Maria Frosini

A Novel Approach for Predicting P-glycoprotein (ABCB1) Inhibition Using Molecular Interaction Fields

P-glycoprotein (Pgp or ABCB1) is an ABC transporter protein involved in intestinal absorption, drug metabolism, and brain penetration, and its inhibition can seriously alter a drugs bioavailability and safety. In addition, inhibitors of Pgp can be used to overcome multidrug resistance. Given this dual purpose, reliable in silico procedures to predict Pgp inhibition are of great interest. A large and accurate literature collection yielded more than 1200 structures; a model was then constructed using various molecular interaction field-based technologies, considering pharmacophoric features and those physicochemical properties related to membrane partitioning. High accuracy was demonstrated internally with two different validation sets and, moreover, using a number of molecules, for which Pgp inhibition was not experimentally available but was evaluated in-house. All of the validations confirmed the robustness of the model and its suitability to help medicinal chemists in drug discovery. The information derived from the model was rationalized as a pharmacophore for competitive Pgp inhibition.

Potentiation of mitochondrial Ca2+ sequestration by taurine

The effects of taurine (2-aminoethanesulphonic acid) and its analogues, 2-aminoethylarsonic acid, 2-hydroxyethanesulphonic (isethionic) acid, 3-aminopropanesulphonic acid, 2-aminoethylphosphonic acid, and N,N-dimethyltaurine, were studied on the transport of Ca2+ by mitochondria isolated from rat liver. Taurine enhanced Ca2+ uptake in an apparently saturable process, with a Km value of about 2.63 mM. Taurine behaved as an uncompetitive activator of Ca2+ uptake, increasing both the apparent Km and Vmax values of the process. This effect was not modified in the presence of cyclosporin A (CsA). N,N-Dimethyltaurine also stimulated Ca2+ uptake at higher concentrations, but there was no evidence that the process was saturable over the concentration range used (1-10 mM). Aminoethylarsonate was a weak inhibitor of basal Ca2+ uptake, but inhibited that stimulated by taurine in an apparently competitive fashion (Ki = 0.05 mM). The other analogues had no significant effects on this process. Taurine either in the presence or the absence of CsA had no effect on Ca2+ release induced by 200 nM ruthenium red. Thus, the mechanism of taurine-enhanced Ca2+ accumulation appears to involve stimulation of Ca2+ uptake via the uniport system rather than inhibition of Ca2+ release via the ion (Na+/Ca2+ and/or H+/Ca2+) exchangers or by taurine modulating the permeability transition of the mitochondrial inner membrane. Overall, these findings indicate an interaction of taurine with an as yet unidentified mitochondrial site which might regulate the activity of the uniporter. The unique role of taurine in modulating mitochondrial Ca2+ homeostasis might be of particular importance under pathological conditions that are characterised by cell Ca2+ overload, such as ischaemia and oxidative stress.

Interactions of taurine and structurally related analogues with the GABAergic system and taurine binding sites of rabbit brain

The aim of this study was to find taurinergic compounds that do not interact with brain GABA ergic systems. Washed synaptic membranes (SM) from whole rabbit brain were able to bind [3H]muscimol. Saturation experiments of the binding of [3H]GABA to GABAB receptors showed that SM possess two binding components; twice Triton X‐100‐treated SM contained 0.048 mmol endogenous taurine/kg protein and bound [3H]taurine in a saturable manner (Kd=249.0±6.3 nM and Bmax=3.4±1.0 pmol mg−1 prot). Among the 19 structural analogues of taurine, 6‐aminomethyl‐3‐methyl‐4H‐1,2,4‐benzothiadiazine 1,1‐dioxide (TAG), 2‐aminoethylarsonic (AEA), 2‐hydroxyethanesulfonic (ISE) and (±)cis‐2‐aminocyclohexane sulfonic acids (CAHS) displaced [3H]taurine binding (Ki=0.13, 0.13, 13.5 and 4.0 μM, respectively). These analogues did not interact with GABAA and GABAB receptors and did not affect taurine‐ and GABA‐uptake systems and GABA‐transaminase activity. 3‐Aminopropanesulfonic acid (OMO), β‐alanine, pyridine‐3‐sulfonic acid, N,N,N‐trimethyltaurine (TMT), 2‐(guanidino)ethanesulfonic acid (GES), ethanolamine‐O‐sulphate, N,N‐dimethyltaurine (DMT), taurine and (±)piperidine‐3‐sulfonic acid (PSA) inhibited [3H]muscimol binding to GABAA receptors with different affinities (Ki=0.013, 7.9, 24.6, 47.5, 52.0, 91.0, 47.5, 118.1 and 166.3 μM, respectively). Taurine, 2‐aminoethylphosphonic acid, DMT, TMT and OMO inhibited the binding of [3H]GABA to GABAB receptors with Kis in the μM range (0.8, 3.5, 4.4, 11.3 and 5.0, respectively). GES inhibited taurine uptake (IC50=3.72 μM) and PSA GABA transaminase activity (IC50=103.0 μM). In conclusion, AEA, TAG, ISE and CAHS fulfill the criteria for taurinergic agents.

Rectal temperature and prostaglandin E2 increase in cerebrospinal fluid of conscious rabbits after intracerebroventricular injection of hemoglobin

Abstract Fever accompanies subarachnoid hemorrhage (SAH) in the majority of patients. In a previous study, hemoglobin (Hb) was shown to catalyze in vitro, under aerobic conditions, the conversion of arachidonic acid to prostaglandin E2 (PGE2) and prostaglandin F2α. The aim of the present work was to assess whether this pathway also operates in vivo and to provide a mechanism to explain post-SAH fever. To this end, PGE2 concentration was determined in cerebrospinal fluid (CSF) of conscious rabbits chronically cannulated in the lateral ventricle and cisterna magna, following intracerebroventricular (i.c.v.) injection of 10 µg or 100 µg of commercial rabbit bicrystallized Hb as a model of SAH. Before i.c.v. injection, Hb solutions were filtered on a polimixin-B column to remove substantially, by over 90%, endotoxin-like substances. Results show that in nine rabbits injection of 10 µg Hb did not significantly modify body temperature or significantly alter CSF PGE2 content. On the contrary, in nine rabbits, injection of 100 µg Hb produced a significant increase in core temperature which was accompanied by a significant increase in CSF PGE2. When data related to these two parameters from the 9 control and 18 Hb-treated rabbits were analyzed as a single group, a linear, positive, and highly significant correlation was found. These findings indicate that, once Hb is released into the subarachnoid space during SAH, it enhances CSF PGE2 content and elicits hyperthermia, thus offering an explanation for the fever that is an aggravating condition in most SAH patients.

Synthesis and biological evaluation of amidine, guanidine, and thiourea derivatives of 2-amino(6-trifluoromethoxy)benzothiazole as neuroprotective agents potentially useful in brain diseases.

A series of amidine, thiourea, and guanidine derivatives of 2-amino-6-(trifluoromethoxy)benzothiazole termed 2, 3, and 4, respectively, and structurally related to riluzole, a neuroprotective drug in many animal models of brain disease, have been synthesized. The biological activity of compounds 2a-e, 3a-f, and 4a,b was preliminarily tested by means of an in vitro protocol of ischemia/reperfusion injury. The results demonstrated that 2c and 3a-d significantly attenuated neuronal injury. Selected for testing of their antioxidant properties, compounds 3a-d were shown to be endowed with a direct ROS scavenging activity. Compounds 3b and 3d were also evaluated for their activity on voltage-dependent Na(+) and Ca(2+) currents in neurons from rat piriform cortex. At 50 microM, compound 3b inhibited the transient Na(+) current to a much smaller extent than riluzole, whereas 3d was almost completely ineffective.

Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 4. Identification of New Potent and Selective Ligands for the Cannabinoid Type 2 Receptor with Diverse Substitution Patterns and Antihyperalgesic Effects in Mice

Experimental evidence suggests that selective CB2 receptor modulators may provide access to antihyperalgesic agents devoid of psychotropic effects. Taking advantage of previous findings on structure-activity/selectivity relationships for a class of 4-quinolone-3-carboxamides, further structural modifications of the heterocyclic scaffold were explored, leading to the discovery of the 8-methoxy derivative 4a endowed with the highest affinity and selectivity ever reported for a CB2 ligand. The compound, evaluated in vivo in the formalin test, behaved as an inverse agonist by reducing at a dose of 6 mg/kg the second phase of the formalin-induced nocifensive response in mice.

A specific taurine recognition site in the rabbit brain is responsible for taurine effects on thermoregulation

Taurine and GABA are recognized as endogenous cryogens. In a previous study, some structural analogues of taurine, namely 6‐aminomethyl‐3‐methyl‐4H‐1,2,4‐benzothiadiazine 1,1‐dioxide (TAG), 2‐aminoethylarsonic (AEA), 2‐hydroxyethanesulfonic (ISE) and (±)cis‐2‐aminocyclohexane sulfonic acids (CAHS) have been shown to displace [3H]taurine binding from rabbit brain synaptic membrane preparations, without interacting either with GABA‐ergic systems, nor with taurine uptake mechanism, thus behaving like direct taurinergic agents. To answer the question whether the role of taurine as an endogenous cryogen depends on the activation of GABA receptors or that of specific taurine receptor(s), taurine or the above structural analogues were injected intracerebroventricularly in conscious, restrained rabbits singularly or in combination and their effects on rectal (RT)‐ and ear–skin temperature and gross motor behavior (GMB) were monitored. Taurine (1.2 × 10−6–4.8 × 10−5 mol) induced a dose‐related hypothermia, vasodilation at ear vascular bed and inhibition of GMB. CAHS, at the highest dose tested (4.8 × 10−5 mol) induced a taurine‐like effect either on RT or GMB. On the contrary ISE, injected at the same doses of taurine, induced a dose‐related hyperthermia, vasoconstriction and excitation of GMB. AEA and TAG caused a dose‐related hyperthermia, but at doses higher than 1.2 × 10−7 mol caused death within 24 h after treatment. CAHS (4.8 × 10−5 mol) antagonized the hyperthermic effect induced by TAG (1.2 × 10−6 mol), AEA (1.2 × 10−8 mol) or ISE (4.8 × 10−5 mol). In conclusion, these findings may indicate the existence of a recognition site specific for taurine, responsible for its effects on thermoregulation.

N,N-bis(cyclohexanol)amine aryl esters: a new class of highly potent transporter-dependent multidrug resistance inhibitors.

A new series of Pgp-dependent MDR inhibitors having a N,N-bis(cyclohexanol)amine scaffold was designed on the basis of the frozen analogue approach. The scaffold chosen gives origin to different geometrical isomers. The new compounds showed a wide range of potencies and efficacies on doxorubicin-resistant erythroleukemia K562 cells in the pirarubicin uptake assay. The most interesting compounds (isomers of 3) were studied further evaluating their action on the ATPase activity present in rat small intestine membrane vesicles and doxorubicin cytotoxicity potentiation on K562 cells. The latter assay was performed also on the isomers of 4. The four isomers of each set present different behavior in each of these tests. Compound 3d shows the most promising properties as it was able to completely reverse Pgp-dependent pirarubicin extrusion at low nanomolar concentration, inhibited ATPase activity at 5 x 10(-9) and increased the cytotoxicity of doxorubicin with a reversal fold (RF) of 36.4 at 3 microM concentration.

Changes in rectal temperature and ECoG spectral power of sensorimotor cortex elicited in conscious rabbits by i.c.v. injection of GABA, GABAA and GABAB agonists and antagonists

In order to ascertain whether both GABAA and GABAB, or only GABAB receptors, directly modulate thermoregulation in conscious rabbits, GABAA/GABAB agonist and antagonist agents were injected intracerebroventricularly in conscious rabbits while monitoring changes in rectal temperature (RT), gross motor behaviour (GMB) and electrocorticogram (ECoG) power spectra (ps) from sensorimotor cortices. GABA (48 μmol), nipecotic acid (50 nmol), THIP (60 nmol), muscimol (18 nmol) and baclofen (8 nmol) induced hypothermia (−ΔRTmax values of 1.70±0.1, 1.4±0.2, 1.0±0.4, 1.1±0.2 and 1.6±0.3°C, respectively), accompanied by inhibition of GMB and ECoG synchronization. THIP increased ps at δ frequency band (1.1−3.3 Hz), while GABA, nipecotic acid, muscimol and baclofen did the same at both δ and θ (4.6−6.5 Hz) frequency bands. ECoG ps changes were concomitant or even preceded hypothermia. Bicuculline (1.8 nmol) induced hyperthermia (ΔRTmax 1.2±0.5°C) and slight excitation of GMB, while CGP35348 (1.2 μmol) did not affect RT nor GMB. Both compounds did not affect ECoG ps. Bicuculline potentiated muscimol‐induced hypothermia, inhibition of GMB and synchronization of ECoG, while CGP35348 fully antagonized these effects. In conclusion, the present results, while confirming the prevailing role of GABAB, also outline a direct involvement of GABAA receptors in the central mechanisms of thermoregulation. Ascending inhibition towards discrete cortical areas controlling muscular activity and thermogenesis may result from GABA receptor activation in neurones proximal to the ventricles, thus contributing to hypothermia, although hypothermia‐induced reduction of neuronal activity of these cortical areas cannot be ruled out.

Role of intracellular Ca2+ and calmodulin/MAP kinase kinase/extracellular signal-regulated protein kinase signalling pathway in the mitogenic and antimitogenic effect of nitric oxide in glia- and neurone-derived cell lines.

To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen‐activated protein kinases [extracellular signal‐regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)‐1‐[2‐aminiethyl]‐N‐[2‐ammonioethyl]amino]diazen‐1‐ium‐1,2diolate (DETA/NO) concentrations (10 µm) determined a gradual, moderate elevation in [Ca2+]i (46.8 ± 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 ± 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 ± 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down‐regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50–300 µm) of DETA/NO negatively regulated cell proliferation via a Ca2+‐independent mechanism.