Membrane Transport Discovery Lab (Group leader Prof. em. Matthias A. Hediger) (https://www.bioparadigms.org/) — Our research focuses on membrane transport proteins and ion channels that play key roles in vital physiological processes in the human body. These proteins are the gatekeepers in membranes of cells and organelles and their dysfunction contributes to the pathogenesis of a wide variety of human diseases. Our emphasis is to develop unique therapeutic treatment strategies and precision medicine tools based on new knowledge obtained from these proteins. Recently, our group has undertaken the identification and classification of Solute Carrier (SLC) transporter proteins in mammalian proteomes (see Figure showing a circular dendrogram of the clustering of all known human transmembrane transporter of the SLC solute carrier series). By synthesizing information from various publicly available databases and scientific literature, we have recently identified ~150 putative new SLCs from human, of which ~25 have known transport function. SLC transporter play important roles in the maintenance of biological barriers in brain, kidney, intestine and many other organs, as well as intracellular organelles. Additionally, currently around 40% of SLCs are orphans, i.e. their physiological substrates and function are still not known. Compared to their physiological importance, SLCs are still relatively unexploited as therapeutic targets, and their characterization can potentially open the way for novel personalized therapeutic applications. Ref.: César-Razquin A, et al., Cell. 2015, 162(3):478-87.
This project aims to decipher biological and pharmaceutical aspects of the SARS-CoV-2 virus pandemic. Using a combination of biochemical assays such as micro-scale thermophoresis (MST) to determine SARS-CoV-2 receptor binding domain (RBD) binding affinity to the ACE2 virus receptor and the SARS-CoV-2 pseudovirus entry assay to reveal viral load, we will clarify the roles of specific allelic variants of viral host genes in conferring COVID-19 severity. In addition, we will screen for blockers of viral susceptibility as hit/lead compounds for the development of novel treatment strategies.
The figure on the right shows the steps involved in SARS-CoV-2 infection in epithelial cells of lung, intestine and kidney, and project strategy outline. Using a combination of binding and pseudovirus entry assays, we will determine the effects of genetic host and viral variants on virus infectivity. Furthermore, we will screen for blockers of viral infection that may serve as future hit/lead compounds for the development of novel antiviral therapies, e.g. by administering identified agents using an oral or nasal spray, as an alternative to COVID-19 vaccination. Abbreviations: ACE2, angiotensin-converting enzyme 2 (serves as virus receptor); TMPRSS2, transmembrane protease (activates SARS-CoV-2); SLC6-AAT, SLC6 family amino acid transporter (transports peptide cleavage products of ACE2 into epithelial cells).
As part of the initial activities of the NCCR TransCure network, we have been working with the group of Jean-Louis Reymond to generate specific TRPV6 inhibitors. Our effort has led to the generation of a series of novel inhibitors with great specificity and IC50 values in the nanomolar range (Angew Chem Int Ed Engl. 2015, 54:14748-52; RSC Med Chem 2020, 11, 1032-1040). In our recent collaboration with Alexander Sobolevsky (University of Columbia) and Christoph Romanin (University of Linz), we combined structural data with mutagenesis and functional/computational analyses to clarify the binding site of these compounds within the open pore of TRPV6. Our study shows that binding converts the channel into a nonconducting state, thereby mimicking the action of calmodulin, which causes inactivation of TRPV6 channels under physiological conditions. This mechanism of inhibition explains the high selectivity and potency of these channel inhibitors and opens up unexplored avenues for the design of future-generation biomimetic drugs (Sci Adv. 2020 Nov 27; 6). Furthermore, in collaboration with Jean-Louis Reymond and Christoph Romanin, we developed a novel photoswitchable inhibitor of TRPV6 that is expected to serve as a versatile tool compound to deepen our understanding of TRPV6 (ACS Med Chem Lett. 2019; 10:1341-1345 ). In addition, we published a paper on capsaicin derivatives and their effects on TRPV1 and TRPV6 channel function (Bioorg Med Chem. 2019; 27:2893-2904). In addition, a review entitled “TRPV5 and TRPV6 Calcium-Selective Channels” was published as a book chapter in “Calcium Entry Channels in Non-Excitable Cells” (Kozak JA, Putney JW Jr., editors, CRC Press/Taylor & Francis; 2018; Chapter 13).
In year 2015, our laboratory got awarded the Sinergia Swiss National Science Foundation (SNSF) interdisciplinary research grant, entitled “Store-operated calcium channels in health and disease” (October 1, 2015–November 30, 2018), together with Nicolas Demaurex (University of Geneva) and Martin Lochner (University of Bern). The first study on this topic we published in collaboration with Nicolas Demaurex, showing that ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain (J Physiol. 2019 597:561-582). Thereafter, as a collaboration with Christoph Romanin, we reported the unveiling of a novel STIM1-Orai1 gating interface that is essential for CRAC channel activation (Cell Calcium. 2019; 79: 57-67). In collaboration with Martin Lochner, we characterized novel 2-aminoethyl diphenylborinate (2-APB) derivatives with respect to inhibition of store-operated calcium entry (SOCE) (Int J Mol Sci. 2020 Aug 5; 21(16):5604). In another recent study, we described a novel role for Ca2+-calmodulin in SCDI of Orai1 (Cell Physiol Biochem 2020; 54:252-270). In addition, we completed our collaboration with Ivan Bogeski (University of Göttingen) and Rainer Schindl (University of Graz), showing that STIM2 cytosolic cysteine residues are targeted by reactive oxygen species to modulate SOCE (Cell Rep. 2020; 33(3):108292). Related to this work, we published a review entitled “Redox modulation of STIM-ORAI signaling” (Cell Calcium. 2016; 60:142-52).
In year 2018, we got awarded a new SNSF Sinergia grant (September 1, 2018–August 31, 2022), which focusses on the role of mitochondrial carriers in metabolic tuning and reprogramming by calcium flow across membrane contact sites, a collaborative effort together with Edmund Kunji (University of Cambridge) and Martin Lochner. A review entitled “Sequence Features of Mitochondrial Transporter Protein Families” has recently been published (Biomolecules. 2020 28; 10(12):E1611).
After the molecular discovery of the divalent metal ion transporter by our group (Nature. 1997, 388:482-8), Raimund Dutzler, as part of the NCCR TransCure iron project, reported the 3D structure of DMT1/SLC11A2 from Staphylococcus capitis (ScaDMT), unveiling it as a LeuT-fold transporter. As a follow-up, we employed molecular dynamics simulations and site-directed mutagenesis and discovered a novel H+ transfer mechanism in DMT1. Our molecular dynamics simulations provided first insight into how H+-translocation through E193 is allosterically linked to intracellular gating, revealing a novel H+-coupling mechanism that is distinct from that of other H+-transporters (Sci Rep. 2017 ;7:6194). The mechanistic base of the inhibition of DMT1 has recently been elucidated for bis-isothiourea substituted compounds in collaboration with the groups of Raimund Dutzler (University of Zürich) and Jean-Louis Reymond (University of Bern) (eLife 2019; 8: e51913).
A combination of in silico and in vitro techniques involving structural modeling, mutagenesis and functional characterization was employed to unravel the structural elements of pH sensitivity and substrate binding in the human zinc transporter SLC39A2 (ZIP2). This work provides the first structural evidence for the previously observed pH and voltage modulation of ZIP2-mediated metal transport (J Biol Chem. 2019; 294:8046-8063). An inhibitor against the SLC39A8/ZIP8 zinc transporter has recently been generated and is being tested using an in vitro osteoarthritis cellular system, looking for beneficial disease treatment effects.
The figure on the right shows the role of amino acid transporters in mTOR activation, energy metabolism, nutritional stress and tumor progression (Trends Biochem Sci. 2018, 43(10):752-789). Our recent data reveal that oncogenic mutations boost amino acid delivery into colorectal cancer (CRC) cells via hippo-mediated upregulation of specific amino acid transporters (manuscript in preparation). Briefly, we investigated the impact of oncogenic and tumor suppressor mutations on the regulation of expression of amino acid transporters in CRC. We found that KRAS and BRAF oncogenic mutations present in different CRC subtypes upregulate the expression and functions of specific amino acid transporters. Our findings suggest that inhibiting amino acid transporters could represent a novel approach for personalized cancer treatment in KRAS/BRAF-mutant tumors.
The orphan endosomal peptide/histidine transporter SLC15A4 is currently being evaluated with respect to application to the treatment of autoimmune diseases such systemic lupus erythematosus (SLE) and inflammatory bowel disease (IBD). The SLC15 family includes four different members (SLC15A1-A4). SLC15A1 (intestinal PepT1) and SLC15A2 (renal PepT2) have previously been extensively studied by our laboratory (Nature. 1994, 368(6471):563-6). SLC15A3 and SLC15A4 have low sequence similarities to SLC15A1 and SLC15A2 and their substrate selectivity is thought to be restricted to histidine, certain oligopeptides and muramyl peptides that represent fragments of peptidoglycan from pathogens, e.g. the bacterial cell wall. SLC15A4 is a peptide/histidine transporter essential component of the inflammatory response system triggered by Toll-like receptors (TLR) pathway. Ligand binding and signaling of TLRs are modulated by the content of histidine and the pH inside the lysosomes (see Figure). The histidine level in lysosomes is thought to be maintained within a defined concentration range by SLC15A4 to warrant maximal functional efficiency of the lysosomal components, including TLR maturation and function. The absence of SLC15A4 leads to failure in the homeostasis of the lysosomal environment, which could explain the disruption of the TLR signaling pathway in SLC15A4-deficient cells. Additionally, SLC15A4 seems to be involved in the transport of the peptidoglycan-like NOD1 ligand, tri-DAP, and the NOD2 cognate ligand muramyl dipeptide (MDP) from lysosomes to the cytosol. Our goal is to generate SLC15A4 inhibitors as a therapeutic treatment strategy for SLE and IBD. Using microscale thermophoresis (MST), we have screened a diverse library of 1600 compounds and identified several hits that are currently being investigated.
Our recent collaboration with Rodan, Lance, Children’s Hospital, Harvard Medical School revealed novel glutamate transporter mutations that cause epilepsy via a dominant negative mechanism. We found that these mutations are localized in the trimerization domain of the glutamate transporter trimers (Ann Neurol. 2019, 85:921-926).