Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common among children. AML is characterized by aberrant proliferation of myeloid blasts in the bone marrow and impaired normal hematopoiesis. Despite the introduction of new drugs and allogeneic bone marrow transplantation, patients have poor overall survival rate with relapse as the major challenge, driving the demand for new therapeutic strategies. AML patients with high expression of the very long/long chain fatty acid transporter CD36 have poorer survival and very long chain fatty acid metabolism is critical for AML cell survival. Here we show that fatty acids are transferred from human primary adipocytes to AML cells upon co-culturing. A drug-like small molecule (SMS121) was identified by receptor-based virtual screening and experimentally demonstrated to target the lipid uptake protein CD36. SMS121 reduced the uptake of fatty acid into AML cells that could be reversed by addition of free fatty acids and caused decreased cell viability. The data presented here serves as a framework for the development of CD36 inhibitors to be used as future therapeutics against AML.

Cancer invasion and metastasis are known to be potentiated by the expression of aquaporins (AQPs). Likewise, the expression levels of AQPs have been shown to be prognostic for survival in patients and have a role in tumor growth, edema, angiogenesis, and tumor cell migration. Thus, AQPs are key players in cancer biology and potential targets for drug development. Here, we present the single-particle cryo-EM structure of human AQP7 at 3.2-Å resolution in complex with the specific inhibitor compound Z433927330. The structure in combination with MD simulations shows that the inhibitor binds to the endofacial side of AQP7. In addition, cancer cells treated with Z433927330 show reduced proliferation. The data presented here serve as a framework for the development of AQP inhibitors.

Glucose transporters (GLUTs) are responsible for transporting hexose molecules across cellular membranes. In adipocytes, insulin stimulates glucose uptake by redistributing GLUT4 to the plasma membrane. In unstimulated adipose‐like mouse cell lines, GLUT4 is known to be retained intracellularly by binding to TUG protein, while upon insulin stimulation, GLUT4 dissociates from TUG. Here, we report that the TUG homolog in human, ASPL, exerts similar properties, i.e., forms a complex with GLUT4. We describe the structural details of complex formation by combining biochemical assays with cross‐linking mass spectrometry and computational modeling. Combined, the data suggest that the intracellular domain of GLUT4 binds to the helical lariat of ASPL and contributes to the regulation of GLUT4 trafficking by cooperative binding.

Superantigens (SAgs) are bacterial enterotoxins produced by Staphylococcus aureus. Staphylococcal enterotoxin type A (SEA), a staphylococcal superantigen, has been shown to bind to the cytokine signalling receptor glycoprotein 130 (gp130). The structural details, as well as the exact physiological role of this interaction, remain unclear. Here, we describe the structural details of the SEA–gp130 complex by combining crosslinking mass spectrometry and computational modelling. Interestingly, SEA is not able to bind gp130‐homologues from rat and mouse. Our data suggest that SEA may interact with human gp130 in a different manner than other known gp130‐ligands. Moreover, the fact that SEA does not bind mouse or rat gp130 suggests that SAgs have additional mechanisms of action in humans.

A key alteration in cancer metabolism is an increase in glucose uptake mediated by the glucose transporters (GLUT). Otto Warburg observed already in the 1950s that glycolysis was increased in many tumors, and this is now called the Warburg effect. Interestingly, a distinct glucose metabolic signature was recently described for acute myeloid leukemia (AML), showing that enhanced glycolysis correlates with decreased sensitivity for chemotherapy (cytarabine, Ara-C) and poor prognosis. AML is the most common acute leukemia in adults and is associated with poor survival, especially in patients >60 years, an age group in which only 5-15% are cured. Moreover, older patients who cannot tolerate intensive chemotherapy have a median overall survival of only 510 months. Thus, novel therapeutic approaches are needed to improve the cure rates of AML. Interestingly, defective GLUT1-mediated glucose uptake was shown to impair AML cell proliferation, and transplantation of GLUT1-deleted murine AML cells attenuated AML development in mice, suggesting that GLUT1 plays an important role in AML. Thus, targeting GLUT1 may represent a novel therapeutic vulnerability in AML by overcoming Ara-C resistance. However, there are still no clinically available drugs targeting GLUT, which may partly be due to the lack of suitable in vitro drug-screening systems. Here we present a detailed structural and functional analysis of compounds that inhibit glucose transporters and sensitize AML cells for chemotherapy. GLUT1 is an integral membrane protein consisting of 12 transmembrane helices and an intracellular domain, which transports glucose depending on the concentration gradient (Figure 1A). Measuring the activity of membrane proteins such as GLUT1, which transport uncharged substrates, is challenging due to the lack of an easily accessible readout. However, we have developed a system by which purified glucose transporters are reconstituted in vitro into giant vesicles reporting their transport activity using fluorescence microscopy. This allows glucose uptake to be measured without any interference from other proteins by having the purified transporters imbedded in a lipid-bilayer mimicking the size and curvature of mammalian cells. Applying this method, putative GLUT1 inhibitors PGL-13, PGL-14 and PGL-27 (Figure 1B), were validated and benchmarked against the wellknown GLUT1 inhibitors WZB-117 and cytochalasin B (CB). A clear decrease in glucose uptake was detected for PGL-13 and PGL-14, but not for PGL-27 (Figure 1C). To rationalize these results, molecular modeling studies including docking, molecular dynamics (MD) simulations and ligand-protein binding energy evaluations were carried out. The structure of GLUT1 has previously been determined in complex with CB and phenylalanine amide-based inhibitor displaying binding to the central substrate-binding site (Figure 1A). To evaluate whether PGL-13 and PGL-14 also interact at the substrate-binding site, PGL-14 was docked into that site of GLUT1 in an inward-open conformation. The docking solutions could be clustered into three binding poses and for each cluster the docking solution with the best estimated binding energy was selected as a representative potential binding mode. To assess the reliability of the predicted binding modes, the three ligand-protein complexes (complex 1-3, Online Supplementary Figure S1A-C) were subjected to MD simulations. In parallel, the same MD protocol was applied to the GLUT1-PGL-14 complex predicted from our previous docking studies based on the GLUT1 homology model in a partially occluded inward-facing conformation, where the ligand is bound to the intracellular domain of GLUT1 (complex 4, Online Supplementary Figure S1D). In 3 out of 4 GLUT1-PGL-14 complexes studied (complex 2-4), the ligand maintained its binding mode predicted by docking showing an average rootmean square deviation (RMSD) of its disposition during the MD simulations below 1.5 Å (Figure 1D). In particular, the binding disposition of compound PGL-14 in the intracellular site (complex 4) was remarkably stable, with an average RMSD of about 0.7 Å. Combined, these analyses suggest that PGL-14 likely interacts with GLUT1 in a partially occluded, inward-facing conformation at the intracellular domain. This is an interesting feature that distinguishes the PGL from competitive inhibitors of GLUT1, for instance CB that is known to bind to the transmembrane area. However, we cannot exclude the possibility of PGL compounds having two potential GLUT1 binding sites: the transmembrane and the intracellular binding site (Figure 1E). A refined binding mode was generated for PGL-14, revealing that the interactions are essentially as previously described, with a salicylketoxime ring sandwiched between the side chains of E146 and R212, forming a π-π stacking interaction with the latter residue, as well as H-bond interactions by the functional groups of the ligand to the backbone of the protein (Figure 1F). As PGL-27 did not have inhibitory effects (Figure 1C), the presence of a metamethyl group in the terminal phenolic ring of PGL-27 (Figure 1B) could result in less favorable binding affinity as it requires displacement of the water molecule that is forming a water-bridged interaction between the ligand and the protein (Figure 1F), and might additionally result in steric clashes. To experimentally investigate the modeled binding sites, we monitored intrinsic fluorescence quenching for the two binding-site tryptophans upon PGL binding. Measurements showed a decrease in fluorescence intensity, suggesting binding to the transmembrane domain (Figure 1G and H). However, at higher concentrations, a red shift in the emission spectra and an increase in fluorescence intensity was observed (Figure 1G and H). Based on the MD simulations, such behavior could imply PGL binding at two sites (Figure 1D and E) with different affinities. The decrease in fluorescence shows binding in the transmembrane part, while the red shift could result from a secondary conformational event in the intracellular domain, as structural changes accompanying PGL binding could have an overall effect. Indeed, a recent study demonstrated that the intracellular domain of GLUT1 is highly mobile and that its conformational flexibility is strongly coupled to other parts of the protein. To evaluate whether the PGL compounds are specific for GLUT1, and if targeting GLUT1 can sensitize AML cells for chemotherapy, myeloid leukemia-derived cell lines were screened for GLUT1 expression (Online Supplementary Figure S2). THP-1 cells expressed significant amounts of GLUT1 in contrast to KG-1 cells (Figure 2A and B). Thus, comparison between THP-1 and KG-1 cells is an applicable model system to validate the specificity of PGL towards GLUT1 and their sensitization effects for Ara-C treatment. First, the effect on cell viability by Ara-C, PGL-13 and PGL-14 was assessed at increasing concentrations in THP-1 and KG-1 cells (by ATP-assay) and IC25 values were determined (Online Supplementary Figure S3A-C). Subsequently, co-treatments with Ara-C and PGLs at IC25 were evaluated for potential synergistic or additive effects. The combinatory