RESUMO
BPTF (bromodomain and PHD finger containing transcription factor) is a multidomain protein that plays essential roles in transcriptional regulation, T-cell homeostasis and stem cell pluripotency. As part of the chromatin remodeling complex hNURF (nucleosome remodeling factor), BPTF epigenetic reader subunits are particularly important for BPTF cellular function. Here we report the synthesis of NVS-BPTF-1, a previously reported highly potent and selective BPTF-bromodomain inhibitor. Evaluation of the impact of the inhibition of BPTF-bromodomain using NVS-BPTF-1 on selected proteins involved in the antigen processing pathway revealed that exclusively targeting BPTF-bromodomain is insufficient to observe an increase of PSMB8, PSMB9, TAP1 and TAP2 proteins.
Assuntos
Proteínas do Tecido Nervoso/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Antígenos Nucleares , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Bromodomain (BD) and extra-terminal domain containing proteins (BET) are chromatin adapters that bind acetylated histone marks via two tandem BDs, BD1 and BD2, to regulate gene transcription. BET proteins are involved in transcriptional reprogramming in response to inflammatory stimuli. BET BD inhibitors (BETis) that are nonselective for BD1 or BD2 have recognized anti-inflammatory properties in vitro and counter pathology in models of inflammation or autoimmune disease. Although both BD1 and BD2 bind acetylated histone residues, they may independently regulate the expression of BET-sensitive genes. Here we characterized the ability of RVX-297, a novel orally active BETi with selectivity for BD2, to modulate inflammatory processes in vitro, in vivo, and ex vivo. RVX-297 suppressed inflammatory gene expression in multiple immune cell types in culture. Mechanistically, RVX-297 displaced BET proteins from the promoters of sensitive genes and disrupted recruitment of active RNA polymerase II, a property shared with pan-BETis that nonselectively bind BET BDs. In the lipopolysaccharide model of inflammation, RVX-297 reduced proinflammatory mediators assessed in splenic gene expression and serum proteins. RVX-297 also countered pathology in three rodent models of polyarthritis: rat and mouse collagen-induced arthritis, and mouse collagen antibody-induced arthritis. Further, RVX-297 prevented murine experimental autoimmune encephalomyelitis (a model of human multiple sclerosis) disease development when administered prophylactically and reduced hallmarks of pathology when administered therapeutically. We show for the first time that a BD2-selective BETi maintains anti-inflammatory properties and is effective in preclinical models of acute inflammation and autoimmunity.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Proteínas/antagonistas & inibidores , Quinazolinonas/uso terapêutico , Doença Aguda , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos/imunologia , Artrite/induzido quimicamente , Artrite/imunologia , Artrite/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Colágeno/imunologia , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Ratos Endogâmicos Lew , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Células U937RESUMO
Inhibition of bromo and extra-terminal (BET) bromodomains, including BRD4, has emerged as a new exciting epigenetic target for oncology, in particular. Recently, novel alternatives to the traditional use of reversible small molecules have emerged, including proteolytic targeting BET agents and irreversible binding inhibitors. These alternatives to reversible inhibitors may offer some advantage and can be used as tools to further decipher the underlying biology. Supportive pre-clinical data have these novel approaches bound for clinical development in the near future.
Assuntos
Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular , Descoberta de Drogas , Humanos , Proteínas Nucleares/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myc-transgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi.
Assuntos
Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Quinazolinonas/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Sinergismo Farmacológico , Linfoma , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/metabolismoRESUMO
Bromodomains are epigenetic readers that specifically bind to the acetyl lysine residues of histones and transcription factors. Small molecule BET bromodomain inhibitors can disrupt this interaction which leads to potential modulation of several disease states. Here we describe the binding properties of a novel BET inhibitor RVX-297 that is structurally related to the clinical compound RVX-208, currently undergoing phase III clinical trials for the treatment of cardiovascular diseases, but is distinctly different in its biological and pharmacokinetic profiles. We report that RVX-297 preferentially binds to the BD2 domains of the BET bromodomain and Extra Terminal (BET) family of protein. We demonstrate the differential binding modes of RVX-297 in BD1 and BD2 domains of BRD4 and BRD2 using X-ray crystallography, and describe the structural differences driving the BD2 selective binding of RVX-297. The isothermal titration calorimetry (ITC) data illustrate the related differential thermodynamics of binding of RVX-297 to single as well as dual BET bromodomains.
Assuntos
Quinazolinonas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Termodinâmica , Fatores de Transcrição/químicaRESUMO
Bromodomains are key transcriptional regulators that are thought to be druggable epigenetic targets for cancer, inflammation, diabetes and cardiovascular therapeutics. Of particular importance is the first of two bromodomains in bromodomain containing 4 protein (BRD4(1)). Protein-ligand docking in BRD4(1) was used to purchase a small, focused screening set of compounds possessing a large variety of core structures. Within this set, a small number of weak hits each contained a dihydroquinoxalinone ring system. We purchased other analogs with this ring system and further validated the new hit series and obtained improvement in binding inhibition. Limited exploration by new analog synthesis showed that the binding inhibition in a FRET assay could be improved to the low µM level making this new core a potential hit-to-lead series. Additionally, the predicted geometries of the initial hit and an improved analog were confirmed by X-ray co-crystallography with BRD4(1).
Assuntos
Desenho de Fármacos , Ligantes , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Sítios de Ligação , Proteínas de Ciclo Celular , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Quinoxalinas/química , Quinoxalinas/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
PB1 is a bromodomain-containing protein hypothesized to act as the nucleosome-recognition subunit of the PBAF complex. Although PB1 is a key component of the PBAF chromatin remodeling complex, its exact role has not been elucidated due to the lack of potent and selective inhibitors. Chemical probes that target specific bromodomains within the complex would constitute highly valuable tools to characterize the function and therapeutic pertinence of PB1 and of each of its bromodomains. Here, we report the design and synthesis of lead compound LM146, which displays strong stabilization of the second and fifth bromodomains of PB1 as shown by DSF. LM146 does not interact with bromodomains outside of sub-family VIII and binds to PB1(2), PB1(5), and SMARCA2B with K D values of 110, 61, and 2100 nM, respectively, providing a â¼34-fold selectivity profile for PB1(5) over SMARCA2.
RESUMO
Piceatannol is a naturally occurring bioactive stilbene with documented antileukemic properties. It has been extensively used as a Syk-selective protein tyrosine kinase inhibitor for the study of various signaling pathways. Herein, we show that the hydroxystilbene, piceatannol, and related catechol ring-containing compounds are able to induce the loss of the Cbl family of proteins. Normal cellular Cbl-regulatory mechanisms were not involved in this process. Screening of a small library of piceatannol-like compounds indicated that aromaticity and a catechol ring were required for the induction of Cbl loss. Further examination of these two chemical properties showed that the oxidative conversion of the catechol ring of piceatannol into a highly reactive O-benzoquinone was the cause of piceatannol-induced Cbl loss. Characterization of the Cbl selectivity of piceatannol-induced protein loss revealed that this compound was also able to induce the functional loss of specific Cbl-associated proteins involved in signaling pathways commonly associated with cancer. This work uncovers a new, piceatannol-dependent effect and shows a novel way in which this phenomenon can be exploited to inhibit disease-associated signaling pathways.
Assuntos
Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Estilbenos/farmacologia , Células 3T3 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Células Cultivadas , Humanos , Células K562 , Camundongos , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredução/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Descoberta de Drogas , Neoplasias Hematológicas/tratamento farmacológico , Metionina/química , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Antineoplásicos/química , Proteínas de Ciclo Celular , Cristalografia por Raios X , Neoplasias Hematológicas/patologia , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Despite the benefit of statins in reducing cardiovascular risk, a sizable proportion of patients still remain at risk. Since HDL reduces CVD risk through a process that involves formation of pre-beta particles that facilitates the removal of cholesterol from the lipid-laden macrophages in the arteries, inducing pre-beta particles, may reduce the risk of CVD. A novel BET bromodomain antagonist, RVX-208, was reported to raise apoA-I and increase preß-HDL particles in non-human primates and humans. In the present study, we investigated the effect of RVX-208 on aortic lesion formation in hyperlipidemic apoE(-/-) mice. Oral treatments of apoE(-/-) mice with 150 mg/kg b.i.d RVX-208 for 12 weeks significantly reduced aortic lesion formation, accompanied by 2-fold increases in the levels of circulating HDL-C, and â¼50% decreases in LDL-C, although no significant changes in plasma apoA-I were observed. Circulating adhesion molecules as well as cytokines also showed significant reduction. Haptoglobin, a proinflammatory protein, known to bind with HDL/apoA-I, decreased >2.5-fold in the RVX-208 treated group. With a therapeutic dosing regimen in which mice were fed Western diet for 10 weeks to develop lesions followed by switching to a low fat diet and concurrent treatment with RVX-208 for 14 weeks, RVX-208 similarly reduced lesion formation by 39% in the whole aorta without significant changes in the plasma lipid parameters. RVX-208 significantly reduced the proinflammatory cytokines IP-10, MIP1(®) and MDC. These results show that the antiatherogenic activity of BET inhibitor, RVX-208, occurs via a combination of lipid changes and anti-inflammatory activities.
Assuntos
Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Betaína-Homocisteína S-Metiltransferase/antagonistas & inibidores , Hiperlipidemias/tratamento farmacológico , Quinazolinas/uso terapêutico , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Apolipoproteína A-I/sangue , Apolipoproteínas E/deficiência , Aterosclerose/sangue , Aterosclerose/etiologia , Aterosclerose/patologia , Linhagem Celular , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Citocinas/sangue , Dieta com Restrição de Gorduras , Dieta Ocidental/efeitos adversos , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais , Perfilação da Expressão Gênica , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/complicações , Hiperlipidemias/dietoterapia , Hiperlipidemias/genética , Inflamação/sangue , Inflamação/prevenção & controle , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinazolinas/farmacologia , Quinazolinonas , RNA Mensageiro/análise , Células U937RESUMO
The structures of the two predominant metabolites (M4 and M5) of RVX-208, observed both in in vitro human and animal liver microsomal incubations, as well as in plasma from animal in vivo studies, were determined. A panel of biocatalytic systems was tested to identify biocatalysts suitable for milligram scale production of metabolite M4 from RVX-208. Rabbit liver S9 fraction was selected as the most suitable system, primarily based on pragmatic metrics such as catalyst cost and estimated yield of M4 (â¼55%). Glucuronidation of RVX-208 catalyzed by rabbit liver S9 fraction was optimized to produce M4 in amounts sufficient for structural characterization. Structural studies using LC/MS/MS analysis and (1)H NMR spectroscopy showed the formation of a glycosidic bond between the primary hydroxyl group of RVX-208 and glucuronic acid. NMR results suggested that the glycosidic bond has the ß-anomeric configuration. A synthetic sample of M4 confirmed the proposed structure. Metabolite M5, hypothesized to be the carboxylate of RVX-208, was prepared using human liver microsomes, purified by HPLC, and characterized by LC/MS/MS and (1)H NMR. The structure was confirmed by comparison to a synthetic sample. Both samples confirmed M5 as a product of oxidation of primary hydroxyl group of RVX-208 to carboxylic acid.
Assuntos
Quinazolinas/isolamento & purificação , Quinazolinas/metabolismo , Animais , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Quinazolinas/química , Quinazolinonas , CoelhosRESUMO
Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.
Assuntos
Apolipoproteína A-I/biossíntese , Proteínas Nucleares/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Animais , Apolipoproteína A-I/genética , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular , Cristalografia por Raios X , Epigênese Genética/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Quinazolinas/química , Quinazolinonas , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Based on the naturally occurring stilbene, Resveratrol, a series of novel stilbene derivatives were synthesized, which have the ability to induce the expression of the ApoA-I gene. Several compounds equally or more potent than Resveratrol were identified. trans-4,4'-dihydroxy-2-methoxystilbene was the most potent (4.6x more potent than Resveratrol). These compounds provide an early lead into new drugs to treat atherosclerosis.
Assuntos
Apolipoproteína A-I/genética , Estilbenos/química , Estilbenos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Células CACO-2 , Humanos , Estrutura Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Estereoisomerismo , Estilbenos/síntese química , Relação Estrutura-Atividade , Transcrição Gênica/genética , Regulação para Cima/genéticaRESUMO
OBJECTIVES: The aim of this study was to determine whether a novel small molecule RVX-208 affects apolipoprotein (apo)A-I and high-density lipoprotein cholesterol (HDL-C) levels in vitro and in vivo. BACKGROUND: Increased apoA-I and HDL-C levels are potential therapeutic targets for reducing atherosclerotic disease. METHODS: HepG2 cells were treated with 0 to 60 mumol/l RVX-208 followed by assays for apoA-I and HDL-C production. For in vivo studies, African green monkeys (AGMs) received 15 to 60 mg/kg/day RVX-208, and the serum was analyzed for lipoprotein levels, HDL-subparticle distribution, cholesterol efflux, and activity of lipid-modifying enzymes. A phase I clinical trial was conducted in healthy volunteers (given 1 to 20 mg/kg/day of RVX-208) to assess safety, tolerability, and pharmacokinetics. RESULTS: The RVX-208 induced apoA-I messenger ribonucleic acid and protein synthesis in HepG2 cells, leading to increased levels of pre-beta-migrating and alpha-lipoprotein particles containing apoA-I (LpA-I) in spent media. Similarly, in AGMs, RVX-208 treatment for 63 days increased serum apoA-I and HDL-C levels (60% and 97%, respectively). In addition, the levels of pre-beta(1)-LpA-I and alpha1-LpA-I HDL-subparticles were increased as well as adenosine triphosphate binding cassette AI, adenosine triphosphate binding cassette G1, and scavenger receptor class B type I-dependent cholesterol efflux. These changes were not mediated by cholesteryl-ester-transfer protein. Treatment of humans for 1 week with oral RVX-208 increased apoA-I, pre-beta-HDL, and HDL functionality. CONCLUSIONS: RVX-208 increases apoA-I and HDL-C in vitro and in vivo. In AGMs, RVX-208 raises serum pre-beta(1)-LpA-I and alpha-LpA-I levels and enhances cholesterol efflux. Data in humans point to beneficial features of RVX-208 that might be useful for treating atherosclerosis.
Assuntos
Apolipoproteína A-I/sangue , Apolipoproteína A-I/efeitos dos fármacos , HDL-Colesterol/sangue , HDL-Colesterol/efeitos dos fármacos , Quinazolinas/farmacologia , Animais , Apolipoproteína A-I/biossíntese , Apolipoproteína A-I/metabolismo , Células Cultivadas , Chlorocebus aethiops , HDL-Colesterol/metabolismo , Cricetinae , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Seguimentos , Células Hep G2/efeitos dos fármacos , Células Hep G2/metabolismo , Humanos , Técnicas In Vitro , Macaca fascicularis , Masculino , Peso Molecular , Probabilidade , Quinazolinas/química , Quinazolinonas , Distribuição Aleatória , Medição de RiscoRESUMO
Peptidase-catalyzed formation of macrocyclic lactams on solid phase identifies ring systems that are favorably bound in the enzyme active site. We evaluated several cyclic peptide motifs linked by ester bonds between the P2 and P1' or the P1 and P2' side chains. The depsipeptide represented by structure 5 was readily generated by a variety of peptidases from precursor omega-amino acids or omega-amino esters. This strategy for identifying ring systems for potential macrocyclic transition state analogues was demonstrated with the serine peptidases trypsin and chymotrypsin, with the aspartic peptidase pepsin, and with the zinc peptidase thermolysin.
Assuntos
Lactamas/síntese química , Peptídeo Hidrolases/química , Amidas/química , Aminoácidos/química , Ácido Aspártico Endopeptidases/química , Sítios de Ligação , Ciclização , Dipeptidases/química , Modelos Químicos , Peptídeos Cíclicos/química , Serina Endopeptidases/química , Especificidade por Substrato , Termolisina/química , Zinco/químicaRESUMO
Specific hydroxy groups of the terminal disaccharide unit of globotriaosyl ceramide (Gb(3)Cer) were identified from binding studies with deoxyGb(3)Cer and verotoxins (VTs) [Nyholm, Magnusson, Zheng, Norel, Binnington-Boyd and Lingwood (1996) Chem. Biol. 3, 263-275]. Four such hydroxy groups (2", 4", 6" and 6') were each substituted with an amino group and the corresponding deoxyamino globotrioses were conjugated to a ceramide-like aglycone which contained an adamantyl group instead of an acyl chain. Such aglycone modification significantly enhanced the water-solubility of the glycoconjugates [Mylvaganam and Lingwood (1999) Biochem. Biophys. Res. Commun. 257, 391-394]. The inhibitory potential of these soluble aminodeoxy conjugates on the binding of VT(1) to Gb(3)Cer immobilized on an ELISA plate was evaluated. Only the 2" and the 6' deoxyamino conjugates were effective inhibitors (IC(50) 10 microM); the 4" and 6" conjugates were ineffective up to 10 mM. To evaluate the importance of incorporating a rigid adamantyl hydrocarbon group into the ceramide aglycone, globotriaose was conjugated to a t- butylacetamido or an adamantaneacetamido aglycone. By similar ELISAs, only the adamantaneacetamido conjugate inhibited the binding of VT(1) to Gb(3)Cer. When deoxyamino conjugates were adsorbed to silica on TLC plates, only the 2" and 6" conjugates bound VT(1) and VT(2). By a similar TLC assay, acetamido derivatives of 2" and 6' deoxyamino conjugates showed less binding to VT(1) and VT(2). Neither the crystallographically determined structure of the VT(1)-globotriaose complex nor modelling studies fully explain the binding patterns shown by these deoxyamino glycoconjugates. Enhanced solvation of the ammonium group of the deoxyamino conjugate could enforce greater constraints in the binding interactions.