BBA - General Subjects (v.1800, #9)

Ubiquitination and SUMOylation of annexin A1 and helicase activity by Fusao Hirata; Lisa M. Thibodeau; Aiko Hirata (899-905).
While annexin A1 in nuclei is proposed to be involved in cell transformation, its functions remain poorly understood. Since annexin A1 has the consensus motif, 160LKRD, for SUMOylation as well as Ks, acceptors for ubiquitination that regulates localization and functions of proteins, we investigated SUMOylation and ubiquitination of annexin A1.SUMOylation and ubiquitination of bovine annexin A1 were biochemically tested in vitro by purified proteins, and were confirmed by cell experiments with L5178 lymphoma cells. Effects of the modifications on DNA helicase activity were measured by ssDNA binding activity and by dsDNA unwinding activity.SUMOylation of annexin A1 was catalyzed by Ubc9, while its ubiquitination was by Rad6-Rad 18. Ubiquitinated annexin A1 had higher affinity for damaged DNA, and promoted in vitro translesion DNA synthesis by Pol β. In mouse lymphoma L5178Y tk(+/) cells, levels of SUMOylated annexin A1 decreased by DNA damaging agents, MMS or As3+, whereas those of ubiquitinated annexin A1 increased under the same conditions.These observations suggest but do not necessarily prove that ubiquitinated annexin A1 in nuclei may be involved in DNA damage response, while SUMOylated annexin A1 functions in proliferation–differentiation.Ubiquitination of annexin A1 may play an important role in mutagenesis, an initial step of cell transformation.
Keywords: Rad6; Ubc9; nucleus; DNA damage; cell transformation;

Pharmacological profiles of the murine gastric and colonic H,K-ATPases by Jiahong Shao; Michelle L. Gumz; Brian D. Cain; Shen-Ling Xia; Gary E. Shull; Ian R. van Driel; Charles S. Wingo (906-911).
The H,K-ATPase, consisting of α and β subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα1 and HKα2 encoded by different genes. The ouabain-resistant gastric HKα1-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα2-H,K-ATPase from different species shows poor primary structure conservation of the HKα2 subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKα1, HKα2, or HKβ genes.Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα1, HKα2, or HKβ knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined.The colonic H,K-ATPase demonstrated slightly greater affinity for K+ than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKα2 KO but was observed in HKβ KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKα1-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα2-H,K-ATPase was not inhibited by these compounds.These data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride.Characterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.
Keywords: Targeted gene disruption; Potassium transport; Proton transport; P-type ATPase; Distal colon; Amiloride;

Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt/GSK3β pathways by Xiuli Sun; Yi Zhang; Jianliu Wang; Lihui Wei; Hui Li; Gregory Hanley; Miaoqing Zhao; Yi Li; Deling Yin (912-918).
Resveratrol is emerging as a novel anticancer agent. However, the mechanism(s) by which resveratrol exerts its effects on endometrial cancer (EC) are unknown. We previously reported that β-arrestin 2 plays a critical role in cell apoptosis. The role of β-arrestin 2 in resveratrol modulation of endometrial cancer cell apoptosis remains to be established.EC cells HEC1B and Ishikawa were transfected with either β-arrestin 2 RNA interfering (RNAi) plasmid or β-arrestin 2 full-length plasmid and control vector. The cells were then exposed to differing concentrations of resveratrol. Apoptotic cells were detected by TUNEL assay. Expression of total and phosphorylated Akt (p-Akt), total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β), and caspase-3 were determined by Western blot analysis. Our data demonstrate that inhibition of β-arrestin 2 increases the number of apoptotic cells and caspase-3 activation. Additionally β-arrestin 2 exerted an additive effect on resveratrol-reduced levels of p-Akt and p-GSK3β. Overexpression of β-arrestin 2 decreased the percentage of apoptosis and caspase-3 activation and attenuated resveratrol-reduced levels of p-Akt and p-GSK3β. Taken together, our studies demonstrate for the first time that β-arrestin 2 mediated signaling plays a critical role in resveratrol-induced apoptosis in EC cells.Resveratrol primes EC cells to undergo apoptosis by modulating β-arrestin 2 mediated Akt/GSK3β signaling pathways.These inspiring findings would provide a new molecular basis for further understanding of cell apoptotic mechanisms mediated by β-arrestin 2 and may provide insights into a potential clinical relevance in EC.
Keywords: Endometrial cancer; β-Arrestin 2; Resveratrol; Apoptosis; Akt; GSK3β;

The methylerythritol phosphate pathway for isoprenoid biosynthesis is an attractive target for the design of new specific antibiotics for the treatment of gastrointestinal diseases associated with the presence of the bacterium Helicobacter pylori since this pathway which is essential to the bacterium is absent in humans.This work reports the molecular cloning of one of the genes of the methylerythritol phosphate pathway form H. pylori (ispDF; HP_1440) its expression in Escherichia coli and the functional characterization of the recombinant enzyme. As shown by genetic complementation and in vitro functional assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate synthase from E. coli.Designing inhibitors that affect at the same time both enzyme activities of the H. pylori bifunctional enzyme (i.e. by disrupting protein oligomerization) would result in more effective antibiotics which would be able to continue their action even if the bacterium acquired a resistance to another antibiotic directed against one of the individual activities.The bifunctional enzyme would be an excellent target for the design of new, selective antibiotics for the treatment of H. pylori associated diseases.
Keywords: 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase; 2-C-methyl-d-erythritol-2,4-cyclodiphosphatesynthase; Helicobacter pylori; Isoprenoid biosynthesis; Methylerythritol phosphate pathway;

Nitric oxide (NO) plays a major role in physiology as a biological mediator. NO has been identified in nervous, immune and vascular systems and is a critical parameter in numerous pathologies, such as cancer. This article describes the electrochemical biomeasurements of NO synthase (NOS) activity from cultured endothelial cells using a multiple microelectrode array.Firstly, the effect of biocompatible fibronectin coating on electrochemical measurements was investigated. Secondly, endothelial cells were deposited on the fibronectin coated sensor and NO release was triggered with vascular endothelial growth factor (VEGF). NG-nitro-l-arginine methyl ester (L-NAME) was used as an inhibitor of NO production, and different kinase blockers were investigated. Change in NOS activity was quantified using differential pulse voltammetry before and after addition of VEGF.Our results show that carefully applied layers of fibronectin have a very limited effect on electrochemistry and that VEGF induces an increase in NOS activity that is mainly mediated through the phosphatidylinositol 3 kinase (PI-3), and not by the extracellular signal-regulated kinases 1/2. Results obtained using electrochemical sensors were supported by wound healing assay demonstrating the critical role of phosphatidylinositol 3 kinase and extracellular signal-regulated kinases 1/2 for angiogenesis.Electrochemical study of the intracellular transduction of the VEGF signal leading to NO synthesis was achieved, showing the critical role of PI-3 kinase.This study presents an electrochemical sensor allowing measurements of NOS activity in cell cultures and tissue samples.
Keywords: Biomeasurement; Nitric oxide; Electrochemistry; Biocompatibility; Vascular endothelial growth factor; Fibronectin;

Comparative study of mature and zymogen mite cysteine protease stability and pH unfolding by Andy Chevigné; Marie-Eve Dumez; Mireille Dumoulin; André Matagne; Alain Jacquet; Moreno Galleni (937-945).
Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes.To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1.We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen.Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis.
Keywords: pH-induced unfolding; Propeptide; Der p 1; Cysteine protease; Stability; Mechanism of activation; Fluorescence quenching; Thermal denaturation; Allergen; Zymogen;

Signal transduction pathways associated with ATP-induced proliferation of colon adenocarcinoma cells by Natalia Buzzi; Ricardo Boland; Ana Russo de Boland (946-955).
In previous work, we have demonstrated that extracellular adenosine 5′-triphosphate (ATP) acts on intestinal Caco-2 cell P2Y receptors promoting a rapid increase in the phosphorylation of ERK1/2, p46 JNK and p38 MAP kinases (MAPKs).In this study, we investigated whether the extracellular ATP-P2Y receptor signalling pathways were required for the proliferation of Caco-2 cells. Confocal microscopy and immunobloting studies showed that ERK1/2 and JNK translocate into the nucleus of the cells stimulated by ATP, where they participate, together with p38 MAPK, in the phosphorylation of JunD, ATF-1 and ATF-2 transcription factors. In addition, ATP through the activation of MAPKs induces the expression of the immediate early genes products of the Jun family, c-Fos and MAP kinase phosphatase-1 (MKP-1). Moreover, ERK1/2 and p38 MAPK are involved in the phosphorylation of MKP-1 in Caco-2 cells. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, ATP increased Caco-2 cell proliferation, and this effect was blocked by UO126, SB203580 and SP600125, the specific inhibitors of ERK1/2, p38 MAPK and JNK1/2, respectively.Extracellular ATP induces proliferation of Caco-2 human colonic cancer cells by activating MAPK cascades and modulation of transcription factors.These findings and identification of the specific P2Y subtype receptors involved in the mitogenic effect of ATP on Caco-2 cells might be relevant for understanding tumor cell development, resistance to treatment regimens and the design of new therapeutic strategies.
Keywords: Caco-2 cell; ATP; Signal transduction; Proliferation;

Effective culture conditions for the induction of pluripotent stem cells by Minoru Okada; Masahiro Oka; Yoshihiro Yoneda (956-963).
Induced pluripotent stem (iPS) cells, which are functionally comparable to embryonic stem (ES) cells, can be generated from mouse fibroblasts by expression of a defined set of transcription factors Oct4, Sox2, Klf4, and c-Myc. Since iPS cells are generated from somatic cells, they provide an invaluable source of pluripotent stem cells for cell transplantation therapy that does not present ethical problems. However, the reprogramming efficiency is extremely low, and optimal culture conditions for iPS cell derivation have not been clearly defined.To generate iPS cells efficiently, we tested 10 different culture conditions: DMEM supplemented with 15% fetal bovine serum (FBS), Knockout DMEM with 15% FBS from Invitrogen, Equitech, or HyClone, DMEM with 15% Knockout Serum Replacement (KSR), and Knockout DMEM with 10%, 15%, 20%, 25%, or 35% KSR. These media all contain 2 mM l-glutamine, 100 μM nonessential amino acids, 100 μM beta-mercaptoethanol, 1000 units ml 1 leukemia inhibitory factor (LIF), 50 units ml 1 penicillin, and 50 μg ml 1 streptomycin.Medium containing Knockout DMEM with 20% KSR permits efficient induction of iPS cells from both mouse embryonic fibroblasts (MEFs) and adult tail tip fibroblasts (TTFs). Mouse iPS cells generated in the condition express ES cell marker genes such as Oct4, Sox2, Rex1, and Nanog at levels comparable to those of ES cells. Furthermore, iPS cells derived form MEFs and adult TTFs can contribute to adult chimeras.Our iPS cell induction efficiency is greater than that described in other reports.These findings provide an important catalyst for examining different culture environments for the generation of iPS cells.
Keywords: Induced pluripotent stem cells; Reprogramming; Culture condition;

Nitric oxide (NO) plays a vital role in maintaining the survivability of circulating erythrocytes. Here we have investigated whether NO depletion associated with visceral leishmaniasis (VL) is responsible for the reduced survival of erythrocytes observed during the disease.Infected hamsters were treated with standard anti-leishmanial sodium stibogluconate (SAG) and NO donor isosorbide dinitrate (ISD). Erythrophagocytosis by macrophages was determined by labelling the cells with FITC followed by flow cytometry. Aggregation of band3 was estimated from band3 associated EMA fluorescence. Caspase 3 activity was measured using immunosorbent assay kit. Phosphatidylserine (PS) externalization and cell shrinkage were determined using annexin V. Aminophspholipid translocase and scramblase activities were measured following NBD-PS and NBD-PC internalization, respectively.Impairment of both synthesis and uptake of NO resulted in decreased bioavailability of this signaling molecule in erythrocytes in VL. NO level was replenished after simultaneous treatment with ISD and SAG. Combination treatment decreased red cell apoptosis in infected animals by deactivating caspase 3 through s-nitrosylation. Drug treatment prevented infection-mediated ATP depletion and altered calcium homeostasis in erythrocytes. Improved metabolic environment effectively amended dysregulation of aminophospholipid translocase and scramblase, which in turn reduced cell shrinkage, and exposure of phosphatidylserine on the cell surface under the diseased condition.In this study, we have identified NO depletion to be an important factor in promoting premature hemolysis with the progress of leishmanial infection. The study implicates NO to be a possible target for future drug development towards the promotion of erythrocyte survival in VL.
Keywords: Nitric oxide (NO); Visceral leishmaniasis (VL); Erythrophagocytosis; Caspase 3; Calcium homeostasis; Transbilayer lipid asymmetry;

Regulation of post-translational protein arginine methylation during HeLa cell cycle by Chongtae Kim; Yongchul Lim; Byong Chul Yoo; Nam Hee Won; Sangduk Kim; Gieun Kim (977-985).
Post-translational arginine methylation which modifies protein-arginyl residues by protein arginine methyltransferase (PRMT) was investigated during synchronized HeLa cell cycle.The lysates of cells synchronized at each stage were subjected to one and/or two dimensional electrophoresis followed by Western immunoblot using against anti-asymmetric-dimethyl-arginine (ASYM24), anti-symmetric-dimethyl-arginine (SYM10), and subclasses of PRMTs, including PRMT1, PRMT3, PRMT4 (CARM1), PRMT5, PRMT6, and PRMT7 antibodies.Proteins with approximate molecular masses of 80 kDa, 68 kDa, and 64 kDa, containing asymmetric-dimethyl-arginine (aDMA) were increased at G0/G1 to G1, which lasted until S phase. In addition, 25 kDa protein of symmetric-dimethyl-arginine (sDMA) was also markedly up-regulated from G0/G1 to G1. The levels of PRMT3, PRMT6 and PRMT7 were concurrently increased during the cell cycle. Two-dimensional gel electrophoresis followed by MALDI-TOF-MS was identified as aDMA-80 kDa and aDMA-68 kDa proteins as heterogeneous nuclear ribonucleoprotein R (hnRNPR), aDMA-64 kDa proteins as cleavage stimulation factor 64 kDa subunit (CstF-64), and sDMA-25 kDa protein as triosephosphate isomerase (TPI). The levels of increased aDMA of hnRNPR were reduced, when HeLa cells were transfected with siRNA for PRMT1, and the aDMA of CstF-64 with siRNA for PRMT3, while depletion of PRMT5 down-regulated sDMA of TPI.Protein arginine dimethylations of hnRNPR, CstF-64, and TPI were regulated during HeLa cell cycle by respective PRMTs.These results suggest that regulation of arginine dimethylation of hnRNPR, CstF-64, and TPI at G0/G1 to G1 are most likely to modulate the cellular growth and proliferation in HeLa cell cycle.
Keywords: ω-N G ,N G -asymmetric and ω-N G ,N′ G -symmetric dimethyl-arginine; HeLa cell cycle; Protein arginine methyltransferases; Heterogeneous nuclear ribonucleoprotein R; Cleavage stimulation factor 64 kDa subunit; Triosephosphate isomerase;

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin by Keisuke Ito; Taishi Sugawara; Ayako Koizumi; Ken-ichiro Nakajima; Akiko Shimizu-Ibuka; Mitsunori Shiroishi; Hidetsugu Asada; Takami Yurugi-Kobayashi; Tatsuro Shimamura; Tomiko Asakura; Katsuyoshi Masuda; Masaji Ishiguro; Takumi Misaka; So Iwata; Takuya Kobayashi; Keiko Abe (986-992).
Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown.Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure–function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed.As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL.The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL.The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.
Keywords: Miraculin; Sweet protein; Taste; Yeast expression system; N-glycan; Steric hindrance;

Proliferative and anti-proliferative effects of retinoic acid at doses similar to endogenous levels in Leydig MLTC-1/R2C/TM-3 cells by Mariarita Perri; Attilio Pingitore; Erika Cione; Emma Vilardi; Valentina Perrone; Giuseppe Genchi (993-1001).
Vitamin A is suggested to be protective against oxidative stress. However, different authors observed pro-oxidant effects of retinoids both in experimental works and clinical trials. These discordances are the bases for the investigation of the proliferative and anti-proliferative properties of retinoic acid (RA) in biological systems.Cell viability is determined with the MTT assay. Oxidative stress parameters are detected measuring catalase (CAT) and glutathione S-transferase (GST) enzymatic activities. FABP5 mRNA levels are measured by RT-PCR. Autophagy and apoptosis are analyzed by Monodansylcadaverine (MDC) staining and TUNEL assay, respectively.RA, at nutraceutic/endogenous doses (10–200 nM), increases cell viability of testes tumor Leydig cell lines (MLTC-1 and R2C) and modulates antioxidant enzyme activities, as CAT and GST. RA is able to induce proliferation through non-classical and redox-dependent mechanisms accompanied by increased levels of FABP5 mRNA. The redox environment of the cell is currently thought to be extremely important for controlling either apoptosis or autophagy. Apoptosis occurs at pharmacological doses, while autophagy, which plays a critical role in removing damaged or surplus organelles in order to maintain cellular homeostasis, is triggered at the critical concentration of 500 nM RA, both in normal and tumoral cells. Slight variations of RA concentrations are evaluated as a threshold value to distinguish between the proliferative or anti-proliferative effects.Although retinoids have a promising role as antineoplastic agents, physiological levels of RA play a key role in Leydig cancer progression, fostering proliferation and growth of testicular tumoral mass.
Keywords: Retinoic acid; Leydig cell; Proliferation; Apoptosis; Autophagy;

Selective detection of Cathepsin E proteolytic activity by Wael R. Abd-Elgaliel; Ching-Hsuan Tung (1002-1008).
Aspartic proteases Cathepsin (Cath) E and D are two different proteases, but they share many common characteristics, including molecular weight, catalytic mechanism, substrate preferences, proteolytic conditions and inhibition susceptibility. To define the biological roles of these proteases, it is necessary to elucidate their substrate specificity. In the present study, we report a new peptide–substrate that is only sensitive to Cath E but not Cath D.Substrate e , Mca-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Lys(Dnp)-DArg-CONH2, designed in such a way that due to the close proximity of a Mca-donor and a Dnp-acceptor, near complete intramolecular quenching effect was achieved in its intact state. After the proteolytic cleavage of the hydrophobic motif of peptide substrate, both Mca and Dnp would be further apart, resulting in bright fluorescence.Substrate e showed a 265 fold difference in the net fluorescence signals between Cath E and D. This Cath E selectivity was established by having -Leu**Pro- residues at the scissile peptide bond. The confined cleavage site of substrate e was confirmed by LC-MS. The catalytic efficiency (Kcat/KM) of Cath E for substrate e was 16.7 μM 1  S 1. No measurable catalytic efficiency was observed using Cath D and no detectable fluorescent changes when incubated with Cath S and Cath B.This study demonstrated the promise of using the developed fluorogenic substrate e as a selective probe for Cath E proteolytic activity measurement.This study forms the foundation of Cath E specific inhibitor development in further studies.
Keywords: Cathepsin E; Cathepsin D; Fluorescent; Peptide; Substrate; Aspartic protease;

Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondria-specific therapies and probes by Carolyn M. Porteous; Angela Logan; Cameron Evans; Elizabeth C. Ledgerwood; David K. Menon; Franklin Aigbirhio; Robin A.J. Smith; Michael P. Murphy (1009-1017).
Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential.Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function.To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration.AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10–30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration.These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.
Keywords: Mitochondrion; Antioxidant; MitoQ; Phosphonium cation; Oxidative stress;

A monovalent agonist of TrkA tyrosine kinase receptors can be converted into a bivalent antagonist by Fouad Brahimi; Jing Liu; Andrey Malakhov; Shafinaz Chowdhury; Enrico O. Purisima; Ljubica Ivanisevic; Antoine Caron; Kevin Burgess; H. Uri Saragovi (1018-1026).
Receptor tyrosine kinases (RTK) act through dimerization. Previously it was thought that only bivalent ligands could be agonistic, whereas monovalent ligands should be antagonistic. This notion changed after the demonstration that monovalent ligands can be agonistic, including our report of a small molecule monovalent ligand “D3” that is a partial agonist of the NGF receptor TrkA. A bivalent “D3-linker-D3” was expected to increase agonism.Dimeric analogs were synthesized and tested in binding, biochemical, and biological assays.One analog, 1-ss, binds TrkA with higher affinity than D3 and induces or stabilizes receptor dimers. However, 1-ss exhibited antagonistic activity, through two mechanisms. One mechanism is that 1-ss blocks NGF binding, unlike D3 which is non-competitive. Inhibition of NGF binding may be due to the linker of 1-ss filling the inter-receptor space that NGF traverses before docking. In a second mechanism, 1-ss acts as a pure antagonist, inhibiting NGF-independent TrkA activity in cells over-expressing receptors. Inhibition is likely due to 1-ss “freezing” the TrkA dimer in the inactive state.Dimerization of an RTK can result in antagonism, through two independent mechanisms.we report a small molecule monovalent agonist being converted to a bivalent antagonist.
Keywords: Neurotrophin; Ligand; Tyrosine kinase receptor; Linker; Agonist; Antagonist;