New chemical tools for studying Endolysosomal Two-pore Channels

Two-pore channels (TPCs) are endolysosomal ion channels of physiological and pathophysiological significance. However, fundamental properties concerning ion permeability and activation mechanisms are ambiguous. Also, as the likely targets for Ca2+-mobilizing messenger NAADP, the role of TPCs in cell-wide Ca2+ signalling is ill-defined. Importantly, their pharmacology is limited to cell-impermeable activators and a few non-selective inhibitors, which brings challenges for characterizing TPCs. In this thesis, I address the above issues. I began by examining the mechanism of action of the lysosomotropic agent, glycyl-L- phenylalanine 2-naphthylamide (GPN), which has long been appreciated for mediating Ca2+ signals from lysosomes and for probing TPC function. Its action on lysosomes has recently been questioned. However, using fibroblasts, here I show that GPN mobilises Ca2+ from acidic organelles. I move on to characterise two cell-permeable and selective TPC2 activators (A1 and H07). Additionally, I confirm that approved drugs targeting estrogen and dopamine receptors are selective TPC2 inhibitors. I go on to show that A1 and H07 activate TPC2 differentially. A1 induced larger and quicker Ca2+ signals than H07 but similar Na+ signals. A1 and H07 targeted distinct sites on TPC2. Besides, H07 but not A1-induced Ca2+ signals were regulated by external (luminal) pH. The implication is that TPC2 may be regulated in an agonist- specific manner. Finally, by using GPN and inhibiting TPC activity with novel inhibitors or siRNA knockdown, I show that TPCs are required for histamine- but not bradykinin-induced Ca2+ signals. More specifically, histamine-mediated Ca2+ signals were reduced upon TPC2 but not TPC1 knockdown. Thus, TPCs are implicated in global Ca2+ signalling evoked by physiological stimuli likely in an isoform-dependent manner. Collectively, this research has provided novel TPC modulators with which to further characterize fundamental properties and physiological roles of TPCs

Targeting viral entry as a strategy for broad-spectrum antivirals

The process of entry into a host cell is a key step in the life cycle of most viruses. In recent years, there has been a significant increase in our understanding of the routes and mechanisms of entry for a number of these viruses. This has led to the development of novel broad-spectrum antiviral approaches that target host cell proteins and pathways, in addition to strategies focused on individual viruses or virus families. Here we consider a number of these approaches and their broad-spectrum potential

COVID-19: Drug targets and potential treatments

92 p.-22 fig.-1 tab.-1 graph. abst.Currently, we are immersed in a pandemic caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which severely threatens public health worldwide. Until now, no drug or vaccine has been approved to treat the severe disease caused by this coronavirus, COVID-19. We will focus on the main virus-based and host-based targets that can guide medicinal chemistry efforts to discover new drugs for this devastating disease. In principle, all CoVs enzymes and proteins involved in viral replication and the control of host cellular machineries are potentially druggable targets in the search for therapeutic options for SARS-CoV-2. This perspective provides an overview of the main targets from a structural point of view, together with reported therapeutic compounds with activity against SARS-CoV-2 and/or other CoVs. Also, the role of innate immune response to coronavirus infection and the related therapeutic options will be presented.Funding from CSIC (201980E024 and 202020E103) is acknowledged. This research was partially supported through "la Caixa" Banking Foundation (HR18-00469), Instituto de Salud Carlos III (ISCIII-COV20/01007), Spanish Ministry of Science and Innovation (RTI2018-097305-R-I00), CONICYT-PCI (REDES190074 to D. R. and A. M.) and FONDECYT (11180604 to D.R.). I. M. was funded by H2020-MSCA-ITN-2017 (grant no. 765912), V. N. holds a pre-doctoral FPU grant (FPU16/04466) and J. U. was financed by FPI-SGIT2018-04.Peer reviewe

Essential requirement for JPT2 in NAADP-evoked Ca²⁺ signaling

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a “clickable” NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry

Novel chemical tools to target two-pore channel 2, P-glycoprotein and histone deacetylase 6 in cancer

The identification of previously unknown targets as well as the development of efficacious inhibitors for known targets are key factors to make more patients benefit from tumor therapy. For instance, the role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of liver cancer cells in vitro, affects their energy metabolism and successfully abrogates tumor growth in vivo. Concurrently, we have identified novel, simplified analogues of the alkaloid tetrandrine (SG-005 and SG-094) as potent TPC2 inhibitors by screening a library of benzyltetrahydroisoquinoline derivatives using cell proliferation assays, endolysosomal patch clamp and calcium imaging. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against several cancer cell lines and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing the first TPC2 inhibitor with antitumor efficacy in mice (SG-094). Hence, our study unveils TPC2 as valid target for cancer therapy and provides novel, easily accessible tetrandrine analogues as promising option for effective pharmacological interference. Furthermore, in-depth studies were conducted to investigate a postulated mechanism of metabolic toxification of tetrandrine. A combined medicinal chemistry and cell biology approach showed that a reduction of the toxicity of tetrandrine cannot be achieved by replacing or eliminating the hypothesized metabolically instable functional group, clearly indicating that the proposed pathway is not the primary cause for the in vitro toxicity of tetrandrine and related alkaloids. Moreover, we have uncovered that the simplified tetrandrine congeners SG-005 and SG-094 additionally inhibit P-glycoprotein (P-gp), a drug efflux pump associated with multidrug resistance and treatment failure in tumor therapy. Since no approved molecules targeting P-gp are currently available, SG-005 and SG-094 might represent promising candidates to treat drug-resistant cancers owing to their favorable drug-like properties. Generally, the dual mode of action of isoquinoline-based TPC2/P-gp antagonists is mentioned here for the first time. Based on this, the known third-generation P-gp inhibitor elacridar was exemplarily studied for its potential to block TPC2, revealing another potent TPC2 blocker and thereby challenging the assumption of elacridar specifically acting on efflux pumps. Hence, on the one hand, a new lead structure (elacridar) for the development of prospective TPC2 blockers is provided. On the other hand, hints for common structural motifs on TPC2 and P-gp are given, which can facilitate the search for additional TPC2 antagonists. We further uncovered that TPC2 and P-gp do not only share mutual small molecule inhibitors, but also seem to be functionally connected. This is reflected by the higher sensitivity of TPC2-deficient, drug-resistant leukemia cells to vincristine, opening the stage for further studying the implication of TPC2 in processes related to (P-gp-mediated) chemoresistance. Summarizing, this work clearly illustrates that the endolysosomal cation channel TPC2 is a suitable target for tumor therapy. Additionally, synthetically accessible, potent TPC2 blockers were developed as promising preclinical candidates, making TPC2 a druggable protein target. Further, an implication of TPC2 and blockers of this channel in chemoresistance was uncovered, both by TPC2 promoting chemoresistance as well as by the dual action of isoquinolines on TPC2 and the drug efflux pump P-gp. Histone deacetylase 6 (HDAC6) is another protein that has gained attention as target for tumor therapy. HDAC6 is primarily located to the cytoplasm, where it deacetylates several non-histone proteins and thereby alters critical cancer-related pathways. Selective targeting of HDAC6 is aimed to reduce the toxicity associated with pan-HDAC inhibition and, along this line, we have developed and characterized potent and selective HDAC6 inhibitors (KV-46, KV-70, KV-181) with a phenothiazine system as cap group and a benzhydroxamic acid moiety as zinc-binding group. In accordance with effects of specific HDAC6 inhibition, KV-46, KV-70 and KV-181 are relatively non-toxic to healthy liver cells and moderately effective at reducing cancer cell proliferation and inducing apoptosis. Further, KV-46, KV-70 and KV-181 exposure increases the expression of critical protein markers of the unfolded protein response and the immune response, suggesting a potential benefit of combining HDAC6 inhibitors with proteasome inhibitors or immunomodulatory agents

Novel chemical tools to target two-pore channel 2, P-glycoprotein and histone deacetylase 6 in cancer

The identification of previously unknown targets as well as the development of efficacious inhibitors for known targets are key factors to make more patients benefit from tumor therapy. For instance, the role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of liver cancer cells in vitro, affects their energy metabolism and successfully abrogates tumor growth in vivo. Concurrently, we have identified novel, simplified analogues of the alkaloid tetrandrine (SG-005 and SG-094) as potent TPC2 inhibitors by screening a library of benzyltetrahydroisoquinoline derivatives using cell proliferation assays, endolysosomal patch clamp and calcium imaging. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against several cancer cell lines and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing the first TPC2 inhibitor with antitumor efficacy in mice (SG-094). Hence, our study unveils TPC2 as valid target for cancer therapy and provides novel, easily accessible tetrandrine analogues as promising option for effective pharmacological interference. Furthermore, in-depth studies were conducted to investigate a postulated mechanism of metabolic toxification of tetrandrine. A combined medicinal chemistry and cell biology approach showed that a reduction of the toxicity of tetrandrine cannot be achieved by replacing or eliminating the hypothesized metabolically instable functional group, clearly indicating that the proposed pathway is not the primary cause for the in vitro toxicity of tetrandrine and related alkaloids. Moreover, we have uncovered that the simplified tetrandrine congeners SG-005 and SG-094 additionally inhibit P-glycoprotein (P-gp), a drug efflux pump associated with multidrug resistance and treatment failure in tumor therapy. Since no approved molecules targeting P-gp are currently available, SG-005 and SG-094 might represent promising candidates to treat drug-resistant cancers owing to their favorable drug-like properties. Generally, the dual mode of action of isoquinoline-based TPC2/P-gp antagonists is mentioned here for the first time. Based on this, the known third-generation P-gp inhibitor elacridar was exemplarily studied for its potential to block TPC2, revealing another potent TPC2 blocker and thereby challenging the assumption of elacridar specifically acting on efflux pumps. Hence, on the one hand, a new lead structure (elacridar) for the development of prospective TPC2 blockers is provided. On the other hand, hints for common structural motifs on TPC2 and P-gp are given, which can facilitate the search for additional TPC2 antagonists. We further uncovered that TPC2 and P-gp do not only share mutual small molecule inhibitors, but also seem to be functionally connected. This is reflected by the higher sensitivity of TPC2-deficient, drug-resistant leukemia cells to vincristine, opening the stage for further studying the implication of TPC2 in processes related to (P-gp-mediated) chemoresistance. Summarizing, this work clearly illustrates that the endolysosomal cation channel TPC2 is a suitable target for tumor therapy. Additionally, synthetically accessible, potent TPC2 blockers were developed as promising preclinical candidates, making TPC2 a druggable protein target. Further, an implication of TPC2 and blockers of this channel in chemoresistance was uncovered, both by TPC2 promoting chemoresistance as well as by the dual action of isoquinolines on TPC2 and the drug efflux pump P-gp. Histone deacetylase 6 (HDAC6) is another protein that has gained attention as target for tumor therapy. HDAC6 is primarily located to the cytoplasm, where it deacetylates several non-histone proteins and thereby alters critical cancer-related pathways. Selective targeting of HDAC6 is aimed to reduce the toxicity associated with pan-HDAC inhibition and, along this line, we have developed and characterized potent and selective HDAC6 inhibitors (KV-46, KV-70, KV-181) with a phenothiazine system as cap group and a benzhydroxamic acid moiety as zinc-binding group. In accordance with effects of specific HDAC6 inhibition, KV-46, KV-70 and KV-181 are relatively non-toxic to healthy liver cells and moderately effective at reducing cancer cell proliferation and inducing apoptosis. Further, KV-46, KV-70 and KV-181 exposure increases the expression of critical protein markers of the unfolded protein response and the immune response, suggesting a potential benefit of combining HDAC6 inhibitors with proteasome inhibitors or immunomodulatory agents

The role of cellular chloride channels during human respiratory syncytial virus infection

Human respiratory syncytial virus (HRSV) is a common cause of respiratory tract infections (RTIs) globally. Of those infected, 25%–40% aged ≤1 year develop severe lower RTIs leading to pneumonia and bronchiolitis, with ~10% requiring hospitalisation. There is currently no HRSV vaccine and clinically approved treatments are only moderately effective. New and more effective anti-HRSV strategies are urgently required. It is established that viruses require cellular ion channels to infect cells. Ion channels are a diverse class of transmembrane proteins that selectively allow ions across membranes, influencing a multitude of cellular processes. Modulation of these channels by viruses is an important host-pathogen interaction that regulates critical stages of the virus multiplication cycle including entry, replication, and egress. Cellular chloride (Cl-) channels are large family of ion channels which were historically overlooked, however the importance of these proteins, especially within the respiratory tract, is now being revealed. This thesis examined the role of Cl- channels during HRSV infection. Utilising GFP-expressing HRSV in combination with an extensive panel of channel-specific pharmacological inhibitors, a critical requirement for calcium (Ca2+)-activated chloride channels (CaCCs) during HRSV infection was highlighted. For the first time, a role for TMEM16A as a host-factor was revealed and the channel was implicated as a post-exposure antiviral target. An investigation into the mechanisms underpinning the relationship between HRSV and TMEM16A revealed that the channel was involved at the genome replication and/or transcription stage of infection, and evidence suggested that this interaction may occur at or near the Golgi, in HRSV replication factories. Lastly, a role for TMEM16A was described within the HRSV-mediated production of antiviral protein interferon γ-induced protein 10 (IP-10), which supported a hypothesis wherein HRSV sequestered TMEM16A for genome replication, and simultaneously prevented the cellular antiviral response. Therefore, these findings have revealed TMEM16A as an exciting target for future host-directed antiviral therapeutics

Convergent activation of Ca2+ permeability in two-pore channel 2 through distinct molecular routes

TPC2 is a pathophysiologically relevant lysosomal ion channel that is activated directly by the phosphoinositide PI(3,5)P2 and indirectly by the calcium ion (Ca2+)-mobilizing molecule NAADP through accessory proteins that associate with the channel. TPC2 toggles between PI(3,5)P2-induced, sodium ion (Na+)-selective and NAADP-induced, Ca2+-permeable states in response to these cues. To address the molecular basis of polymodal gating and ion-selectivity switching, we investigated the mechanism by which NAADP and its synthetic functional agonist, TPC2-A1-N, induced Ca2+ release through TPC2 in human cells. Whereas NAADP required the NAADP-binding proteins JPT2 and LSM12 to evoke endogenous calcium ion signals, TPC2-A1-N did not. Residues in TPC2 that bind to PI(3,5)P2 were required for channel activation by NAADP but not for activation by TPC2-A1-N. The cryptic voltage-sensing region of TPC2 was required for the actions of TPC2-A1-N and PI(3,5)P2 but not for those of NAADP. These data mechanistically distinguish natural and synthetic agonist action at TPC2 despite convergent effects on Ca2+ permeability and delineate a route for pharmacologically correcting impaired NAADP-evoked Ca2+ signals

Known and novel members of the endolysosomal transportome/channelome as candidates to rescue lysosomal storage diseases (LSDs)

The project presented herein addresses our limited understanding of organellar pharmacology. Specifically, the work was conceived to elucidate the biological relevance of the endolysosomal cation channels (mucolipins/TRPMLs and two-pore channels/TPCs) using novel, selective pharmacological modulators. On one hand, we developed a first-in-field selective TRPML2 agonist, ML2-SA1, which activates the TRPML2 ion channel on early endosomes, recycling endosomes, and lysosomes. We demonstrate how TRPML2 accelerates endosomal traffic, enhancing chemokine secretion and macrophage chemoattraction. TRPML2 activity is particularly important in the rapidly recycling pathway, where it mediates cargo transit directly from sorting endosomes to the plasma membrane. This function is largely conferred by its unqiue activation by membrane stretching, a feature we have shown to rely on a single amino acid in the TRPML2 phosphoinositide binding-pocket (L314). Mutation of L314 into its TRPML1/TRPML3 counterpart (L314R) abrogates TRPML2 osmosensitivity, and impedes the rapidly recycling pathway. These findings provide biological and structural information about TRPML2 function, laying the foundation for future endeavors modulating immune cell response and inflammation through the immune cell-restricted, druggable ion channel. Our primary motivation for investigating the endolysosomal ion channels is development of new treatments for diseases currently lacking therapies. The lysosomal storage diseases (LSDs) represent one such family of diseases, where endolysosomal protein defects result in lysosomal dysfunction and (often) neurodegeneration. Mucolipidosis type IV (MLIV) is caused by dysfunction of the lysosomal TRPML1 ion channel, causing blindness and early-onset neurodegeneration. Aiming to treat LSDs such as MLIV, we investigated the related lysosomal ion channel TPC2. We characterized various TPC2 polymorphisms that increase its activity, and developed agonists for TPC2 that either facilitate high Ca2+ fluxes arresting endosomal motility or Na+ fluxes facilitating lysosomal exocytosis and enhancing autophagy. We used CRISPR/Cas9 to develop new induced pluripotent stem cell (iPSC) models for Neuronal Ceroid Lipofuscinosis (colloquially termed “childhood dementia“) and MLIV, differentiating these into cortical neurons. We used the diseased human neurons to investigate treatments for LSDs, finding the autophagic enhancer tamoxifen and the two-pore channel 2 agonist TPC2-A1-P to counteract LSD phenotypes. TPC2-A1-P restored excessive lysosomal proteolysis, storage defects, and trafficking abnormalities in human MLIV neurons and patient fibroblasts. Similarly, TPC2-A1-P ameliorated LSD phenotypes in Niemann-Pick Disease type C1 fibroblasts (NPC1, also known as childhood Alzheimer’s Disease), another LSD marked by impaired activity of lysosomal cation channels. We finally performed a proof-of-concept in vivo investigation, treating MLIV mice with TPC2-A1-P. While DMSO-treated MLIV mice exhibited gliosis of the cerebellum and hippocampus, TPC2-A1-P-injected mouse brains featured much fewer glial cells, akin to the wild-type controls. These findings demonstrate that pharmacological modulation of the endolysosomal system can restore physiology in a variety of lysosomal storage diseases in vitro and in vivo