Small-molecule Blockers Research Articles

Small molecule blockers reveal an important role for Orai1-NFAT-orphan receptor pathway in Th17 differentiation (P5169)

Abstract Orai1 is the pore subunit of the Calcium Release Activated Channels (CRAC) that activate downstream calcineurin-NFAT pathway. Loss of function mutation in Orai1 causes lethality due to severe defects in immune system, which can be rescued by bone marrow transplantation. Using a high throughput chemical library screening, we have identified a class of small molecule blockers that inhibit Orai1. One of the small molecules, compound 5D blocked the activity of a constitutively active mutant of Orai1, suggesting a direct block of ion permeation via Orai1. Primary murine Th17 cells showed highest sensitivity to block by this compound. Treatment with compound 5D not only suppressed cytokine production, but also inhibited differentiation of Th17 cells by suppression of the retinoic-acid-receptor-related orphan receptors RORα and RORγt. Exogenous expression of RORα, RORγt or a constitutive active mutant of NFAT (CA-NFAT) rescued the blocking effect, identifying the Orai1-NFAT- ROR signaling pathway to be important for Th17 differentiation. The defects in cytokine production and differentiation were recapitulated in Th17 cells from Orai1-deficient mice suggesting Orai1 as a specific target of the blockers. In vivo administration of Orai1 blockers effectively delayed the onset and reduced the severity of autoimmune disease in mice. These data indicate that derivatives of compound 5D can be used as chemical templates for development of therapeutic agents to alleviate autoimmune diseases.

Z160: A potent and state-dependent, small molecule blocker of N-type calcium channels effective in nonclinical models of neuropathic pain

N-type voltage-gated calcium channels are validated targets for chronic and neuropathic pain, yet the only approved pharmacological agent that directly modulates this target demonstrates significant limitations. Ziconotide potently blocks the channel, but lack of selectivity for nociceptive N-type channels, including effects on peripheral N-type channels that regulate the cardiovascular system, results in severe side effects when administered systemically. Gabapentin and pregabalin, through interaction with the channel alpha-2-delta subunit, are thought to reduce the number and function of calcium channels at synaptic terminals.

Characterization of selective Calcium-Release Activated Calcium channel blockers in mast cells and T-cells from human, rat, mouse and guinea-pig preparations

Loss of function mutations in the two key proteins which constitute Calcium-Release Activated Calcium (CRAC) channels demonstrate the critical role of this ion channel in immune cell function. The aim of this study was to demonstrate that inhibition of immune cell activation could be achieved with highly selective inhibitors of CRAC channels in vitro using cell preparations from human, rat, mouse and guinea-pig. Two selective small molecule blockers of CRAC channels; GSK-5498A and GSK-7975A were tested to demonstrate their ability to inhibit mediator release from mast cells, and pro-inflammatory cytokine release from T-cells in a variety of species. Both GSK-5498A and GSK-7975A completely inhibited calcium influx through CRAC channels. This led to inhibition of the release of mast cell mediators and T-cell cytokines from multiple human and rat preparations. Mast cells from guinea-pig and mouse preparations were not inhibited by GSK-5498A or GSK-7975A; however cytokine release was fully blocked from T-cells in a mouse preparation. GSK-5498A and GSK-7975A confirm the critical role of CRAC channels in human mast cell and T-cell function, and that inhibition can be achieved in vitro. The rat displays a similar pharmacology to human, promoting this species for future in vivo research with this series of molecules. Together these observations provide a critical forward step in the identification of CRAC blockers suitable for clinical development in the treatment of inflammatory disorders.

Obstructing toxin pathways by targeted pore blockage.

The focus of this review is on the biophysical studies of the channel-forming bacterial toxins that shed light upon the mechanisms of their toxicity and propose new approaches to block their virulent action. Extensive data on the structural features of these toxins, the mechanisms of their secretion, proteolytic activation, extracellular receptors, enzymatic intracellular action, and cellular responses will be mostly omitted here. We refer our reader to several excellent specialized books and reviews that cover this material in the finest detail1–15. 1.1. Channel-blocking and channel-forming toxins In the course of evolution, Nature created numerous toxins, which selectively target ion channels of excitable cells16–18. Regardless of which version of Cleopatra's self-poisoning is true, the toxin-triggered modification of channel function was certainly involved. She either suffered from intoxication following blockage of her ligand-gated channels by the Egyptian cobra venom or from poisonous action of the chloride channel inhibitor extracted from cicuta. Numerous studies of the neurotoxins' biological functions allowed not only for deeper understanding of the structural and functional features of several channels of excitable membranes, but also for developing the pharmaceutical approaches to use these toxins for therapeutic purposes. Conversely, among the great variety of virulence factors secreted by different organisms, there is a significant group of toxins (mostly bacterial) that, instead of blocking channels, are able to form ion-conductive pores in membranes of the targeted cells. For most of them, there are no antidotes or antitoxins developed and approved for human use. At the same time, one of the possible ideas to target the bacterial exotoxins is quite simple. Following Nature, which created the channel-inhibiting toxins, it should be possible to design the potent antitoxins specifically blocking the conductive pathways of the channel-forming toxins with an ultimate goal to defend from the cytotoxic action of these agents. This idea is neither preposterous nor new. Indeed, amantadine, the small-molecule blocker of the tetrameric proton-selective M2 channel from the viral envelope of influenza A virus19–21 had been approved for human use back in 1965. Interestingly, even though amantadine is no longer recommended by the CDC for use to treat seasonable influenza due to developed antidrug resistance, the exact mechanism of the amantadine interaction with the M2 channel is still a matter of debates22–29. Even so, the M2 channel is the main target for virtual screening for powerful channel blockers30–34. Therefore, the idea to design and develop the effective blockers of the channels formed by bacterial toxins in target membranes enjoys a well-appreciated attention. Not being able to use the evolutionary path, we can employ the modern single-molecule biophysical approaches to study the pore-forming toxins and their potential inhibitors.

Enzymatic deglycosylation converts pathogenic neuromyelitis optica anti–aquaporin‐4 immunoglobulin G into therapeutic antibody

Neuromyelitis optica (NMO) is caused by binding of pathogenic autoantibodies (NMO-immunoglobulin G [IgG]) to aquaporin-4 (AQP4) on astrocytes, which initiates complement-dependent cytotoxicity (CDC) and inflammation. We recently introduced mutated antibody (aquaporumab) and small-molecule blocker strategies for therapy of NMO, based on prevention of NMO-IgG binding to AQP4. Here, we investigated an alternative strategy involving neutralization of NMO-IgG effector function by selective IgG heavy-chain deglycosylation with bacteria-derived endoglycosidase S (EndoS). Cytotoxicity and NMO pathology were measured in cell and spinal cord slice cultures, and in mice exposed to control or EndoS-treated NMO-IgG. EndoS treatment of NMO patient serum reduced by >95% CDC and antibody-dependent cell-mediated cytotoxicity, without impairment of NMO-IgG binding to AQP4. Cytotoxicity was also prevented by addition of EndoS after NMO-IgG binding to AQP4. The EndoS-treated, nonpathogenic NMO-IgG competitively displaced pathogenic NMO-IgG bound to AQP4, and prevented NMO pathology in spinal cord slice culture and mouse models of NMO. EndoS deglycosylation converts pathogenic NMO-IgG autoantibodies into therapeutic blocking antibodies. EndoS treatment of blood may be beneficial in NMO, and may be accomplished, for example, by therapeutic apheresis using surface-immobilized EndoS.

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction.

Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.

Clinical studies with chemokine receptor-5 (CCR5)-inhibitors

To summarise recently published clinical studies of chemokine receptor-5 (CCR5)-blockers, including the small-molecule blocker, maraviroc (MVC) and CCR5-monoclonal antibodies for HIV. MVC may have immunomodulating properties through CCR5-blockade. MVC appears well tolerated and penetrates the central nervous system. For these reasons, MVC is being investigated in immunodiscordance, prevention of IRIS and in HCV-HIV co-infection. Novel techniques allow tropism assignment via sequencing of proviral DNA; this testing platform is being utilised in MVC switch studies in those with HIV viraemia below the level of quantification. MVC is being utilised in regimen intensification studies for HIV associated neurocognitive disease. MVC has no anti-inflammatory activity in rheumatoid arthritis. MVC appears well tolerated in hepatitis virus co-infected patients and MVC-intensification in HCV-HIV co-infection suggests a favourable impact on liver fibrosis. Early pilot data suggests MVC intensification may have functional benefit in the CNS. There is a growing body of data on tropism testing using proviral DNA; this technology is being utilised in MVC switch studies. CCR5-monoclonal antibodies administered subcutaneously are promising in Phase II development. The place of MVC as an anti-HIV drug in the switch setting and as an immunomodulator is yet to be fully determined.

Locating a Plausible Binding Site for an Open-Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator

High-throughput screening has led to the identification of small-molecule blockers of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, but the structural basis of blocker binding remains to be defined. We developed molecular models of the CFTR channel on the basis of homology to the bacterial transporter Sav1866, which could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker such as N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101), which is thought to act by entering the channel from the extracellular side. As a more-stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual, ligand-docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest-scoring docked position was near two pore-lining residues, Phe337 and Thr338, and the rates of reactions of anionic, thiol-directed reagents with cysteines substituted at these positions were slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (Phe342) was replaced with alanine, the apparent affinity of the blocker was increased ∼200-fold. A molecular mechanics-generalized Born/surface area analysis of GlyH-101 binding predicted that substitution of Phe342 with alanine would substantially increase blocker affinity, primarily because of decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

Small-molecule inhibitors of NMO-IgG binding to aquaporin-4 reduce astrocyte cytotoxicity in neuromyelitis optica.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of spinal cord and optic nerve caused by pathogenic autoantibodies (NMO-IgG) against astrocyte aquaporin-4 (AQP4). We developed a high-throughput screen to identify blockers of NMO-IgG binding to human AQP4 using a human recombinant monoclonal NMO-IgG and transfected Fisher rat thyroid cells stably expressing human M23-AQP4. Screening of ∼60,000 compounds yielded the antiviral arbidol, the flavonoid tamarixetin, and several plant-derived berbamine alkaloids, each of which blocked NMO-IgG binding to AQP4 without affecting AQP4 expression, array assembly, or water permeability. The compounds inhibited NMO-IgG binding to AQP4 in NMO patient sera and blocked NMO-IgG-dependent complement- and cell-mediated cytotoxicity with IC(50) down to ∼5 μM. Docking computations identified putative sites of blocker binding at the extracellular surface of AQP4. The blockers did not affect complement-dependent cytotoxicity caused by anti-GD3 antibody binding to ganglioside GD3. The blockers reduced by >80% the severity of NMO lesions in an ex vivo spinal cord slice culture model of NMO and in mice in vivo. Our results provide proof of concept for a small-molecule blocker strategy to reduce NMO pathology. Small-molecule blockers may also be useful for other autoimmune diseases caused by binding of pathogenic autoantibodies to defined targets.

Identification and Evaluation of Inhibitors of Orai Channels using a Novel Methodology

Store operated calcium channels (SOCs) play a central role in regulating calcium concentrations in non-excitable cells and are critical for many physiological processes including activation of the immune system. SOCs in lymphocytes are mediated by the ion channel Orai that is activated by the endoplasmic reticulum protein STIM through direct binding. Pharmacological blockers of Orai and STIM would be useful tools for basic research in addition to providing potential treatments for autoimmune disorders. We have developed a novel methodology for generating SOC blockers based on identification and targeting the functional domains of STIM and Orai. We have identified a peptide blocker of Orai that is a potent and specific inhibitor of the channel and a small molecule blocker that can inhibit the channel at low micromolar concentrations. We have shown that the small molecule blocker can inhibit T cell activation both in vitro and in vivo. These studies provide an important set of tools for studying STIM and Orai and lead compounds that could be developed into clinically useful agents.

Design, Synthesis, and Structure−Activity Relationship Exploration of 1-Substituted 4-Aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-one Analogues as Inhibitors of the Annexin A2−S100A10 Protein Interaction

S100 proteins are small adaptors that regulate the activity of partner proteins by virtue of direct protein interactions. Here, we describe the first small molecule blockers of the interaction between S100A10 and annexin A2. Molecular docking yielded candidate blockers that were screened for competition of the binding of an annexin A2 peptide to S100A10. Several inhibitory clusters were identified with some containing compounds with potency in the lower micromolar range. We chose 3-hydroxy-1-(2-hydroxypropyl)-5-(4-isopropylphenyl)-4-(4-methylbenzoyl)-1H-pyrrol-2(5H)-one (1a) as a starting point for structure−activity studies. These confirmed the hypothetical binding mode from the virtual screen for this series of molecules. Selected compounds disrupted the physiological complex of annexin A2 and S100A10, both in a broken cell preparation and inside MDA-MB-231 breast cancer cells. Thus, this class of compounds has promising properties as inhibitors of the interaction between annexin A2 and S100A10 and may help to elucidate the cellular function of this protein interaction.

Getting a GRP on asthma

Duke researchers have shown that a small molecule blocker of GRP signaling could attenuate asthma symptoms with an overall effect that is stronger and longer lasting than that of dexamethasone. The group now is working to elucidate the mechanism of their small molecule candidate.

Identification of phase-I metabolites and chronic toxicity study of the Kv1.3 blocker PAP-1 (5-(4-phenoxybutoxy)psoralen) in the rat

PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in preclinical development for psoriasis. This study was undertaken to identify the major phase I metabolites of PAP-1 in Sprague-Dawley (SD) rats.Five phase I metabolites, that is 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)butoxy]psoralen (M3), 5-[4-(3-hydroxyphenoxy)butoxy]psoralen (M4), and 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated from the bile of rats and identified by mass spectrometry and NMR spectroscopy. The last four metabolites are new compounds.Incubation of PAP-1 with SD rat liver microsomes rendered the same five major metabolites in a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner suggesting that cytochrome P450 (CYP) enzymes are involved in PAP-1 metabolism. Inhibitors of rat CYP1A1/2 (alpha-naphthoflavone) and CYP3A (ketoconazole) but not CYP2D6 (quinidine), CYP2E (diethyldithiocarbamate), or CYP2C9 (sulphaphenazole) blocked the metabolism of PAP-1 in rat microsomes.Of the five metabolites M3, M4, and M5 were found to inhibit Kv1.3 currents with nanomolar IC50s, while M1 and M2 were inactive. Our results identified the Kv1.3-inactive M1 as the major phase I metabolite, and suggest that hydroxylation and O-dealkylation are the major pathways of PAP-1 metabolism.We further conducted a 6-month repeat-dose toxicity study with PAP-1 at 50 mg/kg in both male and female Lewis rats and did not observe any toxic effects.

Use of Kv1.3 Blockers for Inflammatory Skin Conditions

Recent results using animal models of inflammatory skin conditions have shown that blockers of the voltage-gated potassium channel, Kv1.3 hold great promise for clinical utility. Kv1.3 blockers act as immunosuppressants by modulating the various subsets of inflammatory T and B cells involved in autoimmune disorders. While peptidic inhibitors based on naturally occurring venoms demonstrate potent and selective Kv1.3 blockade, these require parenteral administration and may face potential immunogenicity problems. Small molecule blockers show considerable diversity, however selectivity over other Kv1-family channels has been difficult to achieve. More recent advances have added to the evidence that Kv1.3 channels are a suitable therapeutic target and that the development of novel and selective agents will herald new drugs for inflammatory skin disorders.

Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (K(Ca)2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K(Ca)2.2 and K(Ca)2.3 with the same high affinity (K(D) approximately 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC(50) values of approximately 100 pM and approximately 5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K(Ca)2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.

Engineering Proteins for Custom Inhibition of CaVChannels

The influx of Ca(2+) ions through voltage-dependent calcium (Ca(V)) channels links electrical signals to physiological responses in all excitable cells. Not surprisingly, blocking Ca(V) channel activity is a powerful method to regulate the function of excitable cells, and this is exploited for both physiological and therapeutic benefit. Nevertheless, the full potential for Ca(V) channel inhibition is not being realized by currently available small-molecule blockers or second-messenger modulators due to limitations in targeting them either to defined groups of cells in an organism or to distinct subcellular regions within a single cell. Here, we review early efforts to engineer protein molecule blockers of Ca(V) channels to fill this crucial niche. This technology would greatly expand the toolbox available to physiologists studying the biology of excitable cells at the cellular and systems level.

The K+ channels K(Ca)3.1 and K(v)1.3 as novel targets for asthma therapy.

Asthma affects 10% of the UK population and is an important cause of morbidity and mortality at all ages. Current treatments are either ineffective or carry unacceptable side effects for a number of patients; in consequence, development of new approaches to therapy are important. Ion channels are emerging as attractive therapeutic targets in a variety of non-excitable cells. Ion channels conducting K(+) modulate the activity of several structural and inflammatory cells which play important roles in the pathophysiology of asthma. Two channels of particular interest are the voltage-gated K(+) channel K(v)1.3 and the intermediate conductance Ca(2+)-activated K(+) channel K(Ca)3.1 (also known as IK(Ca)1 or SK4). K(v)1.3 is expressed in IFNgamma-producing T cells while K(Ca)3.1 is expressed in T cells, mast cells, macrophages, airway smooth muscle cells, fibroblasts and epithelial cells. Both channels play important roles in cell activation, migration, and proliferation through the regulation of membrane potential and calcium signalling. We hypothesize that K(Ca)3.1- and/or K(v)1.3-dependent cell processes are one of the common denominators in asthma pathophysiology. If true, these channels might serve as novel targets for the treatment of asthma. Emerging evidence lends support to this hypothesis. Further validation through the study of the role that these channels play in normal and asthmatic airway cell (patho)physiology and in vivo models will provide further justification for the assessment of small molecule blockers of K(v)1.3 and K(Ca)3.1 in the treatment of asthma.

Small molecule blockers of the Alzheimer Abeta calcium channel potently protect neurons from Abeta cytotoxicity.

Alzheimer's disease (AD) is a common, chronic neurodegenerative disease that is thought to be caused by the neurotoxic effect of the Amyloid beta peptides (Abeta). We have hypothesized that the intrinsic Abeta calcium channel activity of the oligomeric Abeta polymer may be responsible for the neurotoxic properties of Abeta, and that Abeta channel blockers may be candidate AD therapeutics. As a consequence of a rational search paradigm based on the model structure of the Abeta channel, we have identified two compounds of interest: MRS2481 and an enatiomeric species, MRS2485. These are amphiphilic pyridinium salts that both potently block the Abeta channel and protect neurons from Abeta toxicity. Both block the Abeta channel with similar potency (approximately 500 nM) and efficacy (100%). However, we find that inhibition by MRS2481 is easily reversible, whereas inhibition by MRS2485 is virtually irreversible. We suggest that both species deserve consideration as candidates for Alzheimer's disease drug discovery.

High-throughput analysis of drug binding interactions for the human cardiac channel, Kv1.5

The voltage-gated potassium channel Kv1.5 is one of the key regulators of membrane potential repolarization in human atrial myocytes and is considered a potential drug target to treat atrial fibrillation. In this study we sought to determine molecular mechanism of action of DPO-1, a diphenylphosphine oxide derivative recently shown to terminate experimental atrial arrhythmia without affecting ventricular refractory period. In addition, we provided similar analysis for additional two small molecule blockers, representing different structural classes: cyclohexanones (PAC) and nor-triterpenoids (correolide). To rapidly identify the residues within the Kv1.5 channel critical for blocking activity of these molecules, two functional high-throughput ion channel assays were employed together with site-directed mutagenesis. Our study revealed that the residues critical for blocking activity of for DPO-1 include T480, localized at the outer mouth of the pore, and two residues along S6 helix: V505 and I508. The overlapping site was identified for PAC and included residues T480 and V505. In contrast to DPO-1, the I508A mutation resulted in only a modest reduction in the block of Kv1.5 by PAC (9-fold). Correolide, the largest molecule examined, made widespread interactions along the entire length of the pore (from T480 to V516). In summary, we have identified multiple residues involved in forming high affinity binding site for Kv1.5 blockers. Similar approaches of high-throughput ion channel technologies, combined with site-directed mutagenesis, may allow for parallel, rapid and accurate analysis of ion channel interactions with multiple compounds and could facilitate the design of more potent and selective ion channel blockers.

Uncovering the TREM-1-TLR connection

the innate immune system has several large genetically encoded receptor families that interact during the detection and elimination of pathogens. The Toll-like receptor (TLR) family recognizes a diverse group of evolutionarily conserved molecules including lipopolysaccharide (LPS), peptidoglycan,