Giuseppe Carbonara

Carbonic anhydrase inhibitors are specific openers of skeletal muscle BK channel of K+-deficient rats.

Carbonic‐anhydrase (CA) inhibitors are used in the treatment of hypokalaemic periodic paralysis (hypoPP) and related channelopathies but their mechanism of action is unknown. Patch‐clamp experiments and molecular modeling investigations were performed to evaluate the mechanism of actions of CA inhibitors on skeletal muscle Ca2+‐activated‐K+ (BK) channel of K+‐deficient rats used as animal model of hypoPP. CA inhibitors showing different degree of CA inhibition such as acetazolamide (ACTZ), dichlorphenamide (DCP), hydrochlorthiazide (HCT), etoxzolamide (ETX), methazolamide (MTZ), and bendroflumethiazide (BFT), which lacks inhibitory effects on CA enzymes, were tested in vitro on BK channels. The application of ACTZ, BFT, ETX, and DCP to excised patches activated the BK channel with potency: ACTZ(DE50=7.3x10− 6M)>BFT(DE50=5.93x10− 5M)>ETX(DE50=1.17x10− 4M)>>DCP. In contrast, MTZ and HCT failed to activate the BK channel. Molecular modeling studies showed that the capability of CA inhibitors to open the BK channel was related to the presence in their structures of an intra‐molecular hydrogen bond with calculated inter‐atomic distances ranging between 1.82 A° and 3.01 A° and of an aromatic ring poor of electrons. ACTZ, BFT, ETX, and DCP showed these pharmacofores, while MTZ and HCT did not. Our data indicate that the activation of BK channel is a property of CA inhibitors that interact with the channel subunit/s and that this effect is not related to their capability to inhibit the CA enzymes.

Activation and Inhibition of Kidney CLC-K Chloride Channels by Fenamates

CLC-K Cl– channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-of-function mutations of CLC-Kb produce Bartters syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening. Using the voltage-clamp technique on channels expressed in Xenopus laevis oocytes, we found that niflumic acid (NFA) increases CLC-Ka and CLC-Kb currents in the 10 to 1000 μM range. Flufenamic acid (FFA) derivatives or high doses of NFA produced instead an inhibitory effect on CLC-Ka, but not on CLC-Kb, and on blocker-insensitive CLC-Ka mutants, indicating that the activating binding site is distinct from the blocker site. Evaluation of the sensitivity of CLC-Ka to derivatives of NFA and FFA together with a modeling study of these ligands allow us to conclude that one major characteristic of activating compounds is the coplanarity of the two rings of the molecules, whereas block requires a noncoplanar configuration. These molecules provide a starting point for identification of diuretics or drugs useful in the treatment of Bartters syndrome.

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 μM). 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 μM, 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.

Synthesis, Biological Evaluation, and Molecular Modeling Investigation of Chiral Phenoxyacetic Acid Analogues with PPARα and PPARγ 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 PPARα agonists as well as PPARγ 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 PPARα was reversed, and for this reason docking experiments were performed to rationalize this peculiar behavior.

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

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 ± 1 &mgr;mol/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.

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.

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.

Oxidation of tertiary polycycl1c amines by RuO4

Abstract The reactions of certain tertiary polycyclic amines such as N-benzyl-9-azabicy-clo- [3,3,1]-nonane, N-benzyl-5, 6-dihydro-11H-dibenz [b,e] azepine, and N-benzyl-1,2,3,4-tetrahydroisoquinoline with ruthenium tetroxide, taking place in both heterogeneous and homogeneous systems, have made it possible to establish the order of reactivity of the various methylene groups adjacent to the nitrogen with respect to this oxidizing agent.

Carboxylic acids and skeletal muscle chloride channel conductance: Effects on the biological activity induced by the introduction of an aryloxyalkyl group α to the carboxylic function of 4-chloro-phenoxyacetic acid

2-(4-Chloro-phenoxy)propanoic and 2-(4-chloro-phenoxy)butanoic acids are compounds known to block chloride membrane conductance in rat striated muscle by interaction with a specific receptor. In the present study, a series of chiral analogues has been prepared and tested to evaluate the influence of a second aryloxy moiety introduced in the side-chain at a variable distance from the stereogenic centre. The results show that this chemical modification is detrimental for biological activity which, however, is increased by lengthening the alkyl chain up to three methylenic groups, then decreases to remain constant in the next analogues of the series. A possible explanation for this is proposed on the basis of steric effects and/or different approach of the molecules to the receptor.