Ion Channels of Nociception

The Special Issue “Ion Channels of Nociception” contains 13 articles united by a focus on the peripheral mechanisms of pain. The content covers the mechanisms of neuropathic, inflammatory, and dental pain as well as pain in migraine and diabetes; nociceptive roles of P2X3, ASIC, Piezo and TRP channels; pain control through GPCRs and pharmacological agents; and nonpharmacological treatment with electroacupuncture

Fragment based Drug Discovery; Design and Validation of a Fragment Library; Computer-based Fragment Screening and Fragment-to-Lead Expansion

In recent years, fragment screening has become a popular approach to identify new lead structures. Fragments are usually defined by the Astex ‘rule of three’ (RO3). Surface Plasmon Resonance (SPR), Nuclear Magnetic Resonance spectroscopy (NMR), biochemical assays and X-ray crystallography are efficient screening techniques to discover prospective fragments as binders. However, these methods need an assembled fragment library. We designed an in-house fragment library, starting from approx. 380,000 commercially available fragments. During library design, we modified the RO3 and we did no strict filtering of physico-chemical properties during fragment enumeration (e.g. twice the number of H-bond acceptors was allowed). The fragments were stepwise reduced to 4,000 compounds. The last step was a visual inspection of the candidates, which lead to a final fragment library of 364 fragments. To validate the quality of the library, we screened it against endothiapepsin. The biochemical screening suggested 55 hits, which were entered into a crystallographic screen. Eleven complex crystal structures were determined, pointing out the remarkably high hit rate of the designed library. HotspotsX is a program which predicts (based on knowledge-based potentials) the probability of a certain atom type at a certain position in the binding pocket of a target enzyme. The eleven crystal structures obtained before were used to validate the program HotspotsX. Due to chemical diversity and the different binding modes of the fragments observed for the library examples we obtained binding through aromatic- , H-bond donor- , acceptor- , doneptor- and hydrophobic interactions. The calculated HotspotsX maps coincide remarkably well with the crystallographically determined fragment positions inside the binding pocket. The program HotspotsX has also been validated with crystal structures of molecular probes like phenol, urea and methylurea. Crystal structures of these molecular probes were determined with different targets. Overall, the experimental hotspot analysis coincided well with the computed contour maps. Thus, the calculated maps by HotspotsX have an excellent predictive power. Based on the binding modes of the molecular probe phenol to the cAMP-dependent protein kinase A (PKA), we started a fragment growing approach. In the latter complex, three phenol molecules are bound. Two are occupying the ATP binding site and one is sitting on top of the glycine-rich loop (G-loop). A virtual screening, using the hinge binding phenol as constraint, suggested a phenol derivative for which a crystal structure could be determined. Starting from this hit, a hotspot analysis was performed. This analysis indicates that growth in the direction of the G-loop, placing an aromatic portion under the G-loop and an acceptor functionality capable to address Lys72 is desired. The first compound of this de novo design had an affinity of 70 µM. In the following first design cycle, we were able to enhance the affinity to 6.5 µM. In the second design cycle an additional amino function was introduced, which did not improve affinity dramatically, but enhanced ligand efficiency to 0.38. In the last cycle, a spacer of one and two methylene groups was introduced and the affinity could be increased to about 110 nM for a diastereomeric mixture of four compounds. The phenol-PKA complex provides a putative allosteric site of PKA. The G-loop in this structure is in a closed state which is stabilized by two H-bonds. This G-loop conformation is probably induced by the phenol molecule sitting on top of the G-loop. Therefore, several molecular dynamics (MD) studies were performed, lacking different phenol molecules, to get insights into the G-loop opening. The MD studies suggest that after removal of the phenol sitting on top of the G-loop some first side chain movements are initiated that can indicate the first steps of the G-loop opening cascade. In a different project, a virtual screening approach was used to find new inhibitors for aldose reductase. A pre-filtered subset of the ZINC database was used as ligand dataset. For the best hit, a series of five compounds was synthesized. Among them one compound displayed an inhibition of 920 nM. The available assays to detect fragment hits are currently not sufficient. The challenges are the low affinity of the fragments and their poor solubility. Therefore, the known thermal shift assay was applied and adapted to detect fragment hits. To validate the method, it was used to characterize variant mutations of EctD. Lastly, a modeling study was used to get ideas about possible binding modes of arachidonic acid derivatives in a K+ ion channel. One predominant binding pose could not be suggested. The study proposes, however, that one arachidonic acid molecule can occupy the inner pore cavity, which is consistent with experimental data

Expression and characteristics of ion channels in osteoblasts : putative roles for TRP and K+ channels

Bone turnover is regulated by a cocktail of hormones and signalling factors controlling key cell processes such as proliferation, differentiation, mineralisation and apoptosis. Disruption to the overall mineralisation-resorption balance leads to bone disorders, such as osteoporosis - a 'silent' disease affecting around 7 million people in England and Wales. Ion channels that are presumed targets for bone signalling factors include voltage-gated K channels, ATP-dependent K channels and transient receptor potential TRP channels, and several of these channel-types reportedly have roles in cell proliferation, apoptosis, and differentiation in various tissues. This Thesis shows that human osteoblasts express a number of channels in these families, including maxi-K, ATP-dependent K channels, TRPV1 and TRPM7. The maxi-K channel, displaying characteristic electrophysiological hallmarks, is abundant in patch-clamp recordings of primary human osteoblasts implying a functional role, and the Katp agonist pinacidil is shown to promote osteoblast proliferation. Electrophysiological evidence for the TRPVI channel is not found, although the mRNA signal for a TRPVI splice variant TRPVlb may provide an answer, as it renders the channel less sensitive to capsaicin and protons. However, Ca imaging indicates that osteoblastic TRPV1 channels allow Ca2 influx, and are sensitive to 1 µM capsaicin and protons. In functional studies the TRPVI ligands capsaicin and capsazepine do not influence mineralisation, but interestingly the TRPVI agonists capsaicin, resiniferatoxin and anandamide appear to prevent differentiation of osteoblastic pre-cursor cells to adipocytes, and instead encourage maturation along the osteoblast pathway, whilst TRPV1 antagonists do not affect adipocyte differentiation. In conclusion, a number of K channels and the TRPV1 channel are expressed in osteoblasts and may have important putative roles in osteoblast cell function. Further steps are required to confirm this before the channels can be considered targets for drug development to treat bone disorders.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Expression and characteristics of ion channels in osteoblasts : putative roles for TRP and K+ channels.

Bone turnover is regulated by a cocktail of hormones and signalling factors controlling key cell processes such as proliferation, differentiation, mineralisation and apoptosis. Disruption to the overall mineralisation-resorption balance leads to bone disorders, such as osteoporosis - a 'silent' disease affecting around 7 million people in England and Wales. Ion channels that are presumed targets for bone signalling factors include voltage-gated K channels, ATP-dependent K channels and transient receptor potential TRP channels, and several of these channel-types reportedly have roles in cell proliferation, apoptosis, and differentiation in various tissues. This Thesis shows that human osteoblasts express a number of channels in these families, including maxi-K, ATP-dependent K channels, TRPV1 and TRPM7. The maxi-K channel, displaying characteristic electrophysiological hallmarks, is abundant in patch-clamp recordings of primary human osteoblasts implying a functional role, and the Katp agonist pinacidil is shown to promote osteoblast proliferation. Electrophysiological evidence for the TRPVI channel is not found, although the mRNA signal for a TRPVI splice variant TRPVlb may provide an answer, as it renders the channel less sensitive to capsaicin and protons. However, Ca imaging indicates that osteoblastic TRPV1 channels allow Ca2 influx, and are sensitive to 1 uM capsaicin and protons. In functional studies the TRPVI ligands capsaicin and capsazepine do not influence mineralisation, but interestingly the TRPVI agonists capsaicin, resiniferatoxin and anandamide appear to prevent differentiation of osteoblastic pre-cursor cells to adipocytes, and instead encourage maturation along the osteoblast pathway, whilst TRPV1 antagonists do not affect adipocyte differentiation. In conclusion, a number of K channels and the TRPV1 channel are expressed in osteoblasts and may have important putative roles in osteoblast cell function. Further steps are required to confirm this before the channels can be considered targets for drug development to treat bone disorders

Protein Structure

Since the dawn of recorded history, and probably even before, men and women have been grasping at the mechanisms by which they themselves exist. Only relatively recently, did this grasp yield anything of substance, and only within the last several decades did the proteins play a pivotal role in this existence. In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. The aim is that a non-expert can gain some appreciation for the intricacies involved, and in the current state of affairs. The expert meanwhile, we hope, can gain a deeper understanding of the topic

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

Bioinorganic Chemistry

This book covers material that could be included in a one-quarter or one-semester course in bioinorganic chemistry for graduate students and advanced undergraduate students in chemistry or biochemistry. We believe that such a course should provide students with the background required to follow the research literature in the field. The topics were chosen to represent those areas of bioinorganic chemistry that are mature enough for textbook presentation. Although each chapter presents material at a more advanced level than that of bioinorganic textbooks published previously, the chapters are not specialized review articles. What we have attempted to do in each chapter is to teach the underlying principles of bioinorganic chemistry as well as outlining the state of knowledge in selected areas. We have chosen not to include abbreviated summaries of the inorganic chemistry, biochemistry, and spectroscopy that students may need as background in order to master the material presented. We instead assume that the instructor using this book will assign reading from relevant sources that is appropriate to the background of the students taking the course. For the convenience of the instructors, students, and other readers of this book, we have included an appendix that lists references to reviews of the research literature that we have found to be particularly useful in our courses on bioinorganic chemistry

Avancées et nouvelles technologies en Toxinologie

Collection Rencontres en Toxinologie ISSN 1760-6004 ; http://sfet.asso.fr/images/stories/SFET/pdf/Ebook-RT18-2010-signets-110322.pdfInternational audienc