Elucidation of the synergistic action of Mentha Piperita essential oil with common antimicrobials.

Mentha piperita L. essential oil (EO) is employed for external use as antipruritic, astringent, rubefacient and antiseptic. Several studies demonstrated its significant antiviral, antifungal and antibacterial properties. The aim of this work is the study of the synergistic effects of M. piperita EO with antibacterials and antifungals that are widely available and currently prescribed in therapies against infections. The observed strong synergy may constitute a potential new approach to counter the increasing phenomenon of multidrug resistant bacteria and fungi. In vitro efficacy of the association M. piperita EO/drugs was evaluated against a large panel of Gram-positive and Gram-negative bacteria and yeast strains. The antimicrobial effects were studied by checkerboard microdilution method. The synergistic effect of M. piperita EO with gentamicin resulted in a strong growth inhibition for all the bacterial species under study. The synergistic effect observed for M. piperita EO and antifungals was less pronounced.

Repositioning of Endonuclear Receptors Binders as Potential Antibacterial and Antifungal Agents. Eptyloxìm: A Potential and Novel Gyrase B and Cytochrome Cyp51 Inhibitor.

A novel class of antibacterial and antifungal agents is here identified by means of dockings and virtual screening techniques. Biological data proved the initial effort, formulated on the structure similarity of nuclear receptors binders with known quinolones or thiazole derivatives, to reposition PPARs agonists as likely bacterial type II topoisomerases inhibitors.

Synthesis, in vitro evaluation, and molecular modeling investigation of benzenesulfonimide peroxisome proliferator-activated receptors α antagonists.

Recent evidences suggest a moderate activation of Peroxisome Proliferator-Activated Receptors (PPARs) could be favorable in metabolic diseases, reducing side effects given from full agonists. PPAR partial agonists and antagonists represent, to date, interesting tools to better elucidate biological processes modulated by these receptors. In this work are reported new benzenesulfonimide compounds able to block PPARα, synthesized and tested by transactivation assays and gene expression analysis. Some of these compounds showed a dose-dependent antagonistic behavior on PPARα, submicromolar potency, different profiles of selectivity versus PPARγ, and a repressive effect on CPT1A expression. Dockings and molecular dynamics on properly selected benzenesulfonimide derivatives furnished fresh insights into the molecular determinant most likely responsible for PPARα antagonism.

Kidney CLC-K chloride channels inhibitors: structure-based studies and efficacy in hypertension and associated CLC-K polymorphisms.

OBJECTIVE: Alterations in the handling of renal salt reabsorption may contribute to interindividual differences in blood pressure regulation and susceptibility to hypertension. CLC-K chloride channels and their accessory subunit barttin play a pivotal role in kidney by controlling chloride and water absorption. Compounds selective for CLC-Ks, such as the benzofuran derivative MT-189, may have a significant therapeutic potential. Here, we assessed the feasibility of using CLC-K blockers in hypertension and aimed at enhancing drug inhibitory affinity. METHODS AND RESULTS: We demonstrated that acute in-vivo administration of MT-189 to spontaneously hypertensive rats (SHR) caused a reduction of blood pressure and defined the CLC-K/barttin gene expression pattern in kidney of SHR in comparison with normotensive Wistar-Kyoto rats. Based on MT-189, we designed and tested a new series of benzofuran derivatives on CLC-K chloride channels heterologously expressed in HEK293 cells. These studies enabled us to elucidate the causative molecular relationship for obtaining the most potent and selective inhibitor (SRA-36) described so far, with an IC50 of 6.6 ±â€Š1 µmol/l. The biophysical and pharmacological characterization of A447T CLC-Ka and Y315F CLC-Ka, both polymorphisms associated with hypertension, showed that SRA-36 is an efficacious inhibitor of the chloride currents sustained by these polymorphisms. Molecular docking studies allowed hypothesizing an inhibition mechanism for the considered ligands, laying the foundations for the rational design of new and more effective CLC-K inhibitors. CONCLUSION: The SRA-36 molecule represents a new potential therapeutic option for hypertension.

Design, synthesis and biological evaluation of a class of bioisosteric oximes of the novel dual peroxisome proliferator-activated receptor α/γ ligand LT175.

The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARα/γ dual agonist, have been investigated. This modification allowed the identification of new bioisosteric ligands with fairly good activity on PPARα and fine-tuned moderate activity on PPARγ. For the most interesting compound (S)-3, docking studies in PPARα and PPARγ provided a molecular explanation for its different behavior as full and partial agonist of the two receptor isotypes, respectively. A further investigation of this compound was carried out performing gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible mechanism through which this ligand could be useful in the treatment of metabolic disorders. The higher induction of the expression of some genes, compared to selective agonists, seems to confirm the importance of a dual PPARα/γ activity which probably involves a synergistic effect on both receptor subtypes.

Growth hormone secretagogues exert differential effects on skeletal muscle calcium homeostasis in male rats depending on the peptidyl/nonpeptidyl structure.

The orexigenic and anabolic effects induced by ghrelin and the synthetic GH secretagogues (GHSs) are thought to positively contribute to therapeutic approaches and the adjunct treatment of a number of diseases associated with muscle wasting such as cachexia and sarcopenia. However, many questions about the potential utility and safety of GHSs in both therapy and skeletal muscle function remain unanswered. By using fura-2 cytofluorimetric technique, we determined the acute effects of ghrelin, as well as of peptidyl and nonpeptidyl synthetic GHSs on calcium homeostasis, a critical biomarker of muscle function, in isolated tendon-to-tendon male rat skeletal muscle fibers. The synthetic nonpeptidyl GHSs, but not peptidyl ghrelin and hexarelin, were able to significantly increase resting cytosolic calcium [Ca²âº]i. The nonpeptidyl GHS-induced [Ca²âº]i increase was independent of GHS-receptor 1a but was antagonized by both thapsigargin/caffeine and cyclosporine A, indicating the involvement of the sarcoplasmic reticulum and mitochondria. Evaluation of the effects of a pseudopeptidyl GHS and a nonpeptidyl antagonist of the GHS-receptor 1a together with a drug-modeling study suggest the conclusion that the lipophilic nonpeptidyl structure of the tested compounds is the key chemical feature crucial for the GHS-induced calcium alterations in the skeletal muscle. Thus, synthetic GHSs can have different effects on skeletal muscle fibers depending on their molecular structures. The calcium homeostasis dysregulation specifically induced by the nonpeptidyl GHSs used in this study could potentially counteract the beneficial effects associated with these drugs in the treatment of muscle wasting of cachexia- or other age-related disorders.

Molecular determinants for nuclear receptors selectivity: chemometric analysis, dockings and site-directed mutagenesis of dual peroxisome proliferator-activated receptors α/γ agonists.

A series of previously synthesized chiral derivatives of clofibric and phenylacetic acids, acting as dual agonists towards the peroxisome proliferator-activated receptors (PPARs) α and γ, was taken into account, and the efficacy of these compounds was analyzed by means of 2D-, 3D-QSAR and docking studies with the goal to gain deeper insights into the three-dimensional determinants governing ligands selectivity for PPARs. By multiregressional analysis a correlation between the lipophilicity and PPARα activity was found, whereas for PPARγ the correlation was achieved once efficacy was related to the presence of polar groups on agonists scaffold. Docking of these compounds further corroborated this hypothesis, and then provided a valid support for subsequent chemometric analysis and pharmacophore models development for both receptors subtypes. Computational results suggested site directed mutagenesis experiments which confirmed the importance of amino acid residues in PPAR activity, allowing the identification of critical hotspots most likely taking over PPARs selectivity.

Regular CPAP utilization reduces nasal inflammation assessed by nasal cytology in obstructive sleep apnea syndrome.

OBJECTIVES: To analyze nasal inflammation in a group of patients with obstructive sleep apnea syndrome (OSAS) by means of nasal cytology and to describe the changes induced by continuous positive air pressure (CPAP) treatment. SUBJECTS AND METHODS: Thirty-two consecutive patients affected by OSAS (mean age 46.9 years) and 13 control subjects (mean age 49.1 years) were enrolled. Detailed clinical, laboratory, and polysomnographic studies were obtained in all participants and, in particular, nasal cytology was performed; inflammatory cells (neutrophils, eosinophils, mast cells, lymphocytes), bacteria, and spores were counted. A subgroup of 19 OSAS patients underwent regular nasal CPAP for eight weeks while the remaining 13 were noncompliant. Nasal cytology was repeated after eight weeks in all patients and controls. RESULTS: All patients with OSAS were affected by some form of rhinopathy, mostly subclinical, which was not found to influence compliance to CPAP. Regular CPAP treatment induced a significant reduction of cell infiltration (neutrophils, eosinophils, lymphocytes, and muciparous cells), which was not seen in nontreated patients. CONCLUSION: Nasal inflammation/infection is a very frequent finding in OSAS and can be reverted by the regular use of CPAP.

Might the observed α(2A)-adrenoreceptor agonism or antagonism of allyphenyline analogues be ascribed to different molecular conformations?

We recently reported that the α(2)-adrenoreceptor (AR) ligand allyphenyline (9) significantly enhanced morphine analgesia (due to its α(2C)-AR agonism), was devoid of sedative side effects (due to its α(2A)-AR antagonism), prevented and reversed morphine tolerance and dependence. To highlight the molecular characteristics compatible with this behaviour and to obtain novel agents potentially useful in chronic pain and opioid addiction management, the allyl group of 9 was replaced by substituents of moderate steric bulk (MR) and positive or negative lipophilic (π) and electronic (σ) contributions in all the possible combinations. Effective novel α(2C)-agonists/α(2A)-antagonists (2, 3, 10, 12, and 17) were obtained. This study also demonstrated that contradictory combinations of the physicochemical parameters were similarly able to induce the α(2A)-activation. Since we had previously observed that the absolute configuration affected only the potency, but not the functional profile of the ligands, we hypothesized that the α(2A)-activation was governed by a ligand preferred conformation. From a structural overlay investigation it emerged that an extended conformation appeared to be associated with dual α(2C)-agonism/α(2A)-antagonism, whereas a folded conformation associated with α(2C)-/α(2A)-agonism.

Synthesis, biological evaluation and molecular investigation of fluorinated peroxisome proliferator-activated receptors α/γ dual agonists.

PPARs are transcription factors that govern lipid and glucose homeostasis and play a central role in cardiovascular disease, obesity, and diabetes. Thus, there is significant interest in developing new agonists for these receptors. Given that the introduction of fluorine generally has a profound effect on the physical and/or biological properties of the target molecule, we synthesized a series of fluorinated analogs of the previously reported compound 2, some of which turned out to be remarkable PPARα and PPARγ dual agonists. Docking experiments were also carried out to gain insight into the interactions of the most active derivatives with both receptors.

Structural nucleotide analogs are potent activators/inhibitors of pancreatic ß cell KATP channels: an emerging mechanism supporting their use as antidiabetic drugs.

The 2H-1,4-benzoxazine derivatives are novel drugs structurally similar to nucleotides; however, their actions on the pancreatic ß cell ATP-sensitive K+ (KATP) channel and on glucose disposal are unknown. Therefore, the effects of the linear/branched alkyl substituents and the aliphatic/aromatic rings at position 2 of the 2H-1,4-benzoxazine nucleus on the activity of these molecules against the pancreatic ß cell KATP channel and the Kir6.2ΔC36 subunit were investigated using a patch-clamp technique. The effects of these compounds on glucose disposal that followed glucose loading by intraperitoneal glucose tolerance test and on fasting glycemia were investigated in normal mice. The 2-n-hexyl analog blocked the KATP (IC50 = 10.1 × 10⁻9 M) and Kir6.2ΔC36 (IC50 = 9.6 × 10⁻9 M) channels, which induced depolarization. In contrast, the 2-phenyl analog was a potent opener (drug concentration needed to enhance the current by 50% = 0.04 × 10⁻9 M), which induced hyperpolarization. The ranked order of the potency/efficacy of the analog openers was 2-phenyl > 2-benzyl > 2-cyclohexylmethyl. The 2-phenylethyl and 2-isopropyl analogs were not effective as blockers/openers. The 2-n-hexyl (2-10 mg/kg) and 2-phenyl analogs (2-30 mg/kg) reduced and enhanced the glucose areas under the curves, respectively, after glucose loading in mice. These compounds did not affect the fasting glycemia as is observed with glibenclamide. The linear alkyl chain and the aromatic ring at position 2 of the 1,4-benzoxazine nucleus are the determinants, which confer the KATP channel blocking action with glucose-lowering effects and the opening action with increased glucose levels, respectively. The opening/blocking actions of these compounds mimic those that were observed with ATP and ADP. The results support the use of these compounds as novel antidiabetic drugs.

Novel imidazoline compounds as partial or full agonists of D2-like dopamine receptors inspired by I2-imidazoline binding sites ligand 2-BFI.

Based on the well known biological versatility of the imidazoline nucleus, we prepared the novel derivatives 3a-k inspired by 2-BFI scaffold to assess imidazoline molecules as D(2)-like dopamine receptor ligands. Conservative chemical modifications of the lead structure, such as the introduction of an hydroxy group in the aromatic ring alone or associated with N-benzyl substitution, provided partial (3f) or nearly full (3e and 3h) agonists, all endowed with D(2)-like potency comparable to that of dopamine.

Synthesis, biological evaluation, and molecular modeling investigation of chiral 2-(4-chloro-phenoxy)-3-phenyl-propanoic acid derivatives with PPARalpha and PPARgamma agonist activity.

PPARs are ligand-activated transcription factors that govern lipid and glucose homeostasis and play a central role in cardiovascular disease, obesity, and diabetes. Herein, we present screening results for a series of chiral 2-(4-chloro-phenoxy)-3-phenyl-propanoic acid derivatives, some of which are potent PPARgamma agonists as well as PPARalpha agonists. To investigate the binding modes of the most interesting derivatives into the PPARalpha and PPARgamma binding clefts and evaluate their agonist activity, docking experiments, molecular dynamics simulations, and MM-PBSA analysis were performed.

Molecular determinants for the activating/blocking actions of the 2H-1,4-benzoxazine derivatives, a class of potassium channel modulators targeting the skeletal muscle KATP channels.

The 2H-1,4-benzoxazine derivatives are modulators of the skeletal muscle ATP-sensitive-K(+) channels (K(ATP)), activating it in the presence of ATP but inhibiting it in the absence of nucleotide. To investigate the molecular determinants for the activating/blocking actions of these compounds, novel molecules with different alkyl or aryl-alkyl substitutes at position 2 of the 1,4-benzoxazine ring were prepared. The effects of the lengthening of the alkyl chain and of branched substitutes, as well as of the introduction of aliphatic/aromatic rings on the activity of the molecules, were investigated on the skeletal muscle K(ATP) channels of the rat, in excised-patch experiments, in the presence or absence of internal ATP (10(-4) M). In the presence of ATP, the 2-n-hexyl analog was the most potent activator (DE(50) = 1.08 x 10(-10) M), whereas the 2-phenylethyl was not effective. The rank order of efficacy of the openers was 2-n-hexyl > or =2-cyclohexylmethyl >2-isopropyl = 2-n-butyl > or = 2-phenyl > or = 2-benzyl = 2-isobutyl analogs. In the absence of ATP, the 2-phenyl analog was the most potent inhibitor (IC(50) = 2.5 x 10(-11) M); the rank order of efficacy of the blockers was 2-phenyl > or = 2-n-hexyl > 2-n-butyl > 2-cyclohexylmethyl, whereas the 2-phenylethyl, 2-benzyl, and 2-isobutyl 1,4-benzoxazine analogs were not effective; the 2-isopropyl analog activated the K(ATP) channel even in the absence of nucleotide. Therefore, distinct molecular determinants for the activating or blocking actions for these compounds can be found. For example, the replacement of the linear with the branched alkyl substitutes at the position 2 of the 1,4-benzoxazine nucleus determines the molecular switch from blockers to openers. These compounds were 100-fold more potent and effective as openers than other KCO against the muscle K(ATP) channels.

Molecular switch for CLC-K Cl- channel block/activation: optimal pharmacophoric requirements towards high-affinity ligands.

ClC-Ka and ClC-Kb Cl(-) channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka. The major molecular determinant that distinguishes activators from blockers is the level of planarity of the aromatic portions of the molecules: only molecules with perfectly coplanar aromatic groups display potentiating activity. Combining several molecular features of various CLC-K ligands, we discovered that phenyl-benzofuran carboxylic acid derivatives yield the most potent ClC-Ka inhibitors so far described (affinity <10 microM). The increase in affinity compared with 3-phenyl-2-p-chlorophenoxy-propionic acid (3-phenyl-CPP) stems primarily from the conformational constraint provided by the phenyl-benzofuran ring. Several other key structural elements for high blocking potency were identified through a detailed structure-activity relationship study. Surprisingly, some benzofuran-based drugs inhibit ClC-Kb with a similar affinity of <10 microM, thus representing the first inhibitors for this CLC-K isoform identified so far. Based on our data, we established a pharmacophore model that will be useful for the development of drugs targeting CLC-K channels.

Enantiomeric separation of 2-aryloxyalkyl- and 2-arylalkyl-2-aryloxyacetic acids on a Penicillin G Acylase-based chiral stationary phase: influence of the chemical structure on retention and enantioselectivity.

The chiral recognition mechanism of Penicillin G Acylase (PGA) was investigated with a set of 18 new chiral acidic compounds. A series of 2-aryloxyalkyl- and 2-arylalkyl-2-aryloxyacetic acids in which the absolute configuration has been reported to exert a strong influence on pharmacological activity, were synthesized and analysed on PGA-based chiral stationary phase (CSP) and 11 racemates were completely resolved with a mobile phase composed of 50 mM phosphate buffer (pH 7.0). The influence of structural variations of analytes on retention and enantioselectivity was investigated by application of molecular modelling studies. Docking experiments were also carried out to rationalize the observed enantioselective behaviour. The computation approach revealed to be helpful in elucidating the molecular basis of the enantioselectivity observed on PGA-CSP.

Synthesis, biological evaluation, and molecular modeling investigation of chiral phenoxyacetic acid analogues with PPARalpha and PPARgamma agonist activity.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that govern lipid and glucose homeostasis, and play a central role in cardiovascular disease, obesity, and diabetes. Thus, there is significant interest in developing new and specific agonists for these receptors. Herein we present screening results for a series of chiral phenoxyacetic acid analogues, some of which are potent PPARalpha agonists as well as PPARgamma agonists. The stereochemistry of these compounds plays an important role in determining their activity; the S isomers were observed to be more active than the corresponding R isomers. Interestingly, for one of these analogues, the stereoselectivity toward PPARalpha was reversed, and for this reason docking experiments were performed to rationalize this peculiar behavior.

Exploring the molecular basis of the enantioselective binding of penicillin G acylase towards a series of 2-aryloxyalkanoic acids: a docking and molecular dynamics study.

In the present paper, molecular modeling studies were undertaken in order to shed light on the molecular basis of the observed enantioselectivity of penicillin G acylase (PGA), a well known enzyme for its industrial applications, towards 16 racemic 2-aryloxyalkanoic acids, which have been reported to affect several biological systems. With this intention docking calculations and MD simulations were performed. Docking results indicated that the (S)-enantiomers establish several electrostatic interactions with SerB1, SerB386 and ArgB263 of PGA. Conversely, the absence of specific polar interactions between the (R)-enantiomers and ArgB263 seems to be the main reason for the different binding affinities observed between the two enantiomers. Results of molecular dynamics simulations demonstrated that polar interactions are responsible for both the ligand affinity and PGA enantiospecificity. Modeling calculations provided possible explanations for the observed enantioselectivity of the enzyme that rationalize available experimental data and could be the basis for future protein engineering efforts.

Elucidation of the enantioselective recognition mechanism of a penicillin G acylase-based chiral stationary phase towards a series of 2-aryloxy-2-arylacetic acids.

A series of structurally related 2-aryloxy-2-arylacetic acids (1-3, 5-16) together with a thioisostere derivative (4) have been synthesized and characterized by GC-MS and 1H NMR. The designed compounds were analyzed on a Penicillin G Acylase chiral stationary phase (PGA-CSP) and the influence of the structure variations on retention and enantioselectivity was investigated. The chromatographic study includes the direct separation of the enantiomers of the synthesized compounds and the determination of the elution order of selected racemic mixtures. 10 out of 16 racemates were separated; high chromatographic enantioseparation factors (alpha > 2) were achieved for some compounds. For the enantiomers of four compounds whose absolute configuration was known (1, 3, 12, 16), the elution order was R:S with the exception of 2-(4-chloro-phenoxy)phenylacetic acid (1), for which the elution order was reversed. Preliminary molecular modeling studies suggest that both polar and charge-transfer interactions as well as steric effects play an important role in determining the retention factors and the enantioselectivities observed.