Allosterism in the adenosine A2A and cannabinoid CB2 heteromer.

BACKGROUND AND PURPOSE: Allosterism is a regulatory mechanism for GPCRs that can be attained by ligand-binding or protein-protein interactions with another GPCR. We have studied the influence of the dimer interface on the allosteric properties of the A2A receptor and CB2 receptor heteromer. EXPERIMENTAL APPROACH: We have evaluated cAMP production, phosphorylation of signal-regulated kinases (pERK1/2), label-free dynamic mass redistribution, ß-arrestin 2 recruitment and bimolecular fluorescence complementation assays in the absence and presence of synthetic peptides that disrupt the formation of the heteromer. Molecular dynamic simulations provided converging evidence that the heteromeric interface influences the allosteric properties of the A2AR-CB2R heteromer. KEY RESULTS: Apo A2AR blocks agonist-induced signalling of CB2R. The disruptive peptides, with the amino acid sequence of transmembrane (TM) 6 of A2AR or CB2R, facilitate CB2R activation, suggesting that A2AR allosterically prevents the outward movement of TM 6 of CB2R for G protein binding. Significantly, binding of the selective antagonist SCH 58261 to A2AR also facilitated agonist-induced activation of CB2R. CONCLUSIONS AND IMPLICATIONS: It is proposed that the A2AR-CB2R heteromer contains distinct dimerization interfaces that govern its functional properties. The molecular interface between protomers of the A2AR-CB2R heteromer interconverted from TM 6 for apo or agonist-bound A2AR, blocking CB2R activation, to mainly the TM 1/7 interface for antagonist-bound A2AR, facilitating the independent opening of intracellular cavities for G protein binding. These novel results shed light on a different type of allosteric mechanism and extend the repertoire of GPCR heteromer signalling.

Engineered odorant receptors illuminate structural principles of odor discrimination.

A central challenge in olfaction is understanding how the olfactory system detects and distinguishes odorants with diverse physicochemical properties and molecular configurations. Vertebrate animals perceive odors via G protein-coupled odorant receptors (ORs). In humans, ~400 ORs enable the sense of smell. The OR family is composed of two major classes: Class I ORs are tuned to carboxylic acids while Class II ORs, representing the vast majority of the human repertoire, respond to a wide variety of odorants. How ORs recognize chemically diverse odorants remains poorly understood. A fundamental bottleneck is the inability to visualize odorant binding to ORs. Here, we uncover fundamental molecular properties of odorant-OR interactions by employing engineered ORs crafted using a consensus protein design strategy. Because such consensus ORs (consORs) are derived from the 17 major subfamilies of human ORs, they provide a template for modeling individual native ORs with high sequence and structural homology. The biochemical tractability of consORs enabled four cryoEM structures of distinct consORs with unique ligand recognition properties. The structure of a Class I consOR, consOR51, showed high structural similarity to the native human receptor OR51E2 and yielded a homology model of a related member of the human OR51 family with high predictive power. Structures of three Class II consORs revealed distinct modes of odorant-binding and activation mechanisms between Class I and Class II ORs. Thus, the structures of consORs lay the groundwork for understanding molecular recognition of odorants by the OR superfamily.

The Leu/Val6.51 Side Chain of Cannabinoid Receptors Regulates the Binding Mode of the Alkyl Chain of Δ9-Tetrahydrocannabinol.

(-)-Δ9-trans-tetrahydrocannabinol (THC), which is the principal psychoactive constituent of Cannabis, mediates its action by binding to two members of the G-protein-coupled receptor (GPCR) family: the cannabinoid CB1 (CB1R) and CB2 (CB2R) receptors. Molecular dynamics simulations showed that the pentyl chain of THC could adopts an I-shape conformation, filling an intracellular cavity between Phe3.36 and Trp6.48 for initial agonist-induced receptor activation, in CB1R but not in CB2R. This cavity opens to the five-carbon chain of THC by the conformational change of the γ-branched, flexible, Leu6.51 side chain of CB1R, which is not feasible by the ß-branched, mode rigid, Val6.51 side chain of CB2R. In agreement with our computational results, THC could not decrease the forskolin-induced cAMP levels in cells expressing mutant CB1RL6.51V receptor but could activate the mutant CB2RV6.51L receptor as efficiently as wild-type CB1R. Additionally, JWH-133, a full CB2R agonist, contains a branched dimethyl moiety in the ligand chain that bridges Phe3.36 and Val6.51 for receptor activation. In this case, the substitution of Val6.51 to Leu in CB2R makes JWH-133 unable to activate CB2RV6.51L. In conclusion, our combined computational and experimental results have shown that the amino acid at position 6.51 is a key additional player in the initial mechanism of activation of GPCRs that recognize signaling molecules derived from lipid species.

Structural basis of odorant recognition by a human odorant receptor.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1-3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Unique effect of clozapine on adenosine A2A-dopamine D2 receptor heteromerization.

The striatal dopamine D2 receptor (D2R) is generally accepted to be involved in positive symptoms of schizophrenia and is a main target for clinically used antipsychotics. D2R are highly expressed in the striatum, where they form heteromers with the adenosine A2A receptor (A2AR). Changes in the density of A2AR-D2R heteromers have been reported in postmortem tissue from patients with schizophrenia, but the degree to which A2R are involved in schizophrenia and the effect of antipsychotic drugs is unknown. Here, we examine the effect of exposure to three prototypical antipsychotic drugs on A2AR-D2R heteromerization in mammalian cells using a NanoBiT assay. After 16 h of exposure, a significant increase in the density of A2AR-D2R heteromers was found with haloperidol and aripiprazole, but not with clozapine. On the other hand, clozapine, but not haloperidol or aripiprazole, was associated with a significant decrease in A2AR-D2R heteromerization after 2 h of treatment. Computational binding models of these compounds revealed distinctive molecular signatures that explain their different influence on heteromerization. The bulky tricyclic moiety of clozapine displaces TM 5 of D2R, inducing a clash with A2AR, while the extended binding mode of haloperidol and aripiprazole stabilizes a specific conformation of the second extracellular loop of D2R that enhances the interaction with A2AR. It is proposed that an increase in A2AR-D2R heteromerization is involved in the extrapyramidal side effects (EPS) of antipsychotics and that the specific clozapine-mediated destabilization of A2AR-D2R heteromerization can explain its low EPS liability.

The ADORA1 mutation linked to early-onset Parkinson's disease alters adenosine A1-A2A receptor heteromer formation and function.

Adenosine modulates neurotransmission through inhibitory adenosine A1 receptors (A1Rs) and stimulatory A2A receptors (A2ARs). These G protein-coupled receptors are involved in motor function and related to neurodegenerative diseases such as Parkinson's disease (PD). An autosomal-recessive mutation (G2797.44S) within the transmembrane helix (TM) 7 of A1R (A1RG279S) has been associated with the development of early onset PD (EOPD). Here, we aimed at investigating the impact of this mutation on the structure and function of the A1R and the A1R-A2AR heteromer. Our results revealed that the G2797.44S mutation does not alter A1R expression, ligand binding, constitutive activity or coupling to transducer proteins (Gαi, Gαq, Gα12/13, Gαs, ß-arrestin2 and GRK2) in transfected HEK-293 T cells. However, A1RG279S weakened the ability of A1R to heteromerize with A2AR, as shown in a NanoBiT assay, which led to the disappearance of the heteromerization-dependent negative allosteric modulation that A1R imposes on the constitutive activity and agonist-induced activation of the A2AR. Molecular dynamic simulations allowed to propose an indirect mechanism by which the G2797.44S mutation in TM 7 of A1R weakens the TM 5/6 interface of the A1R-A2AR heteromer. Therefore, it is demonstrated that a PD linked ADORA1 mutation is associated with dysfunction of adenosine receptor heteromerization. We postulate that a hyperglutamatergic state secondary to increased constitutive activity and sensitivity to adenosine of A2AR not forming heteromers with A1R could represent a main pathogenetic mechanism of the EOPD associated with the G2797.44S ADORA1 mutation.

Pharmacological targeting of G protein-coupled receptor heteromers.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

A Single Point Mutation Blocks the Entrance of Ligands to the Cannabinoid CB2 Receptor via the Lipid Bilayer.

Molecular dynamic (MD) simulations have become a common tool to study the pathway of ligand entry to the orthosteric binding site of G protein-coupled receptors. Here, we have combined MD simulations and site-directed mutagenesis to study the binding process of the potent JWH-133 agonist to the cannabinoid CB2 receptor (CB2R). In CB2R, the N-terminus and extracellular loop 2 fold over the ligand binding pocket, blocking access to the binding cavity from the extracellular environment. We, thus, hypothesized that the binding pathway is a multistage process consisting of the hydrophobic ligand diffusing in the lipid bilayer to contact a lipid-facing vestibule, from which the ligand enters an allosteric site inside the transmembrane bundle through a tunnel formed between TMs 1 and 7 and finally moving from the allosteric to the orthosteric binding cavity. This pathway was experimentally validated by the Ala2827.36Phe mutation that blocks the entrance of the ligand, as JWH-133 was not able to decrease the forskolin-induced cAMP levels in cells expressing the mutant receptor. This proposed ligand entry pathway defines transient binding sites that are potential cavities for the design of synthetic modulators.

Analysis by real-time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID-19 diagnosis in Santiago of Chile.

Due to the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit mediums, including DNA/RNA Shield™, NAT, VTM-N, Ezmedlab™, and phosphate-buffered saline (PBS), for NPSs from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield™ shows higher relative fluorescence units (RFUs) and lower Cq values for the reference gene. Collectively, our results suggest that the PBS medium could compromise the sample diagnosis because of its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N, and DNA/RNA Shield™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis.

Structure and function of adenosine receptor heteromers.

Adenosine is one of the most ancient signaling molecules and has receptors in both animals and plants. In mammals there are four specific receptors, A1, A2A, A2B, and A3, which belong to the superfamily of G-protein-coupled receptors (GPCRs). Evidence accumulated in the last 20 years indicates that GPCRs are often expressed as oligomeric complexes formed by a number of equal (homomers) or different (heteromers) receptors. This review presents the data showing the occurrence of heteromers formed by A1 and A2A, A2A and A2B, and A2A and A3 receptors highlighting (i) their tetrameric structural arrangements, and (ii) the functional diversity that those heteromers provide to adenosinergic signaling.

Biological Properties and Absolute Configuration of Flavanones From Calceolaria thyrsiflora Graham.

Flavanones (-)-(2S)-5,4'-dihydroxy-7-methoxyflavanone (1) and (-)-(2S)-5,3',4'-trihydroxy-7-methoxyflavanone (2) were isolated from the extracts of Calceolaria thyrsiflora Graham, an endemic perennial small shrub growing in the central zone of Chile. The absolute configuration of these compounds was resolved by optical rotation experiments and in silico calculations. Three analogs (3, 4, and 5) were synthesized to do structure-activity relationships with the biological assays studied. Biological tests revealed that only flavanone 2 exhibited a moderate inhibitory activity against the methicillin-resistant strain S. aureus MRSA 97-77 (MIC value of 50 µg/ml). In addition, flavanone 2 showed a potent, selective, and competitive inhibition of 5-hLOX, which supports the traditional use of this plant as an anti-inflammatory in diseases of the respiratory tract. Also, 2 exhibited cytotoxic and selective effects against B16-F10 (8.07 ± 1.61 µM) but 4.6- and 17-fold lesser activity than etoposide and taxol.

The computational modeling of allosteric modulation of metabotropic glutamate receptors.

Allosteric modulation of GPCRs, especially metabotropic glutamate (mGlu) receptors, has become an important strategy for drug discovery. Positive and negative allosteric modulators (PAM, NAM) are widely reported for the mGlu receptor family with leads mostly originating by high-throughput screening followed by iterative medicinal chemistry. The progression of the field from mutagenesis and homology modeling to elaborate structure-enabled drug discovery is described. We detail how computational methods have delivered new chemical matter and revealed the functional details of PAM and NAM activity. The breakthrough in mGlu receptor 7-transmembrane (7TM) crystal structures enabled recent combined modeling and experimental studies to confirm common binding sites, interactions and the origins of ligand effect on functional activity. Focusing on allosteric modulation of the mGlu2 and mGlu5 receptors, similarities are seen that still accommodate the known differences in binding sites and SAR. This work reveals the promise of a methodical computational approach built upon deep analysis of 7TM receptor simulations and interpretation of results in the context of our current understanding of receptor function. A crucial aspect was the close collaboration between modeling and experiment necessary to build and interrogate the hypotheses.

Novel hollow titanium dioxide nanospheres with antimicrobial activity against resistant bacteria.

The search for and synthesis of new antimicrobial nanostructures is important to reduce microbial incidence that induces infectious diseases and to aid in the antibiotic resistance crisis, which are two of the most pressing issues in global public health. In this work, novel, hollow, calcined titanium dioxide nanospheres (CSTiO2) were successfully synthesized for the first time through the combination of electrospinning and atomic layer deposition techniques. Poly(vinylpyrrolidone) (PVP) electrosprayed spherical particles were double-coated with alumina and titanium dioxide, and after a calcination process, hollow nanospheres were obtained with a radius of approximately 345 nm and shell thickness of 17 nm. The structural characterization was performed using electron microscopy, and X-ray diffraction and small-angle X-ray diffraction evidenced an anatase titanium dioxide crystalline structure. Thermogravimetric analysis and Fourier-transform infrared spectroscopy studies demonstrated the absence of polymer residue after the calcination process. The antimicrobial properties of the developed CSTiO2 hollow nanospheres were evaluated against different bacteria, including resistant E. coli and S. aureus strains, and when compared to commercial TiO2 nanoparticles, CSTiO2 nanospheres exhibited superior performance. In addition, the positive effect of UV irradiation on the antimicrobial activity was demonstrated.

Computational Drug Design Applied to the Study of Metabotropic Glutamate Receptors.

Metabotropic glutamate (mGlu) receptors are a family of eight GPCRs that are attractive drug discovery targets to modulate glutamate action and response. Here we review the application of computational methods to the study of this family of receptors. X-ray structures of the extracellular and 7-transmembrane domains have played an important role to enable structure-based modeling approaches, whilst we also discuss the successful application of ligand-based methods. We summarize the literature and highlight the areas where modeling and experiment have delivered important understanding for mGlu receptor drug discovery. Finally, we offer suggestions of future areas of opportunity for computational work.

Mechanisms Underlying Allosteric Molecular Switches of Metabotropic Glutamate Receptor 5.

The metabotropic glutamate 5 (mGlu5) receptor is a class C G protein-coupled receptor (GPCR) that is implicated in several CNS disorders making it a popular drug discovery target. Years of research have revealed allosteric mGlu5 ligands showing an unexpected complete switch in functional activity despite only small changes in their chemical structure, resulting in positive allosteric modulators (PAM) or negative allosteric modulators (NAM) for the same scaffold. Up to now, the origins of this effect are not understood, causing difficulties in a drug discovery context. In this work, experimental data was gathered and analyzed alongside docking and Molecular Dynamics (MD) calculations for three sets of PAM and NAM pairs. The results consistently show the role of specific interactions formed between ligand substituents and amino acid side chains that block or promote local movements associated with receptor activation. The work provides an explanation for how such small structural changes lead to remarkable differences in functional activity. While this work can greatly help drug discovery programs avoid these switches, it also provides valuable insight into the mechanisms of class C GPCR allosteric activation. Furthermore, the approach shows the value of applying MD to understand functional activity in drug design programs, even for such close structural analogues.

New antibacterial and 5-lipoxygenase activities of synthetic benzyl phenyl ketones: Biological and docking studies.

We investigated twelve benzyl phenyl ketone derivatives which are synthetic precursors of isoflavonoids that are shown be good 5-hLOX inhibitors, especially those that have the catechol group, but these precursors never have been assayed as 5-hLOX inhibitors being a novelty as inhibitors of the enzyme, due to sharing important structural characteristics. Screening assays, half maximal inhibitory concentration (IC50) and kinetic assays of all the studied molecules (5 µg/ml in media assay) showed that 1-(2,4-dihydroxy-3-methylphenyl)-2-(3-chlorophenyl)-ethanone (K205; IC50 = 3.5 µM; Ki = 4.8 µM) and 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-nitrophenyl)-ethanone (K206; IC50 = 2.3 µM; Ki = 0.7 µM) were potent, selective, competitive and nonredox inhibitors of 5-hLOX. Antioxidant behavior was also assayed by DPPH, FRAP, and assessing ROS production, and those with antibacterial and antiproliferative properties relating to 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-chlorophenyl)-ethanone (K208) established it as the most interesting and relevant compound studied, as it showed nearly 100% inhibition of bacterial growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Finally, docking studies were done that helped to characterize how the inhibitor structures correlated to decreased 5-hLOX activity.

Role of sfk1 Gene in the Filamentous Fungus Penicillium roqueforti.

The sfk1 (suppressor of four kinase) gene has been mainly studied in Saccharomyces cerevisiae, where it was shown to be involved in growth and thermal stress resistance. This gene is widely conserved within the phylum Ascomycota. Despite this, to date sfk1 has not been studied in any filamentous fungus. Previously, we found that the orthologous of sfk1 was differentially expressed in a strain of Penicillium roqueforti with an altered phenotype. In this work, we have performed a functional characterization of this gene by using RNAi-silencing technology. The silencing of sfk1 in P. roqueforti resulted in decreased apical growth and the promotion of conidial germination, but interesting, it had no effect on conidiation. In addition, the attenuation of the sfk1 expression sensitized the fungus to osmotic stress, but not to thermal stress. RNA-mediated gene-silencing of sfk1 also affected cell wall integrity in the fungus. Finally, the silencing of sfk1 depleted the production of the main secondary metabolites of P. roqueforti, namely roquefortine C, andrastin A, and mycophenolic acid. To the best of our knowledge this is the first study of the sfk1 gene in filamentous fungi.

Easy and rapid preparation of benzoylhydrazides and their diazene derivatives as inhibitors of 15-lipoxygenase.

Two series of diaza derivatives were prepared by solvent-free condensation of benzoic acid and 4-substituted phenylhydrazines in order to obtain phenylhydrazides (HYD series) and, by oxidation of these compounds, the corresponding benzoyldiazenes (DIA series). Both sets were evaluated as inhibitors of soybean 15-lipoxygenase activity and antioxidant capability in the FRAP and CUPRAC assays. The most potent inhibitors of both series exhibited IC50 values in the low micromolar range. Kinetic studies showed that at least the more active compounds were competitive inhibitors. Docking results indicated that the most potent inhibitor interacts strongly with Ile-839 and iron in the active site.