David L. Saussy

Experimental design for high-throughput screening

Novel methods in molecular biology and advanced technologies have given pharmaceutical research laboratories the capability to test combinatorial libraries rapidly against large numbers of potential targets. Methods to identify optimal assay conditions efficiently are very useful in the development of robotic screening assays where there are numerous variables and potential interactions between the variables. This review discusses the use of statistical experimental design in the development and optimization of high-throughput screening assays. The authors provide a brief introduction to the theoretical basis for experimental design and discuss practical aspects of using these methods in a research laboratory. Two case studies demonstrate the power of the method in solving problems in assay development and illustrate the diversity of potential applications.

Development of scintillation-proximity assays for alpha adrenoceptors

Binding assays have long been used to determine compound affinity and selectivity for various seven-transmembrane receptors. Over time, the degree of complexity has significantly reduced, whereas the throughput of the various assays has greatly increased. In this article, we detail the development of a filter-binding assay and a scintillation-proximity assay (SPA) designed to quantify a compounds affinity for the three alpha1-adrenoceptor subtypes, alpha1A, alpha1B, and alpha1D. The various components of the assays such as ease of assay performance, robustness, cost, and generation of radioactive waste are compared and contrasted. On the basis of the results, the SPA offers many advantages of high-throughput assay formats over the traditional filter-binding assay. To follow up on the success of the alpha1-adrenoceptor SPA, SPAs for the three alpha2-adrenoceptors were developed and are detailed in this article. Affinity data generated for a select number of alpha2 compounds agree with reported literature values. These assays, like those for alpha1 subtypes, are very amenable to high-throughput screening campaigns. In conclusion, scintillation-proximity assays offer significant advantages over filter-binding assays.

α1-Adrenoceptor Agonists: The Identification of Novel α1A Subtype Selective 2′-Heteroaryl-2-(phenoxymethyl)imidazolines

Abstract Novel 2′-heteroaryl-2-(phenoxymethyl)imidazolines have been identified as potent agonists of the cloned human α 1 -adrenoceptors in vitro. The nature of the 2′-heteroaryl group can have significant effects on the potency, efficacy, and subtype selectivity in this series. α 1A Subtype selective agonists have been identified.

α1-Adrenoceptor activation: A comparison of 4-(anilinomethyl)imidazoles and 4-(phenoxymethyl)imidazoles to related 2-imidazolines

Literature reports suggest that disruption of an interhelical salt bridge is critical for alpha(1)-adrenoceptor activation, and the basic amine found in adrenergic receptor ligands is responsible for the disruption. Novel 4-(anilinomethyl)imidazoles and 4-(phenoxymethyl)imidazoles are agonists of the cloned human alpha(1)-adrenoceptors in vitro, and potent, selective alpha(1A)-adrenoceptor agonists have been identified in this series. These imidazoles demonstrate similar potencies and alpha(1)-subtype selectivities as the corresponding 2-substituted imidazolines. The extremely close SAR suggests that, in spite of the large difference in basicity, these imidazoles and imidazolines may establish the same interactions to activate alpha(1)-adrenoceptors.

Synthesis and pharmacologic activity of hydroxyacetophenone-substituted benzophenone/xanthone leukotriene B4 receptor antagonists

Abstract The preparation and pharmacologic activity of two new hybrid LTB4 receptor antagonists, 1 and 2 (LY282210), are reported. These compounds are among the most potent in vitro LTB4 receptor antagonists yet described, and add new insight into the critical pharmacophores of the LTB4 receptor.