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To identify novel D3 dopamine receptor (D3R) agonists, we conducted a high-throughput screen using a β-arrestin recruitment assay. Counterscreening of the hit compounds provided an assessment of their selectivity, efficacy, and potency. The most promising scaffold was optimized through medicinal chemistry resulting in enhanced potency and selectivity. The optimized compound, ML417 (20), potently promotes D3R-mediated β-arrestin translocation, G protein activation, and ERK1/2 phosphorylation (pERK) while lacking activity at other dopamine receptors. Screening of ML417 against multiple G protein-coupled receptors revealed exceptional global selectivity. Molecular modeling suggests that ML417 interacts with the D3R in a unique manner, possibly explaining its remarkable selectivity. ML417 was also found to protect against neurodegeneration of dopaminergic neurons derived from iPSCs. Together with promising pharmacokinetics and toxicology profiles, these results suggest that ML417 is a novel and uniquely selective D3R agonist that may serve as both a research tool and a therapeutic lead for the treatment of neuropsychiatric disorders.

Graphical abstract, The sigma 1 receptor (σ1R) is a unique endoplasmic reticulum membrane protein. Its ligands have been shown to possess therapeutic potential for neurological and substance use disorders among others. The E102Q mutation of σ1R has been found to elicit familial cases of amyotrophic lateral sclerosis (ALS). Despite reports of its downstream signaling consequences, the mechanistic details of the functional impact of E102Q at molecular level are not clear. Here, we investigate the molecular mechanism of the E102Q mutation with a spectrum of biochemical, biophysical, and pharmacological approaches. Our analysis of the interaction network of σ1R indicates that a set of residues near E102 is critical for the integrity of C-terminal ligand-binding domain. However, this integrity is not affected by the E102Q and E102A mutations, which is confirmed by the radioligand binding results. Instead, the E102 mutations disrupt the connection between the C-terminal domain and the N-terminal transmembrane helix (NT-helix). Results from bioluminescence resonance energy transfer and western blot assays demonstrate that these mutations destabilize higher-order σ1R oligomers, while our molecular dynamics simulations based on a σ1R crystal structure reveal a potential mechanism by which the mutations perturb the NT-helix dynamics. Thus, we propose that E102 is at a critical position in propagating the effects of ligand binding from the C-terminal domain to the NT-helix, while the latter may be involved in forming alternative oligomer interfaces, separate from the previously reported trimer interface. Together, these results provide the first account of the molecular mechanism of σ1R dysfunction caused by E102Q.

The serotonin transporter (SERT) terminates serotonin signaling by rapid presynaptic reuptake. SERT activity is modulated by antidepressants, e.g., S-citalopram and imipramine, to alleviate symptoms of depression and anxiety. SERT crystal structures reveal two S-citalopram binding pockets in the central binding (S1) site and the extracellular vestibule (S2 site). In this study, our combined in vitro and in silico analysis indicates that the bound S-citalopram or imipramine in S1 is allosterically coupled to the ligand binding to S2 through altering protein conformations. Remarkably, SERT inhibitor Lu AF60097, the first high-affinity S2-ligand reported and characterized here, allosterically couples the ligand binding to S1 through a similar mechanism. The SERT inhibition by Lu AF60097 is demonstrated by the potentiated imipramine binding and increased hippocampal serotonin level in rats. Together, we reveal a S1-S2 coupling mechanism that will facilitate rational design of high-affinity SERT allosteric inhibitors., The serotonin transporter (SERT) terminates serotonin signaling and its activity is modulated by antidepressants. Here authors reveal the mechanistic details underlying the coupling between the two binding sites in SERT and a high-affinity ligand for the allosteric site.

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., eLife digest 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.

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.

Cocaine, a widely abused psychostimulant, inhibits the dopamine transporter (DAT) by trapping the protein in an outward-open conformation, whereas atypical DAT inhibitors such as benztropine have low abuse liability and prefer less outward-open conformations. Here, we use a spectrum of computational modeling and simulation approaches to obtain the underlying molecular mechanism in atomistic detail. Interestingly, our quantum mechanical calculations and molecular dynamics (MD) simulations suggest that a benztropine derivative JHW007 prefers a different stereoisomeric conformation of tropane in binding to DAT compared to that of a cocaine derivative, CFT. To further investigate the different inhibition mechanisms of DAT, we carried out MD simulations in combination with Markov state modeling analysis of wild-type and Y156F DAT in the absence of any ligand or the presence of CFT or JHW007. Our results indicate that the Y156F mutation and CFT shift the conformational equilibrium toward an outward-open conformation, whereas JHW007 prefers an inward-occluded conformation. Our findings reveal the mechanistic details of DAT inhibition by JHW007 at the atomistic level, which provide clues for rational design of atypical inhibitors.

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

Metadynamics simulations were used to describe the conformational energy landscapes of several helically folded aromatic quinoline carboxamide oligomers bearing a single chiral group at either the C or N terminus. The calculations allowed the prediction of whether a helix handedness bias occurs under the influence of the chiral group and gave insight into the interactions (sterics, electrostatics, hydrogen bonds) responsible for a particular helix sense preference. In the case of camphanyl-based and morpholine-based chiral groups, experimental data confirming the validity of the calculations were already available. New chiral groups with a proline residue were also investigated and were predicted to induce handedness. This prediction was verified experimentally through the synthesis of proline-containing monomers, their incorporation into an oligoamide sequence by solid phase synthesis and the investigation of handedness induction by NMR spectroscopy and circular dichroism.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping., Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a powerful tool for neuroscience, but the standard DREADD ligand, CNO, has significant drawbacks. Here the authors report two novel high-potency DREADD ligands and a novel DREADD radiotracer for imaging purposes.

The serotonin transporter (SERT) is the primary target for the selective serotonin reuptake inhibitors (SSRIs). However, the structural basis for the extraordinarily high binding affinity of the widely-prescribed SSRI, paroxetine, to human SERT (hSERT) has not yet been fully elucidated. Our previous findings unveiled a plausible ambiguity in paroxetine’s binding orientations that may constitute an integral component of this SSRI’s high affinity for hSERT. Herein, we investigate factors contributing to paroxetine’s high affinity by modifying both the ligand and the protein. We generated a series of Bromine (Br)-containing derivatives and found that the one in which the 4-F of paroxetine had been replaced with the chemically-similar but more electron-rich Br atom (13) has the highest affinity. By comparatively characterizing the binding of paroxetine and 13 to both WT and a construct harboring a paroxetine-sensitive mutation in the binding cavity, we identified a mechanistic determinant responsible for the pose ambiguity of paroxetine, which can guide future drug design.

Understanding how crystal structures reflect the range of possible G protein-coupled receptor (GPCR) states is critical for rational drug discovery (RDD). Combining computational simulations with mutagenesis and binding studies, we find that the structure of the dopamine D2 receptor (D2R)/risperidone complex captures an inactive receptor conformation that accommodates some but not all antagonist scaffolds. Indeed, we find that eticlopride binds D2R in a configuration very similar to that seen in the D3R structure, in a pose that is incompatible with the D2R/risperidone structure. Moreover, our simulations reveal that extracellular loops 1 and 2 (EL1 and EL2) are highly dynamic, with spontaneous transitions of EL2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results highlight previously unappreciated conformational diversity and dynamics in the inactive state of a GPCR with potential functional implications. These findings are also of paramount importance for RDD as limiting a virtual screen to one state will miss relevant ligands.

Medication-assisted treatments are unavailable to patients with cocaine use disorders. Efforts to develop potential pharmacotherapies have led to the identification of a promising lead molecule, JJC8-091, that demonstrates a novel binding mode at the dopamine transporter (DAT). Here, JJC8-091 and a structural analogue, JJC8-088, were extensively and comparatively assessed to elucidate neurochemical correlates to their divergent behavioral profiles. Despite sharing significant structural similarity, JJC8-088 was more cocaine-like, increasing extracellular DA concentrations in the nucleus accumbens shell (NAS) efficaciously and more potently than JJC8-091. In contrast, JJC8-091 was not self-administered and was effective in blocking cocaine-induced reinstatement to drug seeking. Electrophysiology experiments confirmed that JJC8-091 was more effective than JJC8-088 at inhibiting cocaine-mediated enhancement of DA neurotransmission. Further, when VTA DA neurons in DAT-cre mice were optically stimulated, JJC8-088 produced a significant leftward shift in the stimulation-response curve, similar to cocaine, while JJC8-091 shifted the curve downward, suggesting attenuation of DA-mediated brain reward. Computational models predicted that JJC8-088 binds in an outward facing conformation of DAT, similar to cocaine. Conversely, JJC8-091 steers DAT towards a more occluded conformation. Collectively, these data reveal the underlying molecular mechanism at DAT that may be leveraged to rationally optimize leads for the treatment of cocaine use disorders, with JJC8-091 representing a compelling candidate for development.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for precision medicine-based clinical theranostics. DREADD ligands developed to date are not appropriate for such translational applications. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine. The second-generation DREADD agonist, Compound 21 (C21), was developed to overcome these limitations. We found that C21 has low brain penetrance, weak affinity, and low in vivo DREADD occupancy. To address these drawbacks, we developed two new DREADD agonists, JHU37152 and JHU37160, and the first dedicated positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons and at their long-range projections, enabling for the first time, noninvasive and longitudinal neuronal projection mapping and potential for neurotheranostic applications.

The serotonin transporter (SERT) is one of the primary targets for medications to treat neuropsychiatric disorders and functions by exploiting pre-existing ion gradients of Na+, Cl-, and K+ to translocate serotonin from the synaptic cleft into the presynaptic neuron. Although recent hSERT crystal structures represent a milestone for structure-function analyses of mammalian neurotransmitter:sodium symporters, they are all derived from thermostabilized but transport-deficient constructs. Two of these structures are in complex with paroxetine, the most potent selective serotonin reuptake inhibitor known. In this study, by carrying out and analyzing the results of extensive and comparative molecular dynamics simulations while also re-evaluating the transport and binding properties of the thermostabilized constructs, we identified functionally important structural elements that are perturbed by these mutations, revealed unexpected dynamics in the central primary binding site of SERT, and uncovered a conceivable ambiguity in paroxetine's binding orientation. We propose that the favored entropy contribution plays a significant role in paroxetine's extraordinarily high affinity for SERT. Our findings lay the foundation for future mechanistic studies and rational design of high-affinity SERT inhibitors. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

The sigma 1 receptor (σ(1)R) is a structurally unique transmembrane protein that functions as a molecular chaperone in the endoplasmic reticulum (ER), and has been implicated in cancer, neuropathic pain, and psychostimulant abuse. Despite physiological and pharmacological significance, mechanistic underpinnings of structure-function relationships of σ(1)R are poorly understood, and molecular interactions of selective ligands with σ(1)R have not been elucidated. The recent crystallographic determination of σ(1)R as a homo-trimer provides the foundation for mechanistic elucidation at the molecular level. Here we report novel bioluminescence resonance energy transfer (BRET) assays that enable analyses of ligand-induced multimerization of σ(1)R and its interaction with BiP. Haloperidol, PD144418, and 4-PPBP enhanced σ(1)R homomer BRET signals in a dose dependent manner, suggesting their significant effects in stabilizing σ(1)R multimerization, whereas (+)-pentazocine and several other ligands do not. In non-denaturing gels, (+)-pentazocine significantly decreased whereas haloperidol increased the fraction of σ(1)R multimers, consistent with the results from the homomer BRET assay. Further, BRET assays examining heteromeric σ(1)R-BiP interaction revealed that (+)-pentazocine and haloperidol induced opposite trends of signals. From molecular modeling and simulations of σ(1)R in complex with the tested ligands, we identified initial clues that may lead to the differed responses of σ(1)R upon binding of structurally diverse ligands. By combining multiple in vitro pharmacological and in silico molecular biophysical methods, we propose a novel integrative approach to analyze σ(1)R-ligand binding and its impact on interaction of σ(1)R with client proteins.

Molecular capsules have been extensively used in catalysis, drug delivery, molecular recognition and protection of ligands from degradation. Novel "apple peel" shaped helical arylamide capsules have been experimentally pursued due to their flexible nature and designability. They were found to encapsulate a variety of small molecules. The apple peel shape of the capsules led to a hypothesis that binding and release of ligands involve partial unfolding. However, the exact mechanism is unknown. Using molecular dynamics simulations with our new aryl-amide force field parameters, we identify two low energy barrier binding/release mechanisms, in which the capsule's helical structure is either minimally disturbed or restored quickly (within 100 ps). Furthermore, we determine the effects of ligand sizes, their chemical nature (hydrogen bonding capabilities), and solvents on binding modes and stabilities. Our findings not only support experimental observations but also provide underlying principles that allow for rational design of foldamer capsules.

Arylamide foldamers have been shown to have a number of biological and medicinal applications. For example, a class of pyrrole-imidazole polyamide foldamers is capable of binding specific DNA sequences and preventing development of various gene disorders, most importantly cancer. Molecular dynamics (MD) simulations can provide crucial details in understanding the atomic level events related to foldamer/DNA binding. An important first step in the accurate simulation of these foldamer/DNA systems is the reparametrization of force field parameters for torsion around the aryl-amide bonds. Here we highlight our Density Functional Theory (DFT) potential energy profiles and derived force field parameters for four aryl-amide bond types for the pyrrole and imidazole building blocks extensively used in foldamer design for the DNA-binding polyamides. These results contribute to developing of computational tools for an appropriate molecular modeling of pyrrole-imidazole polyamide/DNA binding, and provide an insight into the chemical factors that influence the flexibility of the pyrrole-imidazole polyamides, and their binding to DNA.

Psychostimulant abuse leads to debilitating disorders, the treatment of which remains an alarming challenge. Cocaine and amphetamine act on the dopamine transporter (DAT), which belongs to the neurotransmitter:sodium symporter family that terminates neurotransmission by reuptake of neurotransmitters from the synaptic cleft. The reuptake process can be described by the Na+-coupled alternating-access mechanism in which the transporter adopts outward-open, occluded and inward-open conformations. Cocaine is known to inhibit DAT function by trapping the protein in an outward-open conformation. Interestingly, several other DAT inhibitors, such as benztropine, modafinil, and some of their derivatives, appear to have low abuse liability. Compared to cocaine, these atypical DAT inhibitors show a preference for the conformation of DAT that can be stabilized by a mutation at the intracellular gate, Y335A, and were deduced to prefer less outward-open conformations than cocaine. Nevertheless their differential inhibiting mechanisms at the atomistic level is not well understood.We carried out comparatively molecular dynamics simulations of both DAT-WT and DAT-Y335A constructs, stabilized by a variety of DAT inhibitors, and used Markov State Models to analyze the resulting trajectories. This analysis identifies an ensemble of conformational states exhibited spontaneously by the molecule at local equilibrium, and has the advantage in its ability to represent both thermodynamic and kinetic characteristics of protein conformational changes. Thus we capture the conformational states and kinetics of transitions between outward-occluded and outward-open states of DAT. Identification and characterization of the intermediate states that are stabilized by the atypical inhibitors and/or Y335A mutation is crucial in understanding the mechanism of action of these inhibitors. This understanding of how these states eventually trigger different downstream cellular effects from cocaine holds new promises to develop targeted treatment of cocaine dependence.

We report the first atomistic level description of the handedness inversion mechanism for helical arylamide foldamers. The key process in the handedness inversion is the simultaneous unfolding and folding of two adjacent aryl-aryl linkages, propagating from a helix terminus along the strand. Intermediates along the inversion pathway have a common feature - a single unfolded aryl-aryl linkage (through C(aryl)-C(amide) rotation) connecting two helical segments of opposite handedness. This explicit solvent metadynamics study also provides thorough quantitative free energy information for each step of the previously uncharacterized inversion pathway.

Neurotransmitter:Sodium Symporters (NSS) terminate neurotransmission through sodium-driven reuptake of cognate neurotransmitters, and traverse between outward-open and inward-open states. NSS include serotonin and dopamine transporters, which are targets for antidepressants and abused psychostimulants. Crystallographically, whereas both substrates and inhibitors have been found to bind the central binding site (denoted S1) of the NSS proteins, it has been shown that inhibitors for LeuT and SERT can also bind to a binding cavity in the extracellular vestibule (denoted S2). Based on computational and experimental studies in LeuT, it has been found that substrates can bind to S2 as well, and such binding triggers the intracellular release the substrate and Na+ from S1. However, whether such a role of S2 substrate is common for other NSS remains unclear.The newly available crystal structure of MhsT, a bacterial NSS homolog, which was solved in an inward-occluded state, provides a more suitable starting point to study the substrate induced conformational changes, including the S2 substrate induced transition from inward-occluded to inward-open state transition. Here, we use extensive molecular dynamics simulations combined with hidden Markov model (HMM) analysis to investigate the feasibility and kinetics of the substrate L-tryptophan binding to S2 of MhsT. Based on HMM analysis, we identified transition binding positions in the extracellular vestibule that form potential substrate binding pathways, and the conformational changes near the S2 site associated with the two most populated bound states. Our findings shed light on the S2 substrate binding kinetics and the induced conformational changes, which may be critical in triggering the subsequent events in the transport mechanism.

We tested the hypothesis that the constitutive glucose transporter (GLUT 1) in 3T3-L 1 adipocytes belongs to the family of glucose-regulated proteins which are transcriptionally regulated by glucose deprivation. Using cDNA probes for both GRP78 (BiP) and GLUT1, we show that the level of GRP78 mRNA increased by 15-fold within 24 h of glucose deprivation with little change in GLUT1 mRNA. The elevated GRP78 mRNA in turn led to a time-dependent increase in GRP78 protein. While glucose deprivation did not alter the expression of the normal glycoform of GLUT 1, a lower molecular weight glycoform accumulated with extended deprivation. Mannose and fructose, but not galactose, prevented the induction of GRP78 and accumulation of the abnormal GLUT1. Because GRP78 acts as a chaperone in other cell systems, we also sought evidence to support this activity in 3T3-L1 adipocytes. Using the technique of co-immunoprecipitation, we demonstrate that GRP78 bound several proteins unique to the glucose-deprived state. No deprivation-specific proteins could be detected in association with GLUT 1. These data lead us to conclude that GLUTl does not display characteristics of the glucose-regulated proteins, at least in 3T3-L1 adipocytes, a widely used model for differentiation, hormone action, and nutrient control. However, the mechanisms for activating traditional members of this family appear intact.

Despite its versatility and effectiveness in numerous studies, the vaccinia/HeLa cell expression model may not be optimal for the study of all transport proteins. To evaluate an alternative expression model for amino acid transport Systems ASC and XAG−, the mRNA content and transport activity encoded by human hippocampal ASCT1 cDNA and rat hippocampal EAAC1 cDNA, respectively, were measured in pDR2-cDNA-transfected human embryonic kidney 293 cells made competent by stable transfection with the Epstein–Barr neutral antigen-1 (EBNA-1) cDNA (293c18 cells) to evaluate the EBNA-1/293c18 expression system. The results show that (i) the EBNA-1/293c18 expression system results in a larger increase over background of Systems ASCT1 (6.4×) and EAAC1 (39×) transport activity than does the vaccinia/HeLa expression system (2.6× and 22×, respectively); (ii) transfection and hygromycin B selection for the pDR2 vector do not affect the endogenous transport velocities of Systems ASC, X−AG, or A; and (iii) the endogenous transport velocities of Systems ASC and XAG−in 293c18 cells were not affected by the expression of exogenous EAAC1 or ASCT1. We conclude that the EBNA-1/293c18 cell expression model represents a useful transient expression regimen to characterize mammalian amino acid transport proteins, especially for transporters that may exhibit relatively low activity in transient expression systems lacking a selection mechanism.

IMiD-class compounds, including thalidomide, lenalidomide, and pomalidomide, have been developed as racemic mixtures of S- and R-enantiomers. The isolated enantiomers of thalidomide are known to have distinct biological activities. For example, the well-documented sedative effects of thalidomide are correlated with the R-enantiomer (Eriksson et al., 2000), whereas S-thalidomide exhibits enhanced potency for TNF-α inhibition compared to the R-enantiomer (Wnendt et al., 1996; Moreira et al., 1993). We have demonstrated that S-lenalidomide is more potent than racemic or R-lenalidomide in biological activities that are believed to be important for clinical efficacy of lenalidomide. Due to facile in vivo conversion, isolated S-enantiomers of IMiDs have not been developed clinically. Lenalidomide (Revlimid®) is a racemic mixture of S- and R-enantiomers that interconvert through epimerization. Revlimid is labeled for the treatment of 5q-myelodysplastic syndromes (MDS) and multiple myeloma. CTP-221 is a deuterium-modified analog of S-lenalidomide containing deuterium atoms at key positions including lenalidomide's chiral center. Deuterium modification has the potential, albeit unpredictably, to alter the metabolic fate and hence the pharmacokinetic disposition of drugs, especially those that are biotransformed via the cleavage of carbon-hydrogen bonds (Fisher et al, 2006). The effect of deuterium modification on the epimerization rate and pharmacokinetic profile of CTP-221 was investigated in vitro and in vivo in mice, rats and monkeys. The rates of epimerization of CTP-221 and S-lenalidomide were compared in vitro in whole blood from mouse, rat, monkey, and human. It was found that CTP-221 was 2- to 3-fold more stable to epimerization than S-lenalidomide in the four species. To compare the in vivo pharmacokinetic profiles of CTP-221 and racemic lenalidomide, the compounds (10 mg/kg, PO) were administered to mice, rats and monkeys. In all three species, the exposure (AUC) of S- and R-lenalidomide following administration of racemic lenalidomide was about 50-57% and 43-50%, respectively, of the sum of the AUC of the individual enantiomers. However, when CTP-221 was administered, the AUC of CTP-221 and the deuterated R-enantiomers formed in vivo were about 96-99% and 1-4% respectively, of the sum of the AUC of the two enantiomers. Thus, CTP-221 epimerizes to a minimal extent in vivo and its administration provides exposure to very low levels of the R-enantiomer. In conclusion, the stabilization of CTP-221 via deuterium substitution resulted in maximal exposure to the more potent S-enantiomer and minimal exposure to the R-enantiomer. As a result, CTP-221 has the potential for improved potency and therapeutic index in comparison to racemic lenalidomide. Citation Format: Vinita Uttamsingh, Richard Gallegos, Changfu Cheng, Ara Aslanian, Julie Fields Liu, Roger Tung, Lijun Wu. CTP-221, a deuterated S-enantiomer of lenalidomide, is greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3357. doi:10.1158/1538-7445.AM2013-3357

The high-density lipoprotein (HDL) cholesterol level is a strong predictor of cardiovascular events in epidemiologic studies. Until recently, it has been less extensively studied as a therapeutic target.To assess the angiographic and clinical effects of a pharmacologic strategy to increase HDL cholesterol levels.Randomized, double-blind, placebo-controlled trial conducted from 1993 to 1996.Outpatient specialty clinic of a large U.S. military medical center.143 military retirees younger than 76 years of age with low HDL cholesterol levels and angiographically evident coronary disease.Gemfibrozil, niacin, and cholestyramine or corresponding placebos, with aggressive dietary and lifestyle intervention at baseline.Change from baseline to 30 months and a composite measure of clinical events that included hospitalization for angina, myocardial infarction, transient ischemic attack and stroke, death, and cardiovascular procedures.At baseline, mean (+/-SD) lipid values were as follows: total cholesterol, 5.1 +/- 0.8 mmol/L (196 +/- 31 mg/dL); low-density lipoprotein (LDL) cholesterol, 3.3 +/- 0.7 mmol/L (128 +/- 27 mg/dL); and HDL cholesterol, 0.9 +/- 0.2 mmol/L (34 +/- 6 mg/dL). Compared with placebo, the pharmacologically treated group experienced a 20% (95% CI, 14.8% to 24.3%) decrease in total cholesterol level, a 36% (CI, 28.4% to 43.5%) increase in HDL cholesterol level, a 26% (CI, 19.1% to 33.7%) decrease in LDL cholesterol level, and a 50% (CI, 40.5% to 59.2%) reduction in triglyceride levels. Focal coronary stenosis increased by 1.4% in the placebo group but decreased by 0.8% in the drug group (difference, -2.2 percentage points [CI, -4.2 to -0.1 percentage points]). A composite cardiovascular event end point was reached in 26% of patients in the placebo group and 13% of those in the drug group (difference, 13.7 percentage points [CI, 0.9 to 26.5 percentage points]). Side effects, particularly flushing and gastrointestinal intolerance, were more common in the drug group but rarely led to withdrawal from the study.The study was small and used a composite clinical outcome. Whether improvements in angiographic findings were due to reductions in LDL cholesterol or increases in HDL cholesterol was not established. Flushing may have led to inadvertent unblinding in patients who were randomly assigned to active study drugs.A combination regimen aimed at increasing HDL cholesterol levels improves cholesterol profiles, helps prevent angiographic progression of coronary stenosis, and may prevent cardiovascular events in some people who exercise regularly and eat low-fat diets.