Sitagliptin Extends Lifespan of Caenorhabditis elegans by Inhibiting Insulin/Insulin-Like Signaling and Activating Dietary Restriction-Like Signaling Pathways.

INTRODUCTION: The discovery of longevity molecules that delay aging and prolong lifespan has always been a dream of humanity. Sitagliptin phosphate (SIT), an oral dipeptidyl peptidase-4 (DPP-4) inhibitor, is an oral drug commonly used in the treatment of type 2 diabetes (T2D). In addition to being antidiabetic, previous studies have reported that SIT has shown potential to improve health. However, whether SIT plays a role in the amelioration of aging and the underlying molecular mechanism remain undetermined. METHODS: Caenorhabditis elegans (C. elegans) was used as a model of aging. Lifespan assays were performed with adult-stage worms on nematode growth medium plates containing FUdR with or without the specific concentration of SIT. The period of fast body movement, body bending rates, and pharyngeal pumping rates were recorded to assess the healthspan of C. elegans. Gene expression was confirmed by GFP fluorescence signal of transgenic worms and qPCR. In addition, the intracellular reactive oxygen species levels were measured using a free radical sensor H2DCF-DA. RESULTS: We found that SIT significantly extended lifespan and healthspan of C. elegans. Mechanistically, we found that several age-related pathways and genes were involved in SIT-induced lifespan extension. The transcription factors DAF-16/FOXO, SKN-1/NRF2, and HSF-1 played important roles in SIT-induced longevity. Moreover, our findings illustrated that SIT-induced survival benefits by inhibiting the insulin/insulin-like signaling pathway and activating the dietary restriction-related and mitochondrial function-related signaling pathways. CONCLUSION: Our work may provide a theoretical basis for the development of anti-T2D drugs as antiaging drugs, especially for the treatment of age-related disease in diabetic patients.

Promoter engineering-mediated Tuning of esterase and transaminase expression for the chemoenzymatic synthesis of sitagliptin phosphate at the kilogram-scale.

Here, we report a bienzymatic cascade to produce ß-amino acids as an intermediate for the synthesis of the leading oral antidiabetic drug, sitagliptin. A whole-cell biotransformation using recombinant Escherichia coli coexpressing a esterase and transaminase were developed, wherein the desired expression level of each enzyme was achieved by promotor engineering. The small-scale reactions (30 ml) performed under optimized conditions at varying amounts of substrate (100-300 mM) resulted in excellent conversions of 82%-95% for the desired product. Finally, a kilogram-scale enzymatic reaction (250 mM substrate, 220 L) was carried out to produce ß-amino acid (229 mM). Sitagliptin phosphate was chemically synthesized from ß-amino acids with 82% yield and > 99% purity.

Functional Dissection of CD26 and Its Pharmacological Inhibition by Sitagliptin During Skin Wound Healing.

BACKGROUND Skin fibroblasts are primary mediators underlying wound healing and therapeutic targets in scar prevention and treatment. CD26 is a molecular marker to distinguish fibroblast subpopulations and plays an important role in modulating the biological behaviors of dermal fibroblasts and influencing skin wound repair. Therapeutic targeting of specific fibroblast subsets is expected to reduce skin scar formation more efficiently. MATERIAL AND METHODS Skin burn and excisional wound healing models were surgically established in mice. The expression patterns of CD26 during wound healing were determined by immunohistochemical staining, real-time RT-PCR, and western blot assays. Normal fibroblasts from intact skin (NFs) and fibroblasts in wounds (WFs) were isolated and sorted by fluorescence-activated cell sorting (FACS) into 4 subgroups - CD26⁺ NFs, CD26⁻ NFs, CD26⁺ WFs, and CD26⁻ WFs - for comparisons of their capacities of proliferation, migration, and collagen synthesis. Pharmacological inhibition of CD26 by sitagliptin in skin fibroblasts and during wound healing were further assessed both in vitro and in vivo. RESULTS Increased CD26 expression was observed during skin wound healing in both models. The CD26⁺ fibroblasts isolated from wounds had significantly stronger abilities to proliferate, migrate, and synthesize collagen than other fibroblast subsets. Sitagliptin treatment potently diminished CD26 expression, impaired the proliferation, migration, and collagen synthesis of fibroblasts in vitro, and diminished scar formation in vivo. CONCLUSIONS Our data reveal that CD26 is functionally involved in skin wound healing by regulating cell proliferation, migration, and collagen synthesis in fibroblasts. Pharmacological inhibition of CD26 by sitagliptin might be a viable strategy to reduce skin scar formation.

Curtailing Quorum Sensing in Pseudomonas aeruginosa by Sitagliptin.

Pseudomonas aeruginosa coordinates the secretion of virulence factors through quorum sensing. Quorum sensing inhibitors can attenuate the pathogenesis of bacteria and help the immune system to eradicate them without targeting the bacterial growth. This study aimed to explore the anti-quorum sensing and anti-virulence activities of sitagliptin against P. aeruginosa PAO1 strain. Sub-inhibitory concentration of sitagliptin significantly inhibited the virulence factors pyocyanin, hemolysin, protease and elastase in addition to blocking swimming, swarming and twitching motilities and biofilm formation. In silico analysis showed that sitagliptin interacted with LasR receptors by hydrogen bonding and hydrophobic interaction, mainly with the amino acids leucine present at positions 40 and 125, tyrosine at position 56, serine at position 129, tryptophan at position 60, alanine at position 50 and phenyl alanine at position 101. qRT-PCR confirmed the anti-quorum sensing activity by reducing the expression level of QS genes lasI, lasR, rhlI, rhlR, pqsA and pqsR. In conclusion, sitagliptin is a novel anti-quorum sensing agent that can be used for treating P. aeruginosa infections.

Sitagliptin on Carotid Intima-Media Thickness in Type 2 Diabetes Mellitus Patients and Anemia: A Subgroup Analysis of the PROLOGUE Study.

INTRODUCTION: Randomized clinical trials have not shown an additional clinical benefit of sitagliptin treatment over conventional treatment alone. However, studies of sitagliptin treatment have not examined the relationship between anemia and treatment group outcomes. METHODS: The PROLOGUE study is a prospective clinical trial of 442 participants with type 2 diabetes mellitus (T2DM) randomized to sitagliptin treatment or conventional treatment which showed no treatment differences [Estimated mean (± standard error) common carotid intima-media thickness (CIMT) was 0.827 ± 0.007 mm and 0.837 ± 0.007 mm, respectively, with a mean difference of -0.009 mm (97.2% CI -0.028 to 0.011, p = 0.309) at 24 mo of follow-up]. This is a post hoc subanalysis using data obtained from the PROLOGUE study; the study population was divided into anemic groups (n = 94) and nonanemic group (n = 343) based on hemoglobin level. And we analyzed for the changes in each CIMT parameter from baseline to 24 months in subgroups. RESULTS: The treatment group difference in baseline-adjusted mean common carotid artery- (CCA-) IMT at 24 months was -0.003 mm (95% CI -0.022 to 0.015, p = 0.718) in the nonanemic subgroup and -0.007 mm (95% CI -0.043 to 0.030, p = 0.724) in the anemic subgroup. Although there were no significant differences in the other CIMT parameters between the treatment groups in the anemic subgroup, the changes in mean and max ICA-IMT at 24 months in the nonanemic subgroup were significantly lower in the sitagliptin group than the conventional group [-0.104 mm (95% CI -0.182 to -0.026), p = 0.009 and -0.142 mm (-0.252 to -0.033), p = 0.011, respectively]. CONCLUSION: These data suggest that nonanemia may indicate a potentially large subgroup of those with T2DM patients that sitagliptin therapy has a better antiatherosclerotic effect than conventional therapy. Further research is needed to confirm these preliminary observations.

Evaluation of Drug Efficacy of GLP-1 Receptor Agonists and DPP-4 Inhibitors Based on Target Molecular Binding Occupancy.

Glucagon-like peptide-1 (GLP-1) receptor agonists (liraglutide, exenatide, lixisenatide) have recently been used as anti-diabetes drugs. We examined relationships of the binding occupancy of GLP-1 receptors (Φ) and their clinical efficacy after administration of GLP-1 receptor agonists. Next, by focusing on changes of GLP-1 concentration after administration of dipeptidyl peptidase-4 (DPP-4) inhibitors (vildagliptin, alogliptin, sitagliptin, linagliptin), we analyzed the relationship between Φ and clinical efficacy. Furthermore, using Φ as a common parameter, we compared the clinical efficacy elicited by GLP-1 receptor agonists and DPP-4 inhibitors using a theoretical analysis method. The present results showed that GLP-1 receptor agonists produced their clinical effect at a relatively low level of Φ (1.1-10.7%) at a usual dose. Furthermore, it was suggested that the drugs might achieve their full effect at an extraordinarily low level of Φ. It was also revealed that the Φ value of DPP-4 inhibitors (0.83-1.3%) was at the lower end or lower than that of GLP-1 receptor agonists at a usual dose. Accordingly, the predicted value for hemoglobin A1c (HbA1c) reduction after administration of the GLP-1 receptor agonists was higher than that of DPP-4 inhibitors. We clarified the differences between the therapeutic effects associated with GLP-1 receptor agonists and DPP-4 inhibitors theoretically. Together, the present findings provide a useful methodology for proper usage of GLP-1 receptor agonists and DPP-4 inhibitors.

The Effect of Sitagliptin on Lipid Metabolism of Fatty Liver Mice and Related Mechanisms.

BACKGROUND In clinics, patients with type 2 diabetes complicated with non-alcoholic fatty liver disease (NAFLD) have been shown to receive significant improvements in blood glucose levels, lipid levels, and liver function after sitagliptin treatment, although the mechanism of drug action remains poorly understood. This study investigated the possible mechanism of sitagliptin on lipid metabolism of NAFLD mice. MATERIAL AND METHODS Male C57/BL6 mice were induced for NAFLD via 16 weeks of a high-fat diet, and were treated with 15 mg/kg/day sitagliptin for 16 consecutive weeks. Blood lipid levels were measured and samples were stained with hematoxylin and eosin (H&E) and oil red staining for liver pathology and lipid deposition. Serum levels of fibroblast growth factor (FGF)-9 and FGF-21 were quantified by enzyme-linked immunosorbent assay (ELISA). Peroxisome proliferator-activated receptor (PPAR)-α, and cAMP reactive element binding homolog (CREBH) were measured by Western blotting, while fatty acid synthase and carnitine palmitoyltransferase 1 (CPT1) mRNA levels were assayed by RT-PCR. RESULTS Compared to the control group, the NAFLD model mice had liver fatty disease, lower serum FGF-21 and FGF-19 levels, elevated serum lipid levels, depressed PPAR-α, CREBH, and CPT1 expression, and enhanced FAS expression (p<0.05). Sitagliptin treatment depressed blood lipid levels, increased serum FGF-21 and FGF-19 levels, PPAR-α, CREBH, and CPT1 expression, and suppressed FAS expression (p<0.05). CONCLUSIONS Sitagliptin can protect liver tissue and modulate lipid metabolism in NAFLD mice via elevating FGF-21 and FGF-19, upregulating liver PPAR-a and CREBH levels, and mediating expression levels of key enzymes for lipid metabolism.

Highly enantioselective production of a chiral intermediate of sitagliptin by a novel isolate of Pseudomonas pseudoalcaligenes.

OBJECTIVE: To produce (S)-3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one (S)-1 from 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-1-(2,4,5-trifluorophenyl)butan-2-one (2) by microbial bioreduction. RESULTS: A new isolate of Pseudomonas pseudoalcaligenes reduced enantioselectively prochiral ketone 2 to chiral alcohol (S)-1. Whole cells of the bacterium were tolerant towards 20 % (v/v) DMSO and 10 g 2/l. Under the optimal conditions, the preparative-scale bioreduction yielded (S)-1 at 90 % yield and >99 % ee. Cells could be re-used with the yield and ee of product being 45 % and >99 %, respectively, after five cycles. CONCLUSION: Bioreduction using whole cells of P. pseudoalcaligenes is an attractive approach to produce (S)-1, as a chiral intermediate of the anti-diabetic drug, sitagliptin.

Network-based screening identifies sitagliptin as an antitumor drug targeting dendritic cells.

BACKGROUND: Dendritic cell (DC)-mediated antigen presentation is essential for the priming and activation of tumor-specific T cells. However, few drugs that specifically manipulate DC functions are available. The identification of drugs targeting DC holds great promise for cancer immunotherapy. METHODS: We observed that type 1 conventional DCs (cDC1s) initiated a distinct transcriptional program during antigen presentation. We used a network-based approach to screen for cDC1-targeting therapeutics. The antitumor potency and underlying mechanisms of the candidate drug were investigated in vitro and in vivo. RESULTS: Sitagliptin, an oral gliptin widely used for type 2 diabetes, was identified as a drug that targets DCs. In mouse models, sitagliptin inhibited tumor growth by enhancing cDC1-mediated antigen presentation, leading to better T-cell activation. Mechanistically, inhibition of dipeptidyl peptidase 4 (DPP4) by sitagliptin prevented the truncation and degradation of chemokines/cytokines that are important for DC activation. Sitagliptin enhanced cancer immunotherapy by facilitating the priming of antigen-specific T cells by DCs. In humans, the use of sitagliptin correlated with a lower risk of tumor recurrence in patients with colorectal cancer undergoing curative surgery. CONCLUSIONS: Our findings indicate that sitagliptin-mediated DPP4 inhibition promotes antitumor immune response by augmenting cDC1 functions. These data suggest that sitagliptin can be repurposed as an antitumor drug targeting DC, which provides a potential strategy for cancer immunotherapy.

Sitagliptin mitigates hypoxia/reoxygenation (H/R)-induced injury in cardiomyocytes by mediating sirtuin 3 (SIRT3) and autophagy.

Potential ischemia/reperfusion (I/R) injuries are commonly induced during treatment of cardiovascular diseases, such as acute myocardial infarction (AMI). It is reported that oxidative stress and over-autophagy in cardiomyocytes are involved in the pathogenesis of I/R injury. Sitagliptin is an effective inhibitor of dipeptidyl peptidase 4 (DPP-4) for the treatment of diabetes, which is recently reported to regulate oxidative stress and autophagy. The present study is designed to explore the function of Sitagliptin on I/R injury. Hypoxia/reoxygenation (H/R) condition was used to simulate the I/R injury on cardiomyocytes. We found that the declined cell viability and elevated expression level of creatine kinase myocardial band (CK-MB) were observed in the H/R group, accompanied by the increased mitochondrial reactive oxygen species (ROS), elevated cellular malondialdehyde (MDA) level, and mitochondrial dysfunction. After Sitagliptin treatment, the damages in H9c2 cardiomyocytes, oxidative stress, and mitochondrial dysfunction were significantly alleviated. In addition, the overactivated autophagy and mitophagy in H/R-challenged cardiomyocytes were dramatically mitigated by Sitagliptin, accompanied by the upregulation of SIRT3. Lastly, the protective effect of Sitagliptin on H/R-induced mitophagy, autophagy, and damages in cardiomyocytes was dramatically abolished by the knockdown of SIRT3. Taken together, our data reveal that Sitagliptin ameliorated the H/R-induced injury in cardiomyocytes by mediating sirtuin 3 (SIRT3) and autophagy.

Sitagliptin's renoprotective effect in a diabetic nephropathy model in rats: The potential role of PI3K/AKT pathway.

Management of diabetic nephropathy (DN) is far from satisfactory. There is a rising role of the involvement of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway in the pathogenesis of DN. This study aimed at investigating the renoprotective effects of PI3K/AKT pathway via sitagliptin in a rat model of DN. Thirty-two male Wistar rats were divided into four groups (eight rats each): (I) control, (II) sitagliptin, (III) DN, and (IV) DN + sitagliptin. Fasting blood glucose (FBG), kidney index, and kidney function tests in both blood and urine were measured. The levels of superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-ß) and gene expressions of PI3K, pPI3K, AKT, and pAKT in renal tissue were detected. Renal histopathological and immunohistochemical studies were evaluated. DN + sitagliptin group showed significant decrease in FBG and kidney index, improvement in kidney function tests, and a decrease in levels of TNF-α and TGF-ß in renal tissues compared with DN group. This was associated with significant increase in SOD and gene expressions of PI3K and AKT and their phosphorylated active forms in renal tissue in DN + sitagliptin group compared with DN group. Moreover, DN + sitagliptin group showed apparent decrease in amount of collagen fibers and expression of alpha-smooth muscle actin (α-SMA) compared with DN group. This work shows that sitagliptin improved renal functions and histopathological changes, impeded inflammation, and oxidative stress and upregulated PI3K/AKT pathway which highlights its renoprotective effects in a rat model of DN.

Creation of a robust and R-selective ω-amine transaminase for the asymmetric synthesis of sitagliptin intermediate on a kilogram scale.

The creation of an R-selective ω-amine transaminase (ω-ATA) as biocatalyst is crucial for the asymmetric amination of prochiral ketones to produce sitagliptin intermediates because rare ω-ATAs are R-selective in nature and most of them suffer from poor stability and low activity toward bulky prochiral ketones. Here, the gene of an R-selective ω-ATA was cloned from Arthrobacter cumminsii ZJUT212 (AcATA) and expressed in Escherichia coli. The best variants (M1 + M122H and M1+T134 G) were obtained using a semi-rational protein design after screening. These variants not only exhibited improved activity and substrate affinity but also enhanced stability in aqueous phase containing 20 % dimethyl sulfoxide. The conversion of asymmetric amination on 50 g/L pro-sitagliptin ketone PTfpB (1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione) achieved 92 %, with an extremely high e.e. of >99 %, using 2 gDCW/L E. coli cells harboring M1 + M122H as biocatalyst. In the kilogram-scale experiment, approximately 40 kg of (R)-APTfpB (e.e. >99 %) was produced within 30 h when 50 kg PTfpB was used as the substrate. Furthermore, the space-time yield reached ≈32 g/(L·d).

A novel self-sufficient biocatalyst based on transaminase and pyridoxal 5'-phosphate covalent co-immobilization and its application in continuous biosynthesis of sitagliptin.

Transaminases (TAs, EC 2.6.1.X) are a class of promising catalysts for the synthesis of chiral amines. The addition of exogenous expensive cofactor, pyridoxal 5'-phosphate (PLP) leads to the high cost and complicated reaction composition and thus limits the industrial implementation of TAs. In this study, a novel self-sufficient biocatalyst was developed based on transaminase and PLP covalent co-immobilization with the activity recovery of 83.6% and the specific activity of 343.0 U/g catalyst. The self-sufficient BgTA biocatalyst was employed in the continuous biosynthesis of (R)-sitagliptin in a recirculating packed bed reactor (RPBR) for 700 batches reaction without extra addition of PLP. The yield and e.e. of (R)-sitagliptin for each batch were above 90% and 99% respectively. The space-time yield was 40.0 g/(L·h) which was higher than ever reported. This is the first report about the covalent co-immobilization of TA and PLP with high activity and stability, and our work also contributed to the economic flexibility of chiral amine pharmaceutical intermediates biosynthesis.

Efficient Enantioselective Biocatalytic Production of a Chiral Intermediate of Sitagliptin by a Newly Filamentous Fungus Isolate.

(S)-3-Hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one ((S)-HTPP) is a crucial intermediate for the synthesis of Sitagliptin. A fungal strain ZJPH1308, capable of the biocatalysis of ketoamide 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one (OTPP) to (S)-HTPP with excellent enantioselectivity, was isolated from a soil sample and identified as Rhizopus microsporus var. rhizopodiformis ZJPH1308 based on its morphological characteristics and internal transcribed spacer (ITS) sequence. Some key reaction parameters involved in the bioreduction catalyzed by isolate ZJPH1308 were then optimized. It demonstrated that the bioreduction of OTPP was effective conducted at relative high temperature (45 °C), along with distilled water as reaction medium and glycerol-coupling approach for cofactor regeneration. Under the optimal conditions, the preparative-scale bioreduction gave a 93.2 % yield (with >99.9 % of enantiomeric excess (ee)) at 15 mM of OTPP and 45 °C, reaction for 24 h. The results indicated that fungal isolate ZJPH1308 can afford a thermostable carbonyl reductase and is a promising biocatalyst for clean and efficient production of valuable chiral intermediate.