From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Acetylcholine prioritises direct synaptic inputs from entorhinal cortex to CA1 by differential modulation of feedforward inhibitory circuits.

Acetylcholine release in the hippocampus plays a central role in the formation of new memory representations. An influential but largely untested theory proposes that memory formation requires acetylcholine to enhance responses in CA1 to new sensory information from entorhinal cortex whilst depressing inputs from previously encoded representations in CA3. Here, we show that excitatory inputs from entorhinal cortex and CA3 are depressed equally by synaptic release of acetylcholine in CA1. However, feedforward inhibition from entorhinal cortex exhibits greater depression than CA3 resulting in a selective enhancement of excitatory-inhibitory balance and CA1 activation by entorhinal inputs. Entorhinal and CA3 pathways engage different feedforward interneuron subpopulations and cholinergic modulation of presynaptic function is mediated differentially by muscarinic M3 and M4 receptors, respectively. Thus, our data support a role and mechanisms for acetylcholine to prioritise novel information inputs to CA1 during memory formation.

Characterisation of inverse agonism of the orphan-G protein-coupled receptor GPR52 by cannabinoid ligands Cannabidiol and O-1918.

The identification of cannabinoid ligands Cannabidiol and O-1918 as inverse agonists of the orphan receptor GPR52 is reported. Detailed characterisation of GPR52 pharmacology and modelling of the proposed receptor interaction is described. The identification of a novel and further CNS pharmacology for the polypharmacological agent and marketed drug Cannabidiol is noteworthy.

Structure-based discovery and development of metabotropic glutamate receptor 5 negative allosteric modulators.

The metabotropic glutamate (mGlu) receptors are a family of eight class C G protein-coupled receptors (GPCRs) which modulate cell signaling and synaptic transmission to the major excitatory neurotransmitter l-glutamate (l-glutamic acid). Due to their role in modulating glutamate response, their widespread distribution in the central nervous system (CNS) and some evidence of dysregulation in disease, the mGlu receptors have become attractive pharmacological targets. As the orthosteric (glutamate) binding site is highly conserved across the eight mGlu receptors, it is difficult not only to generate ligands with subtype selectivity but, due to the nature of the binding site, with suitable drug-like properties to allow oral bioavailability and CNS penetration. Selective pharmacological targeting of a single receptor subtype can be achieved by targeting alternative (allosteric) binding sites. The nature of the allosteric binding pockets allows ligands to be developed that have good physical chemical properties as evidenced by several allosteric modulators of mGlu receptors entering clinical trials. The first negative allosteric modulators of the metabotropic glutamate 5 (mGlu5) receptor were discovered from high throughput screening activities. An alternative approach to drug discovery is to use structural knowledge to enable structure-based drug design (SBDD), which allows the design of molecules in a more rational, rather than empirical, fashion. Here we will describe the process of SBDD in the discovery of the mGlu5 negative allosteric modulator HTL0014242 and describe how knowledge of receptor structure can also be used to gain insights into the receptor activation mechanisms.

Comparison of Orexin 1 and Orexin 2 Ligand Binding Modes Using X-ray Crystallography and Computational Analysis.

The orexin system, which consists of the two G protein-coupled receptors OX1 and OX2, activated by the neuropeptides OX-A and OX-B, is firmly established as a key regulator of behavioral arousal, sleep, and wakefulness and has been an area of intense research effort over the past two decades. X-ray structures of the receptors in complex with 10 new antagonist ligands from diverse chemotypes are presented, which complement the existing structural information for the system and highlight the critical importance of lipophilic hotspots and water molecules for these peptidergic GPCR targets. Learnings from the structural information regarding the utility of pharmacophore models and how selectivity between OX1 and OX2 can be achieved are discussed.

Methodologies for the Examination of Water in GPCRs.

The following chapter examines some of the current "state-of-the-art" tools for predicting, scoring, and examining explicit water molecules in proteins and protein/ligand complexes, highlighting some of the ways information can be readily examined in a manner that is useful in a drug discovery process.

Potential for the Rational Design of Allosteric Modulators of Class C GPCRs.

Class C G protein-coupled receptors encompass a range of promising therapeutic targets for a variety of diseases, yet to date only two members of this sub-family of GPCRs have been drugged. Recent advances in structural biology have revealed the X-ray crystallographic structures of allosteric ligands bound to two Class C metabotropic glutamate (mGlu) receptors, mGlu1 and mGlu5. Herein, we review how this information can be leveraged to help understand some of the historical challenges of mGlu receptor allosteric modulator drug discovery, and discuss how the structural enablement can be prospectively used for structurebased drug discovery approaches across Class C GPCR targets in general.

The use of conformationally thermostabilised GPCRs in drug discovery: application to fragment, structure and biophysical techniques.

Recent developments in receptor stabilisation have facilitated major advances in G protein-coupled receptor (GPCR) research, notably structural biology, over the past eight years. Here we review the application of fragment, structure and biophysical techniques using stabilised GPCRs (StaR proteins), and their impact in the drug discovery process. These techniques have, most recently, been utilised in the discovery of the non-alkyne mGlu5 negative allosteric modulator HTL14242, in addition to the dual orexin receptor antagonist HTL6641, with differentiated residence time kinetics.

Selective Negative Allosteric Modulation Of Metabotropic Glutamate Receptors ­ A Structural Perspective of Ligands and Mutants.

The metabotropic glutamate receptors have a wide range of modulatory functions in the central nervous system. They are among the most highly pursued drug targets, with relevance for several neurological diseases, and a number of allosteric modulators have entered clinical trials. However, so far this has not led to a marketed drug, largely because of the difficulties in achieving subtype-selective compounds with desired properties. Very recently the first crystal structures were published for the transmembrane domain of two metabotropic glutamate receptors in complex with negative allosteric modulators. In this analysis, we make the first comprehensive structural comparison of all metabotropic glutamate receptors, placing selective negative allosteric modulators and critical mutants into the detailed context of the receptor binding sites. A better understanding of how the different mGlu allosteric modulator binding modes relates to selective pharmacological actions will be very valuable for rational design of safer drugs.

Fragment and Structure-Based Drug Discovery for a Class C GPCR: Discovery of the mGlu5 Negative Allosteric Modulator HTL14242 (3-Chloro-5-[6-(5-fluoropyridin-2-yl)pyrimidin-4-yl]benzonitrile).

Fragment screening of a thermostabilized mGlu5 receptor using a high-concentration radioligand binding assay enabled the identification of moderate affinity, high ligand efficiency (LE) pyrimidine hit 5. Subsequent optimization using structure-based drug discovery methods led to the selection of 25, HTL14242, as an advanced lead compound for further development. Structures of the stabilized mGlu5 receptor complexed with 25 and another molecule in the series, 14, were determined at resolutions of 2.6 and 3.1 Å, respectively.

Structure of Class B GPCRs: new horizons for drug discovery.

Class B GPCRs of the secretin family are important drug targets in many human diseases including diabetes, neurodegeneration, cardiovascular disease and psychiatric disorders. X-ray crystal structures for the glucagon receptor and corticotropin-releasing factor receptor 1 have now been published. In this review, we analyse the new structures and how they compare with each other and with Class A and F receptors. We also consider the differences in druggability and possible similarity in the activation mechanisms. Finally, we discuss the potential for the design of small-molecule modulators for these important targets in drug discovery. This new structural insight allows, for the first time, structure-based drug design methods to be applied to Class B GPCRs.

Unifying family A GPCR theories of activation.

Several new pairs of active and inactive GPCR structures have recently been solved enabling detailed structural insight into the activation process, not only of rhodopsin but now also of the ß2 adrenergic, M2 muscarinic and adenosine A2A receptors. Combined with structural analyses they have enabled us to examine the different recent theories proposed for GPCR activation and show that they are all indeed parts of the same process, and are intrinsically related through their effect on the central hydrophobic core of GPCRs. This new unifying general process of activation is consistent with the identification of known constitutively active mutants and an in-depth conservational analysis of significant residues implicated in the process.

Comparisons of the HBV and HIV polymerase, and antiviral resistance mutations.

The antiviral treatment of chronic hepatitis B is limited by the selection of antiviral resistance mutations. Primary resistance to lamivudine occurs at rtM2041/V in the C Domain of the polymerase. Recently, resistance to adefovir has also been described in the D Domain at rtN236T. The treatment of patients with resistant virus without complete suppression can lead to the further selection of compensatory mutations. Thus, to gain an understanding of the hepatitis B virus (HBV) polymerase and also mutations associated with resistance, a three-dimensional model of the HBV reverse transcriptase core region based on homology with human immunodeficiency virus (HIV) was created. A comparative analysis of the HIV polymerase and the model of HBV polymerase was performed. In addition, the antiviral resistance mutations including potential compensatory mutations were mapped to determine their effect on the HBV polymerase model, especially in the nucleotide binding site.

A consensus neural network-based technique for discriminating soluble and poorly soluble compounds.

BCUT [Burden, CAS, and University of Texas] descriptors, defined as eigenvalues of modified connectivity matrices, have traditionally been applied to drug design tasks such as defining receptor relevant subspaces to assist in compound selections. In this paper we present studies of consensus neural networks trained on BCUTs to discriminate compounds with poor aqueous solubility from those with reasonable solubility. This level was set at 0.1 mg/mL on advice from drug formulation and drug discovery scientists. By applying strict criteria to the insolubility predictions, approximately 95% of compounds are classified correctly. For compounds whose predictions have a lower level of confidence, further parameters are examined in order to flag those considered to possess unsuitable biopharmaceutical and physicochemical properties. This approach is not designed to be applied in isolation but is intended to be used as a filter in the selection of screening candidates, compound purchases, and the application of synthetic priorities to combinatorial libraries.

Molecular mapping in the CNS.

Since ancient times the operation of the brain has elicited more than usual interest. Data mining of the human genome is revealing that many CNS abnormalities have a genetic component. As yet this information can not be used directly to cure or ameliorate specific CNS disorders although this is regarded as having great potential for future therapies. Current CNS drug design and 3D QSAR is based on knowing either the structures of key proteins and how smaller molecules interact with them to obtain a pharmacological response, or on hypothesising about key structural features and interactions by a variety of molecular modelling and computational techniques. Methods used include conformational analyses, pharmacophore development and QSAR which are now being actively applied to increase our understanding of how molecules interact with specific sites within the CNS as a basis for the design of new pharmacologically active compounds. In this review we give an overview of the latest strategies used in 3D-QSAR based drug design and survey the most recent applications of these strategies to the CNS. By way of example, accounts are given of computer-based research aimed at drugs targeting GABA, glutamate, dopamine and opioid receptors.