Solute carrier (SLC) transporters comprise a family of >450 membrane-bound proteins that facilitate the transport of a wide array of substrates across biological membranes. They play a fundamental role in controlling the transport of molecules, such as ions and metabolites, across the cell membranes. Despite the availability of marketed drugs targeting well-known SLC transporters, such as neurotransmitter (i.e. SERT, NET, DAT) and glucose (GLUTs) transporters, most of the SLCs are still under-investigated as therapeutic targets. A major limiting factor in this area is the lack of suitable assays and tools that enable High Throughput Screening (HTS) of large compound collections, aiming to identify novel therapeutics. In the context of the Innovative Medicines Initiative consortium RESOLUTE, we developed cell-based assays for several SLCs employing a variety of scientific approaches and technologies suitable for running fully automated HTS. Here, we describe the functional assays developed for five SLCs: SLC59A1 (MFSD2A), SLC59A2 (MFSD2B), SLC6A8 (CRTR), SLC9B2​ (NHA2) and SLC12A2 (NKCC1). All these transporters play relevant roles in different pathological conditions, but they still lack drugs able to specifically activate or inhibit them. To address this gap, we have developed and optimized in a miniaturized format cellular assays that enable streamlined and high-throughput investigation of compound libraries. These assays offer a valuable platform for the identification of new therapeutic modulators targeting these underexplored SLCs in a fast and efficient manner.

Sphingosine-1-phosphate (S1P) is a signaling lysolipid critical to heart development, immunity, and hearing. Accordingly, mutations in the S1P transporter SPNS2 are associated with reduced white cell count and hearing defects. SPNS2 also exports the S1P-mimicking FTY720-P (Fingolimod) and thereby is central to the pharmacokinetics of this drug when treating multiple sclerosis. Here, we use a combination of cryo-electron microscopy, immunofluorescence, in vitro binding and in vivo S1P export assays, and molecular dynamics simulations to probe SPNS2's substrate binding and transport. These results reveal the transporter's binding mode to its native substrate S1P, the therapeutic FTY720-P, and the reported SPNS2-targeting inhibitor 33p. Further capturing an inward-facing apo state, our structures illuminate the protein's mechanism for exchange between inward-facing and outward-facing conformations. Finally, using these structural, localization, and S1P transport results, we identify how pathogenic mutations ablate the protein's export activity and thereby lead to hearing loss.

With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.

Mitochondria are key regulators of energy supply and cell death. Generation of ATP within mitochondria occurs through oxidative phosphorylation (OXPHOS), a process which utilizes the four complexes (complex I-IV) of the electron transport chain and ATP synthase. Certain oncogenic mutations (e.g., LKB1 or mIDH) can further enhance the reliance of cancer cells on OXPHOS for their energetic requirements, rendering cells sensitive to complex I inhibition and highlighting the potential value of complex I as a therapeutic target. Herein, we describe the discovery of a potent, selective, and species cross-reactive complex I inhibitor. A high-throughput screen of the Bayer compound library followed by hit triaging and initial hit-to-lead activities led to a lead structure which was further optimized in a comprehensive lead optimization campaign. Focusing on balancing potency and metabolic stability, this program resulted in the identification of BAY-179, an excellent in vivo suitable tool with which to probe the biological relevance of complex I inhibition in cancer indications.

Oxidative stress is a key concept in basic, translational, and clinical research to understand the pathophysiology of various disorders, including cardiovascular and renal diseases. Although attempts to directly reduce oxidative stress with redox-active substances have until now largely failed to prove clinical benefit, indirect approaches to combat oxidative stress enzymatically have gained further attention as potential therapeutic strategies. The pantetheinase Vanin-1 is expressed on kidney proximal tubular cells, and its reaction product cysteamine is described to negatively affect redox homeostasis by inhibiting the replenishment of cellular antioxidative glutathione stores. Vanin-1-deficient mice were shown to be protected against oxidative stress damage. The aim of this study was to elucidate whether pharmacological inhibition of Vanin-1 protects mice from oxidative stress-related acute or chronic kidney injury as well. By studying renal ischemia-reperfusion injury in Col4α3−/−(Alport syndrome) mice and in vitro hypoxia-reoxygenation in human proximal tubular cells we found that treatment with a selective and potent Vanin-1 inhibitor resulted in ample inhibition of enzymatic activity in vitro and in vivo. However, surrogate parameters of metabolic and redox homeostasis were only partially and insufficiently affected. Consequently, apoptosis and reactive oxygen species level in tubular cells as well as overall kidney function and fibrotic processes were not improved by Vanin-1 inhibition. We thus conclude that Vanin-1 functionality in the context of cardiovascular diseases needs further investigation and the biological relevance of pharmacological Vanin-1 inhibition for the treatment of kidney diseases remains to be proven.

The Innovative Medicines Initiative Consortium RESOLUTE has started to develop tools and produce data sets to de-orphanize transporters in the solute carrier protein (SLC) superfamily, thereby lowering the barrier for the scientific community to explore SLCs as an attractive drug target class.

Abstract Background and Aims A dysregulated phosphate homeostasis is strongly associated with mortality, cardiovascular events and vascular calcification, particularly in patients suffering from CKD. Inhibition of the tubular phosphate transporter Npt2a provides a novel and unique mechanism to address phosphate homeostasis imbalance and vascular calcification. Method Npt2a activity was measured in a cell-based assay, using a stable CHO cell line with inducible Npt2a expression. Male Wistar rats were used for all experiments. Healthy rats were treated orally with BAY 767, a potent Npt2a inhibitor developed at Bayer AG to assess urinary phosphate excretion. Vascular calcification was induced by administration of a pan-FGFR inh. (25mg/kg) for 10 days. Calcium content in the aorta was measured as surrogate for vascular calcification. Blood samples were withdrawn at the end of the study to determine the plasma levels of FGF-23, parathyroid hormone and phosphate with commercially available assay systems according to the manufactures protocols (FGF-23 and PTH: ELISA Kit, Immuntopics; phosphate: Pentra400 system). Results BAY 767 was identified as potent Npt2a inhibitor, with an IC50 of 2.9/6 nM on rat/ human Npt2a, respectively, selective over Npt2b, Npt2c and Pit-1. Single dose treatments of healthy rats resulted in a significant, dose-dependent increase in urinary phosphate excretion within 16h. In an experimental vascular calcification model, massive vascular calcification and hyperphosphatemia was induced by daily oral administration of a FGFR inh. Simultaneous treatment with the Npt2a inhibitor BAY 767 significantly inhibited vascular calcification in comparison to untreated rats. In this set-up the plasma phosphate level was increased after 10 days up to 3.7 mmol/L in the FGFR inh. treated group compared to 1.9 mmol/L in control animals, whilst treatment with BAY 767 reduced plasma levels to 2.7 mmol/L. In addition, FGF-23 and PTH were also reduced under compound BAY 767 treatment. However, in the same model 2.2% lanthanum carbonate was not beneficial with respect to vascular calcification. Conclusion Our results show for the first time that treatment with a Npt2a inhibitor improves vascular calcification by addressing urinary phosphate excretion and phosphate homeostasis in rats. Npt2a inhibition may provide a new therapeutic principle for patients suffering from disbalanced phosphate homeostasis and vascular calcifications, including CKD patients.

Unterschemmann, Kerstin D.; Ehrmann, Alexander; Herzig, Ina D.; Andreevski, Anna Lena; Schmeck, Carsten; Eitner, Frank; Grundmann, Manuel Author Information

Abstract Mitochondria are key regulators of both energy supply and apoptotic cell death. The mitochondrial electron transport chain (ETC) consists of four enzyme complexes that transfer electrons from NADH to oxygen. During electron transfer, the ETC pumps protons into the inter-membrane space, generating a gradient across the inner mitochondrial membrane that is used by Complex V to drive ATP synthesis. Recent publications have shown that tumor cells harboring specific mutations (LKB1, mIDH and others) are more sensitive to Complex I inhibition, potentially providing an opportunity for selectively targeting tumor cells. Based on a high throughput screen (HTS), we identified new, albeit moderately active, lead structures with cross reactivity between mouse and human Complex 1. SAR elaboration of the lead structure allowed for optimization of the potency, although compounds still suffered from low metabolic stability. Further improvement of the in vitro and in vivo PK properties finally permitted in vivo animal studies. Herein, we report for the first time the preclinical profile and structure of a highly active, optimized, human/mouse cross-reactive Complex I inhibitor that allowed for the further investigation into the therapeutic potential of Complex I inhibition in cancer. Citation Format: Jeffrey Mowat, Sven Christian, Carolyn Sperl, Alexander Ehrmann, Stephan Menz, Judith Guenther, Roman Hillig, Marcus Bauser, Andrea Haegebarth, Wolfgang Schwede. Identification and optimization of a highly active, cross reactive Complex-1 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3248. doi:10.1158/1538-7445.AM2017-3248

Abstract Mitochondria are both key regulators of energy supply and apoptotic cell death. The mitochondrial electron transport chain (ETC) consists of four enzyme complexes that transfer electrons from NADH to oxygen. During electron transfer, the ETC (Complex I to IV) pumps protons into the inter-membrane space, generating a gradient across the inner mitochondrial membrane that is used by Complex V to drive ATP synthesis. Recent publications have shown that tumor cells harboring specific mutations (LKB1, mIDH and others) are more sensitive to Complex I inhibition, compared to cells that do not have these mutations. We have identified an optimized human/mouse cross-reactive Complex I inhibitor that allows profiling of Complex I inhibitors in pharmacological models. We have pursued different approaches based on the literature, an unbiased screen and in-house results generated with the human-specific Complex I inhibitor BAY 872243 to identify sensitive in vivo tumor models. However, using the cross-reactive Complex I inhibitor we were unable to identify sensitive models apart from weakly sensitive LKB1-deficient tumors (A549, G361) when animals were treated at maximum tolerated dose (MTD). In addition, all approaches for combination therapy failed to improve efficacy in vivo. Direct comparison of human-specific Complex I inhibitor BAY 87-2243 and cross-reactive inhibitor BAY179 in a sensitive LKB1-deficient melanoma model, G361, demonstrated that inhibition of Complex I specifically in the tumor is a valid approach as it results in tumor growth inhibition of ∼50%. However, cross-reactive compounds do not reach exposures at MTD to generate comparable effects. Citation Format: Sven Christian, Carolyn Algire, Wolfgang Schwede, Jeffrey S. Mowat, Alexander Ehrmann, Stephan Menz, Marcus Bauser, Andrea Haegebarth. Comparison of human-specific versus cross-reactive Complex I inhibitor for in vivo tumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 223.

Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries and overall 5-year survival rates of less than 5%. The most frequent mutations in PDAC are gain-of-function mutations in KRAS as well as loss-of-function mutations in p53. Both mutations have severe impacts on the metabolism of tumor cells. Many of these metabolic changes are mediated by transporters or channels that regulate the exchange of metabolites and ions between the intracellular compartment and the tumor microenvironment. In the study presented here, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well as novel regulators of OxPhos. The siRNA that resulted in the greatest change in cellular oxygen consumption was targeting the KCNN4 gene, which encodes for the Ca2+-sensitive K+ channel KCa3.1. This channel has not previously been reported to regulate OxPhos. Knock-down experiments as well as the use of a small molecule inhibitor confirmed its role in regulating oxygen consumption, ATP production and cellular proliferation. Furthermore, PDAC cell lines sensitive to KCa3.1 inhibition were shown to express the channel protein in the plasma membrane as well as in the mitochondria. These differences in the localization of KCa3.1 channels as well as differences in the regulation of cellular metabolism might offer opportunities for targeted therapy in subsets of PDAC.

Abstract Biguanides, such as metformin and phenformin, are currently under investigation for their potential use as anti-neoplastic therapy. Recent publications suggest that both metformin and phenformin exert effects through inhibition of Complex 1 in the electron transport chain. We investigated the effects of metformin and phenformin compared to rotenone, in vitro, and known Complex 1 inhibitor BAY 872243, in vitro and in vivo. As expected, rotenone and BAY 872243 showed strong inhibition of Complex I in cell-based and enzymatic assays with IC50 values in the nanomolar range. The high affinity binding to Complex I was also reflected by induction of cellular reactive oxygen species (ROS) with EC50 values in the nanomolar range. In contrast, the biguanides neither inhibited Complex I in cell-based and biochemical assays, nor led to an induction of ROS at concentrations up to 300 μM. In vivo exposure analysis shows that at the maximal tolerated dose (100 mg/kg QD i.p for phenformin and 350 mg/kg i.p QD for metformin), neither metformin nor phenformin had plasma exposure levels over the IC50 for proliferation in vitro. Recent reports have suggested that biguanides, via the inhibition of Complex 1 and subsequent reduction in oxygen consumption, can be used to re-oxygenate tumor areas prior to radiation therapy. Pimonidazole staining demonstrated that metformin and phenformin effectively eliminated hypoxic regions in NCI-H460 xenografts in a time course dependent manner that reflected the exposure. In contrast to the biguanides, BAY 87-2243 effectively eliminated hypoxic regions up to 24 hours post compound administration. Finally, both phenformin and metformin had minimal effects on inhibition of tumor growth, even in LKB1-deleted xenografts which have been reported to be especially sensitive to biguanides. In conclusion, our in vitro experiments on the mode of action of biguanides raise questions as whether the in vivo effects on hypoxic tumor regions are related to direct inhibition of Complex I. Citation Format: Carolyn Algire, Alexander Ehrmann, Sven Christian, Roland Neuhaus, Stephan Menz, Wolfgang Schwede, Michael Haerter, Andrea Haegebarth. Differential effects of metformin and phenformin vs. other complex 1 inhibitors in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1126. doi:10.1158/1538-7445.AM2015-1126

The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel-Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.