Structure–activity relationship of GPR15L peptide analogues and investigation of their interaction with the GPR15 receptor

The 57-mer full-length GPR15L(25-81) peptide has been identified as the principal endogenous agonist of the G protein-coupled receptor GPR15. Its main activity resides in the C-terminal 11-mer GPR15L(71-81), which has full efficacy but ~40-fold lower potency than the full-length peptide. Here, we systematically investigated the structure–activity relationship of GPR15L(71-81) by truncations/extensions, alanine-scanning, and N- and C-terminal capping. The synthesized peptide analogues were tested at GPR15 stably expressed in HEK293A cells using a homogenous time-resolved Förster resonance energy transfer-based G_i cAMP functional assay. We show that the C-terminal α carboxyl group and the residues Leu⁷⁸, Pro⁷⁵, Val⁷⁴, and Trp⁷² are critical for receptor interaction and contribute significantly to the peptide potency. Furthermore, we tested the ability of GPR15L(71-81), C-terminally amidated GPR15L(71-81), and GPR15L(25-81) to activate the three GPR15 receptor mutants in a bioluminescence resonance energy transfer-based G protein activation assay. The results demonstrate that the Lys192 and Glu272 residues in GPR15 are important for the potency of the GPR15L peptide. Overall, our study identifies critical residues in the peptide and receptor sequences for future drug design.     

Opioids and the immune system: what is their mechanism of action?

There is a significant amount of literature showing that morphine and other opioids modulate immune responses. The findings support many mechanisms by which this may occur. In vitro experiments provide evidence for direct actions of opioids on immune cells using a variety of functional end points. When these drugs are given in vivo, a plethora of immune parameters are also altered. The paper in this issue of the journal by Zhang et al. provides new information on morphine alteration of immune cell subsets in the spleen and thymus of mice and the potential role of glucocorticoids in these observed phenomena. This Commentary reviews the in vitro activities of morphine on leucocytes, as well as other documented mechanisms by which morphine can alter immune function in vivo.     

Structure–activity relationships of 44 halogenated compounds for iodotyrosine deiodinase-inhibitory activity

The aim of this study was to investigate the possible influence of halogenated compounds on thyroid hormone metabolism via inhibition of iodotyrosine deiodinase (IYD) activity. The structure-activity relationships of 44 halogenated compounds for IYD-inhibitory activity were examined in vitro using microsomes of HEK-293 T cells expressing recombinant human IYD. The compounds examined were 17 polychlorinated biphenyls (PCBs), 15 polybrominated diphenyl ethers (PBDEs), two agrichemicals, five antiparasitics, two pharmaceuticals and three food colorants. Among them, 25 halogenated phenolic compounds inhibited IYD activity at the concentration of 1 × 10^?4 M or 6 × 10^?4 M. Rose bengal was the most potent inhibitor, followed by erythrosine B, phloxine B, benzbromarone, 4′-hydroxy-2,2′,4-tribromodiphenyl ether, 4-hydroxy-2,3′,3,4′-tetrabromodiphenyl ether, 4-hydroxy-2′,3,4′,5,6′-pentachlorobiphenyl, 4′-hydroxy-2,2′,4,5′-tetrabromodiphenyl ether, triclosan, and 4-hydroxy-2,2′,3,4′,5-pentabromodiphenyl ether. However, among PCBs and PBDEs without a hydroxyl group, including their methoxylated metabolites, none inhibited IYD activity. These results suggest that halogenated compounds may disturb thyroid hormone homeostasis via inhibition of IYD, and that the structural requirements for IYD-inhibitory activity include halogen atom and hydroxyl group substitution on a phenyl ring.     

Structure–effect relationships of amiodarone analogues on the inhibition of thyroxine deiodination

OBJECTIVES: Amiodarone (AMI) has proven to be a potent anti-arrhythmic compound. Due to the structural similarity between AMI and thyroid hormone, it is possible that the drug could inhibit the activity of the 5'-thyroxine-deiodinase. METHODS: AMI analogues resulting from (1) dealkylation, (2) deiodination and (3) deamination were synthesised and used as inhibitors in an in vitro biotransformation reaction of thyroxine (T4) to 3,3',5'-triiodothyronine (T3). Using high-performance liquid chromatography and ultraviolet detection for quantifying T3, it was found that the 5'-T4 deiodinase type I was involved in the reaction. On separate occasions, AMI or an AMI analogue was added to the reaction as an inhibitor. RESULTS: All studied AMI analogues inhibited 5'-T4 deiodination competitively (Ki value range 25-360 microM). In the concentration range of 1-1000 microM, AMI and its N-desethylated, deiodinated analogues inhibited 5'-T4 deiodination very weakly. AMI analogues with a hydroxyl group at the 4-position were strong inhibitors. Moreover, diiodo-AMI analogues inhibited 5'-T4 deiodination more strongly than their corresponding monoiodo- or deiodinated derivatives. CONCLUSION: It is likely that the degraded products of AMI could be responsible for thyroid dysfunction toxicosis in AMI therapy.     

Structure–activity relationships and evaluation of esterified diterpenoid alkaloid derivatives as antiproliferative agents

Journal of Natural Medicines - Diterpenoid alkaloids with remarkable chemical properties and biological activities are frequently found in plants of the genera Aconitum, Delphinium, and Garrya....     

Inhibitory effect of curcumin analogs on tissue factor procoagulant activity and their preliminary structure–activity relationships

With the aim to explore the multifunctional behaviors of curcumin analogs and to discover new small molecular tissue factor inhibitors, twelve mono carbonyl curcumin analogs of three classes were synthesized and their effect on tissue factor procoagulant activity was evaluated in the human monoblastic leukemia THP-1 cells stimulated by LPS. The most potent compounds 2a exhibited the dramatically enhanced activity with the IC₅₀ values of 0.053 nM. Their preliminary structure–activity relationship was also discussed.     

Structure–property relationships in series of natural and synthetic inhibitors of catalytic activity of 15-lipoxygenase

Structural signatures characteristic of high-, medium-, and low-efficiency inhibitors of the catalytic activity of 15-lipoxygenase (15-LOG) have been revealed using the SARD-21 (Structure Activity Relationship & Design) computer system. The degree of their influence on the efficiency of the inhibiting activity was estimated. Based on these data, two models are constructed for predicting the interval of LOG-inhibiting activity of various sulfur-, nitrogen-, and oxygen-containing heterocyclic compounds with an 80% prediction accuracy level for the two recognition methods. The revealed structural features can be used to construct highly selective inhibitors of 15-LOG catalytic activity.     

Using structure biodegradability relationships for environmentally benign design of organosilicons – An experimental comparison of organosilicons and their carbon analogues

Organosilicon substances are ubiquitous in the environment due to their stability and numerous applications in consumer products. Therefore, it is desirable to reduce their environmental persistency. Our study aimed to better understand the impact of silicon atoms in organic compounds on their environmental biodegradability as a contribution to sustainable chemistry. Accordingly, we investigated the biodegradability of organosilicon compounds and their carbon analogues. OECD 301D test was used to assess ready biodegradability. In addition, GC-MS analyses were performed to study the fate of the compounds in the test. Three out of five carbon compounds and no organosilicon compound were found readily biodegradable. In all but one case, higher biodegradation degrees could be observed for the carbon compounds. Hydrolysis was identified as a mandatory step prior to the biodegradation of organosilicon substances. The silicon-free product of hydrolysis determined the rate of biodegradation. The silicon-containing reaction products of hydrolysis were not biodegradable. The high biodegradability of one organosilicon compound can be attributed to faster hydrolysis due to an easily hydrolysable Si–N bond and a high biodegradation rate of the resulting silicon-free hydrolysis product. Insertion of such heteroatoms or functional groups into polysiloxane chains may be a promising approach to benign organosilicon compounds.     

Structure–activity relationships for α-calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1–7); an α-helix (residues 8–18), a region incorporating a β-bend (residues 19–26) and the C-terminal portion (residues 27–37), that is characterized by bends between residues 28–30 and 33–34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix.

LINKED ARTICLES

This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7     

d-cycloserine lowers kynurenic acid formation—New mechanism of action

d-Cycloserine, known as a partial agonist at the glycine modulatory site of the glutamatergic N-methyl-d-aspartate (NMDA) receptor, exerts anticonvulsive activities and improves cognitive function. The present study evaluates the action of d-cycloserine with respect to the biosynthetic machinery of kynurenic acid (KYNA) synthesis e.g. the activity of enzymes synthesizing KYNA, kynurenine aminotransferases I, kynurenine aminotransferase II and kynurenine aminotransferase III (KAT I, KAT II and KAT III) in the rat liver and brain, and human frontal cortex in the presence of the anti-mycobacterial drug d-cycloserine, in an in vitro study. We found that d-cycloserine blocked dose-dependent and significantly KAT I, II and III activities in rat liver and brain homogenates. Furthermore, the inhibitory effect of KYNA formation was observed in the frontal cortex homogenate of human post mortem tissue, as well. d-Cycloserine, at 63.7 µM concentration blocked significantly KAT II, I and III (53.2, 66.1 and 71.3% of control, P<0.001) activities in the human frontal cortex homogenate. Obtained data indicate that d-cycloserine exerts notable biochemical properties to block KYNA synthesis. Lowering of KYNA content due to d-cycloserine inhibition of KATs activities can free up more glycine sites for the actions of d-cycloserine. On the other site, it needs to be clarified, if the postulated mechanism for d-cycloserine to act as a partial agonist at the glycine site of the NMDA receptor could be mainly due to KAT's inhibition. We propose that this mechanism(s) might play a role in the improvement of memory, cognition and/or delusion in Alzheimer's, HIV-1 infected patients and schizophrenia patients.     

Structure—activity relationship of alkyl 9—nitrocamptothecin esters

AIM: To study the structure-activity relationship of alkyl 9-nitrocamptothecin esters. METHODS: Two alkyl 9-nitrocamptothecin (9NC) esters 5g and 5h were prepared by esterification reactions of 9NC with valeric anhydride and heptanoic anhydride, respectively. Eight 9NC esters 5a-5h were tested for cytotoxicity against human leukemia cell lines HL-60 and U-937. Flow cytometry analysis was used to identify the cell cycle phase targeted by the esters and quantify the extent of ester-induced cell death (apoptosis). RESULTS: Esters 5b and 5c demonstrated great abilities to inhibit growth of the leukemia cells followed by induction of apoptosis; esters 5a, 5e, and 5g induced slight perturbations in the cell cycle at high concentrations; and esters 5d, 5f, and 5h were completely inactive against the cell lines tested. Thus these esters showed the cell anti-proliferative activity in an order of 5b approximately 5c>5a approximately 5e approximately 5g>5d approximately 5f approximately 5h. Esters 5b, 5c, and 5e were tested in vivo against various human carcinomas in nude mice grown as xenografts. Only 5b and 5c showed a significant antitumor activity. Particularly, ester 5b demonstrated an antitumor activity against a broad spectrum of human carcinomas including breast, lung, colon, pancreas, stomach, ovarian, and melanoma, etc. CONCLUSION: These esters act like prodrugs of their parental 9-nitrocamptothecin. High drug doses need to be administered to animals in order to inhibit growth, and induce regression, of human tumor xenografts in nude mice. These compounds may be developed into potent anticancer drugs due to their low toxicity.     

Structure–activity relationships of bumetanide derivatives: correlation between diuretic activity in dogs and inhibition of the human NKCC2A transporter

Background and Purpose

The N-K-Cl cotransporters (NKCCs) mediate the coupled, electroneutral movement of Na⁺, K⁺ and Cl⁻ ions across cell membranes. There are two isoforms of this cation co-transporter, NKCC1 and NKCC2. NKCC2 is expressed primarily in the kidney and is the target of diuretics such as bumetanide. Bumetanide was discovered by screening ∼5000 3-amino-5-sulfamoylbenzoic acid derivatives, long before NKCC2 was identified in the kidney. Therefore, structure–activity studies on effects of bumetanide derivatives on NKCC2 are not available.

Experimental Approach

In this study, the effect of a series of diuretically active bumetanide derivatives was investigated on human NKCC2 variant A (hNKCC2A) expressed in Xenopus laevis oocytes.

Key Results

Bumetanide blocked hNKCC2A transport with an IC₅₀ of 4 μM. There was good correlation between the diuretic potency of bumetanide and its derivatives in dogs and their inhibition of hNKCC2A (r² = 0.817; P < 0.01). Replacement of the carboxylic group of bumetanide by a non-ionic residue, for example, an anilinomethyl group, decreased inhibition of hNKCC2A, indicating that an acidic group was required for transporter inhibition. Exchange of the phenoxy group of bumetanide for a 4-chloroanilino group or the sulfamoyl group by a methylsulfonyl group resulted in compounds with higher potency to inhibit hNKCC2A than bumetanide.

Conclusions and Implications

The X. laevis oocyte expression system used in these experiments allowed analysis of the structural requirements that determine relative potency of loop diuretics on human NKCC2 splice variants, and may lead to the discovery of novel high-ceiling diuretics.     

Phytochemicals from Dodonaea viscosa and their antioxidant and anticholinesterase activities with structure–activity relationships

Context Dodonaea viscosa (L.) Jacq (Sapindaceae) has been used in traditional medicine as antimalarial, antidiabetic and antibacterial agent, but further investigations are needed.

Objective This study determines the antioxidant and anticholinesterase activities of six compounds (1–6) and two crystals (1A and 3A) isolated from D. viscosa, and discusses their structure–activity relationships.

Materials and methods Antioxidant activity was evaluated using six complementary tests, i.e., β-carotene-linoleic acid; DPPH^?, ABTS^?+, superoxide scavenging, CUPRAC and metal chelating assays. Anticholinesterase activity was performed using the Elman method.

Results Clerodane diterpenoids (1 and 2) and phenolics (3–6) – together with three crystals (1A, 3A and 7A) – were isolated from the aerial parts of D. viscosa. Compound 3A exhibited good antioxidant activity in DPPH (IC₅₀: 27.44?±?1.06?μM), superoxide (28.18?±?1.35% inhibition at 100?μM) and CUPRAC (A_0.5: 35.89?±?0.09?μM) assays. Compound 5 (IC₅₀: 11.02?±?0.02?μM) indicated best activity in ABTS assay, and 6 (IC₅₀: 14.30?±?0.18?μM) in β-carotene-linoleic acid assay. Compounds 1 and 3 were also obtained in the crystal (1A and 3A) form. Both crystals showed antioxidant activity. Furthermore, crystal 3A was more active than 3 in all activity tests. Phenol 6 possessed moderate anticholinesterase activity against acetylcholinesterase and butyrylcholinesterase enzymes (IC₅₀ values: 158.14?±?1.65 and 111.60?±?1.28?μM, respectively).

Discussion and conclusion This is the first report on antioxidant and anticholinesterase activities of compounds 1, 2, 5, 6, 1A and 3A, and characterisation of 7A using XRD. Furthermore, the structure–activity relationships are also discussed in detail for the first time.     

In vitro effects of brominated flame retardants and metabolites on CYP17 catalytic activity: a novel mechanism of action?

Fire incidents have decreased significantly over the last 20 years due, in part, to regulations requiring addition of flame retardants (FRs) to consumer products. Five major classes of brominated flame retardants (BFRs) are hexabromocyclododecane isomers (HBCDs), tetrabromobisphenol-A (TBBPA) and three commercial mixtures of penta-, octa- and deca-polybrominated diphenyl ether (PBDE) congeners, which are used extensively as commercial FR additives. Furthermore, concentrations of PBDEs have been rapidly increasing during the 1999s in human breast milk and a number of endocrine effects have been reported. We used the H295R human adrenocortical carcinoma cell line to assess possible effects of some of these BFRs (PBDEs and several of their hydroxylated (OH) and methoxylated (CH(3)O) metabolites or analogues), TBBPA and brominated phenols (BPs) on the combined 17alpha-hydroxylase and 17,20-lyase activities of CYP17. CYP17 enzyme catalyzes an important step in sex steroidogenesis and is responsible for the biosynthesis of dehydroepiandrosterone (DHEA) and androstenedione in the adrenals. In order to study possible interactions with BFRs, a novel enzymatic method was developed. The precursor substrate of CYP17, pregnenolone, was added to control and exposed H295R cells, and enzymatic production of DHEA was measured using a radioimmunoassay. In order to avoid pregnenolone metabolism via different pathways, specific chemical inhibitor compounds were used. None of the parent/precursor BFRs had a significant effect (P < 0.05) on CYP17 activity except for BDE-183, which showed significant inhibition of CYP17 activity at the highest concentration tested (10 muM), with no signs of cytotoxicity as measured by mitochondrial toxicity tests (MTT). A strong inhibition of CYP17 activity was found for 6-OH-2,2',4,4'-tetrabromoDE (6-OH-BDE47) with a concentration-dependent decrease of almost 90% at 10 muM, but with a concurrent decrease in cell viability at the higher concentrations. Replacement of the 6-OH group by a 6-CH(3)O group eliminated this cytotoxic effect, but CYP17 activity measured as DHEA production was still significantly inhibited. Other OH- or CH(3)O-PBDE analogues were used to elucidate possible structural properties behind this CYP17 inhibition and associated cytotoxicity, but no distinct structure activity relationship could be determined. These in vitro results indicate that OH and CH(3)O-PBDEs have potential to interfere with CYP17 activity for which the in vivo relevance still has to be adequately determined.     

Tricyclic antidepressants and fibromyalgia: what is the mechanism of action?

Fibromyalgia is a chronic pain disorder of which other clinical features, such as persistent fatigue and disordered sleep, may be a secondary consequence. The initial pharmacological approach to treating the disorder is the management of the pain. Tricyclic antidepressants are the most effective drugs in use so far, especially when administered in combination with other therapies (e.g., selective serotonin re-uptake inhibitors), which suggests modulation of the neurotransmitters serotonin and noradrenaline. The effectiveness of amitriptyline and related tricyclic antidepressants, however, is consistent with the involvement of mechanisms, such as potassium channel modulation and NMDA receptor antagonism, in addition to or in place of the modulation of monoamine neurotransmitters. Investigation of the importance of each of the pharmacological properties of amitriptyline and related molecules in the management of fibromyalgia could provide clues for the rational design of new drugs.     

Tricyclic antidepressants and fibromyalgia: what is the mechanism of action?

Fibromyalgia is a chronic pain disorder of which other clinical features, such as persistent fatigue and disordered sleep, may be a secondary consequence. The initial pharmacological approach to treating the disorder is the management of the pain. Tricyclic antidepressants are the most effective drugs in use so far, especially when administered in combination with other therapies (e.g., selective serotonin re-uptake inhibitors), which suggests modulation of the neurotransmitters serotonin and noradrenaline. The effectiveness of amitriptyline and related tricyclic antidepressants, however, is consistent with the involvement of mechanisms, such as potassium channel modulation and NMDA receptor antagonism, in addition to or in place of the modulation of monoamine neurotransmitters. Investigation of the importance of each of the pharmacological properties of amitriptyline and related molecules in the management of fibromyalgia could provide clues for the rational design of new drugs.     

Kyotorphin analogues containing unnatural amino acids: synthesis,analgesic activity and computer modeling of their interactions with μ-receptor

Kyotorphin (KTP; Tyr-Arg) an endogenous neuropeptide is potently analgesic when delivered directly to CNS. An effort to enhance the potency, enzymatic stability and improving bioavailability of KTP is the modification with unnatural amino acids. The aims of presented study were: (1) To synthesize new analogues of kyotorphin containing unnatural amino acids: norcanavaine (NCav) and norcanaline (NCan), structural analogues of arginine and ornithine, respectively; (2) To understand the influence of the arginine mimetics on the pharmacological properties of KTP analogues, through examination their effects on the paw pressure nociceptive threshold; (3) To find relationship between the structure and obtained biological effects of the all synthesized kyotorhin analogues, by molecular docking with μ-opioid receptor. As a result of our work four new kyotorphin analogues containing NCan and NCav were obtained. A correlation between the data from the in vivo test and docking results was found. This allows a better elucidation of the ligand-receptor interactions, the prediction of biological activity of the newly synthesized compounds, and to generate compounds with increased biological effects.