An assay recapitulating the 3′ processing activity of HIV-1 integrase (IN) was used to screen the Boehringer Ingelheim compound collection. Hit-to-lead and lead optimization beginning with compound 1 established the importance of the C3 and C4 substituent to antiviral potency against viruses with different aa124/ aa125 variants of IN. The importance of the C7 position on the serum shifted potency was established. Introduction of a quinoline substituent at the C4 position provided a balance of potency and metabolic stability. Combination of these findings ultimately led to the discovery of compound 26 (BI 224436), the first NCINI to advance into a phase Ia clinical trial.

No abstract

The first line drug against leishmaniasis consists of pentavalent antimony [Sb(V)], but there is general belief that the active form of the metal is the trivalent form [Sb(III)]. In this study, we have quantified the accumulation of Sb(V) and Sb(III) in Leishmania by using inductively coupled plasma mass spectrometry. The accumulation was studied in three Leishmania species at various life stages, sensitive or resistant to antimony. Both Sb(III) and Sb(V) are accumulated in promastigote and amastigote parasites, but through competition experiments with arsenite, we found that the routes of entry of Sb(V) and Sb(III) are likely to differ in Leishmania. The level of accumulation of either Sb(III) or Sb(V), however, was not correlated with the susceptibility of wild-type Leishmania cells to antimony. This suggests that other factors may also be implicated in the mode of action of the drugs. In contrast to metal susceptibility, resistance to Sb(III) correlated well with decreased antimony accumulation. This phenotype was energy dependent and highlights the importance of transport systems in drug resistance of this protozoan parasite.

From the discovery of competitive hexapeptide inhibitors, potent and selective HCV NS3 protease macrocyclic inhibitors have been identified. Structure-activity relationship studies were performed focusing on optimizing the N-terminal carbamate and the aromatic substituent on the (4R)-hydroxyproline moiety. Inhibitors meeting the potency criteria in the cell-based assay and with improved oral bioavailability in rats were identified. BILN 2061 was selected as the best compound, the first NS3 protease inhibitor reported with antiviral activity in man.

In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes gamma-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance.