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

Division Of Basic Sciences - Nci

Investigators

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Abstract

Objective One: 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. 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. We began this work by using 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. More recently, 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. The biological aspects of this work are being done in collaboration with the NCI laboratory of Dr. Yves Pommier. X-ray crystallographic studies were initially conducted in the NCI laboratory of Dr. Dave Waugh, but more recently this work has shifted to the FNLCR laboratory of Dr. George Lountos. The SMM work was done in collaboration with the NCI laboratory of Dr. Jay Schneekloth. During the reporting period we have elaborated the structure of the parent SMM-derived platform by adding functionality that extends into the peptide and DNA substrate binding regions. We employed 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 using a library of readily obtainable aldehyde reagents. This involved modifying our SMM-derived platforms by adding aminooxy handles to yield 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 were able to achieve TDP1 inhibitors with nanomolar potencies. We obtained the crystal structure of oxime-derived inhibitors bound to the TDP1 catalytic site and observed that they bind in a fashion that is similar to what was predicted by our molecular docking studies. Our current work is using sulfur (VI) fluoride exchange (SuFEx) biocompatible click chemistry reactions to prepare sulfonyl fluoride and fluorosulfate-containing covalent ligands designed to site-specifically target the Tyr204 residue in the catalytic site of TDP1. We have prepared and screened a small library of substituted quinolines with sulfonyl fluorides and fluorosulfate-containing tethers at the 8-position of a parent quinolone platform. Importantly, our cocrystal structures of TDP1 bound to these quinolones suggest covalent bond to the Tyr204 at the catalytic site of TDP1. Work is progressing to advance these agents. Object Two: The LGR5 potentiates canonical Wnt/B-catenin signaling. The well characterized deregulation of Wnt/B-catenin signaling that occurs during the adenoma-carcinoma sequence in CRC and further that specific subpopulations or molecules within a tumor may be therapeutically targeted to prevent relapse and induce long-term remissions. This renders LGR5 as a potential therapeutic target. The protein, R-spondin-1 (RSPO1) serves as a ligand that binds to the extracellular domain of LGR5. RSPO1 mimetics could potentially serve as affinity tags for targeting cancerous tissues during surgical resection of colon cancers. My laboratory is participating in a multi-party collaboration with Dr. Chris Albanese (Center for Translational Imaging, Georgetown University) to develop first-in-class RSPO1 peptide mimetics that can serve as vehicles to deliver near IR (NIR) dyes to light up cancerous tissues during surgical resection of colon cancers. My laboratory is designing and syntgesizing fluorescently tagged LGR5-binding peptide mimetics. Our initial design of RSPO-1 mimetics is based on the crystal structure of RSPO1 bound to LGR5 in a ternary complex with Ring Finger 43 (RNF43). This shows that the major binding interactions between RSPO1 and LGR5 occur via residues F106 and F110 within the bis-disulfide containing sequence K96-K113 located in its C-terminal Furin 2 Domain (FU2). We term this the "Right Side." Secondary binding is mediated by the R87 residue contained within residues P77-I95 in its mono-disulfide N-terminal Furin 1 Domain (FU1). We term this the "Left Side." Binding interactions of the "Right Side" 18-mer are particularly important and emphasis will be placed on optimizing synthesis of the right side by several approaches. The "Left Side" will be synthetically explored. Following optimization of the Right and Left Side peptides, these mimetics were connected to form bivalent constructs using tethering moieties. We have already developed solid-phase peptide synthesis (SPPS) chemistries to synthesize both the bis-disulfide containing 18-mer Right Side and mono-disulfide containing Left Side. We are using azide-alkyne click chemistry to join the constructs to form the entire "37-mer" RSPO1 mimetic, which contains the major RSPO1 binding interactions. Binding interactions of the 37-mer may be viewed as being "bivalent" in nature with the Left and Right Sides representing independent binding moieties that are connected by a bridging segment. The affinity of bivalent ligands can be significantly higher than the affinities of the isolated components. Specific binding of my RSPO1 mimetics to LGR5 is being confirmed by cell staining, knockdown, and competition assays.