Phosphinotripeptidic Inhibitors of Leucylaminopeptidases.

Phosphinate pseudopeptide are analogs of peptides containing phosphinate moiety in a place of the amide bond. Due to this, the organophosphorus fragment resembles the tetrahedral transition state of the amide bond hydrolysis. Additionally, it is also capable of coordinating metal ions, for example, zinc or magnesium ions. These two properties of phosphinate pseudopeptides make them an ideal candidate for metal-related protease inhibitors. This research investigates the influence of additional residue in the P2 position on the inhibitory properties of phosphinopeptides. The synthetic strategy is proposed, based on retrosynthetic analysis. The N-C-P bond formation in the desired compounds is conveniently available from the three-component condensation of appropriate amino components, aldehydes, and hypophosphorous acid. One of the crucial synthetic steps is the careful selection of the protecting groups for all the functionals. Determination of the inhibitor activity of the obtained compounds has been done using UV-Vis spectroscopy and standard substrate L-Leu-p-nitroanilide toward the enzymes isolated from the porcine kidney (SsLAP, Sus scrofa Leucine aminopeptidase) and barley seeds (HvLAP, Hordeum vulgare Leucine aminopeptidase). An efficient procedure for the preparation of phosphinotripeptides has been performed. Activity test shown that introduction of additional residue into P2 position obtains the micromolar range inhibitors of SsLAP and HvLAP. Moreover, careful selection of the residue in the P2 position should improve its selectivity toward mammalian and plant leucyl aminopeptidases.

Biochemical and cellular characterisation of the Plasmodium falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP).

The Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0-8.0), although PfM1AAP also possesses some activity (< 20%) at the lower pH range of 5.0-5.5. The data supports the proposal that PfM1AAP and PfM17LAP function in the cytoplasm of the parasite, likely in the degradation of haemoglobin-derived peptides generated in the DV and transported to the cytosol.

In Silico Screening for Novel Leucine Aminopeptidase Inhibitors with 3,4-Dihydroisoquinoline Scaffold.

Cancers are the leading cause of deaths worldwide. In 2018, an estimated 18.1 million new cancer cases and 9.6 million cancer-related deaths occurred globally. Several previous studies have shown that the enzyme, leucine aminopeptidase is involved in pathological conditions such as cancer. On the basis of the knowledge that isoquinoline alkaloids have antiproliferative activity and inhibitory activity towards leucine aminopeptidase, the present study was conducted a study which involved database search, virtual screening, and design of new potential leucine aminopeptidase inhibitors with a scaffold based on 3,4-dihydroisoquinoline. These compounds were then filtered through Lipinski's "rule of five," and 25 081 of them were then subjected to molecular docking. Next, three-dimensional quantitative structure-activity relationship (3D-QSAR) study was performed for the selected group of compounds with the best binding score results. The developed model, calculated by leave-one-out method, showed acceptable predictive and descriptive capability as represented by standard statistical parameters r2 (0.997) and q2 (0.717). Further, 35 compounds were identified to have an excellent predictive reliability. Finally, nine selected compounds were evaluated for drug-likeness and different pharmacokinetics parameters such as absorption, distribution, metabolism, excretion, and toxicity. Our methodology suggested that compounds with 3,4-dihydroisoquinoline moiety were potentially active in inhibiting leucine aminopeptidase and could be used for further in-depth in vitro and in vivo studies.

Inhibition of the proliferation, migration, and invasion of human breast cancer cells by leucine aminopeptidase 3 inhibitors derived from natural marine products.

Leucine aminopeptidase 3 is involved in the progression and metastasis of several cancers. This study aimed to screen anti-tumor lead compounds targeting leucine aminopeptidase 3. The compounds' suppression effect on enzyme activity and anti-tumor activity were evaluated through a series of assays. Leucine aminopeptidase 3 overexpression K562 cells were used as an enzyme source to screen 43 natural marine compounds. Compounds 5 and 6 exhibited high suppression effect on leucine aminopeptidase 3 activity. Cell activity tests indicated that both compounds have an anti-proliferative effect on triple-negative breast cancer cells. Wound healing assay and transwell invasion assay showed that both compounds could inhibit the migration and invasion of breast cancer cells. Immunoblot analysis exhibited that both compounds could downregulate the expression of metastasis-related proteins fascin and matrix metalloproteinase-2/9. A molecular dynamic simulation process was applied to discover the key features of compounds 5 and 6 in binding to leucine aminopeptidase 3 active site. This study described the anti-tumor effects of two leucine aminopeptidase 3 small molecule inhibitors. Taken together, compounds 5 and 6 could be used as anti-tumor lead compounds targeting leucine aminopeptidase 3.

A new method to evaluate the enzyme-suppressing activity of a leucine aminopeptidase 3 inhibitor.

The expression of leucine aminopeptidase 3 (LAP3) is associated with the prognosis for and malignant transformation of many types of tumors. Therefore, a LAP3 inhibitor may represent a new strategy for cancer therapy. Evaluating the suppression of enzyme activity by an LAP3 inhibitor is essential. Right now, leucine aminopeptidases (LAPs) purified from the porcine kidneys are the only enzymes that can be used to evaluate the suppression of enzyme activity by an LAP3 inhibitor. This approach cannot accurately reflect the suppression of human LAP3 by an inhibitor. The current study developed a new method with which to evaluate the suppression of enzyme activity by an LAP3 inhibitor. Total protein from K562 cells seldom catalyzed the LAP3 substrate. A lentivirus was used to induce K562 cells to overexpress LAP3 (K562-LAP3). After puromycin screening, flow cytometry data indicated that 98.8% of cells expressed green fluorescent protein. The expression of LAP3 in K562-LAP3 cells was also assessed using Western blotting. K562-LAP3 cells were lysed with ultrasonication. Total protein was used as an enzyme source and L-leucine p-nitroaniline hydrochloride was used as a substrate to measure enzyme activity. Total protein from K562-LAP3 cells catalyzed the substrate more than that from K562 cells did. The LAP3 inhibitor ubenimex was used as a positive control to evaluate the suppression of LAP3 enzyme activity. Results indicated that ubenimex significantly inhibited the enzyme activity of LAP3. This approach provides a convenient and accurate way to evaluate the suppression of enzyme activity by an LAP3 inhibitor.

High-Level Expression in Escherichia coli, Purification and Kinetic Characterization of LAPTc, a Trypanosoma cruzi M17-Aminopeptidase.

The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appKM = 74 µM, appkcat = 4.4 s-1). The optimal temperature is 50 °C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a Ki value of 881 nM. The enzyme is a good target for inhibitor identification.

Egg-Hatching Mechanism of Human Liver Fluke, Opisthorchis viverrini: A Role For Leucine Aminopeptidases From the Snail Host, Bithynia siamensis goniomphalos.

The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24-28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.

The aminopeptidase inhibitor, z-L-CMK, is toxic and induces cell death in Jurkat T cells through oxidative stress.

The leucine aminopeptidase inhibitor, benzyloxycarbonyl-leucine-chloromethylketone (z-L-CMK), was found to be toxic and readily induce cell death in Jurkat T cells. Dose-response studies show that lower concentration of z-L-CMK induced apoptosis in Jurkat T cells whereas higher concentration causes necrosis. In z-L-CMK-induced apoptosis, both the initiator caspases (-8 and -9) and effector caspases (-3 and -6) were processed to their respective subunits. However, the caspases remained intact in z-L-CMK-induced necrosis. The caspase inhibitor, z-VAD-FMK inhibited z-L-CMK-mediated apoptosis and caspase processing but has no effect on z-L-CMK-induced necrosis in Jurkat T cells. The high mobility group protein B1 (HMGB1) protein was found to be released into the culture medium by the necrotic cells and not the apoptotic cells. These results indicate that the necrotic cell death mediated by z-L-CMK at high concentrations is via classical necrosis rather than secondary necrosis. We also demonstrated that cell death mediated by z-L-CMK was associated with oxidative stress via the depletion of intracellular glutathione (GSH) and increase in reactive oxygen species (ROS), which was blocked by N-acetyl cysteine. Taken together, the results demonstrated that z-L-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. The toxic side effects in Jurkat T cells mediated by z-L-CMK are associated with oxidative stress via the depletion of GSH and accumulation of ROS.

Near-Infrared Fluorescent Probe with Remarkable Large Stokes Shift and Favorable Water Solubility for Real-Time Tracking Leucine Aminopeptidase in Living Cells and In Vivo.

Leucine aminopeptidase (LAP) is a kind of proteolytic enzymes and associated closely with pathogenesis of cancer and liver injury. Accurate detection of LAP activity with high sensitivity and selectivity is imperative to detect its distribution and dynamic changes for understanding LAP's function and early diagnosing the disease states. However, fluorescent detection of LAP in living systems is challenging. To date, rarely fluorescent probes have been reported for imaging LAP in vivo. In this study, a novel probe (TMN-Leu) was developed by conjugating a near-infrared dicyanoisophorone derivative fluorophore with LAP activatable l-leucine amide moiety for the first time. TMN-Leu featured large Stokes shift (198 nm), favorable water solubility, ultrasensitive sensitivity (detection limit of ∼0.38 ng/mL), good specificity, excellent cell membrane permeability, low toxicity, and a prominent near-infrared emission (658 nm) in response to LAP. TMN-Leu has been successfully applied to track LAP of cancer cells and normal cells, monitor LAP changes in different disease models, and rapidly evaluate LAP inhibitor in cell-based assay. Notably, this probe firstly revealed that HCT116 cells with higher LAP activity were more invasive than LAP siRNA transfected HCT116 cells, suggesting that LAP might serve as an indicator reflecting the intrinsic invasion ability of cancer cells. Finally, TMN-Leu was also employed for in vivo real-time imaging LAP in living tumor-bearing nude mice with low background interference. All together, our probe possesses potential value as a promising tool for diagnostic application, cell-based screening inhibitors and in vivo real-time tracking enzymatic activity in preclinical applications.

Synthesis, Characterization and In Vitro Evaluation of a Novel Glycol Chitosan-EDTA Conjugate to Inhibit Aminopeptidase-Mediated Degradation of Thymopoietin Oligopeptides.

In this study, a novel conjugate consisting of glycol chitosan (GCS) and ethylene diamine tetraacetic acid (EDTA) was synthesized and characterized in terms of conjugation and heavy metal ion chelating capacity. Moreover, its potential application as a metalloenzyme inhibitor was evaluated with three thymopoietin oligopeptides in the presence of leucine aminopeptidase. The results from FTIR and NMR spectra revealed that the covalent attachment of EDTA to GCS was achieved by the formation of amide bonds between the carboxylic acid group of EDTA and amino groups of GCS. The conjugated EDTA lost part of its chelating capacity to cobalt ions compared with free EDTA as evidenced by the results of cobalt ion chelation-mediated fluorescence recovery of calcein. However, further investigation confirmed that GCS-EDTA at low concentrations significantly inhibited leucine aminopeptidase-mediated degradation of all thymopoietin oligopeptides.

Screening and In Vitro Evaluation of Potential Plasmodium falciparum Leucyl Aminopeptidase Inhibitors.

BACKGROUND: Plasmodium falciparum leucyl aminopeptidase (PfA-M17) regulates the intracellular pool of amino acids required for the growth and development of parasites. Thus, PfA-M17 is a promising target for anti-malarial drug development. METHOD: In the present study, structure-based drug design was used to identify novel PfA-M17 inhibitors, which were subsequently validated by in vitro PfA-M17 and human LAP3 enzyme inhibition assay. A library of 3,147,882 compounds was screened using receptor-based virtual screening against the active site of PfA-M17, and three levels of accuracy were used: high-throughput virtual screening, gridbased ligand docking with energetics (Glide standard precision) and Glide extra precision. RESULTS: Seventeen screened compounds were selected and tested in the rPfA-M17 enzyme inhibition assay. Of these nine compounds were found to be effective inhibitors. To test the target activity, all nine PfA-M17 inhibitors were tested against rhLAP3, the human homolog of PfA-M17. One compound (compound 2) was found to be moderately effective against PfA-M17 (Ki = 287 µM) with limited inhibitory activity against hLAP3 (Ki of 4,464 µM). Subsequently, induced fit docking and pharmacophore modelling were used to further understand more precise ligand-protein interactions in the protein-inhibitor complexes. CONCLUSION: Among the 9 effective PfA-M17 inhibitors, 5 compounds were found effective in the P. falciparum schizont maturation inhibition (SMI) assay. A good correlation (r =0.83) was observed between the rPfA-M17 enzyme inhibition concentration and SMI assay.

In silico screening of novel inhibitors of M17 Leucine Amino Peptidase (LAP) of Plasmodium vivax as therapeutic candidate.

M17 LAP (Leucine Amino Peptidase) plays an important role in the hydrolysis of amino acids essential for growth and development of Plasmodium vivax (Pv), the pathogen causing malaria. In this paper a homology model of PvLAP was generated using MODELLER v9.15. From different in-silico methods such as structure based, ligand based and de novo drug designing a total of 90 compounds were selected for docking studies. A final list of 10 compounds was prepared. The study reported the identification of 2-[(3-azaniumyl-2-hydroxy-4-phenylbutanoyl) amino]-4-methylpentanoate as the best inhibitor in terms of docking score and pharmacophoric features. The reliability of the binding mode of the inhibitor is confirmed by molecular dynamics (MD) simulation study with GROMACS software for a simulation time of 20ns in water environment. Finally, in silico ADMET analysis of the inhibitors using MedChem Designer v3 evaluated the drug likeness of the best hits to be considered for industrial pharmaceutical research.

Inhibition of leucine aminopeptidase 3 suppresses invasion of ovarian cancer cells through down-regulation of fascin and MMP-2/9.

Leucine aminopeptidase 3 (LAP3) is a cell surface aminopeptidase that catalyzes the hydrolysis of leucine residues from the amino termini of protein or peptide substrates. The over-expression of LAP3 correlates with prognosis and malignant development of several human cell carcinomas. However, the molecular mechanism remains unknown. In this study, we used ES-2 ovarian cancer cell line as a model system to explore the role of LAP3 in regulation of cancer cell invasion by employing a natural LAP3 inhibitor bestatin and LAP3 siRNA. Bestatin inhibited tumor cell migration and invasion in a dose-dependent manner. More interestingly, bestatin down-regulated expression of fascin protein and inhibited activity of fascin promoter luciferase reporter. Both proteome profiler array and Western blot assay showed that bestatin up-regulated the phosphorylation of Hsp27. Furthermore, LAP3 siRNA could up-regulate the phosphorylation of Hsp27 and down-regulate the expression of fascin. Meanwhile, LAP3 siRNA could also down-regulate the phosphorylation of Akt and the expression of MMP-2/9. Taken together, LAP3 could affect the expression of fascin and MMP-2/9 and may act as a potential anti-metastasis therapeutic target.

Molecular Characterization of Babesia bovis M17 Leucine Aminopeptidase and Inhibition of Babesia Growth by Bestatin.

The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 µM, respectively, while the Ki was 2210 ± 358 µM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 µM] than B. gibsoni [IC50 = 460.8 ± 114.45 µM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this.

Modelling of human leucyl aminopeptidases for in silico off target binding analysis of potential Plasmodium falciparum leucine aminopeptidase (PfA-M17) specific inhibitors.

Malaria is one of the most widespread infectious diseases in the world. Emergence of multi-drug resistant Plasmodium strains makes it crucial to identify new classes of compounds for anti-malarial therapy. Novel anti-malarial compounds from natural sources (Gomphostema niveum) as well as synthetic chemicals (5-aminolevulinic acid) have been reported in recent patents. Plasmodium falciparum leucyl aminopeptidase (PfA-M17) is a validated target for antimalarial drug development. However, known aminopeptidase inhibitors beset with the problem of non-specificity. Therefore, 3D structural models of PfA-M17 human homologs, Leucine aminopeptidase3 (hLAP3) and probable leucine aminopeptidase (hNPEPL1) were predicted for molecular docking based screening of potential inhibitors for their off target activity. Comparison of IC50 and docking scores of highly active hLAP3 inhibitors shows good correlation (r(2)≈ 0.8). Further, docking analysis with potential PfA-M17 inhibitor Compound-X (identified through virtual screening) shows much higher binding affinity towards PfA-M17 (docking score -11.44) than hLAP3 (docking score -4.26) and hNPEPL1 (docking score -5.08). This lead compound, Compound-X can act as a scaffold for further increasing PfA-M17 binding affinity and hLAP3 and hNPEPL1 3D structure models will be useful for screening of PfA-M17 specific inhibitors.

Structure-guided, single-point modifications in the phosphinic dipeptide structure yield highly potent and selective inhibitors of neutral aminopeptidases.

Seven crystal structures of alanyl aminopeptidase from Neisseria meningitides (the etiological agent of meningitis, NmAPN) complexed with organophosphorus compounds were resolved to determine the optimal inhibitor-enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the P1 amino acid residues of well-processed substrates, were used to assess the impact of the absolute configuration and the stereospecific hydrogen bond network formed between the aminophosphonate polar head and the active site residues on the binding affinity. For the hPhe analog, an imperfect stereochemical complementarity could be overcome by incorporating an appropriate P1 side chain. The constitution of P1'-extended structures was rationally designed and the lead, phosphinic dipeptide hPhePψ[CH2]Phe, was modified in a single position. Introducing a heteroatom/heteroatom-based fragment to either the P1 or P1' residue required new synthetic pathways. The compounds in the refined structure were low nanomolar and subnanomolar inhibitors of N. meningitides, porcine and human APNs, and the reference leucine aminopeptidase (LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a reasonable selectivity versus dizinc LAP. Another set of crystal structures containing the NmAPN dipeptide ligand were used to verify and to confirm the predicted binding modes; furthermore, novel contacts, which were promising for inhibitor development, were identified, including a π-π stacking interaction between a pyridine ring and Tyr372.

Purpurin inhibits adipocyte-derived leucine aminopeptidase and angiogenesis in a zebrafish model.

Adipocyte-derived leucine aminopeptidase (A-LAP) is a novel member of the M1 family of zinc metallopeptidases, which has been reported to play a crucial role in angiogenesis. In the present study, we conducted a target-based screening of natural products and synthetic chemical libraries using the purified enzyme to search novel inhibitors of A-LAP. Amongst several hits isolated, a natural product purpurin was identified as one of the most potent inhibitors of A-LAP from the screening. In vitro enzymatic analyses demonstrated that purpurin inhibited A-LAP activity in a non-competitive manner with a Ki value of 20 M. In addition, purpurin showed a strong selectivity toward A-LAP versus another member of M1 family of zinc metallopeptidase, aminopeptidase N (APN). In angiogenesis assays, purpurin inhibited the vascular endothelial growth factor (VEGF)-induced invasion and tube formation of human umbilical vein endothelial cells (HUVEC). Moreover, purpurin inhibited in vivo angiogenesis in zebrafish embryo without toxicity. These data demonstrate that purpurin is a novel specific inhibitor of A-LAP and could be developed as a new anti-angiogenic agent.

Induction of protective antitumor immunity through attenuation of ERAAP function.

The endoplasmic reticulum aminopeptidase associated with Ag processing, ERAAP, plays an important role in the trimming of antigenic peptides for presentation at the cell surface complexed with MHC class I molecules. Tumors express varying levels of ERAAP, highlighting a possible mechanism of immune-evasion through alteration of the peptide repertoire. Using the CT26 tumor model, we investigated the effects of ERAAP modulation on peptide presentation and the use of ERAAP inhibition as an antitumor therapy. We show that generation of the cross-protective tumor Ag GSW11 in the colorectal carcinoma CT26 is increased when ERAAP expression is reduced. BALB/c mice with reduced ERAAP expression challenged with CT26 induced protective immunity that was mediated by CD8(+) T cells. This antitumor immunity also protected mice when rechallenged with wild-type CT26 tumor; strong CD8(+) T cell responses to GSW11 were observed, despite its presentation being considerably lower. Furthermore, boosting the tumor immunogenicity through inhibition of ERAAP function with the small molecule inhibitor leucinethiol in vitro, or in established tumors in vivo, abrogated tumor growth and prolonged survival. Thus, our results highlight the promising possibility of using modulation of ERAAP to generate protective antitumor responses as a strategy for cancer immunotherapy.

The Staphylococcus aureus leucine aminopeptidase is localized to the bacterial cytosol and demonstrates a broad substrate range that extends beyond leucine.

Staphylococcus aureus is a potent pathogen of humans exhibiting a broad disease range, in part due to an extensive repertoire of secreted virulence factors, including proteases. Recently, we identified the first example of an intracellular protease (leucine aminopeptidase, LAP) that is required for virulence in S. aureus. Disruption of pepZ, the gene encoding LAP, had no affect on the growth rate of bacteria; however, in systemic and localized infection models the pepZ mutant had significantly attenuated virulence. Recently, a contradictory report was published suggesting that LAP is an extracellular enzyme and it is required for growth in S. aureus. Here, we investigate these results and confirm our previous findings that LAP is localized to the bacterial cytosol and is not required for growth. In addition, we conduct a biochemical investigation of purified recombinant LAP, identifying optimal conditions for enzymatic activity and substrate preference for hydrolysis. Our results show that LAP has a broad substrate range, including activity against the dipeptide cysteine-glycine, and that leucine is not the primary target of LAP.