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1.
Int J Mol Sci ; 25(15)2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-39125622

RESUMO

Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials.


Assuntos
Antibacterianos , Inibidores Enzimáticos , Escherichia coli , Exorribonucleases , Antibacterianos/farmacologia , Antibacterianos/química , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Domínio Catalítico , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Bactérias/efeitos dos fármacos , Bactérias/enzimologia
2.
Haematologica ; 106(6): 1616-1623, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32354869

RESUMO

Complement C3 binds fibrinogen and compromises fibrin clot lysis thereby enhancing thrombosis risk. We investigated the role of fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analysing: i) consistency in the fibrinolytic properties of C3, ii) binding sites between fibrinogen and C3 and iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modelling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed Affimers, were used to modulate C3-fibrinogen interaction and fibrinolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein [195±105 and 522±166 seconds, respectively (p=0.04)], with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide microarray screening identified 2 potential C3-fibrinogen interactions sites within fibrinogen ß chain (residues 424-433, 435-445). One fibrinogen-binding Affimer was isolated that displayed sequence identity with C3 in an exposed area of the protein. This Affimer abolished C3-induced prolongation of fibrinolysis (728±25.1 seconds to 632±23.7 seconds, p=0.005) and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen ß-chain and disruption of fibrinogen-C3 interaction using Affimer proteins enhances fibrinolysis, which represents a potential novel target tool to reduce thrombosis in high risk individuals.


Assuntos
Fibrinogênio , Trombose , Complemento C3 , Fibrina , Fibrinólise , Humanos , Trombose/tratamento farmacológico , Trombose/etiologia , Trombose/prevenção & controle
3.
Proc Natl Acad Sci U S A ; 115(1): E72-E81, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29247053

RESUMO

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.


Assuntos
Complexo Antígeno-Anticorpo/química , Imunoglobulina G/química , Simulação de Dinâmica Molecular , Receptores de IgG/química , Regulação Alostérica , Complexo Antígeno-Anticorpo/imunologia , Humanos , Imunoglobulina G/imunologia , Receptores de IgG/imunologia
4.
Bioorg Med Chem ; 27(16): 3546-3550, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31257079

RESUMO

Previously we have reported on a series of pyridine-3-carboxamide inhibitors of DNA gyrase and DNA topoisomerase IV that were designed using a computational de novo design approach and which showed promising antibacterial properties. Herein we describe the synthesis of additional examples from this series aimed specifically at DNA gyrase, along with crystal structures confirming the predicted mode of binding and in vitro ADME data which describe the drug-likeness of these compounds.


Assuntos
DNA Girase/genética , Escherichia coli/metabolismo , Inibidores da Topoisomerase II/uso terapêutico , Modelos Moleculares , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/farmacologia
5.
Mol Microbiol ; 106(3): 492-504, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28876489

RESUMO

Clavulanic acid and avibactam are clinically deployed serine ß-lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo ß-lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine ß-lactamase) but not L1 (metallo ß-lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X-ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam-like ß-lactams plus nonclassical ß-lactamase inhibitors, particularly avibactam-like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.


Assuntos
Stenotrophomonas maltophilia/metabolismo , Inibidores de beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Compostos Azabicíclicos/metabolismo , Compostos Azabicíclicos/farmacologia , Aztreonam , Proteínas de Bactérias/metabolismo , Ceftazidima , Cristalografia por Raios X/métodos , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismo
6.
EMBO J ; 33(16): 1831-44, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-24952894

RESUMO

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Hidantoínas/metabolismo , Ligação de Hidrogênio , Ligantes , Micrococcaceae/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Relação Estrutura-Atividade
7.
Artigo em Inglês | MEDLINE | ID: mdl-28115348

RESUMO

ß-Lactamase-mediated resistance is a growing threat to the continued use of ß-lactam antibiotics. The use of the ß-lactam-based serine-ß-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-ß-lactam inhibitor avibactam has extended the utility of ß-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-ß-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-ß-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of "dual-action" inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-ß-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of ß-lactamase, including the class A extended spectrum ß-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of ß-lactams against Gram-negative clinical isolates expressing a variety of ß-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other ß-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-ß-lactamase catalysis.


Assuntos
Antibacterianos/farmacologia , Ácidos Borônicos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Resistência beta-Lactâmica/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , Motivos de Aminoácidos , Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ácidos Borônicos/síntese química , Clonagem Molecular , Cristalografia por Raios X , Ciclização , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Enterobacteriaceae/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Resistência beta-Lactâmica/genética , Inibidores de beta-Lactamases/síntese química , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia
8.
Bioorg Med Chem Lett ; 27(16): 3878-3882, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28669445

RESUMO

Two new tricyclic ß-aminoacrylate derivatives (2e and 3e) have been found to be inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with Ki 0.037 and 0.15µM respectively. 1H and 13C NMR spectroscopic data show that these compounds undergo ready cis-trans isomerisation at room temperature in polar solvents. In silico docking studies indicate that for both molecules there is neither conformation nor double bond configuration which bind preferentially to PfDHODH. This flexibility is favourable for inhibitors of this channel that require extensive positioning to reach their binding site.


Assuntos
Acrilatos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Acrilatos/síntese química , Acrilatos/química , Di-Hidro-Orotato Desidrogenase , Relação Dose-Resposta a Droga , Simulação de Acoplamento Molecular , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Relação Estrutura-Atividade
9.
Nat Chem Biol ; 10(6): 457-62, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24776929

RESUMO

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder ß-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.


Assuntos
AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Fosfatos de Dinucleosídeos/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , Proteínas Musculares/metabolismo , Canais de Potássio/metabolismo , Animais , Sítios de Ligação , Western Blotting , Cristalografia por Raios X , GMP Cíclico/química , GMP Cíclico/metabolismo , Fosfatos de Dinucleosídeos/química , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteínas Musculares/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genética , Nó Sinoatrial/citologia , Nó Sinoatrial/efeitos dos fármacos , Nó Sinoatrial/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Transfecção
10.
Orig Life Evol Biosph ; 46(4): 425-434, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27220497

RESUMO

We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H2P2O52-; PPi(III)], to the phosphate group of 5'-adenosine mono phosphate (5'-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M2+) and by organic co-factors such as acetate (AcO-). In this specific case of P-transfer from PPi(III) to 5'-AMP, we show a synergistic enhancement of transfer in the combined presence of M2+ & AcO-. Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5'-AMP, [P(III)P(V)-5'-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.


Assuntos
Monofosfato de Adenosina/química , Cátions Bivalentes/química , Origem da Vida , Compostos de Fósforo/química , Metais
11.
Blood ; 121(11): 2117-26, 2013 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-23303819

RESUMO

Formation of a stable fibrin clot is dependent on interactions between factor XIII and fibrin. We have previously identified a key residue on the αC of fibrin(ogen) (Glu396) involved in binding activated factor XIII-A(2) (FXIII-A(2)*); however, the functional role of this interaction and binding site(s) on FXIII-A(2)* remains unknown. Here we (1) characterized the functional implications of this interaction; (2) identified by liquid-chromatography-tandem mass spectrometry the interacting residues on FXIII-A(2)* following chemical cross-linking of fibrin(ogen) αC389-402 peptides to FXIII-A(2)*; and (3) carried out molecular modeling of the FXIII-A(2)*/peptide complex to identify contact site(s) involved. Results demonstrated that inhibition of the FXIII-A(2)*/αC interaction using αC389-402 peptide (Pep1) significantly decreased incorporation of biotinamido-pentylamine and α2-antiplasmin to fibrin, and fibrin cross-linking, in contrast to Pep1-E396A and scrambled peptide controls. Pep1 did not inhibit transglutaminase-2 activity, and incorporation of biotinyl-TVQQEL to fibrin was only weakly inhibited. Molecular modeling predicted that Pep1 binds the activation peptide cleft (AP-cleft) within the ß-sandwich domain of FXIII-A(2)* localizing αC cross-linking Q366 to the FXIII-A(2)* active site. Our findings demonstrate that binding of fibrin αC389-402 to the AP-cleft is fundamental to clot stabilization and presents this region of FXIII-A(2)* as a potential site involved in glutamine-donor substrate recognition.


Assuntos
Domínio Catalítico , Fator XIII/química , Fator XIII/metabolismo , Fibrinogênio/metabolismo , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Trombina/metabolismo , Animais , Células CHO , Domínio Catalítico/genética , Cricetinae , Cricetulus , Fibrinogênio/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Quaternária de Proteína
12.
Org Biomol Chem ; 12(3): 486-94, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-24276506

RESUMO

An approach for designing bioactive small molecules has been developed in which de novo structure-based ligand design (SBLD) was focused on regions of chemical space accessible using a diversity-oriented synthetic approach. The approach was exploited in the design and synthesis of a focused library of platensimycin analogues in which the complex bridged ring system was replaced with a series of alternative ring systems. The affinity of the resulting compounds for the C163Q mutant of FabF was determined using a WaterLOGSY competition binding assay. Several compounds had significantly improved affinity for the protein relative to a reference ligand. The integration of synthetic accessibility with ligand design enabled focus to be placed on synthetically-accessible regions of chemical space that were relevant to the target protein under investigation.


Assuntos
Acetiltransferases/antagonistas & inibidores , Adamantano/farmacologia , Aminobenzoatos/farmacologia , Anilidas/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Acetiltransferases/genética , Acetiltransferases/metabolismo , Adamantano/síntese química , Adamantano/química , Aminobenzoatos/síntese química , Aminobenzoatos/química , Anilidas/síntese química , Anilidas/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/antagonistas & inibidores , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Ligantes , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade
13.
Parasitology ; 141(1): 17-27, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23768800

RESUMO

In light of the low success rate of target-based genomics and HTS (High Throughput Screening) approaches in anti-infective drug discovery, in silico structure-based drug design (SBDD) is becoming increasingly prominent at the forefront of drug discovery. In silico SBDD can be used to identify novel enzyme inhibitors rapidly, where the strength of this approach lies with its ability to model and predict the outcome of protein-ligand binding. Over the past 10 years, our group have applied this approach to a diverse number of anti-infective drug targets ranging from bacterial D-ala-D-ala ligase to Plasmodium falciparum DHODH. Our search for new inhibitors has produced lead compounds with both enzyme and whole-cell activity with established on-target mode of action. This has been achieved with greater speed and efficiency compared with the more traditional HTS initiatives and at significantly reduced cost and manpower.


Assuntos
Anti-Infecciosos/química , Descoberta de Drogas , Inibidores Enzimáticos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Peptídeo Sintases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Anti-Infecciosos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Di-Hidro-Orotato Desidrogenase , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Humanos , Ligantes , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Simulação de Acoplamento Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interface Usuário-Computador
14.
Bioorg Chem ; 55: 69-76, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24962384

RESUMO

In recent years bacterial resistance has been observed against many of our current antibiotics, for instance most worryingly against the cephalosporins which are typically the last line of defence against many bacterial infections. Additionally the failure of high throughput screening in the discovery of new antibacterial drug leads has led to a decline in the number of antibacterial agents reaching the market. Alternative methods of drug discovery including structure based drug design are needed to meet the threats caused by the emergence of resistance. In this review we explore the latest advancements in the identification of new antibacterial agents through the use of a number of structure based drug design programs.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Desenho de Fármacos , Descoberta de Drogas , Animais , Humanos , Relação Estrutura-Atividade
15.
Mol Membr Biol ; 30(2): 184-94, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22908980

RESUMO

Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.


Assuntos
Sistemas de Transporte de Aminoácidos/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Leucina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo
16.
Growth Horm IGF Res ; 77: 101607, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39033666

RESUMO

Type 2 diabetes is characterised by the disruption of insulin and insulin-like growth factor (IGF) signalling. The key hubs of these signalling cascades - the Insulin receptor (IR) and Insulin-like growth factor 1 receptor (IGF1R) - are known to form functional IR-IGF1R hybrid receptors which are insulin resistant. However, the mechanisms underpinning IR-IGF1R hybrid formation are not fully understood, hindering the ability to modulate this for future therapies targeting this receptor. To pinpoint suitable sites for intervention, computational hotspot prediction was utilised to identify promising epitopes for targeting with point mutagenesis. Specific IGF1R point mutations F450A, R391A and D555A show reduced affinity of the hybrid receptor in a BRET based donor-saturation assay, confirming hybrid formation could be modulated at this interface. These data provide the basis for rational design of more effective hybrid receptor modulators, supporting the prospect of identifying a small molecule that specifically interacts with this target.


Assuntos
Mutagênese Sítio-Dirigida , Receptor IGF Tipo 1 , Receptor de Insulina , Receptor de Insulina/genética , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Humanos , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismo , Multimerização Proteica , Peptídeos Semelhantes à Insulina , Antígenos CD
17.
iScience ; 27(1): 108477, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38205261

RESUMO

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

18.
Pharmaceuticals (Basel) ; 16(2)2023 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-37259406

RESUMO

Increases in antibiotic usage and antimicrobial resistance occurrence have caused a dramatic reduction in the effectiveness of many frontline antimicrobial treatments. Topoisomerase inhibitors including fluoroquinolones are broad-spectrum antibiotics used to treat a range of infections, which stabilise a topoisomerase-DNA cleavage complex via intercalation of the bound DNA. However, these are subject to bacterial resistance, predominantly in the form of single-nucleotide polymorphisms in the active site. Significant research has been undertaken searching for novel bioactive molecules capable of inhibiting bacterial topoisomerases at sites distal to the fluoroquinolone binding site. Notably, researchers have undertaken searches for anti-infective agents that can inhibit topoisomerases through alternate mechanisms. This review summarises work looking at the inhibition of topoisomerases predominantly through non-intercalating agents, including those acting at a novel allosteric site, ATPase domain inhibitors, and those offering unique binding modes and mechanisms of action.

19.
RSC Med Chem ; 13(4): 360-374, 2022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-35647546

RESUMO

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are dimeric disulfide-linked receptor tyrosine kinases, whose actions regulate metabolic and mitogenic signalling pathways inside the cell. It is well documented that in tissues co-expressing the IR and IGF1R, their respective monomers can heterodimerise to form IR-IGF1R hybrid receptors. Increased populations of the IR-IGF1R hybrid receptors are associated with several disease states, including type 2 diabetes and cancer. Recently, progress in the structural biology of IR and IGF1R has given insights into their structure-function relationships and mechanism of action. However, challenges in isolating IR-IGF1R hybrid receptors mean that their structural properties remain relatively unexplored. This review discusses the advances in the structural understanding of the IR and IGF1R, and how these discoveries can inform the design of small-molecule modulators of the IR-IGF1R hybrid receptors to understand their role in cell biology.

20.
RSC Med Chem ; 13(7): 831-839, 2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35919336

RESUMO

By 2050, it is predicted that antimicrobial resistance will be responsible for 10 million global deaths annually, more deaths than cancer, costing the world economy $100 trillion. Clearly, strategies to address this problem are essential as bacterial evolution is rendering our current antibiotics ineffective. The discovery of an allosteric binding site on the established antibacterial target DNA gyrase offers a new medicinal chemistry strategy. As this site is distinct from the fluoroquinolone binding site, resistance is not yet documented. Using in silico molecular design methods, we have designed and synthesised a novel series of biphenyl-based inhibitors inspired by a published thiophene-based allosteric inhibitor. This series was evaluated in vitro against Escherichia coli DNA gyrase and E. coli topoisomerase IV with the most potent compounds exhibiting IC50 values towards the low micromolar range for DNA gyrase and only ∼2-fold less active against topoisomerase IV. The structure-activity relationships reported herein suggest insights to further exploit this allosteric site, offering a pathway to overcome developing fluoroquinolone resistance.

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