Identification and Characterization of ML321: A Novel and Highly Selective D2 Dopamine Receptor Antagonist with Efficacy in Animal Models That Predict Atypical Antipsychotic Activity.

We have developed and characterized a novel D2R antagonist with exceptional GPCR selectivity - ML321. In functional profiling screens of 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R and to a lesser extent the D3R, demonstrating excellent receptor selectivity. The D2R selectivity of ML321 may be related to the fact that, unlike other monoaminergic ligands, ML321 lacks a positively charged amine group and adopts a unique binding pose within the orthosteric binding site of the D2R. PET imaging studies in non-human primates demonstrated that ML321 penetrates the CNS and occupies the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that ML321 can selectively antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus indicating exceptional in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 attenuates both amphetamine- and phencyclidine-induced locomotor activity and restored pre-pulse inhibition (PPI) of acoustic startle in a dose-dependent manner. Surprisingly, using doses that were maximally effective in both the locomotor and PPI studies, ML321 was relatively ineffective in promoting catalepsy. Kinetic studies revealed that ML321 exhibits slow-on and fast-off receptor binding rates, similar to those observed with atypical antipsychotics with reduced extrapyramidal side effects. Taken together, these observations suggest that ML321, or a derivative thereof, may exhibit ″atypical″ antipsychotic activity in humans with significantly fewer side effects than observed with the currently FDA-approved D2R antagonists.

Structure-guided engineering of tick evasins for targeting chemokines in inflammatory diseases.

As natural chemokine inhibitors, evasin proteins produced in tick saliva are potential therapeutic agents for numerous inflammatory diseases. Engineering evasins to block the desired chemokines and avoid off-target side effects requires structural understanding of their target selectivity. Structures of the class A evasin EVA-P974 bound to human CC chemokine ligands 7 and 17 (CCL7 and CCL17) and to a CCL8-CCL7 chimera reveal that the specificity of class A evasins for chemokines of the CC subfamily is defined by conserved, rigid backbone-backbone interactions, whereas the preference for a subset of CC chemokines is controlled by side-chain interactions at four hotspots in flexible structural elements. Hotspot mutations alter target preference, enabling inhibition of selected chemokines. The structure of an engineered EVA-P974 bound to CCL2 reveals an underlying molecular mechanism of EVA-P974 target preference. These results provide a structure-based framework for engineering evasins as targeted antiinflammatory therapeutics.

Systematic Assessment of Chemokine Signaling at Chemokine Receptors CCR4, CCR7 and CCR10.

Chemokines interact with chemokine receptors in a promiscuous network, such that each receptor can be activated by multiple chemokines. Moreover, different chemokines have been reported to preferentially activate different signalling pathways via the same receptor, a phenomenon known as biased agonism. The human CC chemokine receptors (CCRs) CCR4, CCR7 and CCR10 play important roles in T cell trafficking and have been reported to display biased agonism. To systematically characterize these effects, we analysed G protein- and ß-arrestin-mediated signal transduction resulting from stimulation of these receptors by each of their cognate chemokine ligands within the same cellular background. Although the chemokines did not elicit ligand-biased agonism, the three receptors exhibited different arrays of signaling outcomes. Stimulation of CCR4 by either CC chemokine ligand 17 (CCL17) or CCL22 induced ß-arrestin recruitment but not G protein-mediated signaling, suggesting that CCR4 has the potential to act as a scavenger receptor. At CCR7, both CCL19 and CCL21 stimulated G protein signaling and ß-arrestin recruitment, with CCL19 consistently displaying higher potency. At CCR10, CCL27 and CCL28(4-108) stimulated both G protein signaling and ß-arrestin recruitment, whereas CCL28(1-108) was inactive, suggesting that CCL28(4-108) is the biologically relevant form of this chemokine. These comparisons emphasize the intrinsic abilities of different receptors to couple with different downstream signaling pathways. Comparison of these results with previous studies indicates that differential agonism at these receptors may be highly dependent on the cellular context.

Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.

Biased agonism at G protein-coupled receptors describes the phenomenon whereby some drugs can activate some downstream signaling activities to the relative exclusion of others. Descriptions of biased agonism focusing on the differential engagement of G proteins versus ß-arrestins are commonly limited by the small response windows obtained in pathways that are not amplified or are less effectively coupled to receptor engagement, such as ß-arrestin recruitment. At the µ-opioid receptor (MOR), G protein-biased ligands have been proposed to induce less constipation and respiratory depressant side effects than opioids commonly used to treat pain. However, it is unclear whether these improved safety profiles are due to a reduction in ß-arrestin-mediated signaling or, alternatively, to their low intrinsic efficacy in all signaling pathways. Here, we systematically evaluated the most recent and promising MOR-biased ligands and assessed their pharmacological profile against existing opioid analgesics in assays not confounded by limited signal windows. We found that oliceridine, PZM21, and SR-17018 had low intrinsic efficacy. We also demonstrated a strong correlation between measures of efficacy for receptor activation, G protein coupling, and ß-arrestin recruitment for all tested ligands. By measuring the antinociceptive and respiratory depressant effects of these ligands, we showed that the low intrinsic efficacy of opioid ligands can explain an improved side effect profile. Our results suggest a possible alternative mechanism underlying the improved therapeutic windows described for new opioid ligands, which should be taken into account for future descriptions of ligand action at this important therapeutic target.

Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism.

By analyzing and simulating inactive conformations of the highly homologous dopamine D2 and D3 receptors (D2R and D3R), we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands.

Almost a third of prescribed drugs work by acting on a group of proteins known as GPCRs (short for G-protein coupled receptors), which help to transmit messages across the cell's outer barrier. The neurotransmitter dopamine, for instance, can act in the brain and body by attaching to dopamine receptors, a sub-family of GPCRs. The binding process changes the three-dimensional structure (or conformation) of the receptor from an inactive to active state, triggering a series of molecular events in the cell. However, GPCRs do not have a single 'on' or 'off' state; they can adopt different active shapes depending on the activating molecule they bind to, and this influences the type of molecular cascade that will take place in the cell. Some evidence also shows that classes of GPCRs can have different inactive structures; whether this is also the case for the dopamine D₂ and D₃ receptors remained unclear. Mapping out inactive conformations of receptors is important for drug discovery, as compounds called antagonists can bind to inactive receptors and interfere with their activation. Lane et al. proposed that different types of antagonists could prefer specific types of inactive conformations of the dopamine D₂ and D₃ receptors. Based on the structures of these two receptors, the conformations of D₂ bound with the drugs risperidone and eticlopride (two dopamine antagonists) were simulated and compared. The results show that the inactive conformations of D₂ were very different when it was bound to eticlopride as opposed to risperidone. In addition D₂ and D₃ showed a very similar conformation when attached to eticlopride. The two drugs also bound to the inactive receptors in overlapping but different locations. These computational findings, together with experimental validations, suggest that D₂ and D₃ exist in several inactive states that only allow the binding of specific drugs; these states could also reflect different degrees of inactivation. Overall, the work by Lane et al. contributes to a more refined understanding of the complex conformations of GPCRs, which could be helpful to screen and develop better drugs.

Molecular Determinants of the Intrinsic Efficacy of the Antipsychotic Aripiprazole.

Partial agonists of the dopamine D2 receptor (D2R) have been developed to treat the symptoms of schizophrenia without causing the side effects elicited by antagonists. The receptor-ligand interactions that determine the intrinsic efficacy of such drugs, however, are poorly understood. Aripiprazole has an extended structure comprising a phenylpiperazine primary pharmacophore and a 1,2,3,4-tetrahydroquinolin-2-one secondary pharmacophore. We combined site-directed mutagenesis, analytical pharmacology, ligand fragments, and molecular dynamics simulations to identify the D2R-aripiprazole interactions that contribute to affinity and efficacy. We reveal that an interaction between the secondary pharmacophore of aripiprazole and a secondary binding pocket defined by residues at the extracellular portions of transmembrane segments 1, 2, and 7 determines the intrinsic efficacy of aripiprazole. Our findings reveal a hitherto unappreciated mechanism for fine-tuning the intrinsic efficacy of D2R agonists.

A Thieno[2,3- d]pyrimidine Scaffold Is a Novel Negative Allosteric Modulator of the Dopamine D2 Receptor.

Recently, a novel negative allosteric modulator (NAM) of the D2-like dopamine receptors 1 was identified through virtual ligand screening. This ligand comprises a thieno[2,3- d]pyrimidine scaffold that does not feature in known dopaminergic ligands. Herein, we provide pharmacological validation of an allosteric mode of action for 1, revealing that it is a NAM of dopamine efficacy and identify the structural determinants of this allostery. We find that key structural moieties are important for functional affinity and negative cooperativity, while functionalization of the thienopyrimidine at the 5- and 6-positions results in analogues with divergent cooperativity profiles. Successive compound iterations have yielded analogues exhibiting a 10-fold improvement in functional affinity, as well as enhanced negative cooperativity with dopamine affinity and efficacy. Furthermore, our study reveals a fragment-like core that maintains low µM affinity and robust negative cooperativity with markedly improved ligand efficiency.

Key determinants of selective binding and activation by the monocyte chemoattractant proteins at the chemokine receptor CCR2.

Chemokines and their receptors collectively orchestrate the trafficking of leukocytes in normal immune function and inflammatory diseases. Different chemokines can induce distinct responses at the same receptor. In comparison to monocyte chemoattractant protein-1 (MCP-1; also known as CCL2), the chemokines MCP-2 (CCL8) and MCP-3 (CCL7) are partial agonists of their shared receptor CCR2, a key regulator of the trafficking of monocytes and macrophages that contribute to the pathology of atherosclerosis, obesity, and type 2 diabetes. Through experiments with chimeras of MCP-1 and MCP-3, we identified the chemokine amino-terminal region as being the primary determinant of both the binding and signaling selectivity of these two chemokines at CCR2. Analysis of CCR2 mutants showed that the chemokine amino terminus interacts with the major subpocket in the transmembrane helical bundle of CCR2, which is distinct from the interactions of some other chemokines with the minor subpockets of their receptors. These results suggest the major subpocket as a target for the development of small-molecule inhibitors of CCR2.

Novel Fused Arylpyrimidinone Based Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor.

Benzoquinazolinone 1 is a positive allosteric modulator (PAM) of the M1 muscarinic acetylcholine receptor (mAChR), which is significantly more potent than the prototypical PAM, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA). In this study, we explored the structural determinants that underlie the activity of 1 as a PAM of the M1 mAChR. We paid particular attention to the importance of the tricyclic scaffold of compound 1, for the activity of the molecule. Complete deletion of the peripheral fused benzene ring caused a significant decrease in affinity and binding cooperativity with acetylcholine (ACh). This loss of affinity was rescued with the addition of either one or two methyl groups in the 7- and/or 8-position of the quinazolin-4(3H)-one core. These results demonstrate that the tricyclic benzo[h]quinazolin-4(3H)-one core could be replaced with a quinazolin-4(3H)-one core and maintain functional affinity. As such, the quinazolin-4(3H)-one core represents a novel scaffold to further explore M1 mAChR PAMs with improved physicochemical properties.

4-Phenylpyridin-2-one Derivatives: A Novel Class of Positive Allosteric Modulator of the M1 Muscarinic Acetylcholine Receptor.

Positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (M1 mAChR) are a promising strategy for the treatment of the cognitive deficits associated with diseases including Alzheimer's and schizophrenia. Herein, we report the design, synthesis, and characterization of a novel family of M1 mAChR PAMs. The most active compounds of the 4-phenylpyridin-2-one series exhibited comparable binding affinity to the reference compound, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA) (1), but markedly improved positive cooperativity with acetylcholine, and retained exquisite selectivity for the M1 mAChR. Furthermore, our pharmacological characterization revealed ligands with a diverse range of activities, including modulators that displayed both high intrinsic efficacy and PAM activity, those that showed no detectable agonism but robust PAM activity and ligands that displayed robust allosteric agonism but little modulatory activity. Thus, the 4-phenylpyridin-2-one scaffold offers an attractive starting point for further lead optimization.

Enhancing performance of P3HT:TiO2 solar cells using doped and surface modified TiO2 nanorods.

Here we demonstrated an approach to increase performance of P3HT:TiO2 solar cell either by electron deficient boron or electron rich bismuth doping into TiO2 nanorods. The B doping increases the absorption, crystallinity and electron mobility of TiO2 nanorods. The Bi-doped TiO2 has higher J(sc) as compared with B-doped TiO2, mainly due to the improvement of electron density and increased absorption of TiO2 nanorods. The devices were fabricated from TiO2 nanorods being surface modified by organic dye W-4. The dye facilitates the bandgap alignment and compatibility between TiO2 and P3HT. The power conversion efficiency of solar cell has been increased by 1.33 times and 1.30 times for Bi-doped TiO2 and B-doped TiO2, respectively, as compared with that of as-synthesized TiO2. The results suggest the optical and electronic properties of TiO2 can be tuned by various dopants to enhance the device performance.

Proof of concept study for designed multiple ligands targeting the dopamine D2, serotonin 5-HT2A, and muscarinic M1 acetylcholine receptors.

Herein we describe the hybridization of a benzoxazinone M1 scaffold with D2 privileged structures derived from putative and clinically relevant antipsychotics to develop designed multiple ligands. The M1 mAChR is an attractive target for the cognitive deficits in key CNS disorders. Moreover, activity at D2 and 5-HT2A receptors has proven useful for antipsychotic efficacy. We identified 9 which retained functional activity at the target M1 mAChR and D2R and demonstrated high affinity for the 5-HT2AR.

A poly(3-hexylthiophene) block copolymer with macroscopically aligned hierarchical nanostructure induced by mechanical rubbing.

A highly ordered, uniformly aligned nanostructure with good crystallinity was first achieved on a P3HT block copolymer possessing a low weight fraction (19.4 wt%) of a flexible polyisoprene segment via a mechanical rubbing process without the use of solution based fabrication and thermal annealing. The attachment of the short polyisoprene segment to P3HT would significantly promote the main chain mobility to allow the orientation control of P3HT and of the self-assembled nanostructure by rubbing.

Airway strategies for lung isolation in a patient with high-velocity nail gun injuries to the right cardiac ventricle and floor of the mouth: a case report.

INTRODUCTION: We report a case of deliberate self-harm in which three three-inch nails were fired from a nail gun resulting in mandibular fixation and two penetrating injuries to the right cardiac ventricle. This combination of high-velocity penetrating injury has not been previously described. CASE PRESENTATION: A 69-year-old Caucasian man with a medical history of chronic depression was brought to hospital after a failed suicide attempt. The attempt consisted of self-asphyxiation with car exhaust fumes and shooting himself thrice with a three-inch nail gun. He sustained a penetrating nail injury to the floor of his mouth, effectively pinning his mouth closed, and penetrating injuries to the right ventricular free wall and at the junction of the right atrioventricular septum. The patient required emergency surgery with requirements for thoracotomy and sternotomy, lung isolation and cardiopulmonary bypass. CONCLUSIONS: This is the first reported case of a combination high-velocity penetrating nail gun injury to the face and the right cardiac ventricle. This rare case offers airway strategies to accommodate the surgical requirement for lung separation for penetrating chest trauma in a patient with iatrogenically limited mouth opening.

A novel series of histamine H4 receptor antagonists based on the pyrido[3,2-d]pyrimidine scaffold: comparison of hERG binding and target residence time with PF-3893787.

In this work we describe the optimization of a lead compound based on the quinazoline template to give a new series of potent pyrido[3,2-d]pyrimidines as histamine H4 receptor antagonists. The pyrido[3,2-d]pyrimidine ligands have significantly reduced hERG binding compared to clinical stage compound PF-3893787 while showing good affinities at the human and rodent histamine receptors. The receptor residence time of several of these new compounds was determined for the human H4R and compared with JNJ7777120 and PF-3893787. The pyrido[3,2-d]pyrimidines showed residence times lower than JNJ7777120 but comparable to the residence time of PF-3893787. Overall, the pyrido[3,2-d]pyrimidines show an excellent in vitro profile that warrants their further investigation in relevant models of human disease.

Ligand based design of novel histamine H4 receptor antagonists; fragment optimization and analysis of binding kinetics.

The histamine H(4) receptor is a G protein-coupled receptor that has attracted much interest for its role in inflammatory and immunomodulatory functions. In our search for new H(4)R ligands, a low affinity isoquinoline fragment was optimized to 7-(furan-2-yl)-4-(piperazin-1-yl)quinazolin-2-amine (VUF11489), as a new H(4)R antagonist. Analysis of its binding kinetics at the human H(4)R showed this compound to have a very different dissociative half-life in comparison with reference antagonist JNJ7777120.

Several down, a few to go: histamine H3 receptor ligands making the final push towards the market?

INTRODUCTION: The histamine H(3) receptor (H(3)R) plays a pivotal role in a plethora of therapeutic areas. Blocking the H(3)R with antagonists/inverse agonists has been postulated to be of broad therapeutic use. Indeed, H(3)R antagonists/inverse agonists have been extensively evaluated in the clinic. AREAS COVERED: Here, we address new developments, insights obtained and challenges encountered in the clinical evaluations. For recent H(3)R clinical candidates, the status and results of the corresponding clinical trial(s) will be discussed along with preclinical data. MAIN FINDINGS: In all, it becomes evident that clinical evaluation of H(3)R antagonists/inverse agonists is characterized by mixed results. On one hand, Pitolisant has successfully passed several Phase II trials and seems to be the most advanced compound in the clinic now, being in Phase III. On the other hand, some compounds (e.g., PF-03654647 and MK-0249) failed at Phase II clinical level for several indications. EXPERT OPINION: A challenging feature in H(3)R research is the multifaceted role of the receptor at a molecular/biochemical level, which can complicate targeting by small molecules at several (pre)clinical levels. Accordingly, H(3)R antagonists/inverse agonists require further testing to pinpoint the determinants for clinical efficacy and to aid in the final push towards the market.

Molecular determinants of ligand binding modes in the histamine H(4) receptor: linking ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) models to in silico guided receptor mutagenesis studies.

The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

Identification of a novel allosteric binding site in the CXCR2 chemokine receptor.

We have shown previously that different chemical classes of small-molecule antagonists of the human chemokine CXCR2 receptor interact with distinct binding sites of the receptor. Although an intracellular binding site for diarylurea CXCR2 antagonists, such as N-(2-bromophenyl)-N'-(7-cyano-1H-benzotriazol-4-yl)urea (SB265610), and thiazolopyrimidine compounds was recently mapped by mutagenesis studies, we now report on an imidazolylpyrimidine antagonist binding pocket in the transmembrane domain of CXCR2. Using different CXCR2 orthologs, chimeric proteins, site-directed mutagenesis, and in silico modeling, we have elucidated the binding mode of this antagonist. Our in silico-guided mutagenesis studies indicate that the ligand binding cavity for imidazolylpyrimidine compounds in CXCR2 is located between transmembrane (TM) helices 3 (Phe130(3.36)), 5 (Ser217(5.44), Phe220(5.47)), and 6 (Asn268(6.52), Leu271(6.55)) and suggest that these antagonists enter CXCR2 via the TM5-TM6 interface. It is noteworthy that the same interface is postulated as the ligand entry channel in the opsin receptor and is occupied by lipid molecules in the recently solved crystal structure of the CXCR4 chemokine receptor, suggesting a general ligand entrance mechanism for nonpolar ligands to G protein-coupled receptors. The identification of a novel allosteric binding cavity in the TM domain of CXCR2, in addition to the previously identified intracellular binding site, shows the diversity in ligand recognition mechanisms by this receptor and offers new opportunities for the structure-based design of small allosteric modulators of CXCR2 in the future.

N-acetylated Proline-Glycine-Proline induced G-protein dependent chemotaxis of neutrophils is independent of CXCL8 release.

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). In this study, we investigated in more detail the mechanism of action of N-ac-PGP in neutrophilic inflammation. N-ac-PGP was chemotactic for human neutrophils via pertussis toxin sensitive G protein-coupled receptors in vitro and directly activated this cell type, which led to cytosolic calcium mobilization and release of CXCL8. Furthermore, using a selective CXCR2 antagonist confirmed that N-ac-PGP-induced neutrophil chemotaxis is mediated through CXCR2 activation. To determine whether N-ac-PGP was solely responsible for the migration and activation of human neutrophils in vitro and not the released CXCL8 upon stimulation with N-ac-PGP, an antibody directed against CXCL8 was used. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of N-ac-PGP whereas effects of CXCL8 were attenuated. These experiments indicate that N-ac-PGP, in addition to the direct induction of chemotaxis, also directly activates neutrophils to release CXCL8. In vivo, this may lead in the long term to a self-maintaining situation enhanced by both N-ac-PGP and CXCL8, leading to a further increase in neutrophil infiltration and chronic inflammation.