ABSTRACT
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.
Subject(s)
Oxidoreductases Acting on CH-CH Group Donors , Proteins , Antiviral Agents/pharmacology , Immunity, Innate , Oxidoreductases Acting on CH-CH Group Donors/genetics , Proteins/metabolism , Ribosomes/genetics , Ribosomes/metabolism , S-Adenosylmethionine , Virus ReplicationABSTRACT
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.
Subject(s)
COVID-19 , Humans , Molecular Structure , SARS-CoV-2 , Immunity, Innate , Cytosine , Metabolic Networks and Pathways , Antiviral AgentsABSTRACT
Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.
Subject(s)
Bacterial Proteins/genetics , CRISPR-Cas Systems/immunology , Endodeoxyribonucleases/genetics , Gene Expression Regulation, Bacterial/immunology , Quorum Sensing/genetics , Serratia/genetics , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/pharmacology , Bacterial Proteins/immunology , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/immunology , Clustered Regularly Interspaced Short Palindromic Repeats , Endodeoxyribonucleases/immunology , Quorum Sensing/drug effects , Quorum Sensing/immunology , Repressor Proteins/genetics , Repressor Proteins/immunology , Serratia/drug effects , Serratia/immunologyABSTRACT
3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin as part of the innate immune response. ddhCTP has been shown to act as an obligate chain terminator of flavivirus and SARS-CoV-2 RNA-dependent RNA polymerases; however, further biophysical studies have been precluded by limited access to this promising antiviral. Herein, we report a robust and scalable synthesis of ddhCTP as well as the mono- and diphosphates ddhCMP and ddhCDP, respectively. Identification of a 2'-silyl ether protection strategy enabled selective synthesis and facile purification of the 5'-triphosphate, culminating in the preparation of ddhCTP on a gram scale.
Subject(s)
Antiviral Agents , COVID-19 , Cytidine Triphosphate , Humans , Proteins , RNA, Viral , SARS-CoV-2ABSTRACT
OBJECTIVE: To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. RESULTS: Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants-Arg255Ala, Arg255Gly-with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. CONCLUSIONS: Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of 'quorum quenching' enzymes.
Subject(s)
Acyl-Butyrolactones/metabolism , Penicillin Amidase/metabolism , Point Mutation , Pseudomonas aeruginosa/growth & development , Arginine/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Crystallography, X-Ray , Gene Expression Regulation, Bacterial , Models, Molecular , Molecular Docking Simulation , Mutagenesis, Site-Directed , Penicillin Amidase/chemistry , Penicillin Amidase/genetics , Protein Conformation , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Quorum Sensing , Substrate SpecificityABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Biosynthetic Pathways/drug effects , Free Radicals/chemistry , Helicobacter pylori/growth & development , S-Adenosylmethionine/metabolism , Vitamin K 2/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalysis , Electron Spin Resonance Spectroscopy/methods , Helicobacter pylori/drug effects , Helicobacter pylori/enzymology , Molecular Structure , Nucleosides/metabolismABSTRACT
Transition state analogue inhibitor design (TSID) and fragment-based drug design (FBDD) are drug design approaches typically used independently. Methylthio-DADMe-Immucillin-A (MTDIA) is a tight-binding transition state analogue of bacterial 5'-methylthioadenosine nucleosidases (MTANs). Previously, Salmonella enterica MTAN structures were found to bind MTDIA and ethylene glycol fragments, but MTDIA modified to contain similar fragments did not enhance affinity. Seventy-five published MTAN structures were analyzed, and co-crystallization fragments were found that might enhance the binding of MTDIA to other bacterial MTANs through contacts external to MTDIA binding. The fragment-modified MTDIAs were tested with Helicobacter pylori MTAN and Staphylococcus aureus MTANs (HpMTAN and SaMTAN) as test cases to explore inhibitor optimization by potential contacts beyond the transition state contacts. Replacement of a methyl group with a 2'-ethoxyethanol group in MTDIA improved the dissociation constant 14-fold (0.09 nM vs 1.25 nM) for HpMTAN and 81-fold for SaMTAN (0.096 nM vs 7.8 nM). TSID combined with FBDD can be useful in enhancing already powerful inhibitors.
Subject(s)
Adenine/analogs & derivatives , Bacterial Proteins/metabolism , Enzyme Inhibitors/metabolism , Purine-Nucleoside Phosphorylase/metabolism , Pyrrolidines/metabolism , Adenine/chemistry , Adenine/metabolism , Bacteria/enzymology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Catalytic Domain , Enzyme Inhibitors/chemistry , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Protein Binding , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/chemistry , Pyrrolidines/chemistryABSTRACT
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.
Subject(s)
Cadherins/deficiency , Cell Membrane/metabolism , Cell Membrane/pathology , Antigens, CD/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cadherins/genetics , Cell Line, Tumor , Female , Genetic Predisposition to Disease , Germ-Line Mutation , Humans , Protein Transport/physiology , Stomach Neoplasms/genetics , Stomach Neoplasms/pathologyABSTRACT
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.
Subject(s)
Formycins/chemistry , Helicobacter pylori/enzymology , Purine-Nucleoside Phosphorylase/chemistry , S-Adenosylhomocysteine/chemistry , Adenine/analogs & derivatives , Adenine/chemistry , Catalytic Domain/genetics , Crystallography, X-Ray , Deoxyadenosines/chemistry , Helicobacter pylori/chemistry , Hydrogen Bonding , Models, Molecular , Neutrons , Protein Binding , Protons , Purine-Nucleoside Phosphorylase/genetics , Pyrrolidines/chemistry , Substrate Specificity , Thionucleosides/chemistryABSTRACT
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.
Subject(s)
Enzymes/genetics , Lyases/genetics , Alanine/metabolism , Amino Acid Sequence , Catalytic Domain , Escherichia coli/genetics , Genome/genetics , Genome, Bacterial/genetics , Lyases/metabolism , Metabolic Networks and Pathways , Thermotoga maritima/genetics , Wolbachia/geneticsABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/enzymology , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Adenine/analogs & derivatives , Adenine/chemistry , Adenine/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chemistry Techniques, Synthetic , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Helicobacter pylori/drug effects , Microbial Sensitivity Tests , Models, Molecular , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Purine-Nucleoside Phosphorylase/chemistry , Purine-Nucleoside Phosphorylase/metabolism , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Structural Homology, Protein , Structure-Activity RelationshipABSTRACT
The rational design and implementation of enantiodivergent enamine catalysis is reported. A simple secondary amine catalyst, 2-methyl-l-proline, and its tetrabutylammonium salt function as an enantiodivergent catalyst pair delivering the enantiomers of α-functionalized aldehyde products in excellent enantioselectivities. This novel concept of designed enantiodivergence is applied to the enantioselective α-amination, aldol, and α-aminoxylation/α-hydroxyamination reactions of aldehydes.
ABSTRACT
HisA is a (ßα)8 barrel enzyme that catalyzes the Amadori rearrangement of N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N'-((5'-phosphoribulosyl) formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the histidine biosynthesis pathway, and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo-state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp-7 acts as the catalytic base, and Asp-176 acts as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen bond network including His-47, Asp-129, Thr-171, and Ser-202, all shown to be functionally important. The closed conformation structure is highly similar to the bifunctional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.
Subject(s)
Aldose-Ketose Isomerases/chemistry , Aldose-Ketose Isomerases/metabolism , Biocatalysis , Aldose-Ketose Isomerases/genetics , Amino Acid Sequence , Apoenzymes/chemistry , Apoenzymes/genetics , Apoenzymes/metabolism , Catalytic Domain , Imidazoles/metabolism , Kinetics , Ligands , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Ribonucleotides/metabolism , Saccharomyces cerevisiae/enzymologyABSTRACT
Transition state analogs mimic the geometry and electronics of the transition state of enzymatic reactions. These molecules bind to the active site of the enzyme much tighter than substrate and are powerful noncovalent inhibitors. Immucillin-H (ImmH) and 4'-deaza-1'-aza-2'-deoxy-9-methylene Immucillin-H (DADMe-ImmH) are picomolar inhibitors of human purine nucleoside phosphorylase (hPNP). Although both molecules are electronically similar to the oxocarbenium-like dissociative hPNP transition state, DADMe-ImmH is more potent than ImmH. DADMe-ImmH captures more of the transition state binding energy by virtue of being a closer geometric match to the hPNP transition state than ImmH. A consequence of these similarities is that the active site of hPNP exerts greater distortional forces on ImmH than on DADMe-ImmH to "achieve" the hPNP transition state geometry. By using magic angle spinning solid-state NMR to investigate stable isotope-labeled ImmH and DADMe-ImmH, we have explored the difference in distortional binding of these two inhibitors to hPNP. High-precision determinations of internuclear distances from NMR recoupling techniques, rotational echo double resonance, and rotational resonance, have provided unprecedented atomistic insight into the geometric changes that occur upon binding of transition state analogs. We conclude that hPNP stabilizes conformations of these chemically distinct analogs having distances between the cation and leaving groups resembling those of the known transition state.
Subject(s)
Models, Molecular , Protein Conformation , Purine-Nucleoside Phosphorylase/chemistry , Humans , Magnetic Resonance Imaging , Molecular Structure , Protein Binding , Purine-Nucleoside Phosphorylase/metabolismABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , Helicobacter pylori/drug effects , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Models, Molecular , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Adenine/analogs & derivatives , Escherichia coli/enzymology , Plasmodium falciparum/enzymology , Pseudomonas aeruginosa/enzymology , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Adenine/chemistry , Adenine/pharmacology , Drug Discovery , Escherichia coli/drug effects , Humans , Models, Molecular , Neoplasms/drug therapy , Neoplasms/enzymology , Plasmodium falciparum/drug effects , Protein Binding , Pseudomonas aeruginosa/drug effects , Purine-Nucleoside Phosphorylase/metabolism , StereoisomerismABSTRACT
The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5'-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5' groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5'-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2'-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5'-methylthioinosine and similarly 5'-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa.
Subject(s)
Enzyme Inhibitors/chemistry , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Purine-Nucleoside Phosphorylase/chemistry , Purine-Nucleoside Phosphorylase/metabolism , Toxoplasma/enzymology , Catalysis , Catalytic Domain , Crystallography, X-Ray , Kinetics , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Purine Nucleosides/chemistry , Purine Nucleosides/metabolism , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/genetics , Pyrimidinones/chemistry , Substrate Specificity , Toxoplasma/chemistry , Toxoplasma/geneticsABSTRACT
The survival and proliferation of Plasmodium falciparum parasites and human cancer cells require de novo pyrimidine synthesis to supply RNA and DNA precursors. Orotate phosphoribosyltransferase (OPRT) is an indispensible component in this metabolic pathway and is a target for antimalarials and antitumor drugs. P. falciparum (Pf) and Homo sapiens (Hs) OPRTs are characterized by highly dissociative transition states with ribocation character. On the basis of the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states, analogues were designed, synthesized, and tested as inhibitors. Iminoribitol mimics of the ribocation transition state in linkage to pyrimidine mimics using methylene or ethylene linkers gave dissociation constants (Kd) as low as 80 nM. Inhibitors with pyrrolidine groups as ribocation mimics displayed slightly weaker binding affinities for OPRTs. Interestingly, p-nitrophenyl riboside 5'-phosphate bound to OPRTs with Kd values near 40 nM. Analogues designed with a C5-pyrimidine carbon-carbon bond to ribocation mimics gave Kd values in the range of 80-500 nM. Acyclic inhibitors with achiral serinol groups as the ribocation mimics also displayed nanomolar inhibition against OPRTs. In comparison with the nucleoside derivatives, inhibition constants of their corresponding 5'-phosphorylated transition state analogues are largely unchanged, an unusual property for a nucleotide-binding site. In silico docking of the best inhibitor into the HsOPRT active site supported an extensive hydrogen bond network associated with the tight binding affinity. These OPRT transition state analogues identify crucial components of potent inhibitors targeting OPRT enzymes. Despite their tight binding to the targets, the inhibitors did not kill cultured P. falciparum.
Subject(s)
Malaria/enzymology , Metabolic Networks and Pathways , Orotate Phosphoribosyltransferase/chemistry , Plasmodium falciparum/chemistry , Pyrimidines/biosynthesis , Antimalarials/chemistry , Binding Sites , Humans , Hydrogen Bonding , Kinetics , Malaria/drug therapy , Malaria/parasitology , Nucleosides , Orotate Phosphoribosyltransferase/genetics , Orotate Phosphoribosyltransferase/metabolism , Plasmodium falciparum/enzymology , Plasmodium falciparum/metabolism , Protein Conformation , Pyrimidines/chemistry , Pyrrolidines/pharmacology , Substrate SpecificityABSTRACT
Human 5'-methylthioadenosine phosphorylase (MTAP) is solely responsible for 5'-methylthioadenosine (MTA) metabolism to permit S-adenosylmethionine salvage. Transition-state (TS) analogues of MTAP are in development as anticancer candidates. TS analogues of MTAP incorporate a cationic nitrogen and a protonated 9-deazaadenine leaving group, which are mimics of the ribocation transition state. MT-ImmA and MT-DADMe-ImmA are two examples of these TS analogues. Thermodynamic analysis of MTA, inhibitor, and phosphate binding reveals the cationic nitrogen to provide -2.6 and -3.6 kcal/mol binding free energy for MT-ImmA and MT-DADMe-ImmA, respectively. The protonated deazaadenine provides an additional -1.3 (MT-ImmA) to -1.7 kcal/mol (MT-DADMe-ImmA). MT-DADMe-ImmA is a better match in TS geometry than MT-ImmA and is thermodynamically favored. Binding of TS analogues to the MTAP/phosphate complex is fully entropic, in contrast to TS analogue binding to the related human purine nucleoside phosphorylase/phosphate complex, which is fully enthalpic (Guan, R., Ho, M. C., Brenowitz, M., Tyler, P. C., Evans, G. B., Almo, S. C., and Schramm, V. L. (2011) Biochemistry 50, 10408-10417). The binding thermodynamics of phosphate or TS analogues alone to MTAP are fully dominated by enthalpy. Phosphate anchored in the catalytic site forms an ion pair with the cationic TS analogue to cause stabilization of the enzyme structure in the ternary complex. The ternary-induced conformational changes convert the individual enthalpic binding energies to entropy, resulting in a presumed shift of the protein architecture toward the transition state. Formation of the ternary TS analogue complex with MTAP induces a remarkable increase in thermal stability (ΔTm 28 °C). The enthalpic, entropic, and protein-stability features of TS analogue binding to human MTAP are resolved in these studies.
Subject(s)
Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Adenine/analogs & derivatives , Adenine/pharmacology , Catalytic Domain , Deoxyadenosines/metabolism , Deoxyadenosines/pharmacology , Enzyme Stability , Humans , Protein Structure, Tertiary , Pyrrolidines/pharmacology , Thermodynamics , Thionucleosides/metabolism , Thionucleosides/pharmacologyABSTRACT
The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C-nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme.