Tod P. Holler

Trypanosoma brucei glycogen synthase kinase-3, a target for anti-trypanosomal drug development: a public-private partnership to identify novel leads.

Background Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3β (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT. Methodology/Principal Findings A subset of over 16,000 inhibitors of HsGSK-3 β from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed. Conclusions/Significance We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds.

Nonpeptidic HIV protease inhibitors: 6-Alkyl-5, 6-dihydropyran-2-ones possessing achiral 3-(4-amino/carboxamide-2-t-butyl, 5-methylphenyl thio) moiety: Antiviral activities and pharmacokinetic properties

Dihydropyran-2-ones possessing amino and carboxamide functionalities on 3-SPh (2-tert-butyl, 5-methyl) ring were synthesized and evaluated for their antiviral activities. Both the enantiomers of inhibitor 15 were synthesized. The in vitro resistance profile, inhibitory activities against cytochrome P450 isozymes and pharmacokinetic properties of inhibitor 15S will be discussed.

Structural biology approaches to antibacterial drug discovery.

Antibacterial drug discovery has undertaken a major experiment in the 12 years since the first bacterial genomes were sequenced. Genome mining has identified hundreds of potential targets that have been distilled to a relatively small number of broad-spectrum targets (‘low-hanging fruit’) using the genetics tools of modern microbiology. Prosecuting these targets with high-throughput screens has led to a disappointingly small number of lead series that have mostly evaporated under closer scrutiny. In the meantime, multi-drug resistant pathogens are becoming a serious challenge in the clinic and the community and the number of pharmaceutical firms pursuing antibacterial discovery has declined. Filling the antibacterial development pipeline with novel chemical series is a significant challenge that will require the collaboration of scientists from many disciplines. Fortunately, advancements in the tools of structural biology and of in silico modeling are opening up new avenues of research that may help deal with the problems associated with discovering novel antibiotics.

In vivo labeling: a glimpse of the dynamic proteome and additional constraints for protein identification.

Identities ascribed to the intact protein ions detected in MALDI-MS of whole bacterial cells or from other complex mixtures are often ambiguous. Isolation of candidate proteins can establish that they are of correct molecular mass and sufficiently abundant, but by itself is not definitive. An in vivo labeling strategy replacing methionine with selenomethionine has been employed to deliver an additional constraint for protein identification, i.e., number of methionine residues, derived from the shift in mass of labeled versus unlabeled proteins. By stressing a culture and simultaneously labeling, it was possible to specifically image the cells’ response to the perturbation. Because labeled protein is only synthesized after application of the stress, it provides a means to view dynamic changes in the cellular proteome. These methods have been applied to identify a 15,879 Da protein ion from E. coli that was induced by an antibacterial agent with an unknown mechanism of action as SpY, a stress protein produced abundantly in spheroplasts. It has also allowed us to propose protein identities (and eliminate others from consideration) for many of the ions observed in MALDI (and ESI-MS) whole cell profiling at a specified growth condition.