Biochimie. 2015 Aug;115:187-93. doi: 10.1016/j.biochi.2015.06.003.

Identification of phlorizin binding domains in sodium-glucose cotransporter family: SGLT1 as a unique model system.

Raja M1, Kinne RK2.
  • 1Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany; Department of Molecular Structure and Function, The Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada. Electronic address:
  • 2Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany. Electronic address:



The sodium glucose cotransporter SGLT1 expressed mainly in the intestine and kidney has been explored extensively for understanding the mechanism of sugar cotransport and its inhibition by a classical competitive inhibitor, phlorizin (Pz). It has been shown that inhibition of SGLT1 by Pz involves its interaction followed by major conformational changes in the Pz binding domain (PBD) in C-terminal loop 13. However, the mechanism of Pz inhibition and its interaction with other members of SGLT is not known. In this hypothesis, we performed molecular modeling of SGLT1-loop 13 with Pz and carried out primary sequence analyses and secondary structure predictions to determine qualitatively similar PBDs in C-termini of human SGLT2-4, except for vSGLT, which contains an unstructured short C-terminus. The ranking of predictions of Pz interaction strongly agrees with the following ranking of previously reported Pz inhibition: SGLT2>SGLT1>SGLT4>SGLT3>>vSGLT. In addition, the sugar binding residues were found to be quite conserved among all SGLT members investigated here. Based on these preliminary analyses, we propose that other Pz-sensitive SGLTs are also inhibited via mechanism similar to SGLT1 where an aglucone of Pz, phloretin, interacts with PBD and glucoside moiety with sugar binding residues. Our hypothesis sets the stage for future analyses on investigation of Pz interaction with SGLT family and further suggests that Pz modeling may be explored to design novel inhibitors targeting several SGLT members.

KEYWORDS: C-terminal loop 13; Modeling; Phlorizin binding domain; SGLT inhibition; SGLT2 inhibitors; Sodium glucose cotransporter SGLT1

PMID: 26086341



Inhibition of renal glucose reabsorption by sodium glucose cotransport (SGLT) inhibitors is an attractive strategy in the management of hyperglycemia or diabetes. A competitive SGLT inhibitor, phlorizin (Pz), has been well known for its crucial roles in renal physiology and diabetes [1]. Therefore, determining structure-function relationship of SGLTs has become an utter prerequisite for designing new drugs targeting these cotransporters in disease states. Although, the crystal structure of bacterial vibrio parahaemolyticus Na+/galactose (vSGLT) cotransporter provides useful insights into secondary structural model for human SGLT1 [2], glucose binding pocket and sugar transport mechanism it fails to exhibit a proper Pz binding domain (PBD) due to lack of an extended C-terminus, which is usually found in human SGLTs. We initially designed experiments to explore the PBD in a fragment of C-terminus (known as loop 13) of SGLT1 [3]. It has been well established that SGLT1-loop 13 is critically involved in recognition of several substrates as well as cotransport inhibitors, like Pz or phloretin (Fig. 1A) [3-5]. Because Pz exhibits poor absorption in the gastro┬şintestinal tract, pharmacologically feasible analogs of Pz, like dapagliflozin and canagliflozin (Fig. 1A), have been developed that exhibit even higher selectivity for both SGLT1 and 2 in the kidney [6]. Besides, there are other SGLTs in other organs with different functions. However, the specificity of inhibitors for these SGLTs is highly desirable. Yet, the binding pockets for Pz or its high affinity analogs need to be discovered in SGLT2 and other SGLTs that also transport mannose or fructose as substrates, in addition to glucose.


Figure 1-Supp

Fig. 1. (A) Chemical structures of SGLT competitive inhibitors, Pz, phloretin, high affinity Pz derivatives (SGLT2 inhibitors). (B) Modeling of interaction between an aglucone of Pz (in grey) via rings A and B. A glucoside moiety is pointing towards the sugar binding site.


To get insight into a precise protein-drug interaction mechanism, we first performed modeling of interaction of Pz in loop 13 of SGLT1 by utilizing vSGLT structure as template (Fig. 1B). We have shown in a number of previous studies that an aglucone of Pz (ring A) interacts with the amino acid region between L606 and D611, which apparently is located in between the two helices H1 and H2. Another part of aglucone (ring B) also interacts within the same region at/around R602 [3], while the glucoside points away from the aglucone binding pocket and interacts with sugar binding residues. This interaction leads to a condensed form of loop 13, similar to intact human SGLT1 [7,8], which is nicely reflected by biophysical/biochemical techniques and computer-modeling program [3]. Most interestingly, the presence of structural elements (30-40% ╬▒-helical residues) in loop 13 [3] strongly indicates a prerequisite of proper interaction of Pz within its binding pocket.

Considering the above-mentioned characteristics of Pz-binding pocket or PBD in SGLT1, we sought to seek residues and putative structural elements in C-termini of other Pz-sensitive SGLT members. We predicted an increased hydrophobicity (helices H1, 2 and 3) of SGLT2-PBD that might cause even higher specificity for Pz or its analogs as compared to SGLT1 (Fig. 2A). Hence, an additional binding between Pz-B ring and helix H3 can be considered. This assumption agrees well with the high affinity of dapagliflozin [9], which could bind stronger with SGLT2-PBD via its extended CH2-CH3 chain of the B ring (Fig. 1A).


Figure 2-Supp

Fig. 2. (A) Prediction of secondary structures in C-termini/loop 13s of SGLT1-4. Pz-binding residues are highlighted by red dotted arrow and helices H1, H2 or H3 are shown as graphical representation in grey. (B) Schematic representation of Pz interaction in loop 13 of SGLT members predicting qualitatively similar folding patterns of PBDs of SGLT1-4, except for vSGLT, which lacks secondary structures and a defined Pz-pocket. The gradient filled arrow (in grey) represents the magnitude of Pz interaction/inhibition in SGLT members.


Conclusions: Due to challenges in resolving three-dimensional structures of human SGLTs, the precise locations of inhibitor binding pocket(s) have not been identified. We therefore predicted critical residues and domains that might play major roles in determining the inhibition of SGLTs by Pz or its high affinity analogs. Similar to the mechanism of Pz interaction in SGLT1, other SGLTs also exhibit a proper Pz-binding region where the length of C-terminus/loop 13, an aglucone binding residues, sequence motif, and presence of helical contents determine the affinity/specificity of an entire inhibitor. Our modeling studies represent a major step towards designing specific drugs that could effectively modulate the regulatory system of a particular SGLT in a disease state.



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