Paolo Tortorella

Use of readily available chiral compounds related to the betti base in the enantioselective addition of diethylzinc to aryl aldehydes

Abstract Readily available members of the family of chiral non-racemic aminonaphthols related to the Betti base 1 were tested as complexing agents in the catalytic enantioselective addition of diethylzinc to aryl aldehydes. The use of these bases gave high ee values (up to > 99%). The highest ee values were obtained with the tertiary aminonaphthol 2. An important role was played by the solvent. The effect of the nature and the position of the substituents on the aromatic ring of the aldehyde was also investigated.

Insights into the mechanism of partial agonism: crystal structures of the peroxisome proliferator-activated receptor gamma ligand-binding domain in the complex with two enantiomeric ligands.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose and lipid metabolism. They are activated by natural ligands, such as fatty acids, and are also targets of synthetic antidiabetic and hypolipidemic drugs. By using cell-based reporter assays, we studied the transactivation activity of two enantiomeric ureidofibrate-like derivatives. In particular, we show that the R-enantiomer, (R)-1, is a full agonist of PPARγ, whereas the S-enantiomer, (S)-1, is a less potent partial agonist. Most importantly, we report the x-ray crystal structures of the PPARγ ligand binding domain complexed with the R- and the S-enantiomer, respectively. The analysis of the two crystal structures shows that the different degree of stabilization of the helix 12 induced by the ligand determines its behavior as full or partial agonist. Another crystal structure of the PPARγ·(S)-1 complex, only differing in the soaking time of the ligand, is also presented. The comparison of the two structures of the complexes with the partial agonist reveals significant differences and is suggestive of the possible coexistence in solution of transcriptionally active and inactive forms of helix 12 in the presence of a partial agonist. Mutation analysis confirms the importance of Leu465, Leu469, and Ile472 in the activation by (R)-1 and underscores the key role of Gln286 in the PPARγ activity.

Crystal Structure of the Peroxisome Proliferator-Activated Receptor γ (PPARγ) Ligand Binding Domain Complexed with a Novel Partial Agonist: A New Region of the Hydrophobic Pocket Could Be Exploited for Drug Design

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect to the marketed antidiabetic agents thiazolidinediones (TZDs) and the dual-agonists glitazars is highly desired. We report the crystal structure and activity of the two enantiomeric forms of a clofibric acid analogue, respectively complexed with the ligand-binding domain (LBD) of PPARgamma, and provide an explanation on a molecular basis for their different potency and efficacy against PPARgamma. The more potent S-enantiomer is a dual PPARalpha/PPARgamma agonist which presents a partial agonism profile against PPARgamma. Docking of the S-enantiomer in the PPARalpha-LBD has been performed to explain its different subtype pharmacological profile. The hypothesis that partial agonists show differential stabilization of helix 3, when compared to full agonists, is also discussed. Moreover, the structure of the complex with the S-enantiomer reveals a new region of the PPARgamma-LBD never sampled before by other ligands.

Collagenase-2 Deficiency or Inhibition Impairs Experimental Autoimmune Encephalomyelitis in Mice

Matrix metalloproteinases (MMPs) have been implicated in a variety of human diseases, including neuroimmunological disorders such as multiple sclerosis. However, the recent finding that some MMPs play paradoxical protective roles in these diseases has made necessary the detailed study of the specific function of each family member in their pathogenesis. To determine the relevance of collagenase-2 (MMP-8) in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we have performed two different analyses involving genetic and biochemical approaches. First, we have analyzed the development of EAE in mutant mouse deficient in MMP-8, with the finding that the absence of this proteolytic enzyme is associated with a marked reduction in the clinical symptoms of EAE. We have also found that MMP-8-/- mice exhibit a marked reduction in central nervous system-infiltrating cells and demyelinating lesions. As a second approach, we have carried out a pharmacological inhibition of MMP-8 with a selective inhibitor against this protease (IC50 = 0.4 nm). These studies have revealed that the administration of the MMP-8 selective inhibitor to mice with EAE also reduces the severity of the disease. Based on these findings, we conclude that MMP-8 plays an important role in EAE development and propose that this enzyme may be a novel therapeutic target in human neuro-inflammatory diseases such as multiple sclerosis.

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.

Gating of myotonic Na channel mutants defines the response to mexiletine and a potent derivative

Background: Myotonia and periodic paralysis caused by sodium channel mutations show variable responses to the anti-myotonic drug mexiletine. Objective: To investigate whether variability among sodium channel mutants results from differences in drug binding affinity or in channel gating. Methods: Whole-cell sodium currents (INa) were recorded in tsA201 cells expressing human wild-type (WT) and mutant skeletal muscle sodium channels (A1156T, hyperkalemic periodic paralysis; R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). Results: At a holding potential (hp) of −120 mV, mexiletine produced a tonic (TB, 0.33 Hz) and a use-dependent (UDB, 10 Hz) block of peak INa with a potency following the order rank R1448C > WT ≈ A1156T > G1306E. Yet, when assayed from an hp of −180 mV, TB and UDB by mexiletine were similar for the four channels. The different midpoints of channel availability curves found for the four channels track the half-maximum inhibitory value (IC50) measured at −120 mV. Thus differences in the partitioning of channels between the closed and fast-inactivated states underlie the different IC50 measured at a given potential. The mexiletine-derivative, Me7 (α-[(2-methylphenoxy)methyl]-benzenemethanamine), behaved similarly but was ∼5 times more potent than mexiletine. Interestingly, the higher drug concentrations ameliorated the abnormally slower decay rate of myotonic INa. Conclusions: These results explain the basis of the apparent difference in block of mutant sodium channels by mexiletine and Me7, opening the way to a more rationale drug use and to design more potent drugs able to correct specifically the biophysical defect of the mutation in individual myotonic patients.

Biphenyl Sulfonylamino Methyl Bisphosphonic Acids as Inhibitors of Matrix Metalloproteinases and Bone Resorption

A number of matrix metalloproteinases (MMPs), proteins important in the balance of bone remodeling, play a critical role both in cancer metastasis and in bone matrix turnover associated with the presence of cancer cells in bone. Here, we report the synthesis and biological evaluation of a new class of MMP inhibitors characterized by a bisphosphonate function as the zinc binding group. Since the bisphosphonate group is also implicated in osteoclast inhibition and provides a preferential affinity to biological apatite, the new molecules can be regarded as bone‐seeking medicinal agents. Docking experiments were performed to clarify the mode of binding of bisphosphonate inhibitors in the active site of MMP‐2. The most promising of the studied bisphosphonates showed nanomolar inhibition against MMP‐2 and resulted in potent inhibition of osteoclastic bone resorption in vitro.

New 2-Aryloxy-3-phenyl-propanoic Acids As Peroxisome Proliferator-Activated Receptors alpha/gamma Dual Agonists with Improved Potency and Reduced Adverse Effects on Skeletal Muscle Function

The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introduction of a linker into the diphenyl system of the previously reported PPARalpha/gamma dual agonist 1, allowed the identification of new ligands with improved potency on PPARalpha and unchanged activity on PPARgamma. For the most interesting stereoisomers S-2 and S-4, X-ray studies in PPARgamma and docking experiments in PPARalpha provided a molecular explanation for their different behavior as full and partial agonists of PPARalpha and PPARgamma, respectively. Due to the adverse effects provoked by hypolipidemic drugs on skeletal muscle function, we also investigated the blocking activity of S-2 and S-4 on skeletal muscle membrane chloride channel conductance and found that these ligands have a pharmacological profile more beneficial compared to fibrates currently used in therapy.

Synthesis and cytotoxic activity evaluation of 2,3-thiazolidin-4-one derivatives on human breast cancer cell lines

It is well known that resveratrol (RSV) displayed cancer-preventing and anticancer properties but its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. Aim of this work was to synthesize pharmacologically active resveratrol analogs with an enhanced structural rigidity and bioavailability. In particular, we have synthesized a library of 2,3-thiazolidin-4-one derivatives in which a thiazolidinone nucleus connects two aromatic rings. Some of these compounds showed strong inhibitory effects on breast cancer cell growth. Our results indicate that some of thiazolidin-based resveratrol derivatives may become a new potent alternative tool for the treatment of human breast cancer.

Synthesis, Characterization and Biological Evaluation of Ureidofibrate-Like Derivatives Endowed with Peroxisome Proliferator-Activated Receptor Activity

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity. In an adipogenic assay, both enantiomers increased the expression of PPARγ target genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a high fat diet reduced visceral fat content and body weight. Examination of different metabolic parameters showed that R-1 and S-1 are insulin sensitizers. Notably, they also enhanced the expression of hepatic PPARα target genes indicating that their in vivo effects stemmed from an activation of both PPARα and γ. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in colon cancer cell lines was also evaluated.