Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II, DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a number of FAP inhibitors having IC(50) of <100 nM with excellent selectivity over DPP-IV, DPP-II, DPP8, and DPP9 (IC(50) > 100 μM). Compounds 18a, 18b, and 19 are the only known potent and selective FAP inhibitors, which prompts us to further study the physiological role of FAP.

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a b s t r a c tThe dipeptidyl peptidase (DPP) family members, including DPP-IV, DPP8, DPP9 and others, cleave the peptide bond after the penultimate proline residue and are drug target rich. The dimerization of DPP-IV is required for its activity. A propeller loop located at the dimer interface is highly conserved within the family. Here we carried out site-directed mutagenesis on the loop of DPPIV and identified several residues important for dimer formation and enzymatic activity. Interestingly, the corresponding residues on DPP9 have a different impact whereby the mutations decrease activity without changing dimerization. Thus the propeller loop seems to play a varying role in different DPPs. Structured summary of protein interactions:DPP-IV and DPP-IV physically interact by comigration in gel electrophoresis (View interaction: 1, 2, 3, 4) DPP9 and DPP9 bind by circular dichroism (View interaction) DPP-IV and DPP-IV bind by circular dichroism (View interaction: 1, 2, 3, 4, 5) DPP-IV and DPP-IV bind by cosedimentation in solution (View interaction: 1, 2, 3, 4, 5) ADA binds to DPP-IV by surface plasmon resonance (View interaction: 1, 2, 3, 4, 5, 6) DPP9 and DPP9 bind by cosedimentation in solution (View interaction)

Sf-Caspase-1 [Spodoptera frugiperda (fall armyworm) caspase-1] is the most studied effector caspase of Lepidoptera and its activation may lead cells to apoptosis (programmed cell death) when under UV irradiation or baculovirus infection. In the present study, we repressed the expression of Sf-caspase-1 in Sf9 (S. frugiperda 9) cells using constitutive RNA interference, and evaluated the effects of stress responses and the production of proteins in recombinant baculovirus-infected cells. The Sf-caspase-1-repressed stable cells, Sf9/pIBdsCasp-1 and Sf9/pIBdsCasp-2, showed a significant increase in resistance to UV- and baculovirus-induced apoptosis. These cells produced higher levels of both intracellular (luciferase) and extracellular [SEAP (secreted alkaline phosphatase)] recombinant proteins than the parental cells when infected with recombinant baculovirus. Thus Sf-caspase-1-repressed stable cells have a greater ability to adapt to various culture conditions, and also to provide the benefits of high-level protein production.

Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera frugiperda cells (Sf9), we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the baculovirus-mediated luciferase expression. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study.