Structural selectivity of human SGLT inhibitors

Human Na(+)-D-glucose cotransporter (hSGLT) inhibitors constitute the newest class of diabetes drugs, blocking up to 50% of renal glucose reabsorption in vivo. These drugs have potential for widespread use in the diabetes epidemic, but how they work at a molecular level is poorly understood. Here, we use electrophysiological methods to assess how they block Na(+)-D-glucose cotransporter SGLT1 and SGLT2 expressed in human embryonic kidney 293T (HEK-293T) cells and compared them to the classic SGLT inhibitor phlorizin. Dapagliflozin [(1S)-1,5,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-glucitol], two structural analogs, and the aglycones of phlorizin and dapagliflozin were investigated in detail. Dapagliflozin and fluoro-dapagliflozin [(1S)-1,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-4-F-4-deoxy-D-glucitol] blocked glucose transport and glucose-coupled currents with ≈100-fold specificity for hSGLT2 (K(i) = 6 nM) over hSGLT1 (K(i) = 400 nM). As galactose is a poor substrate for SGLT2, it was surprising that galacto-dapagliflozin [(1S)-1,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-galactitol] was a selective inhibitor of hSGLT2, but was less potent than dapagliflozin for both transporters (hSGLT2 K(i) = 25 nM, hSGLT1 K(i) = 25,000 nM). Phlorizin and galacto-dapagliflozin rapidly dissociated from SGLT2 [half-time off rate (t(1/2,Off)) ≈ 20-30 s], while dapagliflozin and fluoro-dapagliflozin dissociated from hSGLT2 at a rate 10-fold slower (t(1/2,Off) ≥ 180 s). Phlorizin was unable to exchange with dapagliflozin bound to hSGLT2. In contrast, dapagliflozin, fluoro-dapagliflozin, and galacto-dapagliflozin dissociated quickly from hSGLT1 (t(1/2,Off) = 1-2 s), and phlorizin readily exchanged with dapagliflozin bound to hSGLT1. The aglycones of phlorizin and dapagliflozin were poor inhibitors of both hSGLT2 and hSGLT1 with K(i) values > 100 μM. These results show that inhibitor binding to SGLTs is composed of two synergistic forces: sugar binding to the glucose site, which is not rigid, and so different sugars will change the orientation of the aglycone in the access vestibule; and the binding of the aglycone affects the binding affinity of the entire inhibitor. Therefore, the pharmacophore must include variations in both the structure of the sugar and the aglycone.     

Synthesis of novel l-rhamnose derived acyclic C-nucleosides with substituted 1,2,3-triazole core as potent sodium-glucose co-transporter (SGLT) inhibitors

Sodium-glucose co-transporter (SGLT) inhibitors are a novel class of therapeutic agents for the treatment of type 2 diabetes by preventing renal glucose reabsorption. In our efforts to identify novel inhibitors of SGLT, we synthesized a series of l-rhamnose derived acyclic C-nucleosides with 1,2,3-triazole core. The key β-ketoester building block 4 prepared from l-rhamnose in five steps, was reacted with various aryl azides to produce the respective 1,2,3-triazole derivatives in excellent yields. Deprotection of acetonide group gave the desired acyclic C-nucleosides 7a–o. All the new compounds were screened for their sodium-glucose co-transporters (SGLT1 and SGLT2) inhibition activity using recently developed cell-based nonradioactive fluorescence glucose uptake assay. Among them, 7m with IC₅₀: 125.9 nM emerged as the most potent SGLT2 inhibitor. On the other hand compound 7d exhibited best selectivity for inhibition of SGLT2 (IC₅₀: 149.1 nM) over SGLT1 (IC₅₀: 693.2 nM). The results presented here demonstrated the utility of acyclic C-nucleosides as novel SGLT inhibitors for future investigations.     

Endothelial Na+-D-glucose cotransporter: no role in insulin-mediated glucose uptake.

A recent report indicates that the Na+-D-glucose cotransporter SGLT1 is present in capillaries of skeletal muscle and is required for insulin-mediated glucose uptake in myocytes. This result is based on the complete inhibition of insulin-mediated muscle glucose uptake by phlorizin, an inhibitor of SGLT1. Using the pump-perfused rat hind limb, we measured glucose uptake, lactate efflux, and radioactive 2-deoxyglucose uptake into individual muscles with saline (control), phlorizin, insulin, and insulin plus phlorizin, as well as with saline and insulin using normal and low Na+ perfusion buffer. Insulin-mediated glucose uptake was not inhibited after correction for phlorizin interference in the glucose assay. Lactate efflux and 2-deoxyglucose uptake by individual muscles were unaffected by phlorizin. Low Na+ buffer did not affect insulin-mediated glucose uptake, lactate efflux, or 2-deoxyglucose uptake. We conclude that endothelial SGLT1 exerts no barrier for glucose delivery to myocytes.     

抗糖尿病药物SGLT抑制剂最新研究进展

钠-葡萄糖协转运蛋白（SGLT）是一类在小肠（SGLT-1）和肾脏近曲小管（SGLT-1、SGLT-2）发现的蛋白基因家族,负责吸收 和重吸收葡萄糖。在肾脏处,SGLT 蛋白,特别是SGLT-2 蛋白将肾小球滤过液中的绝大部分葡萄糖重新转运进入血液,从而维持 体内血糖的稳定与平衡。SGLT 蛋白抑制剂通过阻断该蛋白的转运机制,使葡萄糖随尿液排出从而降低血糖,为糖尿病的治疗提 供了创造性的思路。本文重点阐述了SGLT 蛋白和SGLT 抑制剂的作用机制,以及近年来SGLT-2 抑制剂的研发上市情况。     

Up-regulation of Na-coupled glucose transporter SGLT1 by caveolin-1

The Na⁺-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (I_g), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal I_g without significantly modifying the glucose concentration required to trigger half maximal I_g (K_M). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I_g, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.     

A specific pharmacophore model of sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors

Sodium-dependent glucose co-transporter 2 (SGLT2) plays a pivotal role in maintaining glucose equilibrium in the human body, emerging as one of the most promising targets for the treatment of diabetes mellitus type 2. Pharmacophore models of SGLT2 inhibitors have been generated with a training set of 25 SGLT2 inhibitors using Discovery Studio V2.1. The best hypothesis (Hypo1(SGLT2)) contains one hydrogen bond donor, five excluded volumes, one ring aromatic and three hydrophobic features, and has a correlation coefficient of 0.955, cost difference of 68.76, RMSD of 0.85. This model was validated by test set, Fischer randomization test and decoy set methods. The specificity of Hypo1(SGLT2) was evaluated. The pharmacophore features of Hypo1(SGLT2) were different from the best pharmacophore model (Hypo1(SGLT1)) of SGLT1 inhibitors we developed. Moreover, Hypo1(SGLT2) could effectively distinguish selective inhibitors of SGLT2 from those of SGLT1. These results indicate that a highly predictive and specific pharmacophore model of SGLT2 inhibitors has been successfully obtained. Then Hypo1(SGLT2) was used as a 3D query to screen databases including NCI and Maybridge for identifying new inhibitors of SGLT2. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. And several compounds selected from the top ranked hits have been suggested for further experimental assay studies.     

Synthesis and biological evaluation of 6-hydroxyl C-aryl glucoside derivatives as novel sodium glucose co-transporter 2 (SGLT2) inhibitors

The sodium glucose co-transporter 2 (SGLT2) was considered as an important target for the treatment of type 2 diabetes mellitus in recent years. This report describes the design and synthesis of a series of novel SGLT2 inhibitors (11a–17a) as well as their dehydrate dihydrofuran derivatives (11b–17b), which were prepared by Mitsunobu reaction. Their SGLT2 inhibitory activity was also evaluated, and 16a and 17a were found to be the most potent compounds with IC₅₀ values of 0.63 and 0.81?nM, respectively. However, all the dehydrate derivatives lose the SGLT2 inhibitory activity, with inhibition percentage no more than 66.5% at the concentration of 0.5?μM, which might because of the configuration inversion at C-2 of glucose. In conclusion, the present study improves understanding of the SAR of SGLT2 inhibitors, and provided more information that could be applied to design new molecules.     

Design and synthesis of fluorescent SGLT2 inhibitors

The design and synthesis of the first fluorophore-conjugated SGLT2 inhibitors is described. The mode of linking the fluorophore to the SGLT2 pharmacophore was found to be crucial in achieving optimum potency. Superior potency to phlorizin was provided by examples containing TAMRA, BODIPY, Cy3B and NBD fluorophores.     

Absorption of resveratrol by vascular endothelial cells through passive diffusion and an SGLT1-mediated pathway

Resveratrol is a natural polyphenol that exerts potent effects to suppress atherosclerosis. However, its low concentration in plasma has placed this role in doubt. Thus, resveratrol effects might be dependent on its transport into vascular endothelium, a question not previously addressed in spite of its obvious and fundamental importance. Via high-performance liquid chromatography and liquid chromatography/mass spectrometry, we found that resveratrol was absorbed by human umbilical vein endothelial cells in a temperature-, concentration- and time-dependent manner, suggesting the involvement of passive diffusion and active transport. As determined by confocal laser scanning microscopy, resveratrol primarily distributed throughout the cytoplasm. Furthermore, resveratrol absorption was modulated by serum proteins and sodium-dependent glucose transporter 1 (SGLT1) yet inhibited by glucose (an SGLT1 substrate) and phlorizin (an SGLT1 selective inhibitor), as well as SGLT1 siRNA transfection. Additionally, Sprague–Dawley rats were intragastrically administrated with 100 mg/kg of resveratrol and the concentration of resveratrol in blood vessels declined more slowly up to 24 h compared to that in the blood. Our results suggested that resveratrol uptake by vascular endothelial cells involved both passive diffusion and an SGLT1-mediated process, at least partially. Moreover, the intracellular resveratrol pool may be more important than the serum level in vivo. These provide new insights into the cardiovascular benefits of resveratrol.     

Expression of SGLT1 in Human Hearts and Impairment of Cardiac Glucose Uptake by Phlorizin during Ischemia-Reperfusion Injury in Mice

Objective

Sodium-glucose cotransporter 1 (SGLT1) is thought to be expressed in the heart as the dominant isoform of cardiac SGLT, although more information is required to delineate the subtypes of SGLTs in human hearts. Moreover, the functional role of SGLTs in the heart remains to be fully elucidated. We herein investigated whether SGLT1 is expressed in human hearts and whether SGLTs significantly contribute to cardiac energy metabolism during ischemia-reperfusion injury (IRI) via enhanced glucose utilization in mice.

Methods and Results

We determined that SGLT1 was highly expressed in both human autopsied hearts and murine perfused hearts, as assessed by immunostaining and immunoblotting with membrane fractionation. To test the functional significance of the substantial expression of SGLTs in the heart, we studied the effects of a non-selective SGLT inhibitor, phlorizin, on the baseline cardiac function and its response to ischemia-reperfusion using the murine Langendorff model. Although phlorizin perfusion did not affect baseline cardiac function, its administration during IRI significantly impaired the recovery in left ventricular contractions and rate pressure product, associated with an increased infarct size, as demonstrated by triphenyltetrazolium chloride staining and creatine phosphokinase activity released into the perfusate. The onset of ischemic contracture, which indicates the initiation of ATP depletion in myocardium, was earlier with phlorizin. Consistent with this finding, there was a significant decrease in the tissue ATP content associated with reductions in glucose uptake, as well as lactate output (indicating glycolytic flux), during ischemia-reperfusion in the phlorizin-perfused hearts.

Conclusions

Cardiac SGLTs, possibly SGLT1 in particular, appear to provide an important protective mechanism against IRI by replenishing ATP stores in ischemic cardiac tissues via enhancing availability of glucose. The present findings provide new insight into the significant role of SGLTs in optimizing cardiac energy metabolism, at least during the acute phase of IRI.     

Indole-glucosides as novel sodium glucose co-transporter 2 (SGLT2) inhibitors. Part 2

A series of indole-O-glucosides and C-glucosides was synthesized and evaluated in SGLT1 and SGLT2 cell-based functional assays. Compounds 2a and 2o were identified as potent SGLT2 inhibitors and screened in ZDF rats.     

Detection of myosin light chain phosphorylation--a cell-based assay for screening Rho-kinase inhibitors

Here, we describe the first example of a cell-based myosin light chain phosphorylation assay in 96-well format that allows for the rapid screening of novel Rho-kinase inhibitors. We obtained IC₅₀ values for the prototypic Rho-kinase inhibitors Y-27632 (1.2 ± 0.05 μM) and Fasudil (3.7 ± 1.2 μM) that were similar to those previously published utilizing electrophoresis-based methodologies. H-1152P, a Fasudil analog showed an IC₅₀ value of 77 ± 30 nM. Data derived from a set of 21 novel Rho-kinase inhibitors correlate with those generated by a well-established cell-based phenotypic Rho-kinase inhibition assay (R² = 0.744). These results show that imaging technology measuring changes in myosin light chain phosphorylation can be used to rapidly generate quantitative IC₅₀ values and to screen a larger set of small molecule Rho-kinase inhibitors and suggests that this approach can be broadly applied to other cell lines and signaling pathways.     

Human cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1)

We have quantitatively measured gene expression for the sodium-dependent glucose cotransporters 1 and 2 (SGLT1 and SGLT2) in 23 human tissues using the method of real time PCR. As predicted, our results revealed that the expression of SGLT1 was very high in the small intestine (1.2E + 6 molecules/microg total RNA) relative to that in the kidney (3E + 4 molecules/microg total RNA). Surprisingly, we observed that the expression of SGLT1 in human heart was unexpectedly high (3.4E + 5 molecules/microg total RNA), approximately 10-fold higher than that observed in kidney tissue. DNA sequencing confirmed that the PCR amplified fragment was indeed the human SGLT1 gene. Moreover, in situ hybridization studies using a digoxigenin (DIG)-labeled antisense cRNA probe corresponding to human SGLT1 cDNA confirm that human cardiomyocytes express SGLT1 mRNA. In contrast, the expression of SGLT2 in human tissues appears to be ubiquitous, with levels ranging from 6.7E + 4 molecules/microg total RNA (in skeletal muscle) to 3.2E + 6 molecules/microg total RNA (in kidney), levels 10-100-fold higher than the expression of SGLT1 in the same tissues. Our finding that human cardiomyocytes express high levels of SGLT1 RNA suggests that SGLT1 may have a functional role in cardiac glucose transport. Since several SGLT inhibitors are currently in development as potential anti-diabetic agents, it may be important to assess the functional consequences of inhibition of SGLT1 in the heart.     

Polarity of transport of 2-deoxy-D-glucose and D-glucose by cultured renal epithelia (LLC-PK1).

At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence.     

Regulation of Na(+)-coupled glucose carrier SGLT1 by human papillomavirus 18 E6 protein

Tumor cells utilize preferably glucose for energy production. They accomplish cellular glucose uptake in part through Na⁺-coupled glucose transport mediated by SGLT1 (SLC5A1). This study explored the possibility that the human papillomavirus 18 E6 protein HPV18 E6 (E6) participates in the stimulation of SGLT1 activity. E6 is one of the two major oncoproteins of high-risk human papillomaviruses, which are the causative agent for cervical carcinoma. According to Western blotting, SGLT1 is expressed in the HPV18-positive cervical carcinoma cell line HeLa. To explore whether E6 affects SGLT1 activity, SGLT1 was expressed in Xenopus oocytes with and without E6 and electrogenic glucose transport determined by dual electrode voltage clamp. In SGLT1-expressing oocytes, but not in oocytes injected with water or expressing E6 alone, glucose triggered a current (I_g). I_g was significantly increased by coexpression of E6 but not by coexpression of E2. According to chemiluminescence and confocal microscopy, coexpression of E6 significantly increased the SGLT1 protein abundance in the cell membrane. The decay of I_g following inhibition of carrier insertion by Brefeldine A (5 μM) was not significantly affected E6 coexpression. Accrodingly, E6 was not effective by increasing carrier protein stability in the membrane. In conclusion, HPV18 E6 oncoprotein participates in the upregulation of SGLT1.     

Unexpected similarity of intestinal sugar absorption by SGLT1 and apical GLUT2 in an insect (Aphidius ervi, Hymenoptera) and mammals

Sugars are critical substrates for insect metabolism, but little is known about the transporters and epithelial routes that ensure their constant supply from dietary resources. We have characterized glucose and fructose uptakes across the apical and basolateral membranes of the isolated larval midgut of the aphid parasitoid Aphidius ervi. The uptake of radiolabeled glucose at the basal side of the epithelium was almost suppressed by 200 microM cytochalasin B, uninhibited by phlorizin, and showed the following decreasing rank of specificity for the tested substrates: glucose > glucosamine > fructose, with no recognition of galactose. These functional properties well agree with the expression of GLUT2-like transporters in this membrane. When the apical surface of the epithelium was also exposed to the labeled medium, a cation-dependent glucose uptake, inhibited by 10 microM phlorizin and by an excess of galactose, was detected suggesting the presence in the apical membrane of a cation-dependent cotransporter. Radiolabeled fructose uptakes were only partially inhibited by cytochalasin B. SGLT1-like and GLUT5-like transporters were detected in the apical membranes of the epithelial cell by immunocytochemical experiments. These results, along with the presence of GLUT2-like transporters both in the apical and basolateral cell membranes of the midgut, as we recently demonstrated, allow us to conclude that the model for sugar transepithelial transport in A. ervi midgut appears to be unexpectedly similar to that recently proposed for sugar intestinal absorption in mammals.     

Regulation of Na+-coupled glucose carrier SGLT1 by AMP-activated protein kinase

Abstract

AMP-activated protein kinase (AMPK), a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilization. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na⁺-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active ^γR70QAMPK (α1β1γ1(R70Q)) alone, the addition of glucose to the extracellular bath generated a current (I_g), which was half maximal (K_M) at ≈ 650 μM glucose concentration. Coexpression of ^γR70QAMPK did not affect K_M but significantly enhanced the maximal current (≈ 1.7 fold). Coexpression of wild type AMPK or the kinase dead ^αK45RAMPK mutant (α1(K45R)β1γ1) did not appreciably affect I_g. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 μM) enhanced the SGLT1 protein abundance in the cell membrane of Caco2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na⁺-coupled glucose transport.