Stretch-Induced Down-Regulation of HCN2 Suppresses Contractile Activity.

Although hyperpolarization-activated and cyclic nucleotide-gated 2 channels (HCN2) are expressed in multiple cell types in the gut, the role of HCN2 in intestinal motility is poorly understood. HCN2 is down-regulated in intestinal smooth muscle in a rodent model of ileus. Thus, the purpose of this study was to determine the effects of HCN inhibition on intestinal motility. HCN inhibition with ZD7288 or zatebradine significantly suppressed both spontaneous and agonist-induced contractile activity in the small intestine in a dose-dependent and tetrodotoxin-independent manner. HCN inhibition significantly suppressed intestinal tone but not contractile amplitude. The calcium sensitivity of contractile activity was significantly suppressed by HCN inhibition. Inflammatory mediators did not affect the suppression of intestinal contractile activity by HCN inhibition but increased stretch of the intestinal tissue partially attenuated the effects of HCN inhibition on agonist-induced intestinal contractile activity. HCN2 protein and mRNA levels in intestinal smooth muscle tissue were significantly down-regulated by increased mechanical stretch compared to unstretched tissue. Increased cyclical stretch down-regulated HCN2 protein and mRNA levels in primary human intestinal smooth muscle cells and macrophages. Overall, our results suggest that decreased HCN2 expression induced by mechanical signals, such as intestinal wall distension or edema development, may contribute to the development of ileus.

The Role of Inflammatory Mediators in the Development of Gastrointestinal Motility Disorders.

Feeding intolerance and the development of ileus is a common complication affecting critically ill, surgical, and trauma patients, resulting in prolonged intensive care unit and hospital stays, increased infectious complications, a higher rate of hospital readmission, and higher medical care costs. Medical treatment for ileus is ineffective and many of the available prokinetic drugs have serious side effects that limit their use. Despite the large number of patients affected and the consequences of ileus, little progress has been made in identifying new drug targets for the treatment of ileus. Inflammatory mediators play a critical role in the development of ileus, but surprisingly little is known about the direct effects of inflammatory mediators on cells of the gastrointestinal tract, and many of the studies are conflicting. Understanding the effects of inflammatory cytokines/chemokines on the development of ileus will facilitate the early identification of patients who will develop ileus and the identification of new drug targets to treat ileus. Thus, herein, we review the published literature concerning the effects of inflammatory mediators on gastrointestinal motility.

Dual-Target Compounds against Type 2 Diabetes Mellitus: Proof of Concept for Sodium Dependent Glucose Transporter (SGLT) and Glycogen Phosphorylase (GP) Inhibitors.

A current trend in the quest for new therapies for complex, multifactorial diseases, such as diabetes mellitus (DM), is to find dual or even multi-target inhibitors. In DM, the sodium dependent glucose cotransporter 2 (SGLT2) in the kidneys and the glycogen phosphorylase (GP) in the liver are validated targets. Several (ß-D-glucopyranosylaryl)methyl (het)arene type compounds, called gliflozins, are marketed drugs that target SGLT2. For GP, low nanomolar glucose analogue inhibitors exist. The purpose of this study was to identify dual acting compounds which inhibit both SGLTs and GP. To this end, we have extended the structure-activity relationships of SGLT2 and GP inhibitors to scarcely known (C-ß-D-glucopyranosylhetaryl)methyl arene type compounds and studied several (C-ß-D-glucopyranosylhetaryl)arene type GP inhibitors against SGLT. New compounds, such as 5-arylmethyl-3-(ß-D-glucopyranosyl)-1,2,4-oxadiazoles, 5-arylmethyl-2-(ß-D-glucopyranosyl)-1,3,4-oxadiazoles, 4-arylmethyl-2-(ß-D-glucopyranosyl)pyrimidines and 4(5)-benzyl-2-(ß-D-glucopyranosyl)imidazole were prepared by adapting our previous synthetic methods. None of the studied compounds exhibited cytotoxicity and all of them were assayed for their SGLT1 and 2 inhibitory potentials in a SGLT-overexpressing TSA201 cell system. GP inhibition was also determined by known methods. Several newly synthesized (C-ß-D-glucopyranosylhetaryl)methyl arene derivatives had low micromolar SGLT2 inhibitory activity; however, none of these compounds inhibited GP. On the other hand, several (C-ß-D-glucopyranosylhetaryl)arene type GP inhibitor compounds with low micromolar efficacy against SGLT2 were identified. The best dual inhibitor, 2-(ß-D-glucopyranosyl)-4(5)-(2-naphthyl)-imidazole, had a Ki of 31 nM for GP and IC50 of 3.5 µM for SGLT2. This first example of an SGLT-GP dual inhibitor can prospectively be developed into even more efficient dual-target compounds with potential applications in future antidiabetic therapy.

CXCL1 is upregulated during the development of ileus resulting in decreased intestinal contractile activity.

BACKGROUND: Although the development of ileus is widespread and negatively impacts patient outcomes, the mechanism by which ileus develops remains unclear. The purpose of our study was to examine the contribution of myogenic mechanisms to postoperative ileus development and the involvement of inflammation in mediating intestinal smooth muscle dysfunction. METHODS: Contractile activity and the effects of CXCL1 were studied in a gut manipulation model. KEY RESULTS: Contraction amplitude in the ileum decreased significantly, while tone increased significantly in response to gut manipulation. Differences in contraction amplitude were affected by tetrodotoxin at earlier time points, but not at later time points. Agonist-induced contractions in the small intestine decreased significantly with ileus development. Intestinal transit slowed significantly after the induction of ileus. Myosin light chain phosphorylation was significantly decreased and edema increased significantly in the intestinal wall. Conditioned media from mechanically activated macrophages depressed intestinal contractile activity. CXCL1 (GroA) was significantly increased in the mechanically activated macrophages and intestinal smooth muscle within 1 hour after induction of ileus compared with control cells and sham animals, respectively. Treatment with CXCL1 significantly decreased contraction amplitude and agonist-induced contractile activity and increased tone in the small intestine. In the gut manipulation model, treatment with a CXCR2 antagonist prevented the decrease in agonist-induced contractile activity but not contraction amplitude. CONCLUSIONS & INFERENCES: These data suggest that CXCL1, released from macrophages during intestinal wall stress, can suppress intestinal contractile activity. CXCL1 is a potential target for preventing or treating ileus in trauma patients.

Identification of C-ß-d-Glucopyranosyl Azole-Type Inhibitors of Glycogen Phosphorylase That Reduce Glycogenolysis in Hepatocytes: In Silico Design, Synthesis, in Vitro Kinetics, and ex Vivo Studies.

Several C-ß-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (RP) and Spearman (RS) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(ß-d-glucopyranosyl)-imidazoles and 2-aryl-4-(ß-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki values in the low micromolar range (5c = 1.97 µM; 13b = 4.58 µM). Ten C-ß-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these (5a-c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30-60 µM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form.

Glucopyranosylidene-spiro-imidazolinones, a New Ring System: Synthesis and Evaluation as Glycogen Phosphorylase Inhibitors by Enzyme Kinetics and X-ray Crystallography.

Epimeric series of aryl-substituted glucopyranosylidene-spiro-imidazolinones, an unprecedented new ring system, were synthesized from the corresponding Schiff bases of O-perbenzoylated (gluculopyranosylamine)onamides by intramolecular ring closure of the aldimine moieties with the carboxamide group elicited by N-bromosuccinimide in pyridine. Test compounds were obtained by Zemplén O-debenzoylation. Stereochemistry and ring tautomers of the new compounds were investigated by NMR, time-dependent density functional theory (TDDFT)-electronic circular dichroism, and DFT-NMR methods. Kinetic studies with rabbit muscle and human liver glycogen phosphorylases showed that the (R)-imidazolinones were 14-216 times more potent than the (S) epimers. The 2-naphthyl-substituted (R)-imidazolinone was the best inhibitor of the human enzyme (Ki 1.7 µM) and also acted on HepG2 cells (IC50 177 µM). X-ray crystallography revealed that only the (R) epimers bound in the crystal. Their inhibitory efficacy is based on the hydrogen-bonding interactions of the carbonyl oxygen and the NH of the imidazolinone ring.

Glucopyranosylidene-spiro-benzo[ b][1,4]oxazinones and -benzo[ b][1,4]thiazinones: Synthesis and Investigation of Their Effects on Glycogen Phosphorylase and Plant Growth Inhibition.

Glucopyranosylidene-spiro-benzo[ b][1,4]oxazinones were obtained via the corresponding 2-nitrophenyl glycosides obtained by two methods: (a) AgOTf-promoted glycosylation of 2-nitrophenol derivatives by O-perbenzoylated methyl (α-d-gluculopyranosyl bromide)heptonate or (b) Mitsunobu-type reactions of O-perbenzoylated methyl (α-d-gluculopyranose)heptonate with bulky 2-nitrophenols in the presence of diethyl azodicarboxylate (DEAD) and PPh3. Catalytic hydrogenation (H2-Pd/C) or partial reduction (e.g., H2-Pd/C, pyridine) of the 2-nitro groups led to spiro-benzo[ b][1,4]oxazinones and spiro-benzo[ b][1,4]-4-hydroxyoxazinones by spontaneous ring closure of the intermediate 2-aminophenyl or 2-hydroxylamino glycosides, respectively. The analogous 2-aminophenyl thioglycosides, prepared by reactions of O-perbenzoylated methyl (α-d-gluculopyranosyl bromide)heptonate with 2-aminothiophenols, were cyclized in m-xylene at reflux temperature to the corresponding spiro-benzo[ b][1,4]thiazinones. O-Debenzoylation was effected by Zemplén transesterification in both series. Spiro-configurations were determined by NMR and electronic circular dichroism time-dependent density functional theory (ECD-TDDFT) methods. Inhibition assays with rabbit muscle glycogen phosphorylase b showed (1' R)-spiro{1',5'-anhydro-d-glucitol-1',2-benzo[ b][1,4]oxazin-3(4 H)-one} and (1' R)-spiro{1',5'-anhydro-d-glucitol-1',2-benzo[ b][1,4]thiazin-3(4 H)-one} to be the most efficient inhibitors (27 and 28% inhibition at 625 µM, respectively). Plant growth tests with white mustard and garden cress indicated no effect except for (1' R)-4-hydroxyspiro{1',5'-anhydro-d-glucitol-1',2-benzo[ b][1,4]oxazin-3(4 H)-one} with the latter plant to show modest inhibition of germination (95% relative to control).

Synthesis of C-ß-d-glucopyranosyl derivatives of some fused azoles for the inhibition of glycogen phosphorylase.

Annulated C-ß-d-glucopyranosyl heterocycles were synthesized and tested as inhibitors of glycogen phosphorylase. 2-(ß-d-Glucopyranosyl)-1H-imidazo[4,5-b]pyridine was formed by ring-closure of O-perbenzoylated C-ß-d-glucopyranosyl formic acid with 2,3-diaminopyridine in the presence of triphenylphosphite. Cyclisations of bromomethyl 2,3,4,6-tetra-O-benzoyl-ß-d-glucopyranosyl ketone with a set of 2-aminoheterocycles resulted in constitutionally reversed C-ß-d-glucopyranosyl imidazoles fused by pyridine, pyrimidine, thiazole, 1,3,4-thiadiazole, benzothiazole and benzimidazole. O-Debenzoylation of the above compounds was effected by standard transesterification to get the test compounds. The 1H-imidazo[4,5-b]pyridine proved to be a low micromolar inhibitor (Ki = 21.1 µM) of rabbit muscle glycogen phosphorylase b, while the other heterocycles displayed weak or no inhibition against the same enzyme.

Synthesis of New C- and N-ß-d-Glucopyranosyl Derivatives of Imidazole, 1,2,3-Triazole and Tetrazole, and Their Evaluation as Inhibitors of Glycogen Phosphorylase.

The aim of the present study was to broaden the structure-activity relationships of C- and N-ß-d-glucopyranosyl azole type inhibitors of glycogen phosphorylase. 1-Aryl-4-ß-d-gluco-pyranosyl-1,2,3-triazoles were prepared by copper catalyzed azide-alkyne cycloadditions between O-perbenzylated or O-peracetylated ß-d-glucopyranosyl ethynes and aryl azides. 1-ß-d-Gluco-pyranosyl-4-phenyl imidazole was obtained in a glycosylation of 4(5)-phenylimidazole with O-peracetylated α-d-glucopyranosyl bromide. C-ß-d-Glucopyranosyl-N-substituted-tetrazoles were synthesized by alkylation/arylation of O-perbenzoylated 5-ß-d-glucopyranosyl-tetrazole or from a 2,6-anhydroheptose tosylhydrazone and arenediazonium salts. 5-Substituted tetrazoles were glycosylated by O-peracetylated α-d-glucopyranosyl bromide to give N-ß-d-glucopyranosyl-C-substituted-tetrazoles. Standard deprotections gave test compounds which were assayed against rabbit muscle glycogen phosphorylase b. Most of the compounds proved inactive, the best inhibitor was 2-ß-d-glucopyranosyl-5-phenyltetrazole (IC50 600 µM). These studies extended the structure-activity relationships of ß-d-glucopyranosyl azole type inhibitors and revealed the extreme sensitivity of such type of inhibitors towards the structure of the azole moiety.

A multidisciplinary study of 3-(ß-d-glucopyranosyl)-5-substituted-1,2,4-triazole derivatives as glycogen phosphorylase inhibitors: Computation, synthesis, crystallography and kinetics reveal new potent inhibitors.

3-(ß-d-Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PI = 0.82) and potent inhibitors with Ki's < 10 µM (AU-ROC = 0.86). Accordingly, in silico screening of 2335 new analogues exploiting the ZINC docking database was performed and nine predicted candidates selected for synthesis. The compounds were prepared in O-perbenzoylated forms by either ring transformation of 5-ß-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(ß-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(ß-d-glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low µM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles.