CN108383801B - Inhibitors of SGLT2 proteins and uses thereof - Google Patents

Inhibitors of SGLT2 proteins and uses thereof Download PDF

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CN108383801B
CN108383801B CN201810237106.1A CN201810237106A CN108383801B CN 108383801 B CN108383801 B CN 108383801B CN 201810237106 A CN201810237106 A CN 201810237106A CN 108383801 B CN108383801 B CN 108383801B
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于磊
徐巧枝
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Abstract

An inhibitor of SGLT2 protein, application of the compound in preparing medicine for treating diabetes, and screening method of the inhibitor. Wherein the inhibitor comprises a compound having a structure represented by formula (1):
Figure DDA0001604268930000011
the inhibitor can be used as active lead for treating diabetes.

Description

Inhibitors of SGLT2 proteins and uses thereof
Technical Field
The invention relates to the field of diabetes treatment, and further relates to SGLT2 (Na + Glucose cotransporter, omim: *182381, also called SLC5A 2) protein inhibitors, to the use of a compound for the treatment of diabetes, and to a method for screening SGLT2 protein inhibitors.
Background
Kidneys play an important role in regulating blood glucose levels, where there are two classes of glucose transporters, one of which is sodium-dependent glucose transporter (na+ -glucose cotransporter, SGLT), which consumes energy to transport glucose against concentration gradients in an active manner. Wells et al found a transporter called SGLT2 (OMIM:. 182381, also SLC5A 2) from a cDNA library of human kidneys.
Familial renal diabetes (Familial renal Glucosuria, FRG) is a disease with reduced proximal tubular glucose reabsorption function with normal fasting glucose and glucose tolerance tests, with persistent urine glucose as a major feature, and patients mostly have good prognosis without abnormal tubular function and structure. Expression studies in kidney tissues of patients with familial renal diabetes have been conducted in the prior art, and it has been found that both wild-type and mutant SGLT2 proteins are expressed in tubular epithelial cells, wherein wild-type SGLT2 is normally expressed, and the SGLT2 mutant proteins have abnormal distribution and expression on cell membranes, thereby redefining that familial renal diabetes should be defined as co-dominant inheritance, and incomplete exogenous considerations previously thought to be related to compensation of SGLT2 wild-type proteins.
Recent studies have found that the expression and function of SGLT2 in diabetic patients are significantly up-regulated, and familial renal diabetes patients have a good prognosis, so that specific blocking of proximal tubular SGLT2 transporter and further inhibition of glucose reabsorption to control blood glucose levels are a hotspot in current studies. Phlorezin (phlorezin) is the first SGLT inhibitor isolated from apple bark by french chemists of 1835, and has been found in many subsequent diabetic animal studies to lower blood glucose and restore insulin sensitivity, but its metabolites may inhibit GLUT1 and other protein functions due to low oral bioavailability and low specificity, and has some side effects in the central nervous system and other systems, so it has not been applied to clinical treatment of diabetes.
Disclosure of Invention
First, the technical problem to be solved
Accordingly, the present invention is directed to an inhibitor of SGLT2 protein and application of the compound in preparing a medicament for treating diabetes, and a screening method of SGLT2 protein inhibitor, so as to solve at least one of the above technical problems.
(II) technical scheme
According to an aspect of the present invention, there is provided an inhibitor of SGLT2 protein comprising a compound having a structure represented by formula (1):
Figure BDA0001604268910000021
(1),
wherein a is independently:
Figure BDA0001604268910000022
b is independently:
Figure BDA0001604268910000031
according to a further aspect of the present invention there is provided the use of a compound in the manufacture of a medicament for the treatment of diabetes, wherein the compound has the structure of formula (1) above:
according to still another aspect of the present invention, there is provided a screening method of an SGLT2 protein inhibitor, comprising:
setting docking conditions of SGLT2 protein and compound;
virtual screening is carried out in a database according to the docking conditions;
further screening and scoring the virtual screening result according to the drug-like principle to obtain a compound with a set score or more as a lead compound for treating diabetes;
wherein, setting the docking condition of the SGLT2 protein and the compound comprises:
the effect of the compound and SGLT2 is set to be maintained through strong interaction of two parts, on one hand, a sugar part with relatively strong hydrophilicity forms a relatively stable hydrogen bond with an amino acid residue at the outward part of a pocket; on the other hand, the hydrophobic interaction and pi-pi interaction of the small molecule aromatic ring part and the hydrophobic pocket deeper in the pocket; the logarithmic log P of the lipid water distribution coefficient of the compound is-2-5, the molecular weight is 200-500, the hydrogen bond donor is 0-5, the hydrogen bond acceptor is 0-10, the number of rotatable bonds is 0-10, the number of heavy atoms is 20-70, and the charge value is-2.
(III) beneficial effects
Through the scheme, the beneficial effects of the invention are as follows:
the compound in the SGLT2 protein inhibitor can be used as an active lead compound for treating diabetes.
The compound in the SGLT2 protein inhibitor comprises a hydrophilic group and a hydrophobic pocket, and interacts with a polar pocket and a water pocket of an SGLT2 protein active site to inhibit the expression of the SGLT2 protein.
The compound of the invention can be applied to preparing diabetes medicines.
Drawings
FIG. 1 is a compound of an inhibitor of SGLT2 protein of example of the present invention.
FIG. 2 is a schematic diagram showing the interaction of the inhibitor compounds of the examples of the present invention with SGLT2 protein.
FIG. 3 is a graph showing the mutation sites found in patients with familial renal diabetes according to an embodiment of the present invention.
FIG. 4 is a graph showing the structure-activity relationship between the compound of the present invention and SGLT2 protein.
Fig. 5 is a standard curve in embodiment 1 of the present invention.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
Since prior art studies on SGLT2 expression and function only found 2 mechanisms, the inventors of the present application found that the remaining 4 mechanisms all play an important role in the onset of familial renal diabetes and define the relevant sites. The inventors further found that c.244 c > a/p. (F98L), c.356 c > T/p. (P246S) were ideal target sites for screening for diabetes drugs (SGLT 2 protein inhibitors). Because the exact 3D structure of SGLT2 is still not clear, drug development is looking for derivatives or analogues of phlorizin, which may also affect other glucose transporters to different extents while inhibiting the SGLT2 protein function, and thus, adverse effects are generated, and certain difficulties are caused in the drug development process.
According to an aspect of embodiments of the present invention, there is provided an inhibitor of SGLT2 protein comprising the following compounds:
Figure BDA0001604268910000051
formula (1), wherein a is independently:
Figure BDA0001604268910000052
b is independently:
Figure BDA0001604268910000053
Figure BDA0001604268910000061
the compound of formula (1) shown in fig. 1 is a three-molecule docking structure, and referring to fig. 1, it includes hydrophilic groups and hydrophobic groups (marked in the dashed box) on the left and right sides, and a middle linking moiety (middle dashed box), (SGLT 2 as a target protein, shown in fig. 2, has a distinct polar pocket (e.g., residue Ser460 in fig. 2) and hydrophobic pocket (e.g., trp 289) at the active site.
In fig. 2, these compounds bind to SGLT2 in three main ways: 1. the hydrophilic group of the compound and the residues such as Ser460 form hydrogen bond interaction; 2. the linking moiety of the hydrophilic group and the hydrophobic group stabilizes the binding of the compound by hydrogen bonding of residue Lys 154; 3. the hydrophobic group of the compound enters a hydrophobic pocket to form a hydrophobic effect; particularly when the hydrophobic group is an aromatic ring, a strong pi-pi bond is formed with the Trp289 residue, and furthermore the L286V mutation allows a small molecule benzyl moiety (the structure within the circle in fig. 4) to have sufficient spatial position to interact with the protein.
The inhibitor can specifically inhibit the functions of SGLT2 protein, effectively reduce fasting and postprandial blood sugar, and becomes a novel way for treating diabetes.
The inhibitor can be made into tablet or other medicine by using powder as raw material.
In a further embodiment, the A's may be independently
Figure BDA0001604268910000062
Figure BDA0001604268910000063
Preferably, A can be independently
Figure BDA0001604268910000071
More preferred A may independently be +.>
Figure BDA0001604268910000072
In some embodiments, the B is independently
Figure BDA0001604268910000073
Figure BDA0001604268910000074
Figure BDA0001604268910000075
Preferably, B is independently->
Figure BDA0001604268910000076
Figure BDA0001604268910000077
/>
Figure BDA0001604268910000078
More preferably B is independently->
Figure BDA0001604268910000079
The embodiment of the invention also provides application of the compound in preparing a medicament for treating diabetes, wherein the compound has a structure shown in the formula (1).
According to another aspect of the embodiment of the present invention, there is provided a screening method for an SGLT2 protein inhibitor, including:
setting docking conditions of SGLT2 protein and compound;
virtual screening is carried out in a database according to the docking conditions;
further screening and scoring the virtual screening result according to the principle of drug-like properties to obtain a high-score compound serving as a lead compound for treating diabetes.
In the embodiment of the invention, based on SGLT2 gene mutation found in patients with familial renal diabetes in inner Mongolia, further intensive research on SGLT2 from gene mutation to protein expression and function is carried out, clinical and basic research evidence and theoretical basis are provided for revealing key roles and action mechanisms of SGLT2 in the pathogenesis of familial renal diabetes (see figure 3), partial screening results are eliminated by virtual screening according to the principle of drug-like properties, and finally small molecular compounds are obtained. Through the interaction relation between the small molecules and the receptor protein, the analysis and selection of the small molecules can be used for later biological experiment verification. Further through cell and animal level verification, a significant target and important clues and basis are provided for designing and screening diabetes therapeutic drugs and developing new drugs.
The screening process comprises the following steps: 1. drug virtual screening: and determining a target spot of drug screening by using the result of the functional study, and further screening the drugs for treating diabetes. Based on the result of the SGLT2 protein function inhibition experiment performed by the known antagonist phlorizin and its derivatives or analogues, a quantitative structure-activity relation model is established, and a three-dimensional pharmacophore model searching method is adopted to perform virtual screening in a compound database (commercial small molecule compound libraries ACD-SC, MDDR and Chinese natural product database CNPD) so as to find active lead compounds. And further screening and scoring are carried out according to the principle of drug properties, and finally, a plurality of high-differentiation compounds are obtained and used as lead compounds for treating diabetes, thereby providing new clues and basis for drug research and development. 2. Virtual screening scheme: by the flexible docking test of proteins with three drug molecules, we can initially determine: the effect of the finished drug small molecule and the protein is likely to be maintained through the stronger interaction of two parts, on the one hand, the sugar part with stronger hydrophilicity forms a more stable hydrogen bond with the amino acid residue at the outward part of the pocket; on the other hand, the hydrophobic interaction and pi-pi interaction of the small molecule aromatic ring part and the hydrophobic pocket deeper in the pocket. And (3) performing virtual screening according to the set docking conditions, and screening by using a virtual screening platform of a Beijing city computing center, wherein the selected small molecule library is a patent medicine database and a providable database C. 3. The pharmacophore screening process specifically comprises: selection of small molecules: the a/small molecules have the commonality of more than or equal to 4 drug molecules, and the established pharmacophore can screen out 5 small molecules in a full mode; b/small molecules distinguish recognition sites specifically for SGLT2 and SGLT 1. The pharmacophore is established and tested, and 5 medicines can be screened out; and setting a decoy test group containing 5 small molecules, and testing and screening out all 5 medicines to be optimal.
Pharmacophore screening the whole drug library on the market and the docking scoring top30% virtual screening work of the available database c.
The small molecules with higher pharmacophore scoring are selected, and the small molecules of the embodiment of the invention are selected according to the specificity of the combination of the visualized small molecules and amino acid residues.
FIG. 4 is a graph showing the structure-activity relationship between the compound of the present invention and SGLT2 protein. The existing three drug molecules are spliced into SGLT2 protein, and the three small molecules can be found to have a certain rule in the splicing result. As shown in FIG. 4, the glycosyl portions of three small molecules overlap from the intramembrane region of the protein to the deep side of the protein, and the small molecules have multiple hydrophobic functional groups in the hydrophobic region deeper in the pocket. The proportionality model shows that SGLT2 and SGLT1 have mutated amino acid residues at pocket positions, preliminary analysis shows that three drugs may bind specifically to SGLT2 pocket because small molecules are more prone to hydrogen bond interactions with hydrophobic pocket, and that L286V mutation allows sufficient spatial position of the benzyl moiety of small molecule to interact with protein (see circled portion of fig. 4).
Specific example 1 will be listed below, and the effect of the compounds of the examples of the present invention is verified at the cellular level, and a lead compound for diabetes treatment is found as follows:
cell level validation:
1. experimental instrument and reagent
Figure BDA0001604268910000091
2. Experimental method
(1) Cell preparation
1. Cells were digested with 0.25% trypsin to prepare a cell suspension, and the cell density was adjusted to 2×10 5 And each ml.
2. Cell suspensions (500. Mu.L/well) were seeded in 24-well plates and the plates were placed in an incubator (37 ℃,5% CO 2) overnight.
3. The transfection plasmid (SGLT-2 or GFP) was selectively added and the transfection plasmid cells were formed by the transfection reagent P-transter.
4. The culture plate was placed in an incubator for cultivation (cultivation environment 37 ℃,5% CO) 2 )48h。
(2) Drug treatment
After 1.48 h, the culture was changed to serum-free culture based on culture in an incubator (culture environment: 37 ℃,5% CO 2) overnight.
2. The culture medium is replaced by serum-free sugar-free culture medium, and the corresponding inhibitor can be selectively added, and the culture is carried out in an incubator (culture environment: 37 ℃ C., 5% CO 2) for 40min. The inhibitor has a structure represented by formula (1):
Figure BDA0001604268910000101
formula (1), A is->
Figure BDA0001604268910000102
B is->
Figure BDA0001604268910000103
The inhibitor is labeled T.
(3) 2-DG (2-deoxyglucose)
1. Mu.l 2-DG was added to each well. Incubate (conditions: 37 ℃,5% CO 2) in incubator for 20min.
2. The medium was removed and washed 3 times with pre-chilled PBS.
(4) 2-DG is added
1. Preparing a lysis buffer: 1% Triton X-100 was added to NADP Extraction buffer (extraction buffer). 220 μl was prepared per well.
2. Standard curve:
a. preparation of 5. Mu.M 2-DG6P (2-deoxyglucose 6-phosphate): mu.l of 5mM Standard (Standard) was added to 4995. Mu.l of distilled water and mixed well.
b. The dilution method for each standard sample is shown in Table 1 below
TABLE 1
Sequence number Premix+H 2 O 2-DG6P(μM)
1 100μl+0μl 5.0
2 60μl+40μl 3.0
3 30μl+70μl 1.5
4 0μl+100μl 0
3. 200 μl lysis buffer was added to the wells of the 24-well plate and shaken for 5min.
4. Mu.l of the cell lysate sample and 50. Mu.l of the standard sample were each aspirated into a 1.5ml EP tube at 80℃for 10min.
5. Add 50. Mu. l NADPH extraction buffer (extraction buffer) to the cell sample tube and add 50. Mu. l H 2 O was placed in a standard sample tube at-20℃for 5min.
6. Preparing WR1: mu.l Assay buffer, 1. Mu. l G6PDH (glucose 6-phosphate dehydrogenase) Enzyme, 1. Mu.l NADP were added. Each tube was prepared at 12. Mu.l. Mu.l of WR1 was added to each tube at 37℃for 60min.
7. 50 mu l NADPH extraction buffer was added to each tube at 80℃for 15min.
8. 50 mu l NADP extraction buffer-20 ℃ and 5min are added to each tube.
9. Mu.l of each of the cell sample and the standard sample was pipetted into a well of a black 96-well plate.
10. Preparing WR2: mu.l Assay buffer, 1. Mu.l Enzyme A, 1. Mu.l Enzyme B, 10. Mu. l G6P (glucose 6-phosphate) Reagent, 5. Mu.l Probe were added.
11. Mu.l of WR2 was added to each well and absorbance was measured at 530/585 for 0min and 20min, respectively.
3. Experimental grouping
The experimental grouping is shown in table 2 below, with three duplicate wells per group.
TABLE 2
Group of Details of the Drug concentration
C1 Transfecting plasmid (GFP) cells
C2 Transfecting plasmid (SGLT-2) cells
C3 Normal cells
C4 Transfection plasmid (SGLT-2) cells+solvent
T Transfection plasmid (SGLT-2) cell + inhibitor T 1mM
4. Experimental results and analysis
1. Standard curve
TABLE 3 Table 3
0(μM) 1.5(μM) 3(μM) 5(μM)
0min 13286 18528 20563 24000
20min 14764 24054 31384 42060
20-0min 1478 5526 10821 18060
The above table shows the results of absorbance values, which can be fitted to the linear standard curve shown in fig. 5.
2. Glucose uptake data analysis
TABLE 4 Tukey multiple comparison test
Figure BDA0001604268910000131
The Tukey multiple comparison and verification shown in Table 4 is carried out on the glucose uptake amounts of groups C1-C4 and T, so that the glucose uptake amount of the group T is higher than that of the group C1-C4, and the compound has a better inhibition effect and can be used as a lead medicament for treating diabetes.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims (1)

1. Use of a compound for inhibiting expression of SGLT2 protein in the manufacture of a medicament for treating diabetes, wherein the compound has a structure represented by formula (1):
Figure FFW0000024566550000011
wherein a is independently:
Figure FFW0000024566550000012
b is independently:
Figure FFW0000024566550000013
/>
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