The molecular mechanisms of interaction between G(s) and the A(2A) adenosine receptor were investigated using synthetic peptides corresponding to various segments of the Galpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [(3)H]2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxam idoade nosine ([(3)H]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The Galpha(s) peptides stimulated specific binding both in the presence and absence of 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS). Three peptides, Galpha(s)(378-394)C(379)A, Galpha(s)(376-394)C(379)A, and Galpha(s)(374-394)C(379)A, were the most effective. In the presence of GTPgammaS, peptide Galpha(s)(374-394)C(379)A increased specific binding in a dose-dependent fashion. However, the peptide did not stabilize the high-affinity state of the A(2A) adenosine receptor for [(3)H]CGS21680. Binding assays with a radiolabeled selective antagonist, [(3)H]5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH58261), showed that the addition of the Galpha(s) peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPgammaS, the displacement curve was right-shifted, whereas the addition of Galpha(s)(374-394)C(379)A caused a partial left-shift. Both curves were fitted by one-site models. This same Galpha(s) peptide was also able to disrupt G(s)-coupled signal transduction as indicated by inhibition of the A(2A) receptor-stimulated adenylyl cyclase activity without affecting either basal or forskolin-stimulated enzymatic activity in the same membrane preparations. Shorter peptides from Galpha(s) and Galpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide Galpha(s)(374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of Galpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.

The non‐covalent complexes between some DNA‐binding drugs and duplex oligodeoxynucleotides were studied by ionspray mass spectrometry, with the aim of evaluating the suitability of this technique to screen rapidly a series of drugs exerting their activity through non‐covalent binding to specific base sequences of DNA. Two classes of drugs were considered, distamycins (which show affinity for the minor groove of DNA) and anthracyclines (which interact through intercalation between bases). For the former, d(CGCGAATTCGCG)2 was chosen as the model oligodeoxynucleotide. Following optimization of sample preparation and instrumental conditions, the complexes of different distamycins were observed; depending on the ligand considered, 1:1 or 2:1 complexes were formed preferentially. A semi‐quantitative evaluation of the relative affinities was made by measuring the ratio of the complexes signals to those of the duplex, and also by competitive binding with equimolar amounts of distamycin. For anthracyclines, the daunorubicin–d(CGATCG)2 complex was chosen as the model for a preliminary mass spectrometric study; however, the signals of the duplex and the complex were very low compared with the monomer signal. Since the complex was known to be stable in solution, this was ascribed to gas‐phase instability, probably caused by electrostatic repulsion between negatively charged phosphate groups. © 1997 John Wiley & Sons, Ltd.

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The rapid evolution of combinatorial chemistry in recent years has led to a dramatic improvement in synthetic capabilities. The goal is to accelerate the discovery of molecules showing affinity against a target, such as an enzyme or a receptor, through the simultaneous synthesis of a great number of structurally diverse compounds. This is done by generating combinatorial libraries containing as many as hundreds or thousands of compounds. The need to test all these compounds led to the development of high-throughput screening (HTS) techniques, and also high-throughput analytical techniques capable of assessing the occurrence, structure and purity of the products. In order to be applied effectively to the characterization of combinatorial libraries, an analytical technique must be adequately sensitive (to analyse samples which are typically produced in nanomole amounts or less), fast, affordable and easy to automate (to minimize analysis time and operator intervention). Although no method alone can meet all the analytical challenges underlying this task, the recent progress in mass spectrometric (MS) instrumentation renders this technique an essential tool for scientists working in this area. We describe here relevant aspects of the use of MS in combinatorial technologies, such as current methods of characterization, purification and screening of libraries. Some examples from our laboratory deal with the analysis of pooled oligomeric libraries containing n x 324(n = 1, 2) compounds, using both on-line high-performance liquid chromatography/MS with an ion trap mass spectrometer, and direct infusion into a triple quadrupole instrument. In the first approach, MS and product ion MS/MS with automatic selection of the precursor were performed in one run, allowing library confirmation and structural elucidation of unexpected by-products. The second approach used MS scans to characterize the entire library and also precursor ion and neutral loss scans to detect selectively components with given structural characteristics.