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

Division Of Basic Sciences - Nci

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Abstract

Objective One: Targeting protein - protein interactions (PPIs) has emerged as important area of discovery for anticancer therapeutic development. Developing antagonists of the Plk1 PBD can be particularly challenging if one relies solely on interactions within and proximal to the phospho-binding pocket. Fortunately, the affinity of phospho-dependent PPI antagonists can be significantly enhanced by taking advantage of interactions in both the phospho-binding site and hidden cryptic pockets that may be revealed on ligand binding. 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. More recently, we have designed and synthesized macrocyclic peptide mimetics directed against the Plk1 PBD, which are characterized by a new glutamic acid analog that simultaneously serves as a ring-closing junction that provides accesses to a cryptic binding pocket, while at the same time achieving proper orientation of a pT residue for optimal interaction in the signature phospho-binding pocket.. It is noteworthy that this new glutamic acid-based amino acid analog represents the first example of extremely high affinity ligands where access to the cryptic pocket from the pT-2 position is made possible with a residue that is not based on histidine. The concepts employed in the design and synthesis of these new macrocyclic peptide mimetics should be useful for further studies directed against the Plk1 PBD and potentially for ligands directed against other PPI targets. Objective Two: Tyrosyl-DNA phosphodiesterase 1 (TDP1) t is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. Blocking TDP1 function is a potentially attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. In collaboration with the NCI laboratories of Dr. David Waugh, Dr. Yves Pommier and Dr. Jay Schneekloth, we performed a Tdp1 small molecule microarray screen of over 20,000 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. These included 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. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1. However, unlike simpler structures obtained in the earlier fragment screens, these more complex inhibitors orient distinct structural components into both the DNA and peptide substrate-binding regions. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors. Objective Three: Antibody-drug conjugates (ADCs) constitute an important and emerging class of therapeutics. We have a have a long-standing collaboration with the laboratory of Dr. Christoph Rader (Scripps Florida) to develop antibody-drug conjugates (ADCs). One aspect of our collaboration concerned chimeric antigen receptor T cells (CAR-Ts). These constitute a promising class of cancer immunotherapeutic that link antibody-mediated major histocompatibility complex (MHC)-independent recognition of cancer cell surface antigens to the power of T-cell-mediated killing. CAR-Ts are constructed by transducing autologous T cells from cancer patients with chimeric antigen receptors that fuse an extracellular antibody fragment, typically a scFv, to a transmembrane segment, followed by the cytoplasmic signaling domain of a T cell costimulatory receptor and the cytoplasmic signaling domain of the T-cell receptor complex. As such, a CAR-T links antibody-mediated binding to T-cell activation. A major challenge in the development of CAR-Ts is the identification of cell surface receptors that are selectively expressed on cancer cells and can serve as targets for CAR-Ts that do not harm healthy cells and tissues. In this regard, the pocketome of cancer cells comprises thermodynamically favored small molecule binding sites that afford a vast targetable and druggable space that is only accessible to small molecules. However, compared to antibodies, small molecules have inappropriate pharmacokinetic and pharmacodynamic properties for cancer immunotherapy. Chemically programmed bispecific antibodies ae able to address these shortcomings by utilizing small molecules to recruit and activate CAR-Ts. In collaboration with Dr. Rader, we employed a CAR-T platform that uses a chemically programmed antibody fragment (cp-Fab) as on/off switch. In proof-of-concept studies. We found that this cp-Fab/CAR-T system targeting folate binding sites mediated potent and specific eradication of folate receptor-expressing cancer cells in vitro and in vivo. The method harnesses an increasing quantity and quality of cell surfaceome-targeting small molecules derived from virtual and actual small molecule libraries. A second aspect of our collaboration with Dr. Rader utilized a uniquely reactive lysine residue (Lys99) for site-specific attachment of small molecules in the humanized catalytic antibody h38C2. This residue has been used as bioconjugation module for the assembly of chemically programmed antibodies and antibody-drug conjugates. Treatment of h38C2 with beta-lactam-functionalized small molecules has been previously shown to result in covalent conjugation by selective formation of a stable amide bond with the epsilon-amino group of the Lys99 residue. Recently we engaged with Dr. Rader to develop heteroaryl methylsulfonyl-functionalized small molecules that provide an alternative bioconjugation strategy through highly efficient, site-specific and stable arylation of the Lys99 residue of the h38C2. We utilized these constructs to prepare a set of chemically programmed antibodies and antibody-drug conjugates assembled by Lys99 arylation and used these to provide proof-of-concept for the therapeutic utility of this alternative bioconjugation strategy. Having two equally effective methods in place affords additional options for the utilization of catalytic antibody h38C2 as bioconjugation module.