Identification of inhibitors of bacterial transcription/translation machinery utilizing a miniaturized 1536-well format screen.

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC(50) values derived for this assay in a 1536-well format and 384-well format.

Photoreactive analogues of prenyl diphosphates as inhibitors and probes of human protein farnesyltransferase and geranylgeranyltransferase type I.

Photoreactive analogues of prenyl diphosphates have been useful in studying prenyltransferases. The effectiveness of analogues with different chain lengths as probes of recombinant human protein prenyltransferases is established here. A putative geranylgeranyl diphosphate analogue, 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate (DATFP-FPP), was the best inhibitor of both protein farnesyltransferase (PFT) and protein geranylgeranyltransferase-I (PFFT-I). Shorter photoreactive isprenyl diphosphate analogues with geranyl and dimethylallyl moieties and the DATFP-derivative of farnesyl monophosphate were much poorer inhibitors. DATFP-FPP was a competitive inhibitor of both PFT and PGGT-I with Ki values of 100 and 18 nM, respectively. [32P]DATFP-FPP specifically photoradiolabelled the beta-subunits of both PFT and PGGT-I. Photoradiolabelling of PGGT-I was inhibited more effectively by geranylgeranyl diphosphate than farnesyl diphosphate, whereas photoradiolabelling of PFT was inhibited better by farnesyl diphosphate than geranylgeranyl diphosphate. These results lead to the conclusions that DATFP-FPP is an effective probe of the prenyl diphosphate binding domains of PFT and PGGT-I. Furthermore, the beta-subunits of protein prenyltransferases must contribute significantly to the recognition and binding of the isoprenoid substrate.

Solubilization and characterization of dehydrodolichyl diphosphate synthase from the yeast Saccharomyces carlsbergensis.

When the membrane fraction of Saccharomyces carlsbergensis was incubated with radiolabeled isopentenyl diphosphate in the presence of farnesyl diphosphate and Mg2+, phosphorylated and free long-chain polyprenols were formed. The reaction was inhibited by EDTA and heavy metal cations. A series of non-ionic detergents were studied for their efficacy to solubilize the prenyltransferase. The enzyme completely lost its activity in the presence of 0.1% of Triton X-100. n-Octyl-beta-(D)glucopyranoside at the concentration of 0.25-0.5% (10-15 mM) was used to solubilize the prenyltransferase. Both the membrane-bound enzyme and the solubilizate possessed a broad pH optimum shifted to alkaline pH values. The temperature optimum of the solubilizate was somewhat lower than that of the membrane preparation, owing to the significantly lower thermostability of the solubilized enzyme in comparison with the membrane-bound one. The phosphorylated reaction products formed in the presence of the membrane preparation had the same composition as the yeast dolichol synthesized in vivo. Non-phosphorylated polyprenols were formed during the incubation with membranes but not the solubilized enzyme. The composition of the polyprenols was also coincident with that of yeast dolichol, and the individual C80-homolog of the mixture was polyprenol but not dolichol as judged by adsorption HPLC. The results are discussed in relation to the terminal stages in the biosynthesis of dolichol derivatives.