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1.
J Mol Biol ; 430(17): 2709-2721, 2018 08 17.
Article in English | MEDLINE | ID: mdl-29787766

ABSTRACT

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.


Subject(s)
Isaacs Syndrome/genetics , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Peripheral Nervous System Diseases/genetics , Amino Acid Sequence , Crystallography, X-Ray , Histidine/metabolism , Humans , Mutant Proteins/genetics , Nerve Tissue Proteins/genetics , Protein Conformation , Sequence Homology
2.
Mol Pharm ; 14(11): 3987-3997, 2017 11 06.
Article in English | MEDLINE | ID: mdl-28968488

ABSTRACT

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.


Subject(s)
Nerve Tissue Proteins/chemistry , Nucleotides/chemistry , Amides/chemistry , Crystallography, X-Ray , Humans , Phosphoric Acids/chemistry , Substrate Specificity
3.
Biochem Biophys Res Commun ; 491(3): 760-766, 2017 09 23.
Article in English | MEDLINE | ID: mdl-28739258

ABSTRACT

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.


Subject(s)
Metals/chemistry , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/ultrastructure , Analgesics, Opioid/chemistry , Animals , Binding Sites , Catalysis , Cations, Divalent/chemistry , Copper/chemistry , Humans , Ions , Neuralgia/metabolism , Protein Binding , Protein Unfolding
4.
Biochemistry ; 56(28): 3559-3570, 2017 07 18.
Article in English | MEDLINE | ID: mdl-28691797

ABSTRACT

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.


Subject(s)
Antiviral Agents/metabolism , Nerve Tissue Proteins/metabolism , Sofosbuvir/metabolism , Activation, Metabolic , Catalytic Domain , Crystallography, X-Ray , Humans , Kinetics , Models, Molecular , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Point Mutation , Substrate Specificity
5.
J Med Chem ; 60(13): 5507-5520, 2017 07 13.
Article in English | MEDLINE | ID: mdl-28594172

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Biotin/biosynthesis , Mycobacterium tuberculosis/drug effects , Piperazines/pharmacology , Pyridoxal Phosphate/metabolism , Transaminases/antagonists & inhibitors , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Bacterial Proteins/metabolism , Biocatalysis , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Structure , Mycobacterium tuberculosis/metabolism , Piperazines/chemical synthesis , Piperazines/chemistry , Structure-Activity Relationship , Transaminases/metabolism
6.
J Am Chem Soc ; 139(21): 7132-7135, 2017 05 31.
Article in English | MEDLINE | ID: mdl-28510452

ABSTRACT

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).


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/enzymology , Pyridones/pharmacology , Transaminases/antagonists & inhibitors , Bacterial Proteins/metabolism , Enzyme Inhibitors/chemistry , Kinetics , Molecular Structure , Pyridones/chemistry , Transaminases/metabolism
7.
ACS Med Chem Lett ; 7(8): 780-4, 2016 Aug 11.
Article in English | MEDLINE | ID: mdl-27563403

ABSTRACT

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.

8.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 4): 336-7, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27050269

ABSTRACT

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.


Subject(s)
Crystallography, X-Ray , Helicobacter pylori , Crystallization
9.
FEBS Lett ; 589(24 Pt B): 3836-41, 2015 Dec 21.
Article in English | MEDLINE | ID: mdl-26578066

ABSTRACT

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.


Subject(s)
Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Matrix Metalloproteinase Inhibitors/metabolism , Matrix Metalloproteinase Inhibitors/pharmacology , Bacterial Toxins/antagonists & inhibitors , Binding Sites , Ligands , Matrix Metalloproteinase Inhibitors/chemistry , Models, Molecular , Protein Conformation , Structure-Activity Relationship , Tyrosine/metabolism
10.
J Med Chem ; 58(21): 8723-33, 2015 Nov 12.
Article in English | MEDLINE | ID: mdl-26492514

ABSTRACT

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.


Subject(s)
Anthrax/microbiology , Antigens, Bacterial/chemistry , Bacillus anthracis/enzymology , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/chemistry , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Alkylation , Anthrax/drug therapy , Antigens, Bacterial/metabolism , Bacillus anthracis/chemistry , Bacillus anthracis/metabolism , Bacterial Toxins/metabolism , Enzyme Inhibitors/chemistry , Humans , Hydroxamic Acids/chemistry , Models, Molecular , Structure-Activity Relationship
11.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 11): 2813-22, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25372673

ABSTRACT

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.


Subject(s)
Antigens, Bacterial/chemistry , Bacillus anthracis/chemistry , Bacterial Toxins/chemistry , Metalloendopeptidases/chemistry , Anthrax/microbiology , Antigens, Bacterial/metabolism , Bacillus anthracis/metabolism , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/metabolism , Binding Sites , Crystallography, X-Ray , Drug Design , Humans , Ligands , Metalloendopeptidases/metabolism , Models, Molecular , Peptides , Protein Conformation/drug effects , Protein Structure, Tertiary/drug effects , Sulfonamides/chemistry , Sulfonamides/pharmacology
12.
Curr Top Med Chem ; 14(18): 2105-14, 2014.
Article in English | MEDLINE | ID: mdl-25373478

ABSTRACT

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.


Subject(s)
Antidotes/chemistry , Bacterial Toxins/antagonists & inhibitors , Matrix Metalloproteinase 3/chemistry , Matrix Metalloproteinase Inhibitors/chemistry , Research Design/statistics & numerical data , Zinc/chemistry , Anthrax/drug therapy , Anthrax/microbiology , Anthrax/pathology , Antidotes/pharmacology , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/chemistry , Bacillus anthracis/pathogenicity , Bacillus anthracis/physiology , Bacterial Toxins/biosynthesis , Bacterial Toxins/chemistry , Catalytic Domain , High-Throughput Screening Assays , Humans , Matrix Metalloproteinase Inhibitors/pharmacology , Molecular Docking Simulation , User-Computer Interface
13.
FEBS J ; 280(14): 3389-98, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23659632

ABSTRACT

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.


Subject(s)
Mitochondrial Proteins/chemistry , Adenosine Monophosphate/chemistry , Amino Acid Motifs , Catalytic Domain , Crystallography, X-Ray , Humans , Hydrogen Bonding , Models, Molecular , Phylogeny , Protein Binding , Protein Structure, Secondary , Structural Homology, Protein
14.
Methods Mol Biol ; 993: 177-84, 2013.
Article in English | MEDLINE | ID: mdl-23568471

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Toxins/antagonists & inhibitors , Computational Biology/methods , Drug Evaluation, Preclinical/methods , Antigens, Bacterial , Bacillus anthracis/drug effects , User-Computer Interface
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