Quantitative estimation of the contribution of pyrrolcarboxamide groups of the antibiotic distamycin A into specificity of its binding to DNA AT pairs.

Interaction of DNA with the analogs of the antibiotic distamycin A having different numbers of pyrrolcarboxamide groups and labeled with dansyl was studied. The binding isoterms of the analogs to synthetic polydeoxyribonucleotides were obtained. Analysis of the experimental data leads to the following conclusions: (1) the free energy of binding of the analogs to poly(dA).poly(dT) depends linearly on the number of amide groups in the molecule of the analog whereas attachment of each pyrrolcarboxamide group produces changes of 2 kcal/mole in the free energy; (2) attachment of a pyrrolcarboxamide unit to the GC pair results in the free energy change of 0.95 kcal/mole; (3) the binding of analogs to poly(dA).poly(dT) is a cooperative process, presumbly, dependent on conformational changes induced by the binding of analogs to DNA.     

Rapid flip-flop of phospholipids in endoplasmic reticulum membranes studied by a stopped-flow approach

The transbilayer movement of short-chain spin-labeled and fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) phospholipid analogs in rat liver microsomes is measured by stopped-flow mixing of labeled microsomes with bovine serum albumin (BSA) solution. Extraction of analogs from the outer leaflet of microsomes to BSA can be directly monitored in conjunction with electron paramagnetic resonance or fluorescence spectroscopy by taking advantage of the fact that the signal of spin-labeled or fluorescent analogs bound to BSA is different from that of the analogs inserted into membranes. From the signal kinetics, the transbilayer movement and the distribution of analogs in microsomal membranes can be derived provided the extraction of analogs by BSA is much faster in comparison to the transbilayer movement of analogs. Half-times of the back-exchange for spin-labeled and fluorescent analogs were <3.5 and <9.5 s, respectively. The unprecedented time resolution of the assay revealed that the transbilayer movement of spin-labeled analogs is much faster than previously reported. The half-time of the movement was about 16 s or even less at room temperature. Transmembrane movement of NBD-labeled analogs was six- to eightfold slower than that of spin-labeled analogs.     

Binding specificity of the mouse cerebral cortex receptor for small cholecystokinin peptides

Prior studies have shown that the cerebral cortex cholecystokinin (CCK) receptor can bind CCK and gastrin analogs with high affinity. In the present work the brain CCK receptor had approximately a three times greater affinity for CCK₈ than its C-terminal tetrapeptide (CCK₄) while the C-terminal tripeptide (CCK₃) was 1000-fold less potent than CCK₄. Thus the C-terminal tetrapeptide appears to be the minimal C-terminal CCK sequence required for high affinity binding. Since brain membranes degrade various peptides including CCK, we also evaluated the stability of CCK analogs under the conditions used to measure receptor binding by the following three methods: (1) Studies of degradation-resistant analogs in binding assays; (2) analysis of analog degradation by high performance liquid chromatography (HPLC); and (3) determination of the change in potency of CCK analogs in competitive binding studies subsequent to preincubation with brain membranes. These studies indicated that degradation of analogs by the brain membranes although significant did not account for the differences in potency of analogs in competitive binding studies. Therefore, the observed differences in potencies of the analogs tested are due to the receptor affinity and not sensitivity of the analog to degradation.     

Intestinal transport of TRH analogs through PepT1: the role of in silico and in vitro modeling

The present study involves molecular docking, molecular dynamics (MD) simulation studies, and Caco‐2 cell monolayer permeability assay to investigate the effect of structural modifications on PepT1‐mediated transport of thyrotropin releasing hormone (TRH) analogs. Molecular docking of four TRH analogs was performed using a homology model of human PepT1 followed by subsequent MD simulation studies. Caco‐2 cell monolayer permeability studies of four TRH analogs were performed at apical to basolateral and basolateral to apical directions. Inhibition experiments were carried out using Gly‐Sar, a typical PepT1 substrate, to confirm the PepT1‐mediated transport mechanism of TRH analogs. P_app of the four analogs follows the order: NP‐1894 < NP‐2378 < NP‐1896 < NP‐1895. Higher absorptive transport was observed in the case of TRH analogs, indicating the possibility of a carrier‐mediated transport mechanism. Further, the significant inhibition of the uptake of Gly‐Sar by TRH analogs confirmed the PepT1‐mediated transport mechanism. Glide docking scores of all the four analogues were in good agreement with their transport rates, suggesting the role of substrate binding affinity in the PepT1‐mediated transport of TRH analogs. MD simulation studies revealed that the polar interactions with amino acid residues present in the active site are primarily responsible for substrate binding, and a downward trend was observed with the increase in bulkiness at the N‐histidyl moiety of TRH analogs. Copyright © 2014 John Wiley & Sons, Ltd.     

Stimulation of photosynthesis and growth of photoautotrophically cultured plant cells by choline and its analogs

The effects of choline and its analogs, allylcholine and benzylcholine, on the photosynthesis and on the cell growth were examined using photoautotrophically, photomixotrophically and heterotrophically cultured cells. The addition of choline and its analogs stimulated the cellular photosynthetic activity and enhanced the dry weight increase in both photoautotrophic and photomixotrophic cells. However, the growth of heterotrophic cells did not increase by the addition of choline and choline analogs. The photosynthetic electron transport activity in thylakoid membrane was enhanced when cells were treated with choline and choline analogs, suggesting that thylakoid membranes are the initial site of the stimulation of cellular photosynthesis. The stimulatory effect of choline and choline analogs was sustained even after 3 week-culture. Among the choline analogs tested, benzylcholine showed the most quick effect and was effective at a lower concentration (1 mg/l) than choline (10 mg/l).Abbreviations GA₃ gibberellin A₃     

3D-QSAR based optimization of insect neuropeptide allatostatin analogs

Allatostatins (AST) are neuropeptides originally described as inhibitors of juvenile hormone (JH) synthesis in insects. Consequently, they have been considered as potential lead compounds for the discovery of new insect growth regulators (IGRs). In the present work, receptor-based three-dimensional quantitative structure-activity relationship (3D-QSAR) was studied with 48 AST analogs, and a general approach for novel potent bioactive AST analogs is proposed. Hence, six novel AST analogs were designed and synthesized. Bioassays indicated that the majority novel analogs exhibited potent JH inhibitory activity, especially analog A6 (IC₅₀: 3.79?nmol/L), which can be used as lead compound to develop new IGRs.     

Molecular docking analysis of imine stilbene analogs and evaluation of their anti-aging activity using yeast and mammalian cell models

The NAD⁺-dependent histone deacetylase SIRT1 was shown to be associated with aging and longevity. A stilbene, resveratrol (RV) was shown to exert anti-aging activity by stimulating the SIRT1 activity. However, the utility of RV is limited by its low bioavailability and structural instability. It is thus envisaged to test imine stilbene (IMS) analogs of RV for their potential anti-aging activity. In the present study, molecular docking analysis of five IMS analogs (3a, 3b, 3c, 3d and 3e) against the SIRT1 protein has been carried out. All the five IMS analogs displayed enhanced binding affinity towards SIRT1; three out of five IMS analogs (3a, 3?b, 3e) showed significantly higher affinity with lower binding energies (?9.58, ?9.54, and ?9.82?kcal mol^?1) than RV (?8.11?kcal mol^?1). Further, experimental validation of anti-aging activity was performed by measuring the chronological life span in vitro using yeast and cellular replicative senescence (CRS) in mammalian cell line models. All IMS analogs extended the chronological life span in yeast as compared to untreated cells as well as RV treated cells. Enhanced anti-aging activity was also observed in an analogous mammalian cell line model upon treatment with either RV or IMS analogs. The results thus suggest that most of the IMS analogs tested may serve as potent drug lead molecules with anti-aging activity.     

Binding of novel 9-O–N-aryl/arylalkyl amino carbonyl methyl berberine analogs to poly(U)-poly(A)·poly(U) triplex and comparison to the duplex poly(A)-poly(U)

Interaction of the 9-O–N-aryl/arylalkyl amino carbonyl methyl substituted analogs of the anticancer isoquinoline alkaloid berberine with RNA triplex, poly(U)-poly(A)·poly(U) has been studied in comparison to the duplex poly(A)-poly(U), using multiple biophysical techniques. Spectrophotometric and spectrofluorimetric studies established the non-cooperative binding mode of all the analogs with both the duplex and the triplex. However, berberine exhibited cooperative binding with poly(A)-poly(U) and non-cooperative binding with poly(U)-poly(A)·poly(U). Analog BER1 showed the highest affinity to both the duplex and the triplex followed by BER2 and BER3. The overall binding affinity varied as BER1 > BER2 > BER3 > BER. The magnitude of the quantum efficiency values (Q > 1) revealed that energy was transferred from the bases of the triplex and the duplex to the analogs. Comparative ferrocyanide quenching and viscosity studies unambiguously established a stronger intercalative geometry of the analogs to both the triplex and the duplex in comparison to berberine. Circular dichroism studies revealed that the alkaloids perturbed the conformation of both RNA helices. The binding of all the alkaloids was found to be exothermic from isothermal titration studies. Binding of the analogs was highly entropy driven while that of berberine was enthalpy dominated. The results presented here reveal strong and specific binding of these new berberine analogs to the RNA triplex and duplex and highlight the remarkable influence of the 9-substitution on the interaction profile.     

Computational investigation of the molecular conformation-dependent binding mode of (<Emphasis Type="Italic">E</Emphasis>)-<Emphasis Type="Italic">β</Emphasis>-farnesene analogs with a heterocycle to aphid odorant-binding proteins

Odorant-binding proteins (OBPs) play an important role as ligand-transfer filters in olfactory recognition in insects. (E)-β-farnesene (EBF) is the main component of the aphid alarm pheromone and could keep aphids away from crops to prevent damage. Computational insight into the molecular binding mode of EBF analogs containing a heterocycle based on the structure of Megoura viciae OBP 3 (MvicOBP3) was obtained by molecular docking and molecular dynamics simulations. The results showed that high affinity EBF analogs substituted with an aromatic ring present a unique binding conformation in the surface cavity of MvicOBP3. A long EBF chain was located inside the cavity and was surrounded by many hydrophobic residues, while the substituted aromatic ring was exposed to the outside due to limitations from the formation of multiple hydrogen bonds. However, the low activity EBF analogs displayed an exactly inverted binding pose, with EBF loaded on the external side of the protein cavity. The affinity of the recently synthesized EBF analogs containing a triazine ring was evaluated in silico based on the binding modes described above and in vitro through fluorescence competitive binding assay reported later. Compound N1 not only showed a similar binding conformation to that of the high affinity analogs but was also found to have a much higher docking score and binding affinity than the other analogs. In addition, the docking score results correlated well with the predicted logP values for these EBF analogs, suggesting highly hydrophobic interactions between the protein and ligand. These studies provide an in silico screening model for the binding affinity of EBF analogs in order to guide their rational design based on aphid OBPs.     

Inhibition of tubulin polymerization with ribose-modified analogs of GDP and GTP reduced inhibition with microtubule-associated proteins and magnesium

Inhibitory effects of ribose-modified GDP and GTP analogs on tubulin polymerization were examined to explore nucleotide structural requirements at the exchangeable GTP binding site. With microtubule-associated proteins and Mg²⁺, GTP-supported polymerization was only modestly inhibited by GDP, and still weaker inhibitory activity was found with two analogs, dGDP and 9-β-D-arabinofuranosylguanine-5′-diphosphate (araGDP). Omission of Mg²⁺ significantly enhanced the inhibitory effects of GDP, dGDP and araGDP and resulted in weak inhibition of the reaction by several other GDP analogs. The relative inhibitory activity of the GDP analogs had no discernable relationship to the relative activity of cognate GTP analogs in supporting microtubule-associated protein-dependent polymerization. One GTP analog, 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), supports polymerization both with and without microtubule-associated proteins. The inhibitory activity of GDP and GDP analogs in ddGTP-supported polymerization was much greater in the absence of microtubule-associated proteins than in their presence; and both reactions were more readily inhibited than was microtubule-associated protein-dependent, GTP-supported polymerization. Microtubule-associated protein-independent, ddGTP-supported polymerization was also potently inhibited by GTP and a number of GTP analogs. GTP was in fact twice as inhibitory as GDP. The relative inhibitory activity of the GTP analogs was comparable to the relative inhibitory activity of the cognate GDP analogs and very different from their relative activity in supporting polymerization.     

Structure-activity study of endomorphin-2 analogs with C-terminal modifications by NMR spectroscopy and molecular modeling

Endomorphin-2 (EM-2) is a putative endogenous mu-opioid receptor ligand. To get insight into the important role of C-terminal amide group of EM-2, we investigated herein a series of EM-2 analogs by substitution of the C-terminal amide group with -NHNH(2), -NHCH(3), -N(CH(3))(2), -OCH(3), -OCH(2)CH(3), -OC(CH(3))(3), and -CH(2)-OH. Their binding affinity and bioactivity were determined and compared. Despite similar (analogs 1, 4, and 7) or decreased (analogs 2, 3,5, and 6) mu affinity in binding assays, all analogs showed low guinea pig ileum (GPI) and mouse vas deferens (MVD) potencies compared to their parent peptide. Interestingly, as for analogs 2 and 3 (a single and double N-methylation of C-terminal amide), the potency order with the K(i) (mu) values was 2>3; for the C-terminal esterified analogs 4-6, the potency order with the K(i) (mu) values was 4>5>6. Thus, we concluded that the steric hindrance of C-terminus might play an important role in opioid receptor affinity. We further investigated the conformational properties of these analogs by 1D and 2D (1)H NMR spectroscopy and molecular modeling. Evaluating the ratios of cis- and trans-isomers, aromatic interactions, dihedral angles, and stereoscopic views of the most convergent conformers, we found that modifications at the C-terminal amide group of EM-2 affected these analog conformations markedly, therefore changed the opioid receptor affinity and in vitro bioactivity.     

Differential mutagenicity of streptozotocin analogs of the carbohydrate moiety

The mutagenicity of streptozotocin (SZN), 8 of its analogs and N-msthyl-N-nitrosourea (MNU) were compared in Salmonella typhimurium. SZN and its analogs carry MNU attached to the carbohydrate moiety at the C-2 position. The C-1 analogs tested were α- and β-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; in 2 analogs glucose was replaced by α- or β-inositol. When the ability of these compounds to revert the hisG46 auxotroph was compared, they fell into 4 groups which differed by about 10-fold in mutagenicity from one another. The most mutagenic was (i) SZN, followed by (ii) β-methyl-SZN; (iii) α-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; (iv) α and β-inositol-MNU. These results suggest that the presence of the glucose moiety is conducive to a high level of mutagenicity of SZN. Alterations of the glucose moiety by addition of larger alkyl groups, especially in the α position lead to decreased mutagenicity. The least mutagenic analogs are those in which the glucose moiety is replaced by inositols.The mutagenicity of SZN, β-methyl-SZN and of β-butyl-SZN was also compared in a mouse tissue-mediated assay. SZN was about 500-fold more mutagenic than its β-methyl analog, while the β-butyl analog was not mutagenic.Depletion of SZN and 4 of its analogs from the medium in presence of bacteria was determined spectrophotometrically. The more mutagenic compounds were depleted more rapidly but the quantitative differences in mutagenicity between these compounds could not be accounted for by depletion alone.     

Methylated analogs of spermine and spermidine as tools to investigate cellular functions of polyamines and enzymes of their metabolism

Biogenic amines spermine (Spm) and spermidine (Spd) are essential for cell growth. Polyamine analogs are widely used to investigate the enzymes of polyamine metabolism and the functions of spermine and spermidine in vitro and in vivo. It was demonstrated recently that α-methylated derivatives of Spm and Spd are able to fulfill the key cellular functions of polyamines, moreover, in some cases, the effects of (R) and (S) isomers were actually different. Using these α-methylated analogs of Spm and Spd, it turned possible to prevent the development of acute pancreatitis in SSAT-transgenic rats with controllable expression of the Spm/Spd N¹-acetyltransferase gene. The analogs made it possible to reveal dormant stereospecificity of polyamine oxidase, Spm oxidase, and deoxyhypusine synthase. An original approach was suggested to regulate the stereospecificity of polyamine oxidase. Depletion of the intracellular polyamine pool was found to have both hypusine-related consequences and consequences unrelated to posttranslational modification of the eukaryotic translation initiation factor eIF5A. Possible applications of a new family of C-methylated polyamine analogs for the investigation and regulation of polyamine metabolism in vitro and in vivo are discussed.     

In vitro and in vivo characterization of opioid activities of C-terminal esterified endomorphin-2 analogs

Previously, we have synthesized a series of endomorphin-2 (EM-2) analogs by the substitution of C-terminal amide group. In the present study, to further our knowledge of the influence of C-terminal esterified modification on the pharmacological activities, we investigated the in vitro and in vivo opioid activities of C-terminal esterified EM-2 analogs 1–3. Our results showed that the ED₅₀ values on contractions of the longitudinal muscle of distal colon induced by analogs 1–3 were about 1.5-fold higher, 2- and 8-fold lower than EM-2, respectively. In addition, intravenous (i.v.) injections of analogs 1 and 2 dose-dependently decreased the system arterial pressure (SAP) and heart rate (HR) in anesthetized rats, but the degree of the hypotension and bradycardia was significantly smaller relative to the parent. Moreover, analog 3 was almost ineffective. Nevertheless, all these analogs produced potent antinociception in the tail-flick test after intracerebroventricular (i.c.v.) injection, and this antinociception was inhibited by naloxone, indicating an opioid mechanism. In summary, these results gave the evidence that the conversion of C-terminal amide to esterified modification may play an important role in the regulation of opioid affinities and pharmacological activities.     

Type II protein kinase A regulates CFTR in airway, pancreatic, and intestinal cells

The type ofprotein kinase A (PKA) responsible for cystic fibrosis transmembraneconductance regulator (CFTR) activation was determined with adenosine3',5'-cyclic monophosphate analogs capable of selectivelyactivating type I or type II PKA. The type II-selective pair stimulatedchloride efflux in airway, pancreatic, and colonic epithelial cells;the type I-selective pair only stimulated a calcium-dependent efflux inairway cells. The type II-selective analogs activated larger increasesin CFTR-mediated current than did the type I-selective analogs.Measurement of soluble PKA activity demonstrated similar levelsstimulated by type I- and type II-selective analogs, creating anapparent paradox regarding PKA activity and current generated. Also,addition of forskolin after the type I-selective analogs resulted in anincrease in current; little increase was seen after the typeII-selective analogs. Measurement of insoluble PKA activity stimulatedby the analogs resolved this paradox. Type II-selective analogsstimulated three times as much insoluble PKA activity as the typeI-selective pair, indicating that differential activation of PKA incellular compartments is important in CFTR regulation.

     

Semi-synthetic salinomycin analogs exert cytotoxic activity against human colorectal cancer stem cells

Salinomycin, a polyether antibiotic, is a well-known inhibitor of human cancer stem cells. Chemical modification of the allylic C20 hydroxyl of salinomycin has enabled access to synthetic analogs that display increased cytotoxic activity compared to the native structure. The aim of this study was to investigate the activity of a cohort of C20-O-acyl analogs of salinomycin on human colorectal cancer cell lines in vitro. Two human colorectal cancer cell lines (SW480 and SW620) were exposed to three C20-O-acylated analogs and salinomycin. The impact of salinomycin and its analogs on tumor cell number, migration, cell death, and cancer stem cell specifity was analyzed. Exposure of human colorectal cancer cells to the C20-O-acylated analogs of salinomycin resulted in reduced tumor cell number and impaired tumor cell migration at lower concentrations than salinomycin. When used at higher (micromolar) concentrations, these effects were accompanied by induction of apoptotic cell death. Salinomycin analogs further expose improved activity against cancer stem cells compared to salinomycin.     

Specificities of rabbit anti-(2',5')oligoadenylate antibodies towards phosphorothioate and seco analogs of oligoadenylates.

Rabbit antibodies to 2',5'-linked triadenylate were prepared by immunization with (2',5')A3 conjugated via the 2'3'-levulinic group, (2'5')A3-Lev, to BSA. New radioimmunoassay for (2',5')oligoadenylates was developed using 125I thyrosine labeled derivative of (2',5')A3-Lev. Reactivity of antibodies with phosphorothioate and seco analogs of oligoadenylates was studied. It was found that (i) stereospecific substitution of the diastereotopic oxygens with sulfur in the internucleotide phosphodiester linkages changes the immunoreactivity of such analogs; (ii) the seco analogs of oligoadenylates display in some cases a rather high reactivity.     

Selective Toxins and Analogs Produced by Helminthosporium sacchari: Production, Characterization, and Biological Activity

Helminthosporium sacchari toxin and several lower molecular weight, nontoxic analogs were isolated from culture filtrates. Three isomers of the toxin (A, B, and C), each with four galactose units, were separated by high performance liquid chromatography. Isomer C had the highest and isomer A had the lowest toxicity to H. sacchari-susceptible sugarcane; resistant clones were not affected. Each toxin isomer was partially digested with a β-galactofuranosidase and the resulting analogs (seven from each toxin isomer) were separated by reverse phase high performance liquid chromatography and identified. Each isomer of the analogs with 3 galactose units per mole also was partially digested and the arrangement of galactose units was determined. The compound with one galactose attached to position 2 of the bicyclic sesquiterpene and with 2 galactose units attached to position 13 (analog A_1,2) was highly toxic to some but not to all clones of H. sacchari-susceptible sugarcane. Toxin analogs protected sensitive tissue against active toxin; protective effects of the analogs differed, but at least a 10-fold excess of analog was required. Analog C_2,1 was more effective at preventing toxin C-induced electrolyte losses than was any other analog. Each of the 3-galactose analog isomers protected better than did any of the 2-galactose compounds. The 1,1 analogs did not protect as well as did the 2,0 or 0,2 analogs. Thus, the sesquiterpene isomer, the number of galactose units, and the galactose arrangement pattern determine the effectiveness of the compound in induction of electrolyte loss and in prevention of toxininduced loss from sugarcane tissues.     

Convergent synthesis, chiral HPLC, and vitamin D receptor affinity of analogs of 1,25-dihydroxycholecalciferol

A series of analogs of 1,25-dihydroxycholecalciferol was obtained with an additional chiral center at the terminus of the aliphatic side chain (C-25). The analogs were obtained from (+)-(R)- and (-)-(S)-2-methylglycidols, by opening of the oxirane ring with the carbanions derived from vitamin D C23a,24- or C22-sulfones. The diastereomeric purity of the analogs was determined by high-performance liquid chromatography on a chiral stationary phase. The binding affinity of analogs for the calf thymus intracellular vitamin D receptor (VDR) was two orders of magnitude lower than that of the lead compound of this group, 24a,24b-dihomo-1,25-dihydroxycholecalciferol, and it was comparable to the affinity of analogs of 24-nor-1,25-dihydroxycholecalciferol. However, a twofold difference was observed for analogs diastereomeric at C-25 in their affinity for VDR. The diastereodifferentiation of the binding affinity was found to be specific for vitamin D vicinal 25,26-diols as it disappears for analogs where 26-hydroxyl, neighboring the C-25 chiral center, is replaced with methyl.