Counterscreens

First, in addition to the primary target, related enzymes may need to be studied as counterscreens to avoid unwanted side effects due to collateral inhibition of the... 

In order to determine whether compounds identified in the primary HTS screen are specific, a counterscreen is required to identify and eliminate false positives that will arise in the primary screen. For protein—protein interaction screens, it is preferable to test an unrelated protein pair that uses the same mode of detection. For our purposes, we adapted a previously described TR-FRET assay that monitors the interaction between bacterial Staphylococcus aureus Dnal and phage protein 77ORF104 (Liu et al., 2004). 

Eliminate inhibitors of luciferase enzyme activity or light quenchers in counterscreen... 

Compounds not eliminated by the counterscreen are re-tested in a secondary assay involving in vitro translation of FF/HCV/Ren mRNA in Krebs-2 extracts using 35S-methionine followed by SDS-PAGE analysis to monitor protein synthesis... 

We have found that many compounds identified in our screen are nonspecific inhibitors of luciferase enzyme activity. To eliminate these, we test the hits in a luciferase enzyme-based counterscreen. Firefly and renilla luciferase are produced in vitro by programming Krebs-2 extracts with FF/HCV/Ren mRNA and allowing the translations to proceed at 30° for 1 h. Ten microliters are then pipetted into a 96-well plate and compound is added to a final concentration of 20 /iM (1% DMSO). Luciferase activity is then determined as described previously in step 3. Since compound is added only after the translation reaction is complete, inhibitors of translation should not score positively in this assay. Typically, a 1-ml in vitro translation reaction is sufficient to screen 45 candidate hits in duplicate for nonspecific luciferase inhibitory activity. Compounds that inhibit in this counterscreen are eliminated from future analysis. 

Further efforts in related series afforded structurally similar DPP-4 inhibitors such as acid 13 (E. Parmee, unpublished results), thiazole 14 [35] and cyclopentylglycine 15 (Figure 17.3) [36], Although 15 did not show improved selectivity over DPP-8/9,13 and 14 showed some improvement in selectivity over these counterscreens. Nevertheless, none of these compounds exhibited sufficient selectivity to merit further pursuit zwitterion 13 also possesses low oral bioavailability in rats (F < 1%). Consequently, focus in the a-amino acid series shifted to acyclic derivatives. 

Several benzothiadiazepines, as potent and selective TACE inhibitors, have been synthesized with variation in P1 and PI and evaluated versus porcine TACE, and the initial selectivity was assessed with counterscreens of MMP-1, MMP-2, and MMP-9. Several potent and selective inhibitors were discovered, 440 being the most active against porcine TACE. Most compounds were assessed in the human peripheral blood mononuclear cell (PBMC) assay and the human whole blood assay (WBA) to determine their ability to suppress tumor necrosis factor alpha (TNF-a). Compound 441 was found to be the most potent in the PBMC assay (IC50 = 0.35 pM), while 442 was the most active in the WBA (IC50 = 1.4 pM) <2003JME1811>. 

Primary hits are usually cherry-picked for rescreening in triplicate. In order to prioritize compounds for which dry powders will be obtained for retesting, we also recommend assaying serial compound dilutions and discarding hits from further consideration that yield dose-response curves with Hill slopes far from I. In particular, compounds with very steep IC50 curves (Hill slope >4) should be avoided, because unspecific effects are likely at play. Once inhibitors have been confirmed from repurchased powders, compounds with reasonable IC50 values (e.g., <20 pM) should be tested in counterscreens against a panel of additional PTPs, in order to weed out... 

CRC, Counterscreen, Orthogonal Assay, Secondary Assay IC50... 

Finally, the imidazopyridazine series elaborated as inhibitors of PfPK7 in Bouloc et al.,4S was progressed from an initial 11 pM hit to the final compound (compound 4) of 131 nM. The most potent compounds exhibited some capacity to inhibit the proliferation of live parasites in erythrocytes, albeit at levels an order of magnitude higher than those exhibited on the enzyme. The major problem, however, was that counterscreening them on a panel of 80 Human kinases showed that they are promiscuous inhibitors of many potentially undesirable off-target kinases. 

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