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

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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. In addition to its catalytic KD, Plk1 also contains a non-catalytic polo-box domain (PBD), which binds to the enzyme physiological substrates and localizes the enzyme to discrete locations within the kinetochore. PBD inhibitors target a structurally unique domain found in only four proteins (Plk1-3 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. In further work we discovered chemistry to install functionality at the His N(tau)-nitrogen using phospho-directed on-resin Mitsunobu alkylation conditions to produce peptidomimetics containing N(pi),N(tau)-bis-alkylated His residues. Importantly, the cationic bis-alkyl imidazolium species may increase membrane penetration via intramolecular "charge masking" of the anionic phosphate moiety. The X-ray co-crystal structures of bis-alkylated His-containing peptides bound to the PBD indicated that the His N(tau)-nitrogen is within 6 angstroms of the C-terminal carboxamide. We envisioned cyclic ligands could be prepared in which the N(pi),N(tau)-bis-alkylated His could serve as a bifurcated ring junction. We employed methylene linkers of various size between the N(tau)-nitrogen of imidazole and the C-terminus, utilizing an amide forming macrocyclization reaction. We eventually found that we were able to achieve a tripeptide macrocycle that retained high PBD-binding affinity. Inhibition data from the bis-alkyl His-containing cyclic ligands suggested that cyclization between the C-terminus and the pThr(-2) position is beneficial to activity. With this in mind, we investigated new functionality at the pThr(-2) position that could incorporate the -(CH2)8Ph required for high-affinity ligands and also an orthogonally-protected functional group for on-resin macrocyclization. We developed a new non-His-based amino acid that could serve as a macrocycle ring junction while accessing the critical cryptic pocket. Importantly, this new amino acid analog could be incorporated into SPPS on Rink resin to produce macrocyclic ligands that retained high PBD-binding affinities. In further work, we designed a series of probes based on the active pharmacophore of the Plk1 kinase inhibitor, BI2536 tethered to a fluorescent moiety. The probes provided a fluorescence-based measure of binding affinity, which could be used to determine the affinities of candidate Plk1 kinase inhibitors. We found that the assays were able to provide IC50 values of type 1 kinase inhibitors (inhibitors that compete with ATP-binding in the active conformation of the catalytic pocket) that are in accordance with values obtained from enzymatic assays. However, the probe was insensitive to other classes of kinase inhibitors. This rendered it potentially useful in conjunction with enzymatic kinase assays to distinguish type 1 inhibitors from these other classes of inhibitors. The assay may afford a facile means for initial screening of type 1 ATP-competitive Plk1 inhibitors that offers distinct advantages over kinase assays in terms of cost, speed and ease of handling. Objective Two: Tdp1 removes DNA 3-prime 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. Using structural insights into fragment binding, we prepared several fragment derivatives, some of which exhibited significantly higher Tdp1 inhibitory potencies than the parent molecules. 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. We identified 109 hits from 21,000 compounds (0.5% hit rate) and arrived at a preferred Tdp1-binding motif. Further structure activity relationship (SAR) work achieved a class of small molecules that showed low micromolar Tdp1-inhibitory potencies. X-ray co-crystal structures in the Waugh Laboratory showed that the promising leads bind at the catalytic site of Tdp1. In agreement with previous crystal structures of Tdp1-bound phthalic acid-containing fragments, these structures confirmed that the bis-carboxylic moieties recapitulate aspects of phosphate binding to the key catalytic residues. 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. These are the first crystal structures of small molecules accessing the catalytic site as well as both the DNA substrate and peptide-binding regions. Objective Three: Antibody-drug conjugates (ADCs) constitute an important and emerging class of therapeutics. In a fourth area of research focus, we have a have a long-standing collaboration with the laboratory of Dr. Christoph Rader (Scripps Florida) to develop antibody-drug conjugates (ADCs). This capitalizes on our expertise in small molecule and peptide mimetic chemistry. Aspects of our approach employ monoclonal antibodies and antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec). In other work, we use the "catalytic antibody" h38C2 to effect selective covalent conjugation using azetidinone and beta-diketone-containing drug payloads. We are also contributing to the development of a platform of chemically programmed bispecific antibodies (biAbs). These endow target cell-binding small molecules with the ability to recruit and redirect cytotoxic T cells to eliminate cancer cells. In collaboration with Dr. Adrian Wiestner (NHLBI), we have developed a novel strategy for targeted cytotoxic therapy of chronic lymphocytic leukemia (CLL). This employs IgM-based Fc(mu)R-targeted antibody-drug delivery to effect potent and specific therapeutic activity against CLL. In parallel, we are using chimeric antigen receptor ("CAR")-engineered T cells that include the hapten-binding site of h38C2. These are being chemically programmed through covalent binding of the reactive h38C2 Lys residue with 1,3-diketone or beta-lactam moieties tethered by variable PEG spacers to cyclic RGDfK groups. This endows the CARs with the ability to bind to human integrin alpha4,beta3 with high affinity and specificity. These are anticipated to kill human integrin alpha4,beta3 - expressing tumor cells and tumor endothelial cells. We have also examined trifunctional folate receptor 1 (FOR1)-targeting moieties.