Ion channels are desirable therapeutic targets, yet ion channel-directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies-small, single-domain antibody fragments-may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5'-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1β (IL-1β). To further explore its function, we generated and characterized nanobodies against mouse P2X7 that effectively blocked (13A7) or potentiated (14D5) gating of the channel. Systemic injection of nanobody 13A7 in mice blocked P2X7 on T cells and macrophages in vivo and ameliorated experimental glomerulonephritis and allergic contact dermatitis. We also generated nanobody Dano1, which specifically inhibited human P2X7. In endotoxin-treated human blood, Dano1 was 1000 times more potent in preventing IL-1β release than small-molecule P2X7 antagonists currently in clinical development. Our results show that nanobody technology can generate potent, specific therapeutics against ion channels, confirm P2X7 as a therapeutic target for inflammatory disorders, and characterize a potent new drug candidate that targets P2X7.

Abstract:The long-lasting interest in bioactive molecules (namely toxins) produced by (microalga) dinoflagellates has risen in recent years. Exhibiting wide diversity and complexity, said compounds are well-recognized for their biological features, with great potential for use as pharmaceutical therapies and biological research probes. Unfortunately, provision of those compounds is still far from sufficient, especially in view of an increasing demand for preclinical testing. Despite the difficulties to establish dinoflagellate cultures and obtain reasonable productivities of such compounds, intensive research has permitted a number of advances in the field. This paper accordingly reviews the characteristics of some of the most important biotoxins (and other bioactive substances) produced by dinoflagellates. It also presents and discusses (to some length) the main advances pertaining to dinoflagellate production, from bench to large scale-with an emphasis on material published since the latest review available on the subject. Such advances encompass improvements in nutrient formulation and light supply as major operational conditions; they have permitted adaptation of classical designs, and aided the development of novel configurations for dinoflagellate growth-even though shearing-related issues remain a major challenge.

Respiratory chain complex I of the fungus Neurospora crassa contains at least 39 polypeptide subunits, of which 35 are conserved in mammals. The 11.5 kDa and 14 kDa proteins, homologues of bovine IP15 and B16.6, respectively, are conserved among eukaryotes and belong to the membrane domain of the fungal enzyme. The corresponding genes were separately inactivated by repeat-induced point-mutations, and null-mutant strains of the fungus were isolated. The lack of either subunit leads to the accumulation of distinct intermediates of the membrane arm of complex I. In addition, the peripheral arm of the enzyme seems to be formed in mutant nuo14 but, interestingly, not in mutant nuo11.5. These results and the analysis of enzymatic activities of mutant mitochondria indicate that both polypeptides are required for complex I assembly and function.

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