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1.
J Biol Chem ; : 107851, 2024 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-39357825

RESUMO

Tripartite ATP-independent periplasmic (TRAP) transporters are analogous to ABC transporters in that they use a substrate-binding protein to scavenge metabolites (e.g., N-acetylneuraminate) and deliver them to the membrane components for import. TRAP substrate-binding proteins are thought to bind the substrate using a two-state (open and closed) induced-fit mechanism. We solved the structure of the TRAP N-acetylneuraminate substrate-binding protein from Aggregatibacter actinomycetemcomitans (AaSiaP) in both the open ligand-free and closed liganded conformations. Surprisingly, we also observed an intermediate conformation, where AaSiaP is mostly closed and is bound to a non-cognate ligand, acetate, which hints at how N-acetylneuraminate binding stabilises a fully closed state. AaSiaP preferentially binds N-acetylneuraminate (KD = 0.4 µM) compared to N-glycolylneuraminate (KD = 4.4 µM), which is explained by the closed-N-acetylneuraminate bound structure. Small-angle X-ray scattering data alongside molecular dynamics simulations suggest the AaSiaP adopts a more open state in solution than in crystal. However, the open unliganded conformation can also sample closed conformations. Molecular dynamics simulations also demonstrate the importance of water molecules for stabilising the closed conformation. Although our data is consistent with an induced fit model of binding, we suggest that the open unliganded conformation may sample multiple states capable of binding substrate. The mechanism by which the ligand is released for import remains to be determined.

2.
J Biol Chem ; 297(4): 101113, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34437902

RESUMO

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the ß-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.


Assuntos
Proteínas de Bactérias/química , Carboidratos Epimerases/química , Hexosaminas/química , Staphylococcus aureus/enzimologia , Fosfatos Açúcares/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Carboidratos Epimerases/genética , Catálise , Hexosaminas/genética , Hexosaminas/metabolismo , Mutação de Sentido Incorreto , Conformação Proteica em Folha beta , Domínios Proteicos , Staphylococcus aureus/genética , Fosfatos Açúcares/genética , Fosfatos Açúcares/metabolismo
3.
J Biol Chem ; 295(10): 3301-3315, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31949045

RESUMO

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the "boat" conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.


Assuntos
Proteínas de Bactérias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Staphylococcus aureus/enzimologia , Motivos de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Hexosaminas/química , Hexosaminas/metabolismo , Cinética , Fosfotransferases (Aceptor do Grupo Álcool)/química , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Zinco/química , Zinco/metabolismo
4.
Elife ; 122024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38349818

RESUMO

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.


Assuntos
Haemophilus influenzae , Ácido N-Acetilneuramínico , Haemophilus influenzae/metabolismo , Microscopia Crioeletrônica , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo
5.
Eur J Immunol ; 42(11): 2990-3000, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22837158

RESUMO

The molecular basis underlying T-cell recognition of MHC molecules presenting altered peptide ligands is still not well-established. A hierarchy of T-cell activation by MHC class I-restricted altered peptide ligands has been defined using the T-cell receptor P14 specific for H-2D(b) in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H-2D(b) in complex with the alanine-substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H-2D(b) /gp33 was strictly enthalpy driven, recognition of the weak agonist H-2D(b) /Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H-2D(b) /gp33 compared with H-2D(b) /Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex.


Assuntos
Antígenos Virais/química , Glicoproteínas/química , Antígenos H-2/química , Fragmentos de Peptídeos/química , Peptídeos/química , Proteínas/química , Receptores de Antígenos de Linfócitos T/química , Proteínas Virais/química , Animais , Antígenos Virais/imunologia , Dicroísmo Circular , Cristalografia por Raios X , Glicoproteínas/imunologia , Antígenos H-2/imunologia , Cinética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Peptídeos/imunologia , Proteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Organismos Livres de Patógenos Específicos , Ressonância de Plasmônio de Superfície , Termodinâmica , Proteínas Virais/imunologia
6.
Nature ; 448(7149): 92-6, 2007 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-17611542

RESUMO

Oxygen-evolving photosynthetic organisms regulate carbon metabolism through a light-dependent redox signalling pathway. Electrons are shuttled from photosystem I by means of ferredoxin (Fdx) to ferredoxin-thioredoxin reductase (FTR), which catalyses the two-electron-reduction of chloroplast thioredoxins (Trxs). These modify target enzyme activities by reduction, regulating carbon flow. FTR is unique in its use of a [4Fe-4S] cluster and a proximal disulphide bridge in the conversion of a light signal into a thiol signal. We determined the structures of FTR in both its one- and its two-electron-reduced intermediate states and of four complexes in the pathway, including the ternary Fdx-FTR-Trx complex. Here we show that, in the first complex (Fdx-FTR) of the pathway, the Fdx [2Fe-2S] cluster is positioned suitably for electron transfer to the FTR [4Fe-4S] centre. After the transfer of one electron, an intermediate is formed in which one sulphur atom of the FTR active site is free to attack a disulphide bridge in Trx and the other sulphur atom forms a fifth ligand for an iron atom in the FTR [4Fe-4S] centre--a unique structure in biology. Fdx then delivers a second electron that cleaves the FTR-Trx heterodisulphide bond, which occurs in the Fdx-FTR-Trx complex. In this structure, the redox centres of the three proteins are aligned to maximize the efficiency of electron transfer from the Fdx [2Fe-2S] cluster to the active-site disulphide of Trxs. These results provide a structural framework for understanding the mechanism of disulphide reduction by an iron-sulphur enzyme and describe previously unknown interaction networks for both Fdx and Trx (refs 4-6).


Assuntos
Ferredoxinas/química , Proteínas Ferro-Enxofre/química , Oxirredutases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Transporte de Elétrons , Ferredoxinas/metabolismo , Ferro/química , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Proteínas Recombinantes , Spinacia oleracea , Relação Estrutura-Atividade , Synechocystis , Tiorredoxinas/química , Tiorredoxinas/metabolismo
7.
Nat Commun ; 14(1): 1120, 2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36849793

RESUMO

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism.


Assuntos
Proteínas de Membrana Transportadoras , Ácido N-Acetilneuramínico , Transporte Biológico , Archaea , Trifosfato de Adenosina
8.
ACS Chem Biol ; 17(7): 1890-1900, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35675124

RESUMO

Antibiotic resistance is a major worldwide concern, and new drugs with mechanistically novel modes of action are urgently needed. Here, we report the structure-based drug design, synthesis, and evaluation in vitro and in cellular systems of sialic acid derivatives able to inhibit the bacterial sialic acid symporter SiaT. We designed and synthesized 21 sialic acid derivatives and screened their affinity for SiaT by a thermal shift assay and elucidated the inhibitory mechanism through binding thermodynamics, computational methods, and inhibitory kinetic studies. The most potent compounds, which have a 180-fold higher affinity compared to the natural substrate, were tested in bacterial growth assays and indicate bacterial growth delay in methicillin-resistant Staphylococcus aureus. This study represents the first example and a promising lead in developing sialic acid uptake inhibitors as novel antibacterial agents.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Antibacterianos/química , Cinética , Testes de Sensibilidade Microbiana , Ácido N-Acetilneuramínico/farmacologia
9.
Nat Commun ; 12(1): 1988, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790291

RESUMO

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Ácidos Siálicos/metabolismo , Regulação Alostérica , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Motivos de Nucleotídeos/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Repressoras/genética
10.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 1): 24-33, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19153463

RESUMO

Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe-2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe-2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/química , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Tolueno/metabolismo , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Flavina-Adenina Dinucleotídeo/química , Ferro/química , Modelos Químicos , Complexos Multienzimáticos/química , NAD/química , Ligação Proteica , Pseudomonas putida/enzimologia , Especificidade por Substrato
11.
J Am Chem Soc ; 131(46): 16606-7, 2009 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-19877613

RESUMO

We report the first molecular dynamics simulations of an integral membrane protein in a detergent micelle under vacuum conditions. To mimic the dehydration process in electrospray ionization, the N-terminal outer membrane protein A transmembrane domain (OmpA171) from Escherichia coli embedded in a dodecylphosphocholine (DPC) detergent micelle has been simulated with water shells of varying thickness. Removal of the water molecules leaves the membrane protein relatively unaffected by the vacuum conditions. The major structural change occurs in the surrounding micelle, where the DPC molecules structurally rearrange from a normal-phase micelle with DPC detergents radiating spherically from OmpA171 to a structure where the DPC molecules form a layered onion structure in which the head groups, which strive to interact with each other, form an intermediate layer between the inner layer of tail groups that are expelled to the surface, protruding into the void.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Micelas , Simulação de Dinâmica Molecular , Detergentes/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Estrutura Secundária de Proteína , Vácuo
12.
Artigo em Inglês | MEDLINE | ID: mdl-19574645

RESUMO

7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris-HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3(2)21, with unit-cell parameters a = b = 100.1, c = 196.8 A, and diffracted to 2.7 A resolution.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , HIV-2/química , HIV-2/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Animais , Cromatografia em Gel , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Testes de Neutralização , Estrutura Secundária de Proteína
13.
FEBS Lett ; 593(1): 52-66, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30411345

RESUMO

N-Acetylglucosamine-6-phosphate deacetylase (NagA) and glucosamine-6-phosphate deaminase (NagB) are branch point enzymes that direct amino sugars into different pathways. For Staphylococcus aureus NagA, analytical ultracentrifugation and small-angle X-ray scattering data demonstrate that it is an asymmetric dimer in solution. Initial rate experiments show hysteresis, which may be related to pathway regulation, and kinetic parameters similar to other bacterial isozymes. The enzyme binds two Zn2+ ions and is not substrate inhibited, unlike the Escherichia coli isozyme. S. aureus NagB adopts a novel dimeric structure in solution and shows kinetic parameters comparable to other Gram-positive isozymes. In summary, these functional data and solution structures are of use for understanding amino sugar metabolism in S. aureus, and will inform the design of inhibitory molecules.


Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Staphylococcus aureus/enzimologia , alfa-N-Acetilgalactosaminidase/química , alfa-N-Acetilgalactosaminidase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cinética , Modelos Moleculares , Multimerização Proteica , Espalhamento a Baixo Ângulo , Staphylococcus aureus/química , Ultracentrifugação , Difração de Raios X , Zinco/metabolismo
14.
ACS Med Chem Lett ; 10(4): 437-443, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30996776

RESUMO

Aldo-keto reductase 1C3 (AKR1C3) is an attractive target in drug design for its role in resistance to anticancer therapy. Several nonsteroidal anti-inflammatory drugs such as indomethacin are known to inhibit AKR1C3 in a nonselective manner because of COX-off target effects. Here we designed two indomethacin analogues by proposing a bioisosteric connection between the indomethacin carboxylic acid function and either hydroxyfurazan or hydroxy triazole rings. Both compounds were found to target AKR1C3 in a selective manner. In particular, hydroxyfurazan derivative is highly selective for AKR1C3 over the 1C2 isoform (up to 90-times more) and inactive on COX enzymes. High-resolution crystal structure of its complex with AKR1C3 shed light onto the binding mode of the new inhibitors. In cell-based assays (on colorectal and prostate cancer cells), the two indomethacin analogues showed higher potency than indomethacin. Therefore, these two AKR1C3 inhibitors can be used to provide further insight into the role of AKR1C3 in cancer.

15.
Eur J Med Chem ; 163: 266-280, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529545

RESUMO

Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) has been clinically validated as a target for antimalarial drug discovery, as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. Here, we have identified new hydroxyazole scaffold-based PfDHODH inhibitors belonging to two different chemical series. The first series was designed by a scaffold hopping strategy that exploits the use of hydroxylated azoles. Within this series, the hydroxythiadiazole 3 was identified as the best selective PfDHODH inhibitor (IC50 12.0 µM). The second series was designed by modulating four different positions of the hydroxypyrazole scaffold. In particular, hydroxypyrazoles 7e and 7f were shown to be active in the low µM range (IC50 2.8 and 5.3 µM, respectively). All three compounds, 3, 7e and 7f showed clear selectivity over human DHODH (IC50 > 200 µM), low cytotoxicity, and retained micromolar activity in P. falciparum-infected erythrocytes. The crystallographic structures of PfDHODH in complex with compounds 3 and 7e proved their binding mode, supplying essential data for future optimization of these scaffolds.


Assuntos
Antimaláricos/química , Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Antimaláricos/farmacologia , Azóis/química , Azóis/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Eritrócitos/parasitologia , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Ligação Proteica , Pirazóis/química , Pirazóis/farmacologia , Relação Estrutura-Atividade
16.
Nat Commun ; 9(1): 5245, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30532032

RESUMO

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5.


Assuntos
Glucose/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/metabolismo , Sódio/metabolismo , Simportadores/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Feminino , Humanos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Florizina/metabolismo , Florizina/farmacologia , Ligação Proteica , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo , Transportador 2 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/genética , Xenopus laevis
17.
Biophys Rev ; 10(2): 219-227, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29222808

RESUMO

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.

18.
Eur J Med Chem ; 150: 930-945, 2018 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-29602039

RESUMO

The aldo-keto reductase 1C3 (AKR1C3) isoform plays a vital role in the biosynthesis of androgens and is considered an attractive target in prostate cancer (PCa). No AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid and indomethacine are non-steroidal anti-inflammatory drugs known to inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. Recently, we employed a scaffold hopping approach to design a new class of potent and selective AKR1C3 inhibitors based on a N-substituted hydroxylated triazole pharmacophore. Following a similar strategy, we designed a new series focused around an acidic hydroxybenzoisoxazole moiety, which was rationalised to mimic the benzoic acid role in the flufenamic scaffold. Through iterative rounds of drug design, synthesis and biological evaluation, several compounds were discovered to target AKR1C3 in a selective manner. The most promising compound of series (6) was found to be highly selective (up to 450-fold) for AKR1C3 over the 1C2 isoform with minimal COX1 and COX2 off-target effects. Other inhibitors were obtained modulating the best example of hydroxylated triazoles we previously presented. In cell-based assays, the most promising compounds of both series reduced the cell proliferation, prostate specific antigen (PSA) and testosterone production in AKR1C3-expressing 22RV1 prostate cancer cells and showed synergistic effect when assayed in combination with abiraterone and enzalutamide. Structure determination of AKR1C3 co-crystallized with one representative compound from each of the two series clearly identified both compounds in the androstenedione binding site, hence supporting the biochemical data.


Assuntos
Membro C3 da Família 1 de alfa-Ceto Redutase/antagonistas & inibidores , Antineoplásicos/farmacologia , Benzoxazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Ácido Flufenâmico/farmacologia , Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Benzoxazóis/síntese química , Benzoxazóis/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ácido Flufenâmico/síntese química , Ácido Flufenâmico/química , Humanos , Estrutura Molecular , Antígeno Prostático Específico/antagonistas & inibidores , Antígeno Prostático Específico/metabolismo , Relação Estrutura-Atividade , Testosterona/antagonistas & inibidores , Testosterona/biossíntese
19.
Front Chem ; 6: 233, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30023356

RESUMO

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter.

20.
Microb Biotechnol ; 11(2): 420-428, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29345069

RESUMO

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.


Assuntos
Automação Laboratorial/métodos , Clonagem Molecular/métodos , Enzimas/isolamento & purificação , Escherichia coli/metabolismo , Expressão Gênica , Redes e Vias Metabólicas/genética , Ácido N-Acetilneuramínico/metabolismo , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Testes Genéticos/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
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