TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease.

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.

Side chain determinants of biopolymer function during selection and replication.

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.

Publisher Correction: Side chain determinants of biopolymer function during selection and replication.

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.

Novel, highly potent systemic glucokinase activators for the treatment of Type 2 Diabetes Mellitus.

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.

Identification of a new Corin atrial natriuretic peptide-converting enzyme substrate: Agouti-signaling protein (ASIP).

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.

Rare, Damaging DNA Variants in CORIN and Risk of Coronary Artery Disease: Insights From Functional Genomics and Large-Scale Sequencing Analyses.

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.

Biochemical characterization of cholesteryl ester transfer protein inhibitors.

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.

Development of a novel, sensitive cell-based corin assay.

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.

Inhibitors of bacterial cystathionine beta-lyase: leads for new antimicrobial agents and probes of enzyme structure and function.

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.

An in vitro screen of bacterial lipopolysaccharide biosynthetic enzymes identifies an inhibitor of ADP-heptose biosynthesis.

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.

Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor.

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.

Experimental screening of dihydrofolate reductase yields a "test set" of 50,000 small molecules for a computational data-mining and docking competition.

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.

High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase.

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.

Functional annotation of putative aminoglycoside antibiotic modifying proteins in Mycobacterium tuberculosis H37Rv.

The growing availability of sequences of bacterial genomes has revealed a number of open reading frames predicted by sequence alignment to encode antibiotic resistance proteins. The presence of these putative resistance genes within bacterial genomes raises important questions regarding potential reservoirs of resistance elements and their evolution. Here we examine four gene products encoding predicted aminoglycoside-aminocyclitol antibiotic modifying enzymes, two phosphotransferases and two acetyltransferases, derived from analysis of the genome sequence of Mycobacterium tuberculosis strain H37Rv with the goal of assigning biochemical function by purification of each protein and characterization of their ability to modify aminoglycoside antibiotics. Only one of these enzymes, the previously characterized aminoglycoside acetyltransferase AAC(2')-Ic, displayed compelling aminoglycoside modifying activity. While the putative phosphotransferase encoded by the Rv3225c gene did display low levels of aminoglycoside kinase activity, the predicted kinase encoded by the Rv3817 gene lacked any such activity. A potential aminoglycoside 6'-acetyltransferase, encoded by the Rv1347c gene, did not show antibiotic acylation activity but did demonstrate selective thioesterase activity with numerous acyl-CoAs. This activity, together with the genomic environment of the Rv1347c gene in a likely polyketide synthesis cluster, suggests a role for this protein in secondary metabolism and not in antibiotic modification. It was thus shown that only one of four putative aminoglycosides modifying enzymes derived from the whole genome sequencing of M. tuberculosis H37Rv showed sufficient predicted enzyme activity to be annotated as an aminoglycoside resistance element. This study demonstrates the necessity of biochemical annotation methods as a follow up to in silico sequence alignment-based methods of assigning gene product function.

Ultrasensitive ATP detection using firefly luciferase entrapped in sugar-modified sol-gel-derived silica.

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.

Cystathionine beta-lyase is important for virulence of Salmonella enterica serovar Typhimurium.

The biosynthesis of methionine in bacteria requires the mobilization of sulfur from Cys by the formation and degradation of cystathionine. Cystathionine beta-lyase, encoded by metC in bacteria and STR3 in Schizosaccharomyces pombe, catalyzes the breakdown of cystathionine to homocysteine, the penultimate step in methionine biosynthesis. This enzyme has been suggested to be the target for pyridinamine antimicrobial agents. We have demonstrated, by using purified enzymes from bacteria and yeast, that cystathionine beta-lyase is not the likely target of these agents. Nonetheless, an insertional inactivation of metC in Salmonella enterica serovar Typhimurium resulted in the attenuation of virulence in a mouse model of systemic infection. This result confirms a previous chemical validation of the Met biosynthetic pathway as a target for the development of antibacterial agents and demonstrates that cystathionine beta-lyase is important for bacterial virulence.