Alzheimer disease (AD) is the most common neurodegenerative disease, and with Americans' increasing longevity, it is becoming an epidemic. There are currently no effective treatments for this ...disorder. Abnormalities of Tau track more closely with cognitive decline than the most studied therapeutic target in AD, amyloid-β, but the optimal strategy for targeting Tau has not yet been identified. On the basis of considerable preclinical data from AD models, we hypothesize that interactions between Tau and the Src-family tyrosine kinase, Fyn, are pathogenic in AD. Genetically reducing either Tau or Fyn is protective in AD mouse models, and a dominant negative fragment of Tau that alters Fyn localization is also protective. Here, we describe a new AlphaScreen assay and a live-cell bioluminescence resonance energy transfer (BRET) assay using a novel BRET pair for quantifying the Tau-Fyn interaction. We used these assays to map the binding site on Tau for Fyn to the fifth and sixth PXXP motifs to show that AD-associated phosphorylation at microtubule affinity regulating kinase sites increases the affinity of the Tau-Fyn interaction and to identify Tau-Fyn interaction inhibitors by high-throughput screening. This screen has identified a variety of chemically tractable hits, suggesting that the Tau-Fyn interaction may represent a good drug target for AD.
A series of N-pyridyl benzamide KCNQ2/Q3 potassium channel openers were identified and found to be active in animal models of epilepsy and pain. The best compound 12 ICA-027243, ...N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide has an EC50 of 0.38 μM and is selective for KCNQ2/Q3 channels. This compound was active in several rodent models of epilepsy and pain but upon repeated dosing had a number of unacceptable toxicities that prevented further development. On the basis of the structure−activity relationships developed around 12, a second compound, 51, N-(2-chloro-pyrimidin-5-yl)-3,4-difluoro-benzamide, ICA-069673, was prepared and advanced into a phase 1 clinical study. Herein, we describe the structure−activity relationships that led to the identification of compound 12 and to the corresponding pyrimidine 51.
A scalable process for the manufacture of a potassium ion channel blocker was developed and optimized. Key features of the process include an optimized Grignard reaction, a direct cyanation of the ...intermediate trityl alcohol derivative, and an improved nitrile hydrolysis protocol, relative to the original acidic hydrolysis conditions, to generate the crude active pharmaceutical ingredient (API) with >95% HPLC purity. The Grignard and the cyanation reactions could be telescoped, resulting in an improved throughput compared to the original four-step process. An effective recrystallization of the API was also developed and the process scaled up to manufacture multiple batches at the pilot scale.
Alphaviruses are arthropod-transmitted members of the Togaviridae family that can cause severe disease in humans, including debilitating arthralgia and severe neurological complications. Currently, ...there are no approved vaccines or antiviral therapies directed against the alphaviruses, and care is limited to treating disease symptoms. A phenotypic cell-based high-throughput screen was performed to identify small molecules that inhibit the replication of Venezuelan Equine Encephalitis Virus (VEEV). The compound, 1-(2,3-dihydrobenzob1,4dioxin-6-yl)-N-(3-fluoro-4-methoxybenzyl)ethan-1-amine (1), was identified as a highly active, potent inhibitor of VEEV with an effective concentration for 90% inhibition of virus (EC90) of 0.89 μM and 7.49 log reduction in virus titers at 10 μM concentration. These data suggest that further investigation of compound 1 as an antiviral therapeutic against VEEV, and perhaps other alphaviruses, is warranted. Experiments suggested that the antiviral activity of compound 1 is directed at an early step in the VEEV replication cycle by blocking viral RNA and protein synthesis.
Abstract
Resistance to radiation and chemotherapy in colorectal patients is considered one of the major contributors of refractory disease and disease progression. Dysregulation or aberrant ...activation of the SHH/GLI1 signaling pathway, common to both colorectal cancer and other cancer subtypes, influences the integrity of the DNA damage response (DDR) pathway, which may impact tumor response to chemo- and radiation therapies. The DDR pathway is initiated in part by the recognition of a DNA lesion by a sensor protein and the recruitment NBS1 to that lesion. Subsequent repair is initiated by the Mre11, Rad50, NBS1 (MRN) complex formation. In this study, we investigated the expression of GLI1 and NBS1 in tissues samples from 188 patients diagnosed with colorectal cancer by immunohistochemistry and analyzed the clinical significance and prognostic relevance of elevated GLI1-NBS1 co-expression. GLI1 expression was positively associated with the NBS1 expression, and high expression of both in the biopsied specimens significantly correlated with poor patient survival. The result of a series of biochemical experiments directly links GLI1 transcriptional activity to the DNA damage repair pathway. First, NBS1 mRNA levels and protein were found to be reduced upon treatment with GANT61, a GLI inhibitor, indicating that GLI activity was required for NBS1 transcription. Second, overexpression of NBS1 in HT29 cells rescued GANT61-induced cell death by measure of cleaved caspase-3, indicating a significant role of induced DNA damage in the mechanism of GANT61-induced cell death in oncogenic GLI1 cancers. Third, this was verified by ChIP analysis of GLI1 binding to the NBS1 promotor region containing a putative GLI binding sequence. The efficacy of GLI1 inhibition as therapeutic strategy was then tested in parallel with 5-Fluorouracil (5-FU), a component of the standard-of-care therapy FOLFOX. In vitro, colorectal cell lines with GLI1-driven NBS1 expression demonstrated strong 5-FU resistance. These cells were re-sensitized to 5-FU by silencing the expression of NBS1 with siRNA, or with combination therapy involving 5-FU and GANT61. This suggests that a first-in class chemotherapeutic strategy may provide additional treatment options for patients demonstrating 5-FU resistance during the course of their chemotherapy. While GANT61 is commonly used to inhibit GLI in vitro, it exhibits poor pharmacokinetic properties and is not a viable drug for the clinic. To that end, we present a novel GLI1 antagonist SR38832, which exhibits a more stable pharmacokinetic profile than GANT61 in xenograft mouse models and lower IC50 in colon cancer cell lines. In a human colorectal cancer xenograft mouse model, SR38832 significantly inhibited both tumor growth and proliferation. Not only has the identification of SR38832 provided an in vivo tool to study GLI oncogenic activity, but SR38832 is also structural lead to be optimized through an iterative medicinal chemistry approach.
Citation Format: Ruowen Zhang, Justin Avery, Jinlu Ma, Theresa Nguyen, Mark J Suto, Bo Xu, Rebecca J Boohaker. Targeting GLI1-mediated NBS1 transcription overcomes 5-FU resistance abstract. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A103. doi:10.1158/1535-7163.TARG-19-A103
We investigated the structure−activity relationship studies of N-3,5-bis(trifluoromethyl)phenyl2-chloro-4-(trifluoromethyl)pyrimidin-5-ylcarboxamide (1), an inhibitor of transcription mediated by ...both NF-κB and AP-1 transcription factors, with the goal of improving its potential oral bioavailability. Compounds were examined for cell-based activity, were fit to Lipinski's rule of 5, and were examined for potential gastrointestinal permeability using the intestinal epithelial cell line, Caco-2. Selected groups were substituted at the 2-, 4-, and 5-positions of the pyrimidine ring using solution-phase combinatorial methodology. The introduction of a fluorine in the place of 2-chlorine of 1 resulted in a compound with comparable activity. However, other substitutions at the 2-position resulted in a loss of activity. The trifluoromethyl group at the 4-position could be replaced with a methyl, ethyl, chlorine, or phenyl without a substantial loss of activity. The carboxamide group at the 5-position is critical for activity. If it was moved to the 6-position, the activity was lost. The 2-methyl analogue of 1 (81) showed comparable in vitro activity and improved Caco-2 permeability compared to 1.
Abstract
Aberrant activation of Wnt/β-catenin signaling is a necessary initiating event in the genesis of most colorectal cancers. Loss-of-function mutations in the tumor suppressor gene adenomatous ...polyposis coli (APC) are present in about 80% of all colorectal cancers, and gain-of-function mutations in the oncogene CTNNB1 (β-catenin encoding gene) exist in approximately 10% of colorectal cancers. Both APC and CTNNB1 mutations lead to cytosolic accumulation and nuclear translocation of β-catenin, and therefore constitutive activation of β-catenin/TCF4-mediated gene transcription. As such, the Wnt/β-catenin pathway has emerged as one of the most promising targets for colorectal cancer treatment. Despite tremendous efforts in the past decade, there are no small molecule Wnt inhibitors approved by the FDA for cancer treatment. Recent work in our laboratories has identified a series of benzimidazoles as potent Wnt/β-catenin inhibitors. Here, we show that several benzimidazoles displayed strong activities against Wnt/β-catenin signaling in colorectal cancer cells harboring mutations in CTNNB1 or APC. In particular, SRI36324, the lead compound in this series, inhibited Wnt/β-catenin signaling in colorectal cancer HCT116 and DLD-1 cells with IC50 values of 4.1 and 3.7 nM, respectively. Moreover, the benzimidazole compounds exhibited potent activities against colorectal cancer cell proliferation under both standard cell culture conditions (adherent cells in complete medium containing 10% FBS) and conditions designed to enrich for cancer initiating cells. In addition, the benzimidazole compounds had no off-target effects on other pathways (e.g., STAT3 and mTORC1 signaling) in colorectal cancer cells, and were less antiproliferative to non-cancerous cells. Together, our findings indicate that the benzimidazole compounds are promising candidates for development as novel therapeutic agents for colorectal cancer.
Note: This abstract was not presented at the meeting.
Citation Format: Yonghe Li, Patsy G. Oliver, Wenyan Lu, Vibha Pathak, Corinne E. Augelli-Szafran, Donald J. Buchsbaum, Mark J. Suto. Discovery of a series of benzimidazoles as potent Wnt/β-catenin signaling inhibitors in colorectal cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5173. doi:10.1158/1538-7445.AM2017-5173
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