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Excitatory amino acid transporters (EAATs) represent a protein family that is an emerging drug target with great therapeutic potential for managing central nervous system disorders characterized by dysregulation of glutamatergic neurotransmission. As such, it is of significant interest to discover selective modulators of EAAT2 function. Here, we applied computational methods to identify specific EAAT2 inhibitors. Utilizing a homology model of human EAAT2, we identified a binding pocket at the interface of the transport and trimerization domain. We next conducted a high-throughput virtual screen against this site and identified a selective class of EAAT2 inhibitors that were tested in glutamate uptake and whole-cell electrophysiology assays. These compounds represent potentially useful pharmacological tools suitable for further exploration of the therapeutic potential of EAAT2 and may provide molecular insights into mechanisms of allosteric modulation for glutamate transporters.

The topic of gender equality within the United States workforce is receiving a great deal of attention. The field of chemistry is no exception and is increasingly focused on taking steps to achieve gender diversity within the chemistry workforce. Over the past several years, many computational chemistry groups within large pharmaceutical companies have realized growth in the number of women, and here we discuss the key factors that we believe have played a role in attracting and retaining the authors of this review as computational chemists in pharma. Furthermore, we combine our professional experiences in the context of how computational methodology and technology have evolved over the past decades and how that evolution has facilitated the inclusion of more women into the field. Our hope is to be a part of a solution and provide insight that will allow the chemistry workforce to continue to make steps forward in attaining gender diversity in the workplace.

[Image: see text] Drug discovery building blocks available commercially or within an internal inventory cover a diverse range of chemical space and yet describe only a tiny fraction of all chemically feasible reagents. Vendors will eagerly provide tools to search the former; there is no straightforward method of mining the latter. We describe a procedure and use case in assembling chemical structures not available for purchase but that could likely be synthesized in one robust chemical transformation starting from readily available building blocks. Accessing this vast virtual chemical space dramatically increases our curated collection of reagents available for medicinal chemistry exploration and novel hit generation, almost tripling the number of those with 10 or fewer atoms.

The topic of gender equality within the United States workforce is receiving a great deal of attention. The field of chemistry is no exception and is increasingly focused on taking steps to achieve gender diversity within the chemistry workforce. Over the past several years, many computational chemistry groups within large pharmaceutical companies have realized growth in the number of women, and here we discuss the key factors that we believe have played a role in attracting and retaining the authors of this review as computational chemists in pharma. Furthermore, we combine our professional experiences in the context of how computational methodology and technology have evolved over the past decades and how that evolution has facilitated the inclusion of more women into the field. Our hope is to be a part of a solution and provide insight that will allow the chemistry workforce to continue to make steps forward in attaining gender diversity in the workplace.

The stimulator-of-interferon-genes (STING) protein is involved in innate immunity. It has recently been shown that modulation of STING can lead to an aggressive antitumor response. DMXAA is an antitumor agent that had shown great promise in murine models but failed in human clinical trials. The molecular target of DMXAA was subsequently shown to be murine STING (mSTING); however, human STING (hSTING) is insensitive to DMXAA. Molecular dynamics simulations were employed to investigate the differences between hSTING and mSTING that could influence DMXAA binding. An initial set of simulations was performed to investigate a single lid region mutation G230I in hSTING (corresponding residue in mSTING is an Ile), which rendered the protein sensitive to DMXAA. The simulations found that an Ile side chain was enough to form a steric barrier that prevents exit of DMXAA, whereas in WT hSTING, the Gly residue that lacks a side chain formed a porous lid region that allowed DMXAA to exit. A second set of molecular dynamics simulations compared the tendency of STING to be in an open-inactive conformation or a closed-active conformation. The results show that hSTING prefers to be in an open-inactive conformation even with cGAMP, the native ligand, bound. On the other hand, mSTING prefers a closed-active conformation even without a ligand bound. These results highlight the challenges in translating a mouse active STING compound into a human active compound, while also providing avenues to pursue for designing a small-molecule drug targeting human STING.

At the onset of a drug discovery program, the goal is to identify novel compounds with appropriate chemical features that can be taken forward as lead series. Here, we describe three prospective case studies, Bruton Tyrosine Kinase (BTK), RAR-Related Orphan Receptor γ t (RORγt), and Human Leukocyte Antigen DR isotype (HLA-DR) to illustrate the positive impact of high throughput virtual screening (HTVS) on the successful identification of novel chemical series. Each case represents a project with a varying degree of difficulty due to the amount of structural and ligand information available internally or in the public domain to utilize in the virtual screens. We show that HTVS can be effectively employed to identify a diverse set of potent hits for each protein system even when the gold standard, high resolution structural data or ligand binding data for benchmarking, is not available.

We analyzed an extensive data set of 3000 Janssen kinase inhibitors (spanning some 40 therapeutic projects) profiled at 414 kinases in the DiscoverX KINOME scan to better understand the necessity of using such a full kinase panel versus simply profiling one's compound at a much smaller number of kinases, or mini kinase panel (MKP), to assess its selectivity. To this end, we generated a series of MKPs over a range of sizes and of varying kinase membership using Monte Carlo simulations. By defining the kinase hit index (KHI), we quantified a compound's selectivity based on the number of kinases it hits. We find that certain combinations (rather than a random selection) of kinases can result in a much lower average error. Indeed, we identified a focused MKP with a 45.1% improvement in the average error (compared to random) that yields an overall correlation of R2 = 0.786-0.826 for the KHI compared to the full kinase panel value. Unlike using a full kinase panel, which is both time and cost restrictive, a focused MKP is amenable to the triaging of all early stage compounds. In this way, promiscuous compounds are filtered out early on, leaving the most selective compounds for lead optimization.

A prevalent observation in high-throughput screening and drug discovery programs is the inhibition of protein function by small-molecule compound aggregation. Here, we present the X-ray structural description of aggregation-based inhibition of a protein–protein interaction involving tumor necrosis factor α (TNFα). An ordered conglomerate of an aggregating small-molecule inhibitor (JNJ525) induces a quaternary structure switch of TNFα that inhibits the protein–protein interaction between TNFα and TNFα receptors. SPD-304 may employ a similar mechanism of inhibition.

Here, we report a high-throughput virtual screening (HTVS) study using phosphoinositide 3-kinase (both PI3Kγ and PI3Kδ). Our initial HTVS results of the Janssen corporate database identified small focused libraries with hit rates at 50% inhibition showing a 50-fold increase over those from a HTS (high-throughput screen). Further, applying constraints based on "chemically intuitive" hydrogen bonds and/or positional requirements resulted in a substantial improvement in the hit rates (versus no constraints) and reduced docking time. While we find that docking scoring functions are not capable of providing a reliable relative ranking of a set of compounds, a prioritization of groups of compounds (e.g., low, medium, and high) does emerge, which allows for the chemistry efforts to be quickly focused on the most viable candidates. Thus, this illustrates that it is not always necessary to have a high correlation between a computational score and the experimental data to impact the drug discovery process.

Niacin raises high-density lipoprotein and lowers low-density lipoprotein through the activation of the β-hydroxybutyrate receptor hydroxycarboxylic acid 2 (HCA2) (aka GPR109a) but with an unwanted side effect of cutaneous flushing caused by vascular dilation because of the stimulation of HCA2 receptors in Langerhans cells in skin. HCA1 (aka GPR81), predominantly expressed in adipocytes, was recently identified as a receptor for lactate. Activation of HCA1 in adipocytes by lactate results in the inhibition of lipolysis, suggesting that agonists for HCA1 may be useful for the treatment of dyslipidemia. Lactate is a metabolite of glucose, suggesting that HCA1 may also be involved in the regulation of glucose metabolism. The low potency of lactate to activate HCA1, coupled with its fast turnover rate in vivo, render it an inadequate tool for studying the biological role of lactate/HCA1 in vivo. In this article, we demonstrate the identification of 3-hydroxybenzoic acid (3-HBA) as an agonist for both HCA2 and HCA1, whereas 3,5-dihydroxybenzoic acid (3,5-DHBA) is a specific agonist for only HCA1 (EC(50) ∼150 μM). 3,5-DHBA inhibits lipolysis in wild-type mouse adipocytes but not in HCA1-deficient adipocytes. Therefore, 3,5-DHBA is a useful tool for the in vivo study of HCA1 function and offers a base for further HCA1 agonist design. Because 3-HBA and 3,5-DHBA are polyphenolic acids found in many natural products, such as fruits, berries, and coffee, it is intriguing to speculate that other heretofore undiscovered natural substances may have therapeutic benefits.

Orexin receptor antagonists are being investigated as therapeutic agents for insomnia and addictive disorders. In this study the interactions between the orexin receptors (orexin 1 receptor and orexin 2 receptor), orexin peptides, and small molecule orexin antagonists were explored. To study these phenomena, a variety of mutant orexin receptors was made and tested using receptor binding and functional assays. Domains of the two orexin receptors were exchanged to show the critical ligand binding domains for orexin peptides and representative selective orexin receptor antagonists. Results from domain exchanges between the orexin receptors suggest that transmembrane domain 3 is crucially important for receptor interactions with small molecule antagonists. These data also suggest that the orexin peptides occupy a larger footprint, interacting with transmembrane domain 1, the amino terminus and transmembrane domain 5 as well as transmembrane domain 3. Transmembrane domain 3 has been shown to be an important part of the small molecule binding pocket common to rhodopsin and β2-adrenergic receptors. Additional orexin receptor 2 point mutations were made based on the common arrangement of receptor transmembrane domains shown in the G-protein coupled receptor crystal structure literature and the impact of orexin 2 receptor residue threonine 135 on the ligand selectivity of the 2 orexin receptors. These data support a model of the orexin receptor binding pocket in which transmembrane domains 3 and 5 are prominent contributors to ligand binding and functional activity. The data also illustrate key contact points for ligand interactions in the consensus small molecule pocket of these receptors.

Receptors from distant species may have conserved functions despite significant differences in protein sequences. Whereas the noncritical residues are often changed in distant species, the amino acids critical in receptor functions are often conserved. Studying the conserved residues between receptors from distant species offers valuable information to probe the roles of residues in receptor function. We identified two zebrafish receptors (zGPR81-1 and zGPR81-2) that show approximately 60% identity to human GPR81, GPR109a, and GPR109b but respond only to l-lactate and not to the GPR109a ligands. Protein sequence comparison among zebrafish GPR81s, mammalian GPR81s, GPR109a, and GPR109b identified a common structure (six Cys residues at the extracellular domains that potentially form three disulfide bonds) in this subfamily of receptors. In addition, a number of residues conserved in all GPR81s but not in GPR109s have been identified. Furthermore, we identified a conserved motif, C165-E166-S167-F168, at the second extracellular loop of GPR81. Using site-directed mutagenesis, we showed that Arg71 at the transmembrane domain 2 is very critical for GPR81 function. In addition, we demonstrated that the C165-E166-S167-F168 motif at the second extracellular loop is critical for GPR81 function, and the conserved six Cys residues at the extracellular regions are necessary for GPR81 function. It is important to mention that for those residues important for GPR81 function, the corresponding residues or motifs in GPR109a are also critical for GPR109a function. These findings help us better understand the interaction between lactate and GPR81 and provide useful information for GPR81 ligand design.

Further investigation of the recently reported piperidine-4-yl-aminopyrimidine class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant viruses. Synthesis, structure-activity relationship (SAR), clearance data, and crystallographic evidence for the binding motif are discussed.

Further investigation of the recently reported piperidine-4-yl-aminopyrimidine class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant viruses. Synthesis, structure-activity relationship (SAR), clearance data, and crystallographic evidence for the binding motif are discussed.

Lactic acid is a well known metabolic by-product of intense exercise, particularly under anaerobic conditions. Lactate is also a key source of energy and an important metabolic substrate, and it has also been hypothesized to be a signaling molecule directing metabolic activity. Here we show that GPR81, an orphan G-protein-coupled receptor highly expressed in fat, is in fact a sensor for lactate. Lactate activates GPR81 in its physiological concentration range of 1-20 mM and suppresses lipolysis in mouse, rat, and human adipocytes as well as in differentiated 3T3-L1 cells. Adipocytes from GPR81-deficient mice lack an antilipolytic response to lactate but are responsive to other antilipolytic agents. Lactate specifically induces internalization of GPR81 after receptor activation. Site-directed mutagenesis of GPR81 coupled with homology modeling demonstrates that classically conserved key residues in the transmembrane binding domains are responsible for interacting with lactate. Our results indicate that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. GPR81 may thus be an attractive target for the treatment of dyslipidemia and other metabolic disorders.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of HIV. These regimens are extremely effective in suppressing virus replication. Structure-based optimization of diaryl ether inhibitors led to the discovery of a new series of pyrazolo[3,4-c]pyridazine NNRTIs that bind the reverse transcriptase enzyme of human immunodeficiency virus-1 (HIV-RT) in an expanded volume relative to most other inhibitors in this class.The binding mode maintains the beta13 and beta14 strands bearing Pro236 in a position similar to that in the unliganded reverse transcriptase structure, and the distribution of interactions creates the opportunity for substantial resilience to single point mutations. Several pyrazolopyridazine NNRTIs were found to be highly effective against wild-type and NNRTI-resistant viral strains in cell culture.

In this work, we examine how the reported dual decay processes of rhodopsin and binding site stereospecificity can be accounted for by the recently available crystal structure of rhodopsin. Arguments are presented for possible presence of two rhodopsin “rotamers” that fit within the binding cavity. Directed pathways of decay could account for the observed excited decay processes. We summarize evidence for the possible existence of two different ground-state configurations that give rise to two different excited species.

Epstein-Barr virus-induced molecule 2 (EBI2) (also known as G-protein-coupled receptor 183) is a G-protein-coupled receptor (GPCR) that is best known for its role in B cell migration and localization. Our recent deorphanization effort led to the discovery of 7α,25-dihydroxycholesterol (7α,25-OHC) as the endogenous ligand for EBI2, which provides a tool for mechanistic studies of EBI2 function. Because EBI2 is the first GPCR known to bind and to be activated by an oxysterol, the goal of this study was to understand the molecular and structural bases for its ligand-dependent activation; this was achieved by identifying structural moieties in EBI2 or in 7α,25-OHC that might affect receptor-ligand interactions. By using a series of chemically related OHC analogs, we demonstrated that all three hydroxyl groups in 7α,25-OHC contributed to ligand-induced activation of the receptor. To determine the location and composition of the ligand binding domain in EBI2, we used a site-directed mutagenesis approach and generated mutant receptors with single amino acid substitutions at selected positions of interest. Biochemical and pharmacological profiling of these mutant receptors allowed for structure-function analyses and revealed critical motifs that likely interact with 7α,25-OHC. By using a hybrid β(2)-adrenergic receptor-C-X-C chemokine receptor type 4 structure as a template, we created a homology model for EBI2 and optimized the docking of 7α,25-OHC into the putative ligand binding site, so that the hydroxyl groups interact with residues Arg87, Asn114, and Glu183. This model of ligand docking yields important structural insight into the molecular mechanisms mediating EBI2 function and may facilitate future efforts to design novel therapeutic agents that target EBI2.

Several novel azulene-containing retinoids were prepared and evaluated for their ability to suppress carcinogen-induced neoplastic transformation and to concomitantly up-regulate gap junctional communication in the in vitro mouse fibroblast C3H/10T1/2 cell bioassay. The azulenic retinoids were divided into two groups: compounds 1-6 were modeled after retinoic acid with flexible polyenic side chain whereas retinoids 7-13 featured a benzoic acid moiety analogous to the prototypic retinobenzoate (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)-1-propenyl]benzoic acid (TTNPB). Within this latter group the side chains for compounds 7, 10, and 11 were attached at the 1-, 2-, and 8-positions of the azulenic terminus, respectively. Biological activities were determined for all the new compounds. Two of these novel retinoids, azulenic retinobenzoic acid derivatives 7 and 11, were completely effective inhibitors of transformation at 10(-6) M. The most active azulenic retinoids also enhanced gap junctional communication in untransformed cells; this was associated with up-regulated expression of connexin 43, a structural protein of the gap junction. Two fluorinated analogs were also tested. The azulenic fluoro acid 5 was found to be more potent than the trifluoromethyl analog 6. Azulenic analogs with hydroxyl or carboxaldehyde side chain functional groups were ineffective transformation inhibitors. In general, azulenic retinobenzoic acid analogs structurally akin to TTNPB were more effective than flexible side chain analogs related to retinoic acid.

— An attempt has been made to construct a 3-D model of rhodopsin, a member of G-protein coupled receptors. Sequence homology of rhodopsin with the latter was a factor considered in the modeling procedure. The constructed model has been used to compare currently available specific protein/substrate interaction information, the shape of the binding cavity derived from shape of binding retinal isomers and analogs and challenged to explain recently available results from a series of fluorinated rhodopsins.

This is a response to the letter by Hai-jun Xu (1). Regarding the question of whether l-lactate indeed plays a role in inhibiting lipolysis, we think that we have adequately addressed the question in our original JBC paper. In adipocytes, it is very well studied and documented that reagents such as adrenaline and forskolin (stimulating cAMP synthesis) or isobutylmethylxanthine (IBMX) (inhibiting cAMP hydrolysis) that up-regulate cAMP levels in adipocytes increase lipolysis. In contrast, reagents such as insulin (increase cAMP hydrolysis by stimulating phosphodiesterase) or niacin and propionate etc. (inhibiting cAMP synthesis through Gi-protein-coupled GPCRs) that down-regulate cAMP levels in the adipocytes inhibit lipolysis. That lactate stimulates GPR81 and inhibits cAMP synthesis in GPR81-expressing cells strongly suggests that lactate may play a role in suppressing lipolysis in adipocytes, which express high levels of GPR81. Our results from differentiated 3T3L1 cells as well as primary adipocytes from mouse, rat, and human demonstrated that l-lactate indeed inhibits lipolysis in those adipocytes. This hypothesis is further supported by our results using adipocytes from GPR81 knock-out mice, which are completely unresponsive to l-lactate in lipolysis assays. Of course, our results are in isolated cells and are well controlled. In our experiments we always compare the samples with and without added lactate while keeping all of the other elements the same. However, this does not mean that under physiological conditions both lactate and glycerol concentrations can't be elevated since in vivo lactate certainly is not the only regulator for lipolysis. For instance, under hypoglycemic conditions, it is very well known that epinephrine, glucagon, cortisol, and many other hormones are released. Some of those hormones including epinephrine and glucagon are known to increase lipolysis. Therefore, lactate may still play an inhibitory role for lipolysis in adipocytes even under hypoglycemic conditions, but at the same time, there are many other hormones that are stimulating the lipolysis. In addition, those hormones may also stimulate lipolysis in cells (such as liver cells) that do not express GPR81 and therefore won't be suppressed by l-lactate. Overall, what has been observed under hypoglycemic conditions is a result of the body's response to many different hormones, and we should not conclude that l-lactate is not playing an inhibitory role for lipolysis because both lactate and glycerol concentrations are raised under hypoglycemic conditions. Regarding the question of whether the only role of lactate in lipid metabolism is inhibiting lipolysis, so far we can only conclude that l-lactate inhibits lipolysis in adipocytes that express GPR81. Lactate is a metabolite of glucose. A growing number of studies show that lactate is also an energy source. Lactate can also be a signaling molecule through pathways other than GPR81. Therefore, in addition to GPR81, lactate can certainly affect the body's metabolism (including lipid metabolism) either directly or indirectly. Mr. Xu also proposed a hypothesis that lactate could actually promote lipolysis under certain situations because lactate produced by anaerobic glycolysis might relieve the inhibition of glucose aerobic oxidation on fatty acid oxidation or adjust intracellular redox state via lactate shuttle in skeletal muscle and adipose tissues and thus promote fatty acid oxidation. We think this is an interesting proposal but it needs to be supported by experiments and evidence.

A series of benzyl pyridazinones were evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Several members of this series showed good activity against the wild-type virus and NNRTI-resistant viruses. The binding of inhibitor 5a to HIV-RT was analyzed by surface plasmon resonance spectroscopy. Pharmacokinetic studies of 5a in rat and dog demonstrated that this compound has good oral bioavailability in animal species. The crystal structure of a complex between HIV-RT and inhibitor 4c is also described.

Voltage-gated sodium channel 1.7 (Nav1.7) plays a role in the conduction of action potentials and is involved in the sensation of pain. Spider toxins such as Huwentoxin IV (HwTx-IV) are potent inhibitors of Nav1.7, functioning as a gating modifier trapping the voltage sensor in an inward closed conformation. HwTx-IV interacts with specific residues in the voltage sensor S3-S4 region of domain II. The purpose of the present study was to understand the residues important for interaction between HwTx-IV and Nav1.7. Computationally, molecular dynamics was used to study wild type HwTx-IV along with various alanine and cystine mutations to determine residues important for the stability of toxin and to obtain a model of the interacting surface. A homology model of Nav1.7 was built and the toxin docked to determine key interactions. Experimentally, alanine mutants of HwTx-IV were tested for functional activity using FLIPR, QPatch, and manual patch. Computational and experimental results suggest key residues critical for binding of the toxin to Nav1.7, particularly S25, W30 and K32. These studies provide a more clear idea of the nature of interaction between HwTx-IV and Nav1.7 and may therefore be a useful guide in designing novel peptides with improved selectivity for sodium channel subtypes.

In the G protein-coupled receptor family, a highly conserved aspartic acid located within the third transmembrane domain has been shown to be involved in ligand binding. Within the endothelin (ET) peptide receptor family, this aspartic acid has been replaced by a lysine. To assess the importance of this residue in ET binding, the lysine (position 181) of rat ET type B receptor was replaced by an aspartic acid. The effects on ligand binding and phosphoinositide turnover of both the wild-type and K181D mutant receptors were examined using transient receptor expression in COS-7 cells. Using [125I]ET-1 as the radioactive peptide ligand in displacement binding studies, the wild-type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three ET peptides (ET-1, ET-2, and ET-3). The mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM). The K181D mutant receptor still elicited full inositol phosphate (IP) accumulation responses in the presence of saturating concentrations of ETs (10 nM of ET-1, 100 nM of ET-2, or 1 microM of ET-3), indicating that the mutation did not affect G protein coupling.

Publisher Summary The cloning and sequencing of the G-protein coupled receptors (GPCRs) has revealed a superfamily of GPCR proteins that activate various guanine-nucleotide binding (G) proteins. Throughout this chapter, a distinction is made among the GPCRs on the basis of the chemical nature of their ligands. Bacteriorhodopsin (BR), the major light-sensitive protein of the purple membrane of Halobacterium halobium , has been the first membrane bound protein found to be organized into seven transmembrane spanning domains. Although BR is not a GPCR, the sensory receptors require an intermediary G-protein called transducin to activate the second messenger system. To identify the hydrophobic segments in the GPCR primary sequences, various theoretical methods, such as hydrophobicity profiles, hydrophobic moments, helix amphiphilicity, and secondary structure prediction can be applied. The N-terminal segment length varies to extreme across the GPCR families. Protein chemistry experiments have identified various N-glycosylation sites. The intracellular loops and C-terminal fragments have been shown to affect signal transduction through coupling to the G-protein. Throughout all GPCRs, they are characterized by a heavy content of basic amino acid residues. A number of recently proposed structural models for the non-peptide GPCRs suggest schematic 2-dimensional (2D) representations, 3D models limited to selected helices or highly refined 3D models that sometimes incorporate the connecting loops. Very few modeling reports have been expanded to include the challenge of loop modeling. The internal proline residues impose a curving effect in the helices, molding the transmembrane domains in an overall oblong shape. It has long been speculated that a receptor conformational change, triggered by agonist binding, would be responsible for initiating the resulting transduction mechanism. Variations on the familiar non-peptide GPCR structural theme have been reported in very few peptide receptors.

Six geometric isomers of 2 (3,7,13-trimethyl-10-isopropyl-2,4,6,8,10-tetradecapentaenal) have been isolated and characterized. Of these, four (equivalent to 9- cis , 11- cis , 5,9- dicis , and 5,11- dicis of retinoids) were found to form stable visual pigment analogs. So were four isomers (equivalent to 7- cis , 9- cis , 11- cis , and 9,13- dicis ) of 3 (3,7,11-trimethyl-10-isopropyl-2,4,6,8,10-dodecapentaenal). These analogs suggest that the hydrophobic pocket is more flexible than what was recognized earlier, perhaps capable of accepting analogs with 6- S-trans as well as a 6- S-cis conformers. The presence of bacteriorhodopsin analogs from isomers of 2 was also reported.

The 9-cis, 11-cis, 13-cis, and all-trans isomers of 10-fluoro-, 10-chloro-, 10-methyl-, and 10-ethylretinals have been prepared and characterized. Results of their interaction with bovine opsin are reported. The data have been analyzed in terms of the conformational properties of the retinal isomers and their steric compatibility with the binding site as defined by the two-dimensional map disclosed earlier. The need to expand the active zone and previously undetected restrictions in the third dimension are noted.

Results of the binding interaction of isomers of α-retinal and six substituted α-retinals (5-butyl, 5-phenyl, 5-isopropyl, 5-heptyl, 5-decyl, and 10-fluoro) with bovine opsin are reported. Their implications on the binding site requirements are discussed.