RESUMO
The emergence of drug resistant Mycobacterium tuberculosis, the causative agent of tuberculosis, demands the development of new drugs and new drug targets. We have recently reported that the d-phenylalanine benzoxazole Q112 has potent antibacterial activity against this pathogen with a distinct mechanism of action from other antimycobacterial agents. Q112 and previously reported derivatives were unstable in plasma and no free compound could be observed. Here we expand the structure-activity relationship for antimycobacterial activity and find nonhydrolyzable derivatives with decreased plasma binding. We also show that there is no correlation between antibacterial activity and inhibition of PanG, a putative target for these compounds.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Humanos , Benzoxazóis/farmacologia , Antituberculosos/farmacologia , Antituberculosos/química , Relação Estrutura-Atividade , Testes de Sensibilidade MicrobianaRESUMO
BMS906024, a γ-secretase inhibitor that blocks Notch signaling, was previously shown to inhibit Cryptosporidium parvum growth in vitro. A structure-activity relationship (SAR) analysis of BMS906024 reported herein demonstrates the importance of the stereochemistry of the C-3 benzodiazepine and the succinyl ß-substituent. However, concomitant removal of the succinyl α-substituent and switching the primary amide with secondary amides was tolerated. For example, 32 (SH287) inhibited C. parvum growth in HCT-8 host cells with an EC50 = 6.4 nM and an EC90 = 16 nM; however, blocking C. parvum growth with BMS906024 derivatives was correlative with inhibition of Notch signaling, highlighting that additional SAR analysis will be needed to separate these two activities.
Assuntos
Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , Humanos , Relação Estrutura-AtividadeRESUMO
The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrheal disease and an important contributor to early-childhood mortality. Waterborne outbreaks occur frequently, even in countries with advanced water treatment capabilities, and there is currently no fully effective treatment. Nucleotide pathways are attractive targets for antimicrobial development, and several laboratories are designing inhibitors of these enzymes as potential treatment for Cryptosporidium infections. Here we take advantage of newly available molecular genetics for Cryptosporidium parvum to investigate nucleotide biosynthesis by directed gene ablation. Surprisingly, we found that the parasite tolerates the loss of classical targets including dihydrofolate reductase-thymidylate synthase (DHFR-TS) and inosine monophosphate dehydrogenase (IMPDH). We show that thymidine kinase provides a route to thymidine monophosphate in the absence of DHFR-TS. In contrast, only a single pathway has been identified for C. parvum purine nucleotide salvage. Nonetheless, multiple enzymes in the purine pathway, as well as the adenosine transporter, can be ablated. The resulting mutants are viable under normal conditions but are hypersensitive to inhibition of purine nucleotide synthesis in their host cell. Cryptosporidium might use as-yet undiscovered purine transporters and salvage enzymes; however, genetic and pharmacological experiments led us to conclude that Cryptosporidium imports purine nucleotides from the host cell. The potential for ATP uptake from the host has significant impact on our understanding of parasite energy metabolism given that Cryptosporidium lacks oxidative phosphorylation and glycolytic enzymes are not constitutively expressed throughout the parasite life cycle.
Assuntos
Transporte Biológico/fisiologia , Criptosporidiose/metabolismo , Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , Cryptosporidium parvum/metabolismo , Nucleotídeos/metabolismo , Purinas/metabolismo , Linhagem Celular Tumoral , Humanos , IMP Desidrogenase/metabolismo , Complexos Multienzimáticos/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/metabolismoRESUMO
The remarkable power and specificity of enzyme catalysis rely on the dynamic alignment of the enzyme, substrates, and cofactors, yet the role of dynamics has usually been approached from the perspective of the protein. We have been using an underappreciated NMR technique, subtesla high-resolution field cycling 31P NMR relaxometry, to investigate the dynamics of the enzyme-bound substrates and cofactor on guanosine-5'-monophosphate reductase (GMPR). GMPR forms two dead end, yet catalytically competent, complexes that mimic distinct steps in the catalytic cycle: E·IMP·NADP+ undergoes a partial hydride transfer reaction, while E·GMP·NADP+ undergoes a partial deamination reaction. A different cofactor conformation is required for each partial reaction. Here we report the effects of mutations designed to perturb cofactor conformation and ammonia binding with the goal of identifying the structural features that contribute to the distinct dynamic signatures of the hydride transfer and deamination complexes. These experiments suggest that Asp129 is a central cog in a dynamic network required for both hydride transfer and deamination. In contrast, Lys77 modulates the conformation and mobility of substrates and cofactors in a reaction-specific manner. Thr105 and Tyr318 are part of a deamination-specific dynamic network that includes the 2'-OH of GMP. These residues have comparatively little effect on the dynamic properties of the hydride transfer complex. These results further illustrate the potential of high-resolution field cycling NMR relaxometry for the investigation of ligand dynamics. In addition, exchange experiments indicate that NH3/NH4+ has a high affinity for the deamination complex but a low affinity for the hydride transfer complex, suggesting that the movement of ammonia may gate the cofactor conformational change. Collectively, these experiments reinforce the view that the enzyme, substrates, and cofactor are linked in intricate, reaction-specific, dynamic networks and demonstrate that distal portions of the substrates and cofactors are critical features in these networks.
Assuntos
Coenzimas , GMP Redutase , NADP , Humanos , Amônia/metabolismo , Biocatálise , Coenzimas/química , Coenzimas/metabolismo , GMP Redutase/genética , GMP Redutase/metabolismo , Guanosina Monofosfato/química , Cinética , Conformação Molecular , Mutação , NADP/química , NADP/metabolismo , Ligação ProteicaRESUMO
A microbe's ecological niche and biotechnological utility are determined by its specific set of co-evolved metabolic pathways. The acquisition of new pathways, through horizontal gene transfer or genetic engineering, can have unpredictable consequences. Here we show that two different pathways for coumarate catabolism failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly acquired pathways and the modifications required to overcome these limitations. Both pathways required host mutations to enable effective growth with coumarate, but the necessary mutations differed. In one case, a pathway intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid replacements in IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. Molecular dynamics simulation of the wild type and a representative single-residue mutant provide insight into the structural and dynamic changes that relieve inhibition. These results demonstrate how deleterious interactions can limit pathway transfer, that these interactions can be traced to specific molecular interactions between host and pathway, and how evolution or engineering can alleviate these limitations.
Assuntos
Ácidos Cumáricos/metabolismo , Nucleotídeos de Purina/biossíntese , Acinetobacter baumannii/metabolismo , Escherichia coli/genética , Evolução Molecular , Técnicas de Transferência de Genes , Transferência Genética Horizontal , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Redes e Vias Metabólicas/genética , Simulação de Dinâmica Molecular , Mutação , Nucleotídeos de Purina/antagonistas & inibidores , Nucleotídeos de Purina/genéticaRESUMO
Autosomal dominant progressive external ophthalmoplegia (adPEO) is a late-onset, Mendelian mitochondrial disorder characterised by paresis of the extraocular muscles, ptosis, and skeletal-muscle restricted multiple mitochondrial DNA (mtDNA) deletions. Although dominantly inherited, pathogenic variants in POLG, TWNK and RRM2B are among the most common genetic defects of adPEO, identification of novel candidate genes and the underlying pathomechanisms remains challenging. We report the clinical, genetic and molecular investigations of a patient who presented in the seventh decade of life with PEO. Oxidative histochemistry revealed cytochrome c oxidase-deficient fibres and occasional ragged red fibres showing subsarcolemmal mitochondrial accumulation in skeletal muscle, while molecular studies identified the presence of multiple mtDNA deletions. Negative candidate screening of known nuclear genes associated with PEO prompted diagnostic exome sequencing, leading to the prioritisation of a novel heterozygous c.547G>C variant in GMPR (NM_006877.3) encoding guanosine monophosphate reductase, a cytosolic enzyme required for maintaining the cellular balance of adenine and guanine nucleotides. We show that the novel c.547G>C variant causes aberrant splicing, decreased GMPR protein levels in patient skeletal muscle, proliferating and quiescent cells, and is associated with subtle changes in nucleotide homeostasis protein levels and evidence of disturbed mtDNA maintenance in skeletal muscle. Despite confirmation of GMPR deficiency, demonstrating marked defects of mtDNA replication or nucleotide homeostasis in patient cells proved challenging. Our study proposes that GMPR is the 19th locus for PEO and highlights the complexities of uncovering disease mechanisms in late-onset PEO phenotypes.
Assuntos
DNA Mitocondrial/genética , GMP Redutase/genética , Transtornos de Início Tardio/genética , Músculo Esquelético/enzimologia , Oftalmoplegia/genética , Adenina/metabolismo , Idoso , Células Cultivadas , Deficiência de Citocromo-c Oxidase/metabolismo , Replicação do DNA , DNA Mitocondrial/metabolismo , Feminino , Fibroblastos/enzimologia , GMP Redutase/deficiência , GMP Redutase/metabolismo , Guanina/metabolismo , Células HEK293 , Células HeLa , Heterozigoto , Humanos , Transtornos de Início Tardio/metabolismo , Transtornos de Início Tardio/patologia , Músculo Esquelético/patologia , Oftalmoplegia/enzimologia , Oftalmoplegia/fisiopatologia , Fosforilação Oxidativa , Splicing de RNA , Deleção de Sequência , Sequenciamento do ExomaRESUMO
Inosine-5'-monophosphate dehydrogenase (IMPDH) is a potential target for microorganisms. However, identifying inhibitor design determinants for IMPDH orthologs continues to evolve. Herein, a series of mycophenolic anilide inhibitors of Cryptosporidium parvum and human IMPDHs are reported. Furthermore, molecular docking of 12 (e.g. SH-19; CpIMPDH Ki,app = 0.042 ± 0.015 µM, HsIMPDH2 Ki,app = 0.13 ± 0.05 µM) supports different binding modes with the two enzymes. For CpIMPDH the inhibitor extends into a pocket in an adjacent subunit. In contrast, docking suggests the inhibitor interacts with Ser276 in the NAD binding site in HsIMPDH2, as well as an adjacent pocket within the same subunit. These results provide further guidance for generating IMPDH inhibitors for enzymes found in an array of pathogenic microorganisms, including Mycobacterium tuberculosis.
Assuntos
Anilidas/farmacologia , Antiparasitários/farmacologia , Cryptosporidium parvum/enzimologia , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Anilidas/química , Antiparasitários/química , Sítios de Ligação/efeitos dos fármacos , Criptosporidiose/tratamento farmacológico , Criptosporidiose/parasitologia , Cryptosporidium parvum/metabolismo , Inibidores Enzimáticos/química , Humanos , IMP Desidrogenase/metabolismo , Simulação de Acoplamento Molecular , Fenóis/química , Fenóis/farmacologiaRESUMO
Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In contrast, the effects of RNS damaged metabolites are less appreciated. 5-Amino-3-ß-(d-ribofuranosyl)-3 H-imidazo-[4,5- d][1,3]oxazine-7-one (oxanosine) and its nucleotides are products of guanosine nitrosation. Here we demonstrate that oxanosine monophosphate (OxMP) is a potent reversible competitive inhibitor of IMPDH. The value of Ki varies from 50 to 340 nM among IMPDHs from five different organisms. UV spectroscopy and X-ray crystallography indicate that OxMP forms a ring-opened covalent adduct with the active site Cys (E-OxMP*). Unlike the covalent intermediate of the normal catalytic reaction, E-OxMP* does not hydrolyze, but instead recyclizes to OxMP. IMPDH inhibitors block proliferation and can induce apoptosis, so the inhibition of IMPDH by OxMP presents another potential mechanism for RNS toxicity.
Assuntos
Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Fosfatos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , IMP Desidrogenase/isolamento & purificação , IMP Desidrogenase/metabolismo , Estrutura Molecular , Fosfatos/síntese química , Fosfatos/química , Ribonucleosídeos/síntese química , Ribonucleosídeos/química , Ribonucleosídeos/farmacologiaRESUMO
Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors.
Assuntos
Carbonatos/farmacologia , Enzimas Desubiquitinantes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Carbonatos/síntese química , Carbonatos/química , Enzimas Desubiquitinantes/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Ubiquitinação/efeitos dos fármacosRESUMO
The ability of enzymes to modulate the dynamics of bound substrates and cofactors is a critical feature of catalysis, but the role of dynamics has largely been approached from the perspective of the protein. Here, we use an underappreciated NMR technique, subtesla high-resolution field-cycling 31P NMR relaxometry, to interrogate the dynamics of enzyme bound substrates and cofactors in guanosine-5'-monophosphate reductase (GMPR). These experiments reveal distinct binding modes and dynamic profiles associated with the 31P nuclei in the Michaelis complexes for the deamination and hydride transfer steps of the catalytic cycle. Importantly, the substrate is constrained and the cofactor is more dynamic in the deamination complex E·GMP·NADP+, whereas the substrate is more dynamic and the cofactor is constrained in the hydride transfer complex E·IMP·NADP+. The presence of D2O perturbed the relaxation of the 31P nuclei in E·IMP·NADP+ but not in E·GMP·NADP+, providing further evidence of distinct binding modes with different dynamic properties. dIMP and dGMP are poor substrates, and the dynamics of the cofactor complexes of dGMP/dIMP are disregulated relative to GMP/IMP. The substrate 2'-OH interacts with Asp219, and mutation of Asp219 to Ala decreases the value of Vmax by a factor of 30. Counterintuitively, loss of Asp219 makes both substrates and cofactors less dynamic. These observations suggest that the interactions between the substrate 2'-OH and Asp219 coordinate the dynamic properties of the Michaelis complexes, and these dynamics are important for progression through the catalytic cycle.
Assuntos
GMP Redutase/química , GMP Redutase/fisiologia , Espectroscopia de Ressonância Magnética/métodos , Sítios de Ligação , Catálise , Guanosina/metabolismo , Cinética , Imageamento por Ressonância Magnética , Modelos Moleculares , NADP/metabolismo , Ligação ProteicaRESUMO
Guanosine-5'-monophosphate reductase (GMPR) catalyzes the reduction of GMP to IMP and ammonia with concomitant oxidation of NADPH. Here we investigated the structure and dynamics of enzyme-bound substrates and cofactors by measuring 31P relaxation rates over a large magnetic field range using high resolution field cycling NMR relaxometry. Surprisingly, these experiments reveal differences in the low field relaxation profiles for the monophosphate of GMP compared with IMP in their respective NADP+ complexes. These complexes undergo partial reactions that mimic different steps in the overall catalytic cycle. The relaxation profiles indicate that the substrate monophosphates have distinct interactions in E·IMP·NADP+ and E·GMP·NADP+ complexes. These findings were not anticipated by x-ray crystal structures, which show identical interactions for the monophosphates of GMP and IMP in several inert complexes. In addition, the motion of the cofactor is enhanced in the E·GMP·NADP+ complex. Last, the motions of the substrate and cofactor are coordinately regulated; the cofactor has faster local motions than GMP in the deamination complex but is more constrained than IMP in that complex, leading to hydride transfer. These results show that field cycling can be used to investigate the dynamics of protein-bound ligands and provide new insights into how portions of the substrate remote from the site of chemical transformation promote catalysis.
Assuntos
Coenzimas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , GMP Redutase/química , Biocatálise , Coenzimas/metabolismo , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GMP Redutase/genética , GMP Redutase/metabolismo , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/metabolismo , Inosina Monofosfato/química , Inosina Monofosfato/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , NADP/química , NADP/metabolismo , Ligação ProteicaRESUMO
Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism, growth, and proliferation. mTORC1 has been implicated in many diseases such as cancer, diabetes, and neurodegeneration, and is a target to prolong lifespan. Here we report a small molecule inhibitor (Cbz-B3A) of mTORC1 signaling. Cbz-B3A inhibits the phosphorylation of eIF4E-binding protein 1 (4EBP1) and blocks 68% of translation. In contrast, rapamycin preferentially inhibits the phosphorylation of p70(S6k) and blocks 35% of translation. Cbz-B3A does not appear to bind directly to mTORC1, but instead binds to ubiquilins 1, 2, and 4. Knockdown of ubiquilin 2, but not ubiquilins 1 and 4, decreases the phosphorylation of 4EBP1, suggesting that ubiquilin 2 activates mTORC1. The knockdown of ubiquilins 2 and 4 decreases the effect of Cbz-B3A on 4EBP1 phosphorylation. Cbz-B3A slows cellular growth of some human leukemia cell lines, but is not cytotoxic. Thus Cbz-B3A exemplifies a novel strategy to inhibit mTORC1 signaling that might be exploited for treating many human diseases. We propose that Cbz-B3A reveals a previously unappreciated regulatory pathway coordinating cytosolic protein quality control and mTORC1 signaling.
Assuntos
Arginina/análogos & derivados , Carbamatos/farmacologia , Inibidores Enzimáticos/farmacologia , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Arginina/química , Arginina/farmacologia , Carbamatos/química , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Quinases S6 Ribossômicas/metabolismo , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/genéticaRESUMO
Francisella tularensis is the causative agent of tularemia and a potential biowarfare agent. The virulence of F. tularensis is decreased by deletion of guaB, the gene encoding IMP dehydrogenase (IMPDH), suggesting that this enzyme is a target for antibacterial design. Here we report that F. tularensis growth is blocked by inhibitors of bacterial IMPDHs. Seventeen compounds from two different frameworks, designated the D and Q series, display antibacterial activities with MICs of <1 µM. These compounds are also active against intracellular infections. Surprisingly, antibacterial activity does not correlate with IMPDH inhibition. In addition, the presence of guanine does not affect the antibacterial activity of most compounds, nor does the deletion of guaB These observations suggest that antibacterial activity derives from inhibition of another target(s). Moreover, D compounds display antibacterial activity only against F. tularensis, suggesting the presence of a unique target or uptake mechanism. A ΔguaB mutant resistant to compound D73 contained a missense mutation (Gly45Cys) in nuoB, which encodes a subunit of bacterial complex I. Overexpression of the nuoB mutant conferred resistance to D73 in both wild-type and ΔguaB strains. This strain was not resistant to Q compounds, suggesting that a different off-target mechanism operates for these compounds. Several Q compounds are also effective against Mycobacterium tuberculosis, in which a second target has also been implicated, in addition to IMPDH. The fortuitous presence of multiple targets with overlapping structure-activity relationships presents an intriguing opportunity for the development of robust antibiotics that may avoid the emergence of resistance.
Assuntos
Antibacterianos/farmacologia , Benzoxazóis/farmacologia , Francisella tularensis/efeitos dos fármacos , IMP Desidrogenase/antagonistas & inibidores , Ftalazinas/farmacologia , Animais , Linhagem Celular , Complexo I de Transporte de Elétrons/genética , Humanos , IMP Desidrogenase/genética , Camundongos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Tularemia/tratamento farmacológico , Tularemia/microbiologiaRESUMO
Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The enzyme is an emerging target for antimicrobial therapy. The small molecule inhibitor A110 has been identified as a potent and selective inhibitor of IMPDHs from a variety of pathogenic microorganisms. A recent X-ray crystallographic study reported that the inhibitor binds to the NAD(+) cofactor site and forms a ternary complex with IMP. Here we report a pre-steady-state stopped-flow kinetic investigation of IMPDH from Bacillus anthracis designed to assess the kinetic significance of the crystallographic results. Stopped-flow kinetic experiments defined nine microscopic rate constants and two equilibrium constants that characterize both the catalytic cycle and details of the inhibition mechanism. In combination with steady-state initial rate studies, the results show that the inhibitor binds with high affinity (Kd ≈ 50 nM) predominantly to the covalent intermediate on the reaction pathway. Only a weak binding interaction (Kd ≈ 1 µM) is observed between the inhibitor and E·IMP. Thus, the E·IMP·A110 ternary complex, observed by X-ray crystallography, is largely kinetically irrelevant.
Assuntos
Bacillus anthracis/enzimologia , IMP Desidrogenase/antagonistas & inibidores , Cinética , Modelos MolecularesRESUMO
The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization.
Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Sequência de Aminoácidos , Anti-Infecciosos/química , Bacillus anthracis/efeitos dos fármacos , Bacillus anthracis/enzimologia , Bacillus anthracis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/efeitos dos fármacos , Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/enzimologia , Clostridium perfringens/genética , Cristalografia por Raios X , Inibidores Enzimáticos/química , IMP Desidrogenase/química , IMP Desidrogenase/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Emerging evidence reveals that prion-like structures play important roles to maintain the well-being of cells. Although self-assembly of small molecules also affords prion-like nanofibrils (PriSM), little is known about the functions and mechanisms of PriSM. Previous works demonstrated that PriSM formed by a dipeptide derivative selectively inhibiting the growth of glioblastoma cells over neuronal cells and effectively inhibiting xenograft tumor in animal models. Here we examine the protein targets, the internalization, and the cytotoxicity pathway of the PriSM. The results show that the PriSM selectively accumulate in cancer cells via macropinocytosis to impede the dynamics of cytoskeletal filaments via promiscuous interactions with cytoskeletal proteins, thus inducing apoptosis. Intriguingly, Tau proteins are able to alleviate the effect of the PriSM, thus protecting neuronal cells. This work illustrates PriSM as a new paradigm for developing polypharmacological agents that promiscuously interact with multiple proteins yet result in a primary phenotype, such as cancer inhibition.
Assuntos
Citoesqueleto/metabolismo , Nanopartículas/química , Neoplasias/metabolismo , Príons/química , Antineoplásicos/química , Apoptose , Endocitose , Glioblastoma/tratamento farmacológico , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Nanotecnologia , Transplante de Neoplasias , Nocodazol/química , Paclitaxel/química , Peptídeos/química , Estrutura Terciária de Proteína , Tubulina (Proteína)/química , Proteínas tau/químicaRESUMO
The inosine monophosphate dehydrogenase (IMPDH)/guanosine monophosphate reductase (GMPR) family of (ß/α)(8) enzymes presents an excellent opportunity to investigate how subtle changes in enzyme structure change reaction specificity. IMPDH and GMPR bind the same ligands with similar affinities and share a common set of catalytic residues. Both enzymes catalyze a hydride transfer reaction involving a nicotinamide cofactor hydride, and both reactions proceed via the same covalent intermediate. In the case of IMPDH, this intermediate reacts with water, while in GMPR it reacts with ammonia. In both cases, the two chemical transformations are separated by a conformational change. In IMPDH, the conformational change involves a mobile protein flap while in GMPR, the cofactor moves. Thus reaction specificity is controlled by differences in dynamics, which in turn are controlled by residues outside the active site. These findings have some intriguing implications for the evolution of the IMPDH/GMPR family.
Assuntos
GMP Redutase/química , Guanosina Monofosfato/química , IMP Desidrogenase/química , Amônia/química , Domínio Catalítico , Cátions Monovalentes/química , Humanos , Cinética , Ligantes , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , Água/químicaRESUMO
Cryptosporidium parasites are a major cause of diarrhea and malnutrition in the developing world, a frequent cause of waterborne disease in the developed world, and a potential bioterrorism agent. Currently, available treatment is limited, and Cryptosporidium drug discovery remains largely unsuccessful. As a result, the pharmacokinetic properties required for in vivo efficacy have not been established. We have been engaged in a Cryptosporidium drug discovery program targeting IMP dehydrogenase (CpIMPDH). Here, we report the activity of eight potent and selective inhibitors of CpIMPDH in the interleukin-12 (IL-12) knockout mouse model, which mimics acute human cryptosporidiosis. Two compounds displayed significant antiparasitic activity, validating CpIMPDH as a drug target. The best compound, P131 (250 mg/kg of body weight/day), performed equivalently to paromomycin (2,000 mg/kg/day) when administered in a single dose and better than paromomycin when administered in three daily doses. One compound, A110, appeared to promote Cryptosporidium infection. The pharmacokinetic, uptake, and permeability properties of the eight compounds were measured. P131 had the lowest systemic distribution but accumulated to high concentrations within intestinal cells. A110 had the highest systemic distribution. These observations suggest that systemic distribution is not required, and may be a liability, for in vivo antiparasitic activity. Intriguingly, A110 caused specific alterations in fecal microbiota that were not observed with P131 or vehicle alone. Such changes may explain how A110 promotes parasitemia. Collectively, these observations suggest a blueprint for the development of anticryptosporidial therapy.
Assuntos
Coccidiostáticos/uso terapêutico , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , IMP Desidrogenase/antagonistas & inibidores , Animais , Células CACO-2/parasitologia , Modelos Animais de Doenças , Descoberta de Drogas/métodos , Humanos , Interleucina-12/genética , Camundongos , Camundongos Endogâmicos C57BL/parasitologia , Camundongos Knockout/parasitologiaRESUMO
Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.
Assuntos
GMP Redutase/metabolismo , IMP Desidrogenase/metabolismo , Biocatálise , Cristalografia por Raios X , GMP Redutase/química , Guanosina Monofosfato/biossíntese , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Humanos , IMP Desidrogenase/química , Inosina Monofosfato/química , Inosina Monofosfato/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , NADP/química , NADP/metabolismo , Teoria Quântica , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismoRESUMO
Cryptosporidium parvum (Cp) is a potential biowarfare agent and major cause of diarrhea and malnutrition. This protozoan parasite relies on inosine 5'-monophosphate dehydrogenase (IMPDH) for the production of guanine nucleotides. A CpIMPDH-selective N-aryl-3,4-dihydro-3-methyl-4-oxo-1-phthalazineacetamide inhibitor was previously identified in a high throughput screening campaign. Herein we report a structure-activity relationship study for the phthalazinone-based series that resulted in the discovery of benzofuranamide analogs that exhibit low nanomolar inhibition of CpIMPDH. In addition, the antiparasitic activity of select analogs in a Toxoplasma gondii model of C. parvum infection is also presented.