Hologram quantitative structure-activity relationships for a series of farnesoid X receptor activators

The farnesoid X receptor (FXR) is an attractive drug target for the development of novel therapeutic agents for the treatment of dyslipidemia and cholestasis. Hologram quantitative structure-activity relationship (HQSAR) studies were conducted on a series of potent FXR activators originated from natural product-like libraries. A training set containing 82 compounds served to establish the models. The best HQSAR model was generated using atoms, bonds, connections, chirality, and donor and acceptor as fragment distinction and fragment size default (4-7) with six components. The model was used to predict the potency of 20 test set compounds that were not included in the training set, and the predicted values were in good agreement with the experimental results. The final HQSAR model and the information obtained from HQSAR 2D contribution maps should be useful for the design of novel FXR ligands having improved potency.     

5Alpha-bile alcohols function as farnesoid X receptor antagonists

The farnesoid X receptor (FXR) is a bile acid/alcohol-activated nuclear receptor that regulates lipid homeostasis. Unlike other steroid receptors, FXR binds bile acids in an orientation that allows the steroid nucleus A ring to face helix 12 in the receptor, a crucial domain for coactivator-recruitment. Because most naturally occurring bile acids and alcohols contain a cis-oriented A ring, which is distinct from that of other steroids and cholesterol metabolites, we investigated the role of this 5beta-configuration in FXR activation. The results showed that the 5beta-(A/B cis) bile alcohols 5beta-cyprinol and bufol are potent FXR agonists, whereas their 5alpha-(A/B trans) counterparts antagonize FXR transactivation and target gene expression. Both isomers bound to FXR, but their ability to induce coactivator-recruitment and thereby induce transactivation differed. These findings suggest a critical role for the A-ring orientation of bile salts in agonist/antagonist function.     

Cytohesins are cytoplasmic ErbB receptor activators

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in?vitro and in?vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.     

A novel principle for partial agonism of liver X receptor ligands. Competitive recruitment of activators and repressors

Partial, selective activation of nuclear receptors is a central issue in molecular endocrinology but only partly understood. Using LXRs as an example, we show here that purely agonistic ligands can be clearly and quantitatively differentiated from partial agonists by the cofactor interactions they induce. Although a pure agonist induces a conformation that is incompatible with the binding of repressors, partial agonists such as GW3965 induce a state where the interaction not only with coactivators, but also corepressors is clearly enhanced over the unliganded state. The activities of the natural ligand 22(R)-hydroxycholesterol and of a novel quinazolinone ligand, LN6500 can be further differentiated from GW3965 and T0901317 by their weaker induction of coactivator binding. Using biochemical and cell-based assays, we show that the natural ligand of LXR is a comparably weak partial agonist. As predicted, we find that a change in the coactivator to corepressor ratio in the cell will affect NCoR recruiting compounds more dramatically than NCoR-dissociating compounds. Our data show how competitive binding of coactivators and corepressors can explain the tissue-specific behavior of partial agonists and open up new routes to a rational design of partial agonists for LXRs.     

Bis-aryloxadiazoles as effective activators of the aryl hydrocarbon receptor

Bis-aryloxadiazoles are common scaffolds in medicinal chemistry due to their wide range of biological activities. Previously, we identified a 1,2,4-bis-aryloxadiazole that blocks mammary branching morphogenesis through activation of the aryl hydrocarbon receptor (AHR). In addition to defects in mammary differentiation, AHR stimulation induces toxicity in many other tissues. We performed a structure activity relationship (SAR) study of 1,2,4-bis-aryloxadiazole to determine which moieties of the molecule are critical for AHR activation. We validated our results with a functional biological assay, using desmosome formation during mammary morphogenesis to indicate AHR activity. These findings will aid the design of oxadiazole derivative therapeutics with reduced off-target toxicity profiles.     

A benzothiadiazine derivative and methylprednisolone are novel and selective activators of transient receptor potential canonical 5 (TRPC5) channels

The transient receptor potential canonical channel 5 (TRPC5) is a Ca²⁺-permeable ion channel, which is predominantly expressed in the brain. TRPC5-deficient mice exhibit a reduced innate fear response and impaired motor control. In addition, outgrowth of hippocampal and cerebellar neurons is retarded by TRPC5. However, pharmacological evidence of TRPC5 function on cellular or organismic levels is sparse. Thus, there is still a need for identifying novel and efficient TRPC5 channel modulators.We, therefore, screened compound libraries and identified the glucocorticoid methylprednisolone and N-[3-(adamantan-2-yloxy)propyl]-3-(6-methyl-1,1-dioxo-2H-1λ⁶,2,4-benzothiadiazin-3-yl)propanamide (BTD) as novel TRPC5 activators. Comparisons with closely related chemical structures from the same libraries indicate important substructures for compound efficacy. Methylprednisolone activates TRPC5 heterologously expressed in HEK293 cells with an EC₅₀ of 12 μM, while BTD-induced half-maximal activation is achieved with 5-fold lower concentrations, both in Ca²⁺ assays (EC₅₀ = 1.4 μM) and in electrophysiological whole cell patch clamp recordings (EC₅₀ = 1.3 μM). The activation resulting from both compounds is long lasting, reversible and sensitive to clemizole, a recently established TRPC5 inhibitor. No influence of BTD on homotetrameric members of the remaining TRPC family was observed. On the main sensory TRP channels (TRPA1, TRPV1, TRPM3, TRPM8) BTD exerts only minor activity. Furthermore, BTD can activate heteromeric channel complexes consisting of TRPC5 and its closest relatives TRPC1 or TRPC4, suggesting a high selectivity of BTD for channel complexes bearing at least one TRPC5 subunit.     

Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5–P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds.     

Novel small molecule activators of the Trk family of receptor tyrosine kinases

The Tropomyosin-related kinase (Trk) receptors are a subset of the receptor tyrosine kinase family with an important functionality in the regulation of neurotrophic signaling in the peripheral and central nervous system. As the receptors are able to mediate neuronal survival by associating with their respective neurotrophin ligands, many studies have focused on the therapeutic potential of generating small-molecule mimetic compounds that elicit agonistic effects similar to those of the natural protein ligands. To this end, various structure-based studies have led to the generation of bivalent peptide-based agonists and antibodies that selectively initiate Trk receptor signaling; however, these compounds do not possess the ideal characteristics of a potential drug. Additionally, the reliance of structure-based data to generate the compound libraries, limits the potential identification of novel chemical structures with desirable activity. Therefore, subsequent investigations utilized a cell-based apoptotic screen to facilitate the analysis of large, diverse chemical libraries of small molecules and quickly identify compounds with Trk-dependent anti-apoptotic activity. Herein, we describe the Trk agonists that have been identified by this screening methodology and summarize their in vitro and in vivo neurotrophic activity as well as their efficacy in various neurological disease models, implicating their future utility as therapeutic compounds. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Identification of potential physiological activators of protein phosphatase 5

The protein serine/threonine phosphatase designated PP5 has little basal activity, and physiological activators of the enzyme have never been identified. Purified PP5 can, however, be activated by partial proteolysis or by the binding of supraphysiological concentrations of polyunsaturated long-chain fatty acids to its tetratricopeptide repeat (TPR) domain. To test whether activation of PP5 by polyunsaturated but not saturated fatty acids was an artifact of the lower solubility of saturated fatty acids, the effects of fatty acyl-CoA esters were examined. Saturated and unsaturated long-chain fatty acids are both freely water-soluble when esterified to CoA. Long-chain fatty acyl-CoA esters activated PP5 at physiological concentrations, with the saturated compounds being more effective. We investigated the effects of chain length and of the CoA moiety on PP5 activation. Chains of 16 carbons or more were required for optimal activation, with no activation observed below 10 carbons. On the basis of competition studies using acetyl-CoA, the function of the CoA moiety appeared to be to increase solubility of the fatty acyl moiety rather than to interact with a specific binding site. These data suggested that long-chain fatty acid-CoA esters might be physiological activators of PP5 and point to a potential link between fatty acid metabolism and signal transduction via this enzyme. Because heat shock protein 90 is also known to bind to the TPR domain of PP5 via its C-terminal domain (C90), we investigated its effect on PP5 activity. C90 activated the enzyme approximately 10-fold. Thus, we have identified two potential physiological activators of PP5.     

Participation of farnesol in the biosynthesis of ipomeamarone

Farnesol-2-¹⁴C was readily incorporated into ipomeamarone, oneof the furanoterpenoids produced in sweet potato infected withthe black rot fungus, Ceratocystis fumbriata. This was demonstratedby isolating labeled ipomeamarone and analyzing its radioauto-gramby silica gel thin layer chromatography of the extracts solublein chloroformmethanol (1: 1 , v/v), after farnesol-2-¹⁴C feedingto the tissue. Further proof for farnesol-2-¹⁴C incorporationinto ipomeamarone comes from the fact that the specific radioactivityof ipomeamarone semicarbazone was constant throughout the crystallizations.Fractionation of the label of farnesol-2-¹⁴C showed that radioactivitywas little distributed in the methanol-water fraction and wasmainly incorporated into ipomeamarone. Accordingly, it is notlikely that farnesol is incorporated into ipomeamarone afterits degradation to a small molecule(s) such as acetate. An additionalexperiment indicatedthat the incorporation of farnesol-2-¹⁴Cinto ipomeamarone markedly decreased under strict anaerobicconditions. This shows that some oxidative reactions are involvedin ipomeamarone biosynthesis from farnesol. ¹ This paper constitutes Part 91 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received February 3, 1971; )     

New and selective ryanodine receptor activators for insect control

Diamide insecticides have emerged as one of the most promising new classes of insecticide chemistry owing to their excellent insecticidal efficacy and high margins of mammalian safety. Chlorantraniliprole and flubendiamide, the first two insecticides from this class, demonstrate exceptional activity across a broad range of pests in the order Lepidoptera. This chemistry has been confirmed to control insects via activation of ryanodine receptors which leads to uncontrolled calcium release in muscle. The high levels of mammalian safety are attributed to a strong selectivity for insect over mammalian receptors.     

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.