid pThr containing peptide, through an iterative sequential process of structural refinement we have been able to increase the Plk1 PBD binding affinity by over 2 - orders of magnitude and show high selectivity for the Plk1 PBD without binding to the related Plk2 or Plk3 PBDs. Three distinct classes of high affinity-binding inhibitors were discovered, which contain new and as yet unreported 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 shown to reveal an entirely unanticipated mode of binding that has never been observed before. The work has resulted in the development of entirely new amino acid analogues and their application to three classes of selective, high affinity Plk1 PBD inhibitors. Unique binding modes exhibited by these inhibitors define an entirely new genre of PBD-binding interactions that should redefine the field of PBD-directed inhibitors. These compounds could potentially provide the basis for a new type anticancer chemotherapeutic. 5. Ubiquitin ligases (E3s) bind to specific target proteins and work with ubiquitin conjugating enzymes (E2s) resulting in the degradation of specific proteins. Many of these targets are important regulatory proteins implicated in diseases including cancer. In other cases these targets may prevent cancer or the spread of cancer. The human E3 gp78 works together with the E2 Ube2g2 to target proteins for degradation specifically from the endoplasmic reticulum. Among the proteins implicated as targets for gp78 are metastasis suppressors, proteins implicated in cystic fibrosis and emphysema, proteins implicated in neurodegenerative diseases and proteins that are associated with atherosclerosis. Our NCI collaborators (Drs. Andrew Byrd and Allan Weisman) have previously discovered a novel domain within gp78 (referred to as the G2BR) that binds specifically to Ube2g2 but not other E2s. The G2BR plays an important role in the activity of gp78, facilitating the degradation of gp78 target proteins. Based on the crystal structure-defined interaction between gp78 and Ube2g2 we have synthesized peptide mimetics intended to bind to the E2 in the same region as the G2BR, but which might either stimulate or inhibit activity. These peptides were designed to have enhanced alpha helical secondary structure in order to promote the helical structure observed for wild-type (WT) G2BR peptide bound to gp78 protein. Induction of helical structure was achieved in two fashions: (1) Conformational stabilization by ring-closing metathesis (RCM macrocyclization;referred to as """"""""hydrocarbon stapling"""""""") and (2) incorporation of additional amino acid residues at the C-terminus of the WT G2BR peptide that are known to favor alpha helical structure. This latter modification was based on the premise that nucleation of alpha helix formation at the C-terminus could promote helix formation in the remaining residues that had been shown to be crucial for p78 - G2BR binding. To date, this work has resulted in macrocyclic peptide constructs that exhibit enhanced gp78-binding affinity.
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