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

Division Of Basic Sciences - Nci

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Abstract

defined as a molecular target for anti-cancer therapy development. 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. This protein is overexpressed in many cancers and its inhibition can result in antiproliferative effects. Plk1 requires the coordinated actions of both an N-terminal kinase domain (KD), which executes its catalytic function and a C-terminal polo-box domain (PBD), which engages in protein - protein interactions (PPIs) with phosphoserine (pS) and phosphothreonine (pT)-containing sequences. Although Plk1 KD-directed agents are currently in clinical trials for the treatment of cancers, issues related to cytotoxicity have arisen that may result from off-target effects. Targeting protein - protein interactions (PPIs) has emerged as an important area for anticancer therapeutic development. In the case of phospho-dependent PPIs, such as the Plk1 PBD, a phosphorylated protein residue can provide high-affinity recognition and bind to target protein hot spots. 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 inhibitory peptides 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, which 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. Multivalency can be a powerful means to achieve highly potent and selective ligand-protein interactions. The selectivity and affinity of protein kinase (PK) inhibitors can be greatly increased by linking an element that binds within the ATP-binding cleft together with a component that binds exterior to the cleft. When the secondary component accesses ancillary regulatory domains, the resulting ligand may be described as being intramolecular "bivalent." We have undertaken work to develop bivalent ligands, designed to simultaneously engage both KD and PBD regions of Plk1. This has resulted in bivalent constructs exhibiting more than 100-fold Plk1 affinity enhancement relative monovalent PBD-binding ligands, which had until this time, exhibited among the highest PBD-binding affinities yet reported. Startlingly, and in contradiction to widely accepted notions of KD-PBD interactions, we have found that extremely high affinities can be retained even with minimal linkers between KD and PBD-binding components. In addition to significantly advancing the development of PBD-binding ligands, our findings may cause a rethinking of the structure-function of Plk1 and potential implications for the physiological roles played by this kinase. Objective Two: Tyrosyl-DNA phosphodiesterase 1 (TDP1) it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of TOP1 and the -phosphate of its DNA substrate. Blocking TDP1 function would be an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. TDP1 inhibitors would represent a new and potentially promising class of anticancer agents that could be used with TOP1 inhibitors in anticancer therapy. Although there have been reports of TDP1 inhibitors, there is a pressing need for the discovery of effective and specific TDP1 inhibitors for which there is validated binding and a defined mechanism of actions. In collaboration with the NCI laboratories of Dr. David Waugh and Dr. Yves Pommier, used an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. More recently, in collaboration with the NCI laboratory of Dr. Jay Schneekloth, we performed a TDP1 small molecule microarray screen of over 21,000 drug-like molecules in a small molecules microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified 109 hits from 21,000 compounds (0.5% hit rate) and arrived at a preferred TDP1-binding motif. Among the hits were structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines, which we demonstrated functioned as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke-Blackburn-Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. We are currently elaborating the structure of the parent SMM-derived platform by adding functionality that extends into the peptide and DNA substrate binding regions. We are using a "click"-based oxime diversification strategy that we have used successfully in several applications to optimize the binding interactions of parent ligands. A key to this approach is its ability to take a single synthetic parent construct and easily diversity it using a library of readily obtainable aldehyde reagents. In this work, we are modifying our SMM-derived platforms by adding aminooxy handles. This yielded two parent aminooxy-containing constructs. The benzoic acid moieties of these constructs are intended to bind within the catalytic site phosphoryl-binding pocket while the aminooxy groups are situated so that the resulting oxime derivatives would access the DNA or peptide substrate-binding channels. In this way, we were able to rapidly interrogate the structures of approximately 500 oxime derivatives. The most promising compounds (low micromolar IC50 values) were further derivatized to increase the chemical stability of the parent oxime linkages. Through this process, we have been able to achieve TDP1 inhibitors with nanomolar potencies. We have recently received the crystal structure of oxime-derived inhibitors bound to the TDP1 catalytic site and it appears that they bind in a fashion that is similar to what was predicted by our molecular docking studies. Going forward our goal is to derive validated inhibitors with defined binding interactions. These inhibitors will provide pharmacological tools for studying the biochemical effects of competitively inhibiting TDP1 function in cellular settings. Our work should advance the general field of TDP1 inhibitor development. A PCT patent application has recently been filed covering aspects of this technology.