Andrea Carotti

Natural and synthetic geiparvarins are strong and selective MAO-B inhibitors. Synthesis and SAR studies.

Natural geiparvarin 1 and a number of its analogues were prepared and tested as inhibitors of both monoamine oxidase isoforms, MAO-B and MAO-A. The desmethyl congener 6 of geiparvarin, proved potent and selective MAO-B inhibitor (pIC(50)=7.55 vs 4.62). X-ray crystallography and molecular modelling studies helped the understanding of the observed structure-activity relationships.

An integrated approach to ligand- and structure-based drug design: development and application to a series of serine protease inhibitors.

A novel approach was developed to rationally interface structure- and ligand-based drug design through the rescoring of docking poses and automated generation of molecular alignments for 3D quantitative structure-activity relationship investigations. The procedure was driven by a genetic algorithm optimizing the value of a novel fitness function, accounting simultaneously for best regressions among binding-energy docking scores and affinities and for minimal geometric deviations from properly established crystal-based binding geometry. The GRID/CPCA method, as implemented in GOLPE, was used to feature molecular determinants of ligand binding affinity for each molecular alignment. In addition, unlike standard procedures, a novel multipoint equation was adopted to predict the binding affinity of ligands in the prediction set. Selectivity was investigated through square plots reporting experimental versus recalculated binding affinities on the targets under examination. The application of our approach to the modeling of affinity data of a large series of 3-amidinophenylalanine inhibitors of thrombin, trypsin, and factor Xa generated easily interpretable and independent models with robust statistics. As a further validation study, our approach was successfully applied to a series of 3,4,7-substituted coumarins, acting as selective MAO-B inhibitors.

Extending SAR of bile acids as FXR ligands: discovery of 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5β-cholan-23-amine.

Within our efforts in the discovery of novel potent and selective ligands for the FXR receptor, 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5β-cholan-23-amine was synthesized and evaluated for its ability to activate and modulate the biological response of the receptor. Alphascreen and RT-PCR revealed that the 6α-ethyl-24-norcholanyl-23-amine derivate behaves as full FXR agonist endowed with high binding affinity and efficacy, representing a promising lead candidate for further optimization. In addition, docking studies provide new insights into the molecular basis governing the partial and full agonist activity at FXR.

Beyond Bile Acids: Targeting Farnesoid X Receptor (FXR) with Natural and Synthetic Ligands

The modulation of FXR receptor remains an attractive area in drug discovery to develop novel therapeutic opportunities for liver and metabolic disorders. Despite the large variety of FXR ligands reported so far, only a very restricted number of agonists have entered in clinical settings. In this review article we provide the reader with an overview on the different classes of natural and synthetic ligands that have been developed by academic groups and pharmaceutical companies to target FXR. We discuss their structure-activity relationships, analyzing the binding modes that some of these compounds adopt to interact with the receptor.

Targeting the conformational transitions of MDM2 and MDMX: insights into dissimilarities and similarities of p53 recognition.

MDM2 and MDMX are oncogenic homologue proteins that regulate the activity and stability of p53, a tumor suppressor protein involved in more than 50% of human cancers. While the large body of experiments so far accumulated has validated MDM2 as a therapeutically important target for the development of anticancer drugs, it is only recently that MDMX has also become an attractive target for the treatment of tumor cells expressing wild type p53. The availability of structural information of the N-terminal domain of MDM2 in complex with p53-derived peptides and inhibitors, and the very recent disclosure of the crystal structure of the N-terminal domain of MDMX bound to a p53 peptide, offer an unprecedented opportunity to provide insight into the molecular basis of p53 recognition and the identification of discriminating features affecting the binding of the tumor suppressor protein at MDM2 and MDMX. By using coarse graining simulations, in this study we report the exploration of the conformational transitions featured in the pathway leading from the apo-MDM2 and apo-MDMX states to the p53-bound MDM2 and p53-bound MDMX states, respectively. The results have enabled us to identify a pool of diverse conformational states of the oncogenic proteins that affect the binding of p53 and the presence of conserved and non-conserved interactions along the conformational transition pathway that may be exploited in the design of selective and dual modulators of MDM2 and MDMX activity.

Pyrazole[3,4-e][1,4]thiazepin-7-one derivatives as a novel class of Farnesoid X Receptor (FXR) agonists

A virtual screening procedure was applied to the discovery of structurally diverse non-steroidal Farnesoid X Receptor (FXR) agonists. From 117 compounds selected by virtual screening, a total of 47 compounds were found to be FXR agonists, with 34 of them showing activity below a concentration of 20 μM. 1H-Pyrazole[3,4-e][1,4]thiazepin-7-one-based hit compound 7 was chosen for hit-to-lead optimization. A large number of 1H-pyrazole[3,4-e][1,4]thiazepin-7-one derivatives was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activity against FXR. Most of them exhibited low micromolar range of potency and very high efficacy.

High affinity central benzodiazepine receptor ligands. Part 3: insights into the pharmacophore and pattern recognition study of intrinsic activities of pyrazolo[4,3-c]quinolin-3-ones

Novel 2-phenyl-2,5-dihydropyrazolo[4,3-c]quinolin-3-(3H)-ones (PQs) endowed with high affinity for central benzodiazepine receptor (BzR) were synthesized. In particular, 9-fluoro-2-(2-fluorophenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one (2(2)) showed binding affinity in the subnanomolar concentration range and proved to be in vitro a potent antagonist. This finding allowed the nature of the hydrogen bonding receptor site H(2) to be established, as located between the N-1 nitrogen of the PQ nucleus and the ortho position of the N-2-aryl group. [35S]tert-Butylbicyclophosphorothionate ([35S]TBPS) binding assays and electrophysiological measurements of the effects on GABA-evoked Cl(-) currents at recombinant human alpha(1)beta(2)gamma(2)(L) GABA(A) receptors, expressed in Xenopus laevis oocytes, were used to assess the intrinsic activities of a large series of PQs. With the aim of extracting discriminant information and distinguishing BzR ligands with different profiles of efficacy, 51 PQ derivatives, including full and partial agonists, antagonists, and inverse agonists, were analyzed in a multidimensional chemical descriptor space, defined by the lipophilicity parameter CLOG P and 3-D molecular WHIM descriptors, by means of principal component analysis, k-nearest neighbors (k-NN) method, and linear discriminant analysis (LDA). The classification methods were applied to subsets of pairs of efficacy classes, and lipophilicity and 3-D size descriptors were detected as the discriminant variables by a stepwise linear discriminant analysis. LDA proved to be superior to k-NN, especially in classifying PQ ligands (60-84% of success in prediction ability) into categories of efficacies which were contiguous and quite overlapped in the hyperspace of variables.

Sequence Variants in Kynurenine Aminotransferase II (KAT II) Orthologs Determine Different Potencies of the Inhibitor S-ESBA

The kynurenine pathway (KP, Figure 1) constitutes the major metabolic route of the essential amino acid l-tryptophan (lTrp) in mammals. KP metabolism is initiated by oxidative opening of the indole ring of l-Trp and leads to the production of NAD . The catabolic intermediates of KP have recently attracted considerable attention for their role in central nervous system (CNS) physiology and in the etiology and progression of neurodegenerative and immunological disorders. The major neuroactive metabolites originate from two competing branches of the pathway, where the central metabolite l-kynurenine (l-Kyn, 1) is transformed into 3-hydroxykynurenine (3-OH-Kyn, 2) and then further into quinolinic acid (QUIN, 3), or into kynurenic acid (KYNA, 4). KYNA is a competitive antagonist of the glycine site of the NMDA receptor complex, a noncompetitive antagonist of the a7 nicotinic acetylcholine receptor, 6] and is endowed with neuroprotective properties. QUIN (3), in contrast, is a neurotoxic agonist of the NMDA receptor complex, and 3-OH-Kyn (2) is a free radical generator that can contribute to neuronal damage in the CNS. In view of the neuroprotective or neurotoxic activities of KP metabolites, enzymes of the KP have long been considered interesting targets for rational therapeutic intervention. One of the most promising targets is kynurenine aminotransferase (KAT), a pyridoxal-5’-phosphate (PLP)-dependent enzyme that catalyzes the irreversible transamination of l-Kyn (1) into KYNA (4). Four isoforms of this enzyme have so far been identified, namely KAT I, KAT II, KAT III, and KAT IV (or mitAAT). Although all four isoforms are present in the mammalian brain, the enzyme activity of KAT III has not been confirmed so far, and only KAT I and KAT II have been thoroughly characterized with regard to their role in cerebral KYNA synthesis. These two isoforms differ in substrate specificity, with KAT I showing a lower specificity for l-Kyn (1) than KAT II. 13] The development of KAT inhibitors may prove useful in disorders associated with learning and memory deficits, where lowering the levels of brain KYNA may counterbalance glutamatergic and cholinergic hypofunction. Recently, we reported the synthesis and biological characterization of (S)-4-ethylsulfonylbenzoylalanine (S-ESBA, 5), the first potent and selective inhibitor of KAT II, which is the dominant KYNA-forming enzyme in the rat brain. Biological assays conducted in vitro showed that S-ESBA (5) inhibits KAT II obtained from partially purified rat liver with an IC50 value of 6.1 mm. In vivo studies support these results, demonstrating that the administration of S-ESBA (5) lowers the extracellular levels of KYNA in the hippocampus of unanesthetized rats. Herein we report a novel and more efficient synthesis of SESBA (5) and an analysis of its inhibitory activity using purified recombinant human KAT II. The data are discussed in light of the crystal structure of human KAT II in complex with its natural substrate l-Kyn (1), and on the basis of conserved and nonconserved residues that feature the sequences of speciesspecific orthologs of KAT II (human, rat, and mouse). Figure 1. The kynurenine pathway (KP) of tryptophan metabolism.

4,5-Diarylisoxazol-3-carboxylic acids: A new class of leukotriene biosynthesis inhibitors potentially targeting 5-lipoxygenase-activating protein (FLAP)

In this article, we report novel leukotriene (LT) biosynthesis inhibitors that may target 5-lipoxygenase-activating protein (FLAP) based on the previously identified isoxazole derivative (8). The design and synthesis was directed towards a subset of 4,5-diaryl-isoxazole-3-carboxylic acid derivatives as LT biosynthesis inhibitors. Biological evaluation disclosed a new skeleton of potential anti-inflammatory agents, exemplified by 39 and 40, which potently inhibit cellular 5-LO product synthesis (IC50 = 0.24 μM, each) seemingly by targeting FLAP with weak inhibition on 5-LO (IC50 ≥ 8 μM). Docking studies and molecular dynamic simulations with 5-LO and FLAP provide valuable insights into potential binding modes of the inhibitors. Together, these diaryl-isoxazol-3-carboxylic acids may possess potential as leads for development of effective anti-inflammatory drugs through inhibition of LT biosynthesis.

Chromatographic separation of free dafachronic acid epimers with a novel triazole click quinidine-based chiral stationary phase.

For the first time, a successful chromatographic method based on the use of a novel triazole click quinidine (QD) derivative anion-exchange chiral stationary phase (CSP) is applied for the epimer separation of dafachronic acids (Δ(4)- and Δ(7)-DAs). The use of a polar-ionic eluent system consisting of 18mM AcOH in ACN furnished an excellent separation of both Δ(4)- (α=1.19, RS=2.52) and Δ(7)-DA (α=1.14, RS=2.06) C-25 epimer couples. The pool of data collected during the chromatographic analyses revealed the prominent role of anion-exchange interactions in governing the analyte (SA) retention and also indicated the occurrence of stereoselective H-bond contacts with the chiral selector (SO) substructural motifs. In any case, molecular modelling studies corroborate the need for sufficient spatial freedom for optimum binding between the SO and the SAs which seems less the case for the immobilised SO unit of the commercialized QD-based CSP.