Timothy L. Foley

Targeting iron assimilation to develop new antibacterials.

Introduction: Since the first application of antibiotics to treat bacterial infections, the development and spread of resistance has been a persistent threat. An ever evolving pipeline of next-generation therapeutics is required for modern medicine to remain one step ahead of pathogens. Areas covered: This review describes recent efforts to develop drugs that interrupt the assimilation of iron by bacteria: a process that is vital to cellular homeostasis and is not currently targeted by antibiotics used in the clinic. This review also covers the mechanisms by which bacteria acquire iron for their environment, and details efforts to intervene in these processes, using small molecule inhibitors that target key steps in these pathways, with a special emphasis on recent advances published during the 2010 – 2012 period. Expert opinion: For decades, the routes used by bacteria to assimilate iron from host and environmental settings have been the subject of intense study. While numerous investigations have identified inhibitors of these pathways, many have stopped short of translating the in vitro results to in vivo proof of concept experiments. The extension of preliminary findings in this manner will significantly increase the impact of the field.

An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis.

A significant gap exists between genetics-based investigations of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways and our understanding of their regulation, interaction, and activity in living systems. To help bridge this gap, here we present an orthogonal active site identification system (OASIS) for the proteomic identification and analysis of PKS/NRPS biosynthetic enzymes. OASIS probes target conserved features of PKS/NRPS active sites to provide activity-based enrichment of modular synthases, followed by analysis through multidimensional protein identification technology (MudPIT) LC-MS/MS analysis. When applied to the model bacterium Bacillus subtilis, this functional proteomics method detects and quantifies all four modular synthases in the organism. Furthermore, tandem application of multiple OASIS probes enhances identification of specific PKS/NRPS modules from complex proteomic mixtures. By expanding the dynamic range of proteomic analysis for PKS/NRPS enzymes, OASIS offers a valuable tool for strain comparison, culture condition optimization, and enzyme discovery.

An Optimized Immunoaffinity Fluorescent Method for Natural Product Target Elucidation

Understanding the mode of action of small molecules is an integral facet of drug discovery. We report an optimized immunoaffinity fluorescent method that allows one to conduct parallel studies at both the cellular and molecular level using a single probe construct. Viability of the method has been evaluated analytically and applied using glycyrrhetic acid as a model.

4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), a potent inhibitor of bacterial phosphopantetheinyl transferase that attenuates secondary metabolism and thwarts bacterial growth.

4′-Phosphopantetheinyl transferases (PPTases) catalyze a post-translational modification essential to bacterial cell viability and virulence. We present the discovery and medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides, which exhibit submicromolar inhibition of bacterial Sfp-PPTase with no activity toward the human orthologue. Moreover, compounds within this class possess antibacterial activity in the absence of a rapid cytotoxic response in human cells. An advanced analogue of this series, ML267 (55), was found to attenuate production of an Sfp-PPTase-dependent metabolite when applied to Bacillus subtilis at sublethal doses. Additional testing revealed antibacterial activity against methicillin-resistant Staphylococcus aureus, and chemical genetic studies implicated efflux as a mechanism for resistance in Escherichia coli. Additionally, we highlight the in vitro absorption, distribution, metabolism, and excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate the potential utility of this small-molecule inhibitor.

Preparation of FRET reporters to support chemical probe development

In high throughput screening (HTS) campaigns, the quality and cost of commercial reagents suitable for pilot studies often create obstacles upon scale-up to a full screen. We faced such challenges in our efforts to implement an HTS for inhibitors of the phosphopantetheinyl transferase Sfp using an assay that had been validated using commercially available reagents. Here we demonstrate a facile route to the synthetic preparation of reactive tetraethylrhodamine and quencher probes, and their application to economically produce fluorescent and quencher-modified substrates. These probes were prepared on a scale that would allow a full, quantitative HTS of more than 350,000 compounds.

Phosphopantetheinyl transferase inhibition and secondary metabolism

Efforts to isolate carrier protein‐mediated synthases from natural product‐producing organisms using reporter‐linked post‐translational modification have been complicated by the efficiency of the endogenous process. To address this issue, we chose to target endogenous phosphopantetheinyl transferases (PPTases) for inhibitor design to facilitate natural product synthase isolation through a chemical genetics approach. Herein we validate secondary metabolism‐associated PPTase for chemical probe development. We synthesized and evaluated a panel of compounds based on the anthranilate 4H‐oxazol‐5‐one pharmacophore previously described to attenuate PPTase activity within bacterial cultures. Through the use of a new high‐throughput Förster resonance energy transfer assay, we demonstrated that these compounds exclusively inhibit fatty acid synthase‐specific PPTases. In vivo, a lead compound within this panel demonstrated selective antibiotic activity in a Bacillus subtilis model. Further evaluation demonstrated that the compound enhances actinorhodin production in Streptomyces coelicolor, revealing the ability of this class of molecules to stimulate precocious secondary metabolite production.

Covalent Small Molecule Inhibitors of Ca(2+)-Bound S100B.

Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca2+-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNAS100B) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B–p53 complex and restore active p53 in this deadly cancer. Using a structure–activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1–3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX–S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an S100B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity.

The unusual macrocycle forming thioesterase of mycolactone

Mycolactone is a polyketide natural product secreted by Mycobacterium ulcerans, the organism responsible for the tropical skin disease Buruli ulcer. The finding that this small molecule virulence factor is sufficient to reconstitute the necrotic pathology associated with Buruli ulcer suggests that a better understanding of mycolactone biosynthesis, particularly the processes which are distinct from those in human metabolism, may provide a unique avenue for the development of selective therapeutics. In the present study we have cloned, expressed, and biochemically characterized the putative macrocycle forming thioesterase for mycolactone, MLSA2 TE. We have evaluated the enzyme both as the truncated thioesterase domain and as a carrier protein-linked didomain construct. The results of these analyses distinguish MLSA2 TE from traditional fatty acid and polyketide synthase TE-domains in terms of its sequence, kinetic parameters, and susceptibility to traditional active-site directed inhibitors. These findings suggest that MLSA2 TE utilizes a unique biochemical mechanism for macrocycle formation.

Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors.

Phosphopantetheinyl transferase (PPTase; E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low-molecular weight CoA substrate to higher-molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of PPTase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human PPTase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict the therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity.

Laetirobin from the parasitic growth of Laetiporus sulphureus on Robinia pseudoacacia.

(+/-)-Laetirobin (1) was isolated as a cytostatic lead from Laetiporus sulphureus growing parasitically on the black locust tree, Robinia pseudoacacia, by virtue of a reverse-immunoaffinity system. Using an LC/MS procedure, milligram quantities of (+/-)-laetirobin (1) were obtained, and the structure of 1 was elucidated by X-ray crystallography and confirmed by NMR spectroscopy. Preliminary cellular studies indicated that (+/-)-laetirobin (1) rapidly enters in tumor cells, blocks cell division at a late stage of mitosis, and invokes apoptosis.