Identification of Selective Inhibitors

The potency (IC50 values) of the hits was in the micromolar range, as typically observed for virtual screening hits [1, 39, 62], for the reasons discussed above. Compounds 1 and 2 were indeed found to have a cathepsin K-selective tendency because their potency difference is 4—15-fold higher for cathepsin K compared to cathepsin S or L (1 SR S/K 4.1 L/K 6.0, 2 SR S/K 4.4 L/K 14.7). Compound 3 had comparable IC50 values for cathepsin K, L, and S and was therefore not selective. 

The relatively low selectivity of compounds 1 and 2 was consistent with their relatively 

Compound 1 had very low structural similarity to known cathepsin inhibitors. Its maximum MACCS Tc value to reference set compounds was only 0.53. The corresponding values for compounds 2 and 3 were 0.79 and 0.80, respectively, which also indicate only limited structural similarity, below the level at which similarity in biological activity is typically expected [22]. Importantly, compound 1 contains no nitrile group and is the first cathepsin inhibitor identified lacking an electrophilic group that covalently (yet reversibly) modifies the enzyme. Hence, it is expected to act by a different noncovalent mechanism, which makes it attractive for further exploration. 

in this case, global similarity-based LEVS methods successfully identified a new inhibitory chemotype that would not have been found had the known pharmacophore constraints taken into account. 

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A yeast two-hybrid screening system was developed to screen for small molecules that inhibit the interaction of the Ras and the Raf proteins. Hyperpermeable yeast strains useful for high-throughput screening (HTS) for the two-hybrid system were created. Differential inhibition of the Ras-Raf vs the hsRPB4-hsRPB7 interaction allowed the identification of selective inhibitors. 

Several additional projects evolved from the initial TNAP project. Thanks to the broad dynamic range of the luminescent assay, several TNAP activator scaffolds were observed during HTS, resulting in an independent project with a potential therapeutic indication for osteomalacia and osteoporosis. Other human APs, intestinal (lAP) and placental (PLAP) isozymes, were initially utilized to establish counter screen assays based on CDP-star. One of the TNAP scaffolds demonstrated inhibition of lAP, and was further optimized to produce a selective chemical probe for human lAP. Later, the assays for lAP and PLAP were utilized for full-scale screening of these isozymes, eventually leading to identification of selective inhibitors for both isozymes. Interestingly, human lAP... 

S. Walker, Identification of selective inhibitors for the glycosyltransferase via high-throughput MurG screening, Chem. Biol. 2004, 11, 703-711. 

Mundt, S. et al. Development and application of a scintillation proximity assay (SPA) for identification of selective inhibitors of 1 ip-hydroxysteroid dehydrogenase type 1. Assay Drug Dev. Technol. 2005, 3, 367-375. 

Adams, J.L. and Lee, D., Recent progress towards the identification of selective inhibitors of serine/threonine protein kinases, Curr. Opin. Drug Discovery Dev., 2, 96, 1999. 

Patterson, A. W., Wood, W. J. L., Hornsby, M., Lesley, S., Spraggon, G., Ellman, J. A. Identification of selective, nonpeptidic nitrile inhibitors of cathepsin S using the substrate activity screening method. J. Med. Chem. 2006,49, 6298-6307. 

The approaches described above (Bauman et al., 2005) present a resourceintensive description of definitive UGT reaction phenotyping. Depending on need, one or more of the described steps may be omitted. The area primed for the greatest advance in the near future is the identification of selective glucuronidation inhibitors. Note that competitive substrates for individual enzymes are not necessarily selective inhibitors of those enzymes, since compounds do not need to be substrates of a UGT enzyme to be an inhibitor of that enzyme (Williams et al., 2002a). Recent advances have also been made in in silica predictions (Sorich et al., 2002). This promising area should be monitored for significant advances. 

Gschwend DA, Sirawaraporn W, Santi DV, Kuntz ID. Specificity in structure-based drug design identification of a novel, selective inhibitor of Pneumocystis carinii dihydrofolate reductase. Proteins Struct Funct Genet 1997 29 59-67. 

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