Patent Review

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Ca2+ release-activated Ca2+ channel inhibitors Paolo Pevarello*1, Silvia Cainarca2, Chiara Liberati2, Paola Tarroni2, Francesco Piscitelli1 & Elda Severi1 Ca2+ release-activated Ca2+ (CRAC) channels are becoming important targets for therapeutic intervention in several areas of disease, including immunology, allergy and cancer. In parallel to the progression towards reliable methods for measuring CRAC currents and their inhibition, patents have been generated by several companies. In this Patent Review, an ana­lysis of the patents in the CRAC channel inhibition filed is presented. A discussion of the biological methods used in the patents is included. The general interest in this area is growing fast with almost 80% of the patents issued after 2010.

In cells of the immune system, Ca 2+ signals are essential for a variety of functions including differentiation, proliferation, effector function and gene transcription. The store-operated Ca 2+ current (SOCC), mediated by Ca 2+ release-activated Ca 2+ (CRAC) channels, is the predominant route of Ca 2+ flux into nonexcitable cells, including immune cells such as lymphocytes and mast cells, as well as blood platelets. Although the presence of store-operated signaling in immune cells has been known for many years, key proteins in the pathway have only recently been identified. The regulatory protein, stromal interaction molecule 1 (STIM1), which senses depletion of Ca 2+ from the endoplasmic reticulum, and Orai1, which forms the pore of the plasma membrane Ca 2+ channel, are now known to together form the CRAC channel. In lymphocytes and mast cells, antigen or Fc receptor activation causes release of Ca 2+ ions from intracellular stores leading to an influx of Ca 2+ ions through CRAC channels in the plasma membrane [1]. The resulting increase in intracellular Ca 2+ levels activates calcineurin, a phosphatase that regulates the transcription factor, nuclear factor of activated T-cells (NFAT), which translocates to the nucleus leading to an active immune response [2]. Individuals who are homozygous for a mutation (Arg91Trp) in the gene encoding Orai1 acquire a form of severe combined immune deficiency syndrome, in which T-cells fail to respond to pathogens. [3]. The therapeutic potential of inhibiting CRAC currents, termed ICRAC, has been further established by the clinical use of calcineurin inhibitors (cyclosporine A and tacrolimus) to prevent rejection of organ transplants. Deregulated Ca 2+ responses have been associated with several autoimmune and inflammatory diseases, including systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and inflammatory bowel disease [4,5]. Literature reports suggest that such molecules may also have applications in the treatment of allergic disorders, asthma, metastatic breast cancer, diabetes, cardiovascular and cerebrovascular diseases, and certain viral infections. Most relevant literature on CRAC channel has been summarized in a 2010 review [6]. Since 2010, several papers have been published: notably, in 2012, the crystal structure of Orai from Drosophila melanogaster was described and revealed that the Ca 2+ channel comprises a hexameric assembly of Orai subunits arranged around a central ion pore. [7]. However, in a subsequent paper, Thompson and Shuttleworth suggested that Orai1 is rather a tetramer [8]. In 2013, a role of CRAC

10.4155/PPA.14.7 © 2014 Future Science Ltd

Pharm. Pat. Anal. (2014) 3(2), 171–182

Axxam Discovery Chemistry, Via Castellino, 111, 80100 Napoli, Italy 2 Axxam Discovery Biology, Via Meucci, 3, 20091 Bresso, Milano, Italy *Author for correspondence: [email protected] 1

ISSN 2046-8954

171

Patent Review

Pevarello, Cainarca, Liberati, Tarroni, Piscitelli & Severi

channels in human glioblastoma invasion has been proposed [9]. Stromal interaction molecule 1: Finally, Gerasimenko et al. recently Protein that in humans is encoded described that pharmacological by the stromal interaction CRAC channel blockade in isomolecule 1 gene. lated mouse pancreatic acinar cells 2+ 2+ Orai1: Ca release-activated Ca was effective against toxic [Ca 2+]i channel protein 1, a Ca2+ selective elevation, necrosis and trypsin/proion channel that in humans is tease activity, and thus potentially encoded by the ORAI1 gene. targeting pancreatitis [10]. ICRAC: Current that flows through For this review on CRAC chanCa2+ release-activated Ca2+ nel inhibition patent activity, we channels. focus our ana­ lysis upon patents IL-2: Type of cytokine signaling (without any publication time limimolecule in the immune system. tation) featuring biological and/or It is a protein that regulates the activities of white blood cells pharmacological data for at least (leukocytes, often lymphocytes) some of the claimed compounds. that are responsible for immunity. The patent literature search was performed using SciFinder® (American Chemical Society, OH, USA), giving as an input CRAC, store-operated Ca 2+ channels, Orai and STIM. Further refinement was applied by document type (patent). Parallel inquiries were done using public (Google Patent Search™ [Google, CA, USA], Espacenet [European Patent Office, Munich, Germany] and Patent Lens [Cambia, Canberra, Australia]) patent search engines. Key terms

Biological assays reported in the patents: a critical appraisal

Measuring CRAC channel inhibition is not a trivial task and the meaning of the output of the different assays can be blurred by the inherent complexity of the target. There are several different methods to measure Ca 2+ influx mediated by CRAC channels, but the most used are patch-clamp and fluorescent systems. The conventional patch-clamp has long been considered the best approach for studying, in general, ion channel function and pharmacology, and in particular ICRAC since it measures ion channel function directly. With the most common configuration, whole-cell voltage-clamp, a specialized amplifier accurately clamps the membrane potential and measures current flow across the membrane of an entire cell. Thanks to this approach, the CRAC channels of T-lymphocytes and mast cells have been extensively characterized in terms of biophysical properties and downstream functions. It has been reported that CRAC channels are distinguished from other SOCCs by an extraordinarily high selectivity for Ca 2+ over monovalent ions (PCa/PNa > 1,000), a very low unitary conductance (

Ca(2+) release-activated Ca(2+) channel inhibitors.

Ca(2+) release-activated Ca(2+) (CRAC) channels are becoming important targets for therapeutic intervention in several areas of disease, including imm...
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