A Robust Multiplex Mass Spectrometric Assay for Screening Small-Molecule Inhibitors of CD73 with Diverse Inhibition Modalities.

CD73/Ecto-5'-nucleotidase is a membrane-tethered ecto-enzyme that works in tandem with CD39 to convert extracellular adenosine triphosphate (ATP) into adenosine. CD73 is highly expressed on various types of cancer cells and on infiltrating suppressive immune cells, leading to an elevated concentration of adenosine in the tumor microenvironment, which elicits a strong immunosuppressive effect. In preclinical studies, targeting CD73 with anti-CD73 antibody results in favorable antitumor effects. Despite initial studies using antibodies, inhibition of CD73 catalytic activity using small-molecule inhibitors may be more effective in lowering extracellular adenosine due to better tumor penetration and distribution. To screen small-molecule libraries, we explored multiple approaches, including colorimetric and fluorescent biochemical assays, and due to some limitations with these assays, we developed a mass spectrometry (MS)-based assay. Only the MS-based assay offers the sensitivity and dynamic range required for screening small-molecule libraries at a substrate concentration close to the Km value of substrate and for evaluating the mode of binding of screening hits. To achieve a throughput suitable for high-throughput screening (HTS), we developed a RapidFire-tandem mass spectrometry (RF-MS/MS)-based multiplex assay. This assay allowed a large diverse compound library to be screened at a speed of 1536 reactions per 40-50 min.

Defining the extended substrate specificity of kallikrein 1-related peptidases.

Human kallikrein 1-related peptidases (KLKs) form a subfamily of 15 extracellular (chymo)tryptic-like serine proteases. KLKs 4, 5, 13 and 14 display altered expression/activity in diverse pathological conditions, including cancer. However, their distinct (patho)physiological roles remain largely uncharacterized. As a step toward distinguishing their proteolytic functions, we attempt to define their primary and extended substrate specificities and identify candidate biological targets. Heterologously expressed KLKs 4, 5, 13 and 14 were screened against fluorogenic 7-amino-4-carbamoylmethylcoumarin positional scanning-synthetic combinatorial libraries with amino acid diversity at the P1-P4 positions. Our results indicate that these KLKs share a P1 preference for Arg. However, each KLK exhibited distinct P2-P4 specificities, attributable to structural variations in their surface loops. The preferred P4-P1 substrate recognition motifs based on optimal subsite occupancy were as follows: VI-QSAV-QL-R for KLK4; YFWGPV-RK-NSFAM-R for KLK5; VY-R-LFM-R for KLK13; and YW-KRSAM-HNSPA-R for KLK14. Protein database queries using these motifs yielded many extracellular targets, some of which represent plausible KLK substrates. For instance, cathelicidin, urokinase-type plasminogen activator, laminin and transmembrane protease serine 3 were retrieved as novel putative substrates for KLK4, 5, 13 and 14, respectively. Our findings may facilitate studies on the role of KLKs in (patho)physiology and can be used in the development of selective KLK inhibitors.

Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity.

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.

Serpin1 of Arabidopsis thaliana is a suicide inhibitor for metacaspase 9.

Metacaspases are distant relatives of animal caspases found in plants, fungi and protozoa. We demonstrated previously that two type II metacaspases of Arabidopsis thaliana, AtMC4 and AtMC9 are Arg/Lys-specific cysteine-dependent proteases. We screened a combinatorial tetrapeptide library of 130,321 substrates with AtMC9. Here, we show that AtMC9 is a strict Arg/Lys-specific protease. Based on the position-specific scoring matrix derived from the substrate library results, the tetrapeptide Val-Arg-Pro-Arg was identified as an optimized substrate. AtMC9 had a kcat/KM of 4.6x10(5) M-1 s-1 for Ac-Val-Arg-Pro-Arg-amido-4-methyl-coumarin, representing a more than 10-fold improvement over existing fluorogenic substrates. A yeast two-hybrid screen with catalytically inactive AtMC9 as bait identified a serine protease inhibitor, designated AtSerpin1, which was found to be a potent inhibitor of AtMC9 activity in vitro through cleavage of its reactive center loop and covalent binding to AtMC9. On the basis of the substrate profiling of AtMC9 and confirmation through site-directed mutagenesis, the inhibitory P4-P1 cleavage site of AtSerpin1 was determined to be Ile-Lys-Leu-Arg351. Further mutagenesis of the AtSerpin1 inhibitory cleavage site modulated AtMC9 inhibition positively or negatively. Both AtMC9 and AtSerpin1 were localized in the extracellular space, suggesting an in vivo interaction as well. To our knowledge, this is the first report of plant protease inhibition by a plant serpin.