MurF Lead Discovery

To efficiently identify compounds of interest for further study in biochemical or cell-based assays, the candidate ligands remaining after promiscuous compound filtering were deconvoluted in small non-mass-redundant mixtures. The eight deconvoluted hits are shown in Table 4.3, along with their enzyme inhibition values. The two best ligands (compounds 4 and 5) also are structurally related and were discovered in different initial screening mixtures. ASMS binding data is shown in Fig. 4.3. Both compounds were ionized in the negative ion mass spectrometry mode, and the characteristic halogen isotope patterns at M + 2 for the monochloro (4) and dichloro (5) functional groups are evident both in primary screening (Fig. 4.3A) and deconvolution testing (Fig. 4.3B, C). Signal intensities are much weaker in primary screening than in deconvolution, most likely because of ionization of the very low levels (>1 pmol) of several thousand nonli-gands remaining in a mixture after affinity selection. Nevertheless, the signal is still adequate so that these and other hits were selected in the primary screen. Subsequent structure-activity relationship (SAR) studies were conducted to increase the potency of this series, and several analogs with IC50 values in the 2070 nM range have been synthesized [36]. Additional biophysical studies using X-ray crystallography and NMR have confirmed the active site binding and specificity of the compound series (data not shown).

ASMS combined with a novel promiscuous ligand filtering procedure led to the discovery of a potent series of MurF inhibitors. The lead discovery methods were highly parallel, robust, and efficient. One key to success of this very straightforward screening process is the large number of compounds in each primary screening mixture. Without this feature, protein consumption would be prohib-

Table 4.3 Comparison of MurF ASMS screening-based binding constants and MurF activities from the radiolabeled phosphate release assay. Included from [10] with permission from SAGE Publications.



KD Estimate (|iM)

MurF IC50 (|uM)b

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