A paradigm for drug discovery employing encoded combinatorial libraries.

Very large combinatorial libraries of small molecules on solid supports can now be synthesized and each library element can be identified after synthesis by using chemical tags. These tag-encoded libraries are potentially useful in drug discovery, and, to test this utility directly, we have targeted carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) as a model. Two libraries consisting of a total of 7870 members were synthesized, and structure-activity relationships based on the structures predicted by the tags were derived. Subsequently, an active representative of each library was resynthesized (2-[N-(4-sulfamoylbenzoyl)-4'-aminocyclohexanespiro]-4-oxo-7 -hydroxy- 2,3-dihydrobenzopyran and [N-(4-sulfamoylbenzoyl)-L-leucyl]piperidine-3-carboxylic acid) and these compounds were shown to have nanomolar dissociation constants (15 and 4 nM, respectively). In addition, a focused sublibrary of 217 sulfamoylbenzamides was synthesized and revealed a clear, testable structure-activity relationship describing isozyme-selective carbonic anhydrase inhibitors.

Oxidative DNA damage and senescence of human diploid fibroblast cells.

Human diploid fibroblast cells cease growth in culture after a finite number of population doublings. To address the cause of growth cessation in senescent IMR-90 human fibroblast cells, we determined the level of oxidative DNA damage by using 8-oxoguanine excised from DNA and 8-oxo-2'-deoxyguanosine in DNA as markers. Senescent cells excise from DNA four times more 8-oxoguanine per day than do early-passage young cells. The steady-state level of 8-oxo-2'-deoxyguanosine in DNA is approximately 35% higher in senescent cells than in young cells. Measurement of protein carbonyls shows that senescent cells did not appear to have elevated protein oxidation. To reduce the level of oxidative damage, we cultured cells under a more physiological O2 concentration (3%) and compared the replicative life span to the cells cultured at the O2 concentration of air (20%). We found that cells grown under 3% O2 achieved 50% more population doublings during their lifetime. Such an extension of life span resulted from the delayed onset of senescence and elevation of growth rate and saturation density of cells at all passages. The spin-trapping agent alpha-phenyl-t-butyl nitrone (PBN), which can act as an antioxidant, also effectively delayed senescence and rejuvenated near senescent cells. The effect is dose-dependent and is most pronounced for cells at the stage just before entry into senescence. Our data support the hypothesis that oxidative DNA damage contributes to replicative cessation in human diploid fibroblast cells.

Detection of one slowly exchanging substrate water molecule in the S3 state of photosystem II.

The exchangeability of the substrate water molecules at the catalytic site of water oxidation in photosystem II has been probed by isotope-exchange measurements using mass spectrometric detection of flash-induced oxygen evolution. A stirred sample chamber was constructed to reduce the lag time between injection of H2(18)O and the detecting flash by a factor of more than 1000 compared to the original experiments by R. Radmer and O. Ollinger [(1986) FEBS Lett. 195, 285-289]. Our data show that there is a slow (t1/2 approximately 500 ms, 10 degrees C) and a fast (t1/2 <25 ms, 10 degrees C) exchanging substrate water molecule in the S3 state of photosystem II. The slow exchange is coupled with an activation energy of about 75 kJ/mol and is discussed in terms of a terminal manganese oxo ligand, while the faster exchanging substrate molecule may represent a water molecule not directly bound to the manganese center.