Development and validation of a sensitive LC-MS/MS assay of GT-14, a novel Gαi2 inhibitor, in rat plasma, and its application in pharmacokinetic study.

A sensitive and selective LC-MS/MS method was developed and validated for the quantitation of a novel Gαi2 inhibitor, GT-14, in rat plasma using a SCIEX 6500+ triple QUAD LC-MS system equipped with an ExionLC UHPLC unit. GT-14 (m/z 265.2 → 134.1) and griseofulvin (Internal Standard, IS) (m/z 353.1 → 285.1) were detected in a positive mode by electrospray ionization (ESI) using multiple reaction monitoring (MRM). The assay was linear in the concentration range of 0.78-1000 ng/mL in rat plasma. Both accuracy and precision values were within the acceptance criteria of ±15 %, as established by FDA guidance. The matrix effect was negligible from plasma, with signal percentages of 98.5-106.9 %. The mean recovery was 104.5 %, indicating complete extraction of GT-14 from plasma. GT-14 was found to be stable under different experimental conditions. The validated method was successfully applied to evaluate plasma protein binding and in vivo pharmacokinetics of GT-14 in rats.

A Novel Liver Cancer-Selective Histone Deacetylase Inhibitor Is Effective Against Hepatocellular Carcinoma and Induces Durable Responses with Immunotherapy.

Hepatocellular cancer (HCC) progression is facilitated by gene-silencing chromatin histone hypoacetylation due to histone deacetylases (HDACs) activation. However, inhibiting HDACs, an effective treatment for lymphomas, has shown limited success in solid tumors. We report the discovery of a class of HDAC inhibitors (HDACi) that demonstrates exquisite selective cytotoxicity against human HCC cells. The lead compound STR-V-53 (3) showed favorable safety profile in mice and robustly suppressed tumor growth in orthotopic xenograft models of HCC. When combined with the anti-HCC drug sorafenib, STR-V-53 showed greater in vivo efficacy. Moreover, STR-V-53 combined with anti-PD1 therapy increased the CD8+ to regulatory T-cell (Treg) ratio and survival in an orthotopic HCC model in immunocompetent mice. This combination therapy resulted in durable responses in 40% of the mice. Collectively, our data demonstrate that the novel HDACi STR-V-53 is an effective anti-HCC agent that can induce profound responses when combined with standard immunotherapy.

Antiandrogen-Equipped Histone Deacetylase Inhibitors Selectively Inhibit Androgen Receptor (AR) and AR-Splice Variant (AR-SV) in Castration-Resistant Prostate Cancer (CRPC).

BACKGROUND: Epigenetic modification influences androgen receptor (AR) activation, often resulting in prostate cancer (PCa) development and progression. Silencing histone-modifying enzymes (histone deacetylases-HDACs) either genetically or pharmacologically suppresses PCa proliferation in preclinical models of PCa; however, results from clinical studies were not encouraging. Similarly, PCa patients eventually become resistant to androgen ablation therapy (ADT). Our goal is to develop dual-acting small molecules comprising antiandrogen and HDAC-inhibiting moieties that may overcome the resistance of ADT and effectively suppress the growth of castration-resistant prostate cancer (CRPC). METHODS: Several rationally designed antiandrogen-equipped HDAC inhibitors (HDACi) were synthesized, and their efficacy on CRPC growth was examined both in vitro and in vivo. RESULTS: While screening our newly developed small molecules, we observed that SBI-46 significantly inhibited the proliferation of AR+ CRPC cells but not AR- CRPC and normal immortalized prostate epithelial cells (RWPE1) or normal kidney cells (HEK-293 and VERO). Molecular analysis confirmed that SBI-46 downregulated the expressions of both AR+ and AR-splice variants (AR-SVs) in CRPC cells. Further studies revealed the downregulation of AR downstream (PSA) events in CRPC cells. The oral administration of SBI-46 abrogated the growth of C4-2B and 22Rv1 CRPC xenograft tumors that express AR or both AR and AR-SV in xenotransplanted nude mice models. Further, immunohistochemical analysis confirmed that SBI-46 inhibits AR signaling in xenografted tumor tissues. CONCLUSION: These results demonstrate that SBI-46 is a potent agent that inhibits preclinical models of CRPC by downregulating the expressions of both AR and AR-SV. Furthermore, these results suggest that SBI-46 may be a potent compound for treating CRPC.

Presence of Bromotyrosine Alkaloids in Marine Sponges Is Independent of Metabolomic and Microbiome Architectures.

Marine sponge holobionts are prolific sources of natural products. One of the most geographically widespread classes of sponge-derived natural products is the bromotyrosine alkaloids. A distinguishing feature of bromotyrosine alkaloids is that they are present in phylogenetically disparate sponges. In this study, using sponge specimens collected from Guam, the Solomon Islands, the Florida Keys, and Puerto Rico, we queried whether the presence of bromotyrosine alkaloids potentiates metabolomic and microbiome conservation among geographically distant and phylogenetically different marine sponges. A multi-omic characterization of sponge holobionts revealed vastly different metabolomic and microbiome architectures among different bromotyrosine alkaloid-harboring sponges. However, we find statistically significant correlations between the microbiomes and metabolomes, signifying that the microbiome plays an important role in shaping the overall metabolome, even in low-microbial-abundance sponges. Molecules mined from the polar metabolomes of these sponges revealed conservation of biosynthetic logic between bromotyrosine alkaloids and brominated pyrrole-imidazole alkaloids, another class of marine sponge-derived natural products. In light of prior findings postulating the sponge host itself to be the biosynthetic source of bromotyrosine alkaloids, our data now set the stage for investigating the causal relationships that dictate the microbiome-metabolome interconnectedness for marine sponges in which the microbiome may not contribute to natural product biogenesis.IMPORTANCE Our work demonstrates that phylogenetically and geographically distant sponges with very different microbiomes can harbor natural product chemical classes that are united in their core chemical structures and biosynthetic logic. Furthermore, we show that independent of geographical dispersion, natural product chemistry, and microbial abundance, overall sponge metabolomes tightly correlate with their microbiomes.

TBK1 regulates regeneration of pancreatic ß-cells.

Small-molecule inhibitors of non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) have shown to stimulate ß-cell regeneration in multiple species. Here we demonstrate that TBK1 is predominantly expressed in ß-cells in mammalian islets. Proteomic and transcriptome analyses revealed that genetic silencing of TBK1 increased expression of proteins and genes essential for cell proliferation in INS-1 832/13 rat ß-cells. Conversely, TBK1 overexpression decreased sensitivity of ß-cells to the elevation of cyclic AMP (cAMP) levels and reduced proliferation of ß-cells in a manner dependent on the activity of cAMP-hydrolyzing phosphodiesterase 3 (PDE3). While the mitogenic effect of (E)3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA) is derived from inhibition of TBK1, PIAA augmented glucose-stimulated insulin secretion (GSIS) and expression of ß-cell differentiation and proliferation markers in human embryonic stem cell (hESC)-derived ß-cells and human islets. TBK1 expression was increased in ß-cells upon diabetogenic insults, including in human type 2 diabetic islets. PIAA enhanced expression of cell cycle control molecules and ß-cell differentiation markers upon diabetogenic challenges, and accelerated restoration of functional ß-cells in streptozotocin (STZ)-induced diabetic mice. Altogether, these data suggest the critical function of TBK1 as a ß-cell autonomous replication barrier and present PIAA as a valid therapeutic strategy augmenting functional ß-cells.

Liver-Targeting Class I Selective Histone Deacetylase Inhibitors Potently Suppress Hepatocellular Tumor Growth as Standalone Agents.

Dysfunctions in epigenetic regulation play critical roles in tumor development and progression. Histone deacetylases (HDACs) and histone acetyl transferase (HAT) are functionally opposing epigenetic regulators, which control the expression status of tumor suppressor genes. Upregulation of HDAC activities, which results in silencing of tumor suppressor genes and uncontrolled proliferation, predominates in malignant tumors. Inhibition of the deacetylase activity of HDACs is a clinically validated cancer therapy strategy. However, current HDAC inhibitors (HDACi) have elicited limited therapeutic benefit against solid tumors. Here, we disclosed a class of HDACi that are selective for sub-class I HDACs and preferentially accumulate within the normal liver tissue and orthotopically implanted liver tumors. We observed that these compounds possess exquisite on-target effects evidenced by their induction of dose-dependent histone H4 hyperacetylation without perturbation of tubulin acetylation status and G0/G1 cell cycle arrest. Representative compounds 2 and 3a are relatively non-toxic to mice and robustly suppressed tumor growths in an orthotopic model of HCC as standalone agents. Collectively, our results suggest that these compounds may have therapeutic advantage against HCC relative to the current systemic HDACi. This prospect merits further comprehensive preclinical investigations.

Small Molecule Inhibitors Targeting Gαi2 Protein Attenuate Migration of Cancer Cells.

Heterotrimeric G-proteins are ubiquitously expressed in several cancers, and they transduce signals from activated G-protein coupled receptors. These proteins have numerous biological functions, and they are becoming interesting target molecules in cancer therapy. Previously, we have shown that heterotrimeric G-protein subunit alphai2 (Gαi2) has an essential role in the migration and invasion of prostate cancer cells. Using a structure-based approach, we have synthesized optimized small molecule inhibitors that are able to prevent specifically the activation of the Gαi2 subunit, keeping the protein in its inactive GDP-bound state. We observed that two of the compounds (13 and 14) at 10 µΜ significantly inhibited the migratory behavior of the PC3 and DU145 prostate cancer cell lines. Additionally, compound 14 at 10 µΜ blocked the activation of Gαi2 in oxytocin-stimulated prostate cancer PC3 cells, and inhibited the migratory capability of DU145 cells overexpressing the constitutively active form of Gαi2, under basal and EGF-stimulated conditions. We also observed that the knockdown or inhibition of Gαi2 negatively regulated migration of renal and ovarian cancer cell lines. Our results suggest that small molecule inhibitors of Gαi2 have potential as leads for discovering novel anti-metastatic agents for attenuating the capability of cancer cells to spread and invade to distant sites.

Publisher Correction: Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Deferiprone: Pan-selective Histone Lysine Demethylase Inhibition Activity and Structure Activity Relationship Study.

Deferiprone (DFP) is a hydroxypyridinone-derived iron chelator currently in clinical use for iron chelation therapy. DFP has also been known to elicit antiproliferative activities, yet the mechanism of this effect has remained elusive. We herein report that DFP chelates the Fe2+ ion at the active sites of selected iron-dependent histone lysine demethylases (KDMs), resulting in pan inhibition of a subfamily of KDMs. Specifically, DFP inhibits the demethylase activities of six KDMs - 2A, 2B, 5C, 6A, 7A and 7B - with low micromolar IC50s while considerably less active or inactive against eleven KDMs - 1A, 3A, 3B, 4A-E, 5A, 5B and 6B. The KDM that is most sensitive to DFP, KDM6A, has an IC50 that is between 7- and 70-fold lower than the iron binding equivalence concentrations at which DFP inhibits ribonucleotide reductase (RNR) activities and/or reduces the labile intracellular zinc ion pool. In breast cancer cell lines, DFP potently inhibits the demethylation of H3K4me3 and H3K27me3, two chromatin posttranslational marks that are subject to removal by several KDM subfamilies which are inhibited by DFP in cell-free assay. These data strongly suggest that DFP derives its anti-proliferative activity largely from the inhibition of a sub-set of KDMs. The docked poses adopted by DFP at the KDM active sites enabled identification of new DFP-based KDM inhibitors which are more cytotoxic to cancer cell lines. We also found that a cohort of these agents inhibited HP1-mediated gene silencing and one lead compound potently inhibited breast tumor growth in murine xenograft models. Overall, this study identified a new chemical scaffold capable of inhibiting KDM enzymes, globally changing histone modification profiles, and with specific anti-tumor activities.

Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

ß-cell proliferation induction is a promising therapeutic strategy to restore ß-cell mass. By screening small molecules in a transgenic zebrafish model of type 1 diabetes, we identified inhibitors of non-canonical IκB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε), as enhancers of ß-cell regeneration. The most potent ß-cell regeneration enhancer was a cinnamic acid derivative (E)-3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA), which, acting through the cAMP-dependent protein kinase A (PKA), stimulated ß-cell-specific proliferation by increasing cyclic AMP (cAMP) levels and mechanistic target of rapamycin (mTOR) activity. A combination of PIAA and cilostamide, an inhibitor of ß-cell-enriched cAMP hydrolyzing enzyme phosphodiesterase (PDE) 3, enhanced ß-cell proliferation, whereas overexpression of PDE3 blunted the mitogenic effect of PIAA in zebrafish. PIAA augmented proliferation of INS-1ß-cells and ß-cells in mammalian islets including human islets with elevation in cAMP levels and insulin secretion. PIAA improved glycemic control in streptozotocin (STZ)-induced diabetic mice with increases in ß-cell proliferation, ß-cell area, and insulin content in the pancreas. Collectively, these data reveal an evolutionarily conserved and critical role of TBK1/IKKε suppression in expanding functional ß-cell mass.

Novel Selective Calpain 1 Inhibitors as Potential Therapeutics in Alzheimer's Disease.

Alzheimer's disease, one of the most important brain pathologies associated with neurodegenerative processes, is related to overactivation of calpain-mediated proteolysis. Previous data showed a compelling efficacy of calpain inhibition against abnormal synaptic plasticity and memory produced by the excess of amyloid-ß, a distinctive marker of the disease. Moreover, a beneficial effect of calpain inhibitors in Alzheimer's disease is predictable by the occurrence of calpain hyperactivation leading to impairment of memory-related pathways following abnormal calcium influxes that might ensue independently of amyloid-ß elevation. However, molecules currently available as effective calpain inhibitors lack adequate selectivity. This work is aimed at characterizing the efficacy of a novel class of epoxide-based inhibitors, synthesized to display improved selectivity and potency towards calpain 1 compared to the prototype epoxide-based generic calpain inhibitor E64. Both functional and preliminary toxicological investigations proved the efficacy, potency, and safety of the novel and selective calpain inhibitors NYC438 and NYC488 as possible therapeutics against the disease.

A structure-activity relationship of non-peptide macrocyclic histone deacetylase inhibitors and their anti-proliferative and anti-inflammatory activities.

Inhibition of the enzymatic activity of histone deacetylase (HDAC) is a promising therapeutic strategy for cancer treatment and several distinct small molecule histone deacetylase inhibitors (HDACi) have been reported. We have previously identified a new class of non-peptide macrocyclic HDACi derived from 14- and 15-membered macrolide skeletons. In these HDACi, the macrocyclic ring is linked to the zinc chelating hydroxamate moiety through a para-substituted aryl-triazole cap group. To further delineate the depth of the SAR of this class of HDACi, we have synthesized series of analogous compounds and investigated the influence of various substitution patterns on their HDAC inhibitory, anti-proliferative and anti-inflammatory activities. We identified compounds 25b and 38f with robust anti-proliferative activities and compound 26f (IC50 47.2 nM) with superior anti-inflammatory (IC50 88 nM) activity relative to SAHA.

Exploiting translational stalling peptides in an effort to extend azithromycin interaction within the prokaryotic ribosome nascent peptide exit tunnel.

The ribosome is the primary protein synthesis machine in the cell and is a target for treatment of a variety of diseases including bacterial infection and cancer. The ribosomal peptide exit tunnel, the route of egress for the nascent peptide, is an inviting site for drug design. Toward a rational engagement of the nascent peptide components for the design of small molecule inhibitors of ribosome function, we designed and disclosed herein a set of N-10 indole functionalized azithromycin analogs. The indole moiety of these compounds is designed to mimic the translation stalling interaction of SecM W155 side-chain with the prokaryotic (Escherichia coli) ribosome A751 residue. Many of these N-10 functionalized compounds have enhanced translation inhibition activities against E. coli ribosome relative to azithromycin while a subset inhibited the growth of representative susceptible bacteria strains to about the same extent as azithromycin. Moreover, the inclusion of bovine serum in the bacterial growth media enhanced the anti-bacterial potency of the N-10 functionalized azithromycin analogs by as high as 10-fold.

Design, synthesis, and optimization of novel epoxide incorporating peptidomimetics as selective calpain inhibitors.

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer's disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.

Isoxazole moiety in the linker region of HDAC inhibitors adjacent to the Zn-chelating group: effects on HDAC biology and antiproliferative activity.

A series of hydroxamic acid based histone deacetylase inhibitors 6-15, containing an isoxazole moiety adjacent to the Zn-chelating hydroxamic acid, is reported herein. Some of these compounds showed nanomolar activity in the HDAC isoform inhibitory assay and exhibited micro molar inhibitory activity against five pancreatic cancer cell lines.

Design and synthesis of aryl ether inhibitors of the Bacillus anthracis enoyl-ACP reductase.

The problem of increasing bacterial resistance to the current generation of antibiotics is well documented. Known resistant pathogens such as methicillin-resistant Staphylococcus aureus are becoming more prevalent, while the potential exists for developing drug-resistant pathogens for use as bioweapons, such as Bacillus anthracis. The biphenyl ether antibacterial agent, triclosan, exhibits broad-spectrum activity by targeting the fatty acid biosynthetic pathway through inhibition of enoyl-acyl carrier protein reductase (ENR) and provides a potential scaffold for the development of new, broad-spectrum antibiotics. We used a structure-based approach to develop novel aryl ether analogues of triclosan that target ENR, the product of the fabI gene, from B. anthracis (BaENR). Structure-based design methods were used for the expansion of the compound series including X-ray crystal structure determination, molecular docking, and QSAR methods. Structural modifications were made to both phenyl rings of the 2-phenoxyphenyl core. A number of compounds exhibited improved potency against BaENR and increased efficacy against both the Sterne strain of B. anthracis and the methicillin-resistant strain of S. aureus. X-ray crystal structures of BaENR in complex with triclosan and two other compounds help explain the improved efficacy of the new compounds and suggest future rounds of optimization that might be used to improve their potency.

Use of the nitrile oxide cycloaddition (NOC) reaction for molecular probe generation: a new class of enzyme selective histone deacetylase inhibitors (HDACIs) showing picomolar activity at HDAC6.

A series of hydroxamate based HDAC inhibitors containing a phenylisoxazole as the CAP group has been synthesized using nitrile oxide cycloaddition chemistry. An HDAC6 selective inhibitor having a potency of approximately 2 picomolar was identified. Some of the compounds were examined for their ability to block pancreatic cancer cell growth and found to be about 10-fold more potent than SAHA. This research provides valuable, new molecular probes for use in exploring HDAC biology.

Sugar amino acids in designing new molecules.

Emulating the basic principles followed by nature to build its vast repertoire of biomolecules, organic chemists are developing many novel multifunctional building blocks and using them to create 'nature-like' and yet unnatural organic molecules. Sugar amino acids constitute an important class of such polyfunctional scaffolds where the carboxyl, amino and hydroxyl termini provide an excellent opportunity to organic chemists to create structural diversities akin to Nature's molecular arsenal. This article describes some of our works on various sugar amino acids and many other related building blocks, like furan amino acids, pyrrole amino acids etc. used in wide-ranging peptidomimetic studies.