Streptococcus mutans employs a key virulence factor, three glucosyltransferase (GtfBCD) enzymes to establish cariogenic biofilms. Therefore, the inhibition of GtfBCD would provide anti-virulence therapeutics. Here a small molecule library of 500,000 small molecule compounds was screened in silico against the available crystal structure of the GtfC catalytic domain. Based on the predicted binding affinities and drug-like properties, small molecules were selected and evaluated for their ability to reduce S. mutans biofilms, as well as inhibit the activity of Gtfs. The most potent inhibitor was further characterized for Gtf binding using OctetRed instrument, which yielded low micromolar KD against GtfB and nanomolar KD against GtfC, demonstrating selectivity towards GtfC. Additionally, the lead compound did not affect the overall growth of S. mutans and commensal oral bacteria, and selectively inhibit the biofilm formation by S. mutans, indicative of its selectivity and non-bactericidal nature. The lead compound also effectively reduced cariogenicity in vivo in a rat model of dental caries. An analog that docked poorly in the GtfC catalytic domain failed to inhibit the activity of Gtfs and S. mutans biofilms, signifying the specificity of the lead compound. This report illustrates the validity and potential of structure-based design of anti-S. mutans virulence inhibitors.

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Overexpression and activation of the murine double minute 2 (MDM2) or nuclear factor of activated T cells 1 (NFAT1) oncoproteins frequently occur in pancreatic cancer. Most MDM2 inhibitors under development target MDM2-p53 binding and have little or no effect on cancers without functional p53, including pancreatic cancer. Some available compounds indirectly inhibit NFAT1 activity by interfering with calcineurin activity, but there are currently no specific inhibitors against NFAT1. Here we performed a high-throughput virtual and cell-based screening to yield a lead compound (MA242) that can directly bind both MDM2 and NFAT1 with high affinity, induce their protein degradation, and inhibit NFAT1-mediated transcription of As a result of this binding, MA242 decreased cell proliferation and induced apoptosis in pancreatic cancer cell lines regardless of p53 status. MA242 alone or in combination with gemcitabine inhibited pancreatic tumor growth and metastasis without any host toxicity. Our data indicate that targeting both MDM2 and NFAT1 represents a novel and effective strategy to treat pancreatic cancer. These findings suggest that pharmacological inhibition of both MDM2 and NFAT1 is a promising strategy for the treatment of pancreatic cancer, even in tumors lacking functional p53. .

We report a novel delivery platform for a highly potent anticancer drug, 7-(benzylamino)-3,4-dihydro-pyrrolo[4,3,2-de]quinolin-8(1H)-one (BA-TPQ), using pH- and redox-sensitive poly(methacrylic acid) (PMAA) hydrogel cubes of micrometer size as the encapsulating matrix. The hydrogels are obtained upon cross-linking PMAA with cystamine in PMAA/poly(N-vinylpyrrolidone) multilayers assembled within mesoporous sacrificial templates. The BA-TPQ-loaded hydrogels maintain their cubical shape and pH-sensitivity after lyophilization, which is advantageous for long-term storage. Conversely, the particles degrade in vitro in the presence of glutathione (5 mM) providing 80% drug release within 24 h. Encapsulating BA-TPQ into hydrogels significantly increases its transport via Caco-2 cell monolayers used as a model for oral delivery where the apparent permeability of BA-TPQ-hydrogel cubes was ~2-fold higher than that of BA-TPQ. BA-TPQ-hydrogel cubes exhibit better anticancer activity against HepG2 (IC50=0.52 μg/mL) and Huh7 (IC50=0.29 μg/mL) hepatoma cells with a 40% decrease in the IC50 compared to the non-encapsulated drug. Remarkably, non-malignant liver cells have a lower sensitivity to BA-TPQ-hydrogel cubes with 2-fold increased IC50 values compared to those of cancer cells. In addition, encapsulating BA-TPQ in the hydrogels amplifies the potency of the drug via down-regulation of MDM2 oncogenic protein and upregulation of p53 (a tumor suppressor) and p21 (cell proliferation suppressor) expression in HepG2 liver cancer cells. Moreover, enhanced inhibition of MDM2 protein expression by BA-TPQ-hydrogel cubes is independent of p53-status in Huh7 cells. This drug delivery platform of non-spherical shape provides a facile method for encapsulation of hydrophobic drugs and can facilitate the enhanced efficacy of BA-TPQ for liver cancer therapy.

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