Inhibitors of Tyrosine Kinase-Dependent Signaling as Anti-Cancer Agents

Division Of Basic Sciences - Nci

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Abstract

Objective One: The Plk1 plays a central role in cell division and upregulation of Plk1 activity appears to be closely associated with aggressiveness and poor prognosis of several cancers. Targeting Plk1 may permit induction of cancer cell selective mitotic block and apoptotic cell death in Plk1 addicted cancers. However, a potential limitation of inhibitors directed at the Plk1 kinase domain (KD) may arise from a lack of specificity due to the high degree of similarity in the ATP binding clefts among kinases, particularly among other members of the Plk family (Plk1 to 5). Improving Plk1 specificity is one of the most pressing concerns to address to accomplish better clinical outcomes with less toxicological problems. In addition to its catalytic KD, Plk1 also contains a non catalytic polo box domain (PBD), which binds to the enzymes physiological substrates and localizes the enzyme to discrete locations within the kinetochore. Unlike ATP competitive inhibitors, whose specificities must be obtained against more than 500 other cellular kinases, PBD inhibitors target a structurally unique domain found in only four proteins (Plk1 to 3 and Plk5). Inhibition of Plk1 PBD function alone is sufficient for effectively imposing mitotic arrest and apoptotic cell death in cancer cells but not in normal cells and inhibitors of PBD binding interactions may serve as a target restricted strategy for developing anti Plk1 therapeutics. Starting from the 5 mer phosphopeptide PLHSpT and in collaboration with the NCI laboratory of Dr. Kyung Lee and the MIT laboratory of Dr. Michael Yaffe, we initially identified peptidic inhibitors that showed from 1000 to more than 10,000 fold improved PBD binding affinity. 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. The cryptic pocket is accessed by means of a phenylalkyl moiety attached to the N(pi) nitrogen of the His imidazole ring. Subsequently, we have optimized these PBD ligand interactions using an oxime ligation based strategy. Most recently, we have utilized on resin azide alkyne cycloaddition reactions to introduce 1,2,3 triazole functionality into potent lead Plk1 PBD inhibitors. The triazole rings were intended either to induce conformational constraint or to serve as His mimetics. Certain of these new ligands retain the high Plk1 PBD binding affinity of the parent peptide, while having enhanced selectivity for the PBD of Plk1 relative to the PBDs of Plk2 and Plk3. It is of note that certain peptides exhibit significantly greater than anticipated reduced affinities in full length Plk1 ELISA assays relative to values obtained with the isolated PBD (160 fold in one case and 480 fold in a second case). The larger differences may indicate a reduced ability of these triazole containing peptides to effectively relieve auto inhibition arising from interdomain interactions between the KD and PBD or to engage the PBD cryptic pocket in the full length construct. This may potentially indicate significant latitude in the structural interactions of the KD and PBD in full length Plk1. Objective Two: Protein tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosine (pTyr) residues within protein substrates. PTPases work in concert with protein tyrosine kinases to regulate signal transduction pathways. Because of the critical involvement of signal transduction pathways in regulating pathological processes in cancer and infectious diseases, PTPases have emerged as important targets for the development of therapeutic inhibitors. We have previously used the EGFR derived peptide VDADEpYL as a display platform for nonhydrolyzable pTyr mimicking residues, which led to the identification of the difluorophosphonomethyl aryl moiety as a starting point for the design of small molecule inhibitors. This moiety could be used to convert a good substrate into a high affinity inhibitor. Most recently, in collaboration with the USAMRIID laboratory of Dr. Robert Ulrich and the NCI laboratory of Dr. David Waugh, we have used this peptide sequence as a scaffold for presenting microarrayed libraries of druglike fragments to identify motifs for inhibitor design. The goal of this work was to devise a method for developing inhibitors of protein protein interactions (PPIs) that are intrinsic to PTPase catalytic specificities. A diversified library was created by incorporating 300 different druglike fragments at six different positions on the EGFR derived peptide. Detailed interactions with the substrate library were examined using PTPases from smallpox virus Variola major H1 (VH1), the plague bacillus Yersinia outer protein H (YopH), and the human dual specificity protein phosphatases (DUSP14 and DUSP22). In order to identify fragments that could be useful in inhibitor design, primary results were obtained from catalytic assays with the microarrayed library and kinetic binding data were obtained from the microarrayed library using a plasmon resonance (SPR) based microarray screen. As proof of principle, a high affinity oxime fragment identified by the two step catalytic and surface was employed to design low molecular weight, non phosphate containing peptides, which were able to inhibit PTP catalysis at low micromolar concentrations. Object Three: TDP1 removes DNA 3 end blocking lesions generated by chain terminating nucleosides and alkylating agents, and by base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors may potentially synergize with topoisomerase inhibitors (TOP1) to enhance selectivity and potency against cancer cells. In collaboration with the NCI laboratories of Dr. David Waugh and Dr. Yves Pommier, a crystallographic fragment screening campaign was performed against the catalytic domain of TDP1 to identify new lead compounds for the construction of TDP1 inhibitors. Crystal structures identified two fragments that bind to the TDP1 active site and exhibit measurable inhibitory activity against TDP1. The binding mode of these fragments is in a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, we prepared several fragment derivatives, some of which exhibited significantly higher TDP1 inhibitory potencies than the parent fragments. In a separate effort, in collaboration with the NCI laboratory of Dr. Jay Schneekloth, we performed a TDP1 small molecule microarray screen of over 20,000 drug like molecules to identify new TDP1 binding motifs. In collaboration with the laboratories of Drs. Pommier and Waugh, we are currently in the process of optimizing these initial leads.