Characterization of CDK(5) Inhibitor, 20-223 (aka CP668863) for Colorectal Cancer Therapy

Colorectal cancer (CRC) remains one of the leading causes of cancer related deaths in the United States. Currently, there are limited therapeutic options for patients suffering from CRC, none of which focus on the cell signaling mechanisms controlled by the popular kinase family, cyclin dependent kinases (CDKs). Here we evaluate a Pfizer developed compound, CP668863, that inhibits cyclin-dependent kinase 5 (CDK5) in neurodegenerative disorders. CDK5 has been implicated in a number of cancers, most recently as an oncogene in colorectal cancers. Our lab synthesized and characterized CP668863 – now called 20-223. In our established colorectal cancer xenograft model, 20-223 reduced tumor growth and tumor weight indicating its value as a potential anti-CRC agent. We subjected 20-223 to a series of cell-free and cell-based studies to understand the mechanism of its anti-tumor effects. In our hands, in vitro 20-223 is most potent against CDK2 and CDK5. The clinically used CDK inhibitor AT7519 and 20-223 share the aminopyrazole core and we used it to benchmark the 20-223 potency. In CDK5 and CDK2 kinase assays, 20-223 was ~3.5-fold and ~65.3-fold more potent than known clinically used CDK inhibitor, AT7519, respectively. Cell-based studies examining phosphorylation of downstream substrates revealed 20-223 inhibits the kinase activity of CDK5 and CDK2 in multiple CRC cell lines. Consistent with CDK5 inhibition, 20-223 inhibited migration of CRC cells in a wound-healing assay. Profiling a panel of CRC cell lines for growth inhibitory effects showed that 20-223 has nanomolar potency across multiple CRC cell lines and was on an average \u3e2-fold more potent than AT7519. Cell cycle analyses in CRC cells revealed that 20-223 phenocopied the effects associated with AT7519. Collectively, these findings suggest that 20-223 exerts anti-tumor effects against CRC by targeting CDK 2/5 and inducing cell cycle arrest. Our studies also indicate that 20-223 is a suitable lead compound for colorectal cancer therapy

Dimers of Isatin Derived Spirocyclic NF-κB Inhibitor Exhibit Potent Anticancer Activity by Inducing UPR Mediated Apoptosis

Activation of NFκB pathway has been implicated in several malignancies and plays a role in many key processes including tumor initiation and progression. The NFκB pathway is activated when TNFα in the tumor microenvironment binds to its receptor, eventually leading to the phosphorylation of the kinase IKKβ. Once active, IKKβ phosphorylates IκBα, a protein that functions to sequester NFκB in the cytosol of resting cells. The phosphorylation of IκBα leads to its degradation in the proteasome and allows NFκB to translocate to the nucleus where it can drive gene transcription. The sulfhydryl groups on solvent-exposed cysteine (Cys) residues of the key NFκB pathway proteins IKKβ and p65 serve as critical targets for NFκB pathway inhibition through covalent modification. The Natarajan lab previously reported an isatin-derived spirocyclic α-methylene-γ-butyrolactone analog 19, that covalently binds to IKKβ and p65 resulting in NFκB pathway inhibition. Analog 19 also showed synergistic tumor growth inhibition with cisplatin in an orthotopic ovarian cancer model without inducing any overt toxicity. To determine the proteome wide targets of analog 19, we conducted pull-down MS with alkyne-tagged 19 treated lysates. This identified \u3e100 proteins as potential targets of analog 19. Based on these results we hypothesized that dimers of analog 19 will crosslink proteins in the IKK complex and inhibit NFκB pathway. Synthesis and evaluation of the dimers showed that dimers n3 and n7 were more potent than analog 19 in inhibiting nuclear translocation of NFkB and the linker length contributed to their efficacy. The molecular basis for this was attributed to the formation of irreversible covalent bonds between the surface exposed cysteine residues of p65 and IKKβ and other proteins within the IKK complex resulting in higher molecular weight protein complexes. Analyses of the pull-down MS data also revealed that analog 19 binds to a number of proteins involved in protein processing in the ER and protein folding. This suggested that the dimers could induce the unfolded protein response (UPR). Indeed, the dimers induced UPR and activated the PERK/eIF2α/CHOP arm of the UPR leading to robust apoptotic cell death in the cancer cells when compared to immortalized non-transformed cells. The dimers were ~3-9 -fold more potent than the analog 19 in inhibiting cancer cell growth. The potency was modulated by the linker length. We also showed that like analog 19 the Michael acceptor in the dimers is critical for activity against cancer cells. In summary studies presented in this dissertation demonstrate that dimers of the isatin-derived spirocyclic NFκB inhibitor analog 19, potently inhibits cancer cell growth and induce apoptosis by crosslinking NFκB pathway proteins and inducing UPR