The ClC-1 chloride channel inhibitor NMD670 improves skeletal muscle function in rat models and patients with myasthenia gravis.

Myasthenia gravis (MG) is a neuromuscular disease that results in compromised transmission of electrical signals at the neuromuscular junction (NMJ) from motor neurons to skeletal muscle fibers. As a result, patients with MG have reduced skeletal muscle function and present with symptoms of severe muscle weakness and fatigue. ClC-1 is a skeletal muscle specific chloride (Cl-) ion channel that plays important roles in regulating neuromuscular transmission and muscle fiber excitability during intense exercise. Here, we show that partial inhibition of ClC-1 with an orally bioavailable small molecule (NMD670) can restore muscle function in rat models of MG and in patients with MG. In severely affected MG rats, ClC-1 inhibition enhanced neuromuscular transmission, restored muscle function, and improved mobility after both single and prolonged administrations of NMD670. On this basis, NMD670 was progressed through nonclinical safety pharmacology and toxicology studies, leading to approval for testing in clinical studies. After successfully completing phase 1 single ascending dose in healthy volunteers, NMD670 was tested in patients with MG in a randomized, placebo-controlled, single-dose, three-way crossover clinical trial. The clinical trial evaluated safety, pharmacokinetics, and pharmacodynamics of NMD670 in 12 patients with mild MG. NMD670 had a favorable safety profile and led to clinically relevant improvements in the quantitative myasthenia gravis (QMG) total score. This translational study spanning from single muscle fiber recordings to patients provides proof of mechanism for ClC-1 inhibition as a potential therapeutic approach in MG and supports further development of NMD670.

Antibodies toward Na+,HCO3--cotransporter NBCn1/SLC4A7 block net acid extrusion and cause pH-dependent growth inhibition and apoptosis in breast cancer.

BACKGROUND: Na+,HCO3--cotransporter NBCn1/Slc4a7 accelerates murine breast carcinogenesis. Lack of specific pharmacological tools previously restricted therapeutic targeting of NBCn1 and identification of NBCn1-dependent functions in human breast cancer. METHODS: We develop extracellularly-targeted anti-NBCn1 antibodies, screen for functional activity on cells, and evaluate (a) mechanisms of intracellular pH regulation in human primary breast carcinomas, (b) proliferation, cell death, and tumor growth consequences of NBCn1 in triple-negative breast cancer, and (c) association of NBCn1-mediated Na+,HCO3--cotransport with human breast cancer metastasis. RESULTS: We identify high-affinity (KD ≈ 0.14 nM) anti-NBCn1 antibodies that block human NBCn1-mediated Na+,HCO3--cotransport in cells, without cross-reactivity towards human NBCe1 or murine NBCn1. These anti-NBCn1 antibodies abolish Na+,HCO3--cotransport activity in freshly isolated primary organoids from human breast carcinomas and lower net acid extrusion effectively in primary breast cancer tissue from patients with macrometastases in axillary lymph nodes. Inhibitory anti-NBCn1 antibodies decelerate tumor growth in vivo by ~50% in a patient-derived xenograft model of triple-negative breast cancer and pH-dependently reduce colony formation, cause G2/M-phase cell cycle accumulation, and increase apoptosis of metastatic triple-negative breast cancer cells in vitro. CONCLUSIONS: Inhibitory anti-NBCn1 antibodies block net acid extrusion in human breast cancer tissue, particularly from patients with disseminated disease, and pH-dependently limit triple-negative breast cancer growth.

Experimental phasing with vanadium and application to nucleotide-binding membrane proteins.

The structure determination of soluble and membrane proteins can be hindered by the crystallographic phase problem, especially in the absence of a suitable homologous structure. Experimental phasing is the method of choice for novel structures; however, it often requires heavy-atom derivatization, which can be difficult and time-consuming. Here, a novel and rapid method to obtain experimental phases for protein structure determination by vanadium phasing is reported. Vanadate is a transition-state mimic of phosphoryl-transfer reactions and it has the advantage of binding specifically to the active site of numerous enzymes catalyzing this reaction. The applicability of vanadium phasing has been validated by determining the structures of three different protein-vanadium complexes, two of which are integral membrane proteins: the rabbit sarcoplasmic reticulum Ca2+-ATPase, the antibacterial peptide ATP-binding cassette transporter McjD from Escherichia coli and the soluble enzyme RNAse A from Bos taurus. Vanadium phasing was successful even at low resolution and despite severe anisotropy in the data. This method is principally applicable to a large number of proteins, representing six of the seven Enzyme Commission classes. It relies exclusively on the specific chemistry of the protein and it does not require any modifications, making it a very powerful addition to the phasing toolkit. In addition to the phasing power of this technique, the protein-vanadium complexes also provide detailed insights into the reaction mechanisms of the studied proteins.

Blockade of Oncogenic NOTCH1 with the SERCA Inhibitor CAD204520 in T Cell Acute Lymphoblastic Leukemia.

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL).

Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2]E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

Structures of the heart specific SERCA2a Ca2+-ATPase.

The sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca2+ and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA-stabilized E2-AlF4- form (3.3 Å) and the Ca2+-occluded [Ca2]E1-AMPPCP form (4.0 Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform-specific residues are acceptor sites for post-translational modifications. In addition, molecular dynamics simulations predict that isoform-specific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform-specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a.

Membrane-protein crystals for neutron diffraction.

Neutron macromolecular crystallography (NMX) has the potential to provide the experimental input to address unresolved aspects of transport mechanisms and protonation in membrane proteins. However, despite this clear scientific motivation, the practical challenges of obtaining crystals that are large enough to make NMX feasible have so far been prohibitive. Here, the potential impact on feasibility of a more powerful neutron source is reviewed and a strategy for obtaining larger crystals is formulated, exemplified by the calcium-transporting ATPase SERCA1. The challenges encountered at the various steps in the process from crystal nucleation and growth to crystal mounting are explored, and it is demonstrated that NMX-compatible membrane-protein crystals can indeed be obtained.

Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering.

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D2 O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca2+ ATPase (SERCA). In all, the synthesis of isotope-substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to nonspecialists.

Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response.

Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 µm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca2+ transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2-4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover, knockdown of two key store-operated Ca2+ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca2+ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca2+ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca2+ drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca2+ levels and SOCE are not required.

Crystal Structure of the Vanadate-Inhibited Ca(2+)-ATPase.

Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca(2+)-ATPase with bound vanadate in the absence of Ca(2+). Vanadate is bound at the catalytic site as a planar VO3(-) in complex with water and Mg(2+) in a dephosphorylation transition-state-like conformation. Validating bound VO3(-) by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl(-) site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca(2+)-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3(-) structure with that in E2·BeF3(-) (E2P ground state analog) reveals multiple binding modes to the Ca(2+)-ATPase.

Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases.

MOTIVATION: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g. antibiotics. There is thus an interest in predicting the compound synthesized by an NRPS from its primary structure (amino acid sequence) alone, as this would enable an in silico search of whole genomes for NRPS enzymes capable of synthesizing potentially useful compounds. RESULTS: NRPS synthesis happens in a conveyor belt-like fashion where each individual NRPS module is responsible for incorporating a specific substrate (typically an amino acid) into the final product. Here, we present a new method for predicting substrate specificities of individual NRPS modules based on occurrences of motifs in their primary structures. We compare our classifier with existing methods and discuss possible biological explanations of how the motifs might relate to substrate specificity. AVAILABILITY AND IMPLEMENTATION: SEQL-NRPS is available as a web service implemented in Python with Flask at http://services.birc.au.dk/seql-nrps and source code available at https://bitbucket.org/dansondergaard/seql-nrps/. CONTACT: micknudsen@gmail.com or cstorm@birc.au.dk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Isolation of H(+),K(+)-ATPase-enriched Membrane Fraction from Pig Stomachs.

Gastric H(+),K(+)-ATPase is an ATP-driven proton pump responsible for the acid secretion. Here, we describe the procedure for the isolation of H(+),K(+)-ATPase-enriched membrane vesicle fractions by Ficoll/sucrose density gradient centrifugation. Further purification by SDS treatment of membrane fractions is also introduced. These procedures allow us to obtain purified protein preparations in a quantity of several tens of milligrams, with the specific activity of ~480 µmol/mg/h. High purity and stability of H(+),K(+)-ATPase in the membrane preparation enable us to evaluate its detailed biochemical properties, and also to obtain 2D crystals for structural analysis.

Preparation of Ca(2+)-ATPase1a Enzyme from Rabbit Sarcoplasmic Reticulum.

The SERCA calcium ATPase, probably the most well-investigated membrane protein both from a biophysical and structural view, can be purified from native source in substantial quantities, making it a favorable target when conducting biochemical experiments and structure determination, e.g., by X-ray crystallography.

ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay.

Enzymatic coupled assays are usually based on the spectrophotometric registration of changes in NADH/NAD(+) or NADPH/NADP(+) absorption at 340 nm accompanying the oxidation/reduction of reactants that by dehydrogenases and other helper enzymes are linked to the activity of the enzymatic reaction under study. The present NADH-ATP-coupled assay for ATPase activity is a seemingly somewhat complicated procedure, but in practice adaptation to performance is easily acquired. It is a more safe and elegant method than colorimetric methods, but not suitable for handling large number of samples, and also presupposes that the activity of the helper enzymes is not severely affected by the chemical environment of the sample in which it is tested.

Coupling Ratio for Ca(2+) Transport by Calcium Oxalate Precipitation.

The SERCA isoform 1a is constructed to transport 2 Ca(2+) ions across the sarcoplasmic reticulum membrane coupled to the hydrolysis of one molecule of MgATP. However, observed coupling ratios for Ca(2+) transported/ATP hydrolzyed are usually less than 2:1, since part of the Ca(2+) accumulated at high intravesicular concentrations by the active transport of Ca(2+) leaks out of the vesicles because of Ca(2+)-induced Ca(2+) exchange. However, in the presence of a high concentration of oxalate (5 mM) Ca(2+) will precipitate as Ca-oxalate inside the vesicles and thereby be prevented from leaking out and, in addition, this treatment will reduce the intravesicular free concentration of Ca(2+) to a level where optimal coupling ratios of 2:1 can be achieved.

Tryptophan Fluorescence Changes Related to Ca(2+)-ATPase Function.

Fluorescence measurements as monitored with either extrinsic or intrinsic probes constitute important ways with which to study the molecular properties of macromolecules. With high-quality spectrofluorimeters, it is, e.g., possible kinetically to follow local conformational changes, induced by ligands and inhibitors, with a sensitivity that is unsurpassed by any other physicochemical technique. We demonstrate here with Ca(2+) and two specific inhibitors of SERCA how this can be done by measurements of the intrinsic fluorescence of the tryptophan residues of SERCA.

Ca(2+) Binding and Transport Studied with Ca(2+)/EGTA Buffers and (45)Ca(2+).

The chapter describes procedures useful for determination of Ca(2+) binding by membranous Ca(2+)-ATPase based on the correction for the removal of Ca(2+) present in a non-bound state in the suspension medium. This is done by a filtration procedure that retains the membranous material on the Millipore filters. With suitable sucking devices it is possible to gently remove without dehydration nearly all medium from the Ca(2+) containing membranes, except that required for wetting of the filters on which they are deposited. Correction for this effect can be done with a double-filter where the radioactive content of the lower (protein-free) filter is subtracted from that present in the upper filter for calculation of Ca(2+) binding. This methodology can be used to study the effect of inhibitors on Ca(2+) binding and -transport, and with Ca(2+)/EGTA buffers to explore the Ca(2+) binding affinities and cooperative aspects of the two transport sites.

Lipid Exchange by Ultracentrifugation.

Lipids play an important role in maintaining P-type ATPase structure and function, and often they are crucial for ATPase activity. When the P-type ATPases are in the membrane, they are surrounded by a mix of different lipid species with varying aliphatic chain lengths and saturation, and the complex interplay between the lipids and the P-type ATPases are still not well understood. We here describe a robust method to exchange the majority of the lipids surrounding the ATPase after solubilisation and/or purification with a target lipid of interest. The method is based on an ultracentrifugation step, where the protein sample is spun through a dense buffer containing large excess of the target lipid, which results in an approximately 80-85 % lipid exchange. The method is a very gently technique that maintains protein folding during the process, hence allowing further characterization of the protein in the presence of a target lipid of interest.

Structural studies of P-type ATPase-ligand complexes using an X-ray free-electron laser.

Membrane proteins are key players in biological systems, mediating signalling events and the specific transport of e.g. ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein-ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

Discovery of Tricyclic Clerodane Diterpenes as Sarco/Endoplasmic Reticulum Ca(2+)-ATPase Inhibitors and Structure-Activity Relationships.

Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 µM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented.