RESUMO
Inherited peripheral neuropathies are a group of neurodegenerative disorders that clinically affect 1 in 2500 individuals. Recently, genetic mutations in human histidine nucleotide-binding protein 1 (hHint1) have been strongly and most frequently associated with patients suffering from axonal neuropathy with neuromyotonia. However, the correlation between the impact of these mutations on the hHint1 structure, enzymatic activity and in vivo function has remained ambiguous. Here, we provide detailed biochemical characterization of a set of these hHint1 mutations. Our findings indicate that half of the mutations (R37P, G93D and W123*) resulted in a destabilization of the dimeric state and a significant decrease in catalytic activity and HINT1 inhibitor binding affinity. The H112N mutant was found to be dimeric, but devoid of catalytic activity, due to the loss of the catalytically essential histidine; nevertheless, it exhibited high affinity to AMP and a HINT1 inhibitor. In contrast to the active-site mutants, the catalytic activity and dimeric structure of the surface mutants, C84R and G89V, were found to be similar to the wild-type enzyme. Taken together, our results suggest that the pathophysiology of inherited axonal neuropathy with neuromyotonia can be induced by conversion of HINT1 from a homodimer to monomer, by modification of select surface residues or by a significant reduction of the enzyme's catalytic efficiency.
Assuntos
Síndrome de Isaacs/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Histidina/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas do Tecido Nervoso/genética , Conformação Proteica , Homologia de SequênciaRESUMO
Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.
Assuntos
Proteínas do Tecido Nervoso/química , Nucleotídeos/química , Amidas/química , Cristalografia por Raios X , Humanos , Ácidos Fosfóricos/química , Especificidade por SubstratoRESUMO
Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for the management of pain. However, the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHint1 contains multiple histidines. Here we have investigated the effect of divalent metal ions (Cd2+, Cu2+, Mg2+, Mn2+, Ni2+, and Zn2+) on the structural integrity and catalytic activity of hHint1. With the exception of Mg2+, all the divalent ions inhibited hHint1, the rank of order was found to be Cu2+ >Zn2+ >Cd2+ ≥Ni2+ >Mn2+ based on their IC50 and kin/KI values. A crystal structure of hHint1 with bound Cu2+ is described to explain the competitive reversible inactivation of hHint1 by divalent cations. All the metal ions exhibited time- and concentration- dependent inhibition, with the rate of inactivation highly dependent on alterations of the C-terminus. With the exception of Cu2+; restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHint1 upon incubation with 10-fold excess of copper. Thus, hHint1 appears to be structurally sensitive to irreversible inactivation by copper, which may be of neurotoxicological and pharmacological significance.
Assuntos
Metais/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/ultraestrutura , Analgésicos Opioides/química , Animais , Sítios de Ligação , Catálise , Cátions Bivalentes/química , Cobre/química , Humanos , Íons , Neuralgia/metabolismo , Ligação Proteica , Desdobramento de ProteínaRESUMO
Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.
Assuntos
Antivirais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sofosbuvir/metabolismo , Ativação Metabólica , Domínio Catalítico , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Mutação Puntual , Especificidade por SubstratoRESUMO
The pyridoxal 5'-phosphate (PLP)-dependent transaminase BioA catalyzes the second step in the biosynthesis of biotin in Mycobacterium tuberculosis (Mtb) and is an essential enzyme for bacterial survival and persistence in vivo. A promising BioA inhibitor 6 containing an N-aryl, N'-benzoylpiperazine scaffold was previously identified by target-based whole-cell screening. Here, we explore the structure-activity relationships (SAR) through the design, synthesis, and biological evaluation of a systematic series of analogues of the original hit using a structure-based drug design strategy, which was enabled by cocrystallization of several analogues with BioA. To confirm target engagement and discern analogues with off-target activity, each compound was evaluated against wild-type (WT) Mtb in biotin-free and -containing medium as well as BioA under- and overexpressing Mtb strains. Conformationally constrained derivative 36 emerged as the most potent analogue with a KD of 76 nM against BioA and a minimum inhibitory concentration of 1.7 µM (0.6 µg/mL) against Mtb in biotin-free medium.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Biotina/biossíntese , Mycobacterium tuberculosis/efeitos dos fármacos , Piperazinas/farmacologia , Fosfato de Piridoxal/metabolismo , Transaminases/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Biocatálise , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/metabolismo , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-Atividade , Transaminases/metabolismoRESUMO
Mechanism-based inhibitors (MBIs) are widely employed in chemistry, biology, and medicine because of their exquisite specificity and sustained duration of inhibition. Optimization of MBIs is complicated because of time-dependent inhibition resulting from multistep inactivation mechanisms. The global kinetic parameters kinact and KI have been used to characterize MBIs, but they provide far less information than is commonly assumed, as shown by derivation and simulation of these parameters. We illustrate an alternative and more rigorous approach for MBI characterization through determination of the individual microscopic rate constants. Kinetic analysis revealed the rate-limiting step of inactivation of the PLP-dependent enzyme BioA by dihydro-(1,4)-pyridone 1. This knowledge was subsequently applied to rationally design a second-generation inhibitor scaffold with a nearly optimal maximum inactivation rate (0.48 min-1).
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Piridonas/farmacologia , Transaminases/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/química , Cinética , Estrutura Molecular , Piridonas/química , Transaminases/metabolismoRESUMO
Hint1 has recently emerged to be an important target of interest due to its involvement in the regulation of a broad range of CNS functions including opioid signaling, tolerance, neuropathic pain, and nicotine dependence. A series of inhibitors were rationally designed, synthesized, and tested for their inhibitory activity against hHint1 using isothermal titration calorimetry (ITC). The studies resulted in the development of the first small-molecule inhibitors of hHint1 with submicromolar binding affinities. A combination of thermodynamic and high-resolution X-ray crystallographic studies provides an insight into the biomolecular recognition of ligands by hHint1. These novel inhibitors have potential utility as molecular probes to better understand the role and function of hHint1 in the CNS.
RESUMO
Inaccuracies in the article, Crystal structure of HINT from Helicobacter pylori by Tarique et al. [(2016) Acta Cryst. F72, 42-48] are presented, and a brief history of HINT nomenclature is discussed.
Assuntos
Cristalografia por Raios X , Helicobacter pylori , CristalizaçãoRESUMO
The Bacillus anthracis lethal factor (LF) is one component of a tripartite exotoxin partly responsible for persistent anthrax cytotoxicity after initial bacterial infection. Inhibitors of the zinc metalloproteinase have been investigated as potential therapeutic agents, but LF is a challenging target because inhibitors lack sufficient selectivity or possess poor pharmaceutical properties. These structural studies reveal an alternate conformation of the enzyme, induced upon binding of specific inhibitors, that opens a previously unobserved deep pocket termed S1'(∗) which might afford new opportunities to design selective inhibitors that target this subsite.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Inibidores de Metaloproteinases de Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Sítios de Ligação , Ligantes , Inibidores de Metaloproteinases de Matriz/química , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Tirosina/metabolismoRESUMO
The lethal factor (LF) enzyme secreted by Bacillus anthracis is a zinc hydrolase that is chiefly responsible for anthrax-related cell death. Although many studies of the design of small molecule LF inhibitors have been conducted, no LF inhibitor is yet available as a therapeutic agent. Inhibitors with considerable chemical diversity have been developed and investigated; however, the LF S2' subsite has not yet been systematically explored as a potential target for lead optimization. Here we present synthesis, experimental evaluation, modeling, and structural biology for a novel series of sulfonamide hydroxamate LF inhibitor analogues specifically designed to extend into, and probe chemical preferences of, this S2' subsite. We discovered that this region accommodates a wide variety of chemical functionalities and that a broad selection of ligand structural modifications directed to this area can be incorporated without significant deleterious alterations in biological activity. We also identified key residues in this subsite that can potentially be targeted to improve inhibitor binding.
Assuntos
Antraz/microbiologia , Antígenos de Bactérias/química , Bacillus anthracis/enzimologia , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/química , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Alquilação , Antraz/tratamento farmacológico , Antígenos de Bactérias/metabolismo , Bacillus anthracis/química , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Inibidores Enzimáticos/química , Humanos , Ácidos Hidroxâmicos/química , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
The secreted anthrax toxin consists of three components: the protective antigen (PA), edema factor (EF) and lethal factor (LF). LF, a zinc metalloproteinase, compromises the host immune system primarily by targeting mitogen-activated protein kinase kinases in macrophages. Peptide substrates and small-molecule inhibitors bind LF in the space between domains 3 and 4 of the hydrolase. Domain 3 is attached on a hinge to domain 2 via residues Ile300 and Pro385, and can move through an angular arc of greater than 35° in response to the binding of different ligands. Here, multiple LF structures including five new complexes with co-crystallized inhibitors are compared and three frequently populated LF conformational states termed `bioactive', `open' and `tight' are identified. The bioactive position is observed with large substrate peptides and leaves all peptide-recognition subsites open and accessible. The tight state is seen in unliganded and small-molecule complex structures. In this state, domain 3 is clamped over certain substrate subsites, blocking access. The open position appears to be an intermediate state between these extremes and is observed owing to steric constraints imposed by specific bound ligands. The tight conformation may be the lowest-energy conformation among the reported structures, as it is the position observed with no bound ligand, while the open and bioactive conformations are likely to be ligand-induced.
Assuntos
Antígenos de Bactérias/química , Bacillus anthracis/química , Toxinas Bacterianas/química , Metaloendopeptidases/química , Antraz/microbiologia , Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ligantes , Metaloendopeptidases/metabolismo , Modelos Moleculares , Peptídeos , Conformação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
The anthrax toxin lethal factor (LF) and matrix metalloproteinase-3 (MMP-3, stromelysin-1) are popular zinc metalloenzyme drug targets, with LF primarily responsible for anthrax-related toxicity and host death, while MMP-3 is involved in cancer- and rheumatic disease-related tissue remodeling. A number of in silico screening techniques, most notably docking and scoring, have proven useful for identifying new potential drug scaffolds targeting LF and MMP-3, as well as for optimizing lead compounds and investigating mechanisms of action. However, virtual screening outcomes can vary significantly depending on the specific docking parameters chosen, and systematic statistical significance analyses are needed to prioritize key parameters for screening small molecules against these zinc systems. In the current work, we present a series of chi-square statistical analyses of virtual screening outcomes for cocrystallized LF and MMP-3 inhibitors docked into their respective targets, evaluated by predicted enzyme-inhibitor dissociation constant and root-mean-square deviation (RMSD) between predicted and experimental bound configurations, and we present a series of preferred parameters for use with these systems in the industry-standard Surflex-Dock screening program, for use by researchers utilizing in silico techniques to discover and optimize new scaffolds.
Assuntos
Antídotos/química , Toxinas Bacterianas/antagonistas & inibidores , Metaloproteinase 3 da Matriz/química , Inibidores de Metaloproteinases de Matriz/química , Projetos de Pesquisa/estatística & dados numéricos , Zinco/química , Antraz/tratamento farmacológico , Antraz/microbiologia , Antraz/patologia , Antídotos/farmacologia , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/química , Bacillus anthracis/patogenicidade , Bacillus anthracis/fisiologia , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/química , Domínio Catalítico , Ensaios de Triagem em Larga Escala , Humanos , Inibidores de Metaloproteinases de Matriz/farmacologia , Simulação de Acoplamento Molecular , Interface Usuário-ComputadorRESUMO
The histidine triad proteins (HITs) constitute a large and ubiquitous superfamily of nucleotide hydrolases. The human histidine triad nucleotide-binding proteins (hHints) are a distinct class of HITs noted for their acyl-AMP hydrolase and phosphoramidase activity. The first high-resolution crystal structures of hHint2 with and without bound AMP are described. The differences between hHint2 and previously known HIT family protein structures are discussed. HIT family enzymes have historically been divided into five classes based on their catalytic specificity: Hint, fragile HIT protein, galactose-1-phosphate uridylyltransferase, DcpS and aprataxin. However, although several structures exist for the enzymes in these classes, the endogenous substrates of many of these enzymes have not been identified or biochemically characterized. To better understand the structural relationships of the HIT enzymes, a structure-based phylogeny was constructed that resulted in the identification of several new putative HIT clades with potential acyl-AMP hydrolase and phosphoramidase activity.
Assuntos
Proteínas Mitocondriais/química , Monofosfato de Adenosina/química , Motivos de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Filogenia , Ligação Proteica , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
Anthrax is an acute infectious disease caused by the spore-forming, gram-positive, rod-shaped bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF) is the primary anthrax toxin component responsible for cytotoxicity and host death and has been a heavily researched target for design of postexposure therapeutics in the event of a bioterror attack. Various computer-aided drug design methodologies have proven useful for pinpointing new antianthrax drug scaffolds, optimizing existing leads and probes, and elucidating key mechanisms of action. We present a selection of in silico virtual screening protocols incorporating docking and scoring, shape-based searching, and pharmacophore mapping techniques to identify and prioritize small molecules with potential biological activity against LF. We also recommend screening parameters that have been shown to increase the accuracy and reliability of these computational results.