Bettina Hinkelmann

Hepatitis C virus complete life cycle screen for identification of small molecules with pro- or antiviral activity.

Infection with the hepatitis C virus represents a global public health threat given that an estimated 170 million individuals are chronically infected and thus at risk for cirrhosis and hepatocellular carcinoma. A number of direct antiviral molecules are in clinical development. However, side effects, drug resistance and viral genotype-specific differences in efficacy may limit these novel therapeutics. Therefore, a combination of well tolerated drugs with distinct mechanisms of action targeting different steps of the viral replication cycle will likely improve viral response rates and therapy success. To identify small molecules that interfere with different steps of the HCV replication cycle, we developed a novel dual reporter gene assay of the complete HCV life cycle and adapted it to 384-well high-throughput format. The system is based on a highly permissive Huh-7 cell line stably expressing a secreted luciferase. Using these cells and an efficient HCV luciferase reporter virus, perturbations of each step of the viral replication cycle as well as cell viability can be easily and quantitatively determined. The system was validated with a selected set of known HCV entry, replication and assembly inhibitors and then utilized to screen a library of small molecules derived from myxobacteria. Using this approach we identified a number of molecules that specifically inhibit HCV cell entry, or primarily virus assembly and release. Moreover, we also identified molecules that increase viral propagation. These compounds may be useful leads for development of novel HCV inhibitors and could be instrumental for the identification of as yet unknown host-derived viral resistance and dependency factors.

Novel peptidomimetic compounds containing redox active chalcogens and quinones as potential anticancer agents.

Many types of cancer cells are associated with a disturbed intracellular redox balance and oxidative stress (OS). Among the various agents employed to modulate the intracellular redox state of cells, certain redox catalysts containing quinone and chalcogen moieties have shown considerable promise. Passerini multicomponent reaction has been developed for the synthesis of agents combining two, three or even four redox centers in one molecule in a good yield. When incubated with cancer cells these agents inhibited cell proliferation and induced apoptotic cell death. Interestingly, some of these redox active compounds exhibited quite low toxicity with normal cells. The cause was obviously OS, which was reflected by significant decrease in reduced glutathione, subsequently cell cycle arrest and induction of apoptosis.

Identification of myxobacteria-derived HIV inhibitors by a high-throughput two-step infectivity assay

BackgroundDrug-resistance and therapy failure due to drug-drug interactions are the main challenges in current treatment against Human Immunodeficiency Virus (HIV) infection. As such, there is a continuous need for the development of new and more potent anti-HIV drugs. Here we established a high-throughput screen based on the highly permissive TZM-bl cell line to identify novel HIV inhibitors. The assay allows discriminating compounds acting on early and/or late steps of the HIV replication cycle.ResultsThe platform was used to screen a unique library of secondary metabolites derived from myxobacteria. Several hits with good anti-HIV profiles were identified. Five of the initial hits were tested for their antiviral potency. Four myxobacterial compounds, sulfangolid C, soraphen F, epothilon D and spirangien B, showed EC50 values in the nM range with SI > 15. Interestingly, we found a high amount of overlapping hits compared with a previous screen for Hepatitis C Virus (HCV) using the same library.ConclusionThe unique structures and mode-of-actions of these natural compounds make myxobacteria an attractive source of chemicals for the development of broad-spectrum antivirals. Further biological and structural studies of our initial hits might help recognize smaller drug-like derivatives that in turn could be synthesized and further optimized.

The Synthesis of Novel Disorazoles

The disorazoles were first isolated by the groups of Reichenbach and H fle in 1999. As highly potent inhibitors of microtubule stabilization they have attracted considerable attention since they show biological activity at picomolar concentrations. Recently, a new member of this family, disorazole Z (1), was reported. This C2-symmetrical compound exhibits the characteristic functionalities of the other disorazoles within a 26-membered macrocycle. The obvious differences compared to other disorazoles are the smaller ring size and the ester moiety which is placed at the site of the geminal dimethyl groups and consequently introduces a quaternary chiral center (Figure 1, configuration was not assigned). As a consequence of the remarkable biological activities, a variety of different synthetic approaches were put forward with only one synthesis completed by the Wipf group. Most of the problems were associated with the construction of the conjugated polyene system and consecutive lactonization protocols. The synthetic challenge of the disorazoles can be described best by the fact that the lack of success by different research groups to complete the synthesis culminated in the speculation that the structure could have been missassigned. This synthetic challenge combined with our interest to abrogate the structure–activity relationship prompted us to initiate a “chemical editing” program aimed at synthesizing the simplified disorazole 2 (Figure 1). The major difference was that disorazole 2 lacks one C2 unit on each hemisphere, just as in disorazole Z, and the characteristic germinal dimethyl groups, framed by an anti diol and the oxazole, are present. Additionally, the construction of symmetrical disorazoles from one precursor for each hemisphere should simplify the synthetic access. Based on the existing disorazoles we decided to incorporate a Z,E,E pattern of the conjugated double bonds. Our retrosynthetic analysis placed one disconnection between C5 and C6, which could be established in the forward direction through a Wittig reaction (Scheme 1). The remaining two double bonds of the triene moiety would be realized through a vinylogous aldol reaction and an E-selective olefination. The synthesis begins with the known Kiyooka aldol reaction to construct the geminal dimethyl group and the alyllic alcohol (8 ; Scheme 2). The developing cationic center at the ester moiety is concomitantly reduced during this step and provides the appropriate oxidation state for additional transformations. The aldehyde is liberated by migration of the TBS group to the newly generated secondary alcohol (9). Next, the asymmetric vinylogous aldol reaction using the Figure 1. Disorazole C1 and its truncated analogue.

The myxobacterial metabolite ratjadone A inhibits HIV infection by blocking the Rev/CRM1-mediated nuclear export pathway

BackgroundThe nuclear export of unspliced and partially spliced HIV-1 mRNA is mediated by the recognition of a leucine-rich nuclear export signal (NES) in the HIV Rev protein by the host protein CRM1/Exportin1. This makes the CRM1-Rev complex an attractive target for the development of new antiviral drugs. Here we tested the anti-HIV efficacy of ratjadone A, a CRM1 inhibitor derived from myxobacteria.ResultsRatjadone A inhibits HIV infection in vitro in a dose-dependent manner with EC50 values at the nanomolar range. The inhibitory effect of ratjadone A occurs around 12 hours post-infection and is specific for the Rev/CRM1-mediated nuclear export pathway. By using a drug affinity responsive target stability (DARTS) assay we could demonstrate that ratjadone A interferes with the formation of the CRM1-Rev-NES complex by binding to CRM1 but not to Rev.ConclusionRatjadone A exhibits strong anti-HIV activity but low selectivity due to toxic effects. Although this limits its potential use as a therapeutic drug, further studies with derivatives of ratjadones might help to overcome these difficulties in the future.

Deciphering intracellular targets of organochalcogen based redox catalysts

Traditionally, the activity of organochalcogen based redox catalysts in cancer cells has been associated with the increase of ROS levels. Using a phenotype guided approach we were able to identify tubulin and actin as targets of tellurium containing redox catalysts. This is the first detection of a defined intracellular target of such compounds.