Modulating the affinity and signaling bias of cannabinoid receptor 1 antagonists.

Cannabinoid receptor 1 (CB1) is a G protein-coupled receptor and a therapeutic target for metabolic disorders. Numerous CB1 antagonists have been developed, but their functional selectivities and bias towards G protein or ß-arrestin signaling have not been systemically characterized. In this study, we analyzed the binding affinities and downstream signaling of two series of pyrazole derivatives bearing 1-aminopiperidine (Series I) or 4-aminothiomorpholine 1,1-dioxide (Series II) moieties, as well as the well-known CB1 antagonists rimonabant and taranabant. Analyses of the results for the Series I and II derivatives showed that minor structure modifications to their functional groups and especially the incorporation of 1-aminopiperidine or 4-aminothiomorpholine 1,1-dioxide motifs can profoundly affect their bias toward G protein or ß-arrestin signaling, and that their binding affinity and functional activity can be disassociated. Docking and molecular dynamics simulations revealed that the binding modes of Series I and II antagonists differed primarily in that Series I antagonists formed an additional hydrogen bond with the receptor, whereas those in Series II formed a water bridge.

4-Bromophenylhydrazinyl benzenesulfonylphenylureas as indoleamine 2,3-dioxygenase inhibitors with in vivo target inhibition and anti-tumor efficacy.

Indoleamine 2,3-dioxygenase is a heme-containing enzyme implicated in the down regulation of the anti-tumor immune response, and considered a promising anti-cancer drug target. Several pharmaceutical companies, including Pfizer, Merck, and Bristol-Myers Squibb, are known to be in pursuit of IDO inhibitors, and Incyte recently reported good results in the phase II clinical trial of the IDO inhibitor Epacadostat. In previous work, we developed a series of IDO inhibitors based on a sulfonylhydrazide core structure, and explored how they could serve as potent IDO inhibitors with good drug profiles. Herein, we disclose the development of the 4-bromophenylhydrazinyl benzenesulfonylphenylurea 5k, a potent IDO inhibitor which demonstrated 25% tumor growth inhibition in a murine CT26 syngeneic model on day 18 with 100 mg/kg oral administration twice daily, and a 30% reduction in tumor weight. Pharmacodynamic testing of 5k found it to cause a 25% and 21% reduction in kyn/trp ratio at the plasma and tumor, respectively. In the CT26 tumor model, 5k was found to slightly increase the percentage of CD3+ T cells and lymphocyte responsiveness, indicating that 5k may have potential in modulating anti-tumor immunity. These data suggest 5k to be worthy of further investigation in the development of anti-tumor drugs.

Discovery and structure-activity relationships of phenyl benzenesulfonylhydrazides as novel indoleamine 2,3-dioxygenase inhibitors.

A novel class of phenyl benzenesulfonylhydrazides has been identified as potent inhibitors of indoleamine 2,3-dioxygenase (IDO), and their structure-activity relationship was explored. Coupling reactions between various benzenesulfonyl chlorides and phenylhydrazides were utilized to synthesize the sulfonylhydrazides bearing various substituents. Compound 3i exhibited 61 nM of IC50 in enzymatic assay and 172 nM of EC50 in the HeLa cell. The computational study of 3i suggested that the major interactions between 3i and IDO protein are the coordination of sulfone and heme iron, the hydrogen bonding and hydrophobic interactions between 3i and IDO. This novel class of IDO inhibitor provides a new direction to discover effective anti-cancer agents.

Analysis of global methylation using a Zta-expressing nasopharyngeal carcinoma cell line.

EBV infects more than 90% of the human population and persists in most individuals as a latent infection where the viral genome is silenced by host-driven methylation. The lytic cycle is initiated when the viral protein Zta binds to methylated BRLF1 and BRRF1 promoters. Although studies reveal the role of Zta and methylation changes in the viral genome upon EBV infection to reactivation, whether Zta plays any role in alteration of methylation in the host genome remains unknown. Using an inducible model, we demonstrate that global DNA methylation, based on whole-genome 5-methylcytosine content, and regional DNA methylation in repetitive elements, imprinting genes and the X chromosome, remains unchanged in response to Zta expression. Expression of DNA methyltransferases was also unaffected by ectopically expressed Zta. Our data imply that alteration of host gene expression following EBV reactivation may reflect methylation-independent Zta-mediated gene activation and not epigenetic modification of the host genome.

Discovery and development of a novel N-(3-bromophenyl)-{[(phenylcarbamoyl)amino]methyl}-N-hydroxythiophene-2-carboximidamide indoleamine 2,3-dioxygenase inhibitor using knowledge-based drug design.

Indoleamine 2,3-dioxygenase-1 (IDO1) is a potential target for the next generation of cancer immunotherapies. We describe the development of two series of IDO1 inhibitors incorporating a N-hydroxy-thiophene-carboximidamide core generated by knowledge-based drug design. Structural modifications to improve the cellular activity and pharmacokinetic (PK) properties of the compounds synthesized, including extension of the side chain of the N-hydroxythiophene-2-carboximidamide core, resulted in compound 27a, a potent IDO1 inhibitor which demonstrated significant (51%) in vivo target inhibition on IDO1 in a human SK-OV-3 ovarian xenograft tumor mouse model. This strategy is expected to be applicable to the discovery of additional IDO1 inhibitors for the treatment of other diseases susceptible to modulation of IDO1.

Biphasic suppression of appetite by cannabinoid CB1 receptor antagonists with distinct functional activities.

A novel alkynylthiophene series of cannabinoid CB1 receptor antagonists has been described to exhibit distinct intrinsic activities with minimal substructure modifications. The three representatives, BPR0432, BPR0568 and BPR0569, functioning as a neutral antagonist, an inverse agonist and a partial agonist, respectively, in GTP binding assay, were further characterized for their downstream signaling activities in relation to in vivo efficacy in appetite suppression to diets of different macronutrients. Interestingly, these three derivatives all behaved as inverse agonists with the potency of BPR0432>BPR0568>BPR0569 in cAMP assay. After administered to non-deprived rats, the potency in appetite suppression was positively related to their strength in intrinsic activity in the first hour of intake. The preferential suppression to high fat and high carbohydrate diets was revealed after 6h and only appeared in the treatment of BPR0568, presumably due to its metabolic stability in addition to its intrinsic activity. These results indicated the suppression of appetite was controlled in a biphasic manner, and these three structurally close but functionally distinct compounds are invaluable tools in elucidating the mechanism of neuronal response to appetite and palatability.

Important Hydrogen Bond Networks in Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitor Design Revealed by Crystal Structures of Imidazoleisoindole Derivatives with IDO1.

Indoleamine 2,3-dioxygenase 1 (IDO1), promoting immune escape of tumors, is a therapeutic target for the cancer immunotherapy. A number of IDO1 inhibitors have been identified, but only limited structural biology studies of IDO1 inhibitors are available to provide insights on the binding mechanism of IDO1. In this study, we present the structure of IDO1 in complex with 24, a NLG919 analogue with potent activity. The complex structure revealed the imidazole nitrogen atom of 24 to coordinate with the heme iron, and the imidazoleisoindole core situated in pocket A with the 1-cyclohexylethanol moiety extended to pocket B to interact with the surrounding residues. Most interestingly, 24 formed an extensive hydrogen bond network with IDO1, which is a distinct feature of IDO1/24 complex structure and is not observed in the other IDO1 complex structures. Further structure-activity relationship, UV spectra, and structural biology studies of several analogues of 24 demonstrated that extensive hydrophobic interactions and the unique hydrogen bonding network contribute to the great potency of imidazoleisoindole derivatives. These results are expected to facilitate the structure-based drug design of new IDO inhibitors.

Phenyl Benzenesulfonylhydrazides Exhibit Selective Indoleamine 2,3-Dioxygenase Inhibition with Potent in Vivo Pharmacodynamic Activity and Antitumor Efficacy.

Tryptophan metabolism has been recognized as an important mechanism in immune tolerance. Indoleamine 2,3-dioxygenase plays a key role in local tryptophan metabolism via the kynurenine pathway and has emerged as a therapeutic target for cancer immunotherapy. Our prior study identified phenyl benzenesulfonyl hydrazide 2 as a potent in vitro (though not in vivo) inhibitor of indoleamine 2,3-dioxygenase. Further lead optimization to improve in vitro potencies and pharmacokinetic profiles resulted in N'-(4-bromophenyl)-2-oxo-2,3-dihydro-1H-indole-5-sulfonyl hydrazide 40, which demonstrated 59% oral bioavailability and 73% of tumor growth delay without apparent body weight loss in the murine CT26 syngeneic model, after oral administration of 400 mg/kg. Accordingly, 40, is proposed as a potential drug lead worthy of advanced preclinical evaluation.

Identification of Substituted Naphthotriazolediones as Novel Tryptophan 2,3-Dioxygenase (TDO) Inhibitors through Structure-Based Virtual Screening.

A structure-based virtual screening strategy, comprising homology modeling, ligand-support binding site optimization, virtual screening, and structure clustering analysis, was developed and used to identify novel tryptophan 2,3-dioxygenase (TDO) inhibitors. Compound 1 (IC50 = 711 nM), selected by virtual screening, showed inhibitory activity toward TDO and was subjected to structural modifications and molecular docking studies. This resulted in the identification of a potent TDO selective inhibitor (11e, IC50 = 30 nM), making it a potential compound for further investigation as a cancer therapeutic and other TDO-related targeted therapy.

Discovery of novel N-ß-D-xylosylindole derivatives as sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors for the management of hyperglycemia in diabetes.

A novel series of N-linked ß-D-xylosides were synthesized and evaluated for inhibitory activity against sodium-dependent glucose cotransporter 2 (SGLT2) in a cell-based assay. Of these, the 4-chloro-3-(4-cyclopropylbenzyl)-1-(ß-D-xylopyranosyl)-1H-indole 19m was found to be the most potent inhibitor, with an EC(50) value similar to that of the natural SGLT2 inhibitor phlorizin. Further studies in Sprague-Dawley (SD) rats indicated that 19m significantly increased urine glucose excretion in a dose-dependent manner with oral administration. The antihyperglycemic effect of 19m was also observed in streptozotocin (STZ) induced diabetic SD rats. These results described here are a good starting point for further investigations into N-glycoside SGLT2 inhibitors.