Ghrelin proteolysis by insulin-degrading enzyme

Here we report proteolysis of synthetic acylated human ghrelin by recombinant human insulin-degrading enzyme (IDE). Kinetic parameters and sites of proteolytic cleavage were evaluated. Ghrelin proteolysis by IDE was inhibited by ethylenediaminetetraacetate (EDTA), a metal chelating agent. Ghrelin proteolysis appears at least somewhat specific to M16 family proteases such as IDE, as the M13 protease neprilysin (NEP) did not exhibit ghrelin proteolysis in this study. A quenched fluorogenic peptide substrate comprising the primary sites of IDE-mediated ghrelin proteolysis (Mca-QRVQQRKESKK(Dnp)-OH; Mca: 7-methoxycoumarin-3-carboxylic acid; Dnp: 2,4-dinitrophenyl) was developed and used to evaluate enzyme specificity and kinetic parameters of proteolysis. Like acyl ghrelin, Mca-QRVQQRKESKK(Dnp)-OH was efficiently cleaved by IDE central to the target sequence. We anticipate that this quenched fluorogenic peptide substrate will be of value to future studies of ghrelin proteolysis by IDE and potentially other peptidases.

Kinetic characterization of inhibitors of histone deacetylases (HDACs) and sirtuins

Histone deacetylase (HDAC) inhibitors are employed for the treatment of lymphoma and are under development against multiple other types of cancer and neurodegenerative diseases. Here, we describe a robust and uncomplicated in vitro assay for HDAC inhibitor kinetic profiling. Enzyme and fluorogenic peptide substrate are incubated together with a small amount of protease “assay developer”, which enables continuous recording of substrate conversion under steady-state conditions. Assay progression curves upon addition of an inhibitors at varying concentrations permit determination of kinetic constants and overall inhibitor potency. This assay helped provide new insight into the kinetic properties of known HDAC inhibitors as well as the kinetic characterization of both inhibitors and substrates of sirtuin enzymes, which are class III HDACs involved in metabolic control and oncogene regulation.

Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening

The target of this study, the PfM18 aspartyl aminopeptidase ( PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository). A cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counterscreen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from high-throughput screening. Two structurally related compounds, CID 6852389 and CID 23724194, yielded micromolar potency and were inactive in CTSL1 titration experiments (IC50 >59.6 µM). As measured by the Ki assay, both compounds demonstrated micromolar noncompetitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrated potency in malaria growth assays (IC50 4 µM and 1.3 µM, respectively).