Probe Substrate Dependencies in CYP3A4 Allosteric Inhibition: A Novel Molecular Mechanism Involving F-F' Loop Perturbations.

The biochemical basis for substrate dependences in apparent inhibition constant values (Ki) remains unknown. Our study aims to elucidate plausible structural determinants underpinning these observations. In vitro steady-state inhibition assays conducted using human recombinant CYP3A4 enzyme and testosterone substrate revealed that fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and pemigatinib noncompetitively inhibited CYP3A4 with apparent Ki values of 10.2 ± 1.1 and 3.3 ± 0.9 µM, respectively. However, when rivaroxaban was adopted as the probe substrate, there were 2.0- and 3.2-fold decreases in its apparent Ki values. To glean mechanistic insights into this phenomenon, erdafitinib and pemigatinib were docked to allosteric sites in CYP3A4. Subsequently, molecular dynamics (MD) simulations of apo- and holo-CYP3A4 were conducted to investigate the structural changes induced. Comparative structural analyses of representative MD frames extracted by hierarchical clustering revealed that the allosteric inhibition of CYP3A4 by erdafitinib and pemigatinib did not substantially modulate its active site characteristics. In contrast, we discovered that allosteric binding of the FGFR inhibitors reduces the structural flexibility of the F-F' loop region, an important gating mechanism to regulate access of the substrate to the catalytic heme. We surmised that the increased rigidity of the F-F' loop engenders a more constrained entrance to the CYP3A4 active site, which in turn impedes access to the larger rivaroxaban molecule to a greater extent than testosterone and culminates in more potent inhibition of its CYP3A4-mediated metabolism. Our findings suggest a potential mechanism to rationalize probe substrate dependencies in Ki arising from the allosteric noncompetitive inhibition of CYP3A4.

Direct and Sequential Bioactivation of Pemigatinib to Reactive Iminium Ion Intermediates Culminates in Mechanism-Based Inactivation of Cytochrome P450 3A.

We recently established the mechanism-based inactivation (MBI) of cytochrome P450 3A (CYP3A) by the fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and infigratinib. Serendipitously, our preliminary data have also revealed that pemigatinib (PEM), another clinically approved FGFR1-3 inhibitor, similarly elicited time-dependent inhibition of CYP3A. This was rather unexpected, as it was previously purported that PEM did not pose any metabolism-dependent liabilities due to the absence of glutathione-related conjugates in metabolic profiling experiments conducted in human liver microsomes. Here, we confirmed that PEM inhibited both CYP3A isoforms in a time-, concentration-, and cofactor-dependent manner consistent with MBI, with inactivator concentration at half-maximum rate constant, maximum inactivation rate constant, and partition ratio of 8.69 and 11.95 µM, 0.108 and 0.042 min-1, and approximately 44 and approximately 47 for CYP3A4 and CYP3A5, respectively. Although the rate of inactivation was diminished by coincubation with an alternative substrate or direct inhibitor of CYP3A, the inclusion of nucleophilic trapping agents afforded no such protection. Furthermore, the lack of catalytic activity recovery following dialysis and oxidation with potassium ferricyanide coupled with the absence of a spectrally resolvable peak in the Soret region collectively implied that the underlying mechanism of inactivation was not elicited via the formation of pseudo-irreversible metabolite-intermediate complexes. Finally, utilizing cyanide trapping and high-resolution mass spectrometry, we illuminated the direct and sequential oxidative bioactivation of PEM and its major O-desmethylated metabolite at its distal morpholine moiety to reactive iminium ion hard electrophilic species that could covalently inactivate CYP3A via MBI. SIGNIFICANCE STATEMENT: This study reports for the first time the covalent MBI of CYP3A by PEM and deciphered its bioactivation pathway involving the metabolic activation of PEM and its major O-desmethylated metabolite to reactive iminium ion intermediates. Following which, a unique covalent docking methodology was harnessed to unravel the structural and molecular determinants underpinning its inactivation. Findings from this study lay the foundation for future investigation of clinically relevant drug-drug interactions between PEM and concomitant substrates of CYP3A.

Computational Modeling of C-Terminal Tails to Predict the Calcium-Dependent Secretion of Endoplasmic Reticulum Resident Proteins.

The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention/retrieval sequence (ERS). The canonical ERS that promotes ER retrieval is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical "KDEL" ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interacts with the KDELRs. The interaction between the ERS and the KDELR extends beyond the final four carboxy terminal residues of the ERS. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.

Differential Reversible and Irreversible Interactions between Benzbromarone and Human Cytochrome P450s 3A4 and 3A5.

Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and ß of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.

Infigratinib Is a Reversible Inhibitor and Mechanism-Based Inactivator of Cytochrome P450 3A4.

Infigratinib (INF) is a promising selective inhibitor of fibroblast growth factor receptors 1-3 that has recently been accorded both orphan drug designation and priority review status by the US Food and Drug Administration for the treatment of advanced cholangiocarcinoma. Its propensity to undergo bioactivation to electrophilic species was recently expounded upon. However, other than causing aberrant idiosyncratic toxicities, these reactive intermediates may elicit mechanism-based inactivation of cytochrome P450 enzymes. In this study, we investigated the interactions between INF and the most abundant hepatic CYP3A. Our findings revealed that, apart from being a potent noncompetitive reversible inhibitor of CYP3A4, INF inactivated CYP3A4 in a time-, concentration- and NADPH-dependent manner with inactivator concentration at half-maximum inactivation rate constant, maximum inactivation rate constant, and partition ratio of 4.17 µM, 0.068 minute-1, and 41, respectively, when rivaroxaban was employed as the probe substrate. Coincubation with testosterone (alternative CYP3A substrate) or ketoconazole (direct CYP3A inhibitor) attenuated the rate of inactivation, whereas the inclusion of glutathione and catalase did not confer such protection. The lack of enzyme activity recovery after dialysis for 4 hours and oxidation with potassium ferricyanide, coupled with the absence of the characteristic Soret peak signature collectively substantiated that inactivation of CYP3A4 by INF was not mediated by the formation of quasi-irreversible metabolite-intermediate complexes but rather through irreversible covalent adduction to the prosthetic heme and/or apoprotein. Finally, glutathione trapping and high-resolution mass spectrometry experimental results unraveled two plausible bioactivation mechanisms of INF arising from the generation of a p-benzoquinonediimine and epoxide reactive intermediate. SIGNIFICANCE STATEMENT: The potential of INF to cause MBI of CYP3A4 was unknown. This study reports the reversible noncompetitive inhibition and irreversible covalent MBI of CYP3A4 by INF and proposes two potential bioactivation pathways implicating p-benzoquinonediimine and epoxide reactive intermediates, following which a unique covalent docking methodology was harnessed to elucidate the structural and molecular determinants underscoring its inactivation. Findings from this study lay the groundwork for future investigation of clinically relevant drug-drug interactions between INF and concomitant substrates of CYP3A4.

Atypical kinetics of cytochrome P450 2J2: Epoxidation of arachidonic acid and reversible inhibition by xenobiotic inhibitors.

Extrahepatic CYP2J2 metabolism of arachidonic acid (AA) to bioactive regioisomeric epoxyeicosatrienoic acids (EETs) is implicated in both physiological and pathological conditions. Here, we aimed to characterize atypical substrate inhibition kinetics of this endogenous metabolic pathway and its reversible inhibition by xenobiotic inhibitors when AA is used as the physiologically-relevant substrate vis-à-vis conventional probe substrate astemizole (AST). As compared to typical Michaelis-Menten kinetics observed for AST, complete substrate inhibition was observed for CYP2J2 metabolism of AA to 14,15-EET whereby velocity of the reaction declined significantly at concentrations of AA above 20-30 µM with an estimated substrate inhibition constant (Ks) of 31 µM. In silico sequential docking of two AA substrates to orthosteric (OBS) and adjacent secondary binding sites (SBS) within a 3-dimensional homology model of CYP2J2 revealed favorable and comparable binding poses of glide-scores -3.1 and -3.8 respectively. Molecular dynamics (MD) simulations ascertained CYP2J2 conformational stability with dual AA substrate binding as time-dependent root mean squared deviation (RMSD) of protein Cα atoms and ligand heavy atoms stabilized to a plateau in all but one trajectory (n=6). The distance between heme-iron and ω6 (C14, C15) double bond of AA in OBS also increased from 7.5 ± 1.4 Å to 8.5 ± 1.8 Å when CYP2J2 was simulated with only AA in OBS versus the presence of AA in both OBS and SBS (p<0.001), supporting the observed in vitro substrate inhibition phenomenon. Poor correlation was observed between inhibitory constants (Ki) determined for a panel of nine competitive and mixed mode xenobiotic inhibitors against CYP2J2 metabolism of AA as compared to AST, whereby 4 out of 9 drugs had a greater than 5-fold difference between Ki values. Nonlinear Eadie-Hofstee plots illustrated that complete substrate inhibition of CYP2J2 by AA was not attenuated even at high concentrations of xenobiotic inhibitors which further corroborates that CYP2J2 may accommodate three or more ligands simultaneously. In light of the atypical kinetics, our results highlight the importance of using physiologically-relevant substrates in in vitro enzymatic inhibition assays for the characterization of xenobiotic-endobiotic interactions which is applicable to other complex endogenous metabolic pathways beyond CYP2J2 metabolism of AA to EETs. The accurate determination of Ki would further facilitate the association of xenobiotic-endobiotic interactions to observed therapeutic or toxic outcomes.

Mechanism-Based Inactivation of Cytochrome P450 3A4 by Benzbromarone.

Benzbromarone (BBR), a potent uricosuric agent for the management of gout, is known to cause fatal fulminant hepatitis. Although the mechanism of BBR-induced idiosyncratic hepatotoxicity remains unelucidated, cytochrome P450 enzyme-mediated bioactivation of BBR to electrophilic reactive metabolites is commonly regarded as a key molecular initiating event. However, apart from causing aberrant toxicities, reactive metabolites may result in mechanism-based inactivation (MBI) of cytochrome P450. Here, we investigated and confirmed that BBR inactivated CYP3A4 in a time-, concentration-, and NADPH-dependent manner with K I, k inact, and partition ratio of 11.61 µM, 0.10 minutes-1, and 110, respectively. Coincubation with ketoconazole, a competitive inhibitor of CYP3A4, attenuated the MBI of CYP3A4 by BBR, whereas the presence of glutathione and catalase did not confer such protection. The lack of substantial recovery of enzyme activity postdialysis and after oxidation with potassium ferricyanide, combined with the absence of a Soret peak in spectral difference scans, implied that MBI of CYP3A4 by BBR did not occur through the formation of quasi-irreversible metabolite-intermediate complexes. Analysis of the reduced CO-difference spectrum revealed an ∼44% reduction in ferrous-CO binding and hinted that inactivation is mediated via irreversible covalent adduction to both the prosthetic heme moiety and the apoprotein. Finally, our in silico covalent docking analysis further suggested the modulation of substrate binding to CYP3A4 via the covalent adduction of epoxide-derived reactive intermediates of BBR to two key cysteine residues (Cys239 and Cys58) vicinal to the entrance of the orthosteric binding site. SIGNIFICANCE STATEMENT: Although the bioactivation of benzbromarone (BBR) to reactive metabolites has been well characterized, its potential to cause mechanism-based inactivation (MBI) of cytochrome P450 has not been fully investigated. This study reports the MBI of CYP3A4 by BBR via irreversible covalent adduction and develops a unique covalent docking methodology to predict the structural molecular determinants underpinning the inactivation for the first time. These findings lay the groundwork for future investigation of clinically relevant drug-drug interactions implicating BBR and mechanisms of BBR-induced idiosyncratic hepatotoxicity.

Recent Advances in Structure, Function, and Pharmacology of Class A Lipid GPCRs: Opportunities and Challenges for Drug Discovery.

Great progress has been made over the past decade in understanding the structural, functional, and pharmacological diversity of lipid GPCRs. From the first determination of the crystal structure of bovine rhodopsin in 2000, much progress has been made in the field of GPCR structural biology. The extraordinary progress in structural biology and pharmacology of GPCRs, coupled with rapid advances in computational approaches to study receptor dynamics and receptor-ligand interactions, has broadened our comprehension of the structural and functional facets of the receptor family members and has helped usher in a modern age of structure-based drug design and development. First, we provide a primer on lipid mediators and lipid GPCRs and their role in physiology and diseases as well as their value as drug targets. Second, we summarize the current advancements in the understanding of structural features of lipid GPCRs, such as the structural variation of their extracellular domains, diversity of their orthosteric and allosteric ligand binding sites, and molecular mechanisms of ligand binding. Third, we close by collating the emerging paradigms and opportunities in targeting lipid GPCRs, including a brief discussion on current strategies, challenges, and the future outlook.

Evidence for a Stereoselective Mechanism for Bitopic Activity by Extended-Length Antagonists of the D3 Dopamine Receptor.

The D3 dopamine receptor (D3R) has been suggested as a drug target for the treatment of a number of neuropsychiatric disorders, including substance use disorders (SUD). Many D3R-selective antagonists are bivalent in nature in that they engage two distinct sites on the receptor-a primary pharmacophore binds to the orthosteric site, where dopamine binds, whereas a secondary pharmacophore interacts with a unique secondary binding pocket (SBP). When engagement of the secondary pocket exerts allosteric activity, the compound is said to be bitopic. We recently reported the synthesis and characterization of two bitopic antagonists of the D3R, (±)-VK04-87 and (±)-VK05-95, which incorporated a racemic trans-cyclopropylmethyl linking chain. To gain a better understanding of the role of chirality in determining the pharmacology of such compounds, we resolved the enantiomers of (±)-VK04-87. We found that the (+)-isomer displays higher affinity for the D3R and exhibits greater selectivity versus the D2R than the (-)-isomer. Strikingly, using functional assays, we found that (+)-VK04-87 inhibits the D3R in a noncompetitive manner, while (-)-VK04-87 behaves as a purely competitive antagonist, indicating that the apparent allosteric activity of the racemate is due to the (+)-isomer. Molecular dynamic simulations of (+)-VK04-87 and (-)-VK04-87 binding to the D3R suggest that the (+)-isomer is able to interact with the SBP of the receptor whereas the (-)-isomer bends away from this pocket, thus potentially explaining their differing pharmacology. These results emphasize the importance of the linker, and its isomeric conformations, within extended-length molecules for their positioning and engagement within GPCR binding pockets.

A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity.

Signaling bias is the propensity for some agonists to preferentially stimulate G protein-coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein-biased agonist of the D2 dopamine receptor (D2R) that results in impaired ß-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with ß-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired ß-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the ß2-adrenergic receptor (ß2R) to build ß2R-WT and ß2R-Y1995.38A models in complex with the full ß2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in ß2R-Y1995.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in ß2R-Y1995.38A, which is predicted to affect its interactions with ß-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.

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.

Widening cracks in patriarchy: mothers and daughters navigating gender norms in a Mumbai slum.

Inequitable gender norms can be harmful to girls' and boys' health and sexuality. Programmatic approaches that help renegotiate gendered power relationships are sorely needed. This qualitative study reveals how Parivartan, a sport-based intervention in a Mumbai informal settlement, helped families resist inequitable gender norms that limited girls' mobility in public spaces. Fifteen girl athletes were interviewed in two rounds of face-to-face in-depth interviews. Results identify the strategies girls' mothers used to support their daughters' participation in the programme when they feared their husbands' disapproval. Rather than openly confronting their husbands, mothers worked from within the patriarchal gender order, through its 'cracks', for instance initially hiding their daughters' participation from their husbands. At an appropriate moment, girls' mothers revealed to their husbands about their daughters playing sports, convincing them of the usefulness of the programme. Girls' participation profoundly and positively affected relationships between daughters, mothers and fathers. Over time, parents' trust that girls would not compromise family honour increased, eventually changing the acceptability of girls' playing sport in public in spite of the patriarchal gender order. Concluding remarks offer key implications for effective interventions, highlighting the historical nature of gender transformation processes.

Presence of Intra-helical Salt-Bridge in Loop E Half-Helix Can Influence the Transport Properties of AQP1 and GlpF Channels: Molecular Dynamics Simulations of In Silico Mutants.

Major intrinsic protein (MIP) superfamily contains water-transporting AQP1 and glycerol-specific GlpF belonging to two major phylogenetic groups, namely aquaporins (AQPs) and aquaglyceroporins (AQGPs). MIP channels have six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). LE region contributes two residues to the aromatic/arginine (Ar/R) selectivity filter (SF) within the MIP channel. Bioinformatics analyses have shown that all AQGPs have an intra-helical salt-bridge (IHSB) in LE half-helix and all AQGPs and majority of AQPs have helix destabilizing Gly and/or Pro in the same region. In this paper, we mutated in silico the acidic and basic residues in GlpF to Ser and introduced salt-bridge interaction in AQP1 LE half-helix by substituting Ser residues at the equivalent positions with acidic and basic residues. We investigated the influence of IHSB in LE half-helix on the transport properties of GlpF and AQP1 mutant channels using molecular dynamics simulations. With IHSB abolished in LE half-helix, the GlpF mutant exhibited a significantly reduced water transport. In contrast, the introduction of IHSB in the two AQP1 mutants has increased water transport. Absence of salt-bridge in LE half-helix alters the SF geometry and results in a higher energy barrier for the solutes in the Ar/R selectivity filter. Presence/absence of IHSB in LE half-helix influences the channel transport properties and it is evident especially for the AQGPs. By modulating its helical flexibility, LE half-helix can perhaps play a regulatory role in transport either on its own or in conjunction with other extracellular regions.

Gs- versus Golf-dependent functional selectivity mediated by the dopamine D1 receptor.

The two highly homologous subtypes of stimulatory G proteins Gαs (Gs) and Gαolf (Golf) display contrasting expression patterns in the brain. Golf is predominant in the striatum, while Gs is predominant in the cortex. Yet, little is known about their functional distinctions. The dopamine D1 receptor (D1R) couples to Gs/olf and is highly expressed in cortical and striatal areas, making it an important therapeutic target for neuropsychiatric disorders. Using novel drug screening methods that allow analysis of specific G-protein subtype coupling, we found that, relative to dopamine, dihydrexidine and N-propyl-apomorphine behave as full D1R agonists when coupled to Gs, but as partial D1R agonists when coupled to Golf. The Gs/Golf-dependent biased agonism by dihydrexidine was consistently observed at the levels of cellular signaling, neuronal function, and behavior. Our findings of Gs/Golf-dependent functional selectivity in D1R ligands open a new avenue for the treatment of cortex-specific or striatum-specific neuropsychiatric dysfunction.

The structural determinants of the bitopic binding mode of a negative allosteric modulator of the dopamine D2 receptor.

SB269652 is a negative allosteric modulator of the dopamine D2 receptor (D2R) yet possesses structural similarity to ligands with a competitive mode of interaction. In this study, we aimed to understand the ligand-receptor interactions that confer its allosteric action. We combined site-directed mutagenesis with molecular dynamics simulations using both SB269652 and derivatives from our previous structure activity studies. We identify residues within the conserved orthosteric binding site (OBS) and a secondary binding pocket (SBP) that determine affinity and cooperativity. Our results indicate that interaction with the SBP is a requirement for allosteric pharmacology, but that both competitive and allosteric derivatives of SB269652 can display sensitivity to the mutation of a glutamate residue (E952.65) within the SBP. Our findings provide the molecular basis for the differences in affinity between SB269652 derivatives, and reveal how changes to interactions made by the primary pharmacophore of SB269652 in the orthosteric pocket can confer changes in the interactions made by the secondary pharmacophore in the SBP. Our insights provide a structure-activity framework towards rational optimization of bitopic ligands for D2R with tailored competitive versus allosteric properties.

The E2.65A mutation disrupts dynamic binding poses of SB269652 at the dopamine D2 and D3 receptors.

The dopamine D2 and D3 receptors (D2R and D3R) are important targets for antipsychotics and for the treatment of drug abuse. SB269652, a bitopic ligand that simultaneously binds both the orthosteric binding site (OBS) and a secondary binding pocket (SBP) in both D2R and D3R, was found to be a negative allosteric modulator. Previous studies identified Glu2.65 in the SBP to be a key determinant of both the affinity of SB269652 and the magnitude of its cooperativity with orthosteric ligands, as the E2.65A mutation decreased both of these parameters. However, the proposed hydrogen bond (H-bond) between Glu2.65 and the indole moiety of SB269652 is not a strong interaction, and a structure activity relationship study of SB269652 indicates that this H-bond may not be the only element that determines its allosteric properties. To understand the structural basis of the observed phenotype of E2.65A, we carried out molecular dynamics simulations with a cumulative length of ~77 µs of D2R and D3R wild-type and their E2.65A mutants bound to SB269652. In combination with Markov state model analysis and by characterizing the equilibria of ligand binding modes in different conditions, we found that in both D2R and D3R, whereas the tetrahydroisoquinoline moiety of SB269652 is stably bound in the OBS, the indole-2-carboxamide moiety is dynamic and only intermittently forms H-bonds with Glu2.65. Our results also indicate that the E2.65A mutation significantly affects the overall shape and size of the SBP, as well as the conformation of the N terminus. Thus, our findings suggest that the key role of Glu2.65 in mediating the allosteric properties of SB269652 extends beyond a direct interaction with SB269652, and provide structural insights for rational design of SB269652 derivatives that may retain its allosteric properties.

The action of a negative allosteric modulator at the dopamine D2 receptor is dependent upon sodium ions.

Sodium ions (Na+) allosterically modulate the binding of orthosteric agonists and antagonists to many class A G protein-coupled receptors, including the dopamine D2 receptor (D2R). Experimental and computational evidences have revealed that this effect is mediated by the binding of Na+ to a conserved site located beneath the orthosteric binding site (OBS). SB269652 acts as a negative allosteric modulator (NAM) of the D2R that adopts an extended bitopic pose, in which the tetrahydroisoquinoline moiety interacts with the OBS and the indole-2-carboxamide moiety occupies a secondary binding pocket (SBP). In this study, we find that the presence of a Na+ within the conserved Na+-binding pocket is required for the action of SB269652. Using fragments of SB269652 and novel full-length analogues, we show that Na+ is required for the high affinity binding of the tetrahydroisoquinoline moiety within the OBS, and that the interaction of the indole-2-carboxamide moiety with the SBP determines the degree of Na+-sensitivity. Thus, we extend our understanding of the mode of action of this novel class of NAM by showing it acts synergistically with Na+ to modulate the binding of orthosteric ligands at the D2R, providing opportunities for fine-tuning of modulatory effects in future allosteric drug design efforts.

Comparison of two different doses of intrathecal dexmedetomidine as adjuvant with isobaric ropivacaine in lower abdominal surgery.

BACKGROUND: To augment the subarachnoid block utility, the efficacy of newer molecules as an adjuvant is investigated constantly. Considering the favorable profile of dexmedetomidine, it could have a potential role as an adjuvant to ropivacaine. AIM: We evaluated the efficacy of two different doses of dexmedetomidine as an adjuvant to isobaric ropivacaine, intrathecally. METHODS: Ninety patients scheduled for lower abdominal surgery under spinal anesthesia were randomized into three groups to receive 2.5 ml of isobaric ropivacaine (0.75%, 7.5 mg/ml) added to 5 µg (10 µg/ml) or 10 µg (20 µg/ml) of dexmedetomidine or 0.5 ml of normal saline in group A, B or C, respectively. Block characteristics were compared as a primary outcome. STATISTICAL ANALYSIS: One-way analysis of variance test, Fisher's exact test/Chi-square test, whichever appropriate. A P < 0.05 was considered significant. RESULTS: Time to achieve desired block was least in group B and maximum in group C. The sensory-motor blockade remained significantly prolonged in group B compared to other groups. Hemodynamic parameters remained stable in all three groups. CONCLUSION: Among the investigated doses, dexmedetomidine augments the efficacy of intrathecal ropivacaine in a dose-dependent manner, without any untoward side effects.

Major intrinsic protein superfamily: channels with unique structural features and diverse selectivity filters.

Members of the superfamily of major intrinsic proteins (MIPs) facilitate water and solute permeability across cell membranes and are found in sources ranging from bacteria to humans. Aquaporin and aquaglyceroporin channels are the prominent members of the MIP superfamily. Experimental studies show that MIPs are involved in important physiological processes in mammals and plants. They are implicated in several human diseases and are considered to be attractive drug targets for a wide range of diseases such as cancer, brain edema, epilepsy, glaucoma, and congestive heart failure. Three-dimensional structures of MIP channels from diverse sources reveal that MIPs adopt a unique conserved hourglass helical fold consisting of six transmembrane helices (TM1-TM6) and two half-helices (LB and LE). Conserved NPA motifs near the center and the aromatic/arginine selectivity filter (Ar/R SF) toward the extracellular side constitute two narrow constriction regions within the channel. Structural knowledge combined with simulation studies have helped to investigate the role of these two constriction regions in the transport and selectivity of the solutes. With the availability of many genome sequences from diverse species, a large number of MIP genes have been identified. Homology models of 1500 MIP channels have been used to derive structure-based sequence alignment of TM1-TM6 helices and the two half-helices LB and LE. Thirteen residues are highly conserved in different transmembrane helices and half-helices. High group conservation of small and weakly polar residues is observed in 27 positions at the interface of two interacting helices. Thus, although the MIP sequences are diverse, the hourglass helical fold is maintained during evolution with the conservation of these 40 positions within the transmembrane region. We have proposed a generic structure-based numbering scheme for the MIP channels that will facilitate easier comparison of the MIP sequences. Analysis of Ar/R SF in all 1500 MIPs indicates the extent of diversity in the four residues that form this narrow region. Certain residues are completely avoided in the SF, even if they have the same chemical nature as that of the most frequently observed residues. For example, arginine is the most preferred residue in a specific position of Ar/R SF, whereas lysine is almost always avoided in any of the four positions. MIP channels with highly hydrophobic or hydrophilic Ar/R SF have been identified. Similarly, there are examples of MIP channels in which all four residues of Ar/R SF are bulky, thus almost occluding the pore. Many plant MIPs possess small residues at all SF positions, resulting in a larger pore diameter. A majority of MIP channels are yet to be functionally characterized, and their in vivo substrates are not yet identified. A complete understanding of the relationship between the nature of Ar/R SF and the solutes that are transported is required to exploit MIP channels as potential drug targets.

Intra-helical salt-bridge and helix destabilizing residues within the same helical turn: Role of functionally important loop E half-helix in channel regulation of major intrinsic proteins.

The superfamily of major intrinsic proteins (MIPs) includes aquaporin (AQP) and aquaglyceroporin (AQGP) and it is involved in the transport of water and neutral solutes across the membrane. Diverse MIP sequences adopt a unique hour-glass fold with six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). Loop E contains one of the two conserved NPA motifs and contributes two residues to the aromatic/arginine selectivity filter. Function and regulation of majority of MIP channels are not yet characterized. We have analyzed the loop E region of 1468 MIP sequences and their structural models from six different organism groups. They can be phylogenetically clustered into AQGPs, AQPs, plant MIPs and other MIPs. The LE half-helix in all AQGPs contains an intra-helical salt-bridge and helix-breaking residues Gly/Pro within the same helical turn. All non-AQGPs lack this salt-bridge but have the helix destabilizing Gly and/or Pro in the same positions. However, the segment connecting LE half-helix and TM6 is longer by 10-15 residues in AQGPs compared to all non-AQGPs. We speculate that this longer loop in AQGPs and the LE half-helix of non-AQGPs will be relatively more flexible and this could be functionally important. Molecular dynamics simulations on glycerol-specific GlpF, water-transporting AQP1, its mutant and a fungal AQP channel confirm these predictions. Thus two distinct regions of loop E, one in AQGPs and the other in non-AQGPs, seem to be capable of modulating the transport. These regions can also act in conjunction with other extracellular residues/segments to regulate MIP channel transport.