Identification of a novel adiponectin receptor and opioid receptor dual acting agonist as a potential treatment for diabetic neuropathy.

Diabetic neuropathy (DN) is a long-term complication of diabetes mellitus, affecting different periphery nerve systems including sensory and motor neurons. Hyperglycemia is the major cause of DN with symptoms such as weakness of balance or coordination, insensitivity to sensation, weakness of the muscles as well as numbness and pain in limbs Analgesic drug such as opioids can be effective to relief neuropathy pain but there is no effective treatment. Adiponectin is an anti-diabetic adipokine, which possesses insulin-sensitizing and neuroprotective effects. In this project, we aim to identify an agent which is dual acting to opioid and adiponectin receptors. Within a virtual screening repositioning campaign, a large collection of compounds with different structures comprehensive of adipoRon-like piperidine derivatives was screened by docking. Recently developed opioid receptor benzomorphanic agonists finally emerged as good ligands to adiponectin receptors showing some 2D and 3D structural similarities with AdipoRon. Particularly, we have identified (+)-MML1017, which has high affinity to the same binding domain of AdipoR1 and AdipoR2 as AdipoRon. Our western blot results indicate (+)-MML1017 activates AMPK phosphorylation through both adipoR1 and adipoR2 in neuronal cell line. Moreover, pretreatment of (+)-MML1017 can improve the cell viability with motor neurons under hyperglycermic conditions. The (+)-MML1017 also activates µ-opioid receptor cells in a concentration-dependent manner. Our study identified a novel compound having dual activity on opioid receptors and adiponectin receptors that may have analgesic effects and neuroprotective effects to treat diabetic neuropathy.

Peptidomimetics in Silico.

Endogenous peptides as part of physiological processes are targets of interest when it comes to finding desirable therapeutics which are able to modulate molecular interactions. The major limits presented by peptides when they are used as drugs have motivated the research of the synthesis of peptidomimetics obtained through chemical modification and the use of in silico approaches. Here recent works on the discovery of peptidomimetics by computational methods are reported. Together with molecular dynamic simulations, the use of pharmacophore research simulations helps to gain insight into and understand the molecular determinants underlying the physiological processes.

Parameters for Irreversible Inactivation of Monoamine Oxidase.

The irreversible inhibitors of monoamine oxidases (MAO) slow neurotransmitter metabolism in depression and neurodegenerative diseases. After oxidation by MAO, hydrazines, cyclopropylamines and propargylamines form a covalent adduct with the flavin cofactor. To assist the design of new compounds to combat neurodegeneration, we have updated the kinetic parameters defining the interaction of these established drugs with human MAO-A and MAO-B and analyzed the required features. The Ki values for binding to MAO-A and molecular models show that selectivity is determined by the initial reversible binding. Common to all the irreversible inhibitor classes, the non-covalent 3D-chemical interactions depend on a H-bond donor and hydrophobic-aromatic features within 5.7 angstroms apart and an ionizable amine. Increasing hydrophobic interactions with the aromatic cage through aryl halogenation is important for stabilizing ligands in the binding site for transformation. Good and poor inactivators were investigated using visible spectroscopy and molecular dynamics. The initial binding, close and correctly oriented to the FAD, is important for the oxidation, specifically at the carbon adjacent to the propargyl group. The molecular dynamics study also provides evidence that retention of the allenyl imine product oriented towards FADH- influences the formation of the covalent adduct essential for effective inactivation of MAO.

Pharmacological properties and biochemical mechanisms of µ-opioid receptor ligands might be due to different binding poses: MD studies.

Background: Central and peripheral analgesia without adverse effects relies on the identification of µ-opioid agonists that are able to activate 'basal' antinociceptive pathways. Recently developed µ-selective benzomorphan agonists that are not antagonized by naloxone do not activate G-proteins and ß-arrestins. Which pathways do µ receptors activate? How can each of them be selectively activated? What role is played by allosteric binding sites? Methodology & results: Molecular modeling studies characterize the amino acid residues involved in the interaction with various classes of endogenous and exogenous ligands and with agonists and antagonists. Conclusions: Critical binding differences between various classes of agonists with different pharmacological profiles have been identified. MML series binding poses may be relevant in the search for an antinociception agent without side effects.

Quantum Chemical and Molecular Dynamics Studies of MUC1 Calix[4,8]arene Scaffold Based Anticancer Vaccine Candidates.

Functional antitumor vaccine constructs are the basis for active tumor immunotherapy, which is useful in the treatment of many types of cancers. MUC1 is one key glycoprotein for targeting and designing new strategies for multicomponent vaccines. Two self-adjuvant tetravalent vaccine candidates were prepared by clustering four or eight PDTRP MUC1 core epitope sequences on calixarene scaffolds. In this work, the different activities of two molecules with calix[4]arene and calix[8]arene skeleton are rationalized. Quantum mechanics, docking, and molecular dynamics structural optimization were first carried out followed by metadynamics to calculate the energy profiles. Further insights were obtained by complementarity studies of molecular fields. The molecular modeling results are in strong agreement with the experimental in vivo immunogenicity data. In conclusion, the overall data shows that, in the designing of anticancer vaccines, scaffold flexibility has a pivotal role in obtaining a suitable electrostatic, hydrophobic, and steric complementarity with the biological target.

Molecular modeling and biological studies show that some µ-opioid receptor agonists might elicit analgesia acting as MMP-9 inhibitors.

Aim: Despite the serious side effects, analgesics acting on opioid receptors are still considered the best way to get antinociception. Matrix metalloproteinases, a large family of zinc-dependent proteases implicated in many pathological conditions, such as diabetes and osteoarthritis, are also involved in inflammation and pain. Methodology & results: Looking for evidence of possible interactions of opioid pathways and inflammation mediators, molecular modeling studies of a series of recently developed µ-opioid receptor benzomorphanic agonists together with biological data on pain and inflammation molecular targets, allowed us to hypothesize a possible correlation between µ-opioid receptor system and MMP-9. Conclusion: A new compound, (-)-MML1017, emerged as a possible dual-acting agent able to interact selectively and potently with the two molecular targets.

Exploring the Biological Activity of a Library of 1,2,5-Oxadiazole Derivatives Endowed With Antiproliferative Activity.

BACKGROUND/AIM: The identification of a series of oxadiazole-based compounds, as promising antiproliferative agents, has been previously reported. The aim of this study was to explore the SAR of newly-synthesized oxadiazole derivatives and identify their molecular targets. MATERIALS AND METHODS: A small library of 1,2,5-oxadiazole derivatives was synthetized and their antiproliferative activity was tested by the MTT assay. Their interaction with topoisomerase I was evaluated and a molecular docking study was performed. RESULTS: Several candidates showed cytotoxicity towards two human tumor cell lines, HCT-116 (colorectal carcinoma) and HeLa (cervix adenocarcinoma). Some derivatives exhibited inhibitory effects on the catalytic activity of topoisomerase I and this effect was supported by docking studies. CONCLUSION: The enzyme inhibition results, although not directly related to cytotoxicity, suggest that a properly modified 1,2,5 oxadiazole scaffold could be considered for the development of new anti-topoisomerase agents.

In silico screening for human norovirus antivirals reveals a novel non-nucleoside inhibitor of the viral polymerase.

Human norovirus causes approximately 219,000 deaths annually, yet there are currently no antivirals available. A virtual screening of commercially available drug-like compounds (~300,000) was performed on the suramin and PPNDS binding-sites of the norovirus RNA-dependent RNA polymerase (RdRp). Selected compounds (n = 62) were examined for inhibition of norovirus RdRp activity using an in vitro transcription assay. Eight candidates demonstrated RdRp inhibition (>25% inhibition at 10 µM), which was confirmed using a gel-shift RdRp assay for two of them. The two molecules were identified as initial hits and selected for structure-activity relationship studies, which resulted in the synthesis of novel compounds that were examined for inhibitory activity. Five compounds inhibited human norovirus RdRp activity (>50% at 10 µM), with the best candidate, 54, demonstrating an IC50 of 5.6 µM against the RdRp and a CC50 of 62.8 µM. Combinational treatment of 54 and the known RdRp site-B inhibitor PPNDS revealed antagonism, indicating that 54 binds in the same binding pocket. Two RdRps with mutations (Q414A and R419A) previously shown to be critical for the binding of site-B compounds had no effect on inhibition, suggesting 54 interacts with distinct site-B residues. This study revealed the novel scaffold 54 for further development as a norovirus antiviral.

Homology-based Modeling of Rhodopsin-like Family Members in the Inactive State: Structural Analysis and Deduction of Tips for Modeling and Optimization.

Modeling G-Protein Coupled Receptors (GPCRs) is an emergent field of research, since utility of high-quality models in receptor structure-based strategies might facilitate the discovery of interesting drug candidates. The findings from a quantitative analysis of eighteen resolved structures of rhodopsin family "A" receptors crystallized with antagonists and 153 pairs of structures are described. A strategy termed endeca-amino acids fragmentation was used to analyze the structures models aiming to detect the relationship between sequence identity and Root Mean Square Deviation (RMSD) at each trans-membrane-domain. Moreover, we have applied the leave-one-out strategy to study the shiftiness likelihood of the helices. The type of correlation between sequence identity and RMSD was studied using the aforementioned set receptors as representatives of membrane proteins and 98 serine proteases with 4753 pairs of structures as representatives of globular proteins. Data analysis using fragmentation strategy revealed that there is some extent of correlation between sequence identity and global RMSD of 11AA width windows. However, spatial conservation is not always close to the endoplasmic side as was reported before. A comparative study with globular proteins shows that GPCRs have higher standard deviation and higher slope in the graph with correlation between sequence identity and RMSD. The extracted information disclosed in this paper could be incorporated in the modeling protocols while using technique for model optimization and refinement.

A field-based disparity analysis of new 1,2,5-oxadiazole derivatives endowed with antiproliferative activity.

A series of 1,2,5-oxadiazoles were synthesized as new potential antiproliferative agents. The in vitro cytotoxic activity evaluation of title compounds through MTT assay revealed that some of them showed significant activity against the HCT-116 cancer cell line. The field-based disparity analysis provided indications about the electrostatic, hydrophobic, and shape features underlying the cytotoxicity, suggesting that increasing the negative electrostatic field on the heterocyclic core of the structure has positive effects on the activity. The structure-activity relationships (SAR) around a particular compound can be explained allowing for a structural rationale for the differences in activity. The SAR provided by this series of compounds can be exploited to carry out further lead optimization.

Correlating In Vitro Target-Oriented Screening and Docking: Inhibition of Matrix Metalloproteinases Activities by Flavonoids.

Metalloproteases are a family of zinc-containing endopeptidases involved in a variety of pathological disorders. The use of flavonoid derivatives as potential metalloprotease inhibitors has recently increased.Particular plants growing in Sicily are an excellent yielder of the flavonoids luteolin, apigenin, and their respective glycoside derivatives (7-O-rutinoside, 7-O-glucoside, and 7-O-glucuronide).The inhibitory activity of luteolin, apigenin, and their respective glycoside derivatives on the metalloproteases MMP-1, MMP-3, MMP-13, MMP-8, and MMP-9 was assessed and rationalized correlating in vitro target-oriented screening and in silico docking.The flavones apigenin, luteolin, and their respective glucosides have good ability to interact with metalloproteases and can also be lead compounds for further development. Glycones are more active on MMP-1, -3, -8, and -13 than MMP-9. Collagenases MMP-1, MMP-8, and MMP-13 are inhibited by compounds having rutinoside glycones. Apigenin and luteolin are inactive on MMP-1, -3, and -8, which can be interpreted as a better selectivity for both -9 and -13 peptidases. The more active compounds are apigenin-7-O-rutinoside on MMP-1 and luteolin-7-O-rutinoside on MMP-3. The lowest IC50 values were also found for apigenin-7-O-glucuronide, apigenin-7-O-rutinoside, and luteolin-7-O-glucuronide. The glycoside moiety might allow for a better anchoring to the active site of MMP-1, -3, -8, -9, and -13. Overall, the in silico data are substantially in agreement with the in vitro ones (fluorimetric assay).

Design, synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors.

New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC50 value of 43 nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition mode of the more active compounds. Best pose of compound 6c is positioned into the active site with the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.

How to Choose the Suitable Template for Homology Modelling of GPCRs: 5-HT7 Receptor as a Test Case.

G protein-coupled receptors (GPCRs) are a super-family of membrane proteins that attract great pharmaceutical interest due to their involvement in almost every physiological activity, including extracellular stimuli, neurotransmission, and hormone regulation. Currently, structural information on many GPCRs is mainly obtained by the techniques of computer modelling in general and by homology modelling in particular. Based on a quantitative analysis of eighteen antagonist-bound, resolved structures of rhodopsin family "A" receptors - also used as templates to build 153 homology models - it was concluded that a higher sequence identity between two receptors does not guarantee a lower RMSD between their structures, especially when their pair-wise sequence identity (within trans-membrane domain and/or in binding pocket) lies between 25 % and 40 %. This study suggests that we should consider all template receptors having a sequence identity ≤50 % with the query receptor. In fact, most of the GPCRs, compared to the currently available resolved structures of GPCRs, fall within this range and lack a correlation between structure and sequence. When testing suitability for structure-based drug design, it was found that choosing as a template the most similar resolved protein, based on sequence resemblance only, led to unsound results in many cases. Molecular docking analyses were carried out, and enrichment factors as well as attrition rates were utilized as criteria for assessing suitability for structure-based drug design.

Identification of 5-Methoxyflavone as a Novel DNA Polymerase-Beta Inhibitor and Neuroprotective Agent against Beta-Amyloid Toxicity.

Cell-cycle reactivation is a core feature of degenerating neurons in Alzheimer's disease (AD) and Parkinson's disease (PD). A variety of stressors, including ß-amyloid (Aß) in the case of AD, can force neurons to leave quiescence and to initiate an ectopic DNA replication process, leading to neuronal death rather than division. As the primary polymerase (pol) involved in neuronal DNA replication, DNA pol-ß contributes to neuronal death, and DNA pol-ß inhibitors may prove to be effective neuroprotective agents. Currently, specific and highly active DNA pol-ß inhibitors are lacking. Nine putative DNA pol-ß inhibitors were identified in silico by querying the ZINC database, containing more than 35 million purchasable compounds. Following pharmacological evaluation, only 5-methoxyflavone (1) was validated as an inhibitor of DNA pol-ß activity. Cultured primary neurons are a useful model to investigate the neuroprotective effects of potential DNA pol-ß inhibitors, since these neurons undergo DNA replication and death when treated with Aß. Consistent with the inhibition of DNA pol-ß, 5-methoxyflavone (1) reduced the number of S-phase neurons and the ensuing apoptotic death triggered by Aß. 5-Methoxyflavone (1) is the first flavonoid compound able to halt neurodegeneration via a definite molecular mechanism rather than through general antioxidant and anti-inflammatory properties.

Potential of aryl-urea-benzofuranylthiazoles hybrids as multitasking agents in Alzheimer's disease.

New benzofuranylthiazole derivatives containing the aryl-urea moiety were synthesized and evaluated in vitro as dual acetylcholinesterase (AChE)-butyrylcholinesterase (BuChE) inhibitors. In addition, the cupric reducing antioxidant capacities (CUPRAC) and ABTS cation radical scavenging abilities of the synthesized compounds were assayed. The result showed that all the synthesized compounds exhibited inhibitory activity on both AChE and BuChE with 1-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(2-fluorophenyl)urea (e25, IC50 value of 3.85 µM) and 1-(4-iodophenyl)-3-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)urea (e38, IC50 value of 2.03 µM) as the strongest inhibitors against AChE and BuChE, respectively. Compound e38 was 8.5-fold more potent than galanthamine. The selectivity index of e25 and e38 was 2.40 and 0.37 against AChE and BuChE, respectively. Compound e2, e4 and e11 (IC50 = 0.2, 0.5 and 1.13 µM, respectively) showed a better ABTS cation radical scavenging ability than the standard quercetin (IC50 = 1.18 µM). Best poses of compounds e38 on BuChE and e25 on AChE indicate that the thiazole ring and the amidic moiety are important sites of interaction with both ChEs. In addition, the benzofuran ring and phenyl ring are anchored to the side chains of both enzymes by π-π(pi-pi) interactions.

How does binding of imidazole-based inhibitors to heme oxygenase-1 influence their conformation? Insights combining crystal structures and molecular modelling.

Heme oxygenase-1 (HO-1) inhibition is associated with antitumor activity. Imidazole-based analogues show effective and selective inhibitory potency of HO-1. In this work, five single-crystal structures of four imidazole-based compounds are presented, with an in-depth structural analysis. In order to study the influence of the conformation of the ligands on binding to protein, conformational data from crystallography are compared with quantum mechanics analysis and molecular docking studies. Molecular docking of imidazole-based analogues in the active site of HO-1 is in good agreement with the experimental structures. Inhibitors interact with the heme cofactor and a hydrophobic pocket (Met34, Phe37, Val50, Leu147 and Phe214) in the HO-1 binding site. An alternate binding mode can be hypothesized for some inhibitors in the series.

Neuroprotective effects of the monoamine oxidase inhibitor tranylcypromine and its amide derivatives against Aß(1-42)-induced toxicity.

Monoamine oxidase (MAO) enzymes play a central role in the pathogenesis of Alzheimer's disease (AD) and MAO inhibitors (MAOIs) are antidepressant drugs currently studied for their neuroprotective properties in neurodegenerative disorders. In the present work MAOIs such as tranylcypromine [trans-(+)-2-phenylcyclopropanamine, TCP] and its amide derivatives, TCP butyramide (TCP-But) and TCP acetamide (TCP-Ac), were tested for their ability to protect cortical neurons challenged with synthetic amyloid-ß (Aß)-(1-42) oligomers (100 nM) for 48 h. TCP significantly prevented Aß-induced neuronal death in a concentration-dependent fashion and was maximally protective only at 10 µM. TCP-But was maximally protective in mixed neuronal cultures at 1 µM, a lower concentration compared to TCP, whereas the new derivative, TCP-Ac, was more efficacious than TCP and TCP-But and significantly protected cortical neurons against Aß toxicity at nanomolar concentrations (100 nM). Experiments carried out with the Thioflavin-T (Th-T) fluorescence assay for fibril formation showed that TCP and its amide derivatives influenced the early events of the Aß aggregation process in a concentration-dependent manner. TCP-Ac was more effective than TCP-But and TCP in slowing down the Aß(1-42) aggregates formation through a lengthening at the lag phase. In our experimental model co-incubation of Aß(1-42) oligomers with TCP-Ac was able to almost completely prevent Aß-induced neurodegeneration. These results suggest that inhibition of Aß oligomer-mediated aggregation significantly contributes to the overall neuroprotective activity of TCP-Ac and also raise the possibility that TCP, and in particular the new compound TCP-Ac, might represent new pharmacological tools to yield neuroprotection in AD.

Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis.

The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use.

Sequential application of ligand and structure based modeling approaches to index chemicals for their hH4R antagonism.

The human histamine H4 receptor (hH4R), a member of the G-protein coupled receptors (GPCR) family, is an increasingly attractive drug target. It plays a key role in many cell pathways and many hH4R ligands are studied for the treatment of several inflammatory, allergic and autoimmune disorders, as well as for analgesic activity. Due to the challenging difficulties in the experimental elucidation of hH4R structure, virtual screening campaigns are normally run on homology based models. However, a wealth of information about the chemical properties of GPCR ligands has also accumulated over the last few years and an appropriate combination of these ligand-based knowledge with structure-based molecular modeling studies emerges as a promising strategy for computer-assisted drug design. Here, two chemoinformatics techniques, the Intelligent Learning Engine (ILE) and Iterative Stochastic Elimination (ISE) approach, were used to index chemicals for their hH4R bioactivity. An application of the prediction model on external test set composed of more than 160 hH4R antagonists picked from the chEMBL database gave enrichment factor of 16.4. A virtual high throughput screening on ZINC database was carried out, picking ∼ 4000 chemicals highly indexed as H4R antagonists' candidates. Next, a series of 3D models of hH4R were generated by molecular modeling and molecular dynamics simulations performed in fully atomistic lipid membranes. The efficacy of the hH4R 3D models in discrimination between actives and non-actives were checked and the 3D model with the best performance was chosen for further docking studies performed on the focused library. The output of these docking studies was a consensus library of 11 highly active scored drug candidates. Our findings suggest that a sequential combination of ligand-based chemoinformatics approaches with structure-based ones has the potential to improve the success rate in discovering new biologically active GPCR drugs and increase the enrichment factors in a synergistic manner.