Part A: Transporters as targets for cancer therapy
In Project 1, Part A, Matthias Hediger’s group focuses on the elucidation of the role of different transporters and channels in the pathogenesis of cancer. In tight collaboration with TransCure chemists, the group sets out to develop compounds targeting transporters and channels of interest, with the aim to modulate their malfunction in cancer and thus to contribute to the treatment of this disease. In parallel, structural biology efforts going on in his own laboratory and in other groups within TransCure are aiming at the determination of high-resolution protein structures. The structural information gained will aid them in optimizing lead compounds with the help of structure-based drug design.
The following channel/transporter targets are studied within Matthias Hediger’s group:
The calcium selective epithelial channel TRPV6 is responsible for body calcium homeostasis through mechanisms including renal reabsorption, bone resorption and intestinal absorption. Mediating 1,25-vitamin D-regulated calcium uptake in the duodenum, TRPV6 is primarily located within the apical membrane of intestinal enterocytes (Peng and Hediger et al., J Biol Chem, 274, 22739-46, 1999). However, expression profiles have also been verified in the placenta, pancreas, and the prostate gland. In humans, there is a wealth of evidence that upregulation of TRPV6 correlates with the progression of prostate, breast, colon, ovarian, lung and thyroid cancer. Also, animal models have indicated that alterations in expression and function of this channel could lead to pathophysiological conditions, including hypercalciuria with renal stone formation, preeclampsia and osteoporosis (Suzuki and Hediger et al., J Bone Miner Res, 23, 1249-56, 2008; Suzuki and Hediger et al., Annu Rev Physiol, 70, 257-71, 2008). At present, there is no commercially available specific inhibitor or activator of TRPV6 available. The generation of specific modulators of TRPV6 may lead the novel therapeutic strategies. This would be especially important, for example, for the treatment of the advanced, androgen-independent stage of prostate cancer, for which there is currently no specific therapeutic strategy available.
To better understand the role of TRPV6 in calcium homeostasis and cancer progression, a comprehensive effort within TransCure to determine structure and function, as well as drug design of the channel has been launched. The Hediger group, working in collaboration with TransCure chemists (groups Reymond and Lochner) has established a high-throughput compound screening program. Through several rounds of screening and drug design, the potency of lead compounds has improved significantly, opening the door to cancer-related in vitro and in vivo studies. In parallel, structural biology efforts are ongoing in the group of Hediger, with the aim to elucidate the atomic structure of TRPV6 and improve our understanding of how protein, substrate, and drug interactions occur.
- Zinc transporters
Zinc is a trace element essential for a myriad of biological processes within the immune system, protein and DNA synthesis, wound healing, and many general processes in metabolism. Zinc serves as a catalytic cofactor for over 100 biochemical enzymes and as a structural factor for both protein stability and DNA transcription. Despite having many important roles, the cellular available zinc concentration is quite low and homeostasis is tightly controlled to prevent zinc toxicity. In humans, two families of zinc transporters are responsible for this homeostasis, with SLC30 (ZnT) serving to control zinc efflux out of the cytoplasm and SLC39 (ZIP) primarily responsible for zinc entry into the cell. Several pathologies have been directly linked to human ZIP transporters. In many cancers, there is increased demand for zinc as a co-requisite for angiogenesis, proliferation and metastasis. ZIP4, 6, 7 and 10 all show increased expression in breast cancer. Interestingly, zinc levels are elevated in normal prostate, however, down regulation or redistribution of ZIP1, 2, and 3 and subsequent reductions in cytosolic zinc levels have been observed in prostate cancer (Franz and Hediger et al., Mol Aspects Med, 34, 735-41, 2013).
Within TransCure’s cancer project 1, robust screening assays to assess the activity of ZIP transporters in over-expressed mammalian cell lines will be established. After validation with novel single cell ion-selective recordings, the process of generating specific inhibitors and activators will occur simultaneously with pathophysiological experiments in vitro and in vivo to determine the roles of these transporters in cancer and to contribute to the treatment of this disease.
- Amino acid transporters
Various amino acid transporters have been identified as potential targets for inhibition of cancer progression. For example, the activation of mTOR (mammalian Target Of Rapamycin) is a very important process linking growth signals to nutrient availability and is regulated by the uptake of amino acids such as L-glutamine (the obligate nitrogen donor for nucleotide synthesis). Due to altered glutamine metabolism in cancer, tumor cells can develop a “glutamine addiction” which is required to support macromolecular synthesis for cell proliferation. Glutamine uptake is controlled by a bidirectional transport of L-glutamine mediated by various amino acid transporters. Thus, inhibiting glutamine transport and limiting the delivery of this important nutrient is an important strategy to impede tumor progression (El-Gebali and Hediger et al., Mol Aspects Med, 34, 719-34, 2013).
The goal of the Hediger group in collaboration with TransCure chemists (groups Reymond and Lochner) is to study the role of amino acid transporters in cancer and to develop specific inhibitors. As of now, some interesting and promising protein targets have been identified based on in silico analyses, literature data mining and in vitro follow-up validation studies. Moreover, in collaboration with chemists, compound structures relevant for targeting transporters either as drug carriers and/or drug targets and/or chemo-sensitizing drug targets are being identified. In parallel, structural biology approaches on amino acid transporters are ongoing within TransCure (group Fotiadis), aiming at the determination of high-resolution protein structures suitable for structure-based drug design.
- Lactate transporters
Most cancer cells use aerobic glycolysis to obtain sufficient ATP in a hypoxic microenvironment, a phenomena known as ‘‘the Warburg effect’’. As a result, lactate is abundantly synthesized from pyruvate, which is transported by monocarboxylate transporters. The transporters are induced through HIF-1 to avoid cellular acidosis, which triggers apoptosis. It is known that SLC16A3 (MCT4) transports lactate out of the cell and SLC16A1 (MCT1) regulates the entry of lactate into tumor cells. SLC16A3 also acidifies the extracellular matrix leading to increased angiogenesis. On the other hand, lactate is not just a waste product. It was recently identified as a major source of energy in cancer cells. Hypoxia leads cancer cells to upload lactate produced by neighboring hypoxic cells, which feeds aerobic cancer cells through respiration and anabolic functions. Moreover, SLC16 family members have been suggested to directly interact with beta 1 integrin resulting in the regulation of cell migration through the alteration of focal adhesions. Cell migration is influenced by SLC16A3 as it regulates maturation and trafficking of CD147 to the plasma membrane.
Analysis of both data generated by the Hediger group as well as publicly available data suggests that some lactate transporters are significantly overexpressed in various solid tumors as compared to normal tissue. Knock down studies in cancer cell lines show that loss of these transporters significantly reduces cell viability and cell invasion, opening the door to chemistry efforts aiming at the development of highly specific and affine inhibitors. From a structural biology point of view, the group of Fotiadis focuses on the structure determination of close prokaryotic homologues of the lactate transporter family.
- Iron transporters
The project on iron transporters aims to target this important class of membrane proteins in order to control the uptake of iron into cells. The current focus is on the human transporter DMT1 (SLC11A2), a member of the SLC11 family of divalent metal ion transporters. DMT1 is vital for iron uptake in enterocytes and for transferrin associated endosomal iron transport in many other cell types (Montalbetti and Hediger et al., Mol Aspects Med, 34, 270-287, 2013). Dysfunction of human DMT1 is associated with anemia, iron overload disorders, neurodegenerative diseases (e.g., Parkinson's disease and Alzheimer’s disease), breast and colorectal cancer and autoimmune and inflammatory diseases (e.g., rheumatoid arthritis). The involvement of DMT1 in these disorders makes the pharmacological modulation of this protein a promising therapeutic strategy.
The project is well integrated within TransCure, as several groups are addressing different aspects of DMT1. Whereas the Hediger group addresses its pathophysiological characterization in vitro and in vivo with a focus on cancer, the chemists group of Reymond sets out to modulate its function by the development of small molecules. In parallel, the structural characterization of close prokaryotic homologues is ongoing in the group of Dutzler.
- Peptide transporters
Mammalian members of the proton-coupled oligopeptide transporter family (SLC15) are integral membrane proteins that mediate cellular uptake of di/tripeptides and peptide-like drugs (Smith, Clémençon and Hediger, Mol Aspects Med, 34, 323-36, 2013). The oligopeptide transporters PepT1 and PepT2 (SLC15A1 and SLC15A2) are responsible for the absorption peptides in the intestine and kidney, respectively, and for maintaining homeostasis of neuropeptides in the brain. They are also responsible for the absorption and disposition of a number of pharmacologically important compounds. PepT1-mediated transport has been an important paradigm for the development of prodrug strategies and various peptide-like drugs, allowing secondary active absorption of drugs linked to peptides through cellular barriers, e.g. the intestine. Using information about the structure of PepT1, it will be possible to chemically modify the structure of suboptimal drugs to create a transported prodrug that utilizes PepT1 to assure efficient absorption through proton-coupled transport.
The main objective of this project in the Hediger group is to obtain a high-resolution structure of the PepT1 human homologue, in order to identify the binding site of substrates by X-ray crystallography. The identification of each amino acid and their positions in the binding pocket is expected to permit the design of new drugs in collaboration with chemists groups within TransCure. In the future, a therapeutic strategy to be considered may be the design of prodrugs targeting PepT1, based on the unraveled high-resolution structure of its binding site. Due to the higher expression of PepT1 in certain types of cancer, prodrugs may be designed to specifically target tumor cells.
- Vitamin C / nucleobase transporters
Vitamin C is essential for many biochemical processes and protects tissues and cells from oxidative stress. Transporters for vitamin C and its oxidized form, dehydroascorbic acid (DHA), play crucial roles in the maintenance of physiological concentrations of this vitamin in cells. The human SLC23 family encompasses the two Na+-dependent vitamin C transporters SVCT1 (SLC23A1) and SVCT2 (SLC23A2) as well as the orphan transporter SVCT3 (SLC23A3). The murine Slc23a3 gene was originally cloned from mouse yolk sac and subsequent studies could show that it is expressed in the kidney. However, the function of SVCT3 has not been reported and it remains speculative as to whether SVCT3 is a nucleobase transporter. Plasma nucleobases are salvaged into cells in order to sustain the cellular nucleotide synthesis. Therefore, plasma levels of nucleobases are thoroughly maintained, and it was suggested that plasma membrane nucleobase transporters, especially those in the kidney, are intimately involved in this nucleobase homeostasis. Analogues of purines and pyrimidines are used as anti-neoplastic and anti-viral drugs, e.g. 5-Fluoruracil, an anti-neoplastic drug that was shown to be transported by a high affinity nucleobase transport system. Therefore, if SVCT3 turns out to be a nucleobase transporter, it could affect the transport and metabolism of anti-viral and anti-neoplastic drugs, e.g. through allosteric activators increasing the reabsorption of drugs in the kidney, and thus open the door to more effective anti-cancer and anti-viral treatment strategies.
The Hediger group could show that SVCT3 is localized on the apical side of renal proximal straight tubule segments and does not transport ascorbic acid (Bürzle and Hediger et al., Mol Aspects Med, 34, 436-54, 2013). Since nucleobases are known to be reabsorbed in proximal straight tubule segments, it is likely that SVCT3 is responsible for nucleobase reabsorption in order to sustain total body nucleobase homeostasis. If it turns out that SVCT3 indeed transports nucleobases, collaborations with chemists will begin in order to develop allosteric activators capable of increasing the reabsorption of drugs in the kidney and thereby enhancing their therapeutic efficacy. In the discipline structure, efforts are ongoing in the group of Fotiadis on both prokaryotic and eukaryotic SLC23 protein members, in order to maximize their chances of determining a high-resolution structure.
Part B: Glutamate transporters as therapeutic targets
Glutamate is the major excitatory neurotransmitter in the nervous system. Prolonged extracellular accumulation causes excitotoxicity and hence it is crucial that glutamate is removed rapidly from the synaptic cleft after release. The removal is accomplished by high-affinity glutamate transporters (EAATs) from the SLC1 transporter family. The pathophysiologically most relevant isoforms of this family encompass the members SLC1A1 (EAAC1, EAAT3), SLC1A2 (GLT-1, EAAT2) and SLC1A3 (GLAST, EAAT1) that are expressed on the surface of neurons and glial cells. Regulation of glutamate concentration in the synapse is of high interest because its prolonged extracellular accumulation causes excitotoxicity which is presumed to be a factor in numerous neurological diseases, such as Alzheimer’s, epilepsy and the implications of stroke. In the latter case it would be therapeutically beneficial to inhibit neuronal EAAT isoforms (e.g. EAAC1) with selective compounds since there is significant reversal glutamate transport from neurons into the synaptic cleft under ischemic conditions. For amyotrophic lateral sclerosis (ALS) it was found that the function of the glutamate transporters GLT-1 is impaired. Riluzole, the only drug approved to treat ALS, seems to increase glutamate uptake via GLT-1 by an as yet unknown mechanism. Riluzole is far from an ideal drug and thus it could be therapeutically interesting to develop allosteric activators of glutamate transporter isoforms (Kanai and Hediger et al., Mol Aspects Med, 34, 108-120, 2013).
This TransCure project led by Matthias Hediger aims at the identification of the substrate and inhibitor binding sites of glutamate transporters by site-directed mutagenesis experiments and photo-labeling studies (collaboration with the group Lochner). In parallel, computational biology efforts will aim to identify novel selective inhibitors of SLC1 family members using in silico protein structure-based drug design. These studies are relying on homology modeling based on the known structure of the archeal glutamate transporter GltPh. To test developed compounds in cellular systems in vitro, screening assays are established for the glutamate transporters GLAST, GLT-1 and EAAC1.