Highly Selective Inhibitors of Dipeptidyl Peptidase 9 (DPP9) Derived from the Clinically Used DPP4-Inhibitor Vildagliptin.

Dipeptidyl peptidase 9 (DPP9) is a proline-selective serine protease that plays a key role in NLRP1- and CARD8-mediated inflammatory cell death (pyroptosis). No selective inhibitors have hitherto been reported for the enzyme: all published molecules have grossly comparable affinities for DPP8 and 9 because of the highly similar architecture of these enzymes' active sites. Selective DPP9 inhibitors would be highly instrumental to address unanswered research questions on the enzyme's role in pyroptosis, and they could also be investigated as therapeutics for acute myeloid leukemias. Compounds presented in this manuscript (42 and 47) combine low nanomolar DPP9 affinities with unprecedented DPP9-to-DPP8 selectivity indices up to 175 and selectivity indices >1000 toward all other proline-selective proteases. To rationalize experimentally obtained data, a molecular dynamics study was performed. We also provide in vivo pharmacokinetics data for compound 42.

A ubiquitin-independent proteasome pathway controls activation of the CARD8 inflammasome.

CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 inhibitors, including Val-boroPro, accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in Val-boroPro-stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation.

Safety, Tolerability, and Pharmacokinetic Evaluation of Single and Multiple Doses of the Dipeptidyl Peptidase 1 Inhibitor Brensocatib in Healthy Japanese and White Adults.

Brensocatib, an investigational first-in-class, small-molecule, orally bioavailable, selective, and reversible dipeptidyl peptidase 1 inhibitor that blocks activation of neutrophil serine proteases, is currently under clinical development for the treatment of bronchiectasis and other chronic inflammatory diseases. In a 2-part phase 1 study, the safety, tolerability, and pharmacokinetics of brensocatib were evaluated in healthy Japanese and White adults. In part A, participants received single and multiple once-daily doses of brensocatib (10, 25, or 40 mg) or placebo after an overnight fast. In part B, participants received a single oral dose of brensocatib 40 mg on days 1 and 8, with or without food in a crossover fashion. Following a single dose and at steady state, brensocatib exposure was dose dependent, with low to moderate interindividual variability; systemic exposure between Japanese and White participants was similar. Elimination half-life of brensocatib ranged from 22 to 28 hours, resulting in ≈2-fold accumulation in maximum plasma concentration and area under the plasma concentration-time curve at steady state. In both ethnic groups, the presence of food slightly delayed brensocatib absorption with time to maximum plasma concentration increased by 0.7 to 1.7 hours, but it had no significant effect on brensocatib exposure (maximum plasma concentration and area under the plasma concentration-time curve). Brensocatib was well tolerated in Japanese and White participants. The most frequently reported treatment-emergent adverse events were headache and skin exfoliation. No clinically significant vital signs, laboratory abnormalities, or evidence of renal toxicity were observed. The results from this study demonstrate that brensocatib can be administered with or without food and that dose adjustment is unnecessary for Japanese patients when receiving brensocatib treatment.

Unravelling the inhibitory zinc ion binding site and the metal exchange mechanism in human DPP III.

Dipeptidyl peptidase III (DPP III), a zinc-dependent exopeptidase, is widely distributed in organisms and present in almost all human tissues. In addition to its involvement in protein catabolism, it plays a role in oxidative stress and blood pressure regulation, and there is evidence of its involvement in pain modulation. Excess zinc ions have been found to inhibit its hydrolytic activity, but the binding affinity, binding site geometry, and mechanism of inhibitory activity have been unknown. Using several different computational approaches, we determined the inhibitory zinc ion binding site, its coordination and relative binding affinity. During some simulations the translocation of the zinc ion from the inhibitory to the catalytic binding site was observed, accompanied by movement of the catalytic zinc ion toward the exit of the substrate binding site. The traced behavior suggests an associative type of metal ion exchange, in which the formation of the ternary complex between enzyme and two metal ions precedes the exit of the catalytic metal ion. Differently from our previous findings that binding of a peptide induces partial opening of hDPP III, the globularity of the protein did not change in MD simulations of the hermorphin-like peptide bound to hDPP III with two zinc ions. However, the entrance to the interdomain cleft widens during Zn diffusion into the protein and was found to be the highest energy barrier in the process of metal translocation from the solvent to the active site. Finally, we discuss why excess zinc reduces enzyme activity.

Profibrotic mechanisms of DPP8 and DPP9 highly expressed in the proximal renal tubule epithelial cells.

BACKGROUND: DPP8 and DPP9 have been demonstrated to play important roles in multiple diseases. Evidence for increased gene expression of DPP8 and DPP9 in tubulointerstitium was found to be associated with the decline of kidney function in chronic kidney disease (CKD) patients, which was observed in the Nephroseq human database. To examine the role of DPP8 and DPP9 in the tubulointerstitial injury, we determined the efficacy of DPP8 and DPP9 on epithelial-to-mesenchymal transition (EMT) and tubulointerstitial fibrosis (TIF) as well as the underlying mechanisms. METHODS: We conducted the immunofluorescence of DPP8 and DPP9 in kidney biopsy specimens of CKD patients, established unilateral ureteral obstruction (UUO) animal model, treated with TC-E5007 (a specific inhibitor of both DPP8 and DPP9) or Saxagliptin (positive control) or saline, and HK-2 cells model. RESULTS: We observed the significantly increased expression of DPP8 and DPP9 in the renal proximal tubule epithelial cells of CKD patients compared to the healthy control subjects. DPP8/DPP9 inhibitor TC-E5007 could significantly attenuate the EMT and extracellular matrix (ECM) synthesis in UUO mice, all these effects were mediated via interfering with the TGF-ß1/Smad signaling. TC-E5007 treatment also presented reduced renal inflammation and improved renal function in the UUO mice compared to the placebo-treated UUO group. Furthermore, the siRNA for DPP8 and DPP9, and TC-E5007 treatment decreased EMT- and ECM-related proteins in TGF-ß1-treated HK-2 cells respectively, which could be reversed significantly by transduction with lentivirus-DPP8 and lentivirus-DPP9. CONCLUSION: These data obtained provide evidence that the DPP8 and DPP9 could be potential therapeutic targets against TIF.

DPP3/CDK1 contributes to the progression of colorectal cancer through regulating cell proliferation, cell apoptosis, and cell migration.

At present, colorectal cancer (CRC) has become a serious threat to human health in the world. Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase that may be involved in several physiological processes. However, whether DPP3 affects the development and progression of CRC remains a mystery. This study is the first to demonstrate the role of DPP3 in CRC. Firstly, the results of immunohistochemistry analysis showed the upregulation of DPP3 in CRC tissues compared with normal tissues, which is statistically analyzed to be positively correlated with lymphatic metastasis, pathological stage, positive number of lymph nodes. Moreover, the high expression of DPP3 predicts poor prognosis in CRC patients. In addition, the results of cell dysfunction experiments clarified that the downregulation of DPP3 significantly inhibited cell proliferation, colony formation, cell migration, and promoted apoptosis in vitro. DPP3 depletion could induce cell apoptosis by upregulating the expression of BID, BIM, Caspase3, Caspase8, HSP60, p21, p27, p53, and SMAC. In addition, downregulation of DPP3 can reduce tumorigenicity of CRC cells in vivo. Furthermore, CDK1 is determined to be a downstream target of DPP3-mediated regulation of CRC by RNA-seq, qPCR, and WB. The interaction between DPP3 and CDK1 shows mutual regulation. Specifically, downregulation of DPP3 can accentuate the effects of CDK1 knockdown on the function of CRC cells. Overexpression of CDK1 alleviates the inhibitory effects of DPP3 knockdown in CRC cells. In summary, DPP3 has oncogene-like functions in the development and progression of CRC by targeting CDK1, which may be an effective molecular target for the prognosis and treatment of CRC.

Structural basis for an exceptionally strong preference for asparagine residue at the S2 subsite of Stenotrophomonas maltophilia dipeptidyl peptidase 7.

The emergence of drug-resistant bacteria has become a major problem worldwide. Bacterial dipeptidyl peptidases 7 and 11 (DPP7s and DPP11s), belonging to the family-S46 peptidases, are important enzymes for bacterial growth and are not present in mammals. Therefore, specific inhibitors for these peptidases are promising as potential antibiotics. While the molecular mechanisms underlining strict specificity at the S1 subsite of S46 peptidases have been well studied, those of relatively broad preference at the S2 subsite of these peptidases are unknown. In this study, we performed structural and biochemical analyses on DPP7 from Stenotrophomonas maltophilia (SmDPP7). SmDPP7 showed preference for the accommodation of hydrophobic amino acids at the S2 subsite in general, but as an exception, also for asparagine, a hydrophilic amino acid. Structural analyses of SmDPP7 revealed that this exceptional preference to asparagine is caused by a hydrogen bonding network at the bottom of the S2 subsite. The residues in the S2 subsite are well conserved among S46 peptidases as compared with those in the S1 subsite. We expect that our findings will contribute toward the development of a universal inhibitor of S46 peptidases.

Harnessing affinity-based protein profiling to reveal a novel target of nintedanib.

Nintedanib (BIBF1120), a triple angiokinase inhibitor, was first approved for idiopathic pulmonary fibrosis (IPF) therapy and is also efficacious for lung carcinoma, and interstitial lung diseases, far beyond its inhibition of VEGFR/PDGFR/FGFR. We identified tripeptidyl-peptidase 1 (TPP1) as one of the direct targets of nintedanib employing the affinity-based protein profiling (AfBPP) technique. This may be a new mechanism for nintedanib's role different from tyrosine kinase inhibition.

NLRP1 variant M1184V decreases inflammasome activation in the context of DPP9 inhibition and asthma severity.

BACKGROUND: NLRP1 is an innate immune sensor that can form cytoplasmic inflammasome complexes. Polymorphisms in NLRP1 are linked to asthma; however, there is currently no functional or mechanistic explanation for this. OBJECTIVE: We sought to clarify the role of NLRP1 in asthma pathogenesis. METHODS: Results from the GALA II cohort study were used to identify a link between NLRP1 and asthma in Mexican Americans. In vitro and in vivo models for NLRP1 activation were applied to investigate the role of this inflammasome in asthma at the molecular level. RESULTS: We document the association of an NLRP1 haplotype with asthma for which the single nucleotide polymorphism rs11651270 (M1184V) individually is the most significant. Surprisingly, M1184V increases NLRP1 activation in the context of N-terminal destabilization, but decreases NLRP1 activation on dipeptidyl peptidase 9 inhibition. In vitro studies demonstrate that M1184V increases binding to dipeptidyl peptidase 9, which can account for its inhibitory role in this context. In addition, in vivo data from a mouse model of airway inflammation reveal a protective role for NLRP1 inflammasome activation reducing eosinophilia in this setting. CONCLUSIONS: Linking our in vitro and in vivo results, we found that the NLRP1 variant M1184V reduces inflammasome activation in the context of dipeptidyl peptidase 9 inhibition and could thereby increase asthma severity. Our studies may have implications for the treatment of asthma in patients carrying this variant of NLRP1.

Synthesis and Biological Evaluation of N-Aminoimidazolidin-2-one-Containing Angiotensin-(1-7) Peptidomimetics.

N-Aminoimidazolidin-2-one (Aid)-containing peptides with a constrained backbone present a novel class of peptidomimetics for drug discovery. The introduction of Aid residues into peptide sequences has been achieved by intramolecular Mitsunobu cyclization of a serine side chain onto the α-NH of an aza-glycine residue. The effectiveness of this new strategy was demonstrated by synthesizing six Aid-containing analogues of angiotensin-(1-7) on solid support. The Aid analogues of angiotensin-(1-7) exhibited increased peptidase stability against human ACE and DPP3 and improved anti-inflammation and antiproliferation activity.

Decrease of the pro-inflammatory M1-like response by inhibition of dipeptidyl peptidases 8/9 in THP-1 macrophages - quantitative proteomics of the proteome and secretome.

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.

Phase 2 Trial of the DPP-1 Inhibitor Brensocatib in Bronchiectasis.

BACKGROUND: Patients with bronchiectasis have frequent exacerbations that are thought to be related to neutrophilic inflammation. The activity and quantity of neutrophil serine proteases, including neutrophil elastase, are increased in the sputum of patients with bronchiectasis at baseline and increase further during exacerbations. Brensocatib (INS1007) is an oral reversible inhibitor of dipeptidyl peptidase 1 (DPP-1), an enzyme responsible for the activation of neutrophil serine proteases. METHODS: In a phase 2, randomized, double-blind, placebo-controlled trial, we randomly assigned, in a 1:1:1 ratio, patients with bronchiectasis who had had at least two exacerbations in the previous year to receive placebo, 10 mg of brensocatib, or 25 mg of brensocatib once daily for 24 weeks. The time to the first exacerbation (primary end point), the rate of exacerbations (secondary end point), sputum neutrophil elastase activity, and safety were assessed. RESULTS: Of 256 patients, 87 were assigned to receive placebo, 82 to receive 10 mg of brensocatib, and 87 to receive 25 mg of brensocatib. The 25th percentile of the time to the first exacerbation was 67 days in the placebo group, 134 days in the 10-mg brensocatib group, and 96 days in the 25-mg brensocatib group. Brensocatib treatment prolonged the time to the first exacerbation as compared with placebo (P = 0.03 for 10-mg brensocatib vs. placebo; P = 0.04 for 25-mg brensocatib vs. placebo). The adjusted hazard ratio for exacerbation in the comparison of brensocatib with placebo was 0.58 (95% confidence interval [CI], 0.35 to 0.95) in the 10-mg group (P = 0.03) and 0.62 (95% CI, 0.38 to 0.99) in the 25-mg group (P = 0.046). The incidence-rate ratio was 0.64 (95% CI, 0.42 to 0.98) in the 10-mg group, as compared with placebo (P = 0.04), and 0.75 (95% CI, 0.50 to 1.13) in the 25-mg group, as compared with placebo (P = 0.17). With both brensocatib doses, sputum neutrophil elastase activity was reduced from baseline over the 24-week treatment period. The incidence of dental and skin adverse events of special interest was higher with the 10-mg and 25-mg brensocatib doses, respectively, than with placebo. CONCLUSIONS: In this 24-week trial, reduction of neutrophil serine protease activity with brensocatib in patients with bronchiectasis was associated with improvements in bronchiectasis clinical outcomes. (Funded by Insmed; WILLOW ClinicalTrials.gov number, NCT03218917.).

DPP8/9 inhibitors activate the CARD8 inflammasome in resting lymphocytes.

Canonical inflammasomes are innate immune signaling platforms that are formed in response to intracellular pathogen-associated signals and trigger caspase-1-dependent pyroptosis. Inflammasome formation and signaling is thought to mainly occur in myeloid cells, and in particular monocytes and macrophages. Here we show that small molecule inhibitors of dipeptidyl peptidases 8 and 9 (DPP8/9), which activate the related CARD8 and NLRP1 inflammasomes, also activate pyroptosis in human and rodent resting lymphocytes. We found that both CD4+ and CD8+ T cells were particularly sensitive to these inhibitors, although the sensitivity of T cells, like macrophages, varied considerably between species. In human T cells, we show that CARD8 mediates DPP8/9 inhibitor-induced pyroptosis. Intriguingly, although activated human T cells express the key proteins known to be required for CARD8-mediated pyroptosis, these cells were completely resistant to DPP8/9 inhibitors. Overall, these data show that resting lymphoid cells can activate at least one inflammasome, revealing additional cell types and states poised to undergo rapid pyroptotic cell death in response to danger-associated signals.

Inhibition of circulating dipeptidyl-peptidase 3 restores cardiac function in a sepsis-induced model in rats: A proof of concept study.

Sepsis is a global economic and health burden. Dipeptidyl peptidase 3 (DPP3) is elevated in the plasma of septic patients. The highest levels of circulating DPP3 (cDPP3) are found in non-survivor septic shock patients. The aim of this study was to evaluate the benefits of inhibiting cDPP3 by a specific antibody, Procizumab (PCZ), on cardiac function in an experimental model of sepsis, the caecal ligature and puncture (CLP) model. Rats were monitored by invasive blood pressure and echocardiography. Results are presented as mean ± SD, with p <0.05 considered significant. PCZ rapidly restored left ventricular shortening fraction (from 39 ± 4% to 51 ± 2% before and 30 min after PCZ administration (p = 0.004)). Cardiac output and stroke volume were higher in the CLP + PCZ group when compared to the CLP + PBS group (152 ± 33 mL/min vs 97 ± 25 mL/min (p = 0.0079), and 0.5 ± 0.1 mL vs 0.3 ± 1.0 mL (p = 0.009), respectively) with a markedly reduced plasma DPP3 activity (138 ± 70 U/L in CLP + PCZ group versus 735 ± 255 U/L (p = 0.048) in the CLP + PBS group). Of note, PCZ rapidly reduced oxidative stress in the heart of the CLP + PCZ group when compared to those of the CLP + PBS group (13.3 ± 8.2 vs 6.2 ± 2.5 UI, p = 0.005, 120 min after administration, respectively). Our study demonstrates that inhibition of cDPP3 by PCZ restored altered cardiac function during sepsis in rats.

Electrical stimulation inhibits Val-boroPro-induced pyroptosis in THP-1 macrophages via sirtuin3 activation to promote autophagy and inhibit ROS generation.

The incidence of atherosclerosis (AS), a major contributor to cardiovascular disease, is steadily rising along with an increasingly older population worldwide. Pyroptosis, a form of inflammatory programmed cell death, determines the release of pro-inflammatory mediators by endothelial cells, smooth muscle cells, and atheroma-associated macrophages and foam cells, thereby playing a critical role in AS progression. Canonical pyroptosis is mediated by inflammasome formation, activation of caspase-1, and maturation and release of proinflammatory cytokines. Electrical stimulation (ES) is a noninvasive, safe therapy that has been shown to alleviate symptoms in several health conditions. Here, we investigated the anti-inflammatory and anti-pyroptotic effects of ES in human THP-1 macrophages treated with the dipeptidyl peptidase inhibitor Val-boroPro (VbP). We found that ES downregulated NOD-like receptor family protein 3 (NLRP3) inflammasome, ASC, and caspase-1 expression and abrogated the release of Interleukin-1ß (IL-1ß) and Interleukin-18 (IL-18), indicating effective pyroptosis inhibition. These changes were paralleled by a reduction in reactive oxygen species (ROS) production, reversal of VbP-induced sirtuin3 (Sirt3) downregulation, deacetylation of ATG5, and induction of autophagy. These findings suggest that ES may be a viable strategy to counteract pyroptosis-mediated inflammation in AS by raising Sirt3 to promote autophagy and inhibit ROS generation.

Pharmacological evaluation of Rhazya stricta root extract / Evaluación farmacológica del extracto de raíz de Rhazya stricta

The present study aimed to screen the Rhazya stricta Decne root for its antihyperglycemic and antioxidants potential through invitro assays along with phytochemical and elemental analyses. The crude extract was prepared through maceration and fractionated using solvent-solvent extraction technique. The spectroscopic studies indicated the presence of various phytochemical classes in the extract and its fractions. The antioxidant assays showed notable results along with a good concentration of phenolic and flavonoid contents. Enzyme inhibition assays demonstrated glucose-lowering effects by inhibiting the enzyme activity which could reduce post-prandial blood glucose level. The Dipeptidyl peptidase-IV (DPP-IV) inhibition assay results showed the novel DPP-IV inhibition activity of the plant extract and all fractions showed noteworthy enzyme inhibition and antihyperglycemic activity. Conclusively, the Rhazya stricta root extract displayed its antioxidant and antihyperglycemic potential due to the presence of various classes of phytochemicals and micro-nutrients.

El presente estudio tuvo como objetivo examinar la raíz de Rhazya stricta Decne por su potencial antihiperglicémico y antioxidante a través de ensayos in vitro junto con análisis fitoquímicos y elementales. El extracto crudo se preparó por maceración y se fraccionó usando una técnica de extracción solvente-solvente. Los estudios espectroscópicos indicaron la presencia de varias clases fitoquímicas en el extracto y sus fracciones. Los ensayos antioxidantes mostraron resultados notables junto con una importante concentración de contenido fenólico y flavonoide. Los ensayos de inhibición enzimática demostraron efectos reductores de la glucosa al inhibir la actividad enzimática que podría reducir el nivel de glucosa posprandial en sangre. Los resultados del ensayo de inhibición de Dipeptidyl peptidase-IV (DPP-IV) mostraron la nueva actividad de inhibición de DPP-IV del extracto de la planta y todas las fracciones mostraron una notable inhibición enzimática y actividad antihiperglicémica. En conclusión, el extracto de raíz de Rhazya stricta Decne mostró su potencial antioxidante y antihiperglicémico debido a la presencia de varias clases de fitoquímicos y micronutrientes.

Circulating dipeptidyl peptidase 3 is a myocardial depressant factor: dipeptidyl peptidase 3 inhibition rapidly and sustainably improves haemodynamics.

AIMS: Acute heart failure is a high mortality disease and its pathophysiology is not completely understood. Dipeptidyl peptidase 3 (DPP3) is a cytosolic enzyme involved in angiotensin II and enkephalins cleavage. The aim of this study was to investigate the association of circulating DPP3 (cDPP3) levels and mortality in cardiogenic shock patients and to determine the effects of high cDPP3 on organ function in a heart failure (HF) model in mice. METHODS AND RESULTS: cDPP3 was measured in 174 patients in cardiogenic shock and high cDPP3 levels were associated with an increased short-term mortality risk (standardized hazard ratio: 1.4 (1.1-1.8)) and severe organ dysfunction. Additionally, a rapid decrease in cDPP3 in cardiogenic shock patients within 24 h of admission was associated with a favourable outcome. This study showed that injection of DPP3 induced myocardial depression (-10 ± 2% of shortening fraction) and impaired kidney haemodynamics (+0.30 ± 0.02 of renal resistive index) in healthy mice. cDPP3 inhibition by Procizumab, a specific antibody directed against cDPP3, promptly normalized cardiac function and kidney haemodynamics in an acute heart failure mouse model, with a marked reduction in oxidative stress and inflammatory signalling. CONCLUSION: Our study demonstrated cDPP3 is a newly discovered myocardial depressant factor, the levels of which at admission are associated with mortality in severe HF patients. Furthermore, inhibition of cDPP3 by Procizumab improved haemodynamics in a mouse model of HF. Our results suggest that DPP3 could be a new biomarker and biotarget for severe HF.

A nanobody-based nuclear imaging tracer targeting dipeptidyl peptidase 6 to determine the mass of human beta cell grafts in mice.

AIMS/HYPOTHESIS: Type 1 diabetes is characterised by a progressive decline in beta cell mass. This is also observed following implantation of pancreatic islet allografts, but there is no reliable information regarding the time course of beta cell loss. This is due to the limited availability of non-invasive pancreatic islet imaging techniques. We have previously described that dipeptidyl peptidase 6 (DPP6) is an alpha and beta cell-specific biomarker, and developed a camelid antibody (nanobody '4hD29') against it. We demonstrated the possibility to detect DPP6-expressing cells by single-photon emission computed tomography (SPECT)/ computed tomography (CT), but the correlation between the number of cells grafted and the SPECT signal was not assessed. Here, we investigate whether the 4hD29 nanobody allows us to detect different amounts of human pancreatic islets implanted into immune-deficient mice. In addition, we also describe the adaptation of the probe for use with positron emission tomography (PET). METHODS: DPP6 expression was assessed in human samples using tissue arrays and immunohistochemistry. The effect of the 4hD29 nanobody on cell death and glucose-stimulated insulin secretion was measured in EndoC-ßH1 cells and in human islets using Hoechst/propidium iodide staining and an anti-insulin ELISA, respectively. We performed in vivo SPECT imaging on severe combined immunodeficient (SCID) mice transplanted with different amounts of EndoC-ßH1 cells (2 × 106, 5 × 106 and 10 × 106 cells), human islets (1000 and 3000) or pancreatic exocrine tissue using 99mTc-labelled 4hD29 nanobody. This DPP6 nanobody was also conjugated to N-chlorosuccinimide (NCS)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), radiolabelled with either 67Ga (SPECT) or 68Ga (PET) and used in a proof-of-principle experiment to detect DPP6-expressing cells (Kelly neuroblastoma) grafted in SCID mice. RESULTS: The DPP6 protein is mainly expressed in pancreatic islets. Importantly, the anti-DPP6 nanobody 4hD29 allows non-invasive detection of high amounts of EndoC-ßH1 cells or human islets grafted in immunodeficient mice. This suggests that the probe must be further improved to detect lower numbers of islet cells. The 4hD29 nanobody neither affected beta cell viability nor altered insulin secretion in EndoC-ßH1 cells and human islets. The conversion of 4hD29 nanobody into a PET probe was successful and did not alter its specificity. CONCLUSIONS/INTERPRETATION: These findings suggest that the anti-DPP6 4hD29 nanobody may become a useful tool for the quantification of human islet grafts in mice and, pending future development, islet mass in individuals with diabetes.

Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.

Dipeptidyl aminopeptidases (DPAPs) are cysteine proteases that cleave dipeptides from the N-terminus of protein substrates and have been shown to play important roles in many pathologies including parasitic diseases such as malaria, toxoplasmosis and Chagas's disease. Inhibitors of the mammalian homologue cathepsin C have been used in clinical trials as potential drugs to treat chronic inflammatory disorders, thus proving that these enzymes are druggable. In Plasmodium species, DPAPs play important functions at different stages of parasite development, thus making them potential antimalarial targets. Most DPAP inhibitors developed to date are peptide-based or peptidomimetic competitive inhibitors. Here, we used a high throughput screening approach to identify novel inhibitor scaffolds that block the activity of Plasmodium falciparum DPAP1. Most of the hits identified in this screen also inhibit Plasmodium falciparum DPAP3, cathepsin C, and to a lesser extent other malarial clan CA proteases, indicating that these might be general DPAP inhibitors. Interestingly, our mechanism of inhibition studies indicate that most hits are allosteric inhibitors, which opens a completely new strategy to inhibit these enzymes, study their biological function, and potentially develop new inhibitors as starting points for drug development.

Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase.

Antimicrobial resistance is a global public threat and raises the need for development of new antibiotics with a novel mode of action. The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to a new class of serine peptidases, family S46. Because S46 peptidases are not found in mammals, these enzymes are attractive targets for novel antibiotics. However, potent and selective inhibitors of these peptidases have not been developed to date. In this study, a high-resolution crystal structure analysis of PgDPP11 using a space-grown crystal enabled us to identify the binding of citrate ion, which could be regarded as a lead fragment mimicking the binding of a substrate peptide with acidic amino acids, in the S1 subsite. The citrate-based pharmacophore was utilized for in silico inhibitor screening. The screening resulted in an active compound SH-5, the first nonpeptidyl inhibitor of S46 peptidases. SH-5 and a lipophilic analog of SH-5 showed a dose-dependent inhibitory effect against the growth of P. gingivalis. The binding mode of SH-5 was confirmed by crystal structure analysis. Thus, these compounds could be lead structures for the development of selective inhibitors of PgDPP11.