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1.
J Biol Chem ; 299(9): 105077, 2023 09.
Article in English | MEDLINE | ID: mdl-37482279

ABSTRACT

Pathogenic parasites of the Trichomonas genus are causative agents of sexually transmitted diseases affecting millions of individuals worldwide and whose outcome may include stillbirths and enhanced cancer risks and susceptibility to HIV infection. Trichomonas vaginalis relies on imported purine and pyrimidine nucleosides and nucleobases for survival, since it lacks the enzymatic activities necessary for de novo biosynthesis. Here we show that T. vaginalis additionally lacks homologues of the bacterial or mammalian enzymes required for the synthesis of the nicotinamide ring, a crucial component in the redox cofactors NAD+ and NADP. Moreover, we show that a yet fully uncharacterized T. vaginalis protein homologous to bacterial and protozoan nucleoside hydrolases is active as a pyrimidine nucleosidase but shows the highest specificity toward the NAD+ metabolite nicotinamide riboside. Crystal structures of the trichomonal riboside hydrolase in different states reveals novel intermediates along the nucleoside hydrolase-catalyzed hydrolytic reaction, including an unexpected asymmetry in the homotetrameric assembly. The active site structure explains the broad specificity toward different ribosides and offers precise insights for the engineering of specific inhibitors that may simultaneously target different essential pathways in the parasite.


Subject(s)
Hydrolases , Parasites , Trichomonas vaginalis , Animals , Hydrolases/chemistry , Hydrolases/metabolism , NAD/metabolism , Niacinamide/metabolism , Trichomonas vaginalis/enzymology , Crystallography, X-Ray , Substrate Specificity , Protein Structure, Tertiary , Models, Molecular , Protein Binding
2.
Anal Chem ; 95(12): 5300-5306, 2023 03 28.
Article in English | MEDLINE | ID: mdl-36917470

ABSTRACT

Trichomoniasis is the most common nonviral sexually transmitted infection, affecting an estimated 275 million people worldwide. The causative agent is the parasitic protozoan Trichomonas vaginalis. Although the disease itself is typically mild, individuals with trichomonal infections have a higher susceptibility to more serious conditions. The emergence of parasite strains resistant to current therapies necessitates the need for novel treatment strategies. Since T. vaginalis is an obligate parasite that requires nucleoside salvage pathways, essential nucleoside ribohydrolase enzymes are promising new drug targets. Fragment screening and X-ray crystallography have enabled structure-guided design of inhibitors for two of these enyzmes. Linkage of enzymatic and antiprotozoal activity would be a transformative step toward designing novel, mechanism-based therapeutic agents. While a correlation with inhibition of purified enzyme would be mechanistically suggestive, a correlation with inhibition of in-cell enzyme activity would definitively establish this linkage. To demonstrate this linkage, we have translated our NMR-based activity assays that measure the activity of purified enzymes for use in T. vaginalis cells. The 19F NMR-based activity assay for the pyrimidine-specific enzyme translated directly to in-cell assays. However, the 1H NMR-based activity assay for the purine-specific enzyme required a switch from adenosine to guanosine substrate and the use of 13C-editing to resolve the substrate 1H signals from cell and growth media background signals. The in-cell NMR assays are robust and have been demonstrated to provide inhibition data on test compounds. The results described here represent the first direct measurement of enzyme activity in protozoan parasite cells.


Subject(s)
Trichomonas vaginalis , Humans , Nucleosides/metabolism , Guanosine/metabolism , Magnetic Resonance Spectroscopy
3.
Biochemistry ; 61(17): 1853-1861, 2022 09 06.
Article in English | MEDLINE | ID: mdl-35994320

ABSTRACT

Trichomonas vaginalis is the causative parasitic protozoan of the disease trichomoniasis, the most prevalent, nonviral sexually transmitted disease in the world. T. vaginalis is a parasite that scavenges nucleosides from the host organism via catalysis by nucleoside hydrolase (NH) enzymes to yield purine and pyrimidine bases. One of the four NH enzymes identified within the genome of T. vaginalis displays unique specificity toward purine nucleosides, adenosine and guanosine, but not inosine, and atypically shares greater sequence similarity to the pyrimidine hydrolases. Bioinformatic analysis of this enzyme, adenosine/guanosine-preferring nucleoside ribohydrolase (AGNH), was incapable of identifying the residues responsible for this uncommon specificity, highlighting the need for structural information. Here, we report the X-ray crystal structures of holo, unliganded AGNH and three additional structures of the enzyme bound to fragment and small-molecule inhibitors. Taken together, these structures facilitated the identification of residue Asp231, which engages in substrate interactions in the absence of those residues that typically support the canonical purine-specific tryptophan-stacking specificity motif. An altered substrate-binding pose is mirrored by repositioning within the protein scaffold of the His80 general acid/base catalyst. The newly defined structure-determined sequence markers allowed the assignment of additional NH orthologs, which are proposed to exhibit the same specificity for adenosine and guanosine alone and further delineate specificity classes for these enzymes.


Subject(s)
N-Glycosyl Hydrolases , Parasites , Adenosine/chemistry , Animals , Guanosine , Inosine/metabolism , N-Glycosyl Hydrolases/chemistry , Parasites/metabolism , Pyrimidines , Substrate Specificity
4.
ACS Omega ; 4(14): 16226-16232, 2019 Oct 01.
Article in English | MEDLINE | ID: mdl-31592163

ABSTRACT

Trichomoniasis is caused by the parasitic protozoan Trichomonas vaginalis. The increasing prevalence of strains resistant to the current 5-nitroimidazole treatments creates the need for novel therapies. T. vaginalis cannot synthesize purine and pyrimidine rings and requires salvage pathway enzymes to obtain them from host nucleosides. The uridine nucleoside ribohydrolase was screened using an 19F NMR-based activity assay against a 2000-compound fragment diversity library. Several series of inhibitors were identified including scaffolds based on acetamides, cyclic ureas or ureas, pyridines, and pyrrolidines. A number of potent singleton compounds were identified, as well. Eighteen compounds with IC50 values of 20 µM or lower were identified, including some with ligand efficiency values of 0.5 or greater. Detergent and jump-dilution counter screens validated all scaffold classes as target-specific, reversible inhibitors. Identified scaffolds differ substantially from 5-nitroimidazoles. Medicinal chemistry using the structure-activity relationship emerging from the fragment hits is being pursued to discover nanomolar inhibitors.

5.
J Vis Exp ; (148)2019 06 30.
Article in English | MEDLINE | ID: mdl-31305530

ABSTRACT

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 µM and 250 µM, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.


Subject(s)
Escherichia coli/enzymology , Magnetic Resonance Spectroscopy , N-Glycosyl Hydrolases/antagonists & inhibitors , Biological Assay , Drug Discovery , Enzyme Inhibitors , Escherichia coli/metabolism , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , N-Glycosyl Hydrolases/metabolism
6.
ACS Infect Dis ; 5(3): 345-352, 2019 03 08.
Article in English | MEDLINE | ID: mdl-30701958

ABSTRACT

Trichomoniasis is caused by the parasitic protozoan Trichomonas vaginalis and is the most prevalent, nonviral sexually transmitted disease. The parasite has shown increasing resistance to the current 5-nitroimidazole therapies indicating the need for new therapies with different mechanisms. T. vaginalis is an obligate parasite that scavenges nucleosides from host cells and then uses salvage pathway enzymes to obtain the nucleobases. The adenosine/guanosine preferring nucleoside ribohydrolase was screened against a 2000-compound diversity fragment library using a 1H NMR-based activity assay. Three classes of inhibitors with more than five representatives were identified: bis-aryl phenols, amino bicyclic pyrimidines, and aryl acetamides. Among the active fragments were 10 compounds with ligand efficiency values greater than 0.5, including five with IC50 values <10 µM. Jump-dilution and detergent counter screens validated reversible, target-specific activity. The data reveals an emerging SAR that is guiding our medicinal chemistry efforts aimed at discovering compounds with nanomolar potency.


Subject(s)
Antiprotozoal Agents/chemistry , Enzyme Inhibitors/chemistry , N-Glycosyl Hydrolases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Trichomonas vaginalis/enzymology , Antiprotozoal Agents/pharmacology , Enzyme Inhibitors/pharmacology , Female , Humans , Ligands , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Trichomonas Vaginitis/parasitology , Trichomonas vaginalis/chemistry , Trichomonas vaginalis/drug effects , Trichomonas vaginalis/genetics
7.
Chem Biol Drug Des ; 92(4): 1736-1742, 2018 10.
Article in English | MEDLINE | ID: mdl-29808562

ABSTRACT

Trichomonas vaginalis infects approximately 300 million people worldwide annually. Infected individuals have a higher susceptibility to more serious conditions such as cervical and prostate cancer. The parasite has developed increasing resistance to current drug therapies, with an estimated 5% of clinical cases resulting from resistant strains, creating the need for new therapeutic strategies with novel mechanisms of action. Nucleoside salvage pathway enzymes represent novel drug targets as these pathways are essential for the parasite's survival. The guanosine/adenosine/cytidine nucleoside hydrolase (GACNH) may be particularly important as its expression is upregulated under glucose-limiting conditions mimicking those that occur during infection establishment. GACNH was screened against the NIH Clinical Collection to explore its druggability. Seven compounds were identified with IC50 values <20 µM. Extensive overlap was found between inhibitors of GACNH and the adenosine/guanosine nucleoside hydrolase (AGNH), but no overlap was found with inhibitors of the uridine nucleoside hydrolase. The guanosine analog ribavirin was the only compound found to be specific for GACNH. Compounds that inhibit both AGNH and GACNH purine salvage pathway enzymes may prove critical given the role that GACNH appears to play in the early stages of infection.


Subject(s)
N-Glycosyl Hydrolases/metabolism , Protozoan Proteins/metabolism , Trichomonas vaginalis/enzymology , Adenosine/analogs & derivatives , Adenosine/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Inhibitory Concentration 50 , N-Glycosyl Hydrolases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Quercetin/analogs & derivatives , Quercetin/chemistry , Quercetin/metabolism , Ribavirin/chemistry , Ribavirin/metabolism , Structure-Activity Relationship
8.
Bioorg Med Chem Lett ; 25(22): 5036-9, 2015 Nov 15.
Article in English | MEDLINE | ID: mdl-26592812

ABSTRACT

Nucleoside salvage pathway enzymes used by Trichomonas vaginalis are distinct from the pathway involved in activation of existing 5-nitroimidazole drugs. They thus represent excellent targets for developing novel, mechanism-based antitrichomonal agents. The purine-specific adenosine/guanosine preferring ribohydrolase (AGNH) was screened against the NIH Clinical Collection to assess its druggability. Eight compounds, including five flavonoids, were identified with IC50 values ⩽10 µM and confirmed in counter screens run in the presence of detergent. The inhibitors are structurally distinct from inhibitors of the pyrimidine-specific uridine ribohydrolase (UNH) thus indicating that AGNH is a distinct, druggable target from UNH.


Subject(s)
Antitrichomonal Agents/chemistry , Flavonoids/chemistry , N-Glycosyl Hydrolases/antagonists & inhibitors , Adenosine/analogs & derivatives , Adenosine/chemistry , High-Throughput Screening Assays , Quercetin/analogs & derivatives , Quercetin/chemistry , Small Molecule Libraries/chemistry , Stereoisomerism , Trichomonas vaginalis
9.
Bioorg Med Chem Lett ; 24(4): 1080-4, 2014 Feb 15.
Article in English | MEDLINE | ID: mdl-24468412

ABSTRACT

Trichomonas vaginalis continues to be a major health problem with drug-resistant strains increasing in prevalence. Novel antitrichomonal agents that are mechanistically distinct from current therapies are needed. The NIH Clinical Compound Collection was screened to find inhibitors of the uridine ribohydrolase enzyme required by the parasite to scavenge uracil for its growth. The proton-pump inhibitors omeprazole, pantoprazole, and rabeprazole were identified as inhibitors of this enzyme, with IC50 values ranging from 0.3 to 14.5 µM. This suggests a molecular mechanism for the in vitro antitrichomonal activity of these proton-pump inhibitors, and may provide important insights toward structure-based drug design.


Subject(s)
2-Pyridinylmethylsulfinylbenzimidazoles/pharmacology , N-Glycosyl Hydrolases/antagonists & inhibitors , Omeprazole/pharmacology , Proton Pump Inhibitors/pharmacology , Rabeprazole/pharmacology , Trichomonas vaginalis/enzymology , 2-Pyridinylmethylsulfinylbenzimidazoles/chemical synthesis , 2-Pyridinylmethylsulfinylbenzimidazoles/chemistry , Dose-Response Relationship, Drug , Molecular Structure , N-Glycosyl Hydrolases/metabolism , Omeprazole/chemical synthesis , Omeprazole/chemistry , Pantoprazole , Proton Pump Inhibitors/chemical synthesis , Proton Pump Inhibitors/chemistry , Rabeprazole/chemical synthesis , Rabeprazole/chemistry , Structure-Activity Relationship
10.
J Biol Chem ; 285(11): 8340-51, 2010 Mar 12.
Article in English | MEDLINE | ID: mdl-20061378

ABSTRACT

Sirtuins catalyze NAD(+)-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1-7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Heterocyclic Compounds, 4 or More Rings/pharmacology , Sirtuin 1/metabolism , Stilbenes/pharmacology , Acetylation/drug effects , Allosteric Regulation/drug effects , Animals , Blood Glucose/drug effects , Calorimetry , Diabetes Mellitus, Type 2/metabolism , Dietary Fats/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds, 4 or More Rings/chemistry , Humans , Mice , Mice, Obese , Nuclear Magnetic Resonance, Biomolecular , Resveratrol , Rhodamines , Stilbenes/chemistry , Substrate Specificity , Surface Plasmon Resonance , Tumor Suppressor Protein p53/metabolism
11.
Biomol NMR Assign ; 3(2): 183-6, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19888686

ABSTRACT

The sequence specific (1)H, (13)C, and (15)N resonance assignments for stromelysin, a Matrix metalloproteinase, are reported in this article. Almost 70% of assignable backbone and side-chain atoms were assigned in this highly dynamic protein.


Subject(s)
Enzyme Inhibitors/metabolism , Hydroxamic Acids/metabolism , Matrix Metalloproteinase 3/chemistry , Matrix Metalloproteinase 3/metabolism , Sulfones/metabolism , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Matrix Metalloproteinase Inhibitors , Nuclear Magnetic Resonance, Biomolecular , Software , Sulfones/pharmacology
12.
Chem Biol Drug Des ; 73(2): 179-88, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19207420

ABSTRACT

Aberrant activation of the phosphoinositide 3-kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3-phosphoinositide-dependent kinase-1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N-terminal lobe of the 3-phosphoinositide-dependent kinase-1 catalytic domain contains a docking site which recognizes the non-catalytic C-terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so-called PDK1 Interacting Fragment pocket allows interaction with 3-phosphoinositide-dependent kinase-1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3-phosphoinositide-dependent kinase-1 domain construct against a library of chemically diverse fragments in order to identify small, ligand-efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP-site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand-induced changes in 1H-15N TROSY spectra acquired using uniformly 15N-enriched PDK1 provided evidence to distinguish ATP-site from PDK1 Interacting Fragment-site binding. Caliper assay data and 19F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Protein Serine-Threonine Kinases/chemistry , 3-Phosphoinositide-Dependent Protein Kinases , Allosteric Site , Catalytic Domain , Computer Simulation , Humans , Hydrogen/chemistry , Ligands , Nitrogen/chemistry , Protein Conformation , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary
13.
J Am Chem Soc ; 130(18): 5870-1, 2008 May 07.
Article in English | MEDLINE | ID: mdl-18407634

ABSTRACT

19F NMR-based methods have found utility in activity-based screening assays. However, because enzymes catalyze a diverse set of reactions, a large variety of fluorinated substrates would need to be identified to target each one separately. We have developed a more streamlined approach that is applicable to many enzymes that utilize ATP as a substrate. In this method, a fluorine-containing ATP analogue, 2-fluoro-ATP, is used to monitor the reaction. Applications are described for nicotinamide adenine dinucleotide synthetase and 3-phosphoinositide dependent kinase-1. Fragment screening results for the latter indicate that this technique can identify compounds that inhibit as well as activate reactions. The present results, together with previous biochemical studies from other laboratories, have shown that 2-fluoro-ATP can serve as a substrate for nine enzymes that are representative of three of the six enzyme subclasses, namely the transferases, hydrolases, and ligases. This suggests that 2-fluoro-ATP is suitable as a universal tool for screening ATP-requiring enzymes. Importantly, 2-fluoro-ATP has been determined to be a valid substrate for a variety of kinases, including both small molecule and protein kinases, suggesting that it may be useful for investigating the large number of pharmaceutically relevant kinases.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Enzymes/analysis , Nuclear Magnetic Resonance, Biomolecular/methods , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Enzymes/metabolism , Fluorine/chemistry , Nucleoside-Phosphate Kinase/analysis , Nucleoside-Phosphate Kinase/metabolism , Protein Serine-Threonine Kinases/analysis , Protein Serine-Threonine Kinases/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Substrate Specificity
14.
J Biomol Screen ; 12(4): 457-63, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17379856

ABSTRACT

Nicotinamide adenine dinucleotide synthetase (NadE) is an essential enzyme for bacterial pathogens and is thus a promising antibacterial target. It catalyzes the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. Changes in chemical shifts that occur in the nicotinic acid ring as it is converted to nicotinamide can be used for monitoring the reaction. A robust nuclear magnetic resonance-based activity assay was developed using robotically controlled reaction initiation and quenching. The single-enzyme assay has less potential for false positives compared to a coupled activity assay and is especially well suited to the high concentration of compounds in fragment screens. The assay has been used to screen fragment libraries for NadE inhibitors.


Subject(s)
Amide Synthases/chemistry , Amide Synthases/physiology , Magnetic Resonance Spectroscopy , Amide Synthases/genetics , Amino Acid Sequence , Molecular Sequence Data , Niacin/chemistry , Niacin/metabolism , Niacinamide/biosynthesis , Niacinamide/chemistry , Staphylococcus aureus/enzymology , Staphylococcus aureus/genetics
15.
Comb Chem High Throughput Screen ; 6(5): 445-53, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12871051

ABSTRACT

High-throughput ligand-based proton NMR screening performed in the presence of a spy molecule and a control molecule is a valuable tool for identifying drug leads. A limitation of the technique is represented by the severe overlap encountered in the screening of large chemical mixtures. An approach for overcoming this overlap problem is the use of multi-selective R(1) filtered and COSY or TOCSY experiments. Application of this methodology to compounds binding to the Sudlow site I of human serum albumin is presented. The screening is performed by simply monitoring the intensity of two signals. The precise measurement of the relative intensity of the two resonances permits determination of the binding constant of the NMR-hit. For a simple competition binding mechanism, the rapidly-derived NMR binding constants are in good agreement with the values derived from full-titration ITC and fluorescence spectroscopy measurements.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Calorimetry , Fluorescence , Protons
16.
J Am Chem Soc ; 125(25): 7696-703, 2003 Jun 25.
Article in English | MEDLINE | ID: mdl-12812511

ABSTRACT

Competition ligand-based NMR screening experiments have recently been introduced to overcome most of the problems associated with traditional ligand-based NMR screening. Molecules with marginal solubility and high affinity for a given target can be easily identified in a high-throughput manner by screening chemical mixtures against the target in the presence of a weak- to medium-affinity ligand of known binding constant. While the original competition-based approaches utilized (1)H detection, significant advantages are obtained using (19)F detection. The absence of spectral overlap permits the screening of large chemical mixtures and allows for automated analysis of the spectra, even in the presence of protonated buffers, solvents, and detergents. The large chemical shift anisotropy of fluorine and the significant exchange contribution allow for the selection of a weak-affinity spy molecule, thus resulting in a lower binding affinity threshold for the identified NMR hits. The method, labeled FAXS (fluorine chemical shift anisotropy and exchange for screening) is rapid and requires only a limited amount of protein and, therefore, compares favorably with the other established non-NMR techniques used in high-throughput screening. Herein the theoretical aspects of this powerful (19)F-based approach are presented and discussed in detail. The experimental conditions together with the detection limits and binding constant measurements are investigated using human serum albumin as the target.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Serum Albumin/chemistry , Fluorine , Humans , Ligands , Sensitivity and Specificity , Titrimetry
17.
Comb Chem High Throughput Screen ; 5(8): 605-11, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12470257

ABSTRACT

High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection. Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target. A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands. Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits. Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened. Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Protein Serine-Threonine Kinases/chemistry , Anisotropy , Binding, Competitive , Computer Simulation , Drug Design , Escherichia coli/metabolism , Fluorine Compounds/chemistry , Ligands , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism
18.
Comb Chem High Throughput Screen ; 5(8): 645-50, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12470261

ABSTRACT

Many lead molecules that have high affinity for a therapeutic target in vitro exhibit a reduced efficacy in vivo. Drug binding to human serum albumin is a major contributor to this reduction in potency, and many drug discovery programs expand significant resources preparing compounds that have decreased albumin binding. As rational and structure-based approaches have already been demonstrated to design compounds that have reduced affinity for albumin, the ability to rapidly and accurately assess protein binding will be valuable in lead optimization. This review will summarize some of the NMR-based efforts towards developing universal, rapid, accurate, and site-specific assays for estimating protein binding.


Subject(s)
Drug Design , Nuclear Magnetic Resonance, Biomolecular/methods , Serum Albumin/metabolism , Binding Sites , Binding, Competitive , Computer Simulation , Humans , Kinetics , Ligands , Protein Binding , Serum Albumin/chemistry , Structure-Activity Relationship , Tryptophan/chemistry , Tryptophan/metabolism
19.
Anal Biochem ; 309(2): 186-95, 2002 Oct 15.
Article in English | MEDLINE | ID: mdl-12413450

ABSTRACT

Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K(d)'s) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.


Subject(s)
Enzyme Inhibitors/analysis , Hepacivirus/enzymology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Calorimetry/methods , Calorimetry, Differential Scanning , Circular Dichroism , Crystallography, X-Ray , DNA/metabolism , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Kinetics , Ligands , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Denaturation , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Substrate Specificity
20.
J Am Chem Soc ; 124(26): 7702-9, 2002 Jul 03.
Article in English | MEDLINE | ID: mdl-12083923

ABSTRACT

The Achilles heel of ligand-based NMR screening methods is their failure to detect high-affinity ligands and molecules that bind covalently to the receptor. We have developed a novel approach for performing high-throughput screening with NMR spectroscopy that overcomes this limitation. The method also permits detection of potential high-affinity molecules that are only marginally soluble, thus significantly enlarging the diversity of compounds amenable to NMR screening. The techniques developed utilize transverse and/or selective longitudinal relaxation parameters in combination with competition binding experiments. Mathematical expressions are derived for proper setup of the NMR experiments and for extracting an approximate value of the binding constant for the identified ligand from a single-point measurement. With this approach it is possible to screen thousands of compounds in a short period of time against protein or DNA and RNA fragments. The methodology can also be applied for screening plant and fungi extracts.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Binding, Competitive , DNA/chemistry , DNA/metabolism , Fungi/chemistry , Fungi/metabolism , Humans , Kinetics , Plant Extracts/chemistry , Plant Extracts/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , RNA/chemistry , RNA/metabolism , Serum Albumin/chemistry , Serum Albumin/metabolism , Thermodynamics
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