Crystal structure of dopamine D1 receptor in complex with G protein and a non-catechol agonist.

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.

Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket.

The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A2AR-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson's disease.

Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis.

The putative Major Facilitator Superfamily (MFS) transporter, SV2A, is the target for levetiracetam (LEV), which is a successful anti-epileptic drug. Furthermore, SV2A knock out mice display a severe seizure phenotype and die after a few weeks. Despite this, the mode of action of LEV is not known at the molecular level. It would be extremely desirable to understand this more fully in order to aid the design of improved anti-epileptic compounds. Since there is no structure for SV2A, homology modelling can provide insight into the ligand-binding site. However, it is not a trivial process to build such models, since SV2A has low sequence identity to those MFS transporters whose structures are known. A further level of complexity is added by the fact that it is not known which conformational state of the receptor LEV binds to, as multiple conformational states have been inferred by tomography and ligand binding assays or indeed, if binding is exclusive to a single state. Here, we explore models of both the inward and outward facing conformational states of SV2A (according to the alternating access mechanism for MFS transporters). We use a sequence conservation analysis to help guide the homology modelling process and generate the models, which we assess further with Molecular Dynamics (MD). By comparing the MD results in conjunction with docking and simulation of a LEV-analogue used in radioligand binding assays, we were able to suggest further residues that line the binding pocket. These were confirmed experimentally. In particular, mutation of D670 leads to a complete loss of binding. The results shed light on the way LEV analogues may interact with SV2A and may help with the on-going design of improved anti-epileptic compounds.

Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A.

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral ß-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open ß-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

Development of dimethyl sulfoxide solubility models using 163,000 molecules: using a domain applicability metric to select more reliable predictions.

The dimethyl sulfoxide (DMSO) solubility data from Enamine and two UCB pharma compound collections were analyzed using 8 different machine learning methods and 12 descriptor sets. The analyzed data sets were highly imbalanced with 1.7-5.8% nonsoluble compounds. The libraries' enrichment by soluble molecules from the set of 10% of the most reliable predictions was used to compare prediction performances of the methods. The highest accuracies were calculated using a C4.5 decision classification tree, random forest, and associative neural networks. The performances of the methods developed were estimated on individual data sets and their combinations. The developed models provided on average a 2-fold decrease of the number of nonsoluble compounds amid all compounds predicted as soluble in DMSO. However, a 4-9-fold enrichment was observed if only 10% of the most reliable predictions were considered. The structural features influencing compounds to be soluble or nonsoluble in DMSO were also determined. The best models developed with the publicly available Enamine data set are freely available online at http://ochem.eu/article/33409 .

Towards a KCC2 blocker pharmacophore model.

A multi-disciplinary approach was used to identify the first pharmacophore model for KCC2 blockers: several physico-chemical studies such as XRD and NMR were combined to molecular modelling techniques, SAR analysis and synthesis of constrained analogues in order to determine a minimal conformational space regrouping few potential bioactive conformations. These conformations were further compared to the conformational space of a different series of KCC2 blockers in order to identify the common pharmacophoric features. The synthesis of more potent analogues in this second series confirmed the usefulness of this KCC2 blocker pharmacophore model.

Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding.

LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.

Discovery of a new class of non-imidazole oxazoline-based histamine H(3) receptor (H(3)R) inverse agonists.

H(3)R inverse agonists based on an aminopropoxy-phenyloxazoline framework constitute highly valuable druglike lead compounds that display efficacy in a mouse model of recognition memory.

4-benzyl-1H-imidazoles with oxazoline termini as histamine H3 receptor agonists.

Research on the therapeutic applications of the histamine H3 receptor (H3R) has traditionally focused on antagonists/inverse agonists. In contrast, H3R agonists have received less attention despite their potential use in several disease areas. The lower availability of H3R agonists not only hampers their full therapeutic exploration, it also prevents an unequivocal understanding of the structural requirements for H3R activation. In the light of these important issues, we present our findings on 4-benzyl-1H-imidazole-based H3R agonists. Starting from two high throughput screen hits (10 and 11), the benzyl side chain was altered with lipophilic groups using combinatorial and classical chemical approaches (compounds 12-31). Alkyne- or oxazolino-substituents gave excellent affinities and agonist activities up to the single digit nM range. Our findings further substantiate the growing notion that basic ligand sidechains are not necessary for H 3R activation and reveal the oxazolino group as a hitherto unexplored functional group in H3R research.

First dual M3 antagonists-PDE4 inhibitors: synthesis and SAR of 4,6-diaminopyrimidine derivatives.

SAR around 4,6-diaminopyrimidine derivatives allowed the discovery of the first potent dual M(3) antagonists and PDE4 inhibitors. Various chemical modulations around that scaffold led to the discovery of ucb-101333-3 which is characterized by the most interesting profile on both targets.

Metal-organic compounds: a new approach for drug discovery. N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide copper(II) complex as an inhibitor of human immunodeficiency virus 1 protease.

The use of metal-organic complexes is a potentially fruitful approach for the development of novel enzyme inhibitors. They hold the attractive promise of forming stronger attachments with the target by combining the co-ordination ability of metals with the unique stereoelectronic properties of the ligand. We demonstrated that this approach can be successfully used to inhibit the protease of the human immunodeficiency virus (type 1). Several ligands bearing substituents designed to interact with the catalytic site of the enzyme when complexed to Cu(2+) were synthesised. The inhibition pattern of the resulting copper(II) complexes was analysed. We showed that the copper(II) complex of N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide (C1) interacts with the active site of the enzyme leading to competitive inhibition. On the other hand, N2-pyridine-amide ligands and oxazinane carboxamide ligand were found to be poor chelators of the cupric ion under the enzymatic assay conditions. In these cases, the observed inhibition was attributed to released cupric ions which react with cysteine residues on the surface of the protease. While unchelated metal cations are not likely to be useful agents, metal chelates such as C1 should be considered as promising lead compounds for the development of targeted drugs.