Antonella Ciancetta

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC50 = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

Aquaporin inhibition by gold(III) compounds: new insights.

Aquaporins (AQPs) are membrane water/glycerol channels with essential roles in biological systems, as well as being promising targets for therapy and imaging. Using a stopped‐flow method, a series of gold(III), platinum(II) and copper(II) complexes bearing nitrogen donor ligands, such as 1,10‐phenatroline, 2,2′‐bipyridine, 4,4′‐dimethyl‐2,2′‐bipyridine, 4,4′‐diamino‐2,2′‐bipyridine and 2,2′;6′,2“‐terpyridine, were evaluated in human red blood cells expressing AQP1 and AQP3, responsible for water and glycerol movement, respectively. The results showed that the gold(III) complexes selectively modulate AQP3 over AQP1. Molecular modeling and density functional theory (DFT) calculations were subsequently performed to rationalize the observations and to investigate the possible molecular mechanism through which these gold compounds act on their putative target (AQP3). In the absence of any crystallographic data, a previously reported homology model was used for this purpose. Combined, the findings of this study show that potent and selective modulation of these solute channels is possible, however further investigation is required into the selectivity of this class of agents against all AQP isoforms and their potential therapeutic uses.

Understanding allosteric interactions in G protein-coupled receptors using Supervised Molecular Dynamics: A prototype study analysing the human A3 adenosine receptor positive allosteric modulator LUF6000.

The search for G protein-coupled receptors (GPCRs) allosteric modulators represents an active research field in medicinal chemistry. Allosteric modulators usually exert their activity only in the presence of the orthosteric ligand by binding to protein sites topographically different from the orthosteric cleft. They therefore offer potentially therapeutic advantages by selectively influencing tissue responses only when the endogenous agonist is present. The prediction of putative allosteric site location, however, is a challenging task. In facts, they are usually located in regions showing more structural variation among the family members. In the present work, we applied the recently developed Supervised Molecular Dynamics (SuMD) methodology to interpret at the molecular level the positive allosteric modulation mediated by LUF6000 toward the human adenosine A3 receptor (hA3 AR). Our data suggest at least two possible mechanisms to explain the experimental data available. This study represent, to the best of our knowledge, the first case reported of an allosteric recognition mechanism depicted by means of molecular dynamics simulations.

DockBench: An Integrated Informatic Platform Bridging the Gap between the Robust Validation of Docking Protocols and Virtual Screening Simulations

Virtual screening (VS) is a computational methodology that streamlines the drug discovery process by reducing costs and required resources through the in silico identification of potential drug candidates. Structure-based VS (SBVS) exploits knowledge about the three-dimensional (3D) structure of protein targets and uses the docking methodology as search engine for novel hits. The success of a SBVS campaign strongly depends upon the accuracy of the docking protocol used to select the candidates from large chemical libraries. The identification of suitable protocols is therefore a crucial step in the setup of SBVS experiments. Carrying out extensive benchmark studies, however, is usually a tangled task that requires users’ proficiency in handling different file formats and philosophies at the basis of the plethora of existing software packages. We present here DockBench 1.0, a platform available free of charge that eases the pipeline by automating the entire procedure, from docking benchmark to VS setups. In its current implementation, DockBench 1.0 handles seven docking software packages and offers the possibility to test up to seventeen different protocols. The main features of our platform are presented here and the results of the benchmark study of human Checkpoint kinase 1 (hChk1) are discussed as validation test.

2-Arylpyrazolo[4,3-d]pyrimidin-7-amino derivatives as new potent and selective human A3 adenosine receptor antagonists. Molecular modeling studies and pharmacological evaluation.

On the basis of our previously reported 2-arylpyrazolo[4,3-d]pyrimidin-7-ones, a set of 2-arylpyrazolo[4,3-d]pyrimidin-7-amines were designed as new human (h) A3 adenosine receptor (AR) antagonists. Lipophilic groups with different steric bulk were introduced at the 5-position of the bicyclic scaffold (R5 = Me, Ph, CH2Ph), and different acyl and carbamoyl moieties (R7) were appended on the 7-amino group, as well as a para-methoxy group inserted on the 2-phenyl ring. The presence of acyl groups turned out to be of paramount importance for an efficient and selective binding at the hA3 AR. In fact, most of the 7-acylamino derivatives showed low nanomolar affinity (Ki = 2.5-45 nM) and high selectivity toward this receptor. A few selected pyrazolo[4,3-d]pyrimidin-7-amides were effective in counteracting oxaliplatin-induced apoptosis in rat astrocyte cell cultures, an in vitro model of neurotoxicity. Through an in silico receptor-driven approach the obtained binding data were rationalized and the molecular bases of the observed hA3 AR affinity and hA3 versus hA2A AR selectivity were explained.

7-Amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives: Structural investigations at the 5-position to target human A1 and A2A adenosine receptors. Molecular modeling and pharmacological studies

In previous research, several 7-amino-2-arylpyrazolo[4,3-d]pyrimidine derivatives were identified as highly potent and selective antagonists at the human A3 adenosine receptor. Structure-activity relationship studies highlighted that affinity and selectivity depended on the nature of the substituents at the 5- and 7-positions of the pyrazolo[4,3-d]pyrimidine scaffold. In particular, small lipophilic residues at the 5-position and a free amino group at position 7 afforded compounds able to bind all four human (h) adenosine receptors. Hence, to shift affinity toward the hA1 and/or hA(2A) subtypes, alkyl and arylalkyl chains of different length were appended at position 5 of the 2-phenylpyrazolo[4,3-d]pyrimidin-7-amine. Among the new compounds, a dual hA1/hA(2A) receptor antagonist was identified, namely the 5-(3-phenylpropyl) derivative 25, which shows high affinity both at human A1 (K(i) = 5.31 nM) and A(2A) (K(i) = 55 nM) receptors. We also obtained some potent and selective antagonists for the A1 receptor, such as the 5-(3-arylpropyl)-substituted compounds 26-31, whose affinities fall in the low nanomolar range (K(i) = 0.15-18 nM). Through an in silico receptor-driven approach, the obtained binding data were rationalized and the molecular bases of the hA1 and hA(2A) AR affinity and selectivity of derivatives 25-31 are explained.

Deciphering the Complexity of Ligand-Protein Recognition Pathways Using Supervised Molecular Dynamics (SuMD) Simulations.

Molecular recognition is a crucial issue when aiming to interpret the mechanism of known active substances as well as to develop novel active candidates. Unfortunately, simulating the binding process is still a challenging task because it requires classical MD experiments in a long microsecond time scale that are affordable only with a high-level computational capacity. In order to overcome this limiting factor, we have recently implemented an alternative MD approach, named supervised molecular dynamics (SuMD), and successfully applied it to G protein-coupled receptors (GPCRs). SuMD enables the investigation of ligand-receptor binding events independently from the starting position, chemical structure of the ligand, and also from its receptor binding affinity. In this article, we present an extension of the SuMD application domain including different types of proteins in comparison with GPCRs. In particular, we have deeply analyzed the ligand-protein recognition pathways of six different case studies that we grouped into two different classes: globular and membrane proteins. Moreover, we introduce the SuMD-Analyzer tool that we have specifically implemented to help the user in the analysis of the SuMD trajectories. Finally, we emphasize the limit of the SuMD applicability domain as well as its strengths in analyzing the complexity of ligand-protein recognition pathways.

Advances in Computational Techniques to Study GPCR–Ligand Recognition

G-protein-coupled receptors (GPCRs) are among the most intensely investigated drug targets. The recent revolutions in protein engineering and molecular modeling algorithms have overturned the research paradigm in the GPCR field. While the numerous ligand-bound X-ray structures determined have provided invaluable insights into GPCR structure and function, the development of algorithms exploiting graphics processing units (GPUs) has made the simulation of GPCRs in explicit lipid-water environments feasible within reasonable computation times. In this review we present a survey of the recent advances in structure-based drug design approaches with a particular emphasis on the elucidation of the ligand recognition process in class A GPCRs by means of membrane molecular dynamics (MD) simulations.

A3 Adenosine Receptors as Modulators of Inflammation: From Medicinal Chemistry to Therapy

The A3 adenosine receptor (A3AR) subtype is a novel, promising therapeutic target for inflammatory diseases, such as rheumatoid arthritis (RA) and psoriasis, as well as liver cancer. A3AR is coupled to inhibition of adenylyl cyclase and regulation of mitogen‐activated protein kinase (MAPK) pathways, leading to modulation of transcription. Furthermore, A3AR affects functions of almost all immune cells and the proliferation of cancer cells. Numerous A3AR agonists, partial agonists, antagonists, and allosteric modulators have been reported, and their structure–activity relationships (SARs) have been studied culminating in the development of potent and selective molecules with drug‐like characteristics. The efficacy of nucleoside agonists may be suppressed to produce antagonists, by structural modification of the ribose moiety. Diverse classes of heterocycles have been discovered as selective A3AR blockers, although with large species differences. Thus, as a result of intense basic research efforts, the outlook for development of A3AR modulators for human therapeutics is encouraging. Two prototypical selective agonists, N6‐(3‐Iodobenzyl)adenosine‐5′‐N‐methyluronamide (IB‐MECA; CF101) and 2‐chloro‐N6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methyluronamide (Cl‐IB‐MECA; CF102), have progressed to advanced clinical trials. They were found safe and well tolerated in all preclinical and human clinical studies and showed promising results, particularly in psoriasis and RA, where the A3AR is both a promising therapeutic target and a biologically predictive marker, suggesting a personalized medicine approach. Targeting the A3AR may pave the way for safe and efficacious treatments for patient populations affected by inflammatory diseases, cancer, and other conditions.

Exploring the recognition pathway at the human A2A adenosine receptor of the endogenous agonist adenosine using supervised molecular dynamics simulations

Adenosine is a naturally occurring purine nucleoside that exerts a variety of important biological functions through the activation of four G protein-coupled receptor (GPCR) isoforms, namely the A1, A2A, A2B and A3 adenosine receptors (ARs). Recently, the X-ray structure of adenosine-bound hA2A AR has been solved, thus providing precious structural details on receptor recognition and activation mechanisms. To date, however, little is still known about the possible recognition pathway the endogenous agonist might go through while approaching the hA2A AR from the extracellular environment. In the present work, we report the adenosine-hA2A AR recognition pathway through the analysis of a series of Supervised Molecular Dynamics (SuMD) trajectories. Interestingly, a possible energetically stable meta-binding site has been detected and characterized.