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1.
ACS Med Chem Lett ; 14(12): 1746-1753, 2023 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-38116405

RESUMO

The transcription factor YAP-TEAD is the downstream effector of the Hippo pathway which controls cell proliferation, apoptosis, tissue repair, and organ growth. Dysregulation of the Hippo pathway has been correlated with carcinogenic processes. A co-crystal structure of TEAD with its endogenous ligand palmitic acid (PA) as well as with flufenamic acid (FA) has been disclosed. Here we report the development of HC-258, which derives from FA and possesses an oxopentyl chain that mimics a molecule of PA as well as an acrylamide that reacts covalently with TEAD's cysteine. HC-258 reduces the CTGF, CYR61, AXL, and NF2 transcript levels and inhibits the migration of MDA-MB-231 breast cancer cells. Co-crystallization with hTEAD2 confirmed that HC-258 binds within TEAD's PA pocket, where it forms a covalent bond with its cysteine.

2.
Bioinform Adv ; 3(1): vbad136, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37822724

RESUMO

Motivation: The automated data processing provided by the TSA-CRAFT tool enables now to reach high throughput speed analysis of thermal shift assays. While the software is powerful and freely available, it still requires installation process and command line efforts that could be discouraging. Results: To simplify the procedure, we decided to make it available and easy to use by implementing it with a graphical interface via a web server, enabling a cross-platform usage from any web browsers. We developed a web server embedded version of the TSA-CRAFT tool, enabling a user-friendly graphical interface for formatting and submission of the input file and visualization of the selected thermal denaturation profiles. We describe a typical case study of buffer condition optimization of the biologically relevant APH(3')-IIb bacterial protein in a 96 deep-well thermal shift analysis screening. Availability and implementation: wTSA-CRAFT is freely accessible for noncommercial usage at https://bioserv.cbs.cnrs.fr/TSA_CRAFT.

3.
Bioorg Med Chem Lett ; 95: 129488, 2023 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-37770003

RESUMO

The Hippo pathway regulates organ size and tissue homeostasis by controlling cell proliferation and apoptosis. The YAP-TEAD transcription factor, the downstream effector of the Hippo pathway, regulates the expression of genes such as CTGF, Cyr61, Axl and NF2. Aberrant Hippo activity has been identified in multiple types of cancers. Flufenamic acid (FA) was reported to bind in a liphophilic TEAD palmitic acid (PA) pocket, leading to reduction of the expression of Axl and NF2. Here, we show that the replacement of the trifluoromethyl moiety in FA by aromatic groups, directly connected to the scaffold or separated by a linker, leads to compounds with better affinity to TEAD. Co-crystallization studies show that these compounds bind similarly to FA, but deeper within the PA pocket. Our studies identified LM-41 and AF-2112 as two TEAD binders that strongly reduce the expression of CTGF, Cyr61, Axl and NF2. LM-41 gave the strongest reduction of migration of human MDA-MB-231 breast cancer cells.


Assuntos
Ácido Flufenâmico , Neoplasias , Humanos , Ácido Flufenâmico/farmacologia , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Via de Sinalização Hippo , Neoplasias/genética
4.
Protein Sci ; 32(8): e4703, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37338125

RESUMO

Inosine 5'-monophosphate (IMP) dehydrogenase (IMPDH) is an ubiquitous enzyme that catalyzes the NAD+ -dependent oxidation of inosine 5'-monophosphate into xanthosine 5'-monophosphate. This enzyme is formed of two distinct domains, a core domain where the catalytic reaction occurs, and a less-conserved Bateman domain. Our previous studies gave rise to the classification of bacterial IMPDHs into two classes, according to their oligomeric and kinetic properties. MgATP is a common effector but cause to different effects when it binds within the Bateman domain: it is either an allosteric activator for Class I IMPDHs or a modulator of the oligomeric state for Class II IMPDHs. To get insight into the role of the Bateman domain in the dissimilar properties of the two classes, deleted variants of the Bateman domain and chimeras issued from the interchange of the Bateman domain between the three selected IMPDHs have been generated and characterized using an integrative structural biology approach. Biochemical, biophysical, structural, and physiological studies of these variants unveil the Bateman domain as being the carrier of the molecular behaviors of both classes.


Assuntos
Trifosfato de Adenosina , IMP Desidrogenase , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Bactérias/metabolismo , Inosina
5.
Eur J Med Chem ; 246: 114941, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36455355

RESUMO

Nicotinamide adenine dinucleotide kinases (NAD kinases) are essential and ubiquitous enzymes involved in the production of NADP(H) which is an essential cofactor in many metabolic pathways. Targeting NAD kinase (NADK), a rate limiting enzyme of NADP biosynthesis pathway, represents a new promising approach to treat bacterial infections. Previously, we have produced the first NADK inhibitor active against staphylococcal infection. From this linear di-adenosine derivative, namely NKI1, we designed macrocyclic analogues. Here, we describe the synthesis and evaluation of an original series of cyclic diadenosine derivatives as NADK inhibitors of two pathogenic bacteria, Listeria monocytogenes and Staphylococcus aureus. The nature and length of the link between the two adenosine units were examined leading to sub-micromolar inhibitors of NADK1 from L. monocytogenes, including its most potent in vitro inhibitor reported so far (with a 300-fold improvement compared to NKI1).


Assuntos
Adenosina , Fosfotransferases (Aceptor do Grupo Álcool) , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Adenosina/farmacologia , Relação Estrutura-Atividade , Bactérias/metabolismo
6.
FEBS J ; 290(2): 482-501, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36036789

RESUMO

Multidrug resistance is a major public health problem that requires the urgent development of new antibiotics and therefore the identification of novel bacterial targets. The activity of nicotinamide adenine dinucleotide kinase, NADK, is essential in all bacteria tested so far, including many human pathogens that display antibiotic resistance leading to the failure of current treatments. Inhibiting NADK is therefore a promising and innovative antibacterial strategy since there is currently no drug on the market targeting this enzyme. Through a fragment-based drug design approach, we have recently developed a NAD+ -competitive inhibitor of NADKs, which displayed in vivo activity against Staphylococcus aureus. Here, we show that this compound, a di-adenosine derivative, is inactive against the NADK enzyme from the Gram-negative bacteria Pseudomonas aeruginosa (PaNADK). This lack of activity can be explained by the crystal structure of PaNADK, which was determined in complex with NADP+ in this study. Structural analysis led us to design and synthesize a benzamide adenine dinucleoside analogue, active against PaNADK. This novel compound efficiently inhibited PaNADK enzymatic activity in vitro with a Ki of 4.6 µm. Moreover, this compound reduced P. aeruginosa infection in vivo in a zebrafish model.


Assuntos
Antibacterianos , NAD , Pseudomonas aeruginosa , Animais , Antibacterianos/farmacologia , Antibacterianos/química , NAD/análogos & derivados , Fosfotransferases (Aceptor do Grupo Álcool) , Pseudomonas aeruginosa/efeitos dos fármacos , Peixe-Zebra , Desenho de Fármacos
7.
Mol Cell ; 82(17): 3299-3311.e8, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35868311

RESUMO

NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.


Assuntos
Lisina , NAD , Acetilação , Domínio Catalítico , Humanos , Lisina/metabolismo , Proteínas Mitocondriais/metabolismo , NAD/metabolismo , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Prolina/metabolismo
8.
J Med Chem ; 65(8): 5926-5940, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35389210

RESUMO

The Hippo signaling pathway plays a fundamental role in the control of organ growth, cell proliferation, and stem cell characters. TEADs are the main transcriptional output regulators of the Hippo signaling pathway and bind to YAP and TAZ co-activators. TEAD1-4 are expressed differently, depending on the tissue and developmental level, and can be overexpressed in certain pathologies. TEAD ligands mainly target the internal pocket of the C-terminal domain of TEAD, and the first ligands selective for TEAD1 and TEAD3 have been recently reported. In this paper, we focus on the topographic homology of the TEAD C-terminal domain both externally and in the internal pocket to highlight the possibility of rationally designing ligands selective for one of the TEAD family members. We identified a novel TEAD2-specific pocket and reported its first ligand. Finally, AlphaFold2 models of full-length TEADs suggest TEAD autoregulation and emphasize the importance of the interface 2.


Assuntos
Via de Sinalização Hippo , Fatores de Transcrição , Proliferação de Células , Ligantes , Fatores de Transcrição/metabolismo
9.
J Med Chem ; 65(2): 1552-1566, 2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-34958586

RESUMO

Dabrafenib is an anticancer drug currently used in the clinics, alone or in combination. However, dabrafenib was recently shown to potently activate the human nuclear receptor pregnane X receptor (PXR). PXR activation increases the clearance of various chemicals and drugs, including dabrafenib itself. It may also enhance cell proliferation and tumor aggressiveness. Therefore, there is a need for rational design of a potent protein kinase B-Raf inhibitor devoid of binding to the secondary target PXR and resisting rapid metabolism. By determining the crystal structure of dabrafenib bound to PXR and analyzing its mode of binding to both PXR and its primary target, B-Raf-V600E, we were able to derive new compounds with nanomolar activity against B-Raf and no detectable affinity for PXR. The crystal structure of B-Raf in complex with our lead compound revealed a subdomain swapping of the activation loop with potentially important functional implications for a prolonged inhibition of B-Raf-V600E.


Assuntos
Proliferação de Células , Desenho de Fármacos , Imidazóis/farmacologia , Melanoma/tratamento farmacológico , Oximas/farmacologia , Receptor de Pregnano X/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Cristalografia por Raios X , Humanos , Imidazóis/química , Melanoma/patologia , Simulação de Acoplamento Molecular , Oximas/química , Ligação Proteica , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
10.
Eur J Med Chem ; 226: 113835, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34509860

RESUMO

The Hippo pathway is involved in organ size control and tissue homeostasis by regulating cell growth, proliferation and apoptosis. It controls the phosphorylation of the transcription co-activator YAP (Yes associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif) in order to control their nuclear import and their interaction with TEAD (Transcriptional Enhanced Associated Domain). YAP, TAZ and TEADs are dysregulated in several cancers making YAP/TAZ-TEAD interaction a new emerging anti-cancer target. We report the synthesis of a set of trisubstituted pyrazoles which bind to hTEAD2 at the interface 2 revealing for the first time a cryptic pocket created by the movement of the phenol ring of Y382. Compound 6 disrupts YAP/TAZ-TEAD interaction in HEK293T cells and inhibits TEAD target genes and cell proliferation in MDA-MB-231 cells. Compound 6 is therefore the first inhibitor of YAP/TAZ-TEAD targeting interface 2. This molecule could serve with other pan-TEAD inhibitors such as interface 3 ligands, for the delineation of the relative importance of VGLL vs YAP/TAZ in a given cellular model.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Descoberta de Drogas , Pirazóis/farmacologia , Fatores de Transcrição de Domínio TEA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-Atividade , Fatores de Transcrição de Domínio TEA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo
11.
Molecules ; 25(21)2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105870

RESUMO

Nicotinamide adenine dinucleotide (NAD) kinases are essential and ubiquitous enzymes involved in the tight regulation of NAD/nicotinamide adenine dinucleotide phosphate (NADP) levels in many metabolic pathways. Consequently, they represent promising therapeutic targets in cancer and antibacterial treatments. We previously reported diadenosine derivatives as NAD kinase inhibitors with bactericidal activities on Staphylococcus aureus. Among them, one compound (namely NKI1) was found effective in vivo in a mouse infection model. With the aim to gain detailed knowledge about the selectivity and mechanism of action of this lead compound, we planned to develop a chemical probe that could be used in affinity-based chemoproteomic approaches. Here, we describe the first functionalized chemical probe targeting a bacterial NAD kinase. Aminoalkyl functional groups were introduced on NKI1 for further covalent coupling to an activated SepharoseTM matrix. Inhibitory properties of functionalized NKI1 derivatives together with X-ray characterization of their complexes with the NAD kinase led to identify candidate compounds that are amenable to covalent coupling to a matrix.


Assuntos
Adenina/análogos & derivados , Adenosina/síntese química , Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Adenina/síntese química , Adenina/farmacologia , Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Camundongos , Modelos Moleculares , NADP/química , Conformação Proteica , Sefarose/química , Staphylococcus aureus
12.
ACS Infect Dis ; 6(3): 422-435, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32017533

RESUMO

Antibiotic resistance is a worldwide threat due to the decreasing supply of new antimicrobials. Novel targets and innovative strategies are urgently needed to generate pathbreaking drug compounds. NAD kinase (NADK) is essential for growth in most bacteria, as it supports critical metabolic pathways. Here, we report the discovery of a new class of antibacterials that targets bacterial NADK. We generated a series of small synthetic adenine derivatives to screen those harboring promising substituents in order to guide efficient fragment linking. This led to NKI1, a new lead compound inhibiting NADK that showed in vitro bactericidal activity against Staphylococcus aureus. In a murine model of infection, NKI1 restricted survival of the bacteria, including methicillin-resistant S. aureus. Collectively, these findings identify bacterial NADK as a potential drug target and NKI1 as a lead compound in the treatment of staphylococcal infections.


Assuntos
Antibacterianos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Adenina/química , Adenina/farmacologia , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Feminino , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Bibliotecas de Moléculas Pequenas , Staphylococcus aureus/enzimologia , Relação Estrutura-Atividade
13.
Gastroenterology ; 157(5): 1368-1382, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31336123

RESUMO

BACKGROUND & AIMS: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury. METHODS: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif-/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis. RESULTS: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 µmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 µmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif-/- mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. CONCLUSIONS: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.


Assuntos
Inibidores Enzimáticos/farmacologia , Hepatopatias/prevenção & controle , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Peptidil-Prolil Isomerase F/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Peptidil-Prolil Isomerase F/genética , Peptidil-Prolil Isomerase F/metabolismo , Citoproteção , Modelos Animais de Doenças , Humanos , Fígado/enzimologia , Fígado/patologia , Hepatopatias/enzimologia , Hepatopatias/genética , Hepatopatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
14.
Eur J Med Chem ; 167: 124-132, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30769241

RESUMO

Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme in many bacterial pathogens and is considered as a potential drug target for the development of new antibacterial agents. Our recent work has revealed the crucial role of one of the two structural domains (i.e. Bateman domain) in the regulation of the quaternary structure and enzymatic activity of bacterial IMPDHs. Thus, we have screened chemical libraries to search for compounds targeting the Bateman domain and identified first in-class allosteric inhibitors of a bacterial IMPDH. These inhibitors were shown to counteract the activation by the natural positive effector, MgATP, and to block the enzyme in its apo conformation (low affinity for IMP). Our structural studies demonstrate the versatility of the Bateman domain to accommodate totally unrelated chemical scaffolds and pave the way for the development of allosteric inhibitors, an avenue little explored until now.


Assuntos
Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Regulação Alostérica , Apoproteínas/química , Apoproteínas/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Domínios Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas
16.
Artigo em Inglês | MEDLINE | ID: mdl-29760125

RESUMO

Although members of the Flaviviridae display high incidence, morbidity, and mortality rates, the development of specific antiviral drugs for each virus is unlikely. Cyclophilins, a family of host peptidyl-prolyl cis-trans isomerases (PPIases), play a pivotal role in the life cycles of many viruses and therefore represent an attractive target for broad-spectrum antiviral development. We report here the pangenotypic anti-hepatitis C virus (HCV) activity of a small-molecule cyclophilin inhibitor (SMCypI). Mechanistic and modeling studies revealed that the SMCypI bound to cyclophilin A in competition with cyclosporine (CsA), inhibited its PPIase activity, and disrupted the CypA-nonstructural protein 5A (NS5A) interaction. Resistance selection showed that the lead SMCypI hardly selected amino acid substitutions conferring low-level or no resistance in vitro Interestingly, the SMCypI selected D320E and Y321H substitutions, located in domain II of the NS5A protein. These substitutions were previously associated with low-level resistance to cyclophilin inhibitors such as alisporivir. Finally, the SMCypI inhibited the replication of other members of the Flaviviridae family with higher 50% effective concentrations (EC50s) than for HCV. Thus, because of its chemical plasticity and simplicity of synthesis, our new family of SMCypIs represents a promising new class of drugs with the potential for broad-spectrum anti-Flaviviridae activity as well as an invaluable tool to explore the role of cyclophilins in viral life cycles.


Assuntos
Antivirais/farmacologia , Ciclofilina A/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Substituição de Aminoácidos/genética , Ciclofilina A/metabolismo , Ciclosporina/farmacologia , Farmacorresistência Viral/genética , Hepacivirus/crescimento & desenvolvimento , Hepatite C/tratamento farmacológico , Humanos , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos
17.
Structure ; 26(4): 545-554.e4, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29503074

RESUMO

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1,368 amino acid C terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein-kinase fold devoid of catalytic activity, despite a conserved catalytic lysine (K997). By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein-kinase domain is flanked by N- and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. These results reveal a dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation. Further sequence-structure analysis identified an additional member (C19orf35) of the superfamily of dimeric Pragmin/SgK269/PEAK1 pseudo-kinases.


Assuntos
Substituição de Aminoácidos , Proteínas de Transporte/química , Tirosina/química , Quinases da Família src/química , Motivos de Aminoácidos , Sítios de Ligação , Proteína Tirosina Quinase CSK , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tirosina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
18.
Eur J Med Chem ; 124: 1041-1056, 2016 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-27783975

RESUMO

Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus, in the micromolar range. They behave as NAD mimics with the 5',5'-diphosphate group substituted by a 8,5' thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5' position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics.


Assuntos
Adenosina/química , Adenosina/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Listeria monocytogenes/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Adenosina/metabolismo , Sequência de Aminoácidos , Inibidores Enzimáticos/metabolismo , Humanos , Simulação de Acoplamento Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ligação Proteica , Conformação Proteica , Ribose/química , Staphylococcus aureus/enzimologia , Relação Estrutura-Atividade
19.
Nat Commun ; 7: 12777, 2016 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-27652979

RESUMO

Cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyse the interconversion of the peptide bond at proline residues. Several cyclophilins play a pivotal role in the life cycle of a number of viruses. The existing cyclophilin inhibitors, all derived from cyclosporine A or sanglifehrin A, have disadvantages, including their size, potential for side effects unrelated to cyclophilin inhibition and drug-drug interactions, unclear antiviral spectrum and manufacturing issues. Here we use a fragment-based drug discovery approach using nucleic magnetic resonance, X-ray crystallography and structure-based compound optimization to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors with potent in vitro PPIase inhibitory activity and antiviral activity against hepatitis C virus, human immunodeficiency virus and coronaviruses. This family of compounds has the potential for broad-spectrum, high-barrier-to-resistance treatment of viral infections.

20.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 9): 1890-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26327379

RESUMO

Inosine-5'-monophosphate dehydrogenases (IMPDHs), which are the rate-limiting enzymes in guanosine-nucleotide biosynthesis, are important therapeutic targets. Despite in-depth functional and structural characterizations of various IMPDHs, the role of the Bateman domain containing two CBS motifs remains controversial. Their involvement in the allosteric regulation of Pseudomonas aeruginosa IMPDH by Mg-ATP has recently been reported. To better understand the function of IMPDH and the importance of the CBS motifs, the structure of a variant devoid of these modules (ΔCBS) was solved at high resolution in the apo form and in complex with IMP. In addition, a single amino-acid substitution variant, D199N, was also structurally characterized: the mutation corresponds to the autosomal dominant mutant D226N of human IMPDH1, which is responsible for the onset of the retinopathy adRP10. These new structures shed light onto the possible mechanism of regulation of the IMPDH enzymatic activity. In particular, three conserved loops seem to be key players in this regulation as they connect the tetramer-tetramer interface with the active site and show significant modification upon substrate binding.


Assuntos
IMP Desidrogenase/química , Pseudomonas aeruginosa/enzimologia , Regulação Alostérica , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
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