In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of solution-phase ligands in library mixtures that bind to solution-phase receptors. After ligands contained in a library mixture were bound to a macromolecular receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissociated using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissociation constants in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind- and-release experiments demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.

SynopsisA range of linear charge densities of the ordered and disordered forms of DNA or polynucleotides can be obtained experimentally by acid or alkaline titration, or by the investigation of unusual complexes involving protonated bases or three-stranded helices. The variation of melting temperatures with Na+ concentration for various of these systems is known and in some cases is complemented by structural and thermodynamic information. We have extended the condensation-screening theory of Manning [Biopolymers, 11, 937-955 (1972)] to these systems. The stabilizing and destabilizing effects of Na+ (condensation and screening, respectively) can be independently varied, and the theory is successful in predicting the qualitative (in some cases, quantitative) behavior that is observed. Comparison of theory and experiment indicates that the axial phosphate distance b for single-stranded polynucleotides increases with increasing pH. Values of the critical parameter [ are obtained for the various polynucleotide structures. These values are essential for an understanding of ionic effects on charged ligand-polynucleotide interactions.

A method is described whereby a family of homologues is synthesized in a one-pot reaction, without isolation or purification, and the reaction mixture is screened using a competitive binding assay based on pulsed ultrafiltration/electrospray mass spectrometry (PUF/ESMS) to tentatively identify those derivatives having the highest affinity for a target receptor. As a model system to test this approach, a synthetic scheme designed to prepare a series of analogues of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as diastereomeric mixtures, was carried out. Pulsed ultrafiltration screening of the crude reaction mixture against controls without protein detected protonated molecules corresponding to EHNA-type derivatives and three of its linear, alkyl homologues but did not show protonated molecules for an isobutyl or benzylic EHNA derivative, suggesting the latter was inactive. To verify this conclusion, we prepared E/THNA, the linear homologues, and the benzylic derivative (each as a diastereomeric mixture) and bioassayed them for them adenosine deaminase inhibition index ([I]/[S]0.5). The bioassay results for the individually synthesized analogues were in good agreement with that predicted by the observed relative ion enhancement in the PUF experiments. Thus, the PUF protocol might be used as a general method to quickly provide direction to the chemist in search of drug candidates.

Starting from the BBGKY hierarchy and using the method described in paper I, the statistical mechanics of a polyion in a dilute ionic solution is studied. It is shown that if the length of the polyion is much larger than the distance of closest approach, ’’counterion condensation’’ occurs. If the length of the polyion is comparable to the Debye length or larger, then the critical charge density is shown to be independent of the ionic strength. If the length is much smaller than the Debye length, the critical value is shown to depend logarithmically on the ionic strength and the length of the polyion. Expressions are derived for the critical charge density and the distribution of the counterions around the polyion.