Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and enhanced UV resistance.

We investigated whether mutations in the p53 tumor suppressor gene alter UV sensitivity and/or repair of UV-induced DNA damage in primary human skin fibroblasts from patients with Li-Fraumeni syndrome, heterozygous for mutations in one allele of the p53 gene (p53 wt/mut) and sublines expressing only mutant p53 (p53 mut). The p53 mut cells were more resistant than the p53 wt/mut cells to UV cytotoxicity and exhibited less UV-induced apoptosis. DNA repair analysis revealed reduced removal of cyclobutane pyrimidine dimers from overall genomic DNA in vivo in p53 mut cells compared with p53 wt/mut or normal cells. However, p53 mut cells retained the ability to preferentially repair damage in the transcribed strands of expressed genes (transcription-coupled repair). These results suggest that loss of p53 function may lead to greater genomic instability by reducing the efficiency of DNA repair but that cellular resistance to DNA-damaging agents may be enhanced through elimination of apoptosis.

The bend in RNA created by the trans-activation response element bulge of human immunodeficiency virus is straightened by arginine and by Tat-derived peptide.

The trans-activation response element (TAR) found near the 5' end of the viral RNA of the human immunodeficiency virus contains a 3-nt bulge that is recognized by the virally encoded trans-activator protein (Tat), an important mediator of transcriptional activation. Insertion of the TAR bulge into double-stranded RNA is known to result in reduced electrophoretic mobility, suggestive of a bulge-induced bend. Furthermore, NMR studies indicate that Arg causes a change in the structure of the TAR bulge, possibly reducing the bulge angle. However, neither of these effects has been quantified, nor have they been compared with the effects of the TAR-Tat interaction. Recently, an approach for the quantification of bulge-induced bends has been described in which hydrodynamic measurements, employing the method of transient electric birefringence, have yielded precise estimates for the angles of a series of RNA bulges, with the angles ranging from 7 degrees to 93 degrees. In the current study, transient electric birefringence measurements indicate that the TAR bulge introduces a bend of 50 degrees +/- 5 degrees in the absence of Mg2+. Addition of Arg leads to essentially complete straightening of the helix (to < 10 degrees) with a transition midpoint in the 1 mM range. This transition demonstrates specificity for the TAR bulge: no comparable transition was observed for U3 or A3 (control) bulges with differing flanking sequences. An essentially identical structural transition is observed for the Tat-derived peptide, although the transition midpoint for the latter is near 1 microM. Finally, low concentrations of Mg2+ alone reduce the bend angle by approximately 50%, consistent with the effects of Mg2+ on other pyrimidine bulges. This last observation is important in view of the fact that most previous structural/binding studies were performed in the absence of Mg2+.

Efeitos da glutamina e taurina sobre a via de sinalização do NFκB em células Raw 264.7 estimuladas com LPS

O sistema imunológico preserva a integridade do organismo perante o ambiente que ele está inserido. As reações imunológicas são essenciais para controle e eliminação da infecção, no entanto se os mecanismos contra regulatórios da resposta imunológica forem superados, a homeostasia pode falhar levando a um desequilíbrio no processo de reparo do organismo, podendo causar danos, insuficiência de órgão e até a morte. Muitos estudos têm demonstrado a interação entre o sistema imunológico e os aminoácidos. Visto que a glutamina é utilizada como substrato energético para enterócitos, além de fornecer nitrogênio para síntese de purinas e pirimidinas para proliferação celular e a taurina participa da hemostasia, estabilização de membranas, mobilização de cálcio, além de ser importante agente antioxidante, nos propusemos nesse trabalho investigar os efeitos da glutamina e taurina sobre aspectos relacionados a resposta imunológica de células da linhagem Raw 264.7. Para tanto foram avaliadas: viabilidade celular; a capacidade proliferativa e o ciclo celular; a capacidade de síntese de citocinas: IL-1 α, IL-1β, IL-6, IL-10 e TNF-α e a expressão do fator de transcrição NFκB, bem como de seu inibidor IκBα. Foi possível observar aumento da viabilidade e proliferação celular para células tratadas com glutamina, entretanto não foi observado o mesmo efeito para taurina. Quando ambos aminoácidos foram associados, houve prevalência dos efeitos da glutamina. Observamos neste trabalho, que houve uma tendência da diminuição de expressão da relação de p-NFκB/NFκB quando se aumentou a concentração de glutamina. Paralelamente, o mesmo foi encontrado para relação p-IκB/IκB. Esses resultados corroboram com os resultados encontrados na produção das citocinas pró-inflamatórias IL-1α, Il-1β e TNF-α, uma vez que ao aumentarmos a concentração de glutamina bem como glutamina e taurina, observamos menor produção das mesmas. Complementarmente, encontramos resultados opostos para a citocina anti-inflamatória IL-10, a qual teve maior síntese em resposta ao aumento da concentração dos aminoácidos. Portanto concluímos que tanto a glutamina quanto a taurina possuem capacidade de modular aspectos da resposta imunológica de células Raw 264.7.

Desenvolvimento de uma plataforma modelo para a busca de ligantes com potencial leishmanicida baseado na inibição seletiva da enzima diidroorotato desidrogenase

As doenças tropicais negligenciadas (DTNs) causam um imenso sofrimento para a pessoa acometida e em muitos casos podem levar o indivíduo a morte. Elas representam um obstáculo devastador para a saúde e continuam a ser um sério impedimento para a redução da pobreza e desenvolvimento socioeconômico. Das 17 doenças desse grupo, a leishmaniose, incluindo a leishmaniose cutânea, tem grande destaque devido sua alta incidência, os gastos para o tratamento e as complicações geradas em processos de coinfecção. Ainda mais agravante, os investimentos direcionados ao controle, combate e principalmente a inovação em novos produtos é ainda muito limitado. Atualmente, a academia tem um importante papel na luta contra essas doenças através da busca de novos alvos terapêuticos e também de novas moléculas com potencial terapêutico. É nesse contexto que esse projeto teve como meta a implantação de uma plataforma para a identificação de moléculas com atividade leishmanicida. Como alvo terapêutico, optamos pela utilização da enzima diidroorotato desidrogenase de Leishmania Viannia braziliensis (LbDHODH), enzima de extrema importância na síntese de novo de nucleotídeos de pirimidina, cuja principal função é converter o diidroorotato em orotato. Esta enzima foi clonada, expressa e purificada com sucesso em nosso laboratório. Os estudos permitiram que a enzima fosse caracterizada cineticamente e estruturalmente via cristalografia de raios- X. Os primeiros ensaios inibitórios foram realizados com o orotato, produto da catálise e inibidor natural da enzima. O potencial inibitório do orotato foi mensurado através da estimativa do IC50 e a interação proteína-ligante foi caracterizada através de estudos cristalográficos. Estratégias in silico e in vitro foram utilizadas na busca de ligantes, através das quais foram identificados inibidores para a enzima LbDHODH. Ensaios de validação cruzada, utilizando a enzima homóloga humana, permitiram identificar os ligantes com maior índice de seletividade que tiveram seu potencial leishmanicida avaliado in vitro contra as formas promastigota e amastigota de Leishmania braziliensis. A realização do presente projeto permitiu a identificação de uma classe de ligantes que apresentam atividade seletiva contra LbDHODH e que será utilizada no planejamento de futuras gerações de moléculas com atividade terapêutica para o tratamento da leishmaniose. Além disso, a plataforma de ensaios otimizada permitirá a avaliação de novos grupos de moléculas como uma importante estratégia na busca por novos tratamentos contra a leishmaniose

New hybrid materials based on the grafting of Pd(II)-amino complexes on the graphitic surface of AC: preparation, structures and catalytic properties

A novel procedure for the preparation of solid Pd(II)-based catalysts consisting of the anchorage of designed Pd(II)-complexes on an activated carbon (AC) surface is reported. Two molecules of the Ar–S–F type (where Ar is a plane-pyrimidine moiety, F a Pd(II)-ligand and S an aliphatic linker) differing in F, were grafted on AC by π–π stacking of the Ar moiety and the graphene planes of the AC, thus favouring the retaining of the metal-complexing ability of F. Adsorption of Pd(II) by the AC/Ar–S–F hybrids occurs via Pd(II)-complexation by F. After deep characterization, the catalytic activities of the AC/Ar–S–F/Pd(II) hybrids on the hydrogenation of 1-octene in methanol as a catalytic test were evaluated. 100% conversion to n-octane at T = 323.1 K and P = 15 bar, was obtained with both catalysts and most of Pd(II) was reduced to Pd(0) nanoparticles, which remained on the AC surface. Reusing the catalysts in three additional cycles reveals that the catalyst bearing the F ligand with a larger Pd-complexing ability showed no loss of activity (100% conversion to n-octane) which is assigned to its larger structural stability. The catalyst with the weaker F ligand underwent a progressive loss of activity (from 100% to 79% in four cycles), due to the constant aggregation of the Pd(0) nanoparticles. Milder conditions, T = 303.1 K and P = 1.5 bar, prevent the aggregation of the Pd(0) nanoparticles in this catalyst allowing the retention of the high catalytic efficiency (100% conversion) in four reaction cycles.

Tyrosine 394 is phosphorylated in Alzheimer's paired helical filament tau and in fetal tau with c-Abl as the candidate tyrosine kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimer's disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl) pyrazolo[3,4-d] pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimer's disease.

Structure-activity relationships for a new family of sulfonylurea herbicides

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyzes the first common step in branched-chain amino acid biosynthesis. The enzyme is inhibited by several chemical classes of compounds and this inhibition is the basis of action of the sulfonylurea and imidazolinone herbicides. The commercial sulfonylureas contain a pyrimidine or a triazine ring that is substituted at both meta positions, thus obeying the initial rules proposed by Levitt. Here we assess the activity of 69 monosubstituted sulfonylurea analogs and related compounds as inhibitors of pure recombinant Arabidopsis thaliana AHAS and show that disubstitution is not absolutely essential as exemplified by our novel herbicide, monosulfuron (2-nitro-N-(4'-methyl-pyrimidin-2'-yl) phenyl-sulfonylurea), which has a pyrimidine ring with a single meta substituent. A subset of these compounds was tested for herbicidal activity and it was shown that their effect in vivo correlates well with their potency in vitro as AHAS inhibitors. Three-dimensional quantitative structure-activity relationships were developed using comparative molecular field analysis and comparative molecular similarity indices analysis. For the latter, the best result was obtained when steric, electrostatic, hydrophobic and H-bond acceptor factors were taken into consideration. The resulting fields were mapped on to the published crystal structure of the yeast enzyme and it was shown that the steric and hydrophobic fields are in good agreement with sulfonylurea-AHAS interaction geometry.

Molecular and cellular effects of ultraviolet light-induced genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earth's stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.

Immunochemical detection of sequence-specific modifications to DNA induced by UV light

Sequence specificity of antibodies to UV-damaged DNA has not been described previously. The antisera investigated here were specific for UV-modified DNA and were absolutely dependent upon the presence of thymine residues. Using a series of oligonucleotides in competition ELISA, increased inhibition was observed with increasing chain length of UV-polythymidylate. A minimum of three adjacent thymines was required for effective inhibition; alone, dimers of thymine were poor antigens. Although UV-irradiated poly(dC) was not antigenic, cytosines could partially replace thymines within the smallest effective epitope (T-T-T) with a high degree of sequence specificity, not previously described. The main epitope induced by UV was formed from adjacent thymines and either a 3' or a 5' pyrimidine.

Nucleoside and oligonucleotide approaches towards potential HIV chemotherapies

The use of oligonucleotides directed against the mRNA of HIV promises site-specific inhibition of viral replication. In this work, the effect of aralkyl substituents on oligonucleotide duplex stability was studied using model oligonucleotide sequences in an attempt to improve targeting of oligonucleotides to viral mRNA. Arakyl-substituted oligonucleotides were made by solid phase synthesis using either the appropriate aralkyl-substituted phosphoramidite or by post-synthetic substitution of a pentafluorophenoxy substituent by N-methylphenethylamine. The presence of phenethyl or benzoyl substituents invariably resulted in thermodynamic destabilisation of all duplexes studied. The methods which were developed for the synthesis of nucleoside intermediates for oligonucleotide applications were also used to prepare a series of nucleoside analogues derived from uridine, 2'-deoxyuridine and AZT. Crystal structures of six compounds were successfully determined. Anti-HIV activity was observed for most compounds in the series although none were without cytotoxicity. The most active compound of the series was the ribose nucleoside; 1-β-D-erythro-pentofuranosyl-4-pentafluorophenoxy-pyrimidine-2(1H)-one 95, derived directly from uridine. The same series of compounds also displayed very modest anti-cancer activity. To enable synthesis of prooligonucleotides and analogues for possible antisense applications, the properties of a new Silyl-Linked Controlled Pore Glass solid support were investigated. Synthesis of the sequences d(Tp)7T, d(Tps)7T and the base-sensitive d(Tp)3(CBzp)2(Tp)2T was achieved using the silyl-linked solid support in a fluoride-induced cleavage/deprotection strategy.

Design and synthesis of potential antimicrobial agents

Tuberculosis is one of the most devastating diseases in the world primarily due to several decades of neglect and an emergence of multidrug-resitance strains (MDR) of M. tuberculosis together with the increased incidence of disseminated infections produced by other mycobacterium in AIDS patients. This has prompted the search for new antimycobacterial drugs. A series of pyridine-2-, pyridine-3-, pyridine-4-, pyrazine and quinoline-2-carboxamidrazone derivatives and new classes of carboxamidrazone were prepared in an automated fashion and by traditional synthesis. Over nine hundred synthesized compounds were screened for their anti mycobacterial activity against M. fortutium (NGTG 10394) as a surrogate for M. tuberculosis. The new classes of amidrazones were also screened against tuberculosis H37 Rv and antimicrobial activities against various bacteria. Fifteen tested compounds were found to provide 90-100% inhibition of mycobacterium growth of M. tuberculosis H37 Rv in the primary screen at 6.25 μg mL-1. The most active compound in the carboxamidrazone amide series had an MIG value of 0.1-2 μg mL-1 against M. fortutium. The enzyme dihydrofolate reductase (DHFR) has been a drug-design target for decades. Blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infection. The x-ray structure of DHFR from M. tuberculosis and human DHFR were found to have differences in substrate binding site. The presence of glycerol molecule in the Xray structure from M. tuberculosis DHFR provided opportunity to design new antifolates. The new antifolates described herein were designed to retain the pharmcophore of pyrimethamine (2,4- diamino-5(4-chlorophenyl)-6-ethylpyrimidine), but encompassing a range of polar groups that might interact with the M. tuberculosis DHFR glycerol binding pockets. Finally, the research described in this thesis contributes to the preparation of molecularly imprinted polymers for the recognition of 2,4-diaminopyrimidine for the binding the target. The formation of hydrogen bonding between the model functional monomer 5-(4-tert-butyl-benzylidene)-pyrimidine-2,4,6-trione and 2,4-diaminopyrimidine in the pre-polymerisation stage was verified by 1H-NMR studies. Having proven that 2,4-diaminopyrimidine interacts strongly with the model 5-(4-tert-butylbenzylidene)- pyrimidine-2,4,6-trione, 2,4-diaminopyrimidine-imprinted polymers were prepared using a novel cyclobarbital derived functional monomer, acrylic acid 4-(2,4,6-trioxo-tetrahydro-pyrimidin-5- ylidenemethyl)phenyl ester, capable of multiple hydrogen bond formation with the 2,4- diaminopyrimidine. The recognition property of the respective polymers toward the template and other test compounds was evaluated by fluorescence. The results demonstrate that the polymers showed dose dependent enhancement of fluorescence emissions. In addition, the results also indicate that synthesized MIPs have higher 2,4-diaminopyrimidine binding ability as compared with corresponding non-imprinting polymers.

Cyclic and acyclic modifications of 5-aminoimidazole-4-carboxamide

The Introduction gives a brief resume' of the biologically important aspects of 5 -aminoimidazole -4 -carbozamide (1) and explores., in-depth, the synthetic routes to this imidazole. All documented reactions of 5 -aninoimidanole-4 -carboxamide are reviewed in detail, with particular emphasis on the preparation and subsequent coupling reactions of 5 –diazo-imidazole-4 -carboxamide (6). A series of thirteen novel amide 5-amino-2-arylazoimidazole-4-carboxamide derivatives (117-129) were prepared by the coupling of aryldiazonium salts with 5-aminoimidazole-4-carboxamide. Chemical modification of these azo-dyes resulted in the preparation of eight previously unknown acyl derivatives (136-143) Interaction of 5-amino-2-arylazoimidazole-4-carboxides with ethyl formate in sodium ethoxide effected pyrimidine ring closure to the novel 8-arylazohypoxanthines (144 and 145). Several reductive techniques were employed in an effort to obtain the elusive 2,5-diaminoimidazole-4-carboxamide (71),a candidate chemotherapeutic agent, from the arylazoiridazoles. No success can be reported although 5-amino-2-(3-aminoindazol-2-yl) imidazole-4-carboxamide (151) was isolated due to a partial reduction and intramolecular cyclisation of 5-amino72-(2-cyanaphenylazo)imidazole-4-carboxamide (122) .Further possible synthetic approaches to the diaminoimidazole are discussed in Chapter 4. An interesting degradation of a known unstable nitrohydrazone is described in Chapter 5.This resulted in formation of 1, 1-bis(pyrazol--3-ylazo)-1-nitroethane (164) instead of the expected cyclisation to a bicyclic tetrazine N-oxide. An improved preparation of 5-diazoinidazole-4-carboxamide has been achieved, and the diazo-azole formed cycloadducts with isocyanates to yield the hitherto unknown imidazo[5,1-d][1,2,3,5]tetrazin-7(6H)-ones. Eleven derivatives (167-177) of this new ring-system were prepared and characterised. Chemical and spectroscopic investigation showed this ring-system to be unstable under certain conditions, and a comparative study of stability within the group has been made. "Retro-cycloaddition" under protic and photolytic conditions was an unexpected property of 6-substituted imidazo[5,1-d][1,2,3,5]tetrazin--7(0)-ones.Selected examples of the imidazotetrazinone ring-system were tested for antitumour activity. The results of biological evaluation are given in Chapter 7, and have culminated in a Patent application by the collaborating body, May and Baker Ltd. One compound,3-carbamoyl-6-(2-chloro-ethyl)imidazo[5,1-d][1,2,3,5jtetrazin-7(6H)-one (175),shows striking anti-tumour activity in rodent test systems.

Formulation of immunomodulatory agents

Reversed-phase high-performance liquid chromatography procedures were developed for the analysis of pyrimidine-based drugs bropirimine and its derivatives (2-N-acetyl- and 2-N-propanoyl-) and for pyrimethamine and its 2/4- substituted derivatives (2, N-propanoyl and 2,4-N, N-dipropanoyl-) and its 6- substituted (methyl-, ethyl-, propyl- and isopropyl- carboxylates) analogues. Stability studies indicated that these derivatives were not sufficiently labile to act as potential prodrugs. Solubility-pH profiles were constructed from which the dissociation constants were calculated. The physicochemical properties of these compounds were studied and attempts were made to increase the poor aqueous solubility of bropirimine (35μg/mL) by prodrug synthesis, solvate formation (acetic acid, N, N-dimethylformamide and N-methylformamide) and the use of co-solvents and additives. The first two methods proved to be fruitless whereas the latter method resulted in an intravenous formulation incorporating 32mg/mL of bropirimine. An in-vitro method for the detection of precipitation was developed and the results suggested that by using low injection rates (< 0.24mL/min) and high mobile phase flow rates (> 500mL/hr) precipitation could be minimised. Differential scanning calorimetry showed that bropirimine debrominates in the presence of a number of additives commonly used in formulation work but the temperature at which this occurred were usually > 200oC. In-vitro work gave encouraging results for the possibility of rectal delivery of bropirimine but in-vivo work on rabbits showed considerable variations in the resulting plasma levels and pharmacokinetic parameters.

Design, synthesis and biological evaluation of potential antibacterial oligonucleotides

Purine and pyrimidine triplex-forming oligonucleotides (TFOs), as potential antibacterial agents, were designed to bind by Hoogsteen and reverse Hoogsteen hydrogen bonds in a sequence specific manner in the major groove of genomic DNA at specific polypurine sites within the gyrA gene of E. coli and S. pneumoniae. Sequences were prepared by automated synthesis, with purification and characterisation determined by high performance liquid chromatograpy, capillary electrophoresis and mass spectrometry. Triplex stability was assessed using melting curves where the binding of the third strand to the duplex target, was assessed over a temperature range of 0-80°C, and at pH 6.4 and 7.2. The most successful of the unmodified TFOs (6) showed a Tm value of 26 °C at both pH values with binding via reverse Hoogsteen bonds. Binding to genomic DNA was also demonstrated by spectrofluorimetry, using fluorescein-labelled TFOs, from which dissociation constants were determined. Modifications in the form of 5mC, 5' acridine attachment, phosphorothioation, 2'-0-methylation and phosphoramidation, were made in order to. increase Tm values. Phosphoramidate modification was the most with increased Tm values of 42°C. However, the final purity of these sequences was poor due to their difficult syntheses. FACS (fluorescent activated cell sorting) analysis was used to determine the potential uptake of a fluorescently labelled analogue of 6 via passive, coJd shock mediated, and anionic liposome aided, uptake. This was established at 20°C and 37°C. At both temperatures anionic lipid-mediated uptake produced unrivalled fluorescence, equivalent to 20 and 43% at 20 and 37°C respectively. Antibacterial activity of each oligonucleotide was assessed by viable count anaJysis relying on passive uptake, cold shocking techniques, chlorpromazine-mediated uptake, and, cationic and anionic lipid-aided uptake. All oligonucleotides were assessed for their ability to enhance uptake, which is a major barrier to the effectiveness of these agents. Compound 6 under cold shocking conditions produced the greatest consistent decline in colony forming units per ml. Results for this compound were sometimes variable indicating inconsistent uptake by this particular assay method.

Differential impact of amino acid substitutions on critical residues of the human glucagon-like peptide-1 receptor involved in peptide activity and small-molecule allosterys

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor systemis highly complex, able to be activated by both endogenous [GLP-1(1-36)NH2, GLP-1(1-37), GLP-1(7-36)NH2, GLP-1(7-37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tertbutylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptidemediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca2+ mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.