The research group of Chen Lei of the School of Future Technology revealed the inhibition mechanism of the human glucose transporter SGLT1

Sodium-glucose co-transporter (SGLT) is an important transporter responsible for glucose reabsorption in the human body, enabling the reverse concentration gradient transport of glucose using the electrochemical potential of sodium ^ions1
.
In the SGLT protein family in the human body, SGLT1 and SGLT2 are the two proteins that are most critical
for glucose absorption and homeostasis.
Among them, SGLT1 is encoded by the SLC5A1 gene, is mainly expressed in the small intestine and kidney proximal tubular S3 ^segment4, responsible for the reabsorption of glucose from food sources in the intestine and residual glucose in the original urine, and also has the function of water ^channels5
.
Inactivating mutations in SGLT1 can lead to intestinal glucose-galactose malabsorption1; SGLT2 is encoded by the SLC5A2 gene and is mainly expressed in the S1 and S2 segments of the renal proximal convoluted tubule, responsible for 90% glucose reabsorption in the original urine2, and inactivating mutations of SGLT2 lead to familial nephrouria ^sugar2,3
.

As an important target for the treatment of type II diabetes, the idea of using SGLT2 inhibitors to treat diabetes is to inhibit the sugar transport function of SGLT2, so that SGLT2 cannot reabsorb glucose from the original urine, resulting in excess glucose excreted from the urine to achieve the purpose of
indirectly reducing blood sugar.
Many SGLT2 inhibitors such as Empagliflozin, Canagliflozin, Dapagliflozin ⁶ and other small molecule drugs have been developed for the clinical treatment of type II diabetes
.
In addition, recent clinical trials suggest that simultaneous inhibition of SGLT1 and SGLT2 may be superior to SGLT2 inhibitors alone in the treatment of type II ^diabetes7, and that inhibitors targeting SGLT1 may also be effective in treating constipation8
.

SGLT inhibitors are optimized based on the structure of the natural product phlorizin, and these inhibitors can be divided into three classes according to their selectivity: SGLT2-specific inhibitors, such as empagliflozin, canagliflozin, dapagliflozin, etc.
; SGLT1-specific inhibitors, such as KGA-2727, mizagliflozin, and SGLT1-SGLT2 non-selective inhibitors, such as LX2761, sotagliflozin, etc
.
Therefore, understanding how these drugs inhibit the function of SGLT is important for drug development and optimization
.

In December 2021, the research group of Chen Lei, Institute of Molecular Medicine, School of Future Technology, Peking University and Peking University-Tsinghua Joint Center for Life Sciences, first reported in Nature the structure of the binding of the human-derived SGLT2-MAP17 complex to the inhibitor empagliflozin9^, while the research group of Feng Liang of Stanford University in the United States also published the structure of the inward opening of the ligand-free binding state of human-derived SGLT1 in the same journal ¹⁰
。 However, the mode of binding of SGLT1 to inhibitors, and the selective mechanisms of certain SGLT1-specific inhibitors
, remain unknown.

On October 28, 2022, Chen Lei's research group published a research paper entitled "Structural mechanism of SGLT1 inhibitors" in the journal Nature Communications, which analyzed the high-resolution structure
of human SGLT1-MAP17 complex bound to inhibitor LX2761 by single-particle cryo-EM.

Figure 1.
Electron density map of SGLT1-MAP17 interaction and hSGLT1-MAP17 complex

The molecular weight of SGLT1 and SGLT2 are similar, and the sequence similarity is high, so it is challenging
to interpret the structure of SGLT1 protein alone.
The authors found that the amino acids that interacted with MAP17 on TM13 of SGLT1 and SGLT2 were highly consistent, speculating that SGLT1 may also form complexes
with MAP17.
Subsequently, this conjecture was confirmed
experimentally.
On this basis, the authors used the "three-linker strategy" invented in the analysis of SGLT2 structure to analyze the structure of SGLT1: the authors fused GFP in the intracellular region of SGLT1 and GFP nanobodies at the intracellular end of MAP171 to obtain proteins with functions and suitable for cryo-EM research.

The authors analyzed the high-resolution structure of the binding state of the human SGLT1-MAP17 complex to the inhibitor LX2761 by cryo-EM single-particle analysis (Figure 1).

Through structural comparison, it is found that the structure of SGLT1 and SGLT2 is very similar, and they have similar structural characteristics and topological characteristics
.
Unlike SGLT1 in a ligand-free binding state, inhibitor LX2761 locks SGLT1 in an outwardly open conformation (Figure 2).

The glycan head of LX2761 binds to the substrate binding site of SGLT1, and through hydrophobic interaction with the amino acid of the substrate binding site, the long-chain tail extends outward, blocking the channel entrance
of the extracellular region.
The authors mutated L274 and D454 of SGLT1 to alanine, respectively, and found that the mutated SGLT2 reduced binding to inhibitors and weakened the inhibitor's inhibitor inhibition
.

Figure 2.
Binding site of inhibitor LX2761

Figure 3.
There is steric hindrance between the inhibitor mizagliflozin and V157 of SGLT2

To explain the selective mechanism of the SGLT1-specific inhibitor mizagliflozin, the authors compared the inhibitor-binding pockets of SGLT1 and SGLT2 and found that the binding pockets in SGLT1 were slightly larger than SGLT2, and this difference was derived from the difference in amino acids at position 160 of SGLT1: alanine at position 160 of SGLT1 corresponds to valine
at position 157 of SGLT2 。 The authors simulated the binding mode of mizagliflozin to SGLT1 through molecular dynamics simulations, and compared the simulation results with the structure of SGLT2
.
It was found that as the central phenylcyclic group of LX2761 was replaced by the isopropylpyrazole group of mizagliflozin, a spatial conflict was formed at the V157 amino acid of SGLT2, which may have led to a decrease
in the potency of mizagliflozin against SGLT2 。 Therefore, the authors constructed A160V of SGLT1 and V157A mutants of SGLT2, respectively, and found that mutating A to V in SGLT1 reduced the potency of mizagliflozin, while mutating V to A in SGLT2 increased the potency of mizagliflozin (Figure 3).

Therefore, the authors identified A160 of SGLT1 as one of the main sites affecting the selectivity of
Mizagliflozin.

At the same time, since SGLT1 can also function as a water channel, the authors also calculated the water channel structure of SGLT1 in the inhibited state, and found that in the state inhibited by the inhibitor, the water channel in SGLT1 was also blocked by LX2761, and its channel size could not pass through the water molecules
.

Combined with the previously published structure of SGLT1 in the ligand-free binding state, the authors also compared the inward-opening SGLT1 with the outward-open SGLT1 structure, depicting the conformational changes
of SGLT1 between the open and closed states 。 Among them, the author found that the position of some spirals did not change at all, namely TM1, 2, 6, 7, 11, 12, 13, and the author named these spirals constant modules; Other spirals undergo larger conformational changes, including TM0, 3, 4, 5, 8, 9, 10, which are named moving modules
.
Among them, the movement of F453 on TM10 mediates the opening of extracellular gating, and Y290 on TM3 mediates the opening of intracellular gating (Figure 4).

Figure 4.
Conformational changes of SGLT1 in gated areas

In summary, the authors analyzed the structure of SGLT1-MAP17 complex and inhibitor by cryo-EM, found that the inhibitor locked SGLT1 in an open state, determined the binding site and binding mode of SGLT1 inhibitor, explained the mechanism of SGLT1 selective inhibitor to a certain extent, and provided a structural basis
for further optimization of SGLT1 and SGLT2-specific inhibitors.

Yange Niu and Wenhao Cui, doctoral students of the Institute of Molecular Medicine of the School of Future Technology, are the co-first authors of the paper, doctoral student Rui Liu participated in the data collection work, and Chen Lei is the corresponding author
of the paper.
The inhibitor synthesis and molecular dynamics simulation part of this work was completed by Professor Xiaoguang Lei and his doctoral students Sanshan Wang and Han Ke, School of Chemistry and Molecular Engineering
, Peking University.
The preparation, screening and collection of cryo-EM samples was completed on the cryo-EM platform of Peking University, with the help
of Li Xuemei, Guo Zhenxi, Qin Changdong, Pei Xia and Wang Guopeng.
The data processing of this project has obtained hardware and technical support
from the CLS computing platform of Peking University and the unnamed supercomputing platform.
This project is supported
by the Ministry of Science and Technology and the National Natural Science Foundation of China.

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