Interrogating direct NLRP3 engagement and functional inflammasome inhibition using cellular assays.

As a key regulator of the innate immune system, the NLRP3 inflammasome responds to a variety of environmental insults through activation of caspase-1 and release of the proinflammatory cytokines IL-1ß and IL-18. Aberrant NLRP3 inflammasome function is implicated in numerous inflammatory diseases, spurring drug discovery efforts at NLRP3 as a therapeutic target. A diverse array of small molecules is undergoing preclinical/clinical evaluation with a reported mode of action involving direct modulation of the NLRP3 pathway. However, for a subset of these ligands the functional link between live-cell target engagement and pathway inhibition has yet to be fully established. Herein we present a cohort of mechanistic assays to both query direct NLRP3 engagement in cells, and functionally interrogate different nodes of NLRP3 pathway activity. This system enabled the stratification of potency for five confirmed NLRP3 inhibitors, and identification of two reported NLRP3 inhibitors that failed to demonstrate direct pathway antagonism.

DELs enable the development of BRET probes for target engagement studies in cells.

DNA-encoded libraries (DELs) provide unmatched chemical diversity and starting points for novel drug modalities. Here, we describe a workflow that exploits the bifunctional attributes of DEL ligands as a platform to generate BRET probes for live cell target engagement studies. To establish proof of concept, we performed a DEL screen using aurora kinase A and successfully converted aurora DEL ligands as cell-active BRET probes. Aurora BRET probes enabled the validation and stratification of the chemical series identified from primary selection data. Furthermore, we have evaluated the effective repurposing of pre-existing DEL screen data to find suitable leads for BRET probe development. Our findings support the use of DEL workflows as an engine to create cell-active BRET probes independent of structure or compound SAR. The combination of DEL and BRET technology accelerates hit-to-lead studies in a live cell setting.

Biological evaluation and synthesis of calcitroic acid.

We describe the synthesis and broad profiling of calcitroic acid (CTA) as vitamin D receptor (VDR) ligand. The x-ray co-crystal structure of the Danio Rerio VDR ligand binding domain in complex with CTA and peptide MED1 confirmed an agonistic conformation of the receptor. CTA adopted a similar conformation as 1,25(OH)2D3 in the binding pocket. A hydrogen bond with His333 and a water molecule were observed in the binding pocket, which was accommodated due to the shorter CTA side chain. In contrast, 1,25(OH)2D3 interacted with His423 and His333 due to its longer side chain. In vitro, the EC50 values of CTA and CTA-ME for VDR-mediated transcription were 2.89 µM and 0.66 µM, respectively, confirming both compounds as VDR agonists. CTA was further evaluated for interaction with fourteen nuclear receptors demonstrating selective activation of VDR. VDR mediated gene regulation by CTA in intestinal cells was observed for the VDR target gene CYP24A1. CTA at 10 µM upregulated CYP24A1 with similar efficacy as 1,25(OH)2D3 at 20 nM and 100-fold stronger compared to lithocholic acid at 10 µM. CTA reduced the transcription of iNOS and IL-1ß in interferon γ and lipopolysaccharide stimulated mouse macrophages resulting in a reduction of nitric oxide production and secretion of IL-1ß. These observed anti-inflammatory properties of 20 µM CTA were similar to 20 nM 1,25(OH)2D3.

A review of currently identified small molecule modulators of microRNA function.

microRNAs are small, noncoding RNA that negatively regulate gene expression. Since their discovery in 1993, approximately 2500 human mature microRNAs have been discovered and details of their biogenesis, mechanism of action, and function has been studied. Aberrant expression of microRNAs has since been observed in numerous disease states particularly cancer, neurologic disorders, autoimmune diseases, metabolic diseases and cardiovascular diseases. Because of this, a strong interest in developing novel therapies that modulate microRNA function has emerged. Although, several strategies have been employed, small molecule drugs have shown great promise due their inherent stability, bioavailability, and cost-efficiency. In this review, we discuss the microRNA modulating small molecules that have thus far been identified in the literature and highlight the need for continued research in this field.

Inhibition of hedgehog signaling by stereochemically defined des-triazole itraconazole analogues.

Dysregulation of the hedgehog (Hh) signaling pathway is associated with cancer occurrence and development in various malignancies. Previous structure-activity relationships (SAR) studies have provided potent Itraconazole (ITZ) analogues as Hh pathway antagonists. To further expand on our SAR for the ITZ scaffold, we synthesized and evaluated a series of compounds focused on replacing the triazole. Our results demonstrate that the triazole region is amenable to modification to a variety of different moieties; with a single methyl group representing the most favorable substituent. In addition, nonpolar substituents were more active than polar substituents. These SAR results provide valuable insight into the continued exploration of ITZ analogues as Hh pathway antagonists.

Probing the Ligand-Binding Pocket of BTN3A1.

Small-molecule phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate stimulate human Vγ9Vδ2 T cells after binding to the intracellular B30.2 domain of the immune receptor butyrophilin 3 isoform A1 (BTN3A1). To understand the ligand-target interaction in greater detail, we performed molecular docking. Based on the docking results, we synthesized the novel ligand (E)-(7-hydroxy-6-methylhept-5-en-1-yl)phosphonate and mutated proposed binding site residues. We evaluated the impact on butyrophilin binding of existing and novel ligands using a newly developed high-throughput fluorescence polarization assay. We also evaluated the ability of the compounds to stimulate proliferation and interferon-γ production of Vγ9Vδ2 T cells. Mutation of H381 fully blocked ligand binding, whereas mutations to charged surface residues impacted diphosphate interactions. Monophosphonate analogs bind similarly to BTN3A1, although they differ in their antigenicity, demonstrating that binding and efficacy are not linearly correlated. These results further define the structure-activity relationships underlying BTN3A1 ligand binding and antigenicity and support further structure-guided drug design.

Structure-Activity Relationships for Itraconazole-Based Triazolone Analogues as Hedgehog Pathway Inhibitors.

The Food and Drug Administration-approved antifungal agent, itraconazole (ITZ), has been increasingly studied for its novel biological properties. In particular, ITZ inhibits the hedgehog (Hh) signaling pathway and has the potential to serve as an anticancer chemotherapeutic against several Hh-dependent malignancies. We have extended our studies on ITZ analogues as Hh pathway inhibitors through the design, synthesis, and evaluation of novel des-triazole ITZ analogues that incorporate modifications to the triazolone/side chain region of the scaffold. Our overall results suggest that the triazolone/side chain region can be replaced with various functionalities (hydrazine carboxamides and meta-substituted amides) resulting in improved potency when compared to ITZ. Our studies also indicate that the stereochemical orientation of the dioxolane ring is important for both potent Hh pathway inhibition and compound stability. Finally, our studies suggest that the ITZ scaffold can be successfully modified in terms of functionality and stereochemistry to further improve its anti-Hh potency and physicochemical properties.

Development of posaconazole-based analogues as hedgehog signaling pathway inhibitors.

Inhibition of the hedgehog (Hh) signaling pathway has been validated as a therapeutic strategy to treat basal cell carcinoma and holds potential for several other forms of human cancer. Itraconazole and posaconazole are clinically useful triazole anti-fungals that are being repurposed as anti-cancer agents based on their ability to inhibit the Hh pathway. We have previously demonstrated that removal of the triazole from itraconazole does not affect its ability to inhibit the Hh pathway while abolishing its primary side effect, potent inhibition of Cyp3A4. To develop structure-activity relationships for the related posaconazole scaffold, we synthesized and evaluated a series of des-triazole analogues designed through both ligand- and structure-based methods. These compounds demonstrated improved anti-Hh properties compared to posaconazole and enhanced stability without inhibiting Cyp3A4. In addition, we utilized a series of molecular dynamics and binding energy studies to probe specific interactions between the compounds and their proposed binding site on Smoothened. These studies strongly suggest that the tetrahydrofuran region of the scaffold projects out of the binding site and that π-π interactions between the compound and Smoothened play a key role in stabilizing the bound analogues.

Novel VDR antagonists based on the GW0742 scaffold.

The vitamin D receptor is a nuclear hormone receptor that regulates cell proliferation, cell differentiation and calcium homeostasis. The receptor is endogenously activated by 1,25-dihydroxyvitamin D3, which induces transcription of VDR targets genes regulated by coactivator binding. VDR antagonists and partial agonists have been developed based on the secosteroid scaffold of vitamin D. Only a few non-secosteroid VDR antagonists are known. Herein, we report the rational design of non-secosteroid VDR antagonists using GW0742 as a scaffold. GW0742 is a PPARδ agonist previously identified by our group as a VDR antagonist. Several modifications including the replacement of the thiazole ring with an oxazole ring led to compound 7b, which inhibited VDR-mediated transcription (IC50 = 660 nM) without activating PPARδ-mediated transcription. However, inhibition of transcription mediated by other nuclear receptors was observed.

Parallel Chemistry Approach to Identify Novel Nuclear Receptor Ligands Based on the GW0742 Scaffold.

We describe the parallel synthesis of novel analogs of GW0742, a peroxisome proliferator-activated receptor δ (PPARδ) agonist. For that purpose, modified reaction conditions were applied, such as a solid-phase palladium-catalyzed Suzuki coupling. In addition, tetrazole-based compounds were generated as a bioisostere for carboxylic acid-containing ligand GW0742. The new compounds were investigated for their ability to activate PPARδ mediated transcription and their cross-reactivity with the vitamin D receptor (VDR), another member of the nuclear receptor superfamily. We identified many potent PPARδ agonists that were less toxic than GW0742, where ∼65 of the compounds synthesized exhibited partial PPARδ activity (23-98%) with EC50 values ranging from 0.007-18.2 µM. Some ligands, such as compound 32, were more potent inhibitors of VDR-mediated transcription with significantly reduced PPARδ activity than GW0742, however, none of the ligands were completely selective for VDR inhibition over PPARδ activation of transcription.

Methyllysine binding domains: Structural insight and small molecule probe development.

A frequent posttranslational modification that regulates gene expression is the mono-, di-, and/or tri- methylation of lysine residues on the histone tails of chromatin. The recognition of methylated lysine marks is facilitated by specific reader proteins that contain a methyllysine binding domain. This class of reader proteins has emerged as a focus of epigenetic research due to its crucial role in gene regulation, oncogenesis and other disease pathways. The design and synthesis of small molecules that target these domains and disrupt reader/histone protein-protein interactions have demonstrated the druggability of methyllysine binding pockets and provided preliminary evidence that their disruption holds therapeutic potential. In this review, we detail the structures of methyllysine binding domains, highlight the primary roles of these reader proteins in both normal and disease states, and describe the current status of small molecule development against these emerging epigenetic regulators.

Hydrogen peroxide activated quinone methide precursors with enhanced DNA cross-linking capability and cytotoxicity towards cancer cells.

Quinone methide (QM) formation induced by endogenously generated H2O2 is attractive for biological and biomedical applications. To overcome current limitations due to low biological activity of H2O2-activated QM precursors, we are introducing herein several new arylboronates with electron donating substituents at different positions of benzene ring and/or different neutral leaving groups. The reaction rate of the arylboronate esters with H2O2 and subsequent bisquinone methides formation and DNA cross-linking was accelerated with the application of Br as a leaving group instead of acetoxy groups. Additionally, a donating group placed meta to the nascent exo-methylene group of the quinone methide greatly improves H2O2-induced DNA interstrand cross-link formation as well as enhances the cellular activity. Multiple donating groups decrease the stability and DNA cross-linking capability, which lead to low cellular activity. A cell-based screen demonstrated that compounds 2a and 5a with a OMe or OH group dramatically inhibited the growth of various tissue-derived cancer cells while normal cells were less affected. Induction of H2AX phosphorylation by these compounds in CLL lymphocytes provide evidence for a correlation between cell death and DNA damage. The compounds presented herein showed potent anticancer activities and selectivity, which represent a novel scaffold for anticancer drug development.

Synthesis and Characterization of a Novel γ-Aminobutyric Acid Type A (GABAA) Receptor Ligand That Combines Outstanding Metabolic Stability, Pharmacokinetics, and Anxiolytic Efficacy.

1,4-Benzodiazepines are used in the treatment of anxiety disorders but have limited long-term use due to adverse effects. HZ-166 (2) has been shown to have anxiolytic-like effects with reduced sedative/ataxic liabilities. A 1,3-oxazole KRM-II-81 (9) was discovered from a series of six bioisosteres with significantly improved pharmacokinetic and pharmacodynamic properties as compared to 2. Oxazole 9 was further characterized and exhibited improved anxiolytic-like effects in a mouse marble burying assay and a rat Vogel conflict test.

Inhibitors for the Vitamin D Receptor-Coregulator Interaction.

The vitamin D receptor (VDR) belongs to the superfamily of nuclear receptors and is activated by the endogenous ligand 1,25-dihydroxyvitamin D3. The genomic effects mediated by VDR consist of the activation and repression of gene transcription, which includes the formation of multiprotein complexes with coregulator proteins. Coregulators bind many nuclear receptors and can be categorized according to their role as coactivators (gene activation) or corepressors (gene repression). Herein, different approaches to develop compounds that modulate the interaction between VDR and coregulators are summarized. This includes coregulator peptides that were identified by creating phage display libraries. Subsequent modification of these peptides including the introduction of a tether or nonhydrolyzable bonds resulted in the first direct VDR-coregulator inhibitors. Later, small molecules that inhibit VDR-coregulator inhibitors were identified using rational drug design and high-throughput screening. Early on, allosteric inhibition of VDR-coregulator interactions was achieved with VDR antagonists that change the conformation of VDR and modulate the interactions with coregulators. A detailed discussion of their dual agonist/antagonist effects is given as well as a summary of their biological effects in cell-based assays and in vivo studies.

Synthesis and evaluation of vitamin D receptor-mediated activities of cholesterol and vitamin D metabolites.

A systematic study with phase 1 and phase 2 metabolites of cholesterol and vitamin D was conducted to determine whether their biological activity is mediated by the vitamin D receptor (VDR). The investigation necessitated the development of novel synthetic routes for lithocholic acid (LCA) glucuronides (Gluc). Biochemical and cell-based assays were used to demonstrate that hydroxylated LCA analogs were not able to bind VDR. This excludes VDR from mediating their biological and pharmacological activities. Among the synthesized LCA conjugates a novel VDR agonist was identified. LCA Gluc II increased the expression of CYP24A1 in DU145 cancer cells especially in the presence of the endogenous VDR ligand 1,25(OH)2D3. Furthermore, the methyl ester of LCA was identified as novel VDR antagonist. For the first time, we showed that calcitroic acid, the assumed inactive final metabolite of vitamin D, was able to activate VDR-mediated transcription to a higher magnitude than bile acid LCA. Due to a higher metabolic stability in comparison to vitamin D, a very low toxicity, and high concentration in bile and intestine, calcitroic acid is likely to be an important mediator of the protective vitamin D properties against colon cancer.

Antitumor Activity of 3-Indolylmethanamines 31B and PS121912.

AIM: To investigate the in vivo effects of 3-indolylmethanamines 31B and PS121912 in treating ovarian cancer and leukemia, respectively. MATERIALS AND METHODS: Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and western blotting were applied to demonstrate the induction of apoptosis. Xenografted mice were investigated to show the antitumor effects of 3-indolylmethanamines. (13)C-Nuclear magnetic resource (NMR) and western blotting were used to demonstrate inhibition of glucose metabolism. RESULTS: 31B inhibited ovarian cancer cell proliferation and activated caspase-3, cleaved poly (ADP-ribose) polymerase 1 (PARP1), and phosphorylated mitogen-activated protein kinases (MAPK), JUN N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. 31B reduced ovarian cancer xenograft tumor growth and PS121912 inhibited the growth of HL-60-derived xenografts without any sign of toxicity. Compound 31B inhibited de novo glycolysis and lipogenesis mediated by the reduction of fatty acid synthase and lactate dehydrogenase-A expression. CONCLUSION: 3-Indolylmethanamines represent a new class of antitumor agents. We have shown for the first time the in vivo anticancer effects of 3-indolylmethanamines 31B and PS121912.