This chapter contains sections titled: Introduction Compound-Handling Challenges in Drug Discovery Acoustic Drop Ejection –Performance, Quality Assurance, and Platform Validation Acoustic-Assisted Compound Solubilization and Mixing Acoustic Applications in Drug Discovery Emerging Applications References

Compound management faces the daily challenge of providing high-quality samples to drug discovery. The advent of new screening technologies has seen demand for liquid samples move toward nanoliter ranges, dispensed by contactless acoustic droplet ejection. Within AstraZeneca, a totally integrated assay-ready plate production platform has been created to fully exploit the advantages of this technology. This enables compound management to efficiently deliver large throughputs demanded by high-throughput screening while maintaining regular delivery of smaller numbers of compounds in varying plate formats for cellular or biochemical concentration-response curves in support of hit and lead optimization (structure-activity relationship screening). The automation solution, CODA, has the capability to deliver compounds on demand for single- and multiple-concentration ranges, in batch sizes ranging from 1 sample to 2 million samples, integrating seamlessly into local compound and test management systems. The software handles compound orders intelligently, grouping test requests together dependent on output plate type and serial dilution ranges so that source compound vessels are shared among numerous tests, ensuring conservation of sample, reduced labware and costs, and efficiency of work cell logistics. We describe the development of CODA to address the customer demand, challenges experienced, learning made, and subsequent enhancements.

The effects of a novel synthetic macrocyclic polyamine (LY310315) were investigated on recombinant human N-type Ca2+ channels stabley expressed in HEK293 cells. LY310315 proved to be a potent and reversible N-type Ca2+ channel antagonist. Inhibition by this compound was dose-dependent with an IC50 of ∼0.4 μM at pH 7.35. LY310315 blocked very rapidly at all concentrations tested. Upon washout, recovery of the Ca2+ current developed with a time constant of ∼30 s. Use-dependence in the development of block indicated that voltage-dependent transitions in the channel protein were required to permit significant inhibition. Application of >100 times the IC50 dose of LY310315 to the interior of the cell produced no detectable Ca2+ current inhibition. LY310315 had no effects on the kinetics of channel activation or deactivation but did slightly slow the rate of macroscopic inactivation observed during a 300 ms test depolarisation. In the presence of LY310315 the activation curve was significantly shallower. This resulted in a shift in the activation midpoint voltage to a more depolarised levels. LY310315-induced inhibition of human N-type channels was strongly dependent on the extracellular pH, with increased potency seen upon extracellular acidification. Although most effective against N-type Ca2+ channels, LY310315 was also found to inhibit both P-type and L-type Ca2+ channels. LY310315 proved to be a weak blocker of Na+ currents, but produced ∼50% of the K+ currents of AtT20 cells at a concentration of 0.5 μM.

Use of automated synthesis led to the discovery of several 6-membered nitrogen heterocycles as replacements for the N-isoxazolyl substituent present in the 1-naphthalenesulfonamides endothelin-A (ETA) antagonist 5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesu lfo namides (BMS 182874). In each of these heterocycles, a small substituent such as halogen para to the position of attachment to the sulfonamide nitrogen atom was found to be advantageous for ETA receptor affinity. Of these heterocycles, 2-pyrazines offered the greatest scope for improving receptor affinity. Optimization of the substituents at the 3- and 5-positions in the pyrazine ring led to potent, ETA-selective compounds such as 5-(dimethylamino)-N-(5-chloro-3-methoxy-2-pyrazinyl)-1- naphthalenesulfonamides (7m, ETA pIC50 8.1). When dosed orally at 10 mg/kg to conscious, normotensive rats infused with big ET-1, compounds such as 7m showed significant inhibition of the pressor response with a duration of effect lasting for the 5-h course of the experiment.

A series of novel sulphonamides have been discovered which show high affinity and selectivity for the endothelin ETA receptor. N-Methyl-2-[4-(2-methylpropyl)phenyl]-3-(3-methoxy-5-methylpyrazin-2-ylsulfamoyl)benzamide (18) is the most widely investigated compound and is a potent antagonist in vivo when dosed either i. v. or orally, and has prolonged oral duration of action.

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The cDNAs encoding both A and B subtypes of the human endothelin receptor have been inserted into mammalian cell expression vectors that utilize the human globin gene, locus control region. These constructs have been introduced into murine erythroleukemia cells and inducible high level expression of the receptors has been achieved (approximately 1.5-pM/mg membrane protein and approximately 13,500 binding sites/cell for both receptor subtypes). Cell lines expressing these receptors were obtained on a rapid time scale (3-4 weeks), facilitated by the need for the analysis of only small numbers of cell clones/receptor (approximately 6). Competitive binding assays with endothelin-1 gave IC50s of 130 +/- 30 pM for endothelin-A receptor and 160 +/- 30 pM for endothelin-B receptor. Similar studies with the different isoforms of endothelin, sarafatoxin-S6b and -S6c, BQ123 and BQ3020, all gave the expected selectivity profiles. The IC50s for all compounds were in close agreement with those reported for native receptors. Thus, this expression system, which has several advantages over other described expression systems, is capable of rapidly providing large quantities of receptor for detailed pharmacological analyses or drug screening. In addition, the expressed receptors display the expected pharmacological profiles in the absence of any complicating, competing interactions from other subtypes or binding sites.