Informatics in compound library management.

The ability to accurately and efficiently manage inventory is critical to ensure cost-effectiveness and guarantee integrity of samples in drug discovery. While many large companies utilise both customised and off-the-shelf automated systems for compound library management, these systems do not come cheaply. Without doubt, for large pharma the return on investment for one of these systems can be justified; however, the upfront cost is typically prohibitive for smaller businesses looking to stretch their limited cash reserves as far as possible. At Exelgen we have shown that for any business with the combination of fit-for-purpose informatics, relatively inexpensive laboratory hardware and well-constructed SOPs (standard operating procedures), it is possible to undertake cost-effective, large-scale compound library management in a small business environment. The informatics and hardware environment deployed at Exelgen are described in detail.

But-3-ene-1,2-diol: a mechanism-based active site inhibitor for coenzyme B12-dependent glycerol dehydratase.

Coenzyme B(12)-dependent glycerol dehydratase is a radical enzyme that catalyses the conversion of glycerol into 3-hydroxypropanal and propane-1,2-diol into propanal via enzyme-bound intermediate radicals. The substrate analogue but-3-ene-1,2-diol was studied in the expectation that it would lead to the 4,4-dihydroxylbut-2-en-1-yl radical, which is stabilised (allylic) and not reactive enough to retrieve a hydrogen atom from 5'-deoxyadenosine, thereby interrupting the catalytic cycle. Racemic and enantiomerically pure but-3-ene-1,2-diols and their [1,1-(2)H(2)], [2-(2)H] and [4,4-(2)H(2)] isotopomers were synthesised and characterised by NMR spectroscopy. (S)-[4-(14)C]but-3-ene-1,2-diol was also prepared. Kinetic measurements showed but-3-ene-1,2-diol to be a competitive inhibitor of glycerol dehydratase (K(i)=0.21 mM, k(i)=5.0x10(-2) s(-1)). With [4-(14)C]but-3-ene-1,2-diol it was demonstrated that species derived from the diol become tightly bound to the enzyme's active site, but not covalently bound, because the radioactivity could be removed upon denaturation of the enzyme. EPR measurements with propane-1,2-diol as substrate generated sharp signals after 10 s that disappeared after about 1 min. In contrast, EPR resonances appeared and disappeared more slowly when but-3-ene-1,2-diol was incubated with the enzyme. Among the deuterated isotopomers, only [1,1-(2)H(2)]but-3-ene-1,2-diol showed a significantly different EPR spectrum from that of the unlabelled diol; this indicated that coupling between the unpaired electron and a deuterium at C-1 was stronger than with deuterium at C-2 or C-4. The experiments suggest the formation of the 1,2-dihydroxybut-3-en-1-yl radical, which decomposes to unidentified product(s).

Can the peripheral blood monocyte count be used as a marker of CSF resistance to antiretroviral drugs?

Resistance to antiretroviral drugs in human immunodeficiency virus (HIV) disease may be discordant between blood and cerebrospinal fluid (CSF). However, there is no method by which patients who are likely to harbor resistant HIV in the CSF can be noninvasively identified. Activated monocytes are known to traffic through the brain and perivascular microglia are considered to "turnover" regularly from bone marrow-derived monocytes. Monocytes lack certain kinases necessary to metabolize some antiretroviral drugs, making it possible that monocytes, could deliver antiretroviral drugs to the brain. Low monocyte counts in the peripheral blood, however, would be expected to lead to decreased trafficking and turnover of monocytes, with less drug delivery to the brain. The authors hypothesized that this would increase the likelihood of drug resistant HIV in the central nervous system. To test this, 24 matching CSF and plasma samples that had been prospectively collected and stored from patients treated with nucleoside analogue reverse transcriptase inhibitor drugs were assessed for genotypic resistance. Those CSFs, with evidence of resistance mutations, were compared to those without for peripheral blood monocyte count, hemoglobin, CD4 cell count, and zidovudine (ZDV) use. The same analyses were repeated on the plasma samples. There were 11 CSFs with evidence of resistance mutations. The peripheral blood monocyte count was significantly lower in the CSF resistant group (0.29 +/- 0.16) versus (0.52 +/- 0.21) x 10(9)/L (P <.001). There was no difference between the groups according to hemoglobin, CD4 cell count, total white cell count, or use of ZDV. There was no difference between resistant and sensitive plasma samples according to peripheral blood monocyte count. To further test the hypothesis, the authors determined the concentrations of ZDV, stavudine, and abacavir in monocytes after each drug had been added to monocyte cultures. There was a significant decline in the concentration of each drug in the supernatant, implying that it had been "taken up" by the monocytes. These preliminary data suggest that peripheral blood monocytes may be important in delivery of antiretroviral drugs to the brain and the development of resistance.