RESUMO
We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.
Assuntos
COVID-19 , Humanos , Estrutura Molecular , SARS-CoV-2 , Imunidade Inata , Citosina , Redes e Vias Metabólicas , AntiviraisRESUMO
Millions of people are infected by the dengue and Zika viruses each year, resulting in significant morbidity and mortality. Galidesivir is an adenosine nucleoside analog that can attenuate flavivirus replication in cell-based assays and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases and subsequently incorporated into viral RNA by viral RNA polymerases. This has been proposed to lead to the delayed termination of RNA synthesis. Here, we report direct in vitro testing of the effects of Galidesivir triphosphate on dengue-2 and Zika virus polymerase activity. Galidesivir triphosphate was chemically synthesized, and inhibition of RNA synthesis followed using a dinucleotide-primed assay with a homopolymeric poly(U) template. Galidesivir triphosphate was equipotent against dengue-2 and Zika polymerases, with IC50 values of 42 ± 12 µM and 47 ± 5 µM, respectively, at an ATP concentration of 20 µM. RNA primer extension assays show that the dengue-2 polymerase stalls while attempting to add a Galidesivir nucleotide to the nascent RNA chain, evidenced by the accumulation of RNA products truncated immediately upstream of Galidesivir incorporation sites. Nevertheless, Galidesivir is incorporated at isolated sites with low efficiency, leading to the subsequent synthesis of full-length RNA with no evidence of delayed chain termination. The incorporation of Galidesivir at consecutive sites is strongly disfavored, highlighting the potential for modulation of inhibitory effects of nucleoside analogs by the template sequence. Our results suggest that attenuation of dengue replication by Galidesivir may not derive from the early termination of RNA synthesis following Galidesivir incorporation.
Assuntos
Dengue , Infecção por Zika virus , Zika virus , Animais , Antivirais/farmacologia , Adenosina/farmacologia , RNA Viral/genética , Nucleotidiltransferases , Zika virus/genéticaRESUMO
Methylthio-DADMe-immucillin-A (MTDIA) is an 86 picomolar inhibitor of 5'-methylthioadenosine phosphorylase (MTAP) with potent and specific anti-cancer efficacy. MTAP salvages S-adenosylmethionine (SAM) from 5'-methylthioadenosine (MTA), a toxic metabolite produced during polyamine biosynthesis. Changes in MTAP expression are implicated in cancer growth and development, making MTAP an appealing target for anti-cancer therapeutics. Since SAM is involved in lipid metabolism, we hypothesised that MTDIA alters the lipidomes of MTDIA-treated cells. To identify these effects, we analysed the lipid profiles of MTDIA-treated Saccharomyces cerevisiae using ultra-high resolution accurate mass spectrometry (UHRAMS). MTAP inhibition by MTDIA, and knockout of the Meu1 gene that encodes for MTAP in yeast, caused global lipidomic changes and differential abundance of lipids involved in cell signaling. The phosphoinositide kinase/phosphatase signaling network was specifically impaired upon MTDIA treatment, and was independently validated and further characterised via altered localization of proteins integral to this network. Functional consequences of dysregulated lipid metabolism included a decrease in reactive oxygen species (ROS) levels induced by MTDIA that was contemporaneous with changes in immunological response factors (nitric oxide, tumour necrosis factor-alpha and interleukin-10) in mammalian cells. These results indicate that lipid homeostasis alterations and concomitant downstream effects may be associated with MTDIA mechanistic efficacy.
Assuntos
Fosfatidilinositóis , Purina-Núcleosídeo Fosforilase , Animais , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , S-Adenosilmetionina/metabolismo , Oxirredução , Mamíferos/metabolismoRESUMO
Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas , Antivirais/farmacologia , Imunidade Inata , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Proteínas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , S-Adenosilmetionina , Replicação ViralRESUMO
Diffuse gastric cancer and lobular breast cancer are aggressive malignancies that are frequently associated with inactivating mutations in the tumor suppressor gene CDH1. Synthetic lethal (SL) vulnerabilities arising from CDH1 dysfunction represent attractive targets for drug development. Recently, SLEC-11 (1) emerged as a SL lead in E-cadherin-deficient cells. Here, we describe our efforts to optimize 1. Overall, 63 analogues were synthesized and tested for their SL activity toward isogenic mammary epithelial CDH1-deficient cells (MCF10A-CDH1-/-). Among the 26 compounds with greater cytotoxicity, AL-GDa62 (3) was four-times more potent and more selective than 1 with an EC50 ratio of 1.6. Furthermore, 3 preferentially induced apoptosis in CDH1-/- cells, and Cdh1-/- mammary and gastric organoids were significantly more sensitive to 3 at low micromolar concentrations. Thermal proteome profiling of treated MCF10A-CDH1-/- cell protein lysates revealed that 3 specifically inhibits TCOF1, ARPC5, and UBC9. In vitro, 3 inhibited SUMOylation at low micromolar concentrations.
Assuntos
Antineoplásicos/uso terapêutico , Descoberta de Drogas , Neoplasias Gástricas/tratamento farmacológico , Antígenos CD/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caderinas/genética , Linhagem Celular Tumoral , Humanos , Mutação , Neoplasias Gástricas/patologiaRESUMO
Antibiotic resistance continues to spread at an alarming rate, outpacing the introduction of new therapeutics and threatening to globally undermine health care. There is a crucial need for new strategies that selectively target specific pathogens while leaving the majority of the microbiome untouched, thus averting the debilitating and sometimes fatal occurrences of opportunistic infections. To address these challenges, we have adopted a unique strategy that focuses on oxygen-sensitive proteins, an untapped set of therapeutic targets. MqnE is a member of the radical S-adenosyl-l-methionine (RS) superfamily, all of which rely on an oxygen-sensitive [4Fe-4S] cluster for catalytic activity. MqnE catalyzes the conversion of didehydrochorismate to aminofutalosine in the essential menaquinone biosynthetic pathway present in a limited set of species, including the gut pathogen Helicobacter pylori (Hp), making it an attractive target for narrow-spectrum antibiotic development. Indeed, we show that MqnE is inhibited by the mechanism-derived 2-fluoro analogue of didehydrochorismate (2F-DHC) due to accumulation of a radical intermediate under turnover conditions. Structures of MqnE in the apo and product-bound states afford insight into its catalytic mechanism, and electron paramagnetic resonance approaches provide direct spectroscopic evidence consistent with the predicted structure of the radical intermediate. In addition, we demonstrate the essentiality of the menaquinone biosynthetic pathway and unambiguously validate 2F-DHC as a selective inhibitor of Hp growth that exclusively targets MqnE. These data provide the foundation for designing effective Hp therapies and demonstrate proof of principle that radical SAM proteins can be effectively leveraged as therapeutic targets.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Vias Biossintéticas/efeitos dos fármacos , Radicais Livres/química , Helicobacter pylori/crescimento & desenvolvimento , S-Adenosilmetionina/metabolismo , Vitamina K 2/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/enzimologia , Estrutura Molecular , Nucleosídeos/metabolismoRESUMO
Bacterial 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes adenine from its substrates to form S-methyl-5-thioribose and S-ribosyl-l-homocysteine. MTANs are involved in quorum sensing, menaquinone synthesis, and 5'-methylthioadenosine recycling to S-adenosylmethionine. Helicobacter pylori uses MTAN in its unusual menaquinone pathway, making H. pylori MTAN a target for antibiotic development. Human 5'-methylthioadenosine phosphorylase (MTAP), a reported anticancer target, catalyzes phosphorolysis of 5'-methylthioadenosine to salvage S-adenosylmethionine. Transition-state analogues designed for HpMTAN and MTAP show significant overlap in specificity. Fifteen unique transition-state analogues are described here and are used to explore inhibitor specificity. Several analogues of HpMTAN bind in the picomolar range while inhibiting human MTAP with orders of magnitude weaker affinity. Structural analysis of HpMTAN shows inhibitors extending through a hydrophobic channel to the protein surface. The more enclosed catalytic sites of human MTAP require the inhibitors to adopt a folded structure, displacing the phosphate nucleophile from the catalytic site.
Assuntos
Inibidores Enzimáticos/farmacologia , Helicobacter pylori/enzimologia , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Tioléster Hidrolases/antagonistas & inibidores , Domínio Catalítico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Purina-Núcleosídeo Fosforilase/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Tioléster Hidrolases/metabolismoRESUMO
BACKGROUND: The E-cadherin gene (CDH1) is frequently mutated in diffuse gastric cancer and lobular breast cancer, and germline mutations predispose to the cancer syndrome Hereditary Diffuse Gastric Cancer. We are taking a synthetic lethal approach to identify druggable vulnerabilities in CDH1-mutant cancers. METHODS: Density distributions of cell viability data from a genome-wide RNAi screen of isogenic MCF10A and MCF10A-CDH1-/- cells were used to identify protein classes affected by CDH1 mutation. The synthetic lethal relationship between selected protein classes and E-cadherin was characterised by drug sensitivity assays in both the isogenic breast MCF10A cells and CDH1-isogenic gastric NCI-N87. Endocytosis efficiency was quantified using cholera toxin B uptake. Pathway metagene expression of 415 TCGA gastric tumours was statistically correlated with CDH1 expression. RESULTS: MCF10A-CDH1-/- cells showed significantly altered sensitivity to RNAi inhibition of groups of genes including the PI3K/AKT pathway, GPCRs, ion channels, proteosomal subunit proteins and ubiquitinylation enzymes. Both MCF10A-CDH1-/- and NCI-N87-CDH1-/- cells were more sensitive than wild-type cells to compounds that disrupted plasma membrane composition and trafficking, but showed contrasting sensitivities to inhibitors of actin polymerisation and the chloride channel inhibitor NS3728. The MCF10A-CDH1-/- cell lines showed reduced capacity to endocytose cholera toxin B. Pathway metagene analysis identified 20 Reactome pathways that were potentially synthetic lethal in tumours. Genes involved in GPCR signalling, vesicle transport and the metabolism of PI3K and membrane lipids were strongly represented amongst the candidate synthetic lethal genes. CONCLUSIONS: E-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities.
Assuntos
Caderinas/deficiência , Membrana Celular/metabolismo , Membrana Celular/patologia , Antígenos CD/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/genética , Linhagem Celular Tumoral , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Transporte Proteico/fisiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
The rise of antibiotic resistance, coupled with increased expectations for mobility in later life, is creating a need for biofilm inhibitors and delivery systems that will reduce surgical implant infection. A limitation of some of these existing delivery approaches is toxicity exhibited toward host cells. Here, we report the application of a novel inhibitor of the enzyme, methylthioadenosine nucleosidase (MTAN), a key enzyme in bacterial metabolic pathways, which include S-adenosylmethionine catabolism and purine nucleotide recycling, in combination with a poly(vinyl alcohol)-tyramine-based (PVA-Tyr) hydrogel delivery system. We demonstrate that a lead MTAN inhibitor, selected from a screened library of 34 candidates, (2S)-2-(4-amino-5H-pyrrolo3,2-dpyrimidin-7-ylmethyl)aminoundecan-1-ol (31), showed a minimum biofilm inhibitory concentration of 2.2 ± 0.4 µM against a clinical staphylococcal species isolated from an infected implant. We observed that extracellular DNA, a key constituent of biofilms, is significantly reduced when treated with 10 µM compound 31, along with a decrease in biofilm thickness. Compound 31 was incorporated into a hydrolytically degradable photo-cross-linked PVA-Tyr hydrogel and the release profile was evaluated by HPLC studies. Compound 31 released from the PVA-hydrogel system significantly reduced biofilm formation (77.2 ± 8.4% biofilm inhibition). Finally, compound 31 released from PVA-Tyr showed no negative impact on human bone marrow stromal cell (MSC) viability, proliferation, or morphology. The results demonstrate the potential utility of MTAN inhibitors in treating infections caused by Gram-positive bacteria, and the development of a nontoxic release system that has potential for tunability for time scale of delivery.
RESUMO
Campylobacter jejuni is a Gram-negative bacterium responsible for food-borne gastroenteritis and associated with Guillain-Barré, Reiter, and irritable bowel syndromes. Antibiotic resistance in C. jejuni is common, creating a need for antibiotics with novel mechanisms of action. Menaquinone biosynthesis in C. jejuni uses the rare futalosine pathway, where 5'-methylthioadenosine nucleosidase ( CjMTAN) is proposed to catalyze the essential hydrolysis of adenine from 6-amino-6-deoxyfutalosine to form dehypoxanthinylfutalosine, a menaquinone precursor. The substrate specificity of CjMTAN is demonstrated to include 6-amino-6-deoxyfutalosine, 5'-methylthioadenosine, S-adenosylhomocysteine, adenosine, and 5'-deoxyadenosine. These activities span the catalytic specificities for the role of bacterial MTANs in menaquinone synthesis, quorum sensing, and S-adenosylmethionine recycling. We determined inhibition constants for potential transition-state analogues of CjMTAN. The best of these compounds have picomolar dissociation constants and were slow-onset tight-binding inhibitors. The most potent CjMTAN transition-state analogue inhibitors inhibited C. jejuni growth in culture at low micromolar concentrations, similar to gentamicin. The crystal structure of apoenzyme C. jejuni MTAN was solved at 1.25 Å, and five CjMTAN complexes with transition-state analogues were solved at 1.42 to 1.95 Å resolution. Inhibitor binding induces a loop movement to create a closed catalytic site with Asp196 and Ile152 providing purine leaving group activation and Arg192 and Glu12 activating the water nucleophile. With inhibitors bound, the interactions of the 4'-alkylthio or 4'-alkyl groups of this inhibitor family differ from the Escherichia coli MTAN structure by altered protein interactions near the hydrophobic pocket that stabilizes 4'-substituents of transition-state analogues. These CjMTAN inhibitors have potential as specific antibiotic candidates against C. jejuni.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , N-Glicosil Hidrolases/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Antibacterianos/química , Proteínas de Bactérias/química , Campylobacter jejuni/enzimologia , Domínio Catalítico , Inibidores Enzimáticos/química , Cinética , Estrutura Molecular , N-Glicosil Hidrolases/química , Pirimidinas/química , Pirróis/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The Immucillins are chemically stable analogues that mimic the ribocation and leaving-group features of N-ribosyltransferase transition states. Infectious disease agents often rely on ribosyltransferase chemistry in pathways involving precursor synthesis for nucleic acids, salvage of nucleic acid precursors, or synthetic pathways with nucleoside intermediates. Here, we review three infectious agents and the use of the Immucillins to taget enzymes essential to the parasites. First, DADMe-Immucillin-G is a purine nucleoside phosphorylase (PNP) inhibitor that blocks purine salvage and shows clinical potential for treatment for the malaria parasite Plasmodium falciparum, a purine auxotroph requiring hypoxanthine for purine nucleotide synthesis. Inhibition of the PNPs in the host and in parasite cells leads to apurinic starvation and death. Second, Helicobacter pylori, a causative agent of human ulcers, synthesizes menaquinone, an essential electron transfer agent, in a pathway requiring aminofutalosine nucleoside hydrolysis. Inhibitors of the H. pylori methylthioadenosine nucleosidase (MTAN) are powerful antibiotics for this organism. Synthesis of menaquinone by the aminofutalosine pathway does not occur in most bacteria populating the human gut microbiome. Thus, MTAN inhibitors provide high-specificity antibiotics for H. pylori and are not expected to disrupt the normal gut bacterial flora. Third, Immucillin-A was designed as a transition state analogue of the atypical PNP from Trichomonas vaginalis. In antiviral screens, Immucillin-A was shown to act as a prodrug. It is active against filoviruses and flaviviruses. In virus-infected cells, Immucillin-A is converted to the triphosphate, is incorporated into the viral transcript, and functions as an atypical chain-terminator for RNA-dependent RNA polymerases. Immucillin-A has entered clinical trials for use as an antiviral. We also summarize other Immucillins that have been characterized in successful clinical trials for T-cell lymphoma and gout. The human trials support the potential development of the Immucillins in infectious diseases.
Assuntos
Anti-Infecciosos/farmacologia , Anti-Infecciosos/uso terapêutico , Doenças Transmissíveis/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Anti-Infecciosos/química , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Doenças Transmissíveis/etiologia , Doenças Transmissíveis/metabolismo , Inibidores Enzimáticos/química , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Nucleosídeos/metabolismo , Plasmodium/efeitos dos fármacos , Plasmodium/enzimologia , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purinas/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/uso terapêutico , Relação Estrutura-Atividade , Vírus/efeitos dos fármacos , Vírus/enzimologia , Vitamina K 2/metabolismoRESUMO
In the absence of industry partnerships, most academic groups lack the infrastructure to rationally design and build drugs via methods used in industry. Instead, academia needs to work smarter using mechanism-based design. Working smarter can mean the development of new drug discovery paradigms and then demonstrating their utility and reproducibility to industry. The collaboration between Vern Schramm's group at the Albert Einstein College of Medicine, USA and Peter Tyler at the Ferrier Research Institute at The Victoria University of Wellington, NZ has refined a drug discovery process called transition state analogue design. This process has been applied to several biomedically relevant nucleoside processing enzymes. In 2017, Mundesine®, conceived using transition state analogue design, received market approval for the treatment of peripheral T-cell lymphoma in Japan. This short review looks at a brief history of transition state analogue design, the fundamentals behind the development of this process, and the success of enzyme inhibitors produced using this drug design methodology.
RESUMO
Mycobacterium tuberculosis 5'-deoxyadenosine/5'-methylthioadenosine nucleosidase (Rv0091) catalyzes the N-riboside hydrolysis of its substrates 5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'-dAdo). 5'-dAdo is the preferred substrate, a product of radical S-adenosylmethionine-dependent enzyme reactions. Rv0091 is characterized by a ribocation-like transition state, with low N-ribosidic bond order, an N7-protonated adenine leaving group, and an activated but weakly bonded water nucleophile. DADMe-Immucillins incorporating 5'-substituents of the substrates 5'-dAdo and MTA were synthesized and characterized as inhibitors of Rv0091. 5'-Deoxy-DADMe-Immucillin-A was the most potent among the 5'-dAdo transition state analogues with a dissociation constant of 640 pM. Among the 5'-thio substituents, hexylthio-DADMe-Immucillin-A was the best inhibitor at 87 pM. The specificity of Rv0091 for the Immucillin transition state analogues differs from those of other bacterial homologues because of an altered hydrophobic tunnel accepting the 5'-substituents. Inhibitors of Rv0091 had weak cell growth effects on M. tuberculosis or Mycobacterium smegmatis but were lethal toward Helicobacter pylori, where the 5'-methylthioadenosine nucleosidase is essential in menaquinone biosynthesis. We propose that Rv0091 plays a role in 5'-deoxyadenosine recycling but is not essential for growth in these Mycobacteria.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Adenina/análogos & derivados , Adenina/química , Adenina/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Técnicas de Química Sintética , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Helicobacter pylori/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Pirrolidinas/química , Pirrolidinas/farmacologia , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
The first cells probably possessed rudimentary metabolic networks, built using a handful of multifunctional enzymes. The promiscuous activities of modern enzymes are often assumed to be relics of this primordial era; however, by definition these activities are no longer physiological. There are many fewer examples of enzymes using a single active site to catalyze multiple physiologically-relevant reactions. Previously, we characterized the promiscuous alanine racemase (ALR) activity of Escherichia coli cystathionine ß-lyase (CBL). Now we have discovered that several bacteria with reduced genomes lack alr, but contain metC (encoding CBL). We characterized the CBL enzymes from three of these: Pelagibacter ubique, the Wolbachia endosymbiont of Drosophila melanogaster (wMel) and Thermotoga maritima. Each is a multifunctional CBL/ALR. However, we also show that CBL activity is no longer required in these bacteria. Instead, the wMel and T. maritima enzymes are physiologically bi-functional alanine/glutamate racemases. They are not highly active, but they are clearly sufficient. Given the abundance of the microorganisms using them, we suggest that much of the planet's biochemistry is carried out by enzymes that are quite different from the highly-active exemplars usually found in textbooks. Instead, primordial-like enzymes may be an essential part of the adaptive strategy associated with streamlining.
Assuntos
Enzimas/genética , Liases/genética , Alanina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Escherichia coli/genética , Genoma/genética , Genoma Bacteriano/genética , Liases/metabolismo , Redes e Vias Metabólicas , Thermotoga maritima/genética , Wolbachia/genéticaRESUMO
Ribosome inactivating proteins (RIPs) are among the most toxic agents known. More than a dozen clinical trials against refractory cancers have been initiated using modified RIPs with impressive results. However, dose-limiting toxicity due to vascular leak syndrome limits success of the therapy. We have previously reported some tight-binding transition state analogues of Saporin L3 that mimic small oligonucleotide substrates in which the susceptible adenosine has been replaced by a 9-deazaadenyl hydroxypyrrolidinol derivative. They provide the first step in the development of rescue agents to prevent Saporin L3 toxicity on non-targeted cells. Here we report the synthesis, using solution phase chemistry, of these and a larger group of transition state analogues. They were tested for inhibition against Saporin L3 giving Ki values as low as 3.3 nM and indicating the structural requirements for inhibition.
Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/química , Sequência de Bases , Modelos Moleculares , Oligonucleotídeos/genética , Conformação Proteica , RNA/genética , RNA/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , SaporinasRESUMO
MTAN (5'-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.
Assuntos
Formicinas/química , Helicobacter pylori/enzimologia , Purina-Núcleosídeo Fosforilase/química , S-Adenosil-Homocisteína/química , Adenina/análogos & derivados , Adenina/química , Domínio Catalítico/genética , Cristalografia por Raios X , Desoxiadenosinas/química , Helicobacter pylori/química , Ligação de Hidrogênio , Modelos Moleculares , Nêutrons , Ligação Proteica , Prótons , Purina-Núcleosídeo Fosforilase/genética , Pirrolidinas/química , Especificidade por Substrato , Tionucleosídeos/químicaRESUMO
BACKGROUND: Immucillins ImmA (IA), ImmH (IH) and SerMe-ImmH (SMIH) are synthetic deazapurine nucleoside analogues that inhibit Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis multiplication in vitro without macrophage toxicity. Immucillins are compared to the Glucantime standard drug in the chemotherapy of Leishmania (L.) infantum chagasi infection in mice and hamsters. These agents are tested for toxicity and immune system response. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c mice were infected with 107 amastigotes, treated with IA, IH, SMIH or Glucantime (2.5mg/kg/day) and monitored for clinical variables, parasite load, antibody levels and splenocyte IFN-γ, TNF-α, and IL-10 expression. Cytokines and CD4+, CD8+ and CD19+ lymphocyte frequencies were assessed in uninfected controls and in response to immucillins. Urea, creatinine, GOT and GPT levels were monitored in sera. Anti-Leishmania-specific IgG1 antibodies (anti-NH36) increased in untreated animals. IgG2a response, high levels of IFN-γ, TNF-α and lower levels of IL-10 were detected in mice treated with the immucillins and Glucantime. Immucillins permitted normal weight gain, prevented hepato-splenomegaly and cleared the parasite infection (85-89%) without renal and hepatic toxicity. Immucillins promoted 35% lower secretion of IFN-γ in uninfected controls than in infected mice. IA and IH increased the CD4+ T and CD19+ B cell frequencies. SMIH increased only the proportion of CD-19 B cells. IA and IH also cured infected hamsters with lower toxicity than Glucantime. CONCLUSIONS/SIGNIFICANCE: Immucillins IA, IH and SMIH were effective in treating leishmaniasis in mice. In hamsters, IA and IH were also effective. The highest therapeutic efficacy was obtained with IA, possibly due to its induction of a TH1 immune response. Low immucillin doses were required and showed no toxicity. Our results disclose the potential use of IA and IH in the therapy of visceral leishmaniasis.
Assuntos
Adenina/análogos & derivados , Antiprotozoários/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Nucleosídeos de Purina/uso terapêutico , Pirimidinonas/uso terapêutico , Pirrolidinas/uso terapêutico , Adenina/efeitos adversos , Adenina/uso terapêutico , Adenosina/análogos & derivados , Animais , Anticorpos Antiprotozoários/sangue , Antiprotozoários/efeitos adversos , Análise Química do Sangue , Modelos Animais de Doenças , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Expressão Gênica , Imunofenotipagem , Interferon gama/biossíntese , Interleucina-10/biossíntese , Leishmania , Leishmaniose Visceral/patologia , Leucócitos Mononucleares/imunologia , Mesocricetus , Camundongos Endogâmicos BALB C , Carga Parasitária , Nucleosídeos de Purina/efeitos adversos , Pirimidinonas/efeitos adversos , Pirrolidinas/efeitos adversos , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Helicobacter pylori is a Gram-negative bacterium that colonizes the gut of over 50% of the world's population. It is responsible for most peptic ulcers and is an important risk factor for gastric cancer. Antibiotic treatment for H. pylori infections is challenging as drug resistance has developed to antibiotics with traditional mechanisms of action. H. pylori uses an unusual pathway for menaquinone biosynthesis with 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzing an essential step. We validated MTAN as a target with a transition-state analogue of the enzyme [Wang, S.; Haapalainen, A. M.; Yan, F.; et al. Biochemistry 2012, 51, 6892-6894]. MTAN inhibitors will only be useful drug candidates if they can both include tight binding to the MTAN target and have the ability to penetrate the complex cell membrane found in Gram-negative H. pylori. Here we explore structural scaffolds for MTAN inhibition and for growth inhibition of cultured H. pylori. Sixteen analogues reported here are transition-state analogues of H. pylori MTAN with dissociation constants of 50 pM or below. Ten of these prevent growth of the H. pylori with IC90 values below 0.01 µg/mL. These remarkable compounds meet the criteria for potent inhibition and cell penetration. As a consequence, 10 new H. pylori antibiotic candidates are identified, all of which prevent H. pylori growth at concentrations 16-2000-fold lower than the five antibiotics, amoxicillin, metronidazole, levofloxacin, tetracyclin, and clarithromycin, commonly used to treat H. pylori infections. X-ray crystal structures of MTAN cocrystallized with several inhibitors show them to bind in the active site making interactions consistent with transition-state analogues.
Assuntos
Antibacterianos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Transition-state analysis based on kinetic isotope effects and computational chemistry provides electrostatic potential maps to serve as blueprints for the design and chemical synthesis of transition-state analogues. The utility of these molecules is exemplified by potential clinical applications toward leukemia, autoimmune disorders, gout, solid tumors, bacterial quorum sensing and bacterial antibiotics. In some cases, transition-state analogues have chemical features that have allowed them to be repurposed for new indications, including potential antiviral use. Three compounds from this family have entered clinical trials. The transition-state analogues bind to their target proteins with high affinity and specificity. The physical and structural properties of binding teach valuable and often surprising lessons about the nature of tight-binding inhibitors.
Assuntos
Adenina/análogos & derivados , Inibidores Enzimáticos/química , Pirrolidinas/química , Adenina/química , Adenina/metabolismo , Adenosina/análogos & derivados , Animais , Inibidores Enzimáticos/metabolismo , Humanos , N-Glicosil Hidrolases/antagonistas & inibidores , N-Glicosil Hidrolases/metabolismo , Ligação Proteica , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Pirrolidinas/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
MTDIA is a picomolar transition state analogue inhibitor of human methylthioadenosine phosphorylase and a femtomolar inhibitor of Escherichia coli methylthioadenosine nucleosidase. MTDIA has proven to be a non-toxic, orally available pre-clinical drug candidate with remarkable anti-tumour activity against a variety of human cancers in mouse xenografts. The structurally similar compound MTDIH is a potent inhibitor of human and malarial purine nucleoside phosphorylase (PNP) as well as the newly discovered enzyme, methylthioinosine phosphorylase, isolated from Pseudomonas aeruginosa. Since the enantiomers of some pharmaceuticals have revealed surprising biological activities, the enantiomers of MTDIH and MTDIA, compounds 1 and 2, respectively, were prepared and their enzyme binding properties studied. Despite binding less tightly to their target enzymes than their enantiomers compounds 1 and 2 are nanomolar inhibitors.