Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 22
Filter
1.
J Med Chem ; 67(4): 2907-2940, 2024 Feb 22.
Article in English | MEDLINE | ID: mdl-38348661

ABSTRACT

The matrix metalloprotease ADAMTS7 has been identified by multiple genome-wide association studies as being involved in the development of coronary artery disease. Subsequent research revealed the proteolytic function of the enzyme to be relevant for atherogenesis and restenosis after vessel injury. Based on a publicly known dual ADAMTS4/ADAMTS5 inhibitor, we have in silico designed an ADAMTS7 inhibitor of the catalytic domain, which served as a starting point for an optimization campaign. Initially our inhibitors suffered from low selectivity vs MMP12. An X-ray cocrystal structure inspired us to exploit amino acid differences in the binding site of MMP12 and ADAMTS7 to improve selectivity. Further optimization composed of employing 5-membered heteroaromatic groups as hydantoin substituents to become more potent on ADAMTS7. Finally, fine-tuning of DMPK properties yielded BAY-9835, the first orally bioavailable ADAMTS7 inhibitor. Further optimization to improve selectivity vs ADAMTS12 seems possible, and a respective starting point could be identified.


Subject(s)
Atherosclerosis , Coronary Artery Disease , Humans , ADAMTS7 Protein/genetics , ADAMTS7 Protein/metabolism , Genome-Wide Association Study , Matrix Metalloproteinase 12
2.
bioRxiv ; 2023 Apr 27.
Article in English | MEDLINE | ID: mdl-37162877

ABSTRACT

Corin is a transmembrane tethered enzyme best known for processing the hormone atrial natriuretic peptide (ANP) in cardiomyocytes to control electrolyte balance and blood pressure. Loss of function mutations in Corin prevent ANP processing and lead to hypertension. Curiously, Corin loss of function variants also result in lighter coat color pigmentation in multiple species. Corin pigmentation effects are dependent on a functional Agouti locus encoding the agouti-signaling protein (ASIP) based on a genetic interaction. However, the nature of this conserved role of Corin has not been defined. Here we report that ASIP is a direct proteolytic substrate of the Corin enzyme.

3.
Mol Cell Proteomics ; 21(4): 100223, 2022 04.
Article in English | MEDLINE | ID: mdl-35283288

ABSTRACT

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.


Subject(s)
Coronary Artery Disease , Peptide Hydrolases , ADAMTS7 Protein , Animals , Biomarkers , Endopeptidases , Endothelial Cells/metabolism , Mice , Peptide Hydrolases/metabolism , Proteome/chemistry , Tail/metabolism
4.
Circ Genom Precis Med ; 14(5): e003399, 2021 10.
Article in English | MEDLINE | ID: mdl-34592835

ABSTRACT

BACKGROUND: Corin is a protease expressed in cardiomyocytes that plays a key role in salt handling and intravascular volume homeostasis via activation of natriuretic peptides. It is unknown if Corin loss-of-function (LOF) is causally associated with risk of coronary artery disease (CAD). METHODS: We analyzed all coding CORIN variants in an Italian case-control study of CAD. We functionally tested all 64 rare missense mutations in Western Blot and Mass Spectroscopy assays for proatrial natriuretic peptide cleavage. An expanded rare variant association analysis for Corin LOF mutations was conducted in whole exome sequencing data from 37 799 CAD cases and 212 184 controls. RESULTS: We observed LOF variants in CORIN in 8 of 1803 (0.4%) CAD cases versus 0 of 1725 controls (P, 0.007). Of 64 rare missense variants profiled, 21 (33%) demonstrated <30% of wild-type activity and were deemed damaging in the 2 functional assays for Corin activity. In a rare variant association study that aggregated rare LOF and functionally validated damaging missense variants from the Italian study, we observed no association with CAD-21 of 1803 CAD cases versus 12 of 1725 controls with adjusted odds ratio of 1.61 ([95% CI, 0.79-3.29]; P=0.17). In the expanded sequencing dataset, there was no relationship between rare LOF variants with CAD was also observed (odds ratio, 1.15 [95% CI, 0.89-1.49]; P=0.30). Consistent with the genetic analysis, we observed no relationship between circulating Corin concentrations with incident CAD events among 4744 participants of a prospective cohort study-sex-stratified hazard ratio per SD increment of 0.96 ([95% CI, 0.87-1.07], P=0.48). CONCLUSIONS: Functional testing of missense mutations improved the accuracy of rare variant association analysis. Despite compelling pathophysiology and a preliminary observation suggesting association, we observed no relationship between rare damaging variants in CORIN or circulating Corin concentrations with risk of CAD.


Subject(s)
Coronary Artery Disease/genetics , Genomics , Mutation, Missense , Sequence Analysis, DNA , Serine Endopeptidases/genetics , Adult , Coronary Artery Disease/epidemiology , Female , Humans , Italy/epidemiology , Male , Risk Factors
5.
Nat Chem Biol ; 15(5): 550, 2019 05.
Article in English | MEDLINE | ID: mdl-30858597

ABSTRACT

In the version of this article originally published, several data points in Fig. 4c were shifted out of place during production. The corrected version of Fig. 4c is shown below. This error has been corrected in the PDF and HTML versions of the article.

6.
Nat Chem Biol ; 15(4): 419-426, 2019 04.
Article in English | MEDLINE | ID: mdl-30742124

ABSTRACT

The chemical functionalities within biopolymers determine their physical properties and biological activities. The relationship between the side chains available to a biopolymer population and the potential functions of the resulting polymers, however, has proven difficult to study experimentally. Using seven sets of chemically diverse charged, polar, and nonpolar side chains, we performed cycles of artificial translation, in vitro selections for binding to either PCSK9 or IL-6 protein, and replication on libraries of random side chain-functionalized nucleic acid polymers. Polymer sequence convergence, bulk population target binding, affinity of individual polymers, and head-to-head competition among post-selection libraries collectively indicate that polymer libraries with nonpolar side chains outperformed libraries lacking these side chains. The presence of nonpolar groups, resembling functionality existing in proteins but missing from natural nucleic acids, thus may be strong determinants of binding activity. This factor may contribute to the apparent evolutionary advantage of proteins over their nucleic acid precursors for some molecular recognition tasks.


Subject(s)
Biopolymers/chemistry , Biopolymers/physiology , DNA Replication , Humans , Interleukin-6/chemistry , Peptide Library , Polymers/chemistry , Proprotein Convertase 9/chemistry , Proteins/chemistry
7.
Biochem Pharmacol ; 160: 62-70, 2019 02.
Article in English | MEDLINE | ID: mdl-30553787

ABSTRACT

Corin (atrial natriuretic peptide-converting enzyme, EC 3.4.21) is a transmembrane serine protease expressed in cardiomyocytes. Corin exerts its cardioprotective effects via the proteolytic cleavage and activation of pro-atrial natriuretic peptide (pro-ANP) to ANP. We recently described an ANP reporter cell line stably expressing the ANP receptor, a cGMP-dependent cation channel used as a real-time cGMP biosensor, and the Ca2+-sensitive photoprotein aequorin. Here, we describe the generation of a novel reporter cell line expressing the calcium biosensor GCaMP6 instead of aequorin. In contrast to the luminescence-based assay, ANP stimulation of our novel GCaMP6 reporter cell resulted in stable, long-lasting fluorescence signals. Using this novel reporter system, we were able to detect pro-ANP to ANP conversion by purified, soluble wildtype corin (solCorin), but not the active site mutant solCorin(S985A), resulting in left-shifted concentration-response curves. Furthermore, cellular pro-ANPase activity could be detected on HEK 293 cells after transient expression of wildtype corin. In contrast, corin activity was not detected after transfection with the inactive corin(S985A) variant. In supernatants from cardiomyocyte-derived HL-1 cells pro-ANP to ANP conversion could also be detected, while in HL-1 corin knockout cells no conversion was observed. These findings underline the role of corin as the pro-ANP convertase. Our novel fluorescence-based ANP reporter cell line is well-suited for the sensitive detection of corin activity, and may be used for the identification and characterization of novel corin modulators.


Subject(s)
Atrial Natriuretic Factor/metabolism , Cyclic Nucleotide-Gated Cation Channels/metabolism , Receptors, Atrial Natriuretic Factor/metabolism , Serine Endopeptidases/metabolism , Animals , Atrial Natriuretic Factor/genetics , Atrial Natriuretic Factor/pharmacology , Calcium/metabolism , Cell Line , Cyclic GMP/metabolism , Cyclic Nucleotide-Gated Cation Channels/genetics , HEK293 Cells , Humans , Mice , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Receptors, Atrial Natriuretic Factor/genetics , Serine Endopeptidases/genetics
8.
Bioorg Med Chem Lett ; 27(9): 2069-2073, 2017 05 01.
Article in English | MEDLINE | ID: mdl-28284804

ABSTRACT

Glucokinase (GK, hexokinase IV) is a unique hexokinase that plays a central role in mammalian glucose homeostasis. Glucose phosphorylation by GK in the pancreatic ß-cell is the rate-limiting step that controls glucose-stimulated insulin secretion. Similarly, GK-mediated glucose phosphorylation in hepatocytes plays a major role in increasing hepatic glucose uptake and metabolism and possibly lowering hepatic glucose output. Small molecule GK activators (GKAs) have been identified that increase enzyme activity by binding to an allosteric site. GKAs offer a novel approach for the treatment of Type 2 Diabetes Mellitus (T2DM) and as such have garnered much attention. We now report the design, synthesis, and biological evaluation of a novel series of 2,5,6-trisubstituted indole derivatives that act as highly potent GKAs. Among them, Compound 1 was found to possess high in vitro potency, excellent physicochemical properties, and good pharmacokinetic profile in rodents. Oral administration of Compound 1 at doses as low as 0.03mg/kg led to robust blood glucose lowering efficacy in 3week high fat diet-fed mice.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Enzyme Activators/chemistry , Enzyme Activators/therapeutic use , Glucokinase/metabolism , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/therapeutic use , Indoles/chemistry , Indoles/therapeutic use , Allosteric Regulation/drug effects , Animals , Blood Glucose/analysis , Blood Glucose/metabolism , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/metabolism , Drug Design , Enzyme Activation/drug effects , Enzyme Activators/pharmacokinetics , Enzyme Activators/pharmacology , Humans , Hypoglycemic Agents/pharmacokinetics , Hypoglycemic Agents/pharmacology , Indoles/pharmacokinetics , Indoles/pharmacology , Insulin/blood , Insulin/metabolism , Mice , Mice, Inbred C57BL
9.
Bioorg Med Chem Lett ; 27(9): 2063-2068, 2017 05 01.
Article in English | MEDLINE | ID: mdl-28284809

ABSTRACT

Systemically acting glucokinase activators (GKA) have been demonstrated in clinical trials to effectively lower blood glucose in patients with type II diabetes. However, mechanism-based hypoglycemia is a major adverse effect that limits the therapeutic potential of these agents. We hypothesized that the predominant mechanism leading to hypoglycemia is GKA-induced excessive insulin secretion from pancreatic ß-cells at (sub-)euglycemic levels. We further hypothesized that restricting GK activation to hepatocytes would maintain glucose-lowering efficacy while significantly reducing hypoglycemic risk. Here we report the discovery of a novel series of carboxylic acid substituted GKAs based on pyridine-2-carboxamide. These GKAs exhibit preferential distribution to the liver versus the pancreas in mice. SAR studies led to the identification of a potent and orally active hepatoselective GKA, compound 6. GKA 6 demonstrated robust glucose lowering efficacy in high fat diet-fed mice at doses ⩾10mpk, with ⩾70-fold liver:pancreas distribution, minimal effects on plasma insulin levels, and significantly reduced risk of hypoglycemia.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Enzyme Activators/pharmacology , Glucokinase/metabolism , Hypoglycemic Agents/pharmacology , Pyridines/pharmacology , Animals , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/metabolism , Drug Discovery , Enzyme Activators/chemistry , Enzyme Activators/pharmacokinetics , Enzyme Activators/therapeutic use , Humans , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacokinetics , Hypoglycemic Agents/therapeutic use , Insulin/blood , Liver/drug effects , Liver/metabolism , Mice , Mice, Inbred C57BL , Pancreas/drug effects , Pancreas/metabolism , Pyridines/chemistry , Pyridines/pharmacokinetics , Pyridines/therapeutic use
10.
Protein Sci ; 25(11): 2018-2027, 2016 11.
Article in English | MEDLINE | ID: mdl-27534510

ABSTRACT

Circulating low-density lipoprotein cholesterol (LDLc) is regulated by membrane-bound LDL receptor (LDLr). Upon LDLc and LDLr interaction the complex is internalized by the cell, leading to LDLc degradation and LDLr recycling back to the cell surface. The proprotein convertase subtilisin/kexin type 9 (PCSK9) protein regulates this cycling. PCSK9 is secreted from the cell and binds LDLr. When the complex is internalized, PCSK9 prevents LDLr from shuttling back to the surface and instead targets it for degradation. PCSK9 is a serine protease expressed as a zymogen that undergoes autoproteolysis, though the two resulting protein domains remain stably associated as a heterodimer. This PCSK9 autoprocessing is required for the protein to be secreted from the cell. To date, direct analysis of PCSK9 autoprocessing has proven challenging, as no catalytically active zymogen has been isolated. A PCSK9 loss-of-function point mutation (Q152H) that reduces LDLc levels two-fold was identified in a patient population. LDLc reduction was attributed to a lack of PCSK9(Q152H) autoprocessing preventing secretion of the protein. We have isolated a zymogen form of PCSK9, PCSK9(Q152H), and a related mutation (Q152N), that can undergo slow autoproteolysis. We show that the point mutation prevents the formation of the mature form of PCSK9 by hindering folding, reducing the rate of autoproteolysis, and destabilizing the heterodimeric form of the protein. In addition, we show that the zymogen form of PCSK9 adopts a structure that is distinct from the processed form and is unable to bind a mimetic peptide based on the EGF-A domain of the LDLr.


Subject(s)
Peptides/chemistry , Point Mutation , Proprotein Convertase 9/chemistry , Protein Multimerization , Receptors, LDL/chemistry , Amino Acid Substitution , Humans , Peptides/genetics , Peptides/metabolism , Proprotein Convertase 9/genetics , Proprotein Convertase 9/metabolism , Protein Binding , Protein Domains , Receptors, LDL/genetics , Receptors, LDL/metabolism
11.
Nat Biotechnol ; 28(7): 733-7, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20581844

ABSTRACT

The specificity of SCF ubiquitin ligase-mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCF(Cdc4). A co-crystal structure reveals that SCF-I2 inserts itself between the beta-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 A away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.


Subject(s)
Ubiquitin-Protein Ligases/metabolism , Allosteric Regulation , Models, Molecular , Protein Conformation , Ubiquitin-Protein Ligases/antagonists & inhibitors
12.
J Lipid Res ; 51(9): 2739-52, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20458119

ABSTRACT

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.


Subject(s)
Anticholesteremic Agents/chemistry , Cholesterol Ester Transfer Proteins/antagonists & inhibitors , Oxazolidinones/chemistry , Quinolines/chemistry , Sulfhydryl Compounds/chemistry , Amides , Animals , Anticholesteremic Agents/metabolism , Blood Proteins/metabolism , Esters , Humans , Mice , Molecular Structure , Oxazolidinones/metabolism , Quinolines/metabolism , Sulfhydryl Compounds/metabolism
13.
Bioorg Med Chem Lett ; 19(10): 2742-6, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19364644

ABSTRACT

Potent SAH analogues with constrained homocysteine units have been designed and synthesized as inhibitors of human DNMT enzymes. The five membered (2S,4S)-4-mercaptopyrrolidine-2-carboxylic acid, in 1a, was a good replacement for homocysteine, while the corresponding six-member counterpart was less active. Further optimization of 1a, changed the selectivity profile of these inhibitors. A Chloro substituent at the 2-position of 1a, compound 1d, retained potency against DNMT1, while N(6) alkylation, compound 7a, conserved DNMT3b2 activity. The concomitant substitutions of 1a at both 2- and N(6) positions reduced activity against both enzymes.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Homocysteine/analogs & derivatives , Pyrrolidines/chemical synthesis , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/pharmacology , Homocysteine/chemical synthesis , Homocysteine/pharmacology , Humans , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Structure-Activity Relationship
14.
Bioorg Med Chem Lett ; 19(10): 2747-51, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19362833

ABSTRACT

The inhibitory activity of base-modified SAH analogues and the specificity of inhibiting human DNMT1 and DNMT3b2 enzymes was explored. The 6-amino group was essential while the 7-N of the adenine ring of SAH could be replaced by CH- without loss of activity against both enzymes. The introduction of small groups at the 2-position of the adenine moiety favors DNMT1 over DNMT3b2 inhibition whereas alkylation of the N(6)-amino moiety favors the inhibition of DNMT3b2 enzyme.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , S-Adenosylhomocysteine/chemical synthesis , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , S-Adenosylhomocysteine/chemistry , S-Adenosylhomocysteine/pharmacology , Structure-Activity Relationship
15.
J Med Chem ; 50(4): 755-64, 2007 Feb 22.
Article in English | MEDLINE | ID: mdl-17300162

ABSTRACT

The biosynthesis of methionine is an attractive antibiotic target given its importance in protein and DNA metabolism and its absence in mammals. We have performed a high-throughput screen of the methionine biosynthesis enzyme cystathionine beta-lyase (CBL) against a library of 50 000 small molecules and have identified several compounds that inhibit CBL enzyme activity in vitro. These hit molecules were of two classes: those that blocked CBL activity with mixed steady-state inhibition and those that covalently interacted with the enzyme at the active site pyridoxal phosphate cofactor with slow-binding inhibition kinetics. We determined the crystal structure of one of the slow-binding inhibitors in complex with CBL and used this structure as a guide in the synthesis of a small, focused library of analogues, some of which had improved enzyme inhibition properties. These studies provide the first lead molecules for antimicrobial agents that target cystathionine beta-lyase in methionine biosynthesis.


Subject(s)
Anti-Infective Agents/chemical synthesis , Bacteria/enzymology , Benzamides/chemical synthesis , Hydrazines/chemical synthesis , Lyases/antagonists & inhibitors , Lyases/chemistry , Models, Molecular , Quantitative Structure-Activity Relationship , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Benzamides/chemistry , Benzamides/pharmacology , Candida albicans/drug effects , Crystallography, X-Ray , Escherichia coli/enzymology , Hydrazines/chemistry , Hydrazines/pharmacology , Lyases/genetics , Microbial Sensitivity Tests , Salmonella typhi/enzymology
17.
Chem Biol ; 13(4): 437-41, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16632256

ABSTRACT

The lipopolysaccharide (LPS)-rich outer membrane of gram-negative bacteria provides a protective barrier that insulates these organisms from the action of numerous antibiotics. Breach of the LPS layer can therefore provide access to the cell interior to otherwise impermeant toxic molecules and can expose vulnerable binding sites for immune system components such as complement. Inhibition of LPS biosynthesis, leading to a truncated LPS molecule, is an alternative strategy for antibacterial drug development in which this vital cellular structure is weakened. A significant challenge for in vitro screens of small molecules for inhibition of LPS biosynthesis is the difficulty in accessing the complex carbohydrate substrates. We have optimized an assay of the enzymes required for LPS heptose biosynthesis that simultaneously surveys five enzyme activities by using commercially available substrates and report its use in a small-molecule screen that identifies an inhibitor of heptose synthesis.


Subject(s)
Adenosine Diphosphate Sugars/biosynthesis , Enzyme Inhibitors/pharmacology , Glycosyltransferases/antagonists & inhibitors , Lipopolysaccharides/biosynthesis , Cell Membrane/drug effects , Cell Membrane/metabolism , Drug Evaluation, Preclinical , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/metabolism , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/metabolism , Kinetics , Microbial Sensitivity Tests , Multienzyme Complexes/antagonists & inhibitors , Nucleotidyltransferases/antagonists & inhibitors , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Recombinant Proteins/antagonists & inhibitors
18.
J Biomol Screen ; 10(7): 653-7, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16170050

ABSTRACT

High-throughput screening (HTS) generates an abundance of data that are a valuable resource to be mined. Dockers and data miners can use "real-world" HTS data to test and further develop their tools. A screen of 50,000 diverse small molecules was carried out against Escherichia coli dihydrofolate reductase (DHFR) and compared with a previous screen of 50,000 compounds against the same target. Identical assays and conditions were maintained for both studies. Prior to the completion of the second screen, the original screening data were publicly released for use as a "training set", and computational chemists and data analysts were challenged to predict the activity of compounds in this second "test set". Upon completion, the primary screen of the test set generated no potent inhibitors of DHFR activity.


Subject(s)
Computational Biology , Models, Biological , Models, Chemical , Tetrahydrofolate Dehydrogenase/chemistry , Computational Biology/methods , Escherichia coli/enzymology , Folic Acid Antagonists/chemistry , Folic Acid Antagonists/metabolism , Tetrahydrofolate Dehydrogenase/metabolism , Trimethoprim/chemistry , Trimethoprim/metabolism
19.
Chem Biol ; 11(10): 1445-53, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15489171

ABSTRACT

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The main proteinase of SARS-CoV, 3CLpro, is an attractive target for therapeutics against SARS owing to its fundamental role in viral replication. We sought to identify novel inhibitors of 3CLpro to advance the development of appropriate therapies in the treatment of SARS. 3CLpro was cloned, expressed, and purified from the Tor2 isolate. A quenched fluorescence resonance energy transfer assay was developed for 3CLpro to screen the proteinase against 50,000 drug-like small molecules on a fully automated system. The primary screen identified 572 hits; through a series of virtual and experimental filters, this number was reduced to five novel small molecules that show potent inhibitory activity (IC50 = 0.5-7 microM) toward SARS-CoV 3CLpro.


Subject(s)
Antiviral Agents/isolation & purification , Endopeptidases/metabolism , Protease Inhibitors/isolation & purification , Severe acute respiratory syndrome-related coronavirus/drug effects , Severe acute respiratory syndrome-related coronavirus/enzymology , Viral Proteins/antagonists & inhibitors , Viral Proteins/metabolism , Animals , Antiviral Agents/pharmacology , Cattle , Coronavirus 3C Proteases , Cysteine Endopeptidases , Mass Spectrometry/methods , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology
20.
J Am Chem Soc ; 126(22): 6878-9, 2004 Jun 09.
Article in English | MEDLINE | ID: mdl-15174852

ABSTRACT

Firefly luciferase (FL) was entrapped in sol-gel-derived silica containing precursors based on covalent linkage of d-gluconolactone or d-maltonolactone to (aminopropyl)triethoxysilane to form N-(3-triethoxysilylpropyl)gluconamide or N-(3-triethoxysilylpropyl)maltonamide. The enzyme was active and stable in this material and showed catalytic constants close to those in solution. As little as 20 amol ATP could be detected with the entrapped FL, and the entrapped enzyme could be used over several cycles.


Subject(s)
Adenosine Triphosphate/analysis , Carbohydrates/chemistry , Coleoptera/enzymology , Gels/chemistry , Luciferases/metabolism , Silicon Dioxide/chemistry , Animals , Catalysis , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Fluorescence
SELECTION OF CITATIONS
SEARCH DETAIL
...