QSAR-Based Computational Approaches to Accelerate the Discovery of Sigma-2 Receptor (S2R) Ligands as Therapeutic Drugs.

S2R overexpression is associated with various forms of cancer as well as both neuropsychiatric disorders (e.g., schizophrenia) and neurodegenerative diseases (Alzheimer's disease: AD). In the present study, three ligand-based methods (QSAR modeling, pharmacophore mapping, and shape-based screening) were implemented to select putative S2R ligands from the DrugBank library comprising 2000+ entries. Four separate optimization algorithms (i.e., stepwise regression, Lasso, genetic algorithm (GA), and a customized extension of GA called GreedGene) were adapted to select descriptors for the QSAR models. The subsequent biological evaluation of selected compounds revealed that three FDA-approved drugs for unrelated therapeutic indications exhibited sub-1 uM binding affinity for S2R. In particular, the antidepressant drug nefazodone elicited a S2R binding affinity Ki = 140 nM. A total of 159 unique S2R ligands were retrieved from 16 publications for model building, validation, and testing. To our best knowledge, the present report represents the first case to develop comprehensive QSAR models sourced by pooling and curating a large assemblage of structurally diverse S2R ligands, which should prove useful for identifying new drug leads and predicting their S2R binding affinity prior to the resource-demanding tasks of chemical synthesis and biological evaluation.

Novel Sigma 1 Receptor Antagonists as Potential Therapeutics for Pain Management.

The sigma 1 receptor (S1R) is a molecular chaperone protein located in the endoplasmic reticulum and plasma membranes and has been shown to play important roles in various pathological disorders including pain and, as recently discovered, COVID-19. Employing structure- and QSAR-based drug design strategies, we rationally designed, synthesized, and biologically evaluated a series of novel triazole-based S1R antagonists. Compound 10 exhibited potent binding affinity for S1R, high selectivity over S2R and 87 other human targets, acceptable in vitro metabolic stability, slow clearance in liver microsomes, and excellent blood-brain barrier permeability in rats. Further in vivo studies in rats showed that 10 exhibited negligible acute toxicity in the rotarod test and statistically significant analgesic effects in the formalin test for acute inflammatory pain and paclitaxel-induced neuropathic pain models during cancer chemotherapy. These encouraging results promote further development of our triazole-based S1R antagonists as novel treatments for pain of different etiologies.

Identification of an irreversible PPARγ antagonist with potent anticancer activity.

Melanoma is responsible for most skin cancer deaths, and its incidence continues to rise year after year. Different treatment options have been developed for melanoma depending on the stage of the disease. Despite recent advances in immuno- and targeted therapies, advanced melanoma remains incurable and thus an urgent need persists for safe and more effective melanoma therapeutics. In this study, we demonstrate that a novel compound MM902 (3-(3-(bromomethyl)-5-(4-(tert-butyl) phenyl)-1H-1,2,4-triazol-1-yl) phenol) exhibited potent efficacies in inhibiting the growth of different cancer cells, and suppressed tumor growth in a mouse xenograft model of malignant melanoma. Beginning with MM902 instead of specific targets, computational similarity- and docking-based approaches were conducted to search for known anticancer drugs whose structural features match MM902 and whose pharmacological target would accommodate an irreversible inhibitor. Peroxisome proliferator-activated receptor (PPAR) was computationally identified as one of the pharmacological targets and confirmed by in vitro biochemical assays. MM902 was shown to bind to PPARγ in an irreversible mode of action and to function as a selective antagonist for PPARγ over PPARα and PPARδ. It is hoped that MM902 will serve as a valuable research probe to study the functions of PPARγ in tumorigenesis and other pathological processes.

Small molecule inhibitors block Gas6-inducible TAM activation and tumorigenicity.

TAM receptors (Tyro-3, Axl, and Mertk) are a family of three homologous type I receptor tyrosine kinases that are implicated in several human malignancies. Overexpression of TAMs and their major ligand Growth arrest-specific factor 6 (Gas6) is associated with more aggressive staging of cancers, poorer predicted patient survival, acquired drug resistance and metastasis. Here we describe small molecule inhibitors (RU-301 and RU-302) that target the extracellular domain of Axl at the interface of the Ig-1 ectodomain of Axl and the Lg-1 of Gas6. These inhibitors effectively block Gas6-inducible Axl receptor activation with low micromolar IC50s in cell-based reporter assays, inhibit Gas6-inducible motility in Axl-expressing cell lines, and suppress H1299 lung cancer tumor growth in a mouse xenograft NOD-SCIDγ model. Furthermore, using homology models and biochemical verifications, we show that RU301 and 302 also inhibit Gas6 inducible activation of Mertk and Tyro3 suggesting they can act as pan-TAM inhibitors that block the interface between the TAM Ig1 ectodomain and the Gas6 Lg domain. Together, these observations establish that small molecules that bind to the interface between TAM Ig1 domain and Gas6 Lg1 domain can inhibit TAM activation, and support the further development of small molecule Gas6-TAM interaction inhibitors as a novel class of cancer therapeutics.

Micellar and structural stability of nanoscale amphiphilic polymers: Implications for anti-atherosclerotic bioactivity.

Atherosclerosis, a leading cause of mortality in developed countries, is characterized by the buildup of oxidized low-density lipoprotein (oxLDL) within the vascular intima, unregulated oxLDL uptake by macrophages, and ensuing formation of arterial plaque. Amphiphilic polymers (AMPs) comprised of a branched hydrophobic domain and a hydrophilic poly(ethylene glycol) (PEG) tail have shown promising anti-atherogenic effects through direct inhibition of oxLDL uptake by macrophages. In this study, five AMPs with controlled variations were evaluated for their micellar and structural stability in the presence of serum and lipase, respectively, to develop underlying structure-atheroprotective activity relations. In parallel, molecular dynamics simulations were performed to explore the AMP conformational preferences within an aqueous environment. Notably, AMPs with ether linkages between the hydrophobic arms and sugar backbones demonstrated enhanced degradation stability and storage stability, and also elicited enhanced anti-atherogenic bioactivity. Additionally, AMPs with increased hydrophobicity elicited increased atheroprotective bioactivity in the presence of serum. These studies provide key insights for designing more serum-stable polymeric micelles as prospective cardiovascular nanotherapies.

Tartaric acid-based amphiphilic macromolecules with ether linkages exhibit enhanced repression of oxidized low density lipoprotein uptake.

Cardiovascular disease initiates with the atherogenic cascade of scavenger receptor- (SR-) mediated oxidized low-density lipoprotein (oxLDL) uptake. Resulting foam cell formation leads to lipid-rich lesions within arteries. We designed amphiphilic macromolecules (AMs) to inhibit these processes by competitively blocking oxLDL uptake via SRs, potentially arresting atherosclerotic development. In this study, we investigated the impact of replacing ester linkages with ether linkages in the AM hydrophobic domain. We hypothesized that ether linkages would impart flexibility for orientation to improve binding to SR binding pockets, enhancing anti-atherogenic activity. A series of tartaric acid-based AMs with varying hydrophobic chain lengths and conjugation chemistries were synthesized, characterized, and evaluated for bioactivity. 3-D conformations of AMs in aqueous conditions may have significant effects on anti-atherogenic potency and were simulated by molecular modeling. Notably, ether-linked AMs exhibited significantly higher levels of inhibition of oxLDL uptake than their corresponding ester analogues, indicating a dominant effect of linkage flexibility on pharmacological activity. The degradation stability was also enhanced for ether-linked AMs. These studies further suggested that alkyl chain length (i.e., relative hydrophobicity), conformation (i.e., orientation), and chemical stability play a critical role in modulating oxLDL uptake, and guide the design of innovative cardiovascular therapies.

Polyester monomers lack ability to bind and activate both androgenic and estrogenic receptors as determined by in vitro and in silico methods.

The paper presents results from the screening of seven monomers used by Eastman Chemical to make various polymers. Ethylene glycol, diethylene glycol, polytetramethylene glycol, isophthalic acid, monosodium-5-sulfoisophthalic acid, 1,4-cyclohexanedicarboxylic acid, and dimethylcyclohexanedicarboxylate were screened for potential androgenicity or estrogenicity. The following studies were conducted: QSAR for binding to the AR and ER, in vitro Androgen Receptor Binding Assay, in vitro Estrogen Receptor Binding Assays (alpha and beta isoforms), in vitro Androgen Receptor Transactivation Assay in human cells, and in vitro Estrogen Receptor Transactivation Assay in human cells. None of the QSAR models predicted that any of the monomers possessed appreciable binding affinity for either AR or ER. Binding assays showed no evidence of interaction with either the AR or the alpha or beta ER receptors. Similarly, the AR and ER transactivation assays were negative. Moreover, six of the seven monomers have been subjected to 13-week and developmental toxicity studies in rats with no androgen- or estrogen-related effects being noted. Given the negative results of the in vitro screening assays (except PMG which demonstrated cytotoxicity) as well as available repeated dose and developmental and reproductive studies, the data suggest that none of the monomers tested exhibit androgenic or estrogenic hazards.

Novel virtual screening approach for the discovery of human tyrosinase inhibitors.

Tyrosinase is the key enzyme involved in the human pigmentation process, as well as the undesired browning of fruits and vegetables. Compounds inhibiting tyrosinase catalytic activity are an important class of cosmetic and dermatological agents which show high potential as depigmentation agents used for skin lightening. The multi-step protocol employed for the identification of novel tyrosinase inhibitors incorporated the Shape Signatures computational algorithm for rapid screening of chemical libraries. This algorithm converts the size and shape of a molecule, as well its surface charge distribution and other bio-relevant properties, into compact histograms (signatures) that lend themselves to rapid comparison between molecules. Shape Signatures excels at scaffold hopping across different chemical families, which enables identification of new actives whose molecular structure is distinct from other known actives. Using this approach, we identified a novel class of depigmentation agents that demonstrated promise for skin lightening product development.

In silico design of anti-atherogenic biomaterials.

Atherogenesis, the uncontrolled deposition of modified lipoproteins in inflamed arteries, serves as a focal trigger of cardiovascular disease (CVD). Polymeric biomaterials have been envisioned to counteract atherogenesis based on their ability to repress scavenger mediated uptake of oxidized lipoprotein (oxLDL) in macrophages. Following the conceptualization in our laboratories of a new library of amphiphilic macromolecules (AMs), assembled from sugar backbones, aliphatic chains and poly(ethylene glycol) tails, a more rational approach is necessary to parse the diverse features such as charge, hydrophobicity, sugar composition and stereochemistry. In this study, we advance a computational biomaterials design approach to screen and elucidate anti-atherogenic biomaterials with high efficacy. AMs were quantified in terms of not only 1D (molecular formula) and 2D (molecular connectivity) descriptors, but also new 3D (molecular geometry) descriptors of AMs modeled by coarse-grained molecular dynamics (MD) followed by all-atom MD simulations. Quantitative structure-activity relationship (QSAR) models for anti-atherogenic activity were then constructed by screening a total of 1164 descriptors against the corresponding, experimentally measured potency of AM inhibition of oxLDL uptake in human monocyte-derived macrophages. Five key descriptors were identified to provide a strong linear correlation between the predicted and observed anti-atherogenic activity values, and were then used to correctly forecast the efficacy of three newly designed AMs. Thus, a new ligand-based drug design framework was successfully adapted to computationally screen and design biomaterials with cardiovascular therapeutic properties.

Bisphenol A and its analogues activate human pregnane X receptor.

BACKGROUND: Bisphenol A (BPA) is a base chemical used extensively in many consumer products. BPA and its analogues are present in environmental and human samples. Many endocrine-disrupting chemicals, including BPA, have been shown to activate the pregnane X receptor (PXR), a nuclear receptor that functions as a master regulator of xenobiotic metabolism. However, the detailed mechanism by which these chemicals activate PXR remains unknown. OBJECTIVE: We investigated the mechanism by which BPA interacts with and activates PXR and examined selected BPA analogues to determine whether they bind to and activate PXR. METHODS: Cell-based reporter assays, in silico ligand-PXR docking studies, and site-directed mutagenesis were combined to study the interaction between BPA and PXR. We also investigated the influence of BPA and its analogues on the regulation of PXR target genes in human LS180 cells. RESULTS: We found that BPA and several of its analogues are potent agonists for human PXR (hPXR) but do not affect mouse PXR activity. We identified key residues within hPXR's ligand-binding pocket that constitute points of interaction with BPA. We also deduced the structural requirements of BPA analogues that activate hPXR. BPA and its analogues can also induce PXR target gene expression in human LS180 cells. CONCLUSIONS: The present study advances our understanding of the mechanism by which BPA interacts with and activates human PXR. Activation of PXR by BPA may explain some of the adverse effects of BPA in humans.

Application of screening methods, shape signatures and engineered biosensors in early drug discovery process.

PURPOSE: In this study, two unreported estrogen antagonists were identified using a combination of computational screening and a simple bacterial estrogen sensor. METHODS: Molecules here presented were initially part of a group obtained from a library of over a half million chemical compounds, using the Shape Signatures method. The structures within this group were then clustered and compared to known antagonists based on their physico-chemical parameters, and possible binding modes of the compounds to the Estrogen Receptor alpha (ER alpha) were analyzed. Finally, thirteen candidate compounds were purchased, and two of them were shown to behave as potential subtype-selective estrogen antagonists using a set of bacterial estrogen biosensors, which included sensors for ER alpha, ER beta, and a negative control thyroid hormone beta biosensor. These activities were then analyzed using an ELISA assay against activated ER alpha in human MCF-7 cell extract. RESULTS: Two new estrogen receptor antagonists were detected using in silico Shape Signatures method with an engineered subtype-selective bacterial estrogen biosensor and commercially available ELISA assay. Additional thyroid biosensor control experiments confirmed no compounds interacted with human thyroid receptor beta. CONCLUSIONS: This work demonstrates an effective combination of computational analysis and simple bacterial screens for rapid identification of potential hormone-like therapeutics.

The major human pregnane X receptor (PXR) splice variant, PXR.2, exhibits significantly diminished ligand-activated transcriptional regulation.

The pregnane X receptor (PXR; PXR.1) can be activated by structurally diverse lipophilic ligands. PXR.2, an alternatively spliced form of PXR, lacks 111 nucleotides encoding 37 amino acids in the ligand binding domain. PXR.2 bound a classic CYP3A4 PXR response element (PXRE) in electrophoretic mobility shift assays, but transfected PXR.2 failed to transactivate a CYP3A4-promoter-luciferase reporter plasmid in HepG2 cells treated with various PXR ligands. Cotransfection experiments showed that PXR.2 behaved as a dominant negative, interfering with PXR.1/rifampin activation of CYP3A4-PXRE-LUC. In HepG2 and LS180 cells stably transduced with PXR.1, PXR target genes (CYP3A4, MDR1, CYP2B6, and UGT1A1) were higher than mock-transduced cells in the absence of ligand and were further induced in the presence of rifampin. In contrast, PXR.2 stably introduced into the same host cells failed to induce target genes over levels in mock-transfected cells after drug treatment. Our homology modeling suggests that ligands bind PXR.1 more favorably, probably because of the presence of a key disordered loop region, which is missing in PXR.2. Yeast two-hybrid assays revealed that, even in the presence of ligand, the corepressors remain tightly bound to PXR.2, and coactivators are unable to bind at helix 12. In summary, PXR.2 can bind to PXREs but fails to transactivate target genes because ligands do not bind the ligand binding domain of PXR.2 productively, corepressors remain tightly bound, and coactivators are not recruited to PXR.2.

Novel microtubule polymerization inhibitor with potent antiproliferative and antitumor activity.

Microtubule-stabilizing and microtubule-destabilizing agents are commonly used as anticancer agents. Although highly effective, success with these agents has been limited due to their relative insolubility, cumbersome synthesis/purification, toxic side effects, and development of multidrug resistance. Hence, the identification of improved agents that circumvent one or more of these problems is warranted. We recently described the rational design of a series of triazole-based compounds as antimitotic agents. Members of this N-substituted 1,2,4-triazole family of compounds exhibit potent tubulin polymerization inhibition and broad spectrum cellular cytotoxicity. Here, we extensively characterize the in vitro and in vivo effects of our lead compound from the series 1-methyl-5-(3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-4-yl)-1H-indole, designated T115. We show that T115 competes with colchicine for its binding pocket in tubulin, produces robust inhibition of tubulin polymerization, and disrupts the microtubule network system inside the cells. In addition, T115 arrests human cancer cells in the G(2)-M phase of cell cycling, a hallmark of microtubule destabilizing drugs. T115 also inhibits cell viability of several cancer cell lines, including multidrug-resistant cell lines, in the low nanomolar range. No cytotoxicity was observed by T115 against normal human skin fibroblasts cell lines, and acute toxicity studies in normal nontumor-bearing mice indicated that T115 is well-tolerated in vivo (maximum total tolerated dose, 400 mg/kg). In a mouse xenograft model using human colorectal (HT-29) and prostate (PC3) cancer cells, T115 significantly inhibited tumor growth when administered i.p. Taken together, our results suggest that T115 is a potential drug candidate for cancer chemotherapy.

Halogenated ligands and their interactions with amino acids: implications for structure-activity and structure-toxicity relationships.

The properties of chemicals are rooted in their molecular structure. It follows that structural analysis of specific interactions between ligands and biomolecules at the molecular level is invaluable for defining structure-activity relationships (SARs) and structure-toxicity relationships (STRs). This study has elucidated the structural and molecular basis of interactions of biomolecules with alkyl and aryl halides that are extensively used as components in many commercial pesticides, disinfectants, and drugs. We analyzed the protein structures deposited in Protein Data Bank (PDB) for structural information associated with interactions between halogenated ligands and proteins. This analysis revealed distinct patterns with respect to the nature and structural characteristics of halogen interactions with specific types of atoms and groups in proteins. Fluorine had the highest propensity of interactions for glycine, while chlorine for leucine, bromine for arginine, and iodine for lysine. Chlorine, bromine and iodine had the lowest propensity of interactions for cysteine, while fluorine had a lowest propensity for proline. These trends for highest propensity shifted towards the hydrophobic residues for all the halogens when only interactions with the side chain were considered. Halogens had equal propensities of interaction for the halogen bonding partners (nitrogen and oxygen atoms), albeit with different geometries. The optimal angle for interactions with halogens was approximately 120 degrees for oxygen atoms, and approximately 96 degrees for nitrogen atoms. The distance distributions of halogens with various amino acids were mostly bimodal, and the angle distributions were unimodal. Insights gained from this study have implications for the rational design of safer drugs and commercially important chemicals.

Computational discovery of novel low micromolar human pregnane X receptor antagonists.

Very few antagonists have been identified for the human pregnane X receptor (PXR). These molecules may be of use for modulating the effects of therapeutic drugs, which are potent agonists for this receptor (e.g., some anticancer compounds and macrolide antibiotics), with subsequent effects on transcriptional regulation of xenobiotic metabolism and transporter genes. A recent novel pharmacophore for PXR antagonists was developed using three azoles and consisted of two hydrogen bond acceptor regions and two hydrophobic features. This pharmacophore also suggested an overall small binding site that was identified on the outer surface of the receptor at the AF-2 site and validated by docking studies. Using computational approaches to search libraries of known drugs or commercially available molecules is preferred over random screening. We have now described several new smaller antagonists of PXR discovered with the antagonist pharmacophore with in vitro activity in the low micromolar range [S-p-tolyl 3',5-dimethyl-3,5'-biisoxazole-4'-carbothioate (SPB03255) (IC(50), 6.3 microM) and 4-(3-chlorophenyl)-5-(2,4-dichlorobenzylthio)-4H-1,2,4-triazol-3-ol (SPB00574) (IC(50), 24.8 microM)]. We have also used our computational pharmacophore and docking tools to suggest that most of the known PXR antagonists, such as coumestrol and sulforaphane, could also interact on the outer surface of PXR at the AF-2 domain. The involvement of this domain was also suggested by further site-directed mutagenesis work. We have additionally described an FDA approved prodrug, leflunomide (IC(50), 6.8 microM), that seems to be a PXR antagonist in vitro. These observations are important for predicting whether further molecules may interact with PXR as antagonists in vivo with potential therapeutic applications.

Shape signatures: new descriptors for predicting cardiotoxicity in silico.

Shape Signatures is a new computational tool that is being evaluated for applications in computational toxicology and drug discovery. The method employs a customized ray-tracing algorithm to explore the volume enclosed by the surface of a molecule and then uses the output to construct compact histograms (i.e., signatures) that encode for molecular shape and polarity. In the present study, we extend the application of the Shape Signatures methodology to the domain of computational models for cardiotoxicity. The Shape Signatures method is used to generate molecular descriptors that are then utilized with widely used classification techniques such as k nearest neighbors ( k-NN), support vector machines (SVM), and Kohonen self-organizing maps (SOM). The performances of these approaches were assessed by applying them to a data set of compounds with varying affinity toward the 5-HT(2B) receptor as well as a set of human ether-a-go-go-related gene (hERG) potassium channel inhibitors. Our classification models for 5-HT(2B) represented the first attempt at global computational models for this receptor and exhibited average accuracies in the range of 73-83%. This level of performance is comparable to using commercially available molecular descriptors. The overall accuracy of the hERG Shape Signatures-SVM models was 69-73%, in line with other computational models published to date. Our data indicate that Shape Signatures descriptors can be used with SVM and Kohonen SOM and perform better in classification problems related to the analysis of highly clustered and heterogeneous property spaces. Such models may have utility for predicting the potential for cardiotoxicity in drug discovery mediated by the 5-HT(2B) receptor and hERG.

Ligand specificity and evolution of liver X receptors.

Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRalpha and LXRbeta from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity.

Human pregnane X receptor antagonists and agonists define molecular requirements for different binding sites.

The pregnane X receptor (PXR) is an important transcriptional regulator of the expression of xenobiotic metabolism and transporter genes. The receptor is promiscuous, binding many structural classes of molecules that act as agonists at the ligand-binding domain, triggering up-regulation of genes, increasing the metabolism and excretion of therapeutic agents, and causing drug-drug interactions. It has been suggested that human PXR antagonists represent a means to counteract such interactions. Several azoles have been hypothesized to bind the activation function-2 (AF-2) surface on the exterior of PXR when agonists are concurrently bound in the ligand-binding domain. In the present study, we have derived novel computational models for PXR agonists using different series of imidazoles, steroids, and a set of diverse molecules with experimental PXR agonist binding data. We have additionally defined a novel pharmacophore for the steroidal agonist site. All agonist pharmacophores showed that hydrophobic features are predominant. In contrast, a qualitative comparison with the corresponding PXR antagonist pharmacophore models using azoles and biphenyls showed that they are smaller and hydrophobic with increased emphasis on hydrogen bonding features. Azole antagonists were docked into a proposed hydrophobic binding pocket on the outer surface at the AF-2 site and fitted comfortably, making interactions with key amino acids involved in charge clamping. Combining computational and experimental data for different classes of molecules provided strong evidence for agonists and antagonists binding distinct regions on PXR. These observations bear significant implications for future discovery of molecules that are more selective and potent antagonists.

Evaluation of broad spectrum protein kinase inhibitors to probe the architecture of the malarial cyclin dependent protein kinase Pfmrk.

We tested Pfmrk against several naphthalene and isoquinoline sulfonamides previously reported as protein kinase A (PKA) inhibitors. Pfmrk is a Cyclin Dependent protein Kinase (CDK) from Plasmodium falciparum, the causative parasite of the most lethal form of malaria. We find that the isoquinoline sulfonamides are potent inhibitors of Pfmrk and that substitution on the 5 position of the isoquinoline ring greatly influences the degree of potency. Molecular modeling studies suggest that the nitrogen atom in the isoquinoline ring plays a key role in ligand-receptor interactions. Structural analysis suggests that even subtle differences in amino acid composition within the active sites are responsible for conferring specificity of these inhibitors for Pfmrk over PKA.

Unique helical conformation of the fourth cytoplasmic loop of the CB1 cannabinoid receptor in a negatively charged environment.

The proximal portion of the C-terminus of the CB(1) cannabinoid receptor is a primary determinant for G-protein activation. A 17 residue proximal C-terminal peptide (rodent CB1 401-417), the intracellular loop 4 (IL4) peptide, mimicked the receptor's G-protein activation domain. Because of the importance of the cationic amino acids to G-protein activation, the three-dimensional structure of the IL4 peptide in a negatively charged sodium dodecyl sulfate (SDS) micellar environment has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR) spectroscopy and distance geometry calculations. Unambiguous proton NMR assignments were carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints were used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures which were refined using restrained energy minimization and dynamics. In water, the IL4 peptide prefers an extended conformation, whereas in SDS micelles, 3(10)-helical conformation is induced. The predominance of 3(10)-helical domain structure in SDS represents a unique difference compared with structure in alternative environments, which can significantly impact global electrostatic surface potential on the cytoplasmic surface of the CB(1) receptor and might influence the signal to the G-proteins.