Inhibitor Report for: 1G244

BACKGROUND: Cellular peptidases are an emerging target of novel pharmacological strategies in inflammatory diseases and cancer. In this context, the dipeptidyl peptidases 8 and 9 (DPP8/9) have gained special attention due to their activities in the immune cells. However, in spite of more than hundred protein substrates identified to date by mass spectrometry-based analysis, the cellular DPP8/9 functions are still elusive. METHODS: We applied the proteomic approach (iTRAQ-2DLC-MS/MS) to comprehensively analyze the role of DPP8/9 in the regulation of macrophage activation by in-depth protein quantitation of THP-1 proteome and secretome. RESULTS: Cells pre-incubated with DPP8/9 inhibitor (1G244) prior activation (LPS or IL-4/IL-13) diminished the expression levels of M1-like response markers, but not M2-like phenotype features. This was accompanied by multiple intra- and extra-cellular protein abundance changes in THP-1 cells, related to cellular metabolism, mitochondria and endoplasmic reticulum function, as well as those engaged with inflammatory and apoptotic processes, including previously reported and novel DPP8/9 targets. CONCLUSIONS: Inhibition of DPP 8/9 had a profound effect on the THP-1 macrophage proteome and secretome, evidencing the decrease of the pro-inflammatory M1-like response. Presented results are to our best knowledge the first which, among others, highlight the metabolic effects of DPP8/9 inhibition in macrophages.

BACKGROUND: Dipeptidyl peptidase 9 (DPP9) is a relatively new member of the DPPIV family of prolyl dipeptidases which is ubiquitously expressed. Its role in regulation of immune responses and proliferation of epithelial carcinoma cells was reported. There is no data on possible role of DPP9 expressed in skin epithelial cells (keratinocytes) and in dermal fibroblasts. MATERIALS AND
METHODS: Transcriptional and protein expression of DPP9 and DPPIV was examined in fibroblasts and keratinocytes isolated from normal human skin. Localization of DPP9 and its sub-localization in Golgi were determined by immunocytochemistry staining. DPPIV-like enzyme activity was determined in cell lysates and in isolated cell fractions containing membranes (M), cytosol (C) and content of organelles/endosomes/vesicles (V). Relative contribution of DPPIV and DPP8/9 enzyme activity in these fractions was determined by using selective inhibitors: sitagliptin (selective for DPPIV) and 1G244 (selective for DPP9 and a highly homologous DPP8). Possible roles of DPP8/9 via its enzyme activity were analysed by assessment of survival and proliferative capacity of fibroblasts and HaCaT cells of keratinocyte origin in the presence of the inhibitors. Possible role of DPP9 in cell migration and/or adhesion was analysed in fibroblasts and HaCaT cells after DPP9 gene silencing.
RESULTS: Fibroblasts and keratinocytes exerted comparable level of DPP9 both at transcriptional and protein level. Fibroblasts strongly expressed DPPIV, whereas in keratinocytes DPPIV expression was low. DPP9 expression was found in cytosol and in perinuclear area of some fibroblasts, or in scattered pattern of keratinocytes, as well as in nuclei of some cells. Only low level of DPP9 sub-localization within Golgi was observed in fibroblasts and keratinocytes. DPPIV-like enzyme activity was about 5 times higher in lysates of fibroblasts than of HaCaT cells. In fibroblasts DPPIV-like enzyme activity was mainly (65%) found in the fraction containing cell membranes (M) and was predominantly (86.9%) due to DPPIV. In contrast, in HaCaT cells the DPPIV-like enzyme activity was mainly (84.2%) found in cytosol (C) and was predominantly (95.6%) due to DPP8/9. Survival and the proliferative capacity were significantly diminished in the presence of 10muM 1G244, both in fibroblasts and in HaCaT cells, suggesting possible role of DPP8/9 enzyme activity in regulation of survival and proliferation of these cells. DPP9 gene silencing resulted in decreased adhesion of fibroblasts, as well as in decreased migration of fibroblasts and HaCaT cells. Accumulation of DPP9 on the edges of plasma membranes of fibroblasts and keratinocytes adhering to surface supports the idea of possible role of DPP9 in cell adhesion.
CONCLUSIONS: This is the first study showing protein expression, sub-localization and possible biological roles of DPP9 expressed in isolated human skin cells. The data may be relevant for development of new drugs against skin diseases by targeting DPP9 expressed in the skin cells.

DPP-IV (EC 3.4.14.5) is a validated drug target for human type II diabetes. DPP-IV inhibitors without DPP8/9 inhibitory activity have been sought because a possible association has been reported between a "DPP8/9 inhibitor" and severe toxicity in animals. However, at present, it is not known whether the observed toxicity is associated with DPP8/9 inhibition, or an off-target effect induced by the compound. We investigated whether the inhibition of DPP8/9 is the cause of the severe toxicity in animals using a very potent and selective DPP8/9 inhibitor with different pharmacophore, 1G244. By Ki measurement, 1G244 is 15- and 8-fold more potent against DPP8 and DPP9, respectively, than the "DPP8/9 inhibitor". Strikingly, the "DPP8/9 inhibitor" does not penetrate the plasma membrane but remains outside the cells, whereas 1G244 readily enters the cells, even at low doses. By repeatedly exposing Sprague-Dawley rats to 1G244 by intravenous injection for a period of 14 days, we observed no significant toxicological symptoms associated with 1G244. Blood and serum chemistry parameters were all within the normal ranges for the treated animals. Because of the high potency, good membrane penetration and adequate tissue distribution of 1G244, the mild symptoms observed are probably associated with DPP8/9 inhibition.

BACKGROUND: Targeting cell death to induce favorable functional and morphological changes within atherosclerotic plaques has long been postulated as a promising anti-atherosclerotic strategy. In this regard, inhibition of dipeptidyl peptidases 8/9 has received special attention in the context of chronic inflammatory diseases due to its regulatory role in macrophage death in vivo. METHODS: The present study investigates the influence of prolonged treatment with 1G244 - an inhibitor of dipeptidyl peptidases 8/9 - on the development of the advanced atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods. RESULTS: 1G244 administration has led to a reduction in atherosclerotic plaque size in an apoE-knockout mice model. Moreover, it reduced the content of in-plaque macrophages, attributed by immunohistochemical phenotyping to the pro-inflammatory M1-like activation state of these cells. Inhibition of dipeptidyl peptidases 8/9 augmented the lytic form of death response of activated macrophages in-vitro. CONCLUSIONS: In summary, inhibition of DPP 8/9 elicited an anti-atherosclerotic effect in apoE(-/-) mice, which can be attributed to the lytic form of death induction in activated macrophages, as assessed by the in vitro BMDM model. This, in turn, results in a reduction of the plaque area without its transformation towards a rupture-prone morphology.

BACKGROUND: Cellular peptidases are an emerging target of novel pharmacological strategies in inflammatory diseases and cancer. In this context, the dipeptidyl peptidases 8 and 9 (DPP8/9) have gained special attention due to their activities in the immune cells. However, in spite of more than hundred protein substrates identified to date by mass spectrometry-based analysis, the cellular DPP8/9 functions are still elusive. METHODS: We applied the proteomic approach (iTRAQ-2DLC-MS/MS) to comprehensively analyze the role of DPP8/9 in the regulation of macrophage activation by in-depth protein quantitation of THP-1 proteome and secretome. RESULTS: Cells pre-incubated with DPP8/9 inhibitor (1G244) prior activation (LPS or IL-4/IL-13) diminished the expression levels of M1-like response markers, but not M2-like phenotype features. This was accompanied by multiple intra- and extra-cellular protein abundance changes in THP-1 cells, related to cellular metabolism, mitochondria and endoplasmic reticulum function, as well as those engaged with inflammatory and apoptotic processes, including previously reported and novel DPP8/9 targets. CONCLUSIONS: Inhibition of DPP 8/9 had a profound effect on the THP-1 macrophage proteome and secretome, evidencing the decrease of the pro-inflammatory M1-like response. Presented results are to our best knowledge the first which, among others, highlight the metabolic effects of DPP8/9 inhibition in macrophages.

Dipeptidyl peptidases (DPPs) are proteolytic enzymes that are ideal therapeutic targets in human diseases. Indeed, DPP4 inhibitors are widely used in clinical practice as anti-diabetic agents. In this paper, we show that DPP4 inhibitors also induced cell death in multiple human myeloma cells. Among five DPP4 inhibitors, only two of them, vildagliptin and saxagliptin, exhibited apparent cytotoxic effects on myeloma cell lines, without any difference in suppression of DPP4 activity. As these two DPP4 inhibitors are known to have off-target effects against DPP8/9, we employed the specific DPP8/9 inhibitor 1G244. 1G244 demonstrated anti-myeloma effects on several cell lines and CD138+ cells from patients as well as in murine xenograft model. Through siRNA silencing approach, we further confirmed that DPP8 but not DPP9 is a key molecule in inducing cell death induced by DPP8/9 inhibition. In fact, the expression of DPP8 in CD38+ cells from myeloma patients was higher than that of healthy volunteers. DPP8/9 inhibition induced apoptosis, as evidenced by activated form of PARP, caspases-3 and was suppressed by the pan-caspase inhibitor Z-VAD-FMK. Taken together, these results indicate that DPP8 is a novel therapeutic target for myeloma treatment.

BACKGROUND: Dipeptidyl peptidase 9 (DPP9) is a relatively new member of the DPPIV family of prolyl dipeptidases which is ubiquitously expressed. Its role in regulation of immune responses and proliferation of epithelial carcinoma cells was reported. There is no data on possible role of DPP9 expressed in skin epithelial cells (keratinocytes) and in dermal fibroblasts. MATERIALS AND
METHODS: Transcriptional and protein expression of DPP9 and DPPIV was examined in fibroblasts and keratinocytes isolated from normal human skin. Localization of DPP9 and its sub-localization in Golgi were determined by immunocytochemistry staining. DPPIV-like enzyme activity was determined in cell lysates and in isolated cell fractions containing membranes (M), cytosol (C) and content of organelles/endosomes/vesicles (V). Relative contribution of DPPIV and DPP8/9 enzyme activity in these fractions was determined by using selective inhibitors: sitagliptin (selective for DPPIV) and 1G244 (selective for DPP9 and a highly homologous DPP8). Possible roles of DPP8/9 via its enzyme activity were analysed by assessment of survival and proliferative capacity of fibroblasts and HaCaT cells of keratinocyte origin in the presence of the inhibitors. Possible role of DPP9 in cell migration and/or adhesion was analysed in fibroblasts and HaCaT cells after DPP9 gene silencing.
RESULTS: Fibroblasts and keratinocytes exerted comparable level of DPP9 both at transcriptional and protein level. Fibroblasts strongly expressed DPPIV, whereas in keratinocytes DPPIV expression was low. DPP9 expression was found in cytosol and in perinuclear area of some fibroblasts, or in scattered pattern of keratinocytes, as well as in nuclei of some cells. Only low level of DPP9 sub-localization within Golgi was observed in fibroblasts and keratinocytes. DPPIV-like enzyme activity was about 5 times higher in lysates of fibroblasts than of HaCaT cells. In fibroblasts DPPIV-like enzyme activity was mainly (65%) found in the fraction containing cell membranes (M) and was predominantly (86.9%) due to DPPIV. In contrast, in HaCaT cells the DPPIV-like enzyme activity was mainly (84.2%) found in cytosol (C) and was predominantly (95.6%) due to DPP8/9. Survival and the proliferative capacity were significantly diminished in the presence of 10muM 1G244, both in fibroblasts and in HaCaT cells, suggesting possible role of DPP8/9 enzyme activity in regulation of survival and proliferation of these cells. DPP9 gene silencing resulted in decreased adhesion of fibroblasts, as well as in decreased migration of fibroblasts and HaCaT cells. Accumulation of DPP9 on the edges of plasma membranes of fibroblasts and keratinocytes adhering to surface supports the idea of possible role of DPP9 in cell adhesion.
CONCLUSIONS: This is the first study showing protein expression, sub-localization and possible biological roles of DPP9 expressed in isolated human skin cells. The data may be relevant for development of new drugs against skin diseases by targeting DPP9 expressed in the skin cells.

Adipocytes are the primary cells in adipose tissue, and adipocyte dysfunction causes lipodystrophy, obesity and diabetes. The dipeptidyl peptidase (DPP) 4 family includes four enzymes, DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). DPP4 family inhibitors have been used for the treatment of type 2 diabetes patients, but their role in adipocyte formation are poorly understood. Here we demonstrate that the DPP8/9 selective inhibitor 1G244 blocks adipogenesis in preadipocyte 3T3-L1 and 3T3-F422A, while DPP4 and FAP inhibitors have no effect. In addition, knockdown of DPP8 or DPP9 significantly impairs adipocyte differentiation in preadipocytes. We further uncovered that blocking the expression or activities of DPP8 and DPP9 attenuates PPARgamma2 induction during preadipocyte differentiation. Addition of PPARgamma agonist thiazolidinediones (TZDs), or ectopic expression of PPARgamma2, is able to rescue the adipogenic defect caused by DPP8/9 inhibition in preadipocytes. These results indicate the importance of DPP8 and DPP9 on adipogenesis.

Atherosclerosis is a chronic inflammatory disorder of the arterial wall leading to coronary artery disease, stroke, and peripheral arterial disease. Along with the discovery of dipeptidyl peptidase 4 (DPP4) as a therapeutic target in type 2 diabetes, a role for DPP4 in atherosclerosis is emerging. However, until now the expression and role of other DPPs such as DPP8 and DPP9 in atherosclerosis is completely unknown. In the present study, we first investigated DPP expression in human atherosclerotic plaques. DPP4 could only be observed in endothelial cells of plaque neovessels in half of the specimens. In contrast, DPP8 and DPP9 were abundantly present in macrophage-rich regions of plaques. We then focused on DPP expression and function in macrophage differentiation, activation and apoptosis. DPP8/9 was responsible for most of the DPP activity in macrophages. During monocyte to macrophage differentiation, DPP9 was upregulated both in pro-inflammatory M1 (3.7 +/- 0.3-fold increase) and anti-inflammatory M2 macrophages (3.7 +/- 0.4-fold increase) whereas DPP8 expression remained unchanged. Inhibition of DPP8/9 activity with compound 1G244 reduced activation of M1 macrophages (IL-6 88 +/- 16 vs. 146 +/- 19 pg/ml; TNFalpha 3.8 +/- 1.0 vs. 6.6 +/- 1.9 ng/ml in treated vs. untreated cells), but not of M2 macrophages. Likewise, DPP9 silencing reduced TNFalpha and IL-6 secretion, pointing to a DPP9-mediated effect of the inhibitor. DPP8/9 inhibition also enhanced macrophage apoptosis (15 +/- 4 vs. 7 +/- 3 % in untreated cells). Because pro-inflammatory macrophages play a key role in atherogenesis, plaque rupture and subsequent infarction, DPP9 inhibition might provide interesting therapeutic prospects in reducing atherosclerosis and/or in the prevention of plaque rupture.

DPP-IV (EC 3.4.14.5) is a validated drug target for human type II diabetes. DPP-IV inhibitors without DPP8/9 inhibitory activity have been sought because a possible association has been reported between a "DPP8/9 inhibitor" and severe toxicity in animals. However, at present, it is not known whether the observed toxicity is associated with DPP8/9 inhibition, or an off-target effect induced by the compound. We investigated whether the inhibition of DPP8/9 is the cause of the severe toxicity in animals using a very potent and selective DPP8/9 inhibitor with different pharmacophore, 1G244. By Ki measurement, 1G244 is 15- and 8-fold more potent against DPP8 and DPP9, respectively, than the "DPP8/9 inhibitor". Strikingly, the "DPP8/9 inhibitor" does not penetrate the plasma membrane but remains outside the cells, whereas 1G244 readily enters the cells, even at low doses. By repeatedly exposing Sprague-Dawley rats to 1G244 by intravenous injection for a period of 14 days, we observed no significant toxicological symptoms associated with 1G244. Blood and serum chemistry parameters were all within the normal ranges for the treated animals. Because of the high potency, good membrane penetration and adequate tissue distribution of 1G244, the mild symptoms observed are probably associated with DPP8/9 inhibition.