Hydrogel-Encapsulated Biofilm Inhibitors Abrogate the Cariogenic Activity of Streptococcus mutans.

We designed and synthesized analogues of a previously identified biofilm inhibitor IIIC5 to improve solubility, retain inhibitory activities, and to facilitate encapsulation into pH-responsive hydrogel microparticles. The optimized lead compound HA5 showed improved solubility of 120.09 µg/mL, inhibited Streptococcus mutans biofilm with an IC50 value of 6.42 µM, and did not affect the growth of oral commensal species up to a 15-fold higher concentration. The cocrystal structure of HA5 with GtfB catalytic domain determined at 2.35 Å resolution revealed its active site interactions. The ability of HA5 to inhibit S. mutans Gtfs and to reduce glucan production has been demonstrated. The hydrogel-encapsulated biofilm inhibitor (HEBI), generated by encapsulating HA5 in hydrogel, selectively inhibited S. mutans biofilms like HA5. Treatment of S. mutans-infected rats with HA5 or HEBI resulted in a significant reduction in buccal, sulcal, and proximal dental caries compared to untreated, infected rats.

Structural studies of antitumor compounds that target the RING domain of MDM2.

Mouse double minute 2 homolog (MDM2) is an E3 ubiquitin-protein ligase that is involved in the transfer of ubiquitin to p53 and other protein substrates. The expression of MDM2 is elevated in cancer cells and inhibitors of MDM2 showed potent anticancer activities. Many inhibitors target the p53 binding domain of MDM2. However, inhibitors such as Inulanolide A and MA242 are found to bind the RING domain of MDM2 to block ubiquitin transfer. In this report, crystal structures of MDM2 RING domain in complex with Inulanolide A and MA242 were solved. These inhibitors primarily bind in a hydrophobic site centered at the sidechain of Tyr489 at the C-terminus of MDM2 RING domain. The C-terminus of MDM2 RING domain, especially residue Tyr489, is required for ubiquitin discharge induced by MDM2. The binding of these inhibitors at Tyr489 may interrupt interactions between the MDM2 RING domain and the E2-Ubiquitin complex to inhibit ubiquitin transfer, regardless of what the substrate is. Our results suggest a new mechanism of inhibition of MDM2 E3 activity for a broad spectrum of substrates.

Discovery of Potent Inhibitors of Streptococcus mutans Biofilm with Antivirulence Activity.

Dental caries is a bacterial infectious disease characterized by demineralization of the tooth enamel. Treatment of this disease with conventional antibiotics is largely ineffective as the cariogenic bacteria form tenacious biofilms that are resistant to such treatments. The main etiological agent for dental caries is the bacterium Streptococcus mutans. S. mutans readily forms biofilms on the tooth surface and rapidly produces lactic acid from dietary sucrose. Glucosyl transferases (Gtfs) secreted by S. mutans are mainly responsible for the production of exopolysaccharides that are crucial for the biofilm architecture. Thus, inhibiting S. mutans' Gtfs is an effective approach to develop selective biofilm inhibitors that do not affect the growth of oral commensals. Herein, we report a library of 90 analogs of the previously identified lead compound, G43, and exploration of its structure activity relationships (SAR). All compounds were evaluated for the inhibition of S. mutans biofilms and bacterial growth. Selected compounds from this library were further evaluated for enzyme inhibition against Gtfs using a zymogram assay and for growth inhibition against oral commensal bacterial species such as Streptococcus gordonii and Streptococcus sanguinis. This study has led to the discovery of several new biofilm inhibitors with enhanced potency and selectivity. One of the leads, III F1 , showed marked reduction in buccal, sulcal, and proximal caries scores in a rat model of dental caries.

MDM2-NFAT1 dual inhibitor, MA242: Effective against hepatocellular carcinoma, independent of p53.

The overexpression of the MDM2 oncoprotein frequently occurs in hepatocellular carcinoma (HCC). Small molecules that inhibit MDM2-p53 binding show efficacy against p53 wild-type HCC, but most patients have p53-mutant tumors and intrinsic resistance to such MDM2 inhibitors. We have recently discovered that the NFAT1 transcription factor upregulates MDM2 expression, but the role of NFAT1 in HCC is not fully understood. The present study was designed to develop a dual-targeting (MDM2 and NFAT1) strategy for the treatment of HCC. We herein demonstrate that high expression levels of NFAT1 and MDM2 are independent predictors of a poor prognosis in patients with HCC. We have also identified a MDM2 and NFAT1 dual inhibitor (termed MA242) that induces MDM2 auto-ubiquitination and degradation and represses NFAT1-mediated MDM2 transcription. MA242 profoundly inhibits the growth and metastasis of HCC cells in vitro and in vivo, independent of p53. The present efficacy and mechanistic studies provide proof-of-principle data to support the therapeutic value of this dual targeting strategy in future drug discovery.

Recent advances in the development of HBV capsid assembly modulators.

Hepatitis B virus (HBV) infections represent a significant burden on global public health. Current HBV treatments using nucleos(t)ide analogs (NAs) and PEG interferons cannot fully alleviate this burden as they do not affect the transcriptional activity of the tenacious covalently closed circular DNA (cccDNA) responsible for viral persistence. Capsid assembly modulators (CAMs) disrupt the encapsidation of pre-genomic RNA and can cause nucleocapsid disassembly, thereby affecting multiple steps of HBV replication and reduction of cccDNA pools. This review provides a concise overview of the development of CAMs and the progress achieved in understanding their interactions with HBV core proteins.

Discovery and Characterization of Dual Inhibitors of MDM2 and NFAT1 for Pancreatic Cancer Therapy.

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 MDM2 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.Significance: 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. Cancer Res; 78(19); 5656-67. ©2018 AACR.

Inhibition of Streptococcus mutans Biofilms by the Natural Stilbene Piceatannol Through the Inhibition of Glucosyltransferases.

Removal of oral biofilms involves the use of broad-spectrum antimicrobials, which eradicate both pathogenic and protective oral commensal species. Ideal therapeutics for dental caries should be able to selectively inhibit pathogenic biofilms caused by Streptococcus mutans. S. mutans extracellular glucosyltransferases (Gtfs), particularly GtfB and GtfC, synthesize predominantly water-insoluble glucans, which contribute to the structural scaffold of biofilms. The lead stilbene identified through our docking study against the catalytic domain of GtfC is a natural product known as piceatannol, which inhibited S. mutans biofilm formation in a dose-dependent manner, with considerable selectivity over growth inhibition of S. mutans and commensal streptococci. Binding kinetic analysis of piceatannol was performed using Octet RED against both GtfB and GtfC, which produced low micromolar KD values. Piceatannol inhibited S. mutans colonization in an in vivo drosophila model and a rat model of dental caries.

Structure-Based Discovery of Small Molecule Inhibitors of Cariogenic Virulence.

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.

Highly efficient delivery of potent anticancer iminoquinone derivative by multilayer hydrogel cubes.

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 (5mM) providing 80% drug release within 24h. 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=0.52µg/mL) and Huh7 (IC50=0.29µg/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. STATEMENT OF SIGNIFICANCE: Many potent anticancer drugs are hydrophobic and lack tumor selectivity, which limits their application in cancer therapy. Although cubical hydrogels of poly(methacrylic acid) exhibit excellent biocompatibility and versatility, they have not been investigated for hydrophobic drug delivery due to poor mechanical stability and incompatibility between hydrophobic drugs and a hydrophilic hydrogel network. In this study, we provide a facile method to prepare a multilayer hydrogel-based platform with controlled nanostructure, cubical shape and redox-responsiveness for delivery of highly potent anticancer therapeutics, hydrophobic BA-TPQ. The BA-TPQ-hydrogel cubes have exceptional structural stability upon lyophilization which is advantageous for a long-term storage. The greatly enhanced trans-epithelial permeability and amplified anti-tumor activity of BA-TPQ are achieved by encapsulation in these hydrogel cubes. Furthermore, the anticancer BA-TPQ-hydrogel platform retains the selective activity of BA-TPQ to hepatocellular carcinoma cells. Overall, the produced BA-TPQ-hydrogel cubes demonstrate a high potential for clinical liver cancer therapy.

Hydroxychalcone inhibitors of Streptococcus mutans glucosyl transferases and biofilms as potential anticaries agents.

Streptococcus mutans has been implicated as the major etiological agent in the initiation and the development of dental caries due to its robust capacity to form tenacious biofilms. Ideal therapeutics for this disease will aim to selectively inhibit the biofilm formation process while preserving the natural bacterial flora of the mouth. Several studies have demonstrated the efficacies of flavonols on S. mutans biofilms and have suggested the mechanism of action through their effect on S. mutans glucosyltransferases (Gtfs). These enzymes metabolize sucrose into water insoluble and soluble glucans, which are an integral measure of the dental caries pathogenesis. Numerous studies have shown that flavonols and polyphenols can inhibit Gtf and biofilm formation at millimolar concentrations. We have screened a group of 14 hydroxychalcones, synthetic precursors of flavonols, in an S. mutans biofilm assay. Several of these compounds emerged to be biofilm inhibitors at low micro-molar concentrations. Chalcones that contained a 3-OH group on ring A exhibited selectivity for biofilm inhibition. Moreover, we synthesized 6 additional analogs of the lead compound and evaluated their potential activity and selectivity against S. mutans biofilms. The most active compound identified from these studies had an IC50 value of 44µM against biofilm and MIC50 value of 468µM against growth displaying >10-fold selectivity inhibition towards biofilm. The lead compound displayed a dose dependent inhibition of S. mutans Gtfs. The lead compound also did not affect the growth of two commensal species (Streptococcus sanguinis and Streptococcus gordonii) at least up to 200µM, indicating that it can selectively inhibit cariogenic biofilms, while leaving commensal and/or beneficial microbes intact. Thus non-toxic compounds have the potential utility in public oral health regimes.

Cyanobacterial Metabolite Calothrixins: Recent Advances in Synthesis and Biological Evaluation.

The marine environment is host to unparalleled biological and chemical diversity, making it an attractive resource for the discovery of new therapeutics for a plethora of diseases. Compounds that are extracted from cyanobacteria are of special interest due to their unique structural scaffolds and capacity to produce potent pharmaceutical and biotechnological traits. Calothrixins A and B are two cyanobacterial metabolites with a structural assembly of quinoline, quinone, and indole pharmacophores. This review surveys recent advances in the synthesis and evaluation of the biological activities of calothrixins. Due to the low isolation yields from the marine source and the promise this scaffold holds for anticancer and antimicrobial drugs, organic and medicinal chemists around the world have embarked on developing efficient synthetic routes to produce calothrixins. Since the first review appeared in 2009, 11 novel syntheses of calothrixins have been published in the efforts to develop methods that contain fewer steps and higher-yielding reactions. Calothrixins have shown their potential as topoisomerase I poisons for their cytotoxicity in cancer. They have also been observed to target various aspects of RNA synthesis in bacteria. Further investigation into the exact mechanism for their bioactivity is still required for many of its analogs.

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model.

Recent advances in isolation, synthesis, and evaluation of bioactivities of bispyrroloquinone alkaloids of marine origin.

The ocean continues to provide a plethora of unique scaffolds capable of remarkable biological applications. A large number of pyrroloiminoquinone alkaloids, including discorhabdins, epinardins, batzellines, makaluvamines, and veiutamine, have been isolated from various marine organisms. A class of pyrroloiminoquinone-related alkaloids, known as bispyrroloquinones, is the focus of this review article. This family of marine alkaloids, which contain an aryl substituted bispyrroloquinone ring system, includes three subclasses of alkaloids namely, wakayin, tsitsikammamines A-B, and zyzzyanones A-D. Both wakayin and the tsitsikammamines contain a tetracyclic fused bispyrroloiminoquinone ring system, while zyzzyanones contain a fused tricyclic bispyrroloquinone ring system. The unique chemical structures of these marine natural products and their diverse biological properties, including antifungal and antimicrobial activity, as well as the potent, albeit generally nonspecific and universal cytotoxicities, have attracted great interest of synthetic chemists over the past three decades. Tsitsikammamines, wakayin, and several of their analogs show inhibition of topoisomerases. One additional possible mechanism of anticancer activity of tsitsikammamines analogs that has been discovered recently is through the inhibition of indoleamine 2, 3-dioxygenase, an enzyme involved in tumoral immune resistance. This review discusses the isolation, synthesis, and evaluation of bioactivities of bispyrroloquinone alkaloids and their analogs.

Antibacterial and Antibiofilm Activities of Makaluvamine Analogs.

Streptococcus mutans is a key etiological agent in the formation of dental caries. The major virulence factor is its ability to form biofilms. Inhibition of S. mutans biofilms offers therapeutic prospects for the treatment and the prevention of dental caries. In this study, 14 analogs of makaluvamine, a marine alkaloid, were evaluated for their antibacterial activity against S. mutans and for their ability to inhibit S. mutans biofilm formation. All analogs contained the tricyclic pyrroloiminoquinone core of makaluvamines. The structural variations of the analogs are on the amino substituents at the 7-position of the ring and the inclusion of a tosyl group on the pyrrole ring N of the makaluvamine core. The makaluvamine analogs displayed biofilm inhibition with IC50 values ranging from 0.4 µM to 88 µM. Further, the observed bactericidal activity of the majority of the analogs was found to be consistent with the anti-biofilm activity, leading to the conclusion that the anti-biofilm activity of these analogs stems from their ability to kill S. mutans. However, three of the most potent N-tosyl analogs showed biofilm IC50 values at least an order of magnitude lower than that of bactericidal activity, indicating that the biofilm activity of these analogs is more selective and perhaps independent of bactericidal activity.