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1.
Med Chem Res ; 29(7): 1238-1246, 2020 Jul.
Article in English | MEDLINE | ID: mdl-33584083

ABSTRACT

Modification of endogenous proteins by drugs and drug metabolites are thought to be a cause of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP) is a commonly prescribed antibiotic that has been implicated in IADRs; however, there is no known mechanism by which this drug or its metabolites modify proteins. This study describes the results of screening trimethoprim and its primary metabolites for the ability to covalently modify human serum albumin (HSA). The first step of the screen was in vitro reactions of the compounds with HSA followed by western blotting with antisera specific to drug-modified proteins. Compounds with positive signal in the western blot were then screened using an untargeted peptide profiling method to discover modified peptides. This strategy identified two sites in HSA that are modified by incubation with a TMP metabolite, α-hydroxy trimethoprim (Cα-OH-TMP).

2.
Nat Chem Biol ; 10(8): 629-31, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24997604

ABSTRACT

We report that 4-(3-(benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), which behaves as a positive allosteric modulator at the glucagon-like peptide-1 receptor (GLP-1R), covalently modifies cysteines 347 and 438 in GLP-1R. C347, located in intracellular loop 3 of GLP-1R, is critical to the activity of BETP and a structurally distinct GLP-1R ago-allosteric modulator, N-(tert-butyl)-6,7-dichloro-3-(methylsulfonyl)quinoxalin-2-amine. We further show that substitution of cysteine for phenylalanine 345 in the glucagon receptor is sufficient to confer sensitivity to BETP.


Subject(s)
Pyrimidines/chemistry , Receptors, Glucagon/metabolism , Animals , CHO Cells , Cricetulus , Cysteine/chemistry , Glucagon-Like Peptide 1/agonists , Glucagon-Like Peptide-1 Receptor , Humans , Ligands , Pyrimidines/metabolism , Receptors, Glucagon/chemistry
3.
Drug Metab Dispos ; 41(8): 1470-9, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23653442

ABSTRACT

4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP) represents a novel small-molecule activator of the glucagon-like peptide-1 receptor (GLP-1R), and exhibits glucose-dependent insulin secretion in rats following i.v. (but not oral) administration. To explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after i.v. and oral dosing. Failure to detect BETP in circulation after oral administration of a 10-mg/kg dose in rats was consistent with the lack of an insulinotropic effect of orally administered BETP in this species. Likewise, systemic concentrations of BETP in the rat upon i.v. administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma, and liver microsomes from rodents and humans indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of NADP was suggestive of a covalent interaction between BETP and a protein amino acid residue(s) in these matrices. Incubations of BETP with glutathione (GSH) in buffer revealed a rapid nucleophilic displacement of the ethylsulfoxide functionality by GSH to yield adduct M1, which indicated that BETP was intrinsically electrophilic. The structure of M1 was unambiguously identified by comparison of its chromatographic and mass spectral properties with an authentic standard. The GSH conjugate of BETP was also characterized in NADPH- and GSH-supplemented liver microsomes and in plasma samples from the pharmacokinetic studies. Unlike BETP, M1 was inactive as an allosteric modulator of the GLP-1R.


Subject(s)
Pyrimidines/chemistry , Receptors, Glucagon/metabolism , Allosteric Regulation/drug effects , Animals , CHO Cells , Cricetinae , Cricetulus , Drug Stability , Glucagon-Like Peptide-1 Receptor , Glutathione/chemistry , Humans , Male , Mice , Microsomes, Liver/metabolism , Pyrimidines/metabolism , Pyrimidines/pharmacology , Rats , Rats, Wistar
4.
Proc Natl Acad Sci U S A ; 109(22): 8523-7, 2012 May 29.
Article in English | MEDLINE | ID: mdl-22586115

ABSTRACT

Peptide hormones and neuropeptides have important roles in physiology and therefore the regulation of these bioactive peptides is of great interest. In some cases proteolysis controls the concentrations and signaling of bioactive peptides, and the peptidases that mediate this biochemistry have proven to be extremely successful drug targets. Due to the lack of any general method to identify these peptidases, however, the role of proteolysis in the regulation of most neuropeptides and peptide hormones is unknown. This limitation prompted us to develop an advanced peptidomics-based strategy to identify the peptidases responsible for the proteolysis of significant bioactive peptides. The application of this approach to calcitonin gene-related peptide (CGRP), a neuropeptide associated with blood pressure and migraine, revealed the endogenous CGRP cleavage sites. This information was then used to biochemically purify the peptidase capable of proteolysis of CGRP at those cleavage sites, which led to the identification of insulin-degrading enzyme (IDE) as a candidate CGRP-degrading enzyme. CGRP had not been identified as an IDE substrate before and we tested the physiological relevance of this interaction by quantitative measurements of CGRP using IDE null (IDE(-/-)) mice. In the absence of IDE, full-length CGRP levels are elevated in vivo, confirming IDE as an endogenous CGRP-degrading enzyme. By linking CGRP and IDE, this strategy uncovers a previously unknown pathway for CGRP regulation and characterizes an additional role for IDE. More generally, this work suggests that this may be an effective general strategy for characterizing these pathways and peptidases moving forward.


Subject(s)
Calcitonin Gene-Related Peptide/metabolism , Insulysin/metabolism , Peptide Fragments/metabolism , Peptides/metabolism , Amino Acid Sequence , Animals , Binding Sites , Calcitonin Gene-Related Peptide/chemistry , Chromatography, Liquid , Female , Insulysin/chemistry , Insulysin/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptides/chemistry , Protein Binding , Protein Structure, Tertiary , Proteolysis , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spinal Cord/chemistry , Spinal Cord/metabolism , Substrate Specificity , Tandem Mass Spectrometry
5.
Annu Rev Immunol ; 30: 611-46, 2012.
Article in English | MEDLINE | ID: mdl-22449030

ABSTRACT

Stressors ranging from nutrient deprivation to immune signaling can induce the degradation of cytoplasmic material by a process known as autophagy. Increasingly, research on autophagy has begun to focus on its role in inflammation and the immune response. Autophagy acts as an immune effector that mediates pathogen clearance. The roles of autophagy bridge both the innate and adaptive immune systems and include functions in thymic selection, antigen presentation, promotion of lymphocyte homeostasis and survival, and regulation of cytokine production. In this review, we discuss the mechanisms by which autophagy is regulated, as well as the functions of autophagy and autophagy proteins in immunity and inflammation.


Subject(s)
Autophagy/immunology , Immune System/immunology , Adaptive Immunity , Animals , Humans , Immunity, Innate , Infections/immunology , Infections/microbiology , Infections/virology
6.
AAPS J ; 12(4): 483-91, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20552307

ABSTRACT

The prolyl peptidases are a family of enzymes characterized by a biochemical preference for cleaving proline-containing peptides. The members of this enzyme family include prolyl endopeptidase, prolyl endopeptidase-like, dipeptidyl peptidase 4 (DPP4), DPP7, DPP8, DPP9, and fibroblast activation protein. DPP4 is the best studied member of the family, due to its role in physiological glucose tolerance, exerted through the regulation of the insulinotropic peptide glucagon-like peptide-1. While other members of the prolyl peptidase family have also been implicated in various (patho)physiological processes, the underlying peptides and pathways regulated by these enzymes are less clear. The identification of endogenous substrates of the prolyl peptidases is an important step in elucidating the molecular mechanisms of these enzymes. Here, we highlight the utility of liquid chromatography-mass spectrometry-based peptidomics to enable the discovery of endogenous prolyl peptidase substrates directly from tissues, and demonstrate the utility of this information in understanding the biochemical and physiological functions of the prolyl peptidases.


Subject(s)
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Proteomics , Amino Acid Sequence , Animals , Central Nervous System/metabolism , Chromatography, Liquid , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/chemistry , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Humans , Kidney/metabolism , Mass Spectrometry , Mice , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity
7.
Biochemistry ; 48(50): 11971-81, 2009 Dec 22.
Article in English | MEDLINE | ID: mdl-19911840

ABSTRACT

Prolyl endopeptidase (Prep) is a member of the prolyl peptidase family and is of interest because of its unique biochemistry and connections to cognitive function. Using an unbiased mass spectrometry (MS)-based peptidomics platform, we identified Prep-regulated peptides in the central nervous system (CNS) of mice by measuring changes in the peptidome as a function of Prep activity. This approach was validated by the identification of known Prep substrates, such as the neuropeptide substance P and thymosin-beta4, the precursor to the bioactive peptide Ac-SDKP. In addition to these known substrates, we also discovered that Prep regulates many additional peptides, including additional bioactive peptides and proline rich peptides (PRPs). Biochemical experiments confirmed that some of these Prep-regulated peptides are indeed substrates of the enzyme. Moreover, these experiments also supported the known preference of Prep for shorter peptides while revealing a previously unknown cleavage site specificity of Prep when processing certain multi-proline-containing peptides, including PRPs. The discovery of Prep-regulated peptides implicates Prep in new biological pathways and provides insights into the biochemistry of this enzyme.


Subject(s)
Peptides/chemistry , Proteomics/methods , Serine Endopeptidases/chemistry , Spinal Cord/enzymology , Amino Acid Sequence , Animals , Azabicyclo Compounds/pharmacology , COS Cells , Calcitonin Gene-Related Peptide/physiology , Chlorocebus aethiops , Chromatography, Liquid , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Peptides/antagonists & inhibitors , Peptides/metabolism , Prolyl Oligopeptidases , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spinal Cord/drug effects , Substrate Specificity/drug effects , Tandem Mass Spectrometry
8.
Nat Chem Biol ; 5(1): 23-5, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19011639

ABSTRACT

Peptide metabolism is a complex process that involves many proteins working in concert. Mass spectrometry-based global peptide profiling of mice lacking dipeptidyl peptidase 4 (DPP4) identified endogenous DPP4 substrates and revealed an unrecognized pathway during proline peptide catabolism that interlinks aminopeptidase and DPP4 activities. Together, these studies elucidate specific aspects of DPP4-regulated metabolism and, more generally, highlight the utility of global peptide profiling for studying peptide metabolism in vivo.


Subject(s)
Dipeptidyl Peptidase 4/metabolism , Gene Expression Profiling , Gene Expression Regulation/physiology , Amino Acid Sequence , Animals , Dipeptidyl Peptidase 4/chemistry , Dipeptidyl Peptidase 4/genetics , Dipeptidyl-Peptidase IV Inhibitors , Mice , Mice, Knockout , Protein Folding
9.
J Am Chem Soc ; 128(45): 14537-41, 2006 Nov 15.
Article in English | MEDLINE | ID: mdl-17090037

ABSTRACT

We have controllably modulated the drain current (I(D)) and threshold voltage (V(T)) in pseudo metal-oxide-semiconductor field-effect transistors (MOSFETs) by grafting a monolayer of molecules atop oxide-free H-passivated silicon surfaces. An electronically controlled series of molecules, from strong pi-electron donors to strong pi-electron acceptors, was covalently attached onto the channel region of the transistors. The device conductance was thus systematically tuned in accordance with the electron-donating ability of the grafted molecules, which is attributed to the charge transfer between the device channel and the molecules. This surface grafting protocol might serve as a useful method for controlling electronic characteristics in small silicon devices at future technology nodes.

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