Antibacterial Activity of combinatorial treatments composed of transition-metal/antibiotics against Mycobacterium tuberculosis.

Notwithstanding evidence that tuberculosis (TB) is declining, one of the greatest concerns to public health is the emergence and spread of multi-drug resistant strains of Mycobacterium tuberculosis (MDR-TB). MDR-TB are defined as strains which are resistant to at least isoniazid (INH) and rifampicin, the two most potent TB drugs, and their increasing incidence is a serious concern. Recently, notable efforts have been spent on research to pursue novel treatments against MDR-TB, especially on synergistic drug combinations as they have the potential to improve TB treatment. Our research group has previously reported promising synergistic antimicrobial effects between transition-metal compounds and antibiotics in Gram-negative and Gram-positive bacteria. In this work, we evaluated antimycobacterial activity of transition-metals/antibiotics combinatorial treatments against first-line drug resistant strains of Mycobacterium tuberculosis. Our data showed that INH/AgNO3 combinatorial treatment had an additive effect (bactericidal activity) in an isoniazid-resistant clinical strain of Mycobacterium tuberculosis. Moreover, in vitro evaluation of cytotoxicity induced by both, the individual tratments of AgNO3 and INH and the combinatorial treatment of INH/AgNO3 in murine RAW 264.7 macrophages and human A549 lung cells; showed no toxic effects. Together, this data suggests that the INH/AgNO3 combinatorial treatment could be used in the development of new strategies to treat resistant strains of Mycobacterium tuberculosis.

Regulation of eIF4F Translation Initiation Complex by the Peptidyl Prolyl Isomerase FKBP7 in Taxane-resistant Prostate Cancer.

PURPOSE: Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets for overcoming chemoresistance.Experimental Design: We established 4 chemoresistant prostate cancer cell lines and used image-based high-content siRNA functional screening, based on gene-expression signature, to explore mechanisms of chemoresistance and identify new potential targets with potential roles in taxane resistance. The functional role of a new target was assessed by in vitro and in vivo silencing, and mass spectrometry analysis was used to identify its downstream effectors. RESULTS: We identified FKBP7, a prolyl-peptidyl isomerase overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. This is the first study to characterize the function of human FKBP7 and explore its role in cancer. We discovered that FKBP7 was upregulated in human prostate cancers and its expression correlated with the recurrence observed in patients receiving docetaxel. FKBP7 silencing showed that FKBP7 is required to maintain the growth of chemoresistant cell lines and chemoresistant tumors in mice. Mass spectrometry analysis revealed that FKBP7 interacts with eIF4G, a component of the eIF4F translation initiation complex, to mediate the survival of chemoresistant cells. Using small-molecule inhibitors of eIF4A, the RNA helicase component of eIF4F, we were able to kill docetaxel- and cabazitaxel-resistant cells. CONCLUSIONS: Targeting FKBP7 or the eIF4G-containing eIF4F translation initiation complex could be novel therapeutic strategies to eradicate taxane-resistant prostate cancer cells.

Cobalamin malabsorption due to nondegradation of R proteins in the human intestine. Inhibited cobalamin absorption in exocrine pancreatic dysfunction.

In vivo studies demonstrate that the pancreatic enzymes and the ionic environment in the upper gastrointestinal tract are essential determining factors for transport and absorption of cobalamin in man. Jejunal fluid was aspirated from healthy human volunteers after administration of cyano[57Co]cobalamin preparations. Immunochemical analysis of the aspirates demonstrated that all isotopic vitamin was transferred to a protein that is identical to the gastric intrinsic factor in terms of molecular mass (57,500), ionic nature (mean pI, 5.09), and reactivity with anti-intrinsic factor sera. However, in the aspirates from patients with exocrine pancreatic dysfunction the vitamin was found to be coupled > 60% to a protein identical to R proteins in terms of molecular mass (125,000), ionic nature (mean pI, 3.51), and reactivity with anti-R protein and anti-intrinsic factor sera. The preferential transfer of cobalamin to R proteins in the patients and to intrinsic factor in healthy subjects was associated, respectively, with low and normal levels of pancreatic enzymes in the intestine and these in turn were paralleled respectively by impaired and normal ileal absorption of cobalamin. These findings confirm the suggestion that the formation of unabsorbable cobalamin complexes may be the reason of impaired vitamin absorption in exocrine pancreatic insufficiency. Observations made with other selected patients demonstrate: (a) that decreased enzyme activity and nondegradation of R proteins may also be due to nonactivation of pancreatic zymogens in an acidic pH of the intestinal juice the vitamin transported to the jejunum couples to intrinsic factor when pancreatic function is normal, and to intrinsic factor and R protein in exocrine pancreatic insufficiency. The observations made with these selected patients may explain why not all patients with exocrine pancreatic insufficiency develop imparied cobalamin absorption, and also why the malabsorption is corrected by the administration of bicarbonate in certain patients.

In vivo evidence that intrinsic factor-cobalamin complex traverses the human intestine intact.

Ingested cyano[57Co]cobalamin was recovered as coupled to intrinsic factor from the jejunum of healthy humans. This vitamin-protein complex and the analogous complex from patients having exocrine pancreatic insufficiency were indistinguishable from each other in terms of molecular radius (3.30 nm), ionic nature (eight well-defined isoproteins isoelectric at pH 4.71, 4.84, 4.90, 5.13, 5.23, 5.31, 5.40 and 5.69), and antigenic structure. These findings indicate that the pancreatic proteases do not alter the intrinsic factor cobalamin complex in vivo. Purified R type protein-cyano [57Co]cobalamin complex recovered from patients with exocrine pancreatic insufficiency was similar to the analogous gastric protein in terms of molecular radius (alpha = 4.78 nm) and types of isoproteins (seven well-defined isoproteins isoelectric at pH 2.70, 3.03, 3.38, 3.74, 3.87, 4.05 and 4.20). However, this R protein complex from patients and the intrinsic factor complex from both control subjects and from patients was comprised of more of the acidic type of isoproteins, thereby supporting the notion that carbohydrate-rich isoproteins are more resistant to digestion in the intestine.

A derivative of R-type cyanocobalamin binding proteins in the human intestine. A candidate antibacterial molecule.

Human jejunal fluid contains a protein which has a molecular radius of 3.37 nm, an Mr or 60600 and a mean pI of 3.35 and binds cobalamin with a K of 0.1 . 10(9) l/mol. This protein also couples cobalamin analogues lacking the nucleotide moiety, cross-reacts with R-type protein and is resistant to proteolysis in the intestine. These findings refute the hypothesis that cobalamin-analogue binders are present and effectively inhibit the bacterial uptake of analogues in the intestine.

Effect of glycosidases and proteinases on cobalamin binding and physicochemical properties of purified saturated haptocorrin and intrinsic factor.

The effect of exoglycosidase, N-glycanase, trypsin and chymotrypsin was studied on the binding capacity and physicochemical properties of intrinsic factor and of haptocorrin using Superose 6 gel filtration. Intrinsic factor was purified as recently described by us. Haptocorrin was purified 6000-fold from human saliva using thermolabile affinity chromatography and high-performance cationic exchange chromatography with a specific activity of 20.6 nmol of cobalamin (Cbl) per mg protein and a yield of 44.7%. Exoglycosidases provoked a decrease of 54.3 and 78.2% of the Cbl binding capacity of haptocorrin and intrinsic factor, respectively. The sequential incubation of haptocorrin and intrinsic factor wit exoglycosidases and proteinases provoked a decrease of, respectively, 100 and 92.7% of their Cbl binding capacity, whereas the incubation with proteinase decreased the Cbl binding capacity of, respectively, 67.9 and 7.9%. The result of the incubation of [3H]intrinsic factor or [3H]haptocorrin with chymotrypsin and trypsin gave, respectively, no change in the elution position and a shift corresponding to a decrease of 50% of the estimated molecular mass. The estimated molecular mass of Cbl-intrinsic factor and of Cbl-haptocorrin decreased, respectively, to 57.1 kDa and to 88.1 kDa after incubation with exoglycosidases. It was concluded that (1) the carbohydrate core of intrinsic factor protects the whole protein whereas the carbohydrate core of haptocorrin protects only half part of the protein and (2) the carbohydrates are implicated in the formation of the cobalamin binding site of haptocorrin and intrinsic factor.

Purification of intrinsic factor receptor from pig ileum using as affinity medium human intrinsic factor covalently bound to Sepharose.

Intrinsic factor receptor was purified from hog ileum using human intrinsic factor covalently bound to Sepharose. A yield of 49.6% and a specific activity of about 2500 pmol/mg protein were achieved. The purified receptor was very unstable: 24 h of storage or addition of sodium phosphate precipitated it. The association constant of the receptor for the cyano[57Co]cobalamin-intrinsic factor complex was estimated to be 2.1 nM-1. In native polyacrylamide gel electrophoresis it resolved in two 256 and 320 kDa bands; beta-mercaptoethanol treatment cleared it into four bands corresponding to molecular masses of 107, 81.8, 63.5 and 53.2 kDa. An additional 39.3 kDa band was considered to be an artefact due to the presence of Triton X-114. Isoelectric focusing polyacrylamide gel electrophoresis resolved the receptor into two isoproteins isoelectric at pH 4.7 and 5.1. A similar result was obtained in column electrofocusing with the 125I-iodinated receptor. The 125I-labelled receptor did not crossreact with rabbit anti-human intrinsic factor antiserum. The electrophoretic properties of the receptor purified with intrinsic factor covalently bound to Sepharose were compared to those of the receptor purified by the use of the classical cobalamin-affinity medium. It was concluded that a disassembled receptor was produced using the classical method.

Binding assay and physicochemical characteristics of solubilized intrinsic factor receptor in ileal mucosal homogenates using phenyl-Sepharose to separate the saturated receptor from free intrinsic factor.

A radioisotopic assay was set to determine the physicochemical properties of the solubilized intrinsic factor receptor in pig mucosal extracts. In this assay, phenyl-Sepharose was used to separate the receptor-intrinsic factor-labelled cobalamin complex from the free saturated intrinsic factor. The association constant (at pH 7.4) of the receptor-intrinsic factor complex was estimated at 3.4 +/- 0.3 nM-1. Adsorption of the apo-receptor to phenyl-Sepharose allowed its binding site to be made accessible to intrinsic factor with an association constant in order of 6 nM-1. The receptor binding activity obtained with five mucosal extracts was closely correlated with that obtained by gel filtration of the intrinsic factor-receptor complex (r = 0.99). The radioisotope assay was used to detect the unsaturated receptor (apo-receptor) in sucrose density ultracentrifugation and in superose 6 gel filtration. The sedimentation coefficient was 9.5 s. The apo-receptor was eluted in three peaks in gel filtration, corresponding to the formation of oligomers. The peak of the monomer was increased in presence of EDTA. Its molecular mass was estimated at 270 kDa and its Stokes radius at 5.9 nm. It was concluded that calcium is involved in the oligomerisation of the apo-receptor.

Transcobalamin II--cobalamin binding sites are present on rabbit germ cells.

Specific binding sites for rabbit transcobalamin II have been found on isolated adult rabbit germ cells. Scatchard analysis revealed a single class of binding sites for [57Co]cyanocobalamin-transcobalamin II with an association constant (Ka) of 1.3 x 10(10) M-1 and 700 sites per cell. Binding was reversible, saturable and calcium dependent. Electron microscope radioautography following incubation with iodinated transcobalamin II at 4 degrees C led to a detectable labeling mainly restricted to the plasma membrane.

A molecular dynamics study of an archaeal tetraether lipid membrane: comparison with a dipalmitoylphosphatidylcholine lipid bilayer.

Molecular dynamics simulations of an archaeal membrane made up of bipolar tetraether lipids and a dipalmitoylphosphatidylcholine (DPPC) lipid membrane were performed and compared for the first time. The simulated archaeal membrane consists of a pure monolayer of asymmetrical lipids, analogous to the main polar lipid [MPL; Swain, M., Brisson, J.-R., Sprott, G.D., Cooper, F.P., and Patel, G.B., (1997) Identification of beta-L-Gulose as the Sugar Moiety of the Main Polar Lipid of Thermoplasma acidophilum, Biochim. Biophys. Acta 1345, 56-64] found in T. acidophilum, an extremophile archaeal organism. This simulated membrane lipid contains two cyclopentane rings located on one of the two aliphatic chains of the lipid. The archaeal membrane is simulated at 62degreesC, slightly above the optimal growth temperature of T. acidophilum. We compared the organization of this tetraether lipid monolayer with a DPPC bilayer simulated at 50degreesC, both of them being modeled in a partially hydrated state. Our results assess the singularity of the tetraether lipid organization, in particular the influence of the spanning structure on the molecular ordering within the archaeal membrane.

Endocytosis of transcobalamin in male rabbit germ cells: electron microscope radioautography study.

The binding of 125I-iodinated transcobalamin to a suspension of isolated rabbit germ cells was studied by Scatchard plot. The number of binding sites was evaluated to about 1000 per cell, and its association constant (Kass) in order of 14.6 l/nmole. The distribution to structures related to endocytosis was determined by ultrastructural histomorphometric studies. Both coated and uncoated structures were present regardless of maturation stage. The number of coated vesicles was at its highest in the initial maturation steps, whereas the number of uncoated vesicles was highest in the final maturation steps. The endocytosis of 125I-iodinated transcobalamin by the suspension of germ cells was studied by electron microscope radioautography. The tracer was mostly detected over the plasma membrane, coated vesicles and multivesicular bodies of germ cells. The grains were observed mainly over spermatocytes and round spermatids; 31.6% and 32% of these cells, respectively, were labeled. In contrast the tracer was detected in only 8.11% of elongated spermatids. In conclusion, iodinated transcobalamin is internalized in rabbit germ cells by receptor-mediated endocytosis. This phenomenon was predominant in the early stages of germ cell maturation.

Electron microscope radioautographic evidence of in vivo androgen-binding protein internalization in the rat epididymis principal cells.

The androgen-binding protein (ABP) has been purified from rat testes with a yield of 14% using four steps of HPLC and was subsequently iodinated to a specific activity of 0.1 mCi/mg protein. Using a micromanipulator, [125I] iodo-ABP-dihydrotestosterone was injected intraluminally into the proximal caput of the rat epididymis. Epididymides were sampled from 3 to 120 min after the injection of the tracer and processed for transmission electron microscopy autoradiography. Our results showed the accumulation of detectable radioactive sources in the apical cytoplasm of only one of the epithelial cell type lining the ductus, the principal cells. In the interval from 3 to 120 min, the iodinated ABP was mainly present in the supranuclear region and was especially concentrated over coated structures, endosomes, multivesicular bodies, and over the Golgi apparatus. The same pattern was obtained using [3H]dihydrotestosterone-ABP complex instead of iodinated ABP. In addition, there was a negative correlation between the log time and the distribution of the silver grains in the luminal border and in the compartment of the apical vesicles. On the contrary, there was a positive correlation between the log time and the distribution of the silver grains in the Golgi apparatus. These results provide, for the first time, direct histological evidence of the in vivo ABP internalization by the principal cells. Since horseradish peroxidase, a fluid-phase endocytosis marker, when injected under the same conditions was internalized in both apical and principal cells, since labeled radioactive ABP appeared to be bound to the membrane of the endocytic apparatus rather than to its content, and since this binding and uptake could be prevented in the presence of an excess of unlabeled ABP, it is concluded that the internalization of ABP could not be a nonspecific fluid-phase endocytosis but should be dependent on its interaction with the apical plasma membrane of the principal cell. It still remains to be determined if these mechanisms involve the binding of ABP to a specific membrane receptor.

Purification of human intrinsic factor using high-performance ion-exchange chromatography as the final step.

Human intrinsic factor was purified 1430-fold from gastric juice with a yield of 75% using two steps: labile ligand affinity chromatography and high-performance ion-exchange chromatography. Intrinsic factor precipitated in the presence of specific autoantibodies and 15% sodium sulfate, had an estimated Mr of 59 000 in 5% SDS electrophoresis and could bind to the specific ileal receptor in vitro. Its carbohydrate composition could be related to N-lactosaminic and O-glycosidic chains. High-performance ion-exchange chromatography was a mild, rapid and efficient procedure to separate completely intrinsic factor from haptocorrin (another glycoprotein of gastric juice which binds cobalamin) and from other contaminating proteins.

Purification of androgen-binding protein from rat testis using high-performance liquid chromatography and physicochemical properties of the iodinated molecule.

The androgen-binding protein (ABP) has been purified 87,500-fold from rat testis using 4 steps of HPLC, with a yield of 14%. The molecule was 99% pure with a specific activity estimated to 16,600 pmol/mg protein. The iodinated molecule was eluted in 2 peaks in Sephacryl S300 gel filtration with a molecular mass estimated to be 92,600 +/- 3300 and 50,300 +/- 4000 Da. The column isoelectrofocusing of 125I-ABP demonstrated 3 isoproteins isoelectric at pH 4.7, 4.9 and 5.3 and the sedimentation coefficient was estimated to be 4.7 S in sucrose gradient ultracentrifugation. The 125I-ABP had similar physiochemical properties to the non-labelled ABP of epididymis.

Receptor-mediated endocytosis of the intrinsic factor-cobalamin complex in HT 29, a human colon carcinoma cell line.

A HT 29 cell line derived from human colonic carcinoma was shown to express the intrinsic factor receptor, with about 5000 binding sites per cell and an association constant of 20 x 10(9) 1/mol at pH 7.4 and 4 degrees C. The number of binding sites increased dramatically between 7 and 10 days of culture time. Endocytosis of the intrinsic factor-cobalamin-receptor complex was inhibited by two ways: at 4 degrees C and at 37 degrees C by incubating the cells with vinblastine, monensin and chloroquine. The plasma membrane receptor was cross-linked to [57Co]cobalamin-intrinsic factor and solubilized with Triton X-100. The cross-linked complex had a relative molecular mass of 330 kDa in native PAGE.

In vitro and in vivo evidences that the malabsorption of cobalamin is related to its binding on haptocorrin (R binder) in chronic pancreatitis.

The intraluminal transport of cobalamin (Cbl) remains controversial in chronic pancreatitis. We have determined the ability of intestinal juice to degrade the digestive holohaptocorrin (R binder) and the binding of endogenous Cbl in basal intestinal juice from 22 chronic pancreatitis patients and 22 controls. The intestinal juice from patients and controls degraded 34.7 +/- 32.3% and 95.2 +/- 7.2% of holohaptocorrin, respectively. This percentage was correlated with the trypsin output but not with the Schilling test. The unsaturated Cbl-binding capacity was similar in both groups. Respectively, 62.5 +/- 26.6% and 19.6 +/- 11.7% of endogenous Cbl was bound to haptocorrin in intestinal juice from patients and controls. These percentages were correlated with the Schilling test and with the ability of intestinal juice to degrade haptocorrin. We concluded that 1) the sequestration of Cbl to haptocorrin is one of the factors responsible for the malabsorption of crystalline Cbl in patients with chronic pancreatitis and 2) enterohepatic circulation of Cbl can be interrupted in some cases of chronic pancreatitis.

Excretion of cobalamin and haptocorrin in the meconium of cystic fibrosis, premature, and control neonates.

Excretion of haptocorrin (R binder), cobalamin, and other corrinoids was studied in meconium from cystic fibrosis (n = 4), premature (n = 3), and control neonates (n = 13). Corrinoids content was 1.67 +/- 0.92 pmol/mg protein in meconium of cystic fibrosis (CF) neonates but only 0.33 +/- 0.37 and 0.48 +/- 0.47 pmol/mg protein, respectively, in that of prematures and controls. Considering its molecular mass (110,100 +/- 10,100) and its mean isoelectric point (3.67 +/- 0.20), haptocorrin remained undergraded in the meconium of CF neonates whereas it was partially degraded in the meconium of prematures and in most of the meconium from controls. Sequestration of cobalamin by undergraded haptocorrin can explain its increased excretion in CF meconium. Cobalamin-binding capacity of haptocorrin was 22.13 +/- 15.50 pmol/mg protein in CF meconium and about 400-fold lower in meconium of prematures and controls. This may correspond to a fetal intestinal hypersecretion in cases of CF.

Photoaffinity labelled rat androgen-binding protein and human sex hormone steroid-binding protein bind specifically to rat germ cells.

A specific receptor with high affinity for rat androgen-binding protein (rABP) was identified in isolated adult rat germ cells and in the corresponding plasma membrane-enriched preparations. Binding was reversible and time-dependent, with maximum relative binding after 40 min at 4 degrees C; it was pH-dependent, with maximum binding at pH 6-8. Unlabelled rABP and human sex steroid-binding protein (hSBP), but not lactotransferrin, serotransferrin, asialofetuin, fetuin or bovine serum albumin, competed with labelled rABP for binding sites on isolated germ cells. Scatchard analysis revealed a single class of binding site with apparent dissociation constant (Kd) values of 0.78 +/- 0.04 nM and 0.97 +/- 0.05 nM in intact germ cells and plasma membrane preparations respectively. A Kd of 1.72 +/- 0.12 nM for hSBP showed that the receptor binding site was effective for both androgen-carrier molecules. Labelled rABP incubated with solubilized germ cell membrane fractions at pH 7 formed a complex excluded from Superose 6B mini-gels; this complex was not formed at pH 3. The receptor complex was also abolished in the presence of a 100-fold excess of either unlabelled rABP or unlabelled hSBP, or in the presence of 20 mM EDTA. These results suggest that the plasma membrane of rat germ cells contains a receptor which selectively binds rABP and hSBP.

Evidence that androgen-binding protein endocytosis in vitro is receptor mediated in principal cells of the rat epididymis.

We have studied the binding of [125I-iodo]androgen-binding protein (ABP) and of [3H]delta 6-testosterone photoaffinity-labelled ABP to receptors in the plasma membrane of rat epididymal cells in three ways: ABP binding to a Triton X-100-solubilized membrane extract, ABP binding to isolated epithelial cells in suspension and autoradiography of segments of dissected epididymides after in-vitro intraluminal injection of labelled ABP. The binding of iodinated ABP to the receptor was similar to that of photoaffinity-labelled ABP in gel filtration. The ABP-receptor complex was eluted from Superose 6 gels as an aggregate, with a molecular mass of 2000 kDa. It was separated into two peaks by sucrose gradient ultracentrifugation, with respective sedimentation coefficients of 18.4 and 9.0 s. The activity of the receptor (ABP-binding capacity/mg protein) was tenfold higher in the caput than in the cauda. The binding of ABP to the receptor was pH dependent, being almost abolished at pH less than 4. The binding at 4 degrees C of photoaffinity-labelled ABP to epithelial cells corresponded to two types of binding sites. The numbers of high-affinity and low-affinity sites per cell were 1600 and 7700 respectively; the association constants of these sites were 67.9 and 2.8 litres/nM respectively. The binding was decreased by treatment of the cells with trypsin or incubation in the presence of EDTA. The binding in vitro of labelled ABP to the epididymis epithelium reached a maximum after about 20 min at 4 degrees C. In the autoradiographic study the tracer was found to be closely associated with coated pits, coated vesicles, endosomes and pale multivesicular bodies. Treatment of rats with cycloheximide significantly reduced the uptake of the tracer. Perfusion in vitro of epididymides with chloroquine produced a fourfold increase of the tracer in endosomes and multivesicular bodies.

Synthesis of phenalene and acenaphthene derivatives as new conformationally restricted ligands for melatonin receptors.

Conformationally restricted phenalene and acenaphthene derivatives 5 were synthesized from phenalen-1-one and acenaphthen-1-one derivatives using the Horner-Emmons reaction. The amines were prepared through the corresponding isocyanates by the Curtius reaction on the acids or by the reduction of the nitriles. Amido derivatives (R(3) = Me, Et, n-Pr, c-Pr) were prepared by acylation of the amines with the appropriate anhydrides or acid chlorides or by the reductive acylation of the nitriles. The affinities of the compounds for melatonin binding sites were evaluated in vitro in binding assays using chicken brain melatonin and the human mt(1) and MT(2) receptors expressed in HEK-293 cells. The functionality of the compounds was determined by the potency to lighten the skin of Xenopus laevis tadpoles. Highly potent compounds were obtained. The data highlighted the role of the methoxy group located in the ortho position to the ethylamido chain as compounds with picomolar affinities such as 14c were obtained (chicken brain, hmt(1), hMT(2) K(i) values = 0.02, 0.008, 0.069 nM, respectively). Compound 14c was equipotent to the corresponding dimethoxy derivative 15c (chicken brain, hmt(1), hMT(2) K(i) values = 0.07, 0.016, 0.1 nM, respectively). On the other hand, the restricted conformation of the amido chain did not influence selectivity for the cloned hmt(1) and hMT(2) receptors. These compounds were also potent agonists of melanophore aggregation in X. laevis. 15a,c were several hundred fold more potent than melatonin (EC(50) = 0.025, 0.004 nM, respectively). Conformational studies indicated that the minimum energy folded conformation of the ethylamido chain could constitute the putative active form in the receptor site in agreement with previous results.