Identification and Characterization of Novel Inhibitors of Mammalian Aspartyl Aminopeptidase

Abstract

Aspartyl aminopeptidase (DNPEP) has been implicated in the control of angiotensin signaling and endosome trafficking, but its precise biologic roles remain incompletely defined. We performed a high-throughput screen of ∼25,000 small molecules to identify inhibitors of DNPEP for use as tools to study its biologic functions. Twenty-three confirmed hits inhibited DNPEPcatalyzed hydrolysis of angiotensin II with micromolar potency. A counter screen against glutamyl aminopeptidase (ENPEP), an enzyme with substrate specificity similar to that of DNPEP, identified eight DNPEP-selective inhibitors. Structure-activity relationships and modeling studies revealed structural features common to the identified inhibitors, including a metal-chelating group and a charged or polar moiety that could interact with portions of the enzyme active site. The compounds identified in this study should be valuable tools for elucidating DNPEP physiology.

Molecular Basis for Vitamin A Uptake and Storage in Vertebrates

“…LRAT catalyzes the transesterification of retinol utilizing a fatty acyl moiety present in the sn-1 position of phosphatidylcholine [38,39,40], whereas DGAT1 depends on a pre-activated form of fatty acid carried by acyl-CoA [41]. Additionally, in terms of substrate specificity, LRAT activity is limited to retinol and its derivatives such as endogenous apo-carotenoids or xenobiotic retinylamine that share a common structural motif [42,43]. DGAT1, however, exhibits much broader substrate specificity and transfers acyl moieties onto alternative substrates, including 1,2-diacylglycerol, contributing to the formation of triacylglycerols [44].…”

“…Esterase activity of the enzyme is not detectable. Instead, LRAT transfers the acyl chain exclusively from PC onto vitamin A or closely related retinoid-like substrates [40,43]. Moreover, LRAT’s activity is restricted towards the sn-1 fatty acid position in the phospholipids substrate [80].…”

Mammalian Glutamyl Aminopeptidase Genes (ENPEP) and Proteins: Comparative Studies of a Major Contributor to Arterial Hypertension

“…Figure 4 shows RNA-seq gene expression profiles across 53 selected tissues (or tissue segments) were examined from the public database for human ENPEP, based on expression levels for 175 individuals [ 16 ] (Data Source: GTEx Analysis Release V6p (dbGaP Accession phs000424.v6.p1) ( http://www.gtex.org ). These data supported highest levels of gene expression for human ENPEP in the small intestine-terminal ileum and the kidney cortex, which is consistent with the enzyme’s role in digestive tract and renal sodium (Na+) reabsorption and the renin-angiotensin system [ 18 , 34 ].…”

“…RNA-seq gene expression across 53 selected tissues (or tissue segments) that were examined from the public database for human ENPEP, based on expression levels for 175 individuals [ 16 ] (Data Source: GTEx Analysis Release V6p (dbGaP Accession phs000424.v6.p1) ( http://www.gtex.org ).…”

“…The gene encoding ENPEP (ENPEP in humans and most mammals; Enpep in rodents) is expressed at high levels in the epithelial cells of the kidney glomerulus and proximal tubule cells. ENPEP participates in the renin-angiotensin system, by way of the conversion of the biologically active Ang II (angiotensin II) to angiotensin III (Ang III), as a result of the hydrolysis of the N-terminal aspartate (or glutamate) thereby removing biological activity of the Ang peptides [ 15 , 16 ]. In studies of blood pressure control in hypertensive rats, ENPEP is expressed in brain nuclei where ENPEP activity generates angiotensin III, one of the major effector peptides of the brain renin angiotensin system, causing a stimulatory effect on systemic blood pressure [ 7 , 17 ].…”