Daniele Pala

Recent advances in the development of melatonin MT(1) and MT(2) receptor agonists.

Importance of the field: Increasing clinical evidences suggest that melatonin receptor agonists can represent a novel therapeutic approach for the treatment of sleep disturbances and depression. A variety of studies also revealed a role of melatonin and its receptors in different patho-physiological conditions. Due to the multiple effects of this hormone, the design of new agents able to interact selectively with melatonin receptors has become an area of great interest during the last decade. Areas covered in this review: An extensive inspection of patents and scientific literature about MT1 and MT2 melatonin receptor agonists reported from 1999 to early 2010. What the reader will gain: A comprehensive review of structures recently claimed as melatonin receptor agonists and a broad overview of structure–activity relationships for these ligands. Take home message: After 5 decades of research, the field of melatonin receptor agonists comprises a variety of chemical entities, belonging to structurally different classes. Patents filed since 1999 claim new melatonin receptor agonists, characterized either by improved pharmacokinetic or pharmacodynamic properties, compared to those of melatonin receptor agonists already approved for clinical uses. The results of preclinical studies on animal models show that melatonin receptor agonists can be considered promising agents for the treatment of CNS-related pathologies.

Amino Acid Conjugates of Lithocholic Acid As Antagonists of the EphA2 Receptor

The Eph receptor-ephrin system is an emerging target for the development of novel antiangiogenetic agents. We recently identified lithocholic acid (LCA) as a small molecule able to block EphA2-dependent signals in cancer cells, suggesting that its (5β)-cholan-24-oic acid scaffold can be used as a template to design a new generation of improved EphA2 antagonists. Here, we report the design and synthesis of an extended set of LCA derivatives obtained by conjugation of its carboxyl group with different α-amino acids. Structure-activity relationships indicate that the presence of a lipophilic amino acid side chain is fundamental to achieve good potencies. The l-Trp derivative (20, PCM126) was the most potent antagonist of the series disrupting EphA2-ephrinA1 interaction and blocking EphA2 phosphorylation in prostate cancer cells at low μM concentrations, thus being significantly more potent than LCA. Compound 20 is among the most potent small-molecule antagonists of the EphA2 receptor.

Synthesis of (E)-8-(3-Chlorostyryl)caffeine Analogues Leading to 9-Deazaxanthine Derivatives as Dual A2A Antagonists/MAO-B Inhibitors

A systematic modification of the caffeinyl core and substituents of the reference compound (E)-8-(3-chlorostyryl)caffeine led to the 9-deazaxanthine derivative (E)-6-(4-chlorostyryl)-1,3,5,-trimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4-(3H,5H)-dione (17f), which acts as a dual human A(2a) antagonist/MAO-B inhibitor (K(i)(A(2A)) = 260 nM; IC(50)(MAO-B) = 200 nM; IC(50)(MAO-A) = 10 μM) and dose dependently counteracts haloperidol-induced catalepsy in mice from 30 mg/kg by the oral route. The compound is the best balanced A(2A) antagonist/MAO-B inhibitor reported to date, and it could be considered as a new lead in the field of anti-Parkinsons agents. A number of analogues of 17f were synthesized and qualitative SARs are discussed. Two analogues of 17f, namely 18b and 19a, inhibit MAO-B with IC(50) of 68 and 48 nM, respectively, being 5-7-fold more potent than the prototypical MAO-B inhibitor deprenyl (IC(50) = 334 nM).

Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase

Heparanase is a β-d-glucuronidase which cleaves heparan sulfate chains in the extracellular matrix and on cellular membranes. A dysregulated heparanase activity is intimately associated with cell invasion, tumor metastasis and angiogenesis, making heparanase an attractive target for the development of anticancer therapies. SST0001 (roneparstat; Sigma-Tau Research Switzerland S.A.) is a non-anticoagulant 100% N-acetylated and glycol-split heparin acting as a potent heparanase inhibitor, currently in phase I in advanced multiple myeloma. Herein, the kinetics of heparanase inhibition by roneparstat is reported. The analysis of dose-inhibition curves confirmed the high potency of roneparstat (IC50 ≈ 3 nM) and showed, at higher concentrations, a Hill coefficient consistent with the engagement of two molecules of inhibitor. A homology model of human heparanase GS3 construct was built and used for docking experiments with inhibitor fragments. The model has high structural similarity with the recently reported crystal structure of human heparanase. Different interaction schemes are proposed, which support the hypothesis of a complex binding mechanism involving the recruitment of one or multiple roneparstat chains, depending on its concentration. In particular, docking solutions were obtained in which (i) a single roneparstat molecule interacts with both heparin-binding domains (HBDs) of heparanase or (ii) two fragments of roneparstat interact with either HBD-1 or HBD-2, consistent with the possibility of different inhibitor:enzyme binding stoichiometries. This study provides unique insights into the mode of action of roneparstat as well as clues of its interaction with heparanase at a molecular level, which could be exploited to design novel potential inhibitor molecules.

Therapeutic uses of melatonin and melatonin derivatives: a patent review (2012 – 2014)

Introduction: Melatonin is a neurohormone involved in the regulation of circadian rhythms, with potent antioxidant activity. It has a wide functional repertoire, with effects almost on all tissues and organs. It is mainly used as a dietary supplement for sleep regulation and re-synchronization of disrupted circadian rhythms. Melatonin has very low toxicity, but some pharmacokinetic issues, such as limited oral bioavailability and short half-life, limit its tissue availability. Areas covered: Patents and patent applications from 2012 to September 2014 in which melatonin or synthetic analogues are claimed for the prevention or treatment of pathological conditions. Expert opinion: Melatonin is considered a valuable substance that can be safely administered for the prevention and treatment of many diverse diseases. A major trend in 2012 – 2014 patents is the co-administration of melatonin with other drugs to increase the efficacy of the treatment and reduce side-effects. Two different actions have been combined in hybrid ligands (e.g., melatonin-tamoxifen and melatonin-tacrine derivatives). Further experimental evidence is needed to support the usefulness of these approaches. The number of new melatonin analogues has shown a marked decrease in the past 3 years, with claimed applications mainly as hypnotic or antioxidant agents.

Structure-based virtual screening of MT2 melatonin receptor: influence of template choice and structural refinement.

Developing GPCR homology models for structure-based virtual screening requires the choice of a suitable template and refinement of binding site residues. We explored this systematically for the MT2 melatonin receptor, with the aim to build a receptor homology model that is optimized for the enrichment of active melatoninergic ligands. A set of 12 MT2 melatonin receptor models was built using different GPCR X-ray structural templates and submitted to a virtual screening campaign on a set of compounds composed of 29 known melatonin receptor ligands and 2560 drug-like decoys. To evaluate the effect of including a priori information in receptor models, 12 representative melatonin receptor ligands were placed into the MT2 receptor models in poses consistent with known mutagenesis data and with assessed pharmacophore models. The receptor structures were then adapted to the ligands by induced-fit docking. Most of the 144 ligand-adapted MT2 receptor models showed significant improvements in screening enrichments compared to the unrefined homology models, with some template/refinement combinations giving excellent enrichment factors. The discriminating ability of the models was further tested on the 29 active ligands plus a set of 21 inactive or low-affinity compounds from the same chemical classes. Rotameric states of side chains for some residues, presumed to be involved in the binding process, were correlated with screening effectiveness, suggesting the existence of specific receptor conformations able to recognize active compounds. The top MT2 receptor model was able to identify 24 of 29 active ligands among the first 2% of the screened database. This work provides insights into the use of refined GPCR homology models for virtual screening.

ST7612AA1, a thioacetate-ω(γ-lactam carboxamide) derivative selected from a novel generation of oral HDAC inhibitors.

A systematic study of medicinal chemistry aimed at identifying a new generation of HDAC inhibitors, through the introduction of a thiol zinc-binding group (ZBG) and of an amide-lactam in the ω-position of the polyethylene chain of the vorinostat scaffold, allowed the selection of a new class of potent pan-HDAC inhibitors (pan-HDACis). Simple, highly versatile, and efficient synthetic approaches were used to synthesize a library of these new derivatives, which were then submitted to a screening for HDAC inhibition as well as to a preliminary in vitro assessment of their antiproliferative activity. Molecular docking into HDAC crystal structures suggested a binding mode for these thiol derivatives consistent with the stereoselectivity observed upon insertion of amide-lactam substituents in the ω-position. ST7612AA1 (117), selected as a drug candidate for further development, showed an in vitro activity in the nanomolar range associated with a remarkable in vivo antitumor activity, highly competitive with the most potent HDAC inhibitors, currently under clinical trials. A preliminary study of PK and metabolism is also illustrated.

MT1-selective melatonin receptor ligands: synthesis, pharmacological evaluation, and molecular dynamics investigation of N-{[(3-O-substituted)anilino]alkyl}amides.

The design of compounds selective for the MT1 melatonin receptor is still a challenging task owing to the limited knowledge of the structural features conferring selectivity for the MT1 subtype, and only few selective compounds have been reported so far. N‐(Anilinoalkyl)amides are a versatile class of melatonin receptor ligands that include nonselective MT1/MT2 agonists and MT2‐selective antagonists. We synthesized a new series of N‐(anilinoalkyl)amides bearing 3‐arylalkyloxy or 3‐alkyloxy substituents at the aniline ring, looking for new potent and MT1‐selective ligands. To evaluate the effect of substituent size and shape on binding affinity and intrinsic activity, both flexible and conformationally constrained derivatives were prepared. The phenylbutyloxy substituent gave the best result, providing the partial agonist 4 a, which was endowed with high MT1 binding affinity (pKi=8.93) and 78‐fold selectivity for the MT1 receptor. To investigate the molecular basis for agonist recognition, and to explain the role of the 3‐arylalkyloxy substituent, we built a homology model of the MT1 receptor based on the β2 adrenergic receptor crystal structure in its activated state. A binding mode for MT1 agonists is proposed, as well as a hypothesis regarding the receptor structural features responsible for MT1 selectivity of compounds with lipophilic arylalkyloxy substituents.

Homology Models of Melatonin Receptors: Challenges and Recent Advances

Melatonin exerts many of its actions through the activation of two G protein-coupled receptors (GPCRs), named MT1 and MT2. So far, a number of different MT1 and MT2 receptor homology models, built either from the prototypic structure of rhodopsin or from recently solved X-ray structures of druggable GPCRs, have been proposed. These receptor models differ in the binding modes hypothesized for melatonin and melatonergic ligands, with distinct patterns of ligand-receptor interactions and putative bioactive conformations of ligands. The receptor models will be described, and they will be discussed in light of the available information from mutagenesis experiments and ligand-based pharmacophore models. The ability of these ligand-receptor complexes to rationalize structure-activity relationships of known series of melatonergic compounds will be commented upon.

Toward the Definition of Stereochemical Requirements for MT2-Selective Antagonists and Partial Agonists by Studying 4-Phenyl-2-propionamidotetralin Derivatives

New derivatives of 4-phenyl-2-propionamidotetralin (4-P-PDOT) were prepared and tested on cloned MT1 and MT2 receptors, with the purpose of merging previously reported pharmacophores for nonselective agonists and for MT2-selective antagonists. A 8-methoxy group increases binding affinity of both (±)-cis- and (±)-trans-4-P-PDOT, and it can be bioisosterically replaced by a bromine. Conformational analysis of 8-methoxy-4-P-PDOT by molecular dynamics, supported by NMR data, revealed an energetically favored conformation for the (2S,4S)-cis isomer and a less favorable conformation for the (2R,4S)-trans one, fulfilling the requirements of a pharmacophore model for nonselective melatonin receptor agonists. A new superposition model, including features characteristic of MT2-selective antagonists, suggests that MT1/MT2 agonists and MT2 antagonists can share the same arrangement for their pharmacophoric elements. The model correctly predicted the eutomers of (±)-cis- and (±)-trans-4-P-PDOT. The model was validated by preparing three dihydronaphthalene derivatives, either able or not able to reproduce the putative active conformation of 4-P-PDOT.