FIGURE 2.4 (A) ESI-FTICR mass spectrum of a mixture of three RNA targets (Ibis37, 16S, and Ibis15) with a mixture of 25 compounds. No complexes with significant ion abundances were observed. (B) ESI-FTICR mass spectrum of the original mixture, spiked with lividomycin (LV) as in (A). Note the presence of the abundant 16S-LV complex in the presence of the 25 nonbinding compounds and the specificity of the LV for the 16S RNA target vs. the Ibis37 and Ibis15 RNA targets. (Reprinted from Hofstadler and Griffey [22], used with permission. Copyright 2000 by PharmaPress Ltd., UK.)

corresponding to the noncovalent complex of the 16S RNA with lividomycin (Figure 2.4B). Under the low-concentration conditions used (2.5 |xM/target and 50 ^M/ligand) and the low likelihood of a hit (0.01% hit rate), the ligands in the mixture do not interfere with each other in forming the RNA-ligand complexes. This approach for high throughput RNA-drug screening is fully automated, including sample injection, data acquisition, data processing, and data interpretation, and is referred to as multitarget affinity/specificity screening (MASS). In this FTICR-MS experiment, the mass axis can be accurately calibrated (~1-ppm relative error) from the known multiply charged states, and elemental composition of the RNA species and the elemental compositions of the drugs present in the RNA-drug complexes can be confirmed from their exact masses. In addition, the relative binding affinities of drug candidates to RNA can be estimated from the relative abundances of the RNA-drug complex signals, single-point measurements, and can range from very strong (nanomolar) to very weak (millimolar) interactions. Key features for success in the high throughput analysis of noncovalent RNA-drug complexes is the use of volatile buffers (NH4Ac, NH4HCO3), the absence of nonvolatile cations (Na1+, K1+), and optimization of the ESI interface to operate under the most gentle condition to prevent dissociation of the noncovalent complexes. The MASS method was used to screen 100,000 compounds against three RNA targets in less than two weeks by pooling 11 compounds and three targets per well. Recently, a similar high throughput methodology was developed with ion-trap MS instrumentation [28].

An extension of the RNA-drug screening program just described is the use of the hits to identify common structural motifs [29-31]. Derivatives of these compounds are prepared and screened by MASS. Using an iterative process, a structure-activity relationship (SAR) pattern emerges that serves as a guide to elaborate higher-affinity ligands (Figure 2.5). This approach for finding higher-affinity RNA ligands with SAR and MS is referred to as SAR by MS. Despite the fact that structures and activities are correlated, MS alone cannot easily distinguish between specific and nonspecific noncovalent binding. For

Structure-activity relationships by mass spectrometry (SAR by MS) finds high-affinity ligands

RNA subdomain rA

Prepare higher affinity compounds by fusing motifs chemically

Identify new motifs that bind to RNA using MS assay

Observed SAR data provide information about pharmacophore (binding site)

Observed SAR data provide information about pharmacophore (binding site)

Prepare derivatives of interesting binders a

Re screen using MS assay

Source: Eric Swayze and coworfcsrs. Ibis Ttierapeulics

FIGURE 2.5 Schematic diagram for iteratively finding higher-affinity RNA ligands with SARs, with FTICR-MS as the detector, a technique referred to as SAR by MS. (Reprinted from Borman [29], used with permission. Copyright 2000 by the American Chemical Society.)

confirmation of these effects, NMR and X-ray crystallography methods are used.

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