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Large quaternary ammonium (QA) ions block voltage-gated [K.sup.+] (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithio-threitol (SNDTT; [ONSCH.sub.2]CH(OH)CH(OH)[CH.sub.2]SNO) produces qualitatively similar 'open-channel block' in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not [Na.sup.+] or [Ca.sup.2+] channels. Inactivation-removed ShakerB (ShB[Delta]) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT Dose--response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule ([K.sub.d2] = 0.14 mM) is lower than that of the first molecule ([K.sub.d1] = 0.67 mM), indicating cooperativity. The half-blocking concentration ([K.sub.1/2]) is ~0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 [e.sub.0]) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction ([Tau] [approximately equals] 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2. KEY WORDS: voltage-gated potassium channels * ion channel gating * quaternary ammonium compounds * inactivation * nitrosylation

Discovery toxicology focuses on the identification of the most promising drug candidates through the development and implementation of lead optimization strategies and hypothesis-driven investigation of issues that enable rational and informed decision-making. The major goals are to [a] identify and progress the drug candidate with the best overall drug safety profile for a therapeutic area, [b] remove the most toxic drugs from the portfolio prior to entry into humans to reduce clinical attrition due to toxicity, and [c] establish a well-characterized hazard and translational risk profile to enable clinical trial designs. This is accomplished through a framework that balances the multiple considerations to identify a drug candidate with the overall best drug characteristics and provides a cogent understanding of mechanisms of toxicity. The framework components include establishing a target candidate profile for each program that defines the qualities of a successful candidate based on the intended therapeutic area, including the risk tolerance for liabilities; evaluating potential liabilities that may result from engaging the therapeutic target (pharmacology-mediated or on-target) and that are chemical structure-mediated (off-target); and characterizing identified liabilities. Lead optimization and investigation relies upon the integrated use of a variety of technologies and models (in silico, in vitro, and in vivo) that have achieved a sufficient level of qualification or validation to provide confidence in their use. We describe the strategic applications of various nonclinical models (established and new) for a holistic and integrated risk assessment that is used for rational decision-making. While this review focuses on strategies for small molecules, the overall concepts, approaches, and technologies are generally applicable to biotherapeutics.