To accelerate the discovery of novel small molecule central nervous system (CNS) positron emission tomography (PET) ligands, we aimed to define a property space that would facilitate ligand design and prioritization, thereby providing a higher probability of success for novel PET ligand development. Toward this end, we built a database consisting of 62 PET ligands that have successfully reached the clinic and 15 radioligands that failed in late-stage development as negative controls. A systematic analysis of these ligands identified a set of preferred parameters for physicochemical properties, brain permeability, and nonspecific binding (NSB). These preferred parameters have subsequently been applied to several programs and have led to the successful development of novel PET ligands with reduced resources and timelines. This strategy is illustrated here by the discovery of the novel phosphodiesterase 2A (PDE2A) PET ligand 4-(3-[(18)F]fluoroazetidin-1-yl)-7-methyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazine, [(18)F]PF-05270430 (5).

The venom of the funnel-web spider Agelenopsis aperta contains several peptides that paralyze prey by blocking voltage-sensitive calcium channels. Two peptides, omega-Aga-IVB (IVB) and omega-Aga-IVC (IVC), have identical amino acid sequences, yet have opposite absolute configurations at serine 46. These toxins had similar selectivities for blocking voltage-sensitive calcium channel subtypes but different potencies for blocking P-type voltage-sensitive calcium channels in rat cerebellar Purkinje cells as well as calcium-45 influx into rat brain synaptosomes. An enzyme purified from venom converts IVC to IVB by isomerizing serine 46, which is present in the carboxyl-terminal tail, from the L to the D configuration. Unlike the carboxyl terminus of IVC, that of IVB was resistant to the major venom protease. These results show enzymatic activities in A. aperta venom being used in an unprecedented strategy for coproduction of necessary neurotoxins that possess enhanced stability and potency.

Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular

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Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently expressed calcium channels in oocytes showed that DW13.3 had the highest affinity for alpha1A, followed by alpha1B > alpha1C > alpha1E. The affinity of DW13.3 for alpha1B N-type currents varied by 10-fold between expressed channels and native currents. Although block occurred in a similar 1:1 manner for all subtypes, DW13.3 produced a partial block of both alpha1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand omega-conotoxin MVIIC (but not omega-agatoxin IVA) from its binding site in Purkinje neurons suggests that DW13.3 binds to a site close to the pore of the channel. The inhibition of different subtypes of calcium channels by DW13.3 reflects a common "macro" binding site present on all calcium channels except T-type.